INDIAN JOURNAL OF PRACTICAL PEDIATRICSijpp.in/admin/uploadimage/jul-sep-09.pdf2009; 11 (3) : 205...

2009; 11 (3) : 205

INDIAN JOURNAL OFPRACTICAL PEDIATRICS

••••• IJPP is a quarterly subscription journal of the Indian Academy of Pediatricscommitted to presenting practical pediatric issues and managementupdates in a simple and clear manner

••••• Indexed in Excerpta Medica, CABI Publishing.

Vol.11 No.3 JUL.-SEP. 2009

Dr. K.Nedunchelian Dr. S. ThangaveluEditor-in-Chief Executive Editor

CONTENTS

FROM THE EDITOR'S DESK 211

TOPIC OF INTEREST - RATIONAL INVESTIGATIONS IN INFECTIONS

EDITORIAL 214

Conventional blood culture and antimicrobial sensitivity testing 215

- Tanu Singhal, Simantini JogAutomated culture and rapid antigen tests 223

- Camilla RodriguesMarkers of infection 230

- Panna S Patel, Rajal B Prajapati, Baldev S PrajapatiRational investigations in CNS infections 239

- Digant D ShastriRational investigations in community acquired pneumonia andits complications 246

- Nupur GangulyRational investigations to diagnose enteric fever 252

- Tapan Kumar GhoshRational investigations in urinary tract infection 260- Ajay Kalra

Journal Office and address for communications: Dr. K.Nedunchelian, Editor-in-Chief, Indian Journal of PracticalPediatrics, 1A, Block II, Krsna Apartments, 50, Halls Road, Egmore, Chennai - 600 008. Tamil Nadu, India.Tel.No. : 044-28190032 E.mail : [email protected]

1

Indian Journal of Practical Pediatrics 2009; 11(3) : 206

Published by Dr.K.Nedunchelian, Editor-in-Chief, IJPP, on behalf of Indian Academy of Pediatrics,from 1A, Block II, Krsna Apartments, 50, Halls Road, Egmore, Chennai - 600 008. Tamil Nadu, Indiaand printed by Mr. D. Ramanathan, at Alamu Printing Works, 9, Iyyah Street, Royapettah,Chennai - 14.

FOR YOUR KIND ATTENTION

* The views expressed by the authors do not necessarily reflect those of the sponsor orpublisher. Although every care has been taken to ensure technical accuracy, no responsibility isaccepted for errors or omissions.

* The claims of the manufacturers and efficacy of the products advertised in the journal are theresponsibility of the advertiser. The journal does not own any responsibility for the guarantee of theproducts advertised.

* Part or whole of the material published in this issue may be reproduced withthe note "Acknowledgement" to "Indian Journal of Practical Pediatrics" without prior permission.

- Editorial Board

Rational investigations to diagnose leptospirosis 265

- Jaydeep Choudhury

Rational investigations in malaria 272

- Ritabrata Kundu

Rational investigations for Kala - Azar 279

- Nigam Prakash Narain, Neelam Verma

Rational investigation practices in the diagnosis of dengue 283

- Vijay N Yewale

DERMATOLOGY

Tinea Capitis 294

- Madhu R

RADIOLOGIST TALKS TO YOU

Aqueductal stenosis and hydrocephalus 300

- Vijayalakshmi G, Elavarasu E, Venkatesan MD

CASE STUDY

Esophagogastric junctional adenocarcinoma in a child 303

- Radhika, Malathi S, Janani S, Kalaiselvi

Duodenal angiodysplasia - a rare case of upper gastrointestinalbleed in a young child 306

- Sumathi B, Nirmala D, Bhaskar Raju B, Rajalakshmi V

CLIPPINGS 222, 264, 299, 302

NEWS AND NOTES 229, 238, 245, 278, 293

2

2009; 11 (3) : 207

3


4

�1

�F�r�i�d�a�y�,� �A�u�g�u�s�t� �2�0�,� �2�0�1�0� �1�2�:�1�9�:�2�9� �P�M

�5

�F�r�i�d�a�y�,� �A�u�g�u�s�t� �2�0�,� �2�0�1�0� �1�2�:�2�2�:�3�5� �P�M

2009; 11 (3) : 211

FROM THE EDITOR’S DESK

This issue covers articles on “Rationalinvestigations” for various common infections inpediatric age group. The literal meaning of “rational”is “relating to the reason or reasoning”. “Rationalinvestigation” implies, that it is essential to order thetest which is most needed as well as not leaving outany such test where in need.

The diagnostic resources are growing along withsteady increase in selection of test(s) as well. Manyfactors seem to be responsible for increased use ofinvestigations. Considerations other than scientificindications including pressure from patients, fear ofmissing an important diagnosis and desire for opinionsto be backed up by a positive test result are mostlyobserved. With chronic illnesses, patients will haverecurrent vague complaints for which doctors tend toover-investigate. Increasing demand for care, easyavailability of new tests and the urge to use newerinvestigations, etc are also other reasons.

Preliminary requirement for the practice of“Rational investigations” is the correct clinicaldiagnosis and some times differential diagnoses. Thisnecessitates the physician to be well versed with usualsymptoms and signs of common infections andunusual manifestations of otherwise commoninfections. They should be aware of prevailingcommon infections and updated with changingpattern if any, over time. While ordering test(s), it isequally important that the physician should have aclear cut idea on the test to be done like, whichspecimen to be tested, at which stage of the disease,correct interpretation of the test result, etc to avoidmisdiagnosis.

When submitting a patient for evaluation, thedecision whether to do multiple tests sequentially oras cluster of tests simultaneously, may be decidedbased on factors like resources available, time framefor workup and graveness of underlying illness, theprogression of illness, etc. Sequential testing is costeffective which is essential in a resource poor setting,but takes longer time to confirm the diagnosis. A testwith more sensitivity is used initially to select probable

cases, on whom most specific test is done to confirmthe diagnosis. The cluster testing has the advantageof arriving at the diagnosis in short time whencompared to the process of sequential testing whichis cost effective. Here, few tests which when combinedimproves the specificity in confirming the diagnosisstraightaway. Eg. To prove tuberculous etiology of apneumonia, one approach is to consider x-ray, culturetests, etc only in Mantoux positive stable patients(sequential) and the second approach is to doMantoux, x-ray and culture tests simultaneously(cluster) especially when dealing with a sick childwhere we cannot afford to waste time. The over useof investigations increases workload of the laboratoryand expenditure to patients. Interventions focusingon avoidence of inappropriate

testing might reduce

costs while simultaneously improving quality of care.

General practitioners should be sensitized topractice rational use of lab tests or not using particulartest(s). Guidelines, protocols and standards are to beframed. Simply distributing guidelines, alone does notmake clinicians follow them. They should bemotivated to follow guidelines, throughcommunication and information about the contentsand relevance, also make them realize the problemof inappropriate tests. Adequate patient education thatnot all tests give reliable results should be emphasised.Many times the value of investigations, especially inprimary care, is limited. This requires, first of all, thatdoctors know the principles of medical decisionmaking and its relevance to daily practice.

The IJPP team thanks Dr.Tapan Kumar Ghoshfor his untiring effort in coordinating to bring out thisissue and contributing guest editorial.

Apart from the articles on topic of interest, thisissue includes the article “ Aqueductal stenosis andhydrocephalus” under the column “Radiologist talksto you” and another article “Tinea capitis” under“Dermatology” series.

Dr.K.NedunchelianEditor-in-Chief

7


GeneralPrint the manuscript on one side of standard size A4, white bond paper, with margins of at least 2.5 cm (1”)in double space typescript on each side. Use American English using Times New Roman font 12 size.Submit four complete sets of the manuscript.They are considered for publication on the understanding that they are contributed to this journal solely.All pages are numbered at the top of the right corner, beginning with the title page.All manuscripts should be sent to: The Editor-in-Chief, Indian Journal of Practical Pediatrics

Manuscript1st Page –

TitleName of the author and affiliationInstitutionAddress for correspondence (Email, Phone, Fax if any)Word countNo. of figures (colour / black and white)No. of referencesAuthors contribution

2nd Page –Abstract (unstructured, not exceeding 100 words) with key words (not exceeding 4)

3rd Page -AcknowledgementPoints to remember (not more than 5 points)TextReferencesTablesFigures – should be good quality, 4 copies black & white / colour, (4 x 6 inches – Maxi size) G l o s s yprint. (Each colour image will be charged Rs.1,000/- separately)Legends

TextOnly generic names should be usedMeasurements must be in metric units with System International (SI) Equivalents given in parentheses.

ReferencesRecent and relevant references onlyStrictly adhere to Vancouver styleShould be identified in the text by Arabic numerals in parentheses.Type double-space on separate sheets and number consecutively as they appear in the text.Defective references will entail rejection of article

TablesNumbered with Roman numerals and typed on separate sheets.Title should be centered above the table and explanatory notes below the table.

Figures and legendsUnmounted and with figure number, first author’s name and top location indicated on the back of eachfigure.Legends typed double-space on separate sheet. No title on figure.

INSTRUCTIONS TO AUTHORS

8

2009; 11 (3) : 213

Article Categories

Review article

Article should be informative covering the recent and practical aspects in that field. Main articles can be in1500 – 2000 words with 12 – 15 recent references and abstract not exceeding 100 words.

Case report (covering practical importance)

250 – 600 words, 8 – 10 recent references

Clinical spotters section

100 – 150 words write up

With 1 or 2 images of clinically recognizable condition

(of which one could be in the form of clinical photograph / specimen photograph / investigation)

Letters to the Editor

200 – 250 words pertaining to the articles published in the journal or practical viewpoints with scientificbacking and appropriate references in Vancouver style.

Selection procedures

All articles including invited articles will be peer reviewed by two masked reviewers. The decision of theEditorial Board based on the reviewers’ comments is final.

Check List

Covering letter by corresponding author

Declaration (as enclosed) signed by all authors **

Manuscript (4 copies)

Accompanied by a copy in CD / or submit as an email attachment in addition to hard copy.

Failing to comply with the requirement at the time of submission would lead to the rejection of the article.

Author’s contribution / Authorship Criteria

All persons designated as authors should qualify for the authorship. Authorship credit should be based on substantialcontributions to i) concept and design, or collection of data, and interpretation of data;ii) drafting the article or revising it critically for important intellectual content; and iii) final approval of theversion to be published. All conditions i), ii) and iii) must be met.

**Declaration by authors

I/We certify that the manuscript titled ‘……………………………….’ represents valid work and that neitherthis manuscript nor one with substantially similar content under my/our authorship has been published or isbeing considered for publication elsewhere. The author(s) undersigned hereby transfer(s), assign(s), or otherwiseconvey(s) all copyright ownership, including any and all rights incidental thereto, exclusively to the IndianJournal of Practical Pediatrics, in the event that such work is published in Indian Journal of Practical Pediatrics.I / we assume full responsibility for any infringement of copyright or plagiarism.

Authors’ name(s) in order of appearance in the manuscript

Signatures (date)

All manuscripts, which are rejected will not be returned to author. Those submitting articles shouldtherefore ensure that they retain at least one copy and the illustrations, if any.

9


RATIONAL INVESTIGATION

PRACTICES TO ESTABLISH THE

DIAGNOSIS OF INFECTIOUS

DISEASES IN CHILDREN

Of the various issues related to rationalpractice in infectious diseases, properantimicrobial policy is most important. This alsohelps to reduce the development of mono ormulti-drug resistance by the micro-organismswhich commonly infect human beings includingchildren. Similar important issue in infectiousdiseases is the practice of rational investigationpractices. Though we are discussing here ondiagnosis of infectious diseases in children only,it is true for all diseases and for all age groups.Common habit of advising a battery ofinvestigations by the so called specialists doesnot always help to arrive the diagnosis but alsomany times leads to confusion. After listeningto the history and examination of patient, thoughtprocess should be activated first and then onlythe needed investigations should be advised.In many times, the treatment is done in steps,similarly the investigation advice will be madeonly on the need basis according to the step orphase of treatment, eg, advising Widal test in1st week of enteric fever will not help at all butlead to confusion. Similarly advising bloodculture after starting empiric antibiotic therapywill be less useful. Times have changed and thepractice of empiric treatment on presumption isno longer recommended except in serious statesof the disease when saving life is an issue. Allefforts should be taken to diagnose and isolatethe organism. Here the role of rationalinvestigation policy will come. Advising CBC(Complete Blood Count) in seasonal viralinfection, which presents with coryza andmoderate degree fever is totally unnecessary.But with progression of disease, if fever turns

into a long, lasting one the role of someinvestigations may be warranted.

Advice of advanced investigationseg, molecular diagnoses like PCR before the firstline investigations is useless in most of the cases.Rapid diagnostic tests for Plasmodiumfalciparum is important but without advising thinand thick blood film examination foridentification of malaria parasite, advising rapidantigen tests is not a wise decision. If antibioticis to be prescribed in a suspected pyogenicmeningitis case, it is to be advised after doingthe lumbar puncture and sending the CSFspecimen for investigation.

Rational investigation practice apart fromhelping the patient to receive rational therapy,also helps the family financially. At the same timethis helps the medical personnel to developconfidence in himself. He learns to pin point thenecessity of investigation and adviceaccordingly. Debate may arise on this issue butafter all controversies we have to agree that aknowledge of advising investigations in arational manner is very important in day to dayoffice practice and above all it is an art ofpractice.

Lastly, it is to be mentioned that a basicknowledge about the process of investigation isalso important for practicing physician eg, slideWidal test is useless. Only presence of cysts ofEntameba histolytica does not need any treatmentof amebiasis, presence of trophozoites isimportant for that. In blood culture, the bloodshould be inoculated in cultue media at thebedside. This type of knowledge is important.Otherwise rationality of investigations can notbe achieved in practice.

Dr Tapan Kr GhoshScientific Coordinator

Institute of Child Health, Calcutta

EDITORIAL

10

2009; 11 (3) : 215

CONVENTIONAL BLOOD CULTURE

AND ANTIMICROBIAL

SENSITIVITY TESTING

*Tanu Singhal**Simantini Jog

Abstract: Blood culture is an importantinvestigation and the gold standard for diagnosisof many common infectious diseases.Though automated methods offer significantadvantages over conventional blood cultures theyare not widely available. Most clinicians in thecountry have to still resort to conventionalsystems. It is necessary to inculcate the cultureof sending blood cultures. Blood cultures shouldalways be sent from peripheral venipuncture,after thorough cleansing of the skin andpreferably before administration of antibiotics.The volume of blood for culture is importantparticularly in older children and adults.Appropriate processing of the cultures by the laband proper interpretation of the results is crucial.Antimicrobial sensitivity testing (AST) assumesgreat importance in today’s era of risingantimicrobial resistance. Disc diffusion methodsare the most widely used methods for sensitivitytesting. In certain situations dilution methods,automated tests and gradient diffusion methodsmay be used. Here again appropriatemethodology, good quality control and proper

interpretation of the results is paramount.Clinicians should pay due importance to varioushost and pathogen factors in addition to ASTresults before making antimicrobial choices.

Key words: Blood culture, Antimicrobialsensitivity testing

Blood culture is a crucial microbiologicinvestigation for the diagnosis of infectiousdiseases. It is the gold standard for diagnosis ofbacteremias (pneumococcal/ Hib/ staphylo-coccal), infective endocarditis, enteric fever andnosocomial blood stream infections. Apart fromdiagnosis of the etiologic agent, blood culturesallow for assessment of antimicrobial sensitivityof the isolate which is crucial in today’s era ofantimicrobial resistance. This write up addressesconventional blood culture methods.

Method of collection

Process of collection

The chances of contamination of blood forculture should be minimized as contaminatedblood cultures often cause an extension of stayof the patient in hospital with increase in costdue to additional tests and intravenousantibiotics. The rate of contaminated bloodcultures should not exceed 3%.1 In order tominimize contamination rates, the venipuncturesites should preferably be cleaned with threeapplications of the antiseptic agent allowing itto dry in between applications. Options for skinantisepsis include 70% alcohol, 10% aqueouspovidone iodine and then 70% alcohol in thatorder. There is some recent evidence that2% alcoholic chlorhexidine is superior to

* Consultant, Pediatrics and Specialist, InfectiousDiseases,Kokilaben Dhirubhai Ambani Hospital andMedical Research Centre, Mumbai

** Specialist Registrar,Clinical Microbiology & VirologyRoyal Devon & Exeter NHS Foundation Trust

11

RATIONAL INVESTIGATIONS IN INFECTIONS


10% aqueous povidone iodine in reducing bloodculture contamination rates.2 Cultures should bedrawn from venipuncture and not from centralvenous catheters/ arterial catheters. In case acatheter related blood stream infection issuspected, paired cultures should be drawn froma peripheral venipuncture and the catheter sitesimultaneously. The change of the needle usedfor venipuncture to a sterile needle prior toinjecting in the blood culture bottle is notrecommended owing to the risk of needle stickinjury.1 Additionally studies have demonstratedthat change of needles does not significantlyimpact the rate of blood culture contamination.3

Number, volume and timing of collection

The maximal yield is obtained withcollection of three blood culture sets in the first24 hours of illness; each set here denoting twobottles. The yield with one set is around 80%,with the second set is 90% and with the third setis 99%.4 With the exception of communityacquired bacteremia in otherwise normal healthychildren and in suspected neonatal sepsis whereone bottle has been demonstrated as sufficient,solitary blood cultures are not recommended.5,6

If there are resource constraints, at least one setshould be obtained. In the earlier days, each setcomprised of one aerobic and one anaerobicbottle but in the current scenario it isrecommended that each set comprises of twoaerobic bottles unless an anaerobic infection issuspected. This practice of two aerobic bottlesalso enhances the recovery of fungi which havebecome important nosocomial pathogens incurrent day and age.

The volume of blood collected is animportant determinant of the recovery ofpathogens. In adults at least 10-30 ml of bloodshould be drawn per venipuncture; and 10 ml ofblood should be injected in each bottle.Studies show that the rate of isolation increases

by approximately 3% per ml of blood cultured.The ratio of blood to media should ideally be 1:5to dilute any antibacterial properties of blood.Hence the practice of injecting more blood perbottle beyond the prescribed limit should beavoided. In infants and children the volume ofblood per pediatric bottle should be 1-5 ml.Interestingly, in neonates and infants, studieshave shown that even volumes less than 1 ml havesensitivity comparable to larger volumes.6

The cultures should be drawn preferablybefore administration of antimicrobials; howeverjust because a patient is on antibiotics should notpreclude one from obtaining cultures, althoughthis needs to be taken into consideration whenculture results are interpreted. Obtaining theblood cultures one half hour before thetemperature spike is ideal as the highestconcentration of organisms are circulating at thattime. However because the temperature spike isusually unpredictable, an educated guess aboutthe temperature spike suffices. For patients withacute infective endocarditis, three blood culturesshould be drawn from three separatevenipuncture sites within the 1st 2 hours ofevaluation. For subacute bacterial endocarditisthree blood cultures on the 1st day arerecommended spacing venipuncture at least30 minutes apart.

Culture method1

Culture media

The medium used in blood culture bottlesis multipurpose and nutritionally enriched: trypticor trypticase soy, supplemented peptone broth,brain- heart infusion, Columbia broth. All arecommercially available and slightly differ incomposition depending on the manufacturer.Most commercially available blood culture mediacontain the anticoagulant sodium polyanethol-sulfonate (SPS) in concentrations varying from0.025% to 0.05%. Anticoagulation is desirable

12

2009; 11 (3) : 217

as certain bacteria do not survive well within theclot where phagocytosis by neutrophils andmacrophages remains active. Additionally, SPSalso inactivates neutrophils and certainantibiotics and precipitates complementcomponents thus improving recovery. SPS maybe inhibitory for certain bacteria includingmeningococci and gonococci and when theseorganisms are suspected an alternate broth maybe needed or neutralization of SPS antibacterialactivity by 1% gelatin may be needed.Special media may be needed for certain bacteriasuch as brucella. Blood culture bottlesincorporating synthetic antibiotic-removingresins (usually available only in automatedsystems) enhance recovery of organisms inpatients on antibiotics.

Special media is required for fungal andmycobacterial blood cultures. The use of the lysiscentrifugation blood culture system (whereinblood is lysed with saponin in special tubes,centrifuged and sediment sub cultured) hasshown to significantly improve the recovery offungal pathogens but with increased risk ofcontamination.

Processing and incubation of culture media

Blood culture bottles should be incubatedat 35°C and examined visually for evidence ofgrowth (hemolysis, gas production or turbidity)during the first 6-18 hours of collection againstbright fluorescent bulbs or with incandescenttransmitted light. The surface of the sedimentedblood layer should be examined for any discretecolonies. Blind subcultures on agar plates shouldbe made from all blood culture bottles within12 to 24 hours of collection and every alternateday and incubated aerobically at 5-10% CO

2.

The routine microscopic examination ofmacroscopically negative bottles after 24 hoursof incubation is not indicated because the numberof organisms that can be detected by gram stain

(about 105 CFU) varies little from the 106 to 107

CFUs required to produce a visible turbidity ofthe broth. Acridine orange stains are moresensitive detecting 103 to 104 CFUs per ml.In case visual growth is seen, it is recommendedthat both aerobic and anaerobic cultures are setup. Studies have shown that incubatingconventional blood culture bottles beyond 7 daysis unnecessary unless certain fastidiousorganisms such as Eikenella corrodens,Cardiobacterim hominis, and Actinobacillusspecies are suspected.

Interpretation of culture results

A positive blood culture may representcolonization or contamination or true infection.Colonization is likely if cultures are drawnthrough indwelling lines or catheters.Hence when catheter related infections aresuspected paired cultures from the line andperipheral venipuncture should be sent. Only thecentral line culture being positive indicatespossible colonization. Contamination may occurduring the process of collection or inoculationin the culture media. Usual blood culturecontaminants include coagulase negativestaphylococcus and diphtheroids. Sendingpaired cultures helps as contaminants are oftenfound only in one culture and not the other.Also contaminated cultures tend to becomepositive usually after the first 48 hours unliketrue infection which tend to become positiveearlier. Isolation of S. aureus, all gram negativeorganisms and fungi from peripheralvenipuncture samples almost always indicate trueinfection.

Certain organisms including Salmonella,S. aureus are easier to isolate unlike fastidiousorganisms such as pneumococci andH. influenzae where isolation is difficultespecially if antibiotics have been administeredprior to cultures.

13


Antimicrobial sensitivity testing(AST) 7-11

AST is extremely important in the currentday scenario with worldwide development andspread of antimicrobial resistance. It is imperativethat laboratories perform well standardized invitro AST’s and generate reliable reports that aidthe clinicians in optimal management ofinfectious diseases. Reliable AST’s also help incutting down antimicrobial misuse. At the sametime, it is also important that clinicians are welltrained in the interpretation of AST reports sothat they make correct antimicrobial choices.This write up attempts to expound some of thebasic principles behind AST in the laboratory.

Interpretation of AST

Isolates may be classified as susceptible,resistant or of intermediate susceptibility.

Susceptible

There is a high probability that the patientwill respond to treatment with the appropriatedosage of that antimicrobial agent.

Resistant

Treatment with the antimicrobial agent islikely to fail.

Intermediate

With the agents that can be safelyadministered at higher doses, this categoryimplies that higher doses maybe required toensure efficacy or that the agent may proveefficacious if it is normally concentrated in aninfected body fluid, e.g. urine. For bodycompartments where drug penetration isrestricted even in the presence of inflammation,e.g. CSF, it suggests that extreme caution shouldbe taken in the use of the agent. Thus, thiscategory represents a “buffer” zone that prevents

strains with borderline susceptibility from beingincorrectly categorized as resistant.

Labeling a resistant isolate as sensitive isconsidered a very major error in AST reportingwhile characterizing a sensitive isolate asresistant is a major error. Minor errors arecharacterization of a susceptible or resistantisolate as intermediate or characterization of anintermediate isolate as susceptible or resistant.

Methods for antibiotic sensitivitytesting

Disc diffusion is the most commonly usedmethod for AST and gives a qualitative estimationof the sensitivity. Other methods which allowquantitative assessment are, theagar and broth dilution methods, antibioticgradient methods and automated methods.The laboratories worldwide adhere to standardsset and published by Clinical and Lab StandardsInstitute- CLSI (formerly National Committee forClinical Laboratory Standards – NCCLS) and itis important that all laboratories adhere to thesestandards to ensure accuracy and reproducibilityof results.8 The choice of antimicrobial agentsfor AST depends on the organism isolated, thesite of infection, the clinical practice setting inwhich the laboratory functions, the patterns ofantibiotic usage and bacterial resistance in thecommunity. Those agents that are prescribed bythe physicians on a daily basis should be tested.CLSI publishes tables listing the antimicrobialsappropriate for testing various groups of aerobicand fastidious bacteria. These guidelines indicatedrugs that are most appropriate for testing eachorganism group and for treatment based on thespecimen source, for e.g. CSF, blood, urine orfaeces. It also includes a few agents that may betested as surrogates for other agents due to thegreater ability of a particular agent to detectresistance to closely related drugs, for e.g. theuse of oxacillin to predict overall beta lactamresistance in staphylococci.

14

2009; 11 (3) : 219

Disc diffusion susceptibility tests: These arethe most commonly performed susceptibilitytests. Commercially prepared filter paper discsimpregnated with a specified single concentrationof an antimicrobial agent are applied to thesurface of an agar medium inoculated with thetest organism. When the disc comes into contactwith the moist agar surface, water is absorbedinto the filter paper and the antibiotic startsdiffusing into the surrounding medium. As thedistance from the disc increases, theconcentration of the antimicrobial agentdecreases logarithmically, creating a gradient inthe agar medium surrounding each disc.Simultaneous growth of bacteria occurs on theagar surface when a critical cell mass of bacteriais reached and the inhibitory activity of theantibiotic is overcome. The points at which thecritical cell mass is reached appear as a sharplymarginated circle of bacterial growth with themiddle of the disc forming the centre of the circle.The concentration of the antibiotic at thisinterface of growing and inhibited bacteria isknown as the critical concentration.After incubation for 16-18 hrs, the zone diametersare measured around each disc and are interpretedon the basis of CLSI guidelines and the organismsreported as susceptible, intermediate or resistantto the antimicrobial agent tested.

The advantages of disc diffusion testing isthat it is technically simple to perform,reproducible, relatively inexpensive, does notneed special equipment, flexible as regards tothe antimicrobial agents selected for testingrequired and susceptibility results are easilyunderstood by the clinicians. The disadvantagesare that it is standardized for only a limitednumber of organisms, provides a qualitative andnot a quantitative result and is inadequate for thedetection of vancomycin intermediate S. aureus,oxacillin heteroresistant staphylococci, low level(Van-B type) vancomycin resistant enterococciand some gram negative organisms such asP. cepaciae.

Dilution tests: These methods are used todetermine the Minimum Inhibitory Concentration(MIC), which is the lowest concentration of theantimicrobial agent that inhibits the visiblegrowth of the organism. The concentration rangeused varies with the drug, the organism beingtested and also the site of infection. These testsmay be done in solid media (agar dilution tests)or in liquid media (macrodilution/ microdilutionbroth tests). The MIC obtained is then comparedwith the standard MIC sensitive and resistantbreakpoints to classify an organism assusceptible, resistant or intermediate. The MICsensitive breakpoint is the highest MIC at whichthe microbe is usually sensitive, values lower thanthis are susceptible. The MIC resistant breakpointis the lowest MIC at which the microbe is usuallyresistant, values higher than this are resistant.The isolates with MIC in between the sensitiveand resistant breakpoints are of intermediatesusceptibility. The MIC breakpoints areestablished on the basis of 3 parameters;microbiological data (the MIC distribution of alarge number of clinical isolates),pharmacokinetic data (the achievable drugconcentration in various body fluids and tissues)and clinical study data (the clinical efficacy withattainable serum concentrations of the drug).The MIC breakpoints are not fixed and have tobe upgraded from time to time depending on theability of the organism to acquire resistance whenthe antibiotic is in use. It is important that thelatest MIC breakpoints should be used forinterpretation.

The advantages of dilution tests is that theyare well standardized and the reference methodagainst which all methods are evaluated.However they are time consuming and expensiveand therefore are indicated only in certainsituations as detailed below

• Testing isolates from patients withendocarditis or osteomyelitis.

15


• For research purposes, in order to evaluatethe relative degrees of susceptibility ofbacteria to various antimicrobial agents.

• Some species exist where resistantmechanisms might not be reliably detectedby standard disc diffusion testse.g. Vancomycin intermediate S.aureus.

• Some species for which standard discdiffusion test is not well calibratede.g. Stenotrophomonas maltophilia,Burkholderia cepacia.

• Some species for which no disc teststandards exist – Corynebacterium spp,Bacillus spp

• Some species for which MICs are needed toguide selection of therapy and appropriatedosing – penicillin and cephalosporinresistant S. pneumoniae.

Other methods : Other rapid and easyto use MIC determination methods includegradient diffusion methods (E test) and automatedsystems (Vitek and Microscan). These arehowever expensive and should be used whenMIC determination is required (discussedearlier).

Caveats in interpretation of AST

Administration of an antibiotic to whichinvitro susceptibility has been documented byitself does not guarantee a clinical cure. Clinicalcure depends on other host and drug factors someof which are discussed.

1. In vivo versus in vitro susceptibility: Despiteinvitro susceptibility, Trimethoprimsulfamethoxazole (TMP/SMX) is not effectivein vivo to treat Klebsiella infections;Aminoglycosides alone have littleantistreptococcal activity, but when combinedwith a penicillin, aminoglycosides are activeagainst streptococci, including group D

streptococci; S. typhi may show invitrosusceptibility to quinolones but there may beclinical failure to quinolones if the isolate isnalidixic acid resistant.

2. Inadequate blood/tissue levels : In general,there is a step-down in concentration betweenthe blood and tissue levels with beta-lactamantibiotics. With cephalosporins, in most well-vascularized organs (excluding special locations,e.g., the urinary tract, central nervous system,prostate), tissue levels are about one quarter ofsimultaneous serum levels. Adequate tissueconcentrations in most areas of the body areregularly achieved using the full doses ofantibiotics.

3. Antibiotic tissue penetration problems: Mostantibiotics penetrate well-defined abscessespoorly, if at all. Surgical drainage remains thecorner-stone of the therapeutic approach in thepatient with abscesses. Infections involvingforeign implant material usually have aglycocalyx around the foreign body which formsa biofilm and this cannot be eliminated byantimicrobial therapy. Such infections almostalways require removal of the prosthetic device.Infections involving certain anatomic sites in thebody are protected from the effects of mostantibiotics given in the usually recommendeddosages, e.g, the cerebrospinal fluid, prostate.In subacute or chronic prostatitis, however, inwhich inflammation dependent penetration is notvital drugs with high lipid solubility such asdoxycycline, TMP-SMX, quinolones arepreferable.

4. Colonization versus infection: The mererecovery of an organism does not implicate itsetiological role in the underlying infectiousdisease process. Certain organisms are almostalways associated with colonization and onlyrarely cause invasive diseases for e.g. Citrobacterspps. Organisms as Enterobacter spp cause

16

2009; 11 (3) : 221

disease in a narrow range of clinicalcircumstances for e.g. IV line infections, catheterassociated bacteriuria etc. The recovery ofPseudomonas aeruginosa from an endotrachealspecimen in an intubated patient in an intensivecare unit likewise may represent colonization.Treating colonization not only wastes vitalresources but is almost always unsuccessful.

5. Drug fever: Fever may result from thepatient’s reaction with a given medication, andoften it may be the sole manifestation to a givendrug.

Conclusions

It is necessary to inculcate the culture inclinicians of sending appropriate culturesincluding blood cultures when indicated as percorrect protocol. Though automated culturemethods offer significant advantage overconventional culture methods, they may not beuniversally available. Therefore in most settingsconventional blood cultures have an importantrole to play. The laboratory should followstandardized protocols and ensure good qualitycontrol while processing cultures. Finally theclinician should interpret all culture results inlight of the clinical setting and attempt todistinguish true infection from contamination andcolonization.

Antimicrobial sensitivity testing hasassumed great importance in the current day andage of rising antimicrobial resistance. Here againlaboratory standards and quality control areimperative. For most cases, disc diffusion testingsuffices. Dilution and automated methods maybe requested in certain specific settings. Skill ininterpretation of sensitivity testing methods isrequired for optimum antimicrobial therapy.

Points to Remember

• Blood culture is the gold standard fordiagnosis of many common infections.

• Specimen for blood culture is to beprocessed as per standard protocol.

• Culture results to be interpreted alwayswith clinical findings.

• Accurate interpretation of antimicrobialsensitivity leads to optimum antimicrobialtherapy and is mandatory in view of risingantimicrobial resistance.

• Automated culture methods even thoughappear advantageous are not availableuniversally.

References

1. Guidelines for the collection, transport,processing, analysis and reporting of culturesfrom specific specimen sources. In: KonemanEW, Allen SD, Janda WM, Schreckenberger PC,Winn Jr WC, Eds. Colour Atlas and Textbookof Diagnostic Microbiology. 5

th Edn.

Philadelphia: Lippincott; 1997; pp 153-162.

2. Suwanpimolkul G, Pongkumpai M, SuankratayC. A randomized trial of 2% chlorhexidinetincture compared with 10% aqueous povidone-iodine for venipuncture site disinfection: Effectson blood culture contamination rates. J Infect2008;56: 354-359.

3. Isaacman DJ, Karasic RB. Lack of effect ofchanging needles on contamination of bloodcultures. Pediatr Infect Dis J 1990; 9: 274-278.

4. Washington JA, Ilstrup DM. Blood cultures:issues and controversies. Rev Infect Dis 1986;8: 792–802.

5. Paisley JW, Lauer BA. Pediatric blood cultures.Clin Lab Med 1994; 14:17-30.

6. Buttery JP. Blood cultures in newborns andchildren: optimizing an everyday test. Arch DisChild Fetal Neonatal Edn, 2002; 87:F25-F28.

7. Turnidge JD, Ferraro MJ, James H. JorgensenSusceptibility Test Methods: Generalconsiderations. In : Patrick R.Murray, Ellen JoBaron, James H.Jorgensen, Michal A/Pfaller,Robert H.Yolker Eds, Manual of ClinicalMicrobiology, 9

th Edn, ASM Press, Washington

DC., 2003;pp1102-1107.

17


8. Cormican M, Whyte T, Hanahoe B.Antimicrobial Susceptibility Testing in Ireland: An introduction to the methods of the NationalCommittee for Clinical Laboratory Standards(NCCLS) http : //www.amls.ie/ast 110402.htm

9. Elmer W Koneman, Stephen D.Allen, WilliamM.Janda, Paul C.Schreckenberger, WashingtonCwinn.Jr: Colour Atlas and Textbook ofDiagnostic Microbiology, 5

th edn, Lippincott,

Philadelphia, New York, 1997;pp785-856.

10. Jorgensen JH, Turnidge JD. Susceptibility TestMethods: Dilution and Disc: Diffusion MethodsIn: Patrick R.Murray, Ellen Jo Baron, JamesH.Jorgensen, Michal A/Pfaller, RobertH.Yolker: Manual of Clinical Microbiology 9

th

edn, ASM Press, Washington DC, 2003;pp1102-1107.

11. Louie M, Cokerill III FR. Susceptibility Testing:Phenotypic and Genotypic Tests for Bacteriaand Mycobacteria. Infect Dis Clin NAm 2001;15: 1205-1226.

CLIPPINGS

Deworming drugs for treating soil-transmitted intestinal worms in children: effects ongrowth and school performance

About a quarter of the world’s population is infected with one or more soil-transmittedworms. The main soil-transmitted worms are roundworms, hookworms, and whipworms.Infections are widely distributed in tropical and subtropical areas, with most infected peoplehaving more than one type of worm. The burden of disease falls disproportionately on the poor,where there is inadequate sanitation, overcrowding, low levels of education, and lack of accessto health care. These infections cause malnutrition and poor growth for children, and somestudies have suggested an association with poor performance at school. Improved sanitationand hygiene are likely to be helpful. There are also three basic strategies for using drugs to treatthese infections: (1) individual treatment based on a diagnosis of infection (selective treatment);(2) groups at increased risk are treated (targeted treatment); and (3) treating whole communitieswhether people have the infection or not (universal treatment). The targeted treatment is theone generally used. This review of trials looked at targeted treatment with a range of dewormingdrugs for children, particularly focusing on growth and school performance. Thirty-four trialswere identified. These trials either looked at single or multiple doses, but only some assessedschool performance. After just one dose children’s weight improved, and more doses did notseem to improve this further. Only one of the seven trials that assessed school performancefound any positive effect, so it seems unlikely that there is a benefit here. Two trials looked atadverse events, but the trials were small. Further research is needed.

Taylor-Robinson DC, Jones AP, Garner P. Deworming drugs for treating soil-transmitted intestinal worms in children: effects on growth and school performance.Cochrane Database of Systematic Reviews 2007, Issue 4. Art. No.: CD000371. DOI:10.1002/14651858.CD000371.pub3. This version first published online: April 27. 1998.Last assessed as up-to-date: August 13. 2007

18

2009; 11 (3) : 223

AUTOMATED CULTURE AND

RAPID ANTIGEN TESTS

*Camilla Rodrigues

Abstract: Automation offers advantages ofimproved recovery of microorganisms, betteridentification and accurate susceptibility.Apart from automated blood culture systems,there are automation systems used for automationin culture for tuberculosis, microorganismidentification and for antimicrobial susceptibilitytesting. Rapid antigen testing is used forimmediate diagnosis during active infection andreduces the need for waiting for rise in antibodylevels and in culture negative patients onantibiotics for diagnosis of infectious diseases.Various body fluids can be used including CSF,serum, urine, stool and respiratory secretionsfrom representative site of infection.

Key words: Automation, Infection, Rapidantigen detection.

There is a trend in recent years to move awayfrom conventional, manual methods for thedetection and identification of microorganismsto more standardized automated instrument basedmethods. Automation in microbiology offersadvantages of rapid turn around time, improvedrecovery of organisms, precise identification ofisolates and accurate susceptibility testing.Quality control parameters incorporated ensurebetter reporting. With the introduction of the

continuous monitoring blood culture systems,the trend towards automation has accelerated.The increase in cost is justified as improvedisolation of organisms allows for better patientcare, as a correct diagnosis can be made andrational antimicrobial therapy can then beinstituted.

Apart from automated blood culturesystems, there are automation systems inmicrobiology used for automation in liquidcultures for tuberculosis, microorganismidentification and for antimicrobial susceptibilitytesting.

Automated blood culture systems

The continuous monitoring blood culturesystems are modular with a single computercontrolling the incubator units.1 Culture vials areincubated in individual cells and monitored atintervals of 10-15 minutes for evidence ofmicrobial growth. Three such systems, theBACTEC 9250/9120/9050 (BD diagnosticsystems) the BacT/alert (Biomerieux) and theTREK ESP culture systems (TREK Diagnosticsystems) are available in India. Multiple studieshave shown that the bottles need to be incubatedfor only 5 days. Improved and rapid isolation oforganisms in bacteremias as enteric fever,infective endocarditis, sepsis, etc allows foran unequivocal diagnosis to be established.The BACTEC system consists of a self containedincubator, agitator and detection system.As CO2 is produced in each bottle, its sensoremits a fluorescent light that passes an emissionfilter on the way to a light emission diode.Monitoring occurs every 10 minutes1.

* Consultant Microbiologist,PD Hinduja Hospital and Medical ResearchCentre, Mumbai - 400 016.

19



The BacT/alert detects CO2 by a sensor that

changes color in the presence of CO2 with a

permanent end point.An audible or visible alertis generated and the position of the positive bottleis flagged. Advantages in this system includeplastic bottles (safety reasons) and delayed vialentry.1 This implies that the blood can be colloctedin the bottles and loaded in the machine upto 2hrswithout positivity getting affected.

The TREK ESP culture system differs fromthe above two systems as the production of CO

2

is measured manometrically, both gasconsumption and production are monitored andchanges in H

2,O

2 and CO

2 are measured.1

Automated liquid culture methods fortuberculosis

1. BACTEC TB 460 is a sensitive, specificand rapid culture method for respiratory as wellas non-respiratory specimens. Different studieson the sensitivity of TB-Bactec in monitoringmycobacterial growth showed that an inoculumof 200 viable M. tuberculosis bacilli could bedetected in 12-13 days while as few as 20 viablebacilli could be detected if one waited for 14 -17days.2

2. Mycobacterial growth indicator tube(MGIT) 960 TB: The test employs a new state ofthe art fluorescent technology that enables resulttowards positivity as rapidly as 7-10 days. It isbased on oxygen quenching with a fluorescentdye.3

Automated organism identification andantimicrobial susceptibility testing

There are fully automated instrumentsavailable today for rapid and accurateidentification as well as susceptibility testing byminimum inhibitory concentration (MIC).The basis of identification system reactivity isusually based on pH or enzyme profiles but canalso be carbon sourced and visual detection of

growth. They include: (i) Walk-away system(Dade Microscan): based on fluorescence itautomatically adds reagents to conventionalpanels when required and interprets resultswithout operator intervention. (ii) The Vitek 2system (Biomerieux) is an integrated modularsystem that detects bacterial growth andmetabolic changes after short incubation periodswith an accuracy of 93%. Other systems include(iii) Sensititer (TREK Diagnostics), (iv) Phoenix(BD Diagnostics system) and the (v) Omnilog(Biolog) System4. The choice depends on severalvariables including volumes of testing,experience, need for definite identification andcost.

Rapid antigen tests

Today assays are available for rapiddetection of bacterial antigens in various bodyfluids. These are also useful when smear isnegative, prior antibiotics have been initiated andculture is negative. Also rapid antigen methodshave the advantage that since they are antigenbased detection systems,they can be used in theinitial stages of the illness and one does nothave to wait for specific antibody production.The turn around time is faster in these assays -ranging from 10 minutes to a couple of hours.The common methods encompass latexagglutination, Counter current immunoelectrophoresis (CIEP), Immunochromato-graphic assays (ICT)/ Lateral flow formats,Enzyme linked immunosorbent assays (ELISA),Direct immunofluorescence, etc., Increasingnumber of infections can be diagnosed in thisway. Some of these techniques from varioussamples such as cerebrospinal fluid (CSF),serum, urine, stool and nasopharyngeal aspiratesetc. are listed below.

A ) CSF

(i) Latex agglutination techniques are usedto demonstrate the soluble polysaccharide

20

2009; 11 (3) : 225

antigens of Neisseria meningitidis, Haemophilusinfluenzae tybe b and Streptococcus pneumoniaein patients of acute pyogenic meningitis.5,6

In neonatal meningitis the latex agglutinationmethod can be used for detection of E.coli andGroup B streptococci. These tests offer goodspecificity >98% but the sensitivity varies witheach bacterium tested. For N meninigitidisA,C,Y,W135, and N meningitidis B / E.coli K1it is 71% and 65% respectively. ForS pneumoniae it is 88% and for Haemophilusinfluenzae and group B streptococci, it is 67%.This assay can also be used for serum afterappropriate treatment with EDTA.

(ii) Cryptococcus neoformans

Latex agglutination and enzyme linkedimmunoassay are the preferred tests for detectionof cryptococcal antigen. They can be used fordiagnosis as well as therapeutic monitoring.5

The latex agglutination methods have asensitivity of approximately 93% and specificityof 93-100% and are equivalent to ELISA indetecting antigen. The test must be evaluated inconjunction with other clinical and diagnosticfindings. Trichosporon beigelli, Capnocytophagaspps. and stomatococcus have been shown toproduce a cross reacting polysaccharide antigen.Treatment with pronase B reduces the falsepositivity in serum tests in certainrheumatological disorders.

B) Serum

(i) Plasmodium species

Rapid diagnostic tests (RDTs) for malariahave an ability to detect >100 parasites /μL ofblood . These qualitative tests are rapid, simple,easy to perform and interpret. The stability ofthe kit in high environmental temperature andhumidity of tropics should be taken into account.As they cannot quantitate, they are not useful forprognostication. RDTs have decreased sensitivityat lower levels of parasitemia yielding false

negative results in non immune patients with lowlevels of parasitemia.7

There are 3 types of antigens used:(a) pLDH (Parasite Lactate dehydrogenase),(b) HRP II (histidine rich protein) and (c) Parasitealdolase.

(a) pLDH: This enzyme is located in theglycolytic pathway of the malarial parasiteproduced by both sexual and asexual stages ofthe parasite.

Though microscopy is considered the ‘Goldstandard’ method for diagnosis of malaria, itrequires up to 60 minutes time from specimencollection to reporting of result7. The OptiMALtest is a lateral flow format and detects parasitespecific lactate dehydrogenase (pLDH). It isreported to have high sensitivity and specificityin high endemic regions and is a useful adjunctto microscopy. When a single test band (genusspecific band) appears with a control band thetest is considered to be positive for Plasmodiumvivax. When two test bands (genus specific andspecies specific) appear with a control band thetest is considered to be positive for Plasmodiumfalciparum. Some newer kits target P vivaxspecific pLDH for detection of vivax malaria.A mixed infection with Plasmodium falciparumand other plasmodium species is indicated whenboth genus and species specific bands appear andthe genus specific band is much darker and moreintense than the species specific band.

(b) Histidine rich protein II (HRP II)8

Water soluble protein produced by asexual stageand young gamatocytes of P falciparum, HRP IIis generally more sensitive than pLDH but has adisadvantage that the antigen persists atdetectable levels for more than 28 days aftertreatment.

(c) Plasmodium aldolase, also an enzyme of theglycolytic pathway produced by all four species,

21


has been recently developed. In India there aremore than five types of RDT available withvarious combinations of pLDH with or withoutHRP II. The test chosen should be able todistinguish between at least P falciparum andP vivax as both are almost equally prevalent.Differentiation at a species level is vital from atreatment perspective.

(ii) Dengue - Dengue enzyme immuno assay(EIA) NS1 antigen

Detection of the NS1 antigen is valuable asit allows for detection of infection prior toseroconversion and can be detected in serumfrom day 1 after onset of fever upto day 9.This antigen is a marker of early acute activeinfection and early diagnosis of dengue allowsfor earlier implementation of supportive therapyand monitoring9. This reduces the risk ofcomplications such as dengue haemorrhagic feveror dengue shock syndrome. After day 9, onceNS1 antibodies are produced this antigen is nolonger detected. Cross reactivity across theflavivirus group may occur.

(iii) Aspergillus - Enzyme immuno assay(EIA) for galactomannan antigen

Galactomannan is a heteropolysaccharidepresent in the cell wall of most Aspergillus andPenicillium spp. Current data suggests thatgalactomannan production is proportional to thefungal load in tissues.10 Though strictly not arapid assay the decision to offer galactomannantesting depends on resources, institutional indexof invasive aspergillosis and the hospital casemix. In addition galactomannan detection appearsto have prognostic value as a high unremittinglevel of galactomannan despite antifungal therapyis associated with an unfavourable outcome.

This test is best performed before theinitiation of antifungal therapy as otherwisesensitivity is diminished. It is also advised that

twice-weekly tests be performed to enhancesensitivity. The clinical specificity is around 90%but can be lower in infants due to ingestion ofdietary galactomannans. False positive resultsmay be seen in patients on piperacillintazobactam. The early latex agglutination testswere specific but not very sensitive (detectionlimit 25ng/ml). Sandwich ELISA techniques haveenhanced the sensitivity to 1ng/mL.

ß D glucan

1,3-ß-D glucan antigen is present in the cellwall of most fungi including Pneumocystisjirovecii. (exceptions being Cryptococcus spp.and the Zygomycetes). The common features ofglucan assays is the ability of 1,3-ß-D glucan toactivate the coagulation cascade withinamoebocytes derived from the hemolymph ofhorseshoe crabs.10 The EIA assay utilizes serumfor antigen detection. False positive results areseen in hemodialysis, cardiopulmonary bypassand patients on immunoglobulin products.Environmental contamination may compromisespecificity. The cut off of 60 pg/mL is underconsideration.

C) Urine

In patients with community-acquiredpneumonia, antigen testing is useful in patientson treatment to establish the aetiological agent.

S.pneumoniae: The Binax NOW test is arapid imunochromatographic (ICT) assay for thedetection of the S.pneumoniae antigen in urineof patients with pneumonia, and in the CSF ofpatients with meningitis.5 Urinary antigens ofpneumococci are detected and their sensitivitysurpasses that of Gram stain. The clinicalsensitivity and specificity is 86% and 94%respectively. In children the specificity is lowerdue to carriage of S.pneumoniae. Their clinicalusefulness depends on the geographic prevalenceof the specific serogroup detected.

22

2009; 11 (3) : 227

(ii) Similarly for detection Legionellapneumophila serogroup I antigen in urine, an invitro rapid ICT assay is available with BinaxNOW. This antigen is also in the urine and theclinical sensitivity and specificity are more than95%.5

(iii) Histoplasma antigen in urine: Histoplasmaantigen detection in serum, urine and CSF is auseful indicator of systemic infection. ELISAtests can be performed on urine, CSF, serum andbrochoalveolar lavage fluid. The sensitivity ofthe test is approximately 92% in urine specimensand 82% in serum specimens from patients withdisseminated disease. The test has low sensitivityin self limited disease and chronic pulmonaryhistoplasmosis. This test is not readily availablein India due to low prevalence of the disease.

D) Stool

(i) Clostridium difficile antigen: Clostridiumdifficile associated diarrhea (CDAD) is a causeof infectious diarrhea mostly in hospitals.By enzyme immunoassays (EIA) toxin A and Bcan be detected with a specificity approaching100 per cent.

Direct testing of stool sample extracts fortoxin presence using a variety of immunoassaytechniques has largely replaced the culturemethod. Both A and B toxins of C. difficile aredetected by these tests and there are a range ofcommercially available kits in different formats.As strains which only produce either toxin A orB have been reported as pathogenic, methodswhich detect both are preferable5.

Assays can also be presented in a lateral flowformat as single tests which give faster results,normally within a few minutes and with lessoperator skill requirements. Some assays aredesigned to detect C. difficile common antigen(glutamate dehydrogenase or GDH) produced byboth toxigenic and non-toxigenic strains, and can

also have a toxin test included in the panel soboth toxin and organism may be revealed

(ii) Helicobacter pylori : Antigen assays areavailable for detection of Helicobacter pyloriwhich has a sensitivity of 96% and a specificityof 95 per cent.The H pylori Sp antigen assayHpSA (Meridian diagnostics) detects fecalexcretion of H pylori antigens and is a usefulnon invasive test for diagnosis and also forfollow up 7 days to 4 weeks after treatment toascertain complete eradication and outcome ofsuccessful therapy.5

(iii)Rota virus and adenovirus : Rotavirus is aleading cause of gastroenteritis in children underfive years. Adenovirus is an important cause ofdiarrhea in children under two years. Rotavirusand adenovirus antigens can also be demonstratedby latex agglutination tests.5 Ready to usehomogenous ICT systems are also availableincorporating monoclonal antibodies to specificviral antigens. Nitrocellulose membranes pick upspecific proteins of these two viruses. In mostassays the specificity of rotavirus is slightly lessthan that of adenovirus, but the sensitivity is oftenexcellent and usually over 95%. The advantageis that if these assays flag positive, needlessantibiotics can be avoided.

(iv) Cryptosporidial antigen : Stool samples inhuman disease provide for high numbers ofoocysts and relatively simple preparation andprocessing. Non-invasive, acid-fast stainingtechniques are used widely in clinicallaboratories. These techniques also provide datafor other parasitic infections. Antigen producedby the parasites in the gut can be detected fromfaecal specimens quickly and easily using lateralflow assays.11

E) Throat/Nasopharyngeal samples/Sputum/BAL

(i)Respiratory viruses: Viruses can be detected

23


directly from patient specimens and for cultureconfirmation. Respiratory viral screen DFA Kitsutilise fluorescein-labeled monoclonal antibodiesto detect and identify viruses in patient specimensand to screen for the presence of influenza A,influenza B, adenovirus, and RespiratorySyncytial Virus (RSV) in patient specimens andculture confirmation, and to screen forparainfluenza 1,2 and 3 following amplificationin culture.12

(ii) Mycoplasma pneumoniae antigen :Respiratory secretions are used in an ELISAformat for detection in patients suffering withpneumonia and upper respiratory tractinfections.13 This assay has better sensitivity thanM pneumoniae antibody detection as it detectsantigen and the sensitivity is approx 81% with aspecificity of 94%.

Lastly, rapid antigen detection methods alsoinclude sophisticated methods as Gas liquidChromatography (GLC) This involves a samplebeing gassed through a matrix that differentiallyseparates its constitutents and is useful fordetection of anaerobes. The combination ofGLC and mass spectrometry (separation andmeasurement of ions) enables microbiologists toanalyse organisms accurately and rapidly.5

High performance liquid chromtography(HPLC) has special resins that have ion exchangegroups on their surfaces. These retain substancesin the column depending on their charged ionaffinity. It is used today in the rapid identificationof mycobacteria.5

Points to Remember

• Accurate and rapid isolation of bacterialeads to better management of infectiousdisease.

• Automated blood culture systems arebecoming essential in diagnosis ofbacteremias.

• Other automation systems in microbiologyare used for automation in liquid culturesfor tuberculosis, microorganismidentification and for antimicrobialsusceptibility testing.

• Rapid antigen testing encompassesdetection of antigenic components ofbacteria, viruses, fungi or parasitesduring active infection.

• Various body fluids representative of thesite of infection can be exploited in antigendetection within a turn around time of afew minutes to a couple of hours.

References

1. Washington C, Koneman EW, Allen SD, et al.Introduction to Microbiology. In: Koneman’sColor Atlas and Textbook of DiagnosticMicrobiology, 6

th Edn, Lippincott Williams and

Wilkins, 2006; pp67-105.

2. Anargyros P, Astill DSJ, Lim ISL. Comparisonof Improved BACTEC and Lowenstein JensenMedia for culture of Mycobacteria from ClinicalSpecimens. J Clin Microbiol 1990;28:1288-1291.

3. Pfyffer GE, Welsher HM, Kissling P, et al.Comparison of Mycobacterial GrowthInducator Tube ( MGIT) with radiometric andsolid culture for recovery of acid fast bacilli.J Clin Microbiol 1997;35: 364-368.

4. Washington C, Koneman EW, Allen SD, et al.The Enterobacteriaceae In: Koneman’s ColorAtlas and Textbook of Diagnostic Microbiology6

th Edn, Lippincott Williams and Wilkins,

2006; pp211-302.

5. Rodrigues C. Laboratory Diagnosis ofInfections In: API Textbook of Medicine Vol.1,8

th Edn, API, 2008;pp6-8.

6. Kaldor J, Asznowicz R, Buist DGP. Latexagglutination in diagnosis of bacterial infectionswith special reference to patients withmeningitis and septicaemia Am J Clin Pathol1977; 68:284-287.

24

2009; 11 (3) : 229

7. Kundu R, Ganguly N, Gosh TK, et al. Diagnosisand management of malaria in children. IndianPediatr 2005; 42: 1101-1114.

8. Hayward RE, Sullivan DJ, Day KP.Plasmodium falciparum : Histidine richprotein II is expressed during gametocytedevelopment. Exp Parasitol 2000; 96 : 139-146.

9. Alcon S, Talarmin A, Debruyne M, et alEnzyme Linked Immunosorbent assay specificto Dengue virus Type 1 nonstructural proteinNS1 reveals circulation of the antigen in theblood during the acute phase of disease inpatients experiencing primary or secondaryinfections. J Clin Microbiol 2002; 40:376-381.

10. Rodrigues C, Taori S. Laboratory Diagnosis of

Invasive Fungal Infections. In: HematologyUpdate MB Agarwal, Ed 2006;pp309-318.

11. Ortega Y N , Arrowood M . Cryptosporidium,Cyclospora, and Isospora. In:Manual of ClinicalMicrobiology 8

th Edn, ASM Press, Washington

DC, 2003;pp2008-2019.

12. Buschen-Osmond C. Taxonomy andclassification of viruses. In: Manual of ClinicalMicrobiology, 8

th Edn, Vol.2, ASM Press,

Washinton DC, 2003;pp1217-1252.

13. Kleemola M, Raty R, Karjalainen J, et al.Evaluation of an antigen capture Enzymeimmunoassay for rapid diagnosis ofMycoplasma pneumoniae infection.Eur J ClinMicrobiol Infect Dis 1993;12:872-875.

25

IAP Dermatology chapter invites you to the

3rd National Congress of Pediatric Dermatology, DERMPED 2009

at Savera Hotel, Chennai

on 19th & 20th September 2009

Category July 15th August 16th Spot registration

IAPDC member 1500 1700 2500(Dermatology chapter)

Non member 1600 1800 2500

Post graduate 1100 1200 2500

Co Delegate* 1500 1500 2500

*Children above 5 years will be considered as Co Delegates

DD in favour of “DERMPED 2009” payable at Chennai

For details contact :

Dr.V.AnandanOrganising Secretary,223, L Block, 21st Street,Annanagar East, Chennai–102.Mobile: 09841043213

Kindly note that the sessions are for two full days. Registration limited for 200 delegates only.Post graduates must submit a Bonafide Certificate from HOD.

NEWS AND NOTES


MARKERS OF INFECTION

* Panna S Patel** Rajal B Prajapati

** Baldev S Prajapati

Abstract: The systemic changes associated withinflammation especially with infections consistof several clinical manifestations including feverand pathological markers such as cytokines,leucocytes, ESR, C Reactive Protein (CRP),Procalcitonin etc. The bacterial toxins and otherinflammatory stimuli stimulate cytokinesproduction which in turn causes various changeswith leucocytes, CRP, ESR and procalcitonin.They are also called as acute phase reactants.The information regarding these markers ofinfection and appropriate interpretation of theirlaboratory estimation is very useful to theclinicians in their daily practice. The increasedplasma levels of cytokines is the primary hostresponse to an inflammatory insult. They havegood sensitivity and specificity. But their clinicalutility is limited due to their very early and shortterm concentration. CRP is best studied forneonatal sepsis. CRP has positive predictivevalue for septicemia less than 50% while itsnegative predictive value for infection exceeds98%. Therefore, negative CRP response almostexcludes neonatal sepsis and positive responseneeds correlation with other parameters as well

as clinical picture. In these circumstances, it isnot justified to treat the baby with antibiotics onlyon the ground of positive CRP. Procalcitonin, arecently identified marker of infection, is morereliable compared to CRP. It is a reliable markerof late onset sepsis in newborn babies with asensitivity and specificity of almost 100%.The total leucocyte count has a low predictivevalue for diagnosis of infection because of widerange of normal counts. Leucopenia, immatureneutrophil count and thrombocytopenia atvarious stages of illness are useful in clinicalpractice. Elevated ESR is a non specific markerand therefore it does not have much value indiagnosis of infection.

Key words: Markers of infection, Cytokines,CRP, Procalcitonin.

The systemic changes associated withinflammation especially with infections, arecollectively called the Systemic InflammatoryResponse Syndrome (SIRS) or the acute phaseresponse. These changes are reactions tocytokines whose production is stimulated bybacterial products, toxins and other inflammatorystimuli. The acute phase response consists ofseveral clinical manifestations including feverand pathological markers such as changes incytokines and leucocytes, ESR, C ReactiveProtein (CRP), Procalcitonin etc. Informationregarding these markers of infection andappropriate interpretation of their laboratoryestimations is quite useful to the clinicians in theirdaily practice1,2. When clinical picture issupported by markers of infection, appropriateculture specimens are obtained and antibiotictherapy is started. The antibiotic regimen may

* Consultant PediatricianAakanksha Children Hospital and NeonatalNursery, Ahmedabad

** Associate ProfessorSheth LG. General Hospital,Smt. N.H.L. Municipal Medical College,Ahmedabad.

26


2009; 11 (3) : 231

be modified subsequently on the basis of culturereports and response of the patient to thetreatment2.

Leucocyte abnormalities

The total leucocyte count has a lowpredictive value for diagnosis of infectionbecause of wide range of normal counts from8,000 to 20,000 / cu.mm. Leucocytosis is acommon feature of inflammatory reaction,especially those induced by bacterial infections.The leucocyte count usually increases to 15,000to 20,000 cells/cu.mm.1,2 Occasionally, it mayreach to extraordinarily high levels of 40,000to 1,00,000 cells/cu.mm, referred to asleukemoid reaction. This is due to acceleratedrelease of cells from the bone marrow postmitoticreserve pool caused by cytokines includinginterleukin-1 (IL-1) and Tumor Necrosis Factor(TNF). Leukemoid reaction is commonlyassociated with B.pertussis and tuberculousinfections.1,2 Prolonged infection also inducesproliferation of precursors in the bone marrow,caused by increased production of ColonyStimulating Factors (CSFs). Thus, the bonemarrow output of leucocytes is increased tocompensate for the loss of these cells inthe inflammatory reaction.3 Neonatal septicemiais usually associated with leucopenia(< 5000/cu.mm) or absolute neutropenia(<1000/cu.mm).4,5 A band neutrophil is animmature neutrophil, wherein the width of thenarrowest segment of the nucleus is more than

one third of the broadest segment. The band cellcount of more than 20 percent and band count tototal neutrophil count ratio of equal to or morethan 0.2 is considered as 80 to 90 percentsensitive test for diagnosis of neonatalsepticemia.3,4,5 In addition to it, toxic granulationand vacuolization are other morphologicalchanges seen in neutrophils with septicemia.

Viral infections such as infectiousmononucleosis, mumps and rubella produce aleucocytosis by virtue of an absolute increase inthe number of lymphocytes (lymphocytosis).1,2

Hay fever and parasitic infestations are knownfor eosinophilia. Enteric fever is usuallyassociated with low leucocyte count andeosinopenia1. Thrombocytopenia can occurduring the initial phase of sepsis because ofplatelet consumption or vascular sequestration,whereas reactive thrombocytosis can be seen lateras a result of bone marrow stimulation bycytokines. Falciparum malaria, enteric fever,dengue haemorrhagic fever and other infectionsare known for their association withthrombocytopenia and leucopenia (Table 1).In enteric fever platelets usually fall in the secondweek of illness, whereas in others they fall withinthe first week itself. Drop of haemoglobin is thecharacteristic of malaria, while rising hematocritindicates Dengue fever.

Malaria (especially falciparum malaria),acute viral and bacterial infections are known tocause anaemia acutely2. Non specific viral

Parameter Enteric Malaria Dengue Other viral

Hb/Hct N Low N/High N

TLC N/Low N/Low N/Low N/Low

DLC Polys eosinopenia Monocytosis Lymphous Lymphous

Platelets N/Low Low Low N/Low

Table 1. Interpretation of CBC

27


infections can cause coomb’s positive hemolyticanemia. Mycoplasma pneumoniae may presentwith haemolytic anaemia with elevated titres ofcold haemagglutinating antibodies. Transienterythroblastopenia is an acute phase responsepresent during many infections. Hemophagocyticsyndromes leading to pancytopenia can followmany infections especially due to herpes viruses,parvovirus B19, salmonella etc. Chronicinfections like tuberculosis, urinary tractinfection, bacterial endocarditis and chronicosteomyelitis are known to cause anaemia1,2,3.

Erythrocyte Sedimentation Rate(ESR)

Acute phase proteins are plasma proteins,mostly synthesized in the liver, whose plasmaconcentrations may increase several hundred foldas part of the response to inflammatory stimuli.Three of the best known examples of theseproteins are C-reactive protein, fibrinogen andserum amyloid A protein. The rise in fibrinogencauses erythrocytes to states (rouleaux) thatsediment more rapidly at unit gravity than doindividual erythrocytes. This is the basis formeasuring the erythrocyte sedimentation rate(ESR).1,3 ESR is a simple test for the systemicinflammatory response, caused by number ofstimuli including Lipopolysaccharide (LPS).

The ESR is elevated in most bacterial andmycobacterial infections and is normal or mildlyelevated in uncomplicated viral infections.Elevated ESR has poor discriminating power asa single test to predict bacterial infection inchildren with non specific, febrile illness ofshorter duration. ESR is expected to be abnormalif a significant tissue focus of bacterial infectionexists. ESR more than 50 mm/hour providesimpetus for more extensive evaluation that canreveal localized bacterial infection, endocarditis,disseminated tuberculous infection or certainfungal infections. Extreme elevation of ESR

(more than 100 mm/hour) is characteristic ofcertain conditions such as infective endocarditis,miliary tuberculosis, Kawasaki disease,sarcoidosis, malignancy, collagen disorders etc.1,2

In children with infectious diseases,abnormally low ESR is most frequently a sign ofdisseminated intravascular coagulopathy,reflecting low plasma fibrinogen concentration.Abnormally shaped RBCs (sickle cell,spherocytosis) and polycythemia preventcompact rouleaux formation and lower ESR.High dose of steroids and salicylates have beenreported to cause lower ESR.1,2,3

Micro-ESR is a simple marker for neonatalinfection. It is not a very reliable marker.Its normal value is 6 mm during the first 3 daysof life. By the end of first month, maximum fallmay be upto 11 mm. During the neonatal periodvalue of micro ESR more than 15 mm issuggestive of infection. Micro ESR is obtainedby collecting capillary blood in a standard pre-heparinized micro-hematocrit tube with 75 mmlength, internal diameter of 1.1 mm and outerdiameter 1.5 mm.4,5

Those increasing with inflammation.

C-Reactive Protein

Procalcitonin

Cytokines (IL-6 and IL-1ra)

Alpha-1-acid glycoproteins

Heptoglobin (Alpha-2 glycoprotein)

Alpha-1-antitrypsin

Fibrinogen

Those decreasing with inflammation.

Prealbumin

Transferrin

Table 2. Acute phase proteins

28

2009; 11 (3) : 233

Acute phase proteins

Most of the acute phase proteins increasein plasma following inflammatory stimuli.There are two acute phase proteins namelyprealbumin and transferrin called negativereactants as they decrease with infection andreturn to normal with recovery.4 (Table 2)

Cytokines

The important limit of hematological indicesfor early diagnosis of sepsis is the time requiredfor the test to become positive. It takes severalhours for leucocyte indices and acute phasereactants to change significantly after the onsetof reaction.6 (Fig.1)

The cascade of events initiated by thebacterial infection usually begins with theactivation of macrophages and release of

inflammatory cytokines and growth factors.So increased plasma levels of cytokines is theprimary host response to an inflammatory insult.Cytokines are glycoproteins released bymacrophages, monocytes, lymphocytes andendothelial cells.1,6 Tumor necrosis factor-Alpha(TNF-α), interleukin-Beta (IL-β) and Interleukin-6 (IL-6) are major inflammatory cytokines whileinterleukin-3 (IL-3) and Colony StimulatingFactors (CSFs) are important growth factors.Fig.1 shows kinetics of various markers of theinfection following endotoxin challenge inhuman volunteers.

Tumor Necrosis Factor – Alpha(TNF-ααααα)

It is considered the likely initiating factorin the activation of host response and subsequentcytokine release during infection with

Fig. 1. Kinetics of various markers of inflammatory host response afterendotoxin challenge in human volunteers.CRP, C-reative protein; IL, interleukin;PCT, procalcitonin; TNF, tumor necrosis factor

29


concentration increasing to 24 times (828 ng/L)to their preinfection concentration. Difficultiesin using TNF-α for diagnosis of sepsis arise fromits central role in the inflammatory response,short term concentration in response to bacterialinfection and its short half life.6

Interleukin-6 (IL-6)

IL-6 is a pleiotropic cytokine involved inmany aspects of the immune system. It issynthesized by a number of cells such asmonocytes, endothelial cells and fibroblasts afterTNF and IL-1 stimulation. IL-6 is the majorinducer of hepatic acute phase protein synthesisincluding CRP and fibrinogen. In most cases ofneonatal sepsis, IL-6 increases rapidly, severalhours before the increase in the concentration ofCRP and decreases within 24 hours toundetectable levels. The short half life of IL-6 iscaused by binding to plasma proteins, earlyclearance in the liver and inhibition by othercytokines. When used as a marker of infection,IL-6 has good sensitivity and specificity.7,8

A recent study showed that IL-6 andinterleukin-1 receptor antagonist (IL-1ra)increased significantly two days before theclinical diagnosis of sepsis.9 In contrast to CRP,IL-6 is a very early marker, but levels can becomenormal even if infection continues. This leads toan increasing false negative findings whensample is performed later in the course.The simultaneous determination of CRP canobviate this problem, because the rise in plasmaCRP levels occurs 12 to 48 hours after the onsetof infection, at a time when IL-6 levels probablywould have fallen. When these two markers arecombined, the sensitivity becomes 100%.10,11,12

A semi-quantitative IL-6 the quickest test isavailable that provides accurate results within15 minutes.

In addition to the proinflammatoryendothelial and phagocytic activations, anti

inflammatory cytokines also increase duringsepsis. A major focus of current research isIL-10 which strongly inhibits the inflammatorycytokines TNF-α, IL-1, IL-6,IL-12 andIL-18.10,11,12

Granulocyte Colony StimulatingFactor (G-CSF)

G-CSF is a haematopoietic growth factorthat plays a pivotal role in promoting the growthand differentiation of granulocyte precursors andincreasing the functional activities of their matureprogeny. G-CSF production occurs in severaltype of cells such as monocytes, macrophages,epithelial cells and fibroblasts. Polysaccharideis a major stimulus for G-CSF production inneonatal sepsis. Several studies indicate G-CSFhas 95% sensitivity for prediction of sepsis anda specificity of 73% for levels of more than200 pg/ml.3,12,13

C-Reactive Protein (CRP)Physiology and measurement

C-Reactive Protein (CRP) is so calledbecause it can be precipitated by thesomatic C Polysaccharide of Streptococcuspneumoniae.14 It is produced by hepatocytes. Itsconcentration is less than 1 mg/dL in healthyindividuals. It can rise 1000 fold within 24 hoursunder conditions characterized by inflammationwith tissue destruction, especially bacterialinfection.1,2,10,11 Although a specific primary roleof CRP is uncertain, it has been shown to havemultiple effects on immune system and canactivate complement. It is not influenced byserum proteins and RBC chracteristics like ESR.Any tissue destroying event, however, such astrauma, burn injury, ischemia or infarction canelicit production of this acute phase reactant.CRP is degraded rapidly, having half life less than24 hours. Therefore, serial measurements canprovide additional information on the adequacyof treatment.

30

2009; 11 (3) : 235

Extremely high concentrations of CRP canbe present in serum, yielding false negative testresults if only undiluted serum specimen is used.Various methods like latex agglutination,immuno diffusion, enzyme immunoassay andnephelometry have been used for its estimation.Accurate measurement of CRP can be made bylaser nephelometry or single radio-immunodiffusion assay. A semiquantitativebedside latex agglutination technique givesresults within 15 minutes by using capillary bloodsample.4

Clinical usefulness

A number of acute phase proteins serve asuseful indicators of infection in the neonates.The best studied among them is the CRP. In thefirst 4 days of life, apparently healthy neonatescan have mildly elevated CRP (upto 1.5 mg/dL).Non infectious perinatal conditions likemeconium aspiration, birth asphyxia,cephalhematoma and chest tube placement cancause elevated CRP, limiting usefulness of thetest for prediction of neonatal sepsis. Its positivepredictive value for septicemia is less than 50%.In these circumstances, it is not justified to treatthe neonate with antibiotics only on the groundof positive CRP.4,10 Its negative predictive valuefor infection exceeds 98%. Very ill neonates mayfail to mount a CRP response occasionally.In conclusion, negative CRP response almostexcludes neonatal sepsis, but positive responseneeds correlation with other parameters as wellas clinical picture and exclusion of other perinatalconditions.

CRP starts to rise within 12 to 24 hours ofonset of sepsis, earlier than the other acute phasereactants. In a suspected case of sepsis if CRP isnegative, it should be repeated after 12 hours.A semi-quantitative bedside latex agglutinationtechnique gives results within 15 minutes byusing capillary blood sample. A qualitative assayof CRP does not offer significant advantages on

the other indices. On the other hand, quantitativeCRP values, particularly when repeated, are moreuseful.14-20

Because of rapid degradation of CRP,monitoring serial values can provide moreinformation. Serial decline in CRP levels withtherapy is suggestive of adequate response toantibiotics. Usually, it returns to normal within2 to 7 days of successful treatment. Persistentelevation of CRP may indicate persistent bacterialinfection or development of some complication.Persistent elevation of CRP has been found withpersistent bacterial meningitis and abscessformation in necrotizing enterocolitis. Serial CRPlevels are useful in monitoring the response totreatment for osteomyelitis or septic arthritis.21,22

In one of the studies, progressively lowervalues from fourth to sixth day of therapydistinguished children with uneventful casesof acute osteomyelitis from those withcomplications.21 ESR remained elevated in allduring the first week. CRP estimation in CSF isof no value for diagnosis of bacterial meningitis.22

CRP as a marker of cytokine release andactive inflammation can be useful in diagnosisand management of many noninfectiousinflammatory disorders such as Crohn’s disease,rheumatoid disorders, autoimmune vasculitisdiseases, Kawasaki disease, pancreatitis etc.Neoplasms of liver, lymphoma, allograftrejection and graft-versus-host disease are otherconditions associated with high CRP values.

Procalcitonin (PCT)

Procalcitonin is a propeptide of calcitonin,released into the blood 3 to 6 hours afterendotoxin injection and increases upto24 hours.3,23 The increase of procalcitonin doesnot correlate with calcitonin levels and occurseven in subjects who had thyroidectomy. It isphysiologically elevated during first 3 days of

31


life. But it has been found to be a reliable markerof late onset sepsis in newborn babies with asensitivity and specificity of nearly 100%.24-28

The comparative studies have shown that PCTis a more reliable marker of sepsis compared toCRP.26 Quantitative measurement of PCT isperformed, using an immunoluminometric assay

(ILMA) with two monoclonal antibodies.After the initial surge of PCT during first 3 daysof life, the mean normal serum PCT level isaround 2 ng/ml.27 The possibility of bacterialinfection and recommendation of antibiotics withreference to serum procalcitonin level can bepredicted as shown in Table 3.

Table 3. Possibility of bacterial infection and recommendation of antibiot-ics with reference to procalcitonin level

Table 4. Comparison of various inflammation markers in clinical use

Procalcitonin concentration (mg/L)

Possibility of bacterial infection

Recommendation for starting antibiotics

< 0.1 mg No bacterial infection No antibiotics

0.1 to 0.25 Unlikely No antibiotics

0.25 to 0.5 Possible Start antibiotics

> 0.5 Highly indicating Strongly recommended

32

Specific Sensitive Clinical useMarker response for

to inflamma- Advantages Disadvantagesinfection tion

Rapid induction (2 h) Low sensitivity for local infectionsHigh bio stability half High specificity for severe sepsis

PCT ++++ + time 24 h wide biologic and septic shock release in severerange SIRS settings; expensive

Not expensive Low specificitySlow induction time (peak 24 h)

CRP ++ ++ Low biologic rangeNo correlation with severity ofinflammation

High sensitivity High variabilityRapid induction Low bio stability

Cytokines + +++ (minutes) Low correlation with severity ofinflammationExpensive

laucocytes + +++ Simple method Very low specificityHigh sensitivity

2009; 11 (3) : 237

Sensitivity and specificity of variousmarkers of infection is shown in Table 4 withtheir comparison. It is useful to understand theirrelative utility in clinical practice.

Points to Remember

• The acute phase response associated withinflammation especially infection consistsof pathological markers such as changesin cytokines and leucocytes, ESR, CRP,procalcitonin etc.

• The increased plasma levels of cytokinesis the primary host response to aninflammatory insult. They have goodsensitivity and specificity. But theirclinical utility is limited due to their veryearly response and short termconcentration in the body.

• CRP is best studied for neonatal sepsis.Its positive predictive value for septicemiais less than 50% while negative predictivevalue is 98%. Therefore, negative CRPalmost excludes neonatal sepsis andpositive response needs correlation withother parameters and clinical picture. Itis not justified to start the antibiotics onlyon the ground of positive CRP.

• Procalcitonin is a reliable marker of lateonset sepsis in newborns with a sensitivityand specificity of almost 100%.

• Elevated ESR is non specific marker andit does not have much value for diagnosisof infection.

• Total leucocyte count has a low predictivevalue for diagnosis of infection becauseof wide range of normal counts.

References

1. Kumar V, Abbas AK, Fausto N. Acute andchronic inflammation: Robbins and CotranPathologic basis of disease. 7

th edn. New Delhi:

Elsevier, a division of Reed Elsevier India Pvt.Ltd. 2007; pp 47-85.

2. Long SS. Laboratory Manifestations ofInfectious Diseases. In : Long SS, Pickering LK,Prober CG (Eds), Principles and Practice ofPediatric Infectious Diseases, New York :Churchill Livingstone 1997; pp 1530-1570.

3. Katewa S, Bansal D, Sachdeva A, Yadav SP .Markers for sepsis diagnosis : do they help?In : Selected topics in Pediatrics. Pedicon,2008, 17th – 20th January, 2008, Orissa. 2008;pp45-60.

4. Singh M Care of the Newborn (6th edn), NewDelhi : Sagar Publication 2004; 212.

5. Isaacs D, Moxon ER. Investigations forsuspected sepsis. In : Neonatal Infections IstIndian Edn. New Delhi : Jaypee Brothers 1994;pp31-39.

6. Malhotra S, Sachdeva A, Yadav SP, Kler N,Saluja S, Soni A . Role of cytokines in diagnosisof neonatal sepsis In : Selected topics inpediatrics. Pedicon, 2008. 17th-20th January,2008, Orissa, 2008; pp61-66.

7. Buck C, Bundschu J, Gallati H, et al.Interleukin-6 : a sensitive parameter for the earlydiagnosis of neonatal bacterial infection.Pediatrics 1994; 93 : 54-58.

8. Messer J, Eyer D. Donato L, et al : Evaluationof interleukin-6 and soluble receptors of tumournecrosis factor for early diagnosis of neonatalinfection. J Pediatr 1996; 129 : 574-80.

9. Kuster H, Weiss M, Willeitner AE, et al.Interleukin-1 receptor antagonist andinterleukin-6 for early diagnosis of neonatalsepsis 2 days before clinical manifestation.Lancet 1998; 352 : 1271-1277.

10. Puopolo KM. Bacterial and Fungal InfectionsIn : Manual of Neonatal Care. Cloherty JP,Eichenwald EC, Stark AR (Eds) New Delhi,Wolters Kluwer (India) Pvt. Ltd. 2008;pp 274-300.

11. Gabay C, Kushner I . Acute phase proteins andother systemic responses to inflammation.N Engl J Med 1999; 340:448-454.

33


12. Mehr S, Doyle LW . Cytokines as markers ofbacterial sepsis in newborn infants : A review.Pediatr Infect Dis J 2000; 19 : 879-887.

13. Berner R, Niemeyer CM, Leititis JU, et al.Plasma levels and gene expression ofgranulocyte colony-stimulating factor, tumornecrosis factor-alpha, interleukin (IL)-1-betaand soluble intercellular adhesion molecule-1in neonatal early onset sepsis. Pediatr Res 1998;44 : 469-477.

14. Prajapati BS, Prajapati RB, Patel PS.C-Reactive Protein : Current Perspectives.Bulletin ID Chapter of IAP.

15. Ng PC, Cheng SH, Chui KM, et al. Diagnosisof late onset neonatal sepsis with cytokines,adhesion molecules, and C-reactive protein inpreterm very low birth weight infants. Arch DisChild Fetal Neonatal Edn 1997; 77: F221-F227.

16. Berger C, Uehlinger J, Ghelfi D, et al.Comparison of C-reactive protein and whiteblood cell count with differential in neonates atrisk of septicaemia. Eur J Pediatr 1995; 154 :138-144.

17. Da Silva O, Ohlsson A, Kenyon C. Accuracy ofleukocyte indices and C-reactive protein fordiagnosis of neonatal sepsis : a critical review.Pediatr Infect Dis J 1995; 14: 362-366.

18. Laborada G, Rego M, Jain A, Guliano M,Stavola J. Diagnostic value of cytokines and C-reactive proteini n the first 24 hours of neonatalsepsis. Am J Perinatol 2003; 20 : 491-502.

19. Young B, Gleeson M, Cripps AW. C-reactiveprotein : A critical review. Pathology 1991; 23:118-124.

20. Hanson LA, Jodal U, Sabel KG et al :The diagnostic value of C-reactive proteinPediatr Infect D 1983;2: 87-90.

21. Ronie I, Faingezicht I, Argnedas A et al . SerialSerum C-reactive protein to monitor recoveryfrom acute hematogenous osteomyelitis inchildren. Pediatr Infect Dis 1995;14: 40-44.

22. Gerdes LU, Jorgensen PE, Nexo E, Wang P.C-Reactive Protein and bacterial meningitis ameta-analysis. Scand J Clin Invest 1998; 58 :383-393.

23. Dandona P, Nix D, Wilson MF, et al.Procalcitonin increase after endotoxin injectionin normal subjects. J Clin Endocrinol Metab1994; 79 : 1605-1608.

24. Balol C, Sungurtekin H, Gurses E, SungurtekinU, Kaptanoglu B. Usefulness of procalcitoninfor diagnosis of sepsis in the intensive care unit.Crit Care 2003; 7 : 85-90.

25. Ugarte H, Silva E, Mercan D, De Mendonca A,Vincent JL. Procalcitonin used as a marker ofinfection in the intensive care unit. Crit CareMed 1999 ; 27 : 498-504.

26. Luzzani A, Polati E, Dorizzi R, Rungatscher A,Patan R, Merlini A. Comparison ofprocalcitonin and C-reactive protein as markersof sepsis. Crit Care Med 2003 ; 31 : 1737-1741.

27. Chiesa C,Panero A, Rossi N, etal. Reliabilityof procalcitonin concentrations for the diagnosisof sepsis in critically ill neonates. Clin InfectDis 1998; 26 : 664-672.

28. Harbarth S, Holeckova K, Froidevaux C,et al. Diagnostic value of procalcitonin,interleukin-6, and interleukin-8 in critically illpatients admitted with suspected sepsis. Am JRespir Crit Care Med 2001; 164 : 396-402.

NEWS AND NOTES

34

9th Jharkhand Pedicon, Bokaro

November 7th and 8th, 2009Contact :Dr. B. Prasad, Organising Chairman, 1061, Sector 4c, Bokaro Steel city - 827 004.Ph: 09798502749, 06542 232200

2009; 11 (3) : 239

RATIONAL INVESTIGATIONS IN

CNS INFECTIONS

* Digant D Shastri

Abstract: Many infectious agents can causecentral nervous system (CNS) diseases inhumans. Since microbial agents infecting CNSare numerous and have different features,conventional laboratory tests may not besensitive enough to identify and characteriseviruses, bacteria and fungi in human biologicalspecimens. Many laboratory issues addressingthe diagnosis of CNS infection are changing orevolving. These evolutions represent significantimprovements over previous testing. Availabilityof array of tests needs judicious selection of testsdue to their high cost and needs for the newskills for performance and interpretation.The responsibility for proper use of laboratorytesting lies both with the clinician and laboratorytechnician. Though analysis of CSF remain thegold standard for the differential diagnosis,neuroimaging plays a crucial role both in thediagnosis and therapeutic decision making ininfectious diseases of the nervous system.During the past decade tremendous advanceshave been made in the technology used to imagethe central nervous system which has not onlyimproved the clinical outcome but has alsoopened new vistas in case management.

Key words: CNS infections, Laboratorydiagnosis, Neuro imaging.

Infections of the central nervous system(CNS) are a serious threat to the developing brain

and are a major cause of neurologic handicap inthe young children.1 The laboratory diagnosis ofCNS infection is essential for optimal therapy inorder to improve the outcome of the diseases withrapid and accurate detection of the pathogens.The prognosis mainly depends on rapididentification of the site of inflammation andpathogen to install effective antimicrobialtreatment as early as possible.

Many laboratory issues addressing thediagnosis of CNS infection are changing orevolving. These evolving methods for thelaboratory diagnosis of CNS infection representsignificant improvements over previous testing;however, the array of tests available demandsmore attention for appropriate selection, as theyare significantly more expensive and requiresnew skills for performance and interpretation.

Whereas the CSF study, Gram’s stain, fungalstains of direct smears, antigen testing andculture remain the gold standard to identify theinfectious agent, neuroimaging is crucial inclearly depicting inflammatory lesions of brain.Visualization of typical patterns of leasions oftenallows a rapid diagnosis and subsequenttherapeutic decisions. Notably, in opportunisticdisease neuroimaging has a pivotal role not onlyin diagnosis but also in monitoring therapeuticresponse. With the development of thetechnology a new vista has opened up in thediagnosis of various diseases throughneuroimaging.

For the aetiological diagnosis of infectionCSF study, culture and antigen detection testsare performed. CSF study still remains the gold

* Consultant Pediatrician,Killol Children Hospital, Surat, Gujarat.

35



standard for diagnosis. For the diagnosis of CNSinfection following tests are done on sample ofCSF:

1. Routine tests: a) Cell count anddifferential count, b) Bio Chemistry (Glucose,protein), c) Stains/smears (Gram stain, AFB,KOH). 2. Culture and sensitivity and 3. Antigendetection and serology.

Collection, storage and transport ofCSF

CSF should be collected asepticallyand should be transported soon at 37OC.CSF specimen should be stored at 37OC tillmicroscopy is performed and bacterial cultureis inoculated. CSF cell analysis (WBC count)should be done immediately as delay will not givecorrect cell count because of cell lysis. If delayis likely divide the sample in two differentcontainers, one for cytology, gram stain and fordifferent biochemical tests and the second partof the sample in bottle containing few drops ofglucose broth for culture.

CSF cytological examination1,2,3

It is done for differentiation of bacterialmeningitis, tuberculous meningitis, fungalmeningitis and aseptic meningitis. CSF study isalso done for the differentiation of other infectiveconditions like encephalitis(Table 1). It is doneon a WBC counting chamber and for theidentification of cells it is stained with Giemsastain. Normal CSF contains up to 5/mm3 WBCs,and in newborn may have as many as 15/mm3.Polymorphonuclear (PMN) cells are alwaysabnormal in a child, but 1–2/mm3 may be presentin a normal neonate. An elevated PMN countsuggests bacterial meningitis or the earlyphase of an aseptic meningitis. In untreatedbacterial meningitis the CSF WBC count isusually elevated and is in the range of500- 5000 cells/mm3. In bacterial meningitis,

CSF cytological examination usually showspolymorphonuclear preponderance with 80-95%neutrophils. CSF lymphocytosis indicatesaseptic, tuberculous or fungal meningitis;demyelinating diseases; brain or spinal cordtumour; immunologic disorders includingcollagen vascular diseases and chemicalirritation. In TB meningitis the total WBC inCSF is usually < 500 X106 / L with lymphocyticpredominance. Despite the antibiotic therapy, theCSF WBC count remains abnormal. In traumatictap interpretation of CSF cell count is difficultand is not reliable for the diagnosis.

CSF biochemistry1,2,3

Glucose: In healthy child CSF glucose is twothirds of blood sugar. CSF glucose lags behindblood sugar by 30 minutes. So best way is todraw blood glucose and do the lumbar punctureafter about 30 minutes. If blood is not drawnbefore LP, it should be drawn simultaneously,because the stress of the LP procedure itself canincrease the blood sugar. Hypoglycorrhachia isfound in association with bacterial andtuberculous meningitis. Very low CSF glucosesuggests overwhelming infections. The ratio ofCSF glucose : blood glucose is more importantthan low CSF glucose in diagnosing bacterialmeningitis. In children above two months ageCSF glucose : blood glucose ratio less then0.4 mg/dL has 80% sensitivity and96% specificity for distinguishing acute bacterialand acute viral meningitis. In children below twomonths the ratio < 0.6 mg/dL is considered to besuggestive of bacterial meningitis. CSF glucoselevel is found useful in predicting probability ofdeveloping sensorineural hearing loss in patientwith bacterial meningitis. Studies have shownthat patients with sensorineural hearing lossassociated with meningitis CSF glucose levelwere found to be very low.4

Limitation of CSF sugar are : (a) CSF glucose isdecreased in 50% cases only and hence normal

36

2009; 11 (3) : 241

CSF glucose does not rule out infection,(b) In 50% of patients with clinically curedbacterial meningitis, the CSF glucose level maynot return to normal, hence it is not recommendedas a test for cure, (c) CSF sugar may also bedecreased in infections due to syphilis/Lymedisease.

Protein:2,3,5 The normal CSF protein ranges from10–40mg/dL in a child to as high as 120mg/dLin a neonate. The CSF protein falls to the normalchildhood range by 3 months of age. The CSFprotein may be elevated in many conditionsincluding infectious, immunologic, vascular anddegenerative diseases as well as tumours of thebrain and spinal cord. Proteins are found to beelevated in 90% cases of bacterial meningitis.In bacterial meningitis, specially in S pneumoniameningitis the CSF protein level remains in therange of 100- 500 mg/dL. CSF protein levelabove 200/dL, distinguishes bacterial fromaseptic meningitis with 82% sensitivity and100% specificity. In case where antibiotic isstarted prior to obtaining the CSF sample, proteinmay be only slightly elevated. Elevation of CSFimmunoglobulin G (IgG) is observed in subacutesclerosing panencephalitis, postinfectiousencephalomyelitis and in some cases of multiplesclerosis.

Limitation of CSF protein: (a) In 50 % of patientswith clinically cured bacterial meningitis, theCSF protein level may not return to normal,(b) Protein level will be elevated in traumatictap.

CSF Lactate: It is independent of serum lactate.It is also useful for predicting possibility of viralmeningitis from bacterial, TB and fungalmeningitis. If the CSF lactate level is less than3 mmol/L viral meningitis is likely, while if it ismore than 4.2 mmol/L it could be either bacterial,TB and fungal meningitis. Also it candifferentiate partially treated bacterial meningitis

where the level is 3-6 mmol/L with negative gramstain and there is history prior antibiotic therapy.

Limitation: conditions like cerebral hypoxia/ischemia,vascular compromise and anerobicglycolysis also can cause false elevation of lactatein CSF. Hence, it is not recommended in patientssuspected to have community acquired bacterialmeningitis.

CSF Lactate Dehydrogenase (LDH): It is asensitive indicator of meningitis (providedsample is not contaminated with blood). Normal/mild elevation is found in viral meningitis, whilemore marked elevation is seen in bacterialmeningitis.

1. Bacterial meningitis shows elevation ofLDH-4 and LDH-5,

2. Viral meningitis shows elevation of LDH-1and LDH-2,

3. TB meningitis shows elevation of LDH-1,LDH-2 and LDH-3 (specially LDH-3).

Tests for detection of bacteria/bacterial antigen

Gram stain: It is the best single test for rapiddiagnosis of bacterial meningitis. Culture is morereliable than gram stain, but the advantage ofgram stain is that the results are instantaneous.In untreated bacterial meningitis test is positivein 80% of cases. Gram stain can be helpful inidentifying the pathogen in 97% cases whenCSF bacterial concentration is more than105 CFU/ml. Sensitivity of this test is increasedby centrifugation of CSF sample. Sensitivitydecreased to 50% in patients who are alreadystarted on antibiotics. Positivity of gram stain isvariable for different organisms: for pneumococci90%, H influenzae b 85%, meningococci 75%and for Gram negative enteric bacilli 30-50%.On Gram stain study S.pneumoniae appears asgram positive lanceolate diplococci or in short

37


chains, while N.meningitides appears as gramnegative plano convex diplococci.

AFB stain and KOH preparation: These testsare done when tuberculous infection or fungalinfections are suspected respectively.

Petechial fluid: This test is performed mainlyfor diagnosis of meningococcal meningitisin which patient develops petechial lesion.Gram stain from skin scrapping is positive in70% cases of meningococcal meningitis.Petechial lesion is irrigated with 0.2 ml of sterilesaline solution and the irrigated fluid is collectedfor gram stain and culture.

CSF culture4,5: CSF culture should be done inany suspected case of CNS infection. For betteryield the CSF should be inoculated in the culturemedia as soon as possible and preferably shouldbe done at the bedside. If bedside inoculation isnot possible the sample should be stored at 37OCtill bacterial culture is performed. Except formeningitis caused by gram negative entericbacilli previous antibiotic treatment willinvariably decrease yield of bacterial CSF culture.Positive CSF culture has 92% sensitivity and95% specificity. CSF culture is rarely positive incase of brain abscess.

Blood culture: It helps to identify the pathogenwhich has been transmitted by hematogenousroute to brain and has caused CNS infection.In infants and children 1-5 ml of blood issufficient for culture. Preferably the blood cultureshould be collected prior to starting antibiotics.Blood culture is positive only in 10% cases ofbrain abscess.

Antigen detection tests: Indicated whenantibiotics are already started, because previousantibiotic therapy reduces bacterial yield onculture or gram stain, but it does not affect thedetection of bacterial antigen. This is very much

useful in immunocompromized patientswith normal CSF chemistry and cell count.The methods for antigen detection tests aretypically either antibody-based detection ofantigen through immune complex formation orPCR-based detection of organism-specificnucleic acid sequences. Of the available differenttests latex agglutination test is rapid, sensitiveand easy to perform in field conditions. Presentlycommercially available kit can detect antigen ofS. pneumoniae, N. meningitides, H. influenzatype b and group B Streptococcus.

Limitations: (a) Negative result for a specificbacterial antigen can never rule out bacterialmeningitis. (b) Children recently immunized(48hrs) with H influenzae vaccine may get falsepositive latex agglutination in CSF and urine.(c) Since these methods do not distinguishbetween viable and nonviable organisms and thusshould not be relied upon for test of cure.

PCR: Enteroviral meningitis is an importantdiagnostic consideration in patient who haspresented with acute meningitis. In such patientsrapid detection of enterovirus by PCR is a veryuseful technique. Moreover it will be useful inpatients with bacterial meningitis who hasreceived antibiotics . But presently PCR is notrecommended for routine use. PCR amplificationof viral nucleic acid is the diagnostic procedureof choice for HSV, EBV,VZV and Enteroviruses.CSF sample for viral study or PCR should berefrigerated or frozen.

Serological studies and antibody detection:These tests have limited utility for retrospectivediagnosis rather than acute diagnosis becausethe best results of these tests are available afterfirst week of illness. After the end of 1st week ofillness IgM ELISA becomes positive andin sample less than 7 days it will be only50% sensitive. IgG antibodies appears by10-14 days and four fold or greater rise in the

38

2009; 11 (3) : 243

IgG antibody titre in 2 paired samples taken7-14 days apart confirm the presence of infection.

Other laboratory tests

Serum and CSF C- reactive protein: CRP maybe useful for the diagnosis of bacterialmeningitis.8 One should keep in the mind thatthis acute phase reactant can be affected byseveral factors and hence it’s usefulness islimited. The decision of whether to start theantibiotic therapy in patient should not bedetermined by CRP result.

Serum procalcitonin: Serum procalcitonin levelis elevated in cases with bacterial meningitis andhence it is useful for differentiation of bacterialand viral meningitis.

Beta-glucuronidase activity in CSF: This is avery useful test particularly in the early phase ofbacterial meningitis. When the other laboratory

parameters from the CSF remains normal thisenzyme activity is increased. This is a reliableindicator of bacterial meningitis.

Liver function test: SGOT/ SGPT areespecially important in patients with encephalitis.Mild elevation is suggestive of West Nileencephalitis, Human herpes virus 6, Epstein-Barrvirus and Legionnaires disease.

Neuroradiology

Neuroimaging plays a crucial role in thediagnosis and therapeutic decision making ininfectious diseases of the nervous system(Table 2). Following are the conditions where itis indicated: (1) When there are focal signs/seizures, (2) Raised ICT, (3) No significantresponse to medical treatment, (4) Persistence offever or coma, (5) Suspected complications likesubdural empyema, hydrocephalus, etc.

Table 1. Common viral infections of nervous system and their confirmatorylaboratory tests

Virus Disease Laboratory confiramtion

JE Meningitis ELISA for IgM or IgG inEncephalitis serum or CSF

Herpes simplex Encephalitis PCRMeningoencephalitsAseptic MeningitisCerebellitis

Measles Encephalitis SerologySSPE Elevated CSF globulin

Coxsackie and Echovirus Meningitis Culture of Stool, CSFEncephalitis CS PCRCerebellitisPeripheral neuritis

Mumps Meningitis SerologyEncephalitis Culture of CSF

39


Cranial ultrasonography has become animportant radiological modality because of itsportability, low cost, non invasiveness, lack ofradiation hazard and diagnostic image quality.It can demonstrate changes in brain parenchyma,extracerebral fluid and ventricles associated withvarious CNS infections like parenchymalchanges observed with cerebritis, edema andabscess formation; changes in extracerebral fluidinclude effusion and empyema; and ventricularchanges associated with ventriculitis andhydrocephalus. It is useful only in very youngchildren where the anterior fontanel is open.

During the past decade, advances have beenmade in the technology used to image the centralnervous system in the pediatric age group.Although computed tomography (CT) remainsthe first line of imaging for the sick child admittedto the emergency room with fever and alteredmental status, magnetic resonance imaging (MRI)offers superior soft tissue imaging of centralnervous system (CNS) infections.9,10 MRI also isthe gold standard for imaging spinal infections.With the advent of new MRI sequences such asT2-weighted fluid attenuated inversion recovery(FLAIR), diffusion-weighted imaging (DWI) andmagnetic resonance spectroscopy (MRS), we areable to detect early and subtle abnormalities such

as the vasculitis accompanying meningitis andcomplications like subdural/epidural empyema.10

The newer technique of diffusion-weightedimaging (DWI) shows parenchymalcomplica-tions of meningitis earlier and withmore clarity and is of help in differentiation ofcerebral abscess from ring enhancing lesions ofother etiology. Proton magnetic resonancespectroscopy (PMRS) seems to produce specificpeak patterns in cases of abscess. In cases ofsuspected opportunistic infection due totoxoplasma, DWI may be of help in thedifferentiation from lymphoma, showing norestriction of water diffusion. In patients withherpes simplex and more exotic viruses like WestNile and Murray Valley virus DWI allows earlierlesion detection and therapeutic intervention withvirustatic drugs.10,11

Another imaging technique that has beenreported to be helpful in the diagnosis ofencephalitis is single photon emission computedtomography (SPECT).Initial reports suggest thatSPECT is more sensitive than CT. Recently, afew cases of normal MRI scans but abnormalSPECT in patients with herpes simplexencephalitis have been reported. As a generalrule, SPECT should be reserved for cases with

Table 2. Preferred imaging procedures in infectious CNS diseases12

Neurologic disease Imaging procedure

Meningitis or encephalitis Initially CT without and with injection of contrast materialMRI after initial assessment and treatment.

Cerebral or cerbellar abscess CT without and with contrast injection for diagnosis or,if stable, MRI instead of CT;MRI without and with injection of contrast materialsubsequently.

Granuloma MRI without and with injection of contrast material.

40

2009; 11 (3) : 245

normal MRI and a non diagnostic EEG in whichherpes simplex encephalitis is still stronglysuspected.

Radionuclide brain scan: It uses a radioactivematerial such as 99 Tc, which concentrates inregions where the blood-brain barrier has beendisrupted. It is useful in the investigation ofherpes encephalitis and cerebral abscess.

Points to Remember

• CSF cell analysis (WBC count) should bedone immediately after its collection.

• CSF glucose and blood glucose ratio isimportant in diagnosing bacterialmeningitis.

• In 50 % of patients with clinically curedbacterial meningitis, the CSF glucose,protein and WBC count may remainabnormal.

• Gram stain is the single best test for rapiddiagnosis of bacterial meningitis. Antigendetection by latex agglulination is an usualreapid test in partially treated bacterialmeningitis.

• For culture CSF should be inoculated assoon as possible, preferably at bedside.

• Neuroimaging has a crucial role indiagnosis and therapeutic decisionmaking.

References

1. Rajnik M, Ottolini MG. Serious infections ofCNS; encephalitis, meningitis and brain abscess.Adolesc Med 2000;11:401-425.

2. Bonadio WA. The CSF physiologic aspects andalterations associated with bacterial meningitis.Pediatr Infect Dis J 1992;11;423-432.

3. Tunkel AR, Hartman BJ, Kaplan SL, et al.Practice guideline for the management ofbacterial meningitis. Clin Infect Dis 2004:39;1267-1284.

4. Eisenhut M, Meehan T, SL, et al. CSF glucoselevels and sensorineural hearing loss in bacterialmeningitis Infection. 2003; 31:247-250.

5. P Nagbhushana Rao, P Anil Kumar. Bacterialmenaingitis in the post neonatal period. IndianJ Pract Pediatr 2005; 7: 133-156.

6. Perkins MD. False Positive LA result in CSFafter immunisation with H.influenzae b vaccine.Pediatr Infect Dis J 1993;7:614-615

7. Perkins MD, Merrett, Barth L. Rapid bacterialantigen detections is not clinically useful. J ClinMicrobiol 1995;33:1486-1491.

8. Pemde HK, Harish K. C-reactive protein inchildhood meningitis. Indian J Pediatr 1996;63:73-77.

9. Tuncer O, Caksen H. Crainal computedtomography in purulent meningitis of childhood.Int J Neurosci 2004;114: 167- 174.

10. Otsubo H, Snead C. Magnetoencephalographyand magnetic source imaging in children.J Child Neurol 2001;16:227–235.

11. Barkovich AJ. Pediatric Neuroimaging, 4th Edn

Philadelphia: Lippincott- Raven, 2000;pp829-831.

12. Gilman S. Imaging the brain. N Engl J Med1998; 338:812.

41

NEWS AND NOTES

Sixth Annual Training Programme in Pediatric Pulmonologyat Institute of Child Health and Hospital for Children, Chennai

September 14th to 18th, 2009Contact :Dr. D. Vijayasekaran, Prof. and HOD, Dept. of Pulmonology,Institute of Child Health and Hospital for Children, Egmore, Chennai - 8.Mobile : 09884677824, E-mail: [email protected]



COMMUNITY ACQUIRED PNEUMO-

NIA AND ITS COMPLICATIONS

* Nupur Ganguly

Abstract: Diagnosis of community acquiredpneumonia (CAP) in resource poor setting ismainly clinical. Chest radiography should bedone in cases of suspected complication orinadequate response to treatment. Completeblood count, microbiology and serology may beindicated in hospitalized patients. Importantcomplications of CAP are pleural effusion,empyema and lung abscess, which need completeevaluation.

Key words: Community Acquired Pneumonia(CAP), Fast breathing, Chest radiography,Pleural effusion, Empyema, Lung abscess.

Community acquired pneumonia can bedefined clinically as the presence of symptomsand signs of pneumonia in a previously healthychild due to infection which has been acquiredfrom the community. A more practical term-acutelower respiratory tract infection is preferred indeveloping countries due to difficulties inobtaining a chest radiograph.

Pneumonia is the leading cause of death inchildren under 5 years of age. There is anestimated 1.9 million deaths worldwide due topneumonia. The true mortality is under-estimatedin developing countries as death occurs at homewithout true medical diagnosis. In India total

number of under 5 deaths is 2.2 million of whichpneumonia alone contributes to more than 4 lakhs(nearly 20%).1 As pneumonia kills more childrenthan any other illness every effort should be madeto diagnose pneumonia and ensure propertreatment to reduce its death rate.

In resource poor setting often chestradiography and lab tests to confirm pneumoniaare not available. Hence, pneumonia is diagnosedby clinical symptoms of cough associated withfast or difficult breathing.

Table 1 shows the diagnostic criteria forpneumonia, according to the “Integratedmanagement of neonatal and childhood illness(IMNCI)” guidelines.

There are variations in practice regardingthe investigations of pneumonia in children.

Indications for chest radiography

Routine chest Xray is not beneficial in anambulatory child having lower respiratory tractinfection. Those having severe pneumonia maybenefit from them. British Thoracic Societysuggests that a chest radiograph may beconsidered in a child less than 5 years who hasfever (higher than 39OC) of unknown origin andwithout features typical of bronchiolitis.2

It is also indicated if the child isunresponsive after 48hours of treatment insuspected pneumonia or if the condition of thepatient deteriorates.

Radiography as clue to the pathogens

Chest radiography is too insensitive to beuseful in differentiating bacterial and non

* Associate Professor,Institute of Child Health, Kolkata.

42


2009; 11 (3) : 247

bacterial pneumonia. However, viral pneumoniais usually characterized by hyperinflation of thechest with bilateral interstitial infiltrates andperibronchial cuffing.3 The radiographicappearance alone is not diagnostic and otherclinical features must be considered.

Some of the radiological patterns ofcommon etiological agents are given in Table 2.

In staphylococcal pneumonia radiologicalfindings include bronchopneumonia withalveolar infiltrates. The infiltrates may coalesceto form large area of consolidation and cavitation.Destruction of alveolar wall may lead topneumatocele formation and pleural effusion.Empyema is found in 90% of communityacquired staphylococcal pneumonia cases.4

Follow up chest radiography

Follow up radiograph after acuteuncomplicated pneumonia are of no value whenthe patient is asymptomatic.5,6 There are only fewconditions where follow up X ray in children ishelpful : (i) Lobar collapse, (ii) Children withround pneumonia to ensure that tumor massesare not missed, (iii) In case of persistence ofsymptoms despite adequate antimicrobial therapyto detect complication or deterioration.

General investigations

Blood count : Total white cell countincluding differentials, C reactive protein (CRP)and erythrocyte sedimentation rate (ESR) mayhelp to differentiate between bacterial and viral

Chest radiograph Pathogenpattern

Focal, large Usually bacteria.pleural effusion

Cavitary lesion Bacterial, Fungi (inimmuno compromised)TB.

Milliary lesions TB / Fungi.

Rapid progression Pneumococcus,of multiple infiltrates Staphylococcus or

Legionella species

Interstitial Viruses,Pneumocystis jiroveccior Mycoplasmapsittaci.

Mediastinal Inhalation, Anthraxwidening withoutinfiltrate

Table 2. Radiological patterns ofcommon etiological agents

43

Table 1. Diagnostic criteria forpneumonia (IMNCI)

Signs Classify as

Any general Severe pneumoniadanger sign* or orchest wall Very severe diseaseindrawing, stridorin calm child

Fast breathing # Pneumonia

No signs of No pneumonia :pneumonia or Cough or coldvery severe disease

* General danger signs : Symptoms like, childnot able to drink or breast feed, vomitingeverything, convulsions, signs of lethargy orunconsciousness.

# Fast breathing (Respiratory rate per minuteaccording to age) : Less than 2 months:> 60/min; 2 months - upto 12 months: >50/min;12 months - upto 5 years : > 40/min.


pneumonia. In viral pneumonia the WBC countcan be normal or elevated with predominantlymphocytes, where as, in bacterial pneumoniaand occasionally adenoviral pneumonia theWBC count may be in the range of 15,000 to40,000/mm3 with predominant polymorpho-nuclear leucocyte response. The appearance ofacute phase reactant will depend upon the hostresponse. Some viral infections particularlyadenovirus or influenza virus are capable ofcausing invasive infection and may induce hostresponse similar to that of bacterial infection.It can be measured as a base line and may beuseful if the patient does not improve ontreatment as expected.

Urea and electrolytes

Estimation of urea and electrolytes is doneif the patient is seriously ill or has dehydration.Fluid restriction is only indicated if there isSIADH suggested by hyponatremia.

Specific microbiological investigations

There is no role for microbiologicalinvestigation in a child with pneumonia in thecommunity. It may be helpful in patients admittedto the hospital.

Blood culture

The British Thoracic Society guidelinesrecommend blood culture to be performed in allchildren who are hospitalized and bacterialpneumonia is suspected. However blood culturesare positive in 10% - 30% children only. Positivetest will suggest severe invasive disease andidentify the drug sensitivity pattern.

Nasopharyngeal culture

Bacterial growth in the nasopharynx doesnot indicate infection with the same organism inthe lower airways because nasopharyngealcarriage of bacteria like pneumococcus is knownto occur. Viral antigen detection fromnasopharyngeal aspirate is specific for RSV,

parainfluenza virus and adenovirus. Sensitivitiesof these tests are 80% particularly in infants.However, these tests are not available in mostcenters, are laborious and slow and the infectionusually resolves by the time it is confirmed.

Sputum gram stain

In those patients where it can be obtained,an adequate sputum sample is defined as a samplecontaining greater than 25 polymorphs and lessthan 10 squamous epithelial cells per low powerfield. A positive gram stain for pneumococci inan adequate sample has high sensitivity andspecificity for diagnosing pneumococcalpneumonia. In case of pleural effusion ifsignificant fluid is present it should be aspiratedand sent for gram stain examination and cultureas early as possible.

Serologic diagnosis

A number of antigen, antibody andpneumococcal immune complex methods ofserological diagnosis have become available7,8.No single test has specificity and sensitivitysufficiently high to be diagnostic.

Urine antigen detection test forpneumococcus has poor specificitiy in childrendue to nasopharyngeal carriage of pneumococci.

Compliment fixation test

A rise in paired titer is regarded as goldstandard for the diagnosis of Mycoplasmapneumonaie. IgM Elisa reaches a diagnostic levelduring second week of the disease but remainspositive for 6 month to 1 year after infection.This accounts for its poor sensitivity. Coldagglutinins are often used as an acute test. Coldagglutinin titer greater than 1:64 are found in 50%of patients. In children aged 5 to 14 years, thepositive predictive value for mycoplasmapneumonia by rapid cold agglutinin testwas 70%.2

44

2009; 11 (3) : 249

Lung aspiration

It is rarely performed for etiologicaldiagnosis of bacteria. However, the technique issafer than usually presumed and can provide anetiological diagnosis in more than 50% of cases.

Investigations in complicatedpneumonia

The most common complications associatedwith pneumonia are pleural effusion, empyemaand lung abscess.

Pleural effusion and empyema

Pleural effusion can be divided into 3 stages.

1. Exudative stage : It is the initial stagewhen inflammation has just set in, usually lastsfor 1-3 days, pleural fluid has little cellularcontent and is relatively thin. Gram staining andculture of this fluid often yields negative result.Pleural fluid has a pH more than 7.3 with glucosecontent above 60 mg/dl and LDH less than500 IU/L. Adequate treatment in this stage willresult in prevention of any further complication.

2. Fibrinopurulent stage (4-10 days) : There isout pouring of large number of polymorphs inthe pleural space which makes it relatively thick.Pleural fluid pH falls below 7.1 with glucose lessthan 40mg/dl and LDH greater than 1000 IU/L.With thickening of the fluid there are loculationsand the lungs become less expansible.

3. Organizing stage (after14 days) : Fibroblastsgrow into the exudate with laying down of fibrinwhich results in thickening of both the pluralsurfaces. The lung might be encased within thethickened pleura ie, “the peel” and gets entrappedwith inability to expand.

About 40% of hospitalized patients withpneumonia develop parapneumonic effusion asa complication of pneumonia. It might also resultfrom mediastinal lymph node abscess,

retropharyngeal abscess or as a complication ofsubdiaphragmatic infection. Abdominal orthoracic surgery may also lead to empyema.

Investigations : Every child with suspectedpleural effusion should have a chest radiographin erect posture and a lateral decubitus (patientlying on the affected side) view.

Erect film : Earliest sign is obliteration of thecostophrenic angle. With massive effusion theremay be whitening of the hemithorax withmediastinal shift. Small amount of fluid may beconcealed behind the diaphragm. In uprightposture a distance of more than 2 cm betweenthe gastric bubble and the left lung is anotherclue to diagnosis of effusion.9

Lateral decubitus view: On lateral decubitusview, free fluid “layers out” to the dependant partsaspect of thoracic cavity. In case of loculatedfluid and pleural thickening there will be absenceof layering. This view also reveals the smalleffusion which is concealed behind thediaphragm in upright view. As the fluid shiftsaway to the dependant part, underlying lung andmediastinum may be viewed, If the distancebetween the inner border of the thoracic cavityand outer border of the lung is greater than10 mm the pleural effusion is significant andneeds thoracentesis.

Ultrasound examination: Ultrasoundexamination is useful to differentiate loculatedfluid from thickened pleural membrane as bothappear as increased opacity on radiograph.The size, site and echogenicity of pleural effusionis also assessed by ultrasound. Anechoic pleuralfluid may be either a transudate or an exudate,however multiple echogenic foci indicate anexudate or an empyema. In an opacified chestX ray ultrasound helps to differentiate betweenfluid collection, pneumonia, loculated fluid frompleural peel. Finally it is extremely useful inguiding the position of drains and providescontinuous guidance for thoracentesis and biopsy.

45


CT chest: It is used to distinguish pneumonia,lung abscess and pleural effusion. It is alsoindicated when there is hemothorax, chylothoraxor effusion of unclear etiology and if surgery isplanned.

Pleural fluid aspiration : Diagnosticthoracentesis should be performed in everypatient with free pleural fluid that measures morethan 10 mm on decubitus view.10,11 Exceptions tothis are transudative effusion with congestiveheart failure, nephrotic syndrome, etc. or denguehemorthogic fever.

Pleural fluid examination : Naked eyeexamination : It offers little help but if theeffusion consists of pus it is always aparapneumonic effusion. Purulent fluid that isalso sanguinous is typical of empyema due toGroup A streptococci . Staphylococcus aureususually produces thick tan or brown effusion.Anaerobic organisms produce putrid odour.Bloody effusions that do not qualify ashemothorax (pleural fluid hematocrit is less than50% of peripheral blood) indicate Group Astreptococcal empyema, tuberculosis and trauma.

The following findings suggest traumaticthoracentesis : Nonuniform red discoloration offluid during aspiration, clotting of the fluid withinminutes (presence of platelets), and absence ofhemosiderin-laden macrophages.

Biochemical examination : It helps to stagethe effusion. Pleural fluid should be sent forglucose, protein, LDH and pH estimation. Pleural

fluid pH estimation needs usual precaution asarterialpH. Tube for glucose estimation shouldcontain antiglycolytic substance as fluorideoxalate. Exudative fluid should meet at least oneof the Light’s criteria as in Table 3 where astransudative fluid meets none.12

Light’s criteria can misidentify transudativefluid as exudative in 25% cases. If clinically thepatient appears to have transudative effusion butfluid meets the exudative criteria then thedifference of serum and pleural fluid albuminshould be assessed. If this difference exceeds1.2 gram/dL the patient in all probability hastransudative effusion. Empyema is characterizedby, pH <7.1, glucose <40 mg/dL, protein>3gm/dL and LDH>1000IU/L.

Cytological examination : Predominance ofpolymorphonuclear leukocyte indicatesempyema, whereas lymphocytic predominancepoints towards tuberculosis. However thesedifferences are non specific.

Microbiology : Pleural fluid should be sent forgram stain, ZN stain and culture/sensitivity tofind the causative organism. Prior antibiotic usedecreases the yield. One can also test for bacterialantigens.

Factors indicating the need for more invasiveprocedure than thoracentesis are in order ofimportance are : 1) Loculated pleural fluid,2) Pleural fluid pH<7.2, 3) Pleural fluid glucose<60 mg/dl, 4) Positive gram stain or culture ofthe pleural fluid and 5) Presence of gross pus inthe pleural space (empyema).

All children with empyema should beadmitted to hospital. As empyema is essentiallyan abscess of the pleural space, antibiotic aloneis not sufficient for management. Drainageof fluid is required for resolution of infection.The longer the duration between the onset ofinfection and drainage by thoracotomy tubehigher the incidence of loculation and

Table 3. Light’s criteria

1. Pleural fluid protein/serum protein ratio(pleural fluid/serum) > 0.5

2. Pleural fluid LDH/serum LDH ratio(pleural fluid/serum) >0.6

3. Pleural fluid LDH > 2/3rd of normal upperlimit of serum LDH.

46

2009; 11 (3) : 251

progression to the stage of development offibrous peel that encases the lung and preventsits expansion. To avoid such complication it issafest to go for immediate thoracotomy tubedrainage of any exudative pleural effusion.2 It iswell tolerated and preferable to repeatedthoracentesis.

Lung abscess

Chest radiograph may show radiolucentlesion. Cavitation with air fluid level establishesthe diagnosis of lung abscess. However, CT scanis more diagnostic. The most frequent sites areposterior segment of upper and superior segmentof the lower lobe, specially on the right as theseare most dependent areas of the lung in supineposition with aspiration as predisposing event.Ultrasound and CT scan are usually utilized tolocate the lesion for aspiration.

Lab findings : WBCs are increasedwith polymorphonuclear leucocytosis.Nasopharyngeal or sputum samples are notreliable for identification of the pathogen.At times lung abscess may need transthoracicaspiration and fiber optic bronchoscopy.

Points to Remember

• Diagnosis of CAP is clinical, especially byfast breathing and chest indrawing

• Imaging is needed in cases of non-responders and those developingcomplication.

• For pleural effusion and empyemacomplete evaluation including pleural fluidanalysis and imaging are needed.

References

1. Unicef /WHO. Pneumonia: The forgotten killerof children. 2006.

2. BTS Guidelines for the Management ofCommunity Acquired Pneumonia inChildhood.Thorax 2002; 57supplement 1:i1-i24

3. Sectish TC, Prober CG. Pneumonia. In: NelsonText book of pediatrics, Eds, Kliegman RM,Behrman RE, Jenson HB, Stanton BF. 18

th Edn,

Saunders, Philadelphia, 2007; pp1795-1800.

4. Stein RT, Marostica PIC. Community AcquiredBacterial Pneumonia In: Kendig’s disorder ofrespiratory tract in children. Eds,Chernick V,Boat TF, Wilmott RW, Bush A. 7

th Edn. Elsevier,

Philadelphia USA 2006; pp441-452.

5. Gibson NA, Hollman AS, Paton JY. Value ofradiological follow up of childhood pneumonia.Brit Med J 1993:307:1117.

6. Heaton P, Arthur K. The utility of chestradiography in the follow up of pneumonia.N Z Med J 1998;111:315-317.

7. Korppi M, Heiskanen-Kosma T, Leinonen M.Antigen and antibody assay in the etiologicaldiagnosis of respiratory tract in children.Acta Paediatr 1993;82:137-141.

8. Korppi M, Leinonen M. Pneumococcal immunecomplexes in the diagnosis of lower respiratoryinfections in children. Paediatr Infect Dis J1998;17:992-995.

9. Papastamelos C. Pleural effusions In:SchidlowDV, Smith DS, Eds, A Practical guide toPediatric Respiratory Diseases. 1

st Indian Edn,

New Delhi, Jaypee Brothers Medical PublishersLtd, 1995; pp113-120.

10. http://www.mdconsult.com. Approach topatients with pleural effusion. In:Mason RJ,Murray JF, Broaddus VC, Nadal JA, Eds, Textbook of respiratory medicine, 4

th Edn,

Philadelphia, Elseivier, 2005.

11. Singh V, Bhatnagar S. Thoracocentesis/pleuraltap. In : Sachdev HPS, Choudhury P, Bagga A,Chugh K, Ramji S, Puri RK, Eds Priciples ofPediatric and Neonatal Emergencies, 2

nd Edn,

New Delhi : JP Brothers, 2004; pp681-682.

12. Light RW. Disorders of the pleura, mediastinum,diaphragm and chest wall. In : Kasper DL,Braunwald E, Fauci A, Hauser S, Longo D,Jameson JL, Eds. Harrison’s Principles ofInternal Medicine, 16

th Edn, New York.

McGraw Hill Professional 2005; pp1565-1568.

47


RATIONAL INVESTIGATIONS TO

DIAGNOSE ENTERIC FEVER

*Tapan Kumar Ghosh

Abstract: Blood culture is the gold standard fordiagnosis of typhoid fever and it also gives thesensitivity pattern, thereby helping in the choiceof antibiotics. Nalidixic acid sensitivity is asurrogate marker of fluoroquinolone sensitivity.Sensitivity of bone marrow culture is 80 – 95 %even in late disease and despite prior to antibiotictherapy. Widal test has poor sensitivity and poorspecificity. It should be done after 5-7 days offever by tube method and level of 1 in 160 forboth H and O antibodies is usually taken asdiagnostic. Typhidot test detects IgG and IgMagainst outer membrane protein of Salmonellatyphi. This test has been improved in modifiedtyphidot M test which detects only IgM antibodiesdenoting acute infection. Other serological testslike IDL tubex and IgM dipstick test detect onlyIgM antibodies to different S typhi antigens.Molecular methods like PCR are still inexperimental stage and are not useful in day today practice.

Keywords: Typhoid fever, Enteric fever,Children, Investigations.

The correct and rapid diagnosis of entericfever is of paramount importance not only forinstituting appropriate therapy but also foravoiding unnecessary therapy.

Complete blood count (CBC)

For practical purposes the CBC in entericfever is unremarkable. The hemoglobin is normalin the initial stages but drops with progressingillness. Severe anemia is unusual and shouldmake one suspect intestinal hemorrhage orhemolysis or an alternative diagnosis likemalaria. The WBC count is normal in most casesand leukocytosis makes the diagnosis lessprobable. Leukopenia perceived to be animportant feature of typhoid fever has beenreported in only 20-25% cases.1 The differentialcount is usually unremarkable except foreosinopenia. Eosinopenia often absolute may bepresent in 70-80% cases.2,3 Presence of absoluteeosinopenia offers a clue to diagnosis but doesnot differentiate enteric fever from other acutebacterial or viral infections. Conversely a normaleosinophil count does make typhoid fever a lesslikely possibility. Platelet counts are normal tobegin with and fall in some cases by the secondweek of illness. Overall prevalence ofthrombocytopenia is around 10-15%.4

Cultures

Blood Culture

Blood culture is the gold standard diagnosticmethod for diagnosis of enteric fever5. Thesensitivity of blood culture is highest in the firstweek of the illness and reduces with advancingillness.6 Overall sensitivity is around 50 % butdrops considerably with prior antibiotic therapy.5

Failure to isolate the organism may be caused byseveral factors which includes inadequatelaboratory media, the volume of blood taken forculture, the presence of antibiotics and the time

* Scientific Co-ordinator,Institute of Child Health,Kolkata

48


2009; 11 (3) : 253

of collection. It is essential to inoculate bloodinto the cultue media at the time of drawingblood.

Salmonella can be easily cultured in mostmicrobiologic laboratories with use of routineculture media (Hartley’s media, blood agar andMacConkey agar). Automated blood culturesystems such as BACTEC certainly enhance therecovery rate. Sufficient amount of blood shouldbe collected for culture as the median bacterialcount in the peripheral blood is only 0.3 CFU/ml(inter quartile range 0.1 to 10; range, 0.1 to 399)7.At least 10 ml of blood in adults and 5ml inchildren should be collected. Dilution should beappropriate in order to adequately neutralize thebactericidal effect of serum and a ratio of 1:5 to1:10 of blood to broth is recommended.Clot cultures, wherein the inhibitory effect ofserum is obviated have not been found to be ofsuperior sensitivity as compared to blood culturesin several clinical studies8-10. In the laboratory,blood culture bottles should be incubated at 39OCand checked for turbidity, gas formation and otherevidence of growth after 1, 2, 3 and 7 days.For days 1,2 and 3 only bottles showing signs ofpositive growth are cultured on agar plates.On day 7 all bottles should be sub-cultured beforebeing discarded as negative.

There are considerable advantages ofroutine blood cultures while investigating asuspected enteric fever. They are not only 100%specific, but also provide information on theantimicrobial sensitivity of the isolate. This isvital in today’s scenario of multidrug resistance.Moreover alternative methods for diagnosisparticularly serology are rather unsatisfactory.Blood cultures may actually turn out to becheaper and very cost effective in the long run,as positive cultures unequivocally establish thediagnosis of enteric fever and all otherinvestigations for PUO can be safely deferred.

Bone marrow culture : Salmonella typhi is anintracellular pathogen in the reticuloendothelialcells of the body including bone marrow5. Studieshave revealed that the median bacteremia in thebone marrow is 9 CFU/ml (IQR, 1 to 85; range,0.1 to 1,5805) compared to 0.3 CFU/ml(IQR, 0.1 to 10; range, 0.1 to 399) in blood. Thisbone marrow: peripheral blood ratio which isaround 4.8 (IQR, 1 to 27.5) in the first week ofthe illness increases to 158 (IQR, 60 to 397)during the third week owing to disappearance ofbacteria from the peripheral blood7. The overallsensitivity of bone marrow cultures ranges from80 – 95 % and is good even in late disease anddespite prior antibiotic therapy5,11-13.

The invasive nature of bone marrowaspiration deters its use as a first line investigationfor diagnosis of typhoid fever. It is however avery useful and valid investigation in evaluationof PUO wherein the marrow should be inoculatedin the culture bottle at the bedside.

Stool, urine and other cultures

Stool specimen should be collected in asterile wide mouthed container. Specimensshould preferably be processed within 2 hoursafter collection. If there is a delay the specimenshould be stored in a refrigerator at 4OC or in acool box with freezer packs. The sensitivity ofstool culture depends on the amount of fecescultured and the positivity rate increases with theduration of the illness. Rectal swabs should beavoided as these are less successful. Stoolcultures are positive in 30% of patients with acuteenteric fever5. For the detection of carriers,several samples should be examined because ofirregular shedding of salmonella. Urine culturesare not recommended for diagnosis in view ofpoor sensitivity5,14. Other methods such asduodenal string and skin snip culture of rose spotshave been reported to be more efficacious thanblood cultures but are mainly of academicimportance14-16.

49


Antimicrobial sensitivity testing

The crucial issue here pertains tofluoroquinolone susceptibility testing.Fluoroquinolones were introduced in 1989 andduring the past decade there has been aprogressive increase in the MICs of ciprofloxacinin Salmonella typhi and paratyphi5. Since thecurrent MIC’s are still below the NationalCommittee for Clinical Laboratory Standards(NCCLS) susceptibility breakpoint, laboratoryreports will continue to report Salmonella typhi/paratyphi as ciprofloxacin/ofloxacin sensitive17.However use of fluoroquinolones in this scenariois associated with a high incidence of clinicalfailure5,17. It has also been demonstrated thatresistance to nalidixic acid is a surrogate markerfor high ciprofloxacin MIC’s, predictsfluoroquinolone failure and can hence be usedto guide antibiotic therapy (ie, if culture resultsshow resistance to nalidixic acid, irrespective ofthe results of ciprofloxacin/ ofloxacin sensitivity,quinolones should not be used or if used highdoses should be given)18. Since MIC testing isnot within the scope of most laboratories,nalidixic acid susceptibility testing is mandatoryto help guide choice of antibiotics.

Serologic tests

Widal test

This test first described by F Widal in 1896,detects agglutinating antibodies against the O andH antigens of Salmonella typhi and H antigensof paratyphi A and B6,19. The “O” antigen is thesomatic antigen of Salmonella typhi and is sharedby Salmonella paratyphi A, paratyphi B, otherSalmonella species and other members of theEnterobacteriaceae family20. Antibodies againstthe O antigen are predominantly IgM, riseearly in the illness and disappear early20.The H antigens are flagellar antigens ofSalmonella typhi, paratyphi A and paratyphi B.Antibodies to H antigens are both IgM and IgG,

rise late in the illness and persist for a longertime.19,20 Usually, O antibodies appear on day6-8 and H antibodies on days 10-12 after the onsetof disease. The test is usually performed on anacute serum (at first contact with the patient).A convalescent serum should preferably also becollected so that paired titrations can beperformed.

Conventionally, a positive Widal test resultimplies demonstration of rising titers in pairedblood samples 10-14 days apart.19 Unfortunatelythis criterion is purely of academic interest.Decisions about antibiotic therapy cannot waitfor results from two samples. Moreoverantibiotics may dampen the immune responseand prevent a rise in titer even in truly infectedindividuals. Therapeutic decisions have to begenerally based on results of a single acutesample. In endemic areas, baseline anti O andanti H antibodies are present in the populationowing to repeated subclinical infections withSalmonella typhi/paratyphi, infections with otherEnterobacteriaceae and other tropical diseasessuch as dengue and malaria.19-21 These antibodytiters vary with age, socio economic strata, urbanor rural areas and prior immunization with thetyphoid vaccine. Establishing appropriate cutoffs for distinguishing acute from past infectionsis thus important for the population where thetest is applied. In one study from Central India,anti O and anti H titer of more than 1:80 wasseen in 14% and 8% respectively of a studysample of 1200 healthy blood donors.22

While interpreting the results of the Widaltest, both H and O antibodies have to be takeninto account. There is controversy about thepredictive value of O and H antibodies fordiagnosis of enteric fever. Certain authoritiesclaim that O antibodies have superior specificityand positive predictive value (PPV) because theseantibodies decline early after an acute infection.23

Other studies report a poorer positive predictive

50

2009; 11 (3) : 255

value of O antibodies probably due to rise of theseantibodies in other salmonella species, gram-negative infections, in unrelated infection andfollowing TAB vaccination.21 For practicalpurpose and for optimal result this test should bedone after 5-7 days of fever by tube method andlevel of both H and O antibodies of 1 in 160dilution should be taken as cut off value fordiagnosis. H anitbodies once positive can remainpositive for a long time.

The Widal test as a diagnostic modality hassuboptimal sensitivity and specificity.19-21 It canbe negative in up to 30% of culture proven casesof typhoid fever. Sub optimal sensitivity resultsfrom negativity in early infection, prior antibiotictherapy and failure to mount an immune responseby certain individuals.19 Poor specificity, an evengreater problem is a consequence of preexistingbaseline antibodies in endemic areas, crossreactivity with other gram-negative infectionsand non-typhoidal salmonella, anamnesticreactions in unrelated infections and priorparenteral or oral typhoid vaccination. The purityand standardization of antigens used for the Widaltest is a major problem and often results in poorspecificity and poor reproducibility of testresults.19 The slide Widal test should also bediscouraged owing to high rate of falsepositives.20

Not withstanding these problems, the Widaltest may be the only test available in certainresource poor settings for diagnosis of entericfever. In Vietnam, using a cutoff of >1/200 forthe O agglutinin or >1/100 for H agglutinin testperformed on acute-phase serum the Widal testcould correctly diagnose 74% of blood culturepositive typhoid fever, however 14% resultswould be false positive and 10% false negative.21

Hence, it is important to realize the limitationsof the Widal test and interpret the results carefullyin light of endemic titers so that both overdiagnosis and under diagnosis of typhoid feverand the resulting consequences are avoided.24

Other serologic tests

In view of the limitations of the Widal testand need for a cheap and rapid diagnostic method,several attempts to develop alternative serologictests have been made. These include rapiddipstick assays, dot enzyme immunoassays andagglutination inhibition tests.25-27

Enzyme immunoassay (EIA) test or typhidot test:A dot enzyme immunoassay that detects IgG andIgM antibodies against a 50 KD Outer MembraneProtein distinct from the somatic (O), flagellar(H) or capsular (Vi) antigen of Salmonella typhiis commercially available as Typhidot.27

The sensitivity and specificity of this test hasbeen reported to vary from 70%-100% and43% - 90% respectively.28-33 This dot EIA testoffers simplicity, speed, early diagnosis and highnegative and positive predictive values.The detection of IgM reveals acute typhoid inthe early phase of infection, while the detectionof both IgG and IgM suggests acute typhoid inthe middle phase of infection. In areas of highendemicity where the rate of typhoidtransmission is high the detection of specific IgGincreases. Since IgG can persist for more than2 years after typhoid infection34 the detection ofspecific IgG can not differentiate between acuteand convalescent cases. Further more, falsepositive results attributable to previous infectionmay occur. On the other hand IgG positivity mayalso occur in the event of current reinfection.In cases of reinfection there is a secondaryimmune response with a significant boosting ofIgG over IgM, such that the later can not bedetected and its effect masked. A possible strategyfor solving this problem is to enable the detectionof IgM by ensuring that it is unmasked.35 Theoriginal Typhidot test was modified byinactivating the total IgG in the serum samples.Studies with modified test, Typhidot M, haveshown that inactivation of IgG removescompetitive binding and allows the access of theantigen to the specific IgM when it is present.

51


The Typhidot M that detects onlyIgM antibodies of Salmonella typhi has beenreported to be slightly more specific in a coupleof studies.26,33

IDL tubex test : The Tubex test is easy to performand takes approximately 2 minutes time.36

The test is based on detecting antibodies to asingle antigen in S. typhi only. The 09 antigenused in this test is very specific found in onlysero group D salmonellae. A positive resultalways suggest a salmonella infection but notwhich group D salmonella is responsible.Infection by other serotypes like S. paratyphi Agive negative result. This test detects IgMantibodies but not IgG which is further helpfulin the diagnosis of current infections.

IgM dipstick test26 : The test is based on thebinding of S. typhi specific IgM antibodies toS. typhi lipopolysaccharide (LPS) antigen andthe staining of the bound antibodies by anantihuman IgM antibody conjugated to colloidaldye particles. This test will be useful in placeswhere culture facilities are not available as it canbe performed without formal training and in theabsence of specialized equipments. One shouldkeep in mind that specific antibodies appear aweek after the onset of symptoms so thesensitivity of this test increases with time.

Antigen detection tests : Enzyme immunoassays,counterimmune electro-phoresis and co-agglutination tests to detect serum or urinarysomatic/flagellar/Vi antigens of Salmonella typhihave been evaluated.37-40 Sensitivity of Vi antigenhas been found to be superior to somatic andflagellar antigen and has been reported as rangingfrom 50% to 100% in different studies.37-40

Similarly specificity estimates have been reportedto vary from 25% to 90%.37-40 The suboptimaland variable sensitivity and specificity estimates,inability to detect Salmonella paratyphi infectionand Vi antigen negative strains of S typhi areserious limitations of the Vi antigen detectiontests.

Molecular methods

The limitations of cultures and serologictests advocate for development of alternativediagnostic strategies. PCR as a diagnosticmodality for typhoid fever was first evaluated in1993 when Song, et al successfully amplified theflagellin gene of S typhi in all cases of cultureproven typhoid fever and from none of thehealthy controls.41 Moreover some patients withculture negative typhoid fever were PCR positivesuggesting that PCR diagnosis of typhoid mayhave superior sensitivity than cultures. Over thenext 10 years a handful of studies have reportedPCR methods targeting the flagellin gene,somatic gene, Vi antigen gene, 5S-23S spacerregion of the ribosomal RNA gene, invA geneand hilA gene of Salmonella typhi for diagnosisof typhoid fever.42-50 These studies have reportedexcellent sensitivity and specificity whencompared to positive (blood culture proven) andhealthy controls. The turnaround time fordiagnosis has been less than 24 hours.

These reports should be viewed within thecontext of certain limitations. Clinical utility ofPCR tests has been inadequately evaluated.Performance of the test in individuals with febrileillnesses other than typhoid, in those with pasthistory of typhoid, carriers of S typhi, and thosevaccinated with typhoid vaccine is not known.Patients with a clinical diagnosis of typhoid feverwho are culture negative but PCR positive mayin fact be false positives. Comparison of PCR tobone marrow cultures as a gold standard may bea superior way of evaluating the sensitivity andspecificity of these tests, but has not been done.The tests claim to detect as few as 10 organisms,but it should be remembered that in typhoid themedian bacteremia is 0.3 CFU/ml of blood.7

Using small volumes of blood for DNA extractionmay significantly lower the sensitivity of thesetests. The cost and requirement for sophisticatedinstruments is also a potential drawback ofmolecular methods.

52

2009; 11 (3) : 257

Conclusions

The complete blood count is the logical firstinvestigation. Presence of a normal or lowleukocyte count with eosinopenia points topossible enteric fever. It also helps in evaluationof alternative diagnoses such as malaria, dengueand other bacteremias. Blood cultures remain themost effective investigations for diagnosis ofenteric fever till date. They should be sent earlyin the course of the illness and prior to startingantibiotic therapy. Susceptibility testing fornalidixic acid should be routinely done for allisolates to aid choice of antibiotics. Bone marrowculture is a highly sensitive diagnostic test evenin late stages of the illness and with priorantibiotic therapy. It should be performed in allpatients with prolonged pyrexia if routineinvestigations have failed to establish a diagnosis.Widal test has several limitations and should berequested for in the second week of the illnessand its results interpreted with caution. Data onbaseline titers in the local population should begenerated by studies to help in determiningappropriate cut offs for Widal. Modified Widaltest, Typhidot, Tubex and Vi antigen tests needto be evaluated further before their routine usecan be recommended. Molecular methods are stillexperimental.

Points to Remember

• CBC has limited diagnostic value in entericfever.

• Blood culture is the gold standard fordiagnosis of typhoid fever. But it is to bedone before using any antibiotic.Laboratory media and volume of bloodtaken for culture should be adequate(10ml of blood in adults and 5 ml inchildren). Automated blood cultureenhaces recovery rate.

• The invasive nature of bone marrowaspiration deters from its use as a 1st lineinvestigation in enteric fever.

• Widal test has suboptimal sensitivity andspecificity as a diagnostic modality ofenteric fever. For practical purpose andfor optimal result this test should be doneafter 5-7 days of fever by tube method andlevel of both H and O antibodies of 1 in160 dilution should be taken as cut offvalue of diagnosis. H antibodies oncepositive can remain positive for long time.

References

1. Abdool Gaffar MS, Seedat YK, Coovadia YM,Khan Q. The white cell count in typhoid fever.Trop Geogr Med. 1992; 44: 23-27.

2. Deshmukh CT, Nadkarni UB, Karande SC.An analysis of children with typhoid feveradmitted in 1991. J Postgrad Med 1994; 40:204-207.

3. Pandey KK, Srinivasan S, Mahadevan S,Nalini P, Rao RS. Typhoid fever below fiveyears. Indian Pediatr 1990; 27: 153-156.

4. Chiu CH, Tsai JR, Ou JT, Lin TY. Typhoid feverin children: a fourteen-year experience. ActaPediatr Taiwan 2000; 41: 28-32.

5. Parry CM, Hien TT, Dougan G, White NJ,Farrar JJ. Typhoid Fever. N Engl J Med 2002;347: 1770-1782.

6. Ananthanarayan R, Panikar CKJ.Enterobacteriaceae III - Salmonella. Textbookof Microbiology. Chennai: Orient Longman,1999; pp244–249.

7. Wain J, Pham VB, Ha V, et al. Quantitation ofbacteria in bone marrow from patients withtyphoid fever: relationship between counts andclinical features. J Clin Microbiol 2001; 39:1571-1576.

8. Hoffman SL, Edman DC, Punjabi NH, et al.Bone marrow aspirate culture superior tostreptokinase clot culture and 8 ml 1:10 blood-to-broth ratio blood culture for diagnosis oftyphoid fever. Am J Trop Med Hyg 1986; 35:836-839.

9. Simanjuntak CH, Hoffman SL, DarmowigotoR, Lesmana M, Soeprawoto, Edman DC.Streptokinase clot culture compared with whole

53


blood culture for isolation of Salmonella typhiand S. paratyphi A from patients with entericfever. Trans R Soc Trop Med Hyg 1988; 82:340-341.

10. Escamilla J, Florez-Ugarte H, Kilpatrick ME.Evaluation of blood clot cultures for isolationof Salmonella typhi, Salmonella paratyphi-Aand Brucella melitensis. J Clin Microbiol 1986;24: 388-390.

11. Farooqui BJ, Khurshid M, Ashfaq MK,Khan MA. Comparative yield of Salmonellatyphi from blood and bone marrow cultures inpatients with fever of unknown origin. J ClinPathol 1991; 44 : 258-259.

12. Duthie R, French GL. Comparison of methodsfor the diagnosis of typhoid fever. J Clin Pathol1990; 43: 863-865.

13. Akoh JA. Relative sensitivity of blood and bonemarrow cultures in typhoid fever. Trop Doct1991; 21:174-176.

14. Gilman RH, Terminel M, Levine MM,Hernandez-Mendoza P, Hornick RB. Relativeefficacy of blood, urine, rectal swab, bone-marrow, and rose-spot cultures for recovery ofSalmonella typhi in typhoid fever. Lancet 1975;31; 1:1211-1213.

15. Vallenas C, Hernandez H, Kay B, Black R,Gotuzzo E. Efficacy of bone marrow, blood,stool and duodenal contents cultures forbacteriologic confirmation of typhoid fever inchildren.Pediatr Infect Dis 1985 ; 4 : 496-498.

16. Benavente L, Gotuzzo E, Guerra J, Grados O,Guerra H, Bravo N. Diagnosis of typhoid feverusing a string capsule device. Trans R Soc TropMed Hyg 1984; 78: 404-406.

17. Crump JA, Barrett TJ, Nelson JT, Angulo FJ.Reevaluating fluoroquinolone breakpoints forSalmonella enterica serotype typhi and fornon-typhi salmonellae. Clin Infect Dis 2003;37:75-81.

18. Kapil A, Renuka, Das B. Nalidixic acidsusceptibility test to screen ciprofloxacinresistance in Salmonella typhi. Indian J MedRes 2002; 115: 49-54.

19. Olopoenia LA, King AL. Widal agglutinationtest - 100 years later: still plagued by

controversy. Postgrad Med J 2000; 76: 80-84.

20. Rodrigues C. The Widal test more than 100years old: abused but still used. J Assoc PhysIndia 2003; 51:7-8.

21. Parry CM, Hoa NT, Diep TS, et al. Value of asingle-tube Widal test in diagnosis of typhoidfever in Vietnam. J Clin Microbiol 1999; 37:2882-2886.

22. Shukla S, Patel B, Chitnis DS. 100 years ofWIDAL test and its reappraisal in an endemicarea. Indian J Med Res 1997; 105: 53-57.

23. Schoeder SA. Interpretation of serologic testsfor typhoid fever. J Am Med Assoc 1968; 206:839-840.

24. Nsutebu EF, Ndumbe PM, Koulla S. Theincrease in occurrence of typhoid fever inCameroon: overdiagnosis due to misuse of theWidal test? Trans R Soc Trop Med Hyg. 2002;96: 64-67.

25. Jesudason M, Esther E, Mathai E. Typhidot testto detect IgG & IgM antibodies in typhoid fever.Indian J Med Res 2002; 116:70-72.

26. Hatta M, Goris MG, Heerkens E, Gooskens J,Smits HL Simple dipstick assay for the detectionof Salmonella typhi-specific IgM antibodies andthe evolution of the immune response in patientswith typhoid fever. Am J Trop Med Hyg. 2002;66: 416-421.

27. Gasem MH, Smits HL, Goris MG, DolmansWM. Evaluation of a simple and rapid dipstickassay for the diagnosis of typhoid fever inIndonesia. J Med Microbiol 2002; 51: 173-177.

28. Khan E, Azam I, Ahmed S, Hassan R. Diagnosisof typhoid fever by Dot enzyme immunoassayin an endemic region. J Pak Med Assoc 2002;52: 415-417.

29. Cardona-Castro N, Agudelo-Florez P.Immunoenzymatic dot-blot test for the diagnosisof enteric fever caused by Salmonella typhi inan endemic area. Clin Microbiol Infect 1998;4: 64-69.

30. Handojo I, Dewi R. The diagnostic value of theELISA-Ty test for the detection of typhoid feverin children. Southeast Asian J Trop Med PubHlth 2000; 31: 702-707.

54

2009; 11 (3) : 259

31. Bhutta ZA, Mansurali N. Rapid serologicdiagnosis of pediatric typhoid fever in anendemic area: a prospective comparativeevaluation of two dot-enzyme immunoassaysand the Widal test. Am J Trop Med Hyg 1999;61: 654-657.

32. Jackson AA, Ismail A, Ibrahim TA, Kader ZS,Nawi NM. Retrospective review of dot enzymeimmunoassay test for typhoid fever in anendemic area. Southeast Asian J Trop Med PubHlth 1995; 26: 625-630.

33. Choo KE, Davis TM, Ismail A, Tuan IbrahimTA, Ghazali WN. Rapid and reliable serologicaldiagnosis of enteric fever: comparativesensitivity and specificity of Typhidot andTyphidot-M tests in febrile Malaysian children.Acta Tropica 1999; 72: 175-183.

34. Saha SK, Talukdar SY, Islam M, Saha S. Ahighly ceftriaxone resistant Salmonella typhi inBangladesh. The Ped Infect Dis J 1999; 18 :297-303.

35. Bhutta ZA. Impact of age and drug resistanceon mortality in typhoid fever. Arch Dis Child1996; 75 : 214-217.

36. Lim PL, Tam FC, Cheong YM, Jegathesan M.One-step 2-minute test to detect typhoid-specific antibodies based on particle separationin tubes. J Clin Microbiol 1998; 36: 2271-2278.

37. Rao PS, Prasad SV, Arunkumar G, ShivanandaPG. Salmonella typhi Vi antigen co-agglutination test for the rapid diagnosis oftyphoid fever. Indian J Med Sci 1999; 53:7-9.

38. Sharma M, Datta U, Roy P, Verma S, Sehgal S.Low sensitivity of counter-current immuno-electrophoresis for serodiagnosis of typhoidfever. J Med Microbiol 1997; 46: 1039-1042.

39. Pandya M, Pillai P, Deb M. Rapid diagnosis oftyphoid fever by detection of Barber protein andVi antigen of Salmonella serotype typhi. J MedMicrobiol 1995; 43:185-188.

40. Chaicumpa W, Ruangkunaporn Y, Burr D,Chongsa-Nguan M, Echeverria P. Diagnosis oftyphoid fever by detection of Salmonella typhiantigen in urine. J Clin Microbiol. 1992; 30 :2513-2515.

41. Song JH, Cho H, Park MY, Na DS, Moon HB,Pai CH. Detection of Salmonella typhi in theblood of patients with typhoid fever bypolymerase chain reaction. J Clin Microbiol1993; 31:1439-1443.

42. Sanchez-Jimenez MM, Cardona-Castro N.Validation of a PCR for diagnosis of typhoidfever and salmonellosis by amplification of thehilA gene in clinical samples from Colombianpatients. J Med Microbiol 2004; 53: 875-878.

43. Cocolin L, Manzano M, Astori G, Botta GA,Cantoni C, Comi G. A highly sensitive and fastnon-radioactive method for the detection ofpolymerase chain reaction products fromSalmonella serovars, such as Salmonella typhi,in blood specimens. FEMS Immunol MedMicrobiol 1998; 22: 233-239.

44. Chaudhry R, Laxmi BV, Nisar N, Ray K, KumarD. Standardisation of polymerase chain reactionfor the detection of Salmonella typhi in typhoidfever. J Clin Pathol 1997; 50: 437-439.

45. Zhu Q, Lim CK, Chan YN. Detection ofSalmonella typhi by polymerase chain reaction.J Appl Bacteriol 1996; 80: 244-251.

46. Hashimoto Y, Itho Y, Fujinaga Y, et al.Development of nested PCR based on the ViaBsequence to detect Salmonella typhi. J ClinMicrobiol 1995; 33: 775-777.

47. Hirose K, Itoh K, Nakajima H, et al. Selectiveamplification of tyv (rfbE), prt (rfbS), viaB, andfliC genes by multiplex PCR for identificationof Salmonella enterica serovars typhi andParatyphi A. J Clin Microbiol 2002; 40: 633-636.

48. Haque A, Ahmed N, Peerzada A, Raza A, BashirS, Abbas G. Utility of PCR in diagnosis ofproblematic cases of typhoid. Jpn J Infect Dis2001; 54: 237-239.

49. Massi MN, Shirakawa T, Gotoh A, Bishnu A,Hatta M, Kawabata M. Quantitative detectionof Salmonella enterica serovar typhi from bloodof suspected typhoid fever patients by real-timePCR. Int J Med Microbiol 2005; 295:117-120.

50. Prakash P, Mishra OP, Singh AK, Gulati AK,Nath G. Evaluation of nested PCR in diagnosisof typhoid fever. J Clin Microbiol 2005; 43:431-432.

55



URINARY TRACT INFECTION

*Ajay Kalra

Abstract: Key findings pointing towards urinarytracat infection (UTI) include pyuria (which canhave other causes also), hematuria (to excludeother diagnosis such as urethritis and vaginitis),and significant bacteriuria. Urinalysis is helpfulin providing immediate information to suspectUTI and enable initiation of treatment, but thediagnosis is based on confirmation by culturewhich remains the gold standard.

Key Words: UTI, Urinalyses, Culture, Radioimaging.

Urinary tract infection (UTI) is defined asgrowth of a significant number of organisms ofa single species in the urine, in the presence ofsymptoms. It is a common bacterial infection ininfants and children. These infections occur in3 – 5% of girls and 1% of boys. In girls the firstUTI occurs by the age of 5 years with peaksduring infancy and toilet training. After the firstUTI, 60 – 80% of the girls develop a second UTIwithin 18 months. In boys most UTIs occurduring the first year of life and are more commonin uncircumcised boys. During the first year oflife, the male and female ratio is 3 to 5 : 1. Beyond1 to 2 years of life, there is a striking femalepreponderance with male to female ratio of1 : 10. UTI in newborns is commonly a result ofbacteremia or septicemia.1

Laboratory investigations

Urinalysis

The ease of obtaining urine specimens andthe availability of microscopes, dipsticks andfacilities for culture makes the lab diagnosis ofUTI a straight forward feasibility. However, thereis a high risk that urine may get contaminated byperiurethral flora during passage from bladderto the container. A false diagnosis of UTI willunnecessarily subject the child to antibiotictreatment, investigations and follow up. On theother hand, failure to identify the child with UTIis associated with a risk of progressive renaldamage. The specimen for urine culture shouldtherefore be obtained with all due precautions.

Method of urine collection

i) Mid stream urine specimen : A cleancatch of this specimen is the most widely usedmethod. Prior preparation should be done bycleaning of the genitalia with soap and water.The child is asked to void urine in a squattingposition after retraction of prepuce in the malesand separation of labia in females. Antisepticwash and forced prepucial retraction are notadvised.

ii) Suprapubic aspiration would be neededin new borns and infants when mid stream urinesample can not be obtained or an earlier samplereports contamination. The suprapubic region iswashed with soap and water, cleaned withantiseptic solution and the bladder fullness isascertained by percussion. A needle mounted onsterile 2 ml syringe is introduced about

* Professor Pediatrics,S.N. Medical College, Agra.

56


2009; 11 (3) : 261

2 centimetres above the pubic symphysis into thebladder and 1 – 2 ml of urine is drawn andtransferred to the container.

iii) Urethral catheterization is anotheralternative but is usually reserved forunconscious patients already catheterized.Otherwise, only for the purpose of the collectionof urine sample, catheterization should beavoided. The age at which it may be needed isneonates and infants.

iv) Urinary bags : Because of the high riskof contamination, culture of urine specimen froma bag applied to the perineum is notrecommended. However, if used a scanty or nogrowth can be a useful indicator of no infection.

Prompt plating of the urine specimens isimportant. If delay is anticipated the sampleshould be immediately stored in a refrigerator at4OC for upto 24 hours. It is important that thistemperature is also maintained during transportof specimen.

Routine urine analysis and micro-scopic examination

In the presence of UTI, urine analysis mayshow following positive findings :

a) Mild proteinuria

b) Pyuria: More than 10 white cells/cmm3 inuncentrifuged specimen (chamber count ispreferred and counts by high power field are notreliable). Freshly voided urine sample should beexamined as there can be a false negative readingfor WBCs, due to lysis of WBCs on prolongedstanding of hypotonic urine.

c) Bacteria on Gram staining

Doing a specific gravity of the urine is helpful toidentify diluted or hypertonic urine sampleswhich can affect the investigations as broughtout below.

Dipstick analysis of urine

Chemical reagents on separate test pads ofthe dipstick evaluate different properties of urinesuch as pH, glucose and protein content and thepresence or absence of white blood cells (pyuria),red blood cells (hematuria), and significantbacteriuria. Leucocyte esterase test and nitritetest are popular due to rapid results but haveproblems of false positivity and negativity.

The leucocyte esterase test is used to screenfor white blood cells in urine (pyuria).The dipstick is read at one minute and again at5 minutes to increase the sensitivity of the test.False positive tests are usually caused bycontamination, often by vaginal secretions.False negative results can be caused byhypertonic urine, glycosuria and urobilinogen.

The nitrite (Greiss) test is used forsignificant bacteriuria. Normal urine containsnitrates but not nitrites. Gram negative entericbacteria causing UTI can convert urinary nitratesto nitrites. False positive test can result fromsubstances that cause red urine e.g. ingestion ofbeets and urinary analgesics. False negativenitrite can occur in infections caused by bacteriathat do not produce nitrites such as enterococci(streptococci), dilute urine specimens, acid urineor due to rapid emptying of bladder withoutadequate contact time in bladder for the reactionto take place.

A combined positive dipstick test forproteins, leucocyte esterase and nitrite is foundmore sensitive than individual tests.

The combination of presence of less than10 WBC/cu.mm and a positive Gram stain has asensitivity of 75% and specificity of 99% indiagnosing UTI. Any child with positive nitriteand leucocyte esterase results (sensitivity 72%and specificity 96%) or a positive Gram stain(sensitivity 93% and specificity 95%) is likely

57


to have UTI and can be started on empiricalantimicrobial therapy pending urine culturereports.

Urine culture

Determining the number of bacteria isaccomplished by plating out cultures using acalibrated loop. A number of alternative methodshave been developed as dip-slides and pipettescoated with culture media. The interpretation ofthe results of the urine culture depends on themethod of urine collection (besides clinicalbackground). (Table 1).

Table 1. Interpretation of urineculture2,3

Method of Colony Probability ofCollection Count Infection (%)

(CFU/ml)

Suprapubicaspiration Any number 99

Urethralcatheterization > 5 x 104 95

Midstreamclean catch > 1 x 105 90 – 95

Generally, a significant bacteriuria isdefined as colony count of more than 105/ml of asingle species in a clean catch sample.Young infants and children with urinaryfrequency may not always show colony count inthis range and colony count of 104/ml is alsosignificant. Lower colony counts may be foundin patients on antibiotics. Low colony countbetween 103 and 104/ml might be taken assignificant in adolescents with symptomslocalized to urinary bladder and in infections withStaphylococcus saprophyticus and Chlamydiatrachomatis (needs special media for growth).

Growth of multiple organisms on urineculture generally indicates contamination of

sample or faulty technique in collection ofspecimen. In case of isolation of candida species,two consecutive culture reports which comepositive may be necessary before initiatingrelevant therapy. The most common cause of UTIis E.coli. Other organisms can be Proteus,Klebsiella, Enterococci, Pseudomonas and Staphsaprophyticus. Presence of Proteus species mayindicate renal calculi and Pseudomonas infectionis usually nosocomial in nature or because ofinstrumentation of urinary tract. Therefore,further appropriate investigations may be neededin these cases.

Repeating urine culture is essential in thefollowing conditions : (i) Patients who fail torespond clinically within 48 hours of startingantibiotic therapy (reduction of toxicity, fever,systemic symptoms), (ii) Recurrence ofsymptoms suggestive of UTI, (iii) There is mixedgrowth of two or more pathogens or growth oforganism that normally constitutes theperiurethral flora. (lactobacilli in healthy girls andenterococci in infants) and (iv) A borderlinecolony count.

Repeat urine culture also helps to confirmthat bacteria have been eradicated, yet manyclinicians prefer to rely on clinical response only4.

Radio imaging studies

These have revolutionalized the scenario ofinvestigation for UTI in the recent years. Theyare advised for children below 5 years so as tominimize the renal damage in growing kidneys.Therefore every child with UTI should have ultrasonography (USG) evaluation. Children less then2 years should also be advised cystography bymicturating cystourethrogram (MCU) andradionuclide scintigraphy by Dimercaptosuccinicacid (DMSA) scan even if USG is normal.Between the age of 2 and 5 years DMSA scan isnecessary even if USG is normal. MCU isindicated if there is scar on DMSA scan or if

58

2009; 11 (3) : 263

DMSA scan is not available. In the age group ofmore than 5 years with first UTI with normalUSG no further evaluation is usually needed.If USG itself is abnormal, then irrespective ofage, MCU and DMSA scan need to be done5.

USG can detect posterior urethral valve, postvoid residual urine, bladder hypertrophy,dilatation of ureters, ureterocele, hydronephrosis,pelvicalyceal anomalies and under expert handsvesicoureteric reflux (VUR) also.

MCU is useful in detecting VUR, posteriorurethral valve, ureterocele, bladder and urethraldiverticuli. MCU is usually avoided during acuteUTI for the fear of increasing sepsis.After making urine sterile, the child is started onchemoprophylaxis and after 1 – 2 weeks, MCUcan be done.

DMSA scan is becoming gold standard testfor detecting renal cortical scars. DMSA scanshould be done even during acute phase of UTIand renal failure.

Children with recurrent UTI would needUSG, DMSA and MCU irrespective of age.

Intravenous pyleography : With the availabilityof radio imaging studies, the role of intravenouspyleography has declined and may be neededonly where other imaging facilities are notavailable.

Role of urodynamic studies in UTI : Childrenprone to recurrent UTI in the absence ofcongenital anomaly may have subtle bladderdysfunction or voiding disorders. Theseconditions can be diagnosed with the help ofurodynamic studies6.

Points to Remember

• Midstream specimen most preferred andpractical method of urine collection.Urinary bags unreliable.

• Ensure prompt plating or storing at 4ocupto 24 hours after urine collection.

• Proteinurea, pyuria (10 white cells/cmmin uncentrifuged specimen), bacteria onGram’s staining indicate UTI.

• Combined dipstic test for proteins,leucocyte esterase and nitrite moresensitive than individual tests.

• Culture colony count more than 105 /mlof single species considered diagnostic.Lower counts considered significant inpresence of symptoms localised to urinarysystem.

• USG to be done in all cases of UTI.Above 5 years, this will suffice. Between

Table 2. Radio imaging studies in different age groups of children

(First UTI) Less than 2 years 2 – 5 years Over 5 years

Ultrasonography Ultrasonography Ultrasonography

MCU DMSA scan (even if no abnormality DMSA scanDMSA scan (even is detected in ultrasonography) MCU(ifif no abnormality is MCU (if DMSA is not available abnormality isdetected in or if abnormality is detected detected inultrasonography) on DMSA ultrasonography)

Recurrent UTI Undertake detailed evaluation with ultrasonography, DMSA scan and MCUif the child has recurrent UTI

59


2 - 5 years DMSA scan needs to be done. Below 2 years and in all cases withabnormal USG findings (irrespective ofage) MCU and DMSA scan are advised.

• Urodynamic studies needed in childrenprone to recurrent UTI.

References

1. Elder JB. Urinary Tract Infections. In : NelsonText book of Pediatrics, 18

th Edn, Kliegman

RM, Behrman RE, Jenson HB, Stanton B, Eds.Elsevie India Pvt. Ltd, New Delhi, 2008;pp 2223 – 2224.

2. Hellerstein S. Urinary tract infections, PediatrClin North Am. 1995; 42:1433-1457.

3. Indian Pediatric Nephrology Group : IndianAcademy of Pediatrics, Consensus Statementon management of urinary tract infection. IndianPediatr 2001;38:1106 – 1115.

4. Prajapati BS. Rational Management of UrinaryTract Infection in Children. In: RationalAntibiotic Therapy, Shivanand, Yewale,Prajapati, Kundu. Eds, IAP ID ChapterPublication 2009;pp25 – 31.

5. Bagga A. Urinary Tract Infections. In:Ghosh T, Yewale V, Parthasarathy A, Shah NK,Eds. IAP subspecialty series on PediatricInfectious Diseases (under IAP Action Plan2006) 1

st Edn, 2006;pp138 – 145.

6. Bagga A. Urinary tract Infection: DiagnosisIn: Ask IAP, Deepak Ugra, Ed, IAP Publication,2006;pp41 – 48.

Fluoroquinolones for treating typhoid and paratyphoid fever (enteric fever)

Not enough sound evidence for using fluoroquinolones in typhoid and paratyphoidfever compared with the standard antibiotics

The potentially fatal typhoid and paratyphoid fevers are caused by bacterial infectionthat begins in the small intestine (enteric fever). Transmission occurs throughcontaminated food and water, and there are areas where these diseases are endemic, suchas Asia, Africa, and South and Central America. People often relapse or become carriers.Chloramphenicol has been the standard treatment, but the bacteria are becoming resistant.A new group of drugs, the fluoroquinolones, are being tried, but the review of trialsfound there were insufficient numbers of participants in the trials, which were also ofvarying quality, to be able to give recommendations with any degree of certainty, especiallyfor children.

Thaver D, Zaidi AKM, Critchley JA, Azmatullah A, Madni SA, Bhutta ZA.Fluoroquinolones for treating typhoid and paratyphoid fever (enteric fever).Cochrane Database of Systematic Reviews 2008, Issue 4. Art. No.: CD004530. DOI:10.1002/14651858.CD004530.pub3. This version first published online: April 20.2005. Last assessed as up-to-date: April 02. 2008

CLIPPINGS

60

2009; 11 (3) : 265

RATIONAL INVESTIGATIONS TO

DIAGNOSE LEPTOSPIROSIS

* Jaydeep Choudhury

Abstract: Leptospirosis should be diagnosedearly as most of the organ damage starts by theend of the first week. All the serological tests ofleptospirosis become positive in the second week.Microscopic agglutination test (MAT) isconsidered the “gold standard” of serodiagnosis.IgM ELISA is more sensitive than MAT when thefirst sample is taken early in the course of acuteillness. But even with good laboratory backup,diagnosis of leptospirosis is essentially clinical.Modified Faine’s criteria may be used fordiagnosis of leptospirosis.

Key words: Leptospirosis, Diagnosis, Serology.

Leptospirosis is a widespread zoonosiscaused by a spirochete. It infects many speciesof domestic and wild animals but the major hostsare rodents. The organism is passed in their urinefor long periods of time. Humans are usuallyinfected through the direct and indirect contactwith the urine of the infected animals.Leptospirosis is no longer a disease of high riskpopulation. With the crumbling urbaninfrastructure, everyone is at risk of this disease.

In order to adopt the correct approachtowards diagnosis of leptospirosis, it is importantto understand its pathogenesis. After enteringhuman body, leptospira circulate in the blood and

spread to all organs. It causes vasculitis of smallblood vessels by damaging the endothelium.In this septicemic phase, leptospira are presentin various body fluids like blood and CSF anddisappear by the end of first week. During thisinitial phase all the serological tests are negative.The immune phase starts in the second weekwhen inflammatory reaction occurs. During thisphase circulating antibodies appear in bodyfluids.

It is important to diagnose leptospirosis earlyin the course of the disease. Most of the organdamage starts by the end of the first week and allthe severe manifestations are related to theimmune phase, which starts in the second week.1

Ideally antibiotics have to be administered beforethe invading leptospira damage the endotheliumof blood vessels and various other organs ortissues. Serological detection of antibodies is theinvestigation of choice. But it can be done onlyafter symptoms are present for 5-6 days.2

Screening tests of leptospirosis

Screening tests for leptospirosis are usuallydone whenever leptospirosis is suspected.

Macroscopic slide agglutination test (MSAT)

The most widely adopted screening test todiagnose leptospirosis is macroscopic slideagglutination test. The test can be done after5-6 days of illness. This test is an adaptation ofagglutination test. Killed or formalinizedorganisms are used as antigens for this test.It detects only genus specific antibodies andcannot identify the different serovars ofleptospira. Titer results are slightly lower than

* Assistant Professor,Department of PediatricsInstitute of Child Health, Kolkata

61



tests which are done by antibody detection andmore cross reactions occur.3 Commercial kits areavailable for this test.

Other screening tests are Patoc-slideagglutination test (PSAT), microcapsuleagglutination test, latex agglutination tests,dipstick tests and indirect hemagglutination test.

Antibody detection

It is possible only towards the end of thefirst week of illness and titers generally reachpeak levels within 3-4 weeks.4 The titer detectedin blood will depend on the relativeconcentrations and strength of the reactionsbetween antibodies and antigens. The titer canbe measured by preparing dilutions of the serumand determining the highest dilution in which thatreaction can still be detected. The final dilutiongiving a detectable reaction is termed the titer.The titer is also sometimes called “positive titer”or “significant titer”, if it is above a certain level,namely the cut-off point.

The titer of antibodies gradually increasesduring the disease, peaks and then decreases afterrecovery. The interpretation of weak serologicalreactions is not always clear as they mayrepresent either the very early or late phase ofthe immune response or non-specific reactions.In addition, low titers or a delayed response maybe observed in severe cases, in immuno-suppressed patients and when high doses ofantibiotics were administered in the early phaseof the disease.

1. ELISA

It is the popular test and several assays areavailable. It can be performed with commercialkits or with antigen produced “in house”.A broadly reactive genus-specific antigen isgenerally used to detect IgM, and sometimes alsoIgG antibodies. The presence of IgM antibodies

may indicate current or recent leptospirosis, butit should be remembered that IgM-classantibodies may remain detectable for severalyears. IgM ELISA is more sensitive than MATwhen the first sample is taken early in the courseof acute illness.5

Advantages of ELISA : 1. ELISA can detectIgM-class antibody in the early phase of thedisease so that current or recent infection maybe indicated. Where no antibody is detected oronly a low ELISA titer is found, a second serumsample should be examined for seroconversionor a significant rise in titer.2. Only a singleantigen is used, namely the genus-specificantigen, which is shared by pathogenic andsaprophytic leptospires alike.3. Commercialsource of kits are available.

Disadvantages of ELISA : 1. Some ELISA testsystems are less specific than the microscopicagglutination test (MAT) and weak cross-reactions due to the presence of other diseasesmay be observed. ELISA results should thereforebe confirmed by the MAT or a second sampleshould be tested for rising titer. So this willrequire testing a follow-up sample if the initialsample was taken at an early stage in the infectionwhen the ELISA test may be positive, but theMAT negative. 2. Does not indicate the infectingserovar.

2. Microscopic Agglutination Test(MAT)

The microscopic agglutination test (MAT) isconsidered the “gold standard” of serodiagnosisbecause of its unsurpassed diagnostic specificity,both serovar and serogroup, in comparison withother currently available tests.2 But it is notpossible to do MAT routinely. It remains a specialinvestigation mainly for research purposes as itis done in few laboratories due to variouslimitations.

62

2009; 11 (3) : 267

The MAT is a test which determinesagglutinating antibodies in the serum of a patientby mixing it in various dilutions with 12-20different serovars of live leptospires.Antileptospiral antibodies present in the serumcause leptospires to stick together to formclumps.1 This clumping process is calledagglutination and is observed using dark-fieldmicroscopy. Agglutinating antibodies can be ofboth IgM and IgG classes.

As with other serological tests, twoconsecutive serum samples should be examinedto look for seroconversion or a four-fold orgreater rise in titer. A fourfold or greater rise intiter confirms the diagnosis regardless of theinterval between samples.5 The significance oftiters in single serum specimen is a matter ofdebate. In different areas, different cut-offpoint titers are applied. Some consider a titer of1:100 positive, whilst others accept 1:200, 1:400or 1:800 as diagnostic of current or recentleptospirosis. Titers exceeding 1600 would putthe diagnosis beyond doubt.6

Advantages of MAT: The major advantage isits high specificity.

Disadvantages of MAT : 1. The MAT cannot bestandardized because live leptospires are used asantigens. 2. It is a complex test to perform andinterpret. There is need to maintain live culturesof all serovars, so the test is both technicallydemanding and time consuming. There ispossibility of inter-observer variability inreporting results, cross-contamination and riskof exposure to the laboratory personnel.6

3. An obvious shortcoming is that antibodies maynot be detectable when the causative strain is notrepresented in the panel or only a low titer isfound with a serovar that antigenically resemblesthe absent causative serovar. The finding of notiter or a low titer in the MAT does not excludeleptospirosis in these circumstances. 4. It is never

possible to be sure that the panel is complete sincenew, unidentified leptospires may cause disease.

Is positive serology proof of a currentinfection?

Positive serology is not always proof of acurrent infection. Some antibodies may persistfor long periods after an infection so detectionof antibodies is not a proof of current infection.7

What is the diagnostic proof of recent orcurrent infection?

Seroconversion where there is no detectabletiter in the first sample, but there is positive, ie,above the cut-off point titer in the second sampleor a four-fold or higher rise in titer in consecutiveserum samples, is considered to be diagnosticproof of recent or current infection.1,5,7

A high IgM titer, i.e., a titer several-foldabove the cut-off point in a single serum specimenas detected by ELISA or a similar test isconsistent with current or recent leptospirosis,but it should be remembered that IgM classantibodies may remain detectable for severalmonths or even years.7 Serological data areimportant in the diagnostic process but mustalways be considered in conjunction with theclinical presentation and epidemiological data.

Direct Methods

Isolation of pathogenic leptospires is theonly direct and definitive proof of infection7.

1. Culture of leptospires

Leptospires grow in a variety of culturemedia. Their growth is relatively slow, with adoubling time of about 6–8 hours at best. Optimaltemperatures for growth are 28–30OC. Someserovars are, however, more fastidious thanothers in terms of their requirements in culture.7

Blood or other clinical samples should becollected for culture before antibiotics are

63


administered. Cultures from other body fluids orfrom tissues can also be made. Usually 1–3 dropsof blood or other fluids are added to culturemedium, but a small piece of minced tissue canalso be used. The chance of detecting leptospiresin blood cultures declines rapidly when thespecimen is taken after day 4 of illness.2

Growth may occasionally be detectable afterculture for about a week but often takes longer.The culture medium should therefore be checkedfor growth of leptospires at regular intervals fora period of up to 4 months. Dark-field microscopyshould be used for this purpose.

The urine will be negative in the bacteremicphase, so is not worth testing until the illness hasbeen under way for some 10 days, but by thenserological tests will be positive. So it is notworthwhile to examine urine if serum isavailable.2

Advantages of culture : 1. Isolation ofpathogenic leptospires is proof of an infection.2. Isolated leptospires can be typed to identifyserovars.

Disadvantages of culture: Leptospires growslowly so that, by the time they can be identifiedin the culture, the patient will already haveantibodies detectable by serology.

2. Dark-field microscopy

Leptospires are observed as thin, coiled,rapidly moving microorganisms in fluids suchas culture medium, blood or urine usingdark-field microscopy after concentration ofblood or urine by differential centrifugation.1

Advantages of dark-field microscopy :1. Useful for observing agglutination in the MAT.2. For observing leptospires in culture,particularly when they are present in largenumbers.

Disadvantages of dark-field microscopy :1. Dark-field microscopy is technicallydemanding. 2. Recognizing leptospires isdifficult, particularly when only small numbersare present. Artifacts, such as fibrin threads inblood, are easily mistaken for leptospires andfalse-negative diagnoses can occur frequently.Dark-field microscopy is therefore useful onlyto those with considerable experience inobserving leptospires. 3. The results of dark-fieldmicroscopy of clinical material should alwaysbe confirmed by other tests.

Staining of leptospira: Leptospires can bestained only weakly by conventionalGram staining, hence not preferred. It can bestained satisfactorily by silver staining.Immunostaining methods, such as directimmunofluorescence and variants, such asimmunoperoxidase staining, have been used.7

However, all staining methods suffer fromthe same shortcomings as dark-field microscopy,i.e., a high risk of false-positive and false-negative diagnoses. Artifacts are easily confusedwith leptospires, particularly when few arepresent.

3. PCR for leptospirosis

PCR is a method of amplifying specificsegments of leptospiral DNA in clinical samplessuch as blood, to detectable levels.7 Thus, thepresence of leptospires is confirmed by detectingand identifying specific segments of leptospiralDNA.

Advantages of PCR: Bacteria may be identifiedbefore antibody titers are at detectable levels.

Disadvantages of PCR : 1. PCR requires specialequipment and dedicated laboratory space andalso highly skilled personnel. 2. DNA that maycontaminate working areas will give falsepositivity. 3. It may also give false-negative

64

2009; 11 (3) : 269

results because inhibitors are present in theclinical materials that are being examined.

Collection of blood sample

The samples that are useful and most commonlycollected are:

1. Blood with heparin (to prevent clotting) forculture in the first 10 days. Samples for cultureshould be stored and transported at ambienttemperatures, since low temperatures aredetrimental to pathogenic leptospires.

2. Clotted blood or serum for serology:These should preferably be collected twice at aninterval of several days based on the date of onsetof disease and the probable time ofseroconversion. The testing of paired sera isnecessary to detect a rise in titers between thetwo samples or seroconversion, and thus toconfirm the diagnosis of leptospirosis. A negativeserological result in the early phase of the diseasedoes not exclude leptospirosis.

Nonspecific supportive tests forleptospirosis

1. WBC count – Normal or only mildly elevated.Severe leptospirosis may demonstrateleucocytosis with shift to the left. Occasionallyleucopenia may also be present.

2. Platelet count – Usually normal, may bedecreased.

3. ESR – Elevated in anicteric phase.

4. Urine – Proteinuria and pyuria may be present.Sometimes hyaline, granular casts andmicroscopic hematuria may be present.

5. Bilirubin and liver enzymes – Mildly elevated,liver enzymes usually in hundreds. All theparameters are more elevated in ictericleptospirosis. PT and PTT may be prolonged.

6. Serum creatinine – Raised in renal impairment.

7. CPK – Usually elevated.

8. CSF analysis – Raised CSF pressure may bethe only abnormality noted early. CSF analysisduring immune phase may be abnormal in 80%of anicteric leptospirosis and meningeal signsmay be present in 50%cases. CSF cell countshows lymphocytic predominance(50 – 500 cells/cmm), elevated protein(50 –200mg/dl) and CSF glucose is normal.

9. ECG – Findings may be suggestive ofmyocarditis.

10. Chest X ray – Patchy alveolar patternparticularly lower lobes after 3 to 9 days.

In order to treat leptospirosis effectively onehas to diagnose it within the first week. But it isvery difficult to establish the diagnosis ofleptospirosis in the first week of the disease.8

Reasons why leptospirosis is difficult todiagnose in the first week

1. All the serological tests of leptospirosis becomepositive in the second week when circulatingantibodies appear.

2. Though leptospira are present in various bodyfluids in the first week, it is not easy to isolate.

3. Microscopic demonstration of leptospira isdifficult, can be done by dark field microcsopyor immunofluorescence.

4. It is often not possible to identify thisspirochete.

5. Isolation by culture of blood or CSF in thefirst week is difficult.

Even with good laboratory back up thediagnosis of leptospirosis is essentially clinical.A useful tool for diagnosing leptospirosis isFaine’s criteria.9 It consists of three partscomprising of clinical data which is part A,epidemiological factors which is part B and part

65


C covers bacteriological and laboratory finding.Each part has several characters which is givena particular score. Leptospirosis should beconsidered when total score of part A and B is26 or more or when total score of parts A, B and

Table 1. Modified Faine's criteria for diagnosis of leptospirosis

66

Part A Clinical data Score

1 Headache 2

2 Fever 2

3 Temperature > 39OC 2

4 Conjunctival suffusion 4

5 Meningism 4

6 Muscle pain 4

7 Conjunctival suffusion 10+ Meningism+ Muscle pain

8 Jaundice 1

9 Albuminuria / increased blood urea 2

Total score 31

Part B Epidemiological factors Score

1 Rainfall 5

2 Contact with contaminated 4environment

3 Animal contact 1

Total score 10

Part C Bacteriological and laboratory Scorefindings

Isolation of leptospires in culture Diagnosis certain

Positive serology1. ELISA IgM positive 152. PSAT positive 153. MAT single high titer 154. Rising titer 25

Consider leptospirosis A + B > 26A + B + C > 25

C is 25 or more. Considering Indian conditionsShivakumar, et al has suggested modificationsof Faine’s criteria, which can be effectively usedfor diagnosis of leptospirosis.10,11 The modifiedFaine’s criteria are shown in Table 1.

2009; 11 (3) : 271

Points to Remember

• It is important to diagnose leptospirosisearly as most of the organ damage startsby the end of the first week and all thesevere manifestations starts in the secondweek. Leptospira are present in variousbody fluids in the first week, but it is noteasy to isolate. All the serological tests ofleptospirosis become positive in the secondweek.

• Macroscopic slide agglutination test(MSAT) is a commonly adopted screeningtest. Microscopic agglutination test (MAT)is considered the “gold standard” ofserodiagnosis, it is both serovar andserogroup specific. A four fold rise in titerin second sample is confirmative of thediagnosis.

• The presence of IgM ELISA antibodiesmay indicate current or recentleptospirosis. IgM ELISA is more sensitivethan MAT when the first sample is takenearly in the course of acute illness.

• Seroconversion, where there is nodetectable titer in the first sample, butpositive in the second sample or a four-foldor higher rise in titer, is considered to bediagnostic proof of recent infection.

• Even with the best laboratory backup,diagnosis of leptospirosis is essentiallyclinical.

References

1. Feigin RD. Leptospirosis. In: Feigin RD, CherryJD, Demmler GJ, Kaplan SL, Eds, Textbook

of Pediatric Infectious Diseases. 5th

Edn.Philadelphia, Saunders, 2004; pp 1708-1722.

2. Scott G, Coleman TJ. Leptospirosis. In:Cook G, Zumla A, Eds. Manson’s TropicalDiseases. 21

st Edn, London: Saunders, 2003;

pp 1165-1171.

3. Azmi P. Leptospira. In: Kliegman RM,Jenson HB, Behrman RE, Stanton BF, Eds.Nelson Textbook of Pediatrics. 18th edn,Philadelphia, Saunders, 2007; pp 1271-1272.

4. Ahmad SN, Shah S, Ahmad FMH. LaboratoryDiagnosis of Leptospirosis. J Postgrad Med2005; 51: 195-200.

5. Bavdekar SB, Jadhav S, Agarwal R.Leptospirosis. In: Ghosh TK, Yewale V,Parthasarathy A, Shah NK, Eds. Pediatr InfectDis. Mumbai, Indian Academy of Pediatrics2007; pp 331-339.

6. Bavdekar SB, Agarwal R. Leptospirosis. In:Ghosh TK, eds. Infectious Diseases in Childrenand Newer Vaccines Part 1. Kolkata: IAP InfectDis Chapter, 2003; pp 40-48.

7. Human Leptospirosis: Guidance for Diagnosis,Surveillance and Control. Geneva, WorldHealth Organization 2003.

8. Centres for Disease Control and Prevention:Leptospirosis after flooding in universitycampus, Hawaii, 2004. MMWR 2006; 55: 125-127.

9. Faine S, ed. Guidelines for the control ofleptospirosis. Geneva, Switzerland: WorldHealth Organization, 1982; WHO offsetpublication no. 67.

10. Shivakumar S. Leptospirosis evaluation.J Assoc Phys India 2003; 51: 329-330.

11. Shivakumar S, Shareek PS. Diagnosis ofleptospirosis utilizing modified Faine’s criteria.J Assoc Phys India 2004; 52: 678-679.

67



MALARIA

* Ritabrata Kundu

Abstract : Diagnosis of malaria is best basedon either clinical criteria or detection of parasitein blood. Parasitological diagnosis can be bylight microscopy or rapid diagnostic tests (RDTs).Light microscopy has remained a gold standardfor malaria diagnosis. Thick films are moresensitive tests whereas species identification isbetter with thin films. There are immuno-chromatographic tests to detect plasmodiumspecific antigens in blood samples. Two types ofRDTs, HRPII and pLDH are preferred.Other diagnostic tests like fluorescentmicroscopy and polymerase chain reaction arenot suitable for routine use.

Key words : Malaria, Diagnosis, Lightmicroscopy, Thick and thin films, Rapiddiagnostic tests, HRPII, pLDH, QBC,Fluorescent microscopy, PCR.

Malaria diagnosis still remains a challengein most of the countries. Lack of infrastructureand expertise leads to presumptive diagnosisbased exclusively on the clinical symptoms.Various clinical algorithms have bothpoor specificity and positive predictive value.They invariably lead to over treatment of malariain endemic areas and missing the diagnosis inlow transmission areas. Indiscriminate use ofantimalarials leads to increased drug pressurewhich results in widespread resistance to

antimalarials. Hence every effort should be givenfor a parasitological diagnosis of malaria beforecommencing treatment. However, in severe lifethreatening malaria presumptive treatment maybe started before confirmation after collectingblood for examination.

Parasitological diagnosis includes lightmicroscopy and rapid diagnostic tests (RDTs).As treatment of malaria has become expensivedue to use of artemisinin based combinationtherapy (ACT) parasitological diagnosis besidessaving cost has the following advantages1:(i) Improved care owing to certainity ofdiagnosis, (ii) Search for alternative diagnosisin parasitological negative cases, (iii) Reducingunnecessary use of antimalarials,(iv) Confirmation of treatment failure,(v) Improved health information.

MICROSCOPIC DIAGNOSIS

Conventional light microscopy of a wellprepared and stained blood film by an expertmicroscopist remains the “gold standard” fordetecting and identifying malarial parasite.2

Collection of blood sample : Blood should becollected as soon as malaria is suspectedirrespective of fever and not necessarily only atthe height of fever but definitely beforeadministration of antimalarials which alter themorphology of parasites.3 Blood should beobtained from finger tip or earlobe as thesecapillary rich areas contain greater density ofdeveloping parasites.4 Blood obtained by veni-puncture should be preferably, collected in EDTAvials and films should be prepared within 2 hoursfor best result.5

* Professor of Pediatrics,Institute of Child Health, Kolkata.

68


2009; 11 (3) : 273

Examination of blood film : Both thick and thinfilms should be prepared. Smears should beprepared soon after blood collection whichensures better adherence of the films to the slideand causes minimal distortion of parasites andred cells. A minimum of 100 fields should beexamined before concluding the slide to benegative. Once negative, samples may beexamined for at least three consecutive dayswhere clinical suspicion of malaria persists.

Both thin and thick smear should beprepared. Thickness of the thick film should beuniform and correct which may be ascertainedby the legibility of printed text seen through theslide. Thick films are nearly 10 times moresensitive for diagnosis of malaria as largeramount of blood is there in a given area ascompared to thin film.5 In thick film RBCs arelysed which alters the morphology of theparasites making it a good screening test fordiagnosis of malaria. They are much better thanthin film for detection of low levels of parasitemiaand reappearance of circulating parasites duringrecrudescence or relapse. In thin films as fixedmonolayer of RBC are available, morphologicalidentification of the parasite to the species leveland stage of parasite can be determined.

Performance characteristic of microscopy

(i) Skilled microscopist with proper infrastructurecan pick up parasites as low as5-10 parasite/ml of blood.6 However in actualpractice most diagnostic laboratories generallyachieve detection when parasite level is100-500 parasite/ml of blood.7,8

(ii) Species identification can be done which isvital where treatment differs with differentspecies. Stage of parasite can also be ascertainedin the peripheral blood. In general, prognosisworsens with predominance of more matureparasite stage and if more than 50% of theperipheral blood parasites are at the tiny ring

stage (diameter of the nucleus <50% of thediameter of the rim of cytoplasm) the prognosisis relatively good. Presence of pigmentcontaining asexual parasite of P. falciparumindicates bad prognosis if more than 20% of theparasites show it. It indicates mature trophozoitesor schizonts which have been released in theperipheral blood from parasites sequestered inthe capillaries of internal organs.9

(iii) Determination of the number of circulatingparasites (parasite density) is exceedinglyimportant to monitor the severity of malaria,evolution of the disease and assessing therapeuticefficacy. Parasite density can be calculated fromboth thick and thin films and expressed eitheras number of parasites present per micro literof blood or percentage of parasitised RBCs.There is no uniform agreed definition ofhyperparasitemia but parasite count of more than250,000/ml of blood or more than 5% parasitizedred blood cells carry poor prognosis.9 It isimportant that every positive blood film shouldhave parasite density assessed exactly in the sameway on post treatment specimens as on the initialspecimen to judge therapeutic efficacy. Countingparasite in a limited area of thick film isacceptable when large number of parasites areencountered whereas percentage infection ofRBC in thin films are method of choice with lowparasitemia.

(iv) The presence of malarial pigment inpolymorphonuclear leukocyte are diagnostic ofmalaria. It is particularly useful in anemicchildren with severe malaria associated with lowparasitemia. If more than 5% of polymorpho-nuclear leukocytes contain visible pigment theprognosis worsens.9

Disadvantage of microscopy

(i) It is time consuming often requiring morethan 60 minutes from blood collection to the

69


result.2 (ii) It is labour intensive, needs significanttechnical skill and proper infrastructure whichare often unavailable at peripheral health centers,(iii) There is often long delay in providing theresults of microscopy leading to treatmentwithout the benefit of the results, (iv) It cannotdetect parasite sequestered deep in the vascularcompartment which often requires repeated bloodexamination.

Rapid diagnostic tests (RDTs)

These tests were developed with the hopethat it would offer accurate, cheap and rapidresults as compared to traditional diagnosis.Tests are sensitive at parasite level of more than100-500 parasite/ml of blood. However they haveshown limitation in sensitivity in low parasitecount, ability to differentiate between species androbustness under field condition in the tropics.It was expected to give information on parasitedensities, distinguish between viable parasitefrom parasite products not associated with viableparasite and prediction of treatment outcome.

They employ monoclonal antibodiestargeted against the parasite antigens. The testkit contains specific antibody that is labeled witha visually detectable marker. If the antigen underinvestigation is present then antigen-antibodycomplex is formed. The labeled antigen-antibodycomplex will be immobilized at the pre depositedline of capture antibody and will be visuallydetectable. Whether the blood contains antigenor not, the control line will become visible aslabeled antibody is captured by the pre-depositedline of antibody directed against it. The test timevaries from 5 to 15 minutes.

Targeted antigens in currentlyavailable RDTs.

1) Histidine-rich protein II (HRP-II) – This is awater soluble protein produced by asexual stageand young gametocytes of P falciparum.10

2) Parasite lactate dehydrogenase (pLDH) –pLDH is an enzyme needed in the glycolyticpathway of the malarial parasite produced by bothsexual and asexual stages of the parasite. It isfound in all the four species of malaria namelyP falciparum, P vivax, P malariae and P ovaleand is known as pan specific.

Distinct isomers of pLDH for each of thefour plasmodium species infecting humans existand they can be detected.

The next antigen is P falciparum specific,pLDH.11

Some newer kits target P vivax specificpLDH for detection of vivax malaria.11

3) Certain new antigens like plasmodiumaldolase: Plasmodium aldolase also an enzymeof the glycolytic pathway produced by all fourspecies has been recently developed.12

Performance characteristics ofRDTs

It is an important consideration before choosingRDT which include :

1. The test should be able to distinguish betweenmalaria species at least falciparum and vivax.Falciparum and vivax malaria as single speciesinfection occurs in nearly equal number in ourcountry. Differentiation is essential as treatmentof these two differs and hence accurate diagnosisis essential. Table 1 shows currently availableRDTs in India.

In our country where falciparum and vivaxmalarial parasites cocirculate typically occurringas a single species infection, RDT which candetect both falciparum and vivax malaria anddistinguish between them is warranted.13

2. The sensitivity and specificity for detection ofeach of the species should be noted. The World

70

2009; 11 (3) : 275

Health Organization (WHO) has recommendeda minimum standard of 95% sensitivity forP falciparum densities of 100 parasite/ml of bloodand a specificity of 95%.14

RDTs using HRPII are generally moresensitive than RDTs detecting P falciparumspecific PLDH. P vivax specific monoclonalantibodies have undergone limited evaluation.Unfortunately independent peer reviewedevaluation for most commercially available RDTsare not available. In general with high parasitedensity these tests are fairly sensitive but withlow parasite load sensitivity decreases oftenyielding false negative results. False positiveresult may also develop when gametocytes arepresent but asexual stage parasites are eradicatedby therapy.

One of the US FDA approved RDT wasextensively investigated for its performance in atropical country.15 The trial showed overallsensitivity of the test of 82% for detection of anyplasmodium species. The overall sensitivity fordetection of P. falciparum was 95%, with asensitivity of 99% for parasitemia in excess of1000 parasite/ml, dropping to 89% forparasitemia of 100 to 500 parasite/ml of blood.The overall specificity of P falciparum was 94%.

There are some reports of occasional failureof RDTs to detect high parasite densities. Reportsof failure to detect both P falciparum and P vivaxhave been demonstrated even when parasitedensities exceeded 5000/ml of blood.16

3. HRPII antigen persists at detectable levels formore than 28 days even after successful therapy.17

Table 1. Currently available RDT in India

Note:

• Those kits which detect non falciparum malaria cannot distinguish isolated falciparum malariafrom mixed infection.

• As vivax malaria is almost the only nonfalciparum malaria in our country so often they equatenon falciparum malaria with vivax malaria.

Product Antigen Detection of

P falciparum P vivax

OptiMAL-IT PfLDH Yes Non falciparumPLDH (pan specific) malaria

Para HIT f HRP II Yes No

SD Malaria HRP II Yes Non falciparumPLDH (pan specific) malaria

Falci Vax HRP II Yes YesPvLDH

Paramax-3 HRP II Yes YesPvLDHPLDH(pan specific)

Diagnos HRP II Yes YesMalaria card PvLDH

71


Aldolase and PLDH rapidly fall toundetectable levels after initiation of effectivetherapy but these antigens are expressed ingametocytes which may appear after clinicalinfection is cleared.18 So none of the RDTs areuseful for monitoring the response to treatmentfor which microcopy is the investigation ofchoice.

4) Tests are usually simple without much trainingrequirement, easy to interpret, do not needelectricity and results are available rapidly. Thestability of the kit in high environmentaltemperature and humidity of tropics should betaken into account.

Disadvantages of RDTs

1. RDTs are not quantitative but qualitative andgive a ‘yes or no answer’. Hence they are notsuitable for prognostication and cannot be usedfor assessment of therapeutic efficacy ofantimalarial drugs.

2.Persistence of antigenemia after parasiteclearance precludes using the test to monitorresponse to therapy.

3.RDTs have decreased sensitivity at lower levelsof parasitemia yielding false negative results innon immune patients with low levels ofparasitemia.

4. False positive results when gametocytes arepresent but asexual stage parasites are eradicatedby therapy may lead to unnecessary treatment.

5. RDTs cannot determine the stage of parasiteie, early ring form or late schizonts and thus donot help in prognostication.

Advantages of RDTs

1. Relatively easy with minimal training andresults are available quickly.

2. They are able to detect falciparum infectioneven when the parasites are sequestered in the

deep vascular compartment and thus undetectableby microscopic examination of a peripheral bloodsmear.

Role of RDTs in the diagnosis of malaria inour country

In comparison to high transmission areas,malaria in our country occurs less frequently, inall age groups and is almost always symptomatic.Drug resistance including multi drug resistancehas started developing in our country solaboratory confirmation of malaria is an essentialcomponent of disease management. Expertmicroscopic diagnosis is available in centrallevels of health care system like metro cities butit is often unreliable or unavailable in areas withpoor health facilities. So RDTs will be useful infollowing situations in our country :

(i) In far away communities with poor health carefacilities where microscopic diagnosis is notavailable. Also in areas where laboratory serviceis inadequate, of an unacceptable standard or notavailable at odd hours.

(ii) In places where quality microscopy isavailable, RDTs and microscopy can run inparallel. RDTs will provide rapid or screeningdiagnosis whereas microscopy is reserved forresolution of confusing cases, confirmation ofnegative result in RDTs with high clinicalsuspicion of malaria.

(iii) US FDA has approved RDT with a note thatnegative results by the RDT be confirmed bymicroscopy.19

(iv) In some cases of severe and complicatedmalaria peripheral parasitemia may be negativedue to sequestration but RDTs are expected toprovide evidence of antigenemia.

(v) According to the new National drug policyof malaria (2008) that any fever case clinicallysuspected of malaria should preferably beinvestigated for confirmation of malaria by

72

2009; 11 (3) : 277

microscopy or RDT so as to ensure fulltherapeutic dose with appropriate drug to allconfirmed cases.20

Other methods

Fluorescent microscopy : The test is based onthe ability of fluorescent dyes to detect RNA andDNA of the parasite. As mature red blood cellshave no nucleus anything which binds the dye ispresumed to be the parasite. Two flourochromeshave frequently been used for this purposenamely, acridine orange (AO) and benzothio-carboxypurine (BCP). Both are excited at 490nmand exhibit apple green or yellow fluorescence.Microscopist using AO must distinguish betweenstained parasite from other cells and debriscontaining nucleic acid like leucocytes.

Quantitative buffy coat assay : A modificationof fluorescent microscopy in which blood is firstcentrifuged to separate the parasite below thegranulocyte layer. In this test heparinised tube,pre-coated with AO and an anticoagulant,containing a separatory plastic float is used.Blood is taken in the tube and centrifuged andthe area below the buffy coat is examined insitu, under a fluroscent microscope to detectparasitised cells. In field conditions, sensitivityof the test is same as that of conventionalmicroscopy. Gametocytes and late parasite stagetend to localize among the leucocytes of buffycoat and are easily missed. Also speciesidentification other than falciparum is difficultand neither this test gives exact idea about theintensity of infection. The tubes remain readableonly for hours so unlike blood smear cannot beused as parmanent record purpose. The high costof the tube and equipment required prevents itsuse for routine practice.

Polymerase chain reaction (PCR): They areable to identify genetic material of the parasite.The test is highly sensitive which enables todetect even negligible amount of parasite DNA.

It can also identify different species andmutations co-relating to resistance. Howevertheir use is limited to only research laboratories.

Points to Remember

• Thick films are more sensitive testwhereas species identification is betterwith thin films.

• Rapid diagnostic tests (RDTs) areimmunochromatographic tests to detectplasmodium specific antigen in bloodsamples.

• Parasite lactate dehydrogenase (pLDH)is produced by all 4 species ofplasmodium. Monoclonal antibodiesproduced against pLDH are of 3 groups– one specific for P falciparum, onespecific for P vivax and the other ispanspecific antibody which reacts with allthe 4 species of plasmodium but unableto separate them individually.Commercially available kits can detectP vivax and P falciparum and othermalaria but can not differentiate P ovaleand P malarie.

References

1. WHO guidelines for the treatment of malaria2006. WHO/HTM/MAL/2006;1108:8-10.

2. World Health Organization. Approaches to theDiagnosis Malaria, In : New PerspectivesMalaria Diagnosis. Report of a Joint WHO/USAID Informal Consultation 2000;pp10-18.

3. Nandy A. National Antimalarial Drug Policyand its implications in the malaria control inIndia. In: Ganguly N, Kundu R, Ghosh TK, Eds.Common MDR Infections in Children. Typhoid,Tuberculosis, Malaria, New Delhi, CBSPublication, 2005:pp197-207.

4. Gilles H. Diagnostic methods in malaria. In:HM Gilles and D A Warrell eds. Essentialmalariology, 3

rd Edn, P Edward Arnold, London,

UK 1998;p78.

73


5. Houwen B. Blood film preparation and stainingprocedure, Clin Lab Med 2002;22:1-14.

6. World Health Organization, Severe andcomplicated malaria. Trans R Soc Med Hyg1990, 84 (Suppl. 2); 23-25.

7. WHO. Malaria diagnosis: Memorandum froma WHO meeting. Bull WHO 1988; 66 :575-594.

8. Milne LM, Chiodini PL, Warhurst DC.Accuracy of routine laboratory diagnosis ofmalaria in the United Kingdom. J Clin Pathol1994;47: 740-742.

9. World Health Organization. Control of tropicaldisease. Severe and complicated malaria. TransR Soc Trop Med Hyg 1990;84(Suppl 2):1-65.

10. Hayward RE, Sullivan DJ, Day KP. Plasmodiumfalciparum : histidne – rich protein II isexpressed during gametocyte development. ExpParasitol 2000;96 :139-146.

11. Makler MT, Piper RC, Milhous WK. Lactatedehydrogenase and the diagnosis of Malaria.Parasitol Today 1998;14:376-377.

12. Meier B, Dobeli H, Certa U. Stage specificexpression of aldolase isoenzyme in the rodentmalaria parasite Plasmodium bergei. MolBiochem Parasitol 1992;52:15-27.

13. WHO. The role of laboratory diagnosis tosupport malaria disease management : focus onthe use of rapid diagnostic tests in areas of hightransmission. WHO Geneva, Switzerland, 2006.

14. WHO, Western Pacific Region. Towards qualitytesting of malaria rapid diagnostic tests :

evidence and methods. WHO. Western PacificRegion, Manila, Philippines, 2006.

15. Gasser RA Jr. Magill AJ, Ruebush T, et al.Malarial diagnosis : performance of NOW ICTMalaria in a large scale field trial. Abstr 54thAnnu. Meet. Am Soc Trop Med Hyg.2005;Abstr 2338.

16. Iqbal J, Khalid N, Hira PR. Comparison of twocommercial assays with expert microscopy forconfirmation of symptomatically diagnosedmalaria. J Clin Microbial 2002;40:4675-4678.

17. Karbwang J, Tasanor O, Kanda T, WattanagoonY, Ibrahim M, Na-Bangchang K et al. ParaSight– F test for the detection of treatment failure inmultidrug resistant Plasmodium falciparummalaria. Trans R Soc Trop Med Hyg 1996;90:513-515.

18. Mueller L, Betuela I, Ginny M, Reeder JC,Genton B. The sensitivity of the Optimal rapiddiagnostic test to the presence of Plasmodiumfalciparum gametocytes compromises its abilityto monitor treatment outcomes in an area ofPapua New Guinea in which malaria is endemic.J Clin Microbial 2007;45:627-630.

19. http://www.fda.gov/edrh/pdf6/K061542 pdf.Access on 27.01.2009.

20. National Drug Policy on Malaria (2008).Directorate of National Vector Borne DiseaseControl Program. Directorate General of HealthServices. Ministry of Health and FamilyWelfare, 22 Shamnath Marg, Delhi.

74

LAKESIDE EDUCATION TRUST27th Annual CME on Sunday 26th July 2009, At Hotel Atria.

“Pitfalls in Pediatric Practice”For Details ContactDr.H.Paramesh, Chairman, Lakeside Education Trust, Pediatrician-in-Chief & PediatricPulmonologist, C/o. Lakeside Medical Center & Hospital,33/4, Meanee Avenue Road, Near Ulsoor Lakeside Medical Center and Hospital,Bangalore – 560042. Phone: 080-25303677, 25304276, 25566738, 25366723, 25512934Fax: 25303677 Email: [email protected]

NEWS AND NOTES

2009; 11 (3) : 279

RATIONAL INVESTIGATIONS

FOR KALA-AZAR

* Nigam Prakash Narain** Neelam Verma

Abstract: Clinical diagnosis of visceralleishmaniasis (Kala-azar) should be consideredin any patient hailing from endemic areapresenting with a history of prolonged fever,weakness, cachexia, massive splenomegaly andhepatomegaly. But since these clinical featuresare shared by most of other commonly occurringdiseases such as malaria, tuberculosis and asmany of these can be present along with visceralleishmaniasis (in case of co infection), thediagnosis becomes complex. The variousdiagnostic modalities including the demons-tration of the parasite in the bone marrow orsplenic aspirate which remains the gold standardare discussed in this article.

Keywords: Kala-azar, Splenomegaly, Immuno-diagnosis, Bone-marrow examination,rK 39 strip test.

Any child with prolonged fever andsplenomegaly who hails from the Kala-azarendamic areas of Bihar and adjoining places ofthis state should be viewed with a high index ofsuspicion for visceral leishmaniasis (VL). Afterinitial screening of patient with routine bloodtests (complete blood count), liver and renalfunction tests and a X-ray chest are done.

Lab diagnosis of visceral leishmaniasis (VL)can be made by the following.1,2

• Demonstration of parasite in tissue ofrelevance by light microscopic examinationof stained specimen, in-vitro culture oranimal inoculation.

• Immunodiagnosis by detection of parasiticantigen in tissues, blood or urine sample, bydetection of nonspecific or specificantileishmanial antibodies (immuno-globulin) or by assay for Leishmania specificcell mediated immunity.

• Molecular methods by detection of parasiteDNA in tissue samples.

• Indirect evidences supporting the diagnosis.

Although several serodiagnostic tests havebeen developed e.g. ELISA, indirect fluorescentantibody test; none is in practice because ofdrawbacks like high cost, need for electricity,complicated technical procedures andunsatisfactory specificity and sensitivity.Direct agglutination test is a cheap, sensitive andspecific test and is popular in Africa, but it isassociated with drawbacks like batch to batchvariation, instability of the antigen, need forincubation and a cumbersome procedure, andthus is not popular in India. A rapid immuno-chromatographic test based on a 39 aminoacidantigen found in the kinesine region ofamastigotes of L chopasi, has recently beendeveloped and is cheap, easy to use, requires noequipment, and has high sensitivity andspecificity. However, like other antibody-basedtests, it remains positive for long periods after

* Associate Professor,

** Assistant Professor,

Department of Pediatrics,Patna Medical College

75



cure, and cannot be used for predicting cure orrelapses. In several studies from the Indiansubcontinent, the strip test was 100% sensitiveand 93-98% specific. Though there are a fewlimitations, rK39 strip test is a very usefuldiagnostic modality in the field conditions andis being applied increasingly in the Indiansubcontinent. Antigen detection is more specificthan antibody- based immunodiagnostic tests.Unfortunately none of these new tools are fieldfriendly and commercially available.

Direct evidences1,2

Demonstration of parasites is the mostreliable and conventional method for diagnosingVL. The parasite is commonly found in thesplenic or bone marrow aspirate and in the buffycoat of peripheral blood (in HIV co-infection).

(a) Bone marrow examination

The sensitivity of bone marrow smear isonly about 60-85%. It is a safer procedure butcertainly painful. Aspirated material is stainedwith Giemsa or Leishman stain. Amastigotesappear as rods or oval bodies 2-3μ in length andare found intracellularly in monocytes andmacrophages. After identification, parasitedensity can be scored microscopically by meansof a logarithmic scale ranging from 0 (no parasite/field) to +6 (>100 parasite/field).

(b) Splenic puncture

It is one of the most valuable methods fordiagnosis with sensitivity exceeding 95%. But itcarries a risk of fatal haemorrhage ininexperienced hands. Splenic aspiration shouldnot be done if the prothombin time is more than5 seconds longer than that of the control or if theplatelet count is below 40x109/L (40,000/cu mm)

The two important factors for safety are thatthe needle remains within the spleen for less than1 sec and the entry and exit axis of the aspirating

needle are identical to avoid tearing of spleniccapsule.

(C) Liver biopsy

Not usually done. Sensitivity is 70%.

(d) Culture

Blood, bone marrow aspirate or splenicaspirate can also be cultured. Media usedare: Diphasic - (a) NNN medium, (b) Tobiesmedium, (c) RPM9-1640+20% FCS medium.Monophasic - Schneider’s insect medium.

Immunodiagnosis3,4,5

(1) Antigen detection6,7

Recently, a new latex agglutination test(KATEX) has been developed to detectleishmanial antigen in the urine of patients withkala-azar. Evaluation of this test is currentlyunderway in India, Sudan, Nepal and Brazil.DNA detection using polymerase chain reactionhas also been developed by many laboratorieswith a high level of accuracy. A latexagglutination test detecting a heat stable, lowmolecular weight carbohydrate antigen in urineof visceral leishmaniasis patients has shownpromising results. Its specificity and sensitivityare 100% and 48-87% respectively.

Advantage: Antigen can be detected quite earlyduring infection and it declines rapidly followingchemotherapy and hence an useful diagnosticmethod in cases of immunodeficiency(eg. AIDS).

(2) Antibody detection 6,7

Several tests detecting specificantileishmanial antibody have been developedbut all have two major limitations:

(a) May remain detectable up to severalyears after cure (relapse cannot be diagnosed).

76

2009; 11 (3) : 281

(b) A significant proportion of healthyindividuals living in endemic areas are positivefor antileishmanial antibody owing toasymptomatic infection.

The available tests are 8,9

(a) Indirect fluorescent antibody test (IFAT),(b) ELISA, (c) Western blot.

These serological tests have shown highdiagnostic accuracy in most studies but are poorlyadapted to field settings.

(d) DAT: It is a semiquantitative test that usesmicrotitre plate in which increasing dilution ofpatient’s serum or blood mixed with stained killedLeishmania donovani promastigotes. If specificantibodies are positive, agglutination is visibleafter 18 hours with naked eyes. It is 91-100%sensitive and 72-100% specific.

Disadvantages: Costly. No prognostic value.Not user friendly for field conditions

(e) Immuno chromatographic test (ICT): Thisdipstick rapid diagnostic test is based on clonedantigen of a 39-aminoacid gene of L. chopasi.This recombinant antigen rk39 is in the kinesinregion and is specific for antibodies in patientsof visceral leishmaniasis. The sensitivity andspecificity of this test are 93% and 95%respectively.

Advantages: Easy to perform. Rapid (10-20 min).Cheap and reproducible results. Currently bestavailable diagnostic tool for VL for use in remoteareas.

Disadvantage: cannot predict response to therapyor relapse.

Molecular methods (DNA detectionmethod)10

Due to various limitations of other tests, newapproaches such as DNA hybridization have beenattempted. The development of PCR has provided

a powerful tool to the application of molecularbiology techniques in diagnosis of visceralleishmaniasis.The available tests in highercenters are:-

(a) Ln PCR-Leishmania nested polymerasechain reaction.

(b) Kinetoplast DNA(K DNA);seminestedPCR(Sn PCR).

(c) Restriction Fragment Length Polymorphism(RFLP).

A positive PCR is related to the presenceof living parasites, so result is negative after cure.

Advantage: Only test that can differentiatebetween relapse and re-infection.

Indirect evidences (Non leishmanialtests)2

(a) Complete blood count: Anemia,leucopenia, thrombocytopenia, relativelymphocytosis and monocytosis, eosinopenia,(b) Elevated hepatic transaminase level,(c) Polyclonal hypergammaglobulinemia,(d) Leucocyte:Erythrocyte ratio-1:2000 to1:1000(normal is 1:750), (e) Raised ESR.

Diagnosis of HIV- Leishmania coinfection

Atypical clinical presentation of visceralleishmaniasis in HIV infected patients pose aconsiderable diagnostic challenge.

The key issues are: a) Diagnostic principlesremain essentially same. b) Amastigotes may bedemonstrated in buffy coat preparation.c) Amastigotes can be demonstrated in bonemarrow. d) Sensitivity of antibody basedimmunologic tests is low. e) Leshmaniaamastigotes can be found at unexpected locationslike the stomach, colon or lungs. f) PCR analysisof the whole blood or its buffy coat preparationmay prove a useful screening test for thesepatients

77


Approach to diagnosis in HIV- Kala-azarco-infection

(1) For the diagnosis of first clinical episode,the combination of any antibody detectiontechnique (viz-rk39 strip test) plus KATEX testand Ln PCR analysis of peripheral blood yieldbetter results than the traditional invasivediagnostic procedures.

(2) If this test proves negative when clinicalsuspicion persists a Ln PCR of bone marrow isproposed as a reliable diagnostic method used asa test of sterile cure.

(3) KATEX test, bone marrow Novy-McNeil-Nicolle (NNN) culture and microscopybecome negative for all patients during posttreatment control.

(4) Serological antibody tests are usuallypositive even after treatment.

(5) KDNA-SnPCR-RFLP test candistinguish between a relapse and newly acquiredinfection.

Points to Remember

• Among all the tests available today,demonstration of parasite remains thegold standard.

• Bone marrow examination and splenicaspirate examination are first lineinvestigation for establishing a case ofKala-azar.

• Due to its simplicity and availability rk39strip test is now becoming popular as abedside investigation for Kala-azar.

• Katex test, which is still in field trial, is apromising bedside test in near futurebecause it is superior to rk39 in assessingresponse to therapy.

• RFLP is the only test that can differentiatebetween relapse and re-infection.

• Ln PCR analysis of bone marrow is themost sensitive test. This test is able todetect the persistence of parasites andpredict relapse.

Reference

1. Sundar S, Rai M. Laboratory Diagnosis ofVisceral Leishmaniasis. Clin Diag Lab Immunol2002;9:951-958.

2. Sundar S, Reed SG, Singh VP, Kumar PCK,Murrary HW. Rapid accurate field diagnosis ofVisceral leishmaniasis. Lancet 1998;351:563-565.

3. Sundar S. Indian Kala-azar—better toolsneeded for diagnosis and treatment;J Postgraduate Med 2003:49;29-30.

4. Singh S, Sivakumar R. Recent advances in thediagnosis of Leishmaniasis. J Postgraduate Med2003;49:55-60.

5. Zijlstra EE, Nur Y, Desjeux P, Khalil EA, etHussan AM, Groen J. Diagnosis of visceralleishmariasis with the recombinant K39 striptest : experience from Sudan. Trop Med IntHealth 2001;6:108-113.

6. Vipolana C, Blanco S, Dominguiz J, et al.Noninvasive method for diagnosis of Visceralleishmaniasis by a latex agglutination test fordetection of antigens in urine samples. J ClinMinobiol 2004;42:1853-1854.

7. A simple and economical direct agglutinationtest for serodiagnosis and seroepidemiologicalstudies of visceral leishmaniasis. Trans RSocTrop Med Hyg 80:583-587.

8. Bern C, Jha SN, Joshi AB, Thakur GD,Bista MB. Use of the recombinant k39 dipsticktest and the direct agglutination test in a settingendemic for visceral leishmaniasis in Nepal. AmJ Trop Med Hyg 2000;63:153-157.

9. Attar ZJ, Chance ML, el-Safi S, et al. Latexagglutination test for detection of urinaryantigens in visceral leishmaniasis. Acta Trop2001;78:11-16.

10. Osman OFL, Oskam EE, Zijtsra AM,El Hassan DA, El-Naiem, Kager PA. Use ofPolymerase Chain Reaction to assess thesuccess of Visceral leishmaniasis treatment.Trans R SocTrop Med Hyg 1998;92:397-400.

78

2009; 11 (3) : 283

RATIONAL INVESTIGATION

PRACTICES IN THE DIAGNOSIS

OF DENGUE

* Vijay N Yewale

Abstract:Dengue infection presents with aspectrum of clinical syndromes, ranging from abenign undifferentiated fever to a life threateningDengue Shock Syndrome. Diagnosis of dengue,though has improved over the years, still posesa challenge. A high index of suspicion is requiredto diagnose and differentiate various forms ofdengue at the time of presentation. The vascularleakage due to increased capillary permeabilityis very subtle at the onset and it is very crucial torecognize this from management point of view.Repeated clinical assessment and demonstrationof a rising hematocrit with falling platelet count,a feature unique to DHF, form the cornerstoneof diagnosis of DHF . Tourniquet test is a veryuseful clinical tool for early recognition ofincreased vascular permeability and can be usedas an alert in a suspected case. Imaging studieslike x ray chest and ultrasonography of chest andabdomen detect capillary leak before it isclinically evident and before the changes in thehematocrit. Dengue serology, though useful inthe diagnosis does not help differentiate betweenDF and DHF at the time of presentation of theillness and hence an intelligent use of dengueserology is required with due caution in itsinterpretation. PCR and viral isolation are

research tools and researchers are also lookingat the estimation of heparin sulfate in the urineas an early marker of increased capillarypermeability.

Key words : Dengue diagnosis, Dengue serology

It is important to know the outcome of adengue infection and the clinical spectrum of thedisease before we discuss the rationalinvestigation practices in dengue.

Outcome of a dengue infection

Dengue virus infections can cause aspectrum of illness ranging from asymptomaticinfection, undifferentiated fever, to classicaldengue fever (DF), dengue fever withhemorrhagic manifestation, and denguehemorrhagic fever (DHF) with or without shock.(Fig.1)1

Clinically four syndromes are recognized

1. Undifferentiated fever, 2. Dengue fever,3. Dengue hemorrhagic fever (DHF) withoutshock, 4. Dengue shock syndrome(DSS) whichis a severe dengue hemorrhagic fever with shock.

Diagnosis of dengue fever

Though several laboratory tests are used forthe diagnosis of dengue, from the managementpoint of view, a clinical diagnosis is essential forearly intervention.

During rainy season from July to October,in a child living in an area endemic for dengueand having fever with thrombocytopenia, theindex of suspicion for dengue must be high.

* Consultant Pediatrician,Dr. Yewale Hospital and Vashi Criticare,Vashi, Navi Mumbai.

79



Undifferentiated fever

It is the commonest form of dengueinfection, especially in young children andmimics any other viral fever or URI like illness.No laboratory tests are required to diagnose it.It is very often treated as viral URI therefore notdiagnosed as a dengue illness.

Dengue fever

It is again a clinical diagnosis and hence adetailed description of the illness is given here.Typical dengue fever is seen in older childrenand adolescents. Typically, the onset of DF issudden, with a sharp rise in temperatureoccasionally accompanied by chills, and isinvariably associated with severe headache andflushed face.2 Within 24 hours there may be retroorbital pain, particularly on eye movement or eyepressure, photophobia, backache and pain in themuscles and joints/bones of the extremities.The other common symptoms include anorexiaand altered taste sensation, constipation, colickypain and abdominal tenderness, dragging pain in

the inguinal region, sore throat and generaldepression. These symptoms vary in severity andusually persist for several days.

Fever: The body temperature is usually between39oC and 40oC, and the fever may be biphasic,lasting for 5-7 days.

Rash: Diffuse flushing or fleeting pinpointeruptions may be observed on the face, neck andchest during the first half of the febrile period,and a conspicuous rash that may bemaculopapular or scarlatiniform appears onapproximately the third or fourth day. Towardsthe end of the febrile period or immediately afterdefervescence, the generalized rash fades andlocalized clusters of petechiae may appear overthe dorsum of the feet, on the legs, and on thehands and arms. This confluent petechial rash ischaracterized by scattered, pale, round areas ofnormal skin. Occasionally the rash isaccompanied by itching.

Skin hemorrhage: A positive tourniquet test and/or petechiae.

Dengue Virus Infection

Asymptomatic Symptomatic

Undifferentiated Dengue Fever Dengue Haemorrhagic FeverFever Syndrome (Plasma Leakage)

Without Unusual No Shock Dengue ShockHemorrhage Hemorrhage Syndrome

Fig. 1. Spectrum of dengue infection

80

2009; 11 (3) : 285

Course: The relative duration and severity of DFvaries between individuals in a given epidemic,as well as from one epidemic to another.Convalescence may be short and uneventful,but may also be prolonged. Bradycardia iscommon during convalescence. Hemorrhagiccomplications, such as epistaxis, gingivalbleeding, gastrointestinal bleeding, hematuriaand hypermenorrhoea, may accompanyepidemics of DF. Severe bleeding hasoccasionally caused deaths in some epidemics.

Dengue fever with hemorrhagic manifesta-tions must be differentiated from denguehemorrhagic fever.

Clinical laboratory findings

The laboratory findings during an acute DFepisode of illness are as follows: a) Total WBCis usually normal at the onset of fever; thenleucopenia develops and lasts throughout thefebrile period. b) Platelet counts are usuallynormal, as are other components of the bloodclotting mechanism. However, thrombocytopeniais common in some epidemics. c) Liver enzymelevels may be elevated.

Dengue hemorrhagic fever (DHF)and Dengue shock syndrome (DSS)

DHF is a syndrome of increased vascularpermeability accompanied by abnormalhemostasis. Abnormal hemostasis and leakage ofplasma as manifested by thrombocytopenia andrising hematocrit differentiate DHF from DF.

The clinical differentiation between gradesI and II DHF from dengue fever may not be clearbecause of the variation in the baselinehaematocrit in the paediatric population.

Criteria for Clinical Diagnosis ofDHF/DSS3

WHO case definition of DHF (WHO 1975,1986)

All of the following criteria must be present :

1. Fever (high and continuous of 2-7 daysduration), 2. Hemorrhagic diathesis {at leasta positive tourniquet test (except in shock)},3. Thrombocytopenia (less than 100,000/mm3),4. Hemoconcentration (20% or more relative tobaseline) or evidence of plasma leakage(i.e. pleural effusion, ascites and/orhypoproteinemia)

The first two clinical criteria, plusthrombocytopenia and haemoconcentration or arising haematocrit, are sufficient to establish aclinical diagnosis of DHF.

Limitations of definition 4

a) It has a sensitivity and specificity of 82%and 94% respectively. b) Requires different andrepeated clinical tests which is a challenge forcountries with limited resources. c) Does not usethe current threshold for thrombocytopenia whichshould be (<150 000 platelets per mm3) or a levelestablished by risk analysis.

DHF definition paradox

a) Early treatment will prevent fulfillmentof the diagnostic criteria. b) Appropriatelyhydrated cases may not have hemoconcentrationto the extreme required by the definition, andtherefore will not be classified as DHF. c) Chestradiographs or ultrasound are required todocument the capillary leakage criterion and maynot be feasible in all places.

Unusual manifestations

1.Encephalopathy and less oftenencephalitis. 2. Hepatic failure. 3. Cardio-myopathy. 4. Dengue fever with severehemorrhage. 5. Acute respiratory distresssyndrome. 6. Acalculous cholecystitis

These are not identified by the WHO casedefinition and are not rare in all endemiclocations.

81


Three improvements are necessary to standardisethe DHF definition and to allow it to do well indifferent populations: a) Redefine the thresholdfor thrombocytopenia, b) Clarify the standardpractice and value of the tourniquet test,c) Incorporate a criterion to measure intravenousfluid replacement.

Role of tourniquet test

The tourniquet test is performed by inflatinga blood pressure cuff to a point midway betweenthe systolic and diastolic pressures for fiveminutes. The test is considered positive when10 or more petechiae in a square area overforearm of 2.5 cm x 2.5 cm. In DHF the testusually gives a definite positive result with20 petechiae or more. The test may be negativeor only mildly positive during the phase ofprofound shock. It usually becomes positive,sometimes strongly positive, if it is conductedafter recovery from shock.

In children above 4 years of age this is auseful test, as a positive reading alerts theclinician about dengue infection. In Thailand,the tourniquet test is claimed to have a positivepredictive value of 90% for dengue.5 Appropriatesized blood pressure cuff should be used. If thetest is positive the child should be sent to a centrewhich can provide good observation andinvestigation facilities. If the test cannot be doneor is negative, then a full blood count with thewhite cell count, platelet count, hemoglobin andhematocrit should be done. A low white cellcount with relative lymphocytosis suggests a viralinfection.

Laboratory studies

Complete blood cell count findings include thefollowing:

The WBC may be normal, but leucopeniais common initially, with neutrophilspredominating. Towards the end of the febrile

phase there is a drop in the total number of whitecells (Fig.1) as well as in the number ofpolymorpho-nuclear cells. A relativelymphocytosis with more than 15% atypicallymphocytes is commonly observed towards theend of the febrile phase (critical stage) and at theearly stage of shock.

The absence of leukopenia and a lowpercentage of typical lymphocytes predict severedengue illness. Simple hematologic parametersmay be used to reduce unnecessary admissionsof patients with suspected dengue infection inthe absence of more sophisticated predictors.6

A rise in hematocrit greater than 20% is asign of hemoconcentration (Table 1) and precedesshock. The hematocrit level should be monitoredat least every 24 hours to facilitate earlyrecognition of dengue hemorrhagic fever (DHF)and every 3-4 hours in severe cases of DHF ordengue shock syndrome (DSS).

Table 1. Normal haematocrit valuesfor age

Age Range (%) Mean (%)

2 weeks 42 – 66 50

3 months 31 – 41 36

6 months – 6 years 33 – 42 37

7 years – 12 years 34 – 40 38

Male 42 – 52 47

Female 37 – 47 42

Source : Camitta BM. The anemias. In: NelsonText Book of Pediatrics, Eds, Behrman RE,Kliegman RM, Arvin AM, 15th Edn, Prism BookPvt. Ltd, Bangalore, India, 1996;pp1378-1380.

If the baseline hematocrit is not available,the average hematocrit appropriate for age canbe used. An initial hematocrit of > 40% warrants

82

2009; 11 (3) : 287

close monitoring. Thrombocytopenia andhemoconcentration are constant findings in DHF.A drop in platelet count to below 100,000/mm3

is usually found between the third and eighth daysof illness. A rise in hematocrit occurs in allDHF cases, particularly in shock cases.Hemoconcentration with hematocrit increased by20% or more is considered objective evidenceof increased vascular permeability and leakageof plasma.It should be noted that the level ofhematocrit may be affected by early volumereplacement and by bleeding. Platelet counts ofless than 100,000 are seen in DHF or DSS andoccur before defervescence and the onset ofshock.

The platelet count should be monitored atleast every 24 hours to facilitate early recognitionof DHF.

Platelet count

Direct count using a phase-contrastmicroscope (normal 200,000-500,000/mm3).In practice, for outpatients, an approximate countfrom a peripheral blood smear is acceptable.In normal persons, 4-10 platelets per oil-immersion field (the average observed from10 fields is recommended) indicate an adequateplatelet count. An average of 2-3 platelets peroil-immersion field or less is considered low (lessthan 100,000/mm3).

A false low platelet count may occur due toclot formation in the sample due to samplingerror.

In an epidemic setting all patients withDF need regular monitoring by a primarycare physician for early detection of DHF.The primary care physician/health care workershould monitor the patient for clinical featuresof DHF/DSS along with hematocrit and plateletcounts if possible. Any patient developing coldextremities, restlessness, acute abdominal pain,

decreased urine output, bleeding andhemoconcentration should be admitted in ahospital.7

Presence of the following features indicateneed for close monitoring

a) Abdominal pain during defervescence.b) Dropping platelet count (< 50,000).c) Dropping leucocyte count. d) Risinghematocrit (Hb more than 13 gm%/Hct >40; riseof Hb > 1 gm%, Hct by 3). e) Third spacing,puffiness, falling urine output, positive fluidbalance. f) Positive dengue IgG, high titres.

Thrombocytpenia is seen in many illnesses.Platelet count done on cell counter should beconfirmed with the manual count or theperipheral smear, if there is doubt.

Basic metabolic panel findings include thefollowing: a) Hyponatremia is the most commonelectrolyte abnormality observed in patients withDHF or DSS. b) Metabolic acidosis is observedin those with shock, and it must be correctedrapidly. c) Elevated BUN is observed in thosewith shock.

Liver function test findings include thefollowing: a) Mild elevations in transaminaselevels may be seen. b) Low albumin which is asign of hemoconcentration.

Coagulation studies may help guide therapy inpatients with severe hemorrhagic manifestations.Findings are as follows: a) Partialthromboplastin time (PTT) and prothrombintime (PT) are prolonged in about one-half andone-third of DHF cases respectively. Thrombintime is also prolonged in severe cases.b) Deranged prothrombin time (PT) is asignificant risk factor for DSS. A longer durationof shock may be associated with an increasedrisk of hemorrhage. c) Low fibrinogen andelevated fibrin degradation product levels aresigns of disseminated intravascular coagulation.

83


In most cases, assays of coagulation andfibrinolytic factors show reduction in fibrinogen,prothrombin, factor VIII, factor XII, andantithrombin III. A reduction in antiplasmin(plasmin inhibitor) has been noted in some cases.In severe cases with marked liver dysfunction,reduction is observed in the vitamin K-dependentprothrombin family, such as factors V, VII, IXand X. Serum complement levels are reduced.Typing and crossmatching of blood should beperformed in cases of severe DHF or DSSbecause blood products may be required.

Serum specimens should be sent to thelaboratory for serodiagnosis, polymerase chainreaction (PCR), and viral isolation. Because thesigns and symptoms of DF are nonspecific,attempting laboratory confirmation of dengueinfection is important.

Imaging Studies

Chest radiograph: Right-sided pleural effusionis typical. Bilateral pleural effusions are commonin patients with DSS.

Ultrasound examination detects plasma leakagein multiple body compartments around the timeof defervescence. Ultrasonographic signs ofplasma leakage are detectable before changes inhematocrit. Ultrasound is a useful tool fordetecting plasma leakage in dengue infection8

Gallbladder wall thickening measured byultrasound is significantly associated with severedengue, as well as with hallmark features ofthrombocytopenia and hemoconcentration, inchildren with suspected dengue. Gallbladder wallthickening serves as a clinically relevantdiagnostic test and prognostic indicator of severedengue in pediatric populations.9

Other tests

• Arterial blood gas determinations should beperformed in severe cases to assess pH,oxygenation, and ventilation.

• A transient mild albuminuria is sometimesobserved.

• Occult blood is often found in the stool.

Fig.1. Need for serial CBCs in DHF

84

2009; 11 (3) : 289

Definitive diagnosis

Confirmatory lab studies for dengue virusinfection include:

1. Isolation of virus is most definitive butnot feasible and requires time, skills, expertiseand equipments.

2. Demonstration of rising titre of specificdengue antibodies.

3. Demonstration of specific viral antigen/RNA in the tissue or serum.

Virus isolation

Although this is the most definitive methodfor the diagnosis of dengue infection, it is onlyperformed in very few research laboratories. Itsusefulness is in virus surveillance and theprediction of outbreak severity in secondaryinfection.

Detection of dengue ribonucleic acid

The use of polymerase chain reaction todetect dengue RNA is not a routine procedureand should be considered more of a research tool.It is indicated when there is a diagnostic problem.

Serology

Dengue serology10

The serologic tests that have been used fordiagnosis of dengue infections includehemagglutination inhibition (HI) test,complement fixation test (CFT), neutralizationtests (NT), IgM capture ELISA, IgG captureELISA, immunoflourescence assays, dot blotimmunoassays, dipstick enzyme immunoassaysand the rapid immunochromatographic card tests.The HI test, CFT and NT are performed only inreference laboratories like National Institute ofVirology, Pune and in general is unavailable forclinical practice. The ELISA’s and the rapid card

tests are most commonly used for denguediagnosis in current clinical practice.

Serologic response following dengueinfection

In primary dengue infections, 80% ofindividuals demonstrate IgM MAC ELISAantibodies by day 5, 93% by 6-10 days and 99%by day 10-20. IgM antibodies peak at 2 weeksand then decline over the next 2-3 months. IgGantibodies rise later and to lower levels ascompared to IgM, then decline gradually butpersist at low levels for life.

In secondary dengue infections, there is abrisk and rapid IgG response much higher thanthat seen in primary dengue infections, whichpeaks at 2 weeks and then declines slowly overthe next 3-6 months. The IgM response insecondary dengue infections is slower and lowerthan IgG with some individuals showing nodetectable IgM.

Performance characteristics of differentserology tests

With use of paired sera, the sensitivity ofIgM MAC ELISA was reported as 88% andspecificity as 96%. False negative resultsmay be due to secondary dengue infections.False positive results can occasionally occurdue to recent dengue infections in the past2-3 months (as IgM antibodies persist for2-3 months). Cross reactivity with other flaviviruses is rare with IgM MAC ELISA. Thesensitivity and specificity of IgG ELISA withpaired sera is reported as 85% and 96%respectively. Cross reactivity with otherflaviviruses is more with IgG. Combined use ofIgM and IgG in paired sera gives a sensitivity of99% and specificity of 96%. The use of a singleacute sample has a much lower sensitivity (55%).

The rapid immunochromatic card tests anddipstick ELISA’s have become popular as bedside

85


tests. They have been validated againstconventional ELISA’s and found to be inexcellent agreement. It is important to rememberthat sensitivity and specificity are also kitdependent and how well the kit has been validatedin the local population

Clinical use

Therefore the best results are obtained ifacute and convalescent sera are tested for bothIgM and IgG dengue. For reasons of cost and toobviate the repeat visit, a single sample may besent for IgG and IgM dengue after day 5 of illness.If the sample result is negative, a repeatconvalescent sample may be asked for

The HI test was traditionally used fordifferentiating primary from secondary dengueinfection wherein a titer of > 1: 2560 in a singleacute phase/ convalescent sample was considereddiagnostic of secondary dengue. This is notcommercially available.

The commercially available IgG captureELISA is able to differentiate primary fromsecondary dengue. This is because a higher cutoff of IgG is used to define a positive test.This level is reached in secondary infection andnot in past or primary infection. Studies on pairedsera have shown that IgG capture ELISA cancorrectly classify 88% of primary and 98% ofsecondary dengue infections. The rapidimmunochromatographic card test for dengueIgG can also differentiate primary fromsecondary dengue, if the test is so designed to beable to pick up only a high IgG titer.

Recently, determination of IgG avidity hasbeen evaluated as a method of differentiatingprimary from secondary dengue infections withreported sensitivity and specificity of 95% and80% respectively. Primary dengue has lowavidity and secondary dengue has high avidity

Table 2 can serve as a rough guide forinterpretation of dengue serology reports

Table 2. Interpretation of dengueserology

IgM IgG Interpretation

Negative Negative Early sample/not dengue -Repeat

Negative Positive Past dengue infection,(low titer) repeat

Negative Positive Secondary dengue(high titer) infection

Positive Negative Primary dengueinfection

Positive Positive Current/ Recent(low titer) primary dengueinfection

Positive Positive Secondary dengue(high titer) infection

Repeated clinical assessment and serialblood counts are the basis for patientmanagement. In early infection, serology may benegative and hence may impart a false sense ofsecurity. In most cases by the time serology ispositive and is able to differentiate primary fromsecondary infections, the diagnosis is fairlyclinically evident. For a test to be useful in patientmanagement, it should be positive at the time ofpatient presentation and accurately predict thelikely clinical course.

Dengue illnesses have a wide clinicalspectrum ranging from clinically inapparentinfection to fatal shock syndrome: DF, DHF, andDSS are various range of their severity.

Dengue serology though useful in diagnosisof dengue illnesses is ineffectual in differentiatingthese clinical syndromes. While these tests are

86

2009; 11 (3) : 291

capable of differentiating primary fromsecondary responses; it is worth knowing thatsecondary serological responses are notsynonymous with DHF, indeed only six to eightpercent of secondary infections culminate in todengue hemorrhagic fever.

When the child with DHF presents withcritical phase in the first week, clinical diagnosisis more reliable than serology. It will be moredefinitely positive only after the first week, whenthe child is actually recovering.

Repeated physical assessment and serialblood counts are the basis for the clinicaldiagnosis and management of various dengueillnesses. Serology does have a role, albeit a smallone.

Role of repeat serology

If the first test done at the time of patientpresentation is negative/ equivocal and theclinical suspicion for diagnosis of dengueinfection is strong, we need to repeat the test.The repeat test should ideally be done at 2 weeksafter onset of symptoms, as this is the time whenthe antibody levels are peaking. However inmajority of cases the result of repeat test are onlyof academic relevance. It is necessary to repeatthe tests for optimum sensitivity; the sensitivityof a single sample is lower especially if it is doneearly in the illness. Repeating the test alsoimproves the specificity as conversion fromnegative to positive or rise in the optical densityvalues/ indicates a current infection (IgM and IgGantibodies to dengue can persist for severalmonths after infection and hence lead to falsepositive results). The limitations of the repeattest are cost and need for a second visit.The disadvantages of a single test are lowersensitivity and specificity as explained earlier.

Transplacental transmission of the antibodiesto the dengue virus: The IgG antibody against

dengue can be transmitted trans- placentally.A study from Thailand where 97% of pregnantmothers were seropositive for dengueIgG, showed 100% transmission to the babies.The cord blood antibody titers in 53% of infantswere higher than the mothers suggesting activetransport of antibodies through the placenta.The half-life of these antibodies is estimated tobe 41 days with 19% of infants losing antibodiesby 4 months, 72% by 6 months, 99% by ninemonths and 100% by 12 months.

These transplacental antibodies may resultin a false positive IgG test in infancy. Howeversince the IgM will be negative and the titer ofIgG will be low, an acute dengue infection canbe ruled out. Since these antibodies belong toIgG sub class 1 and are complement fixing theycan cause dengue hemorrhagic fever even withprimary dengue infections.

Differentiating the type of dengue virusinvolved by dengue serology: The HI, CFT andNT tests are able to identify the infectingserotype. The commercially available ELISAtests or the rapid immunochromatographic cardtests are not able to identify the infectingserotype. Certain specific ELISA’s may howeveridentify the infecting serotype. A monotypicresponse in a serological test can help inidentifying infecting serotype, however there arelimitations; monotypic responses occur onlyduring primary infections, secondary infectionsgive rise to polytypic responses, moreover it isnot unusual to have polytypic responses evenduring a primary infection

The interpretation of serological resultsmust be carefully considered against clinicalsuspicion and not taken in isolation. Serologicalresults may be negative in early acute bloodspecimen and a second specimen should be testedto confirm or exclude dengue infection.

87


Dengue antigen detection

Dengue Non Structural Protein 1 (NS1)antigen testing is suggested as a helpful tool forthe early diagnosis of dengue infection after theonset of fever. The additional Ig M antibodydetermination increases the diagnostic rates.(The positive rates of patients with DF werehigher than those with DHF but no statisticallysignificant difference was found). However,patients with primary DHF infection hadsignificantly higher positive rates than those withsecondary DHF infection. The positive rates ofIg M antibodies were in reverse proportion tothose of NS1 antigen.

Conclusion

Dengue infection presents with a spectrumof clinical syndromes, ranging from a benignundifferentiated fever to a life threateningDengue Shock Syndrome.A high index ofsuspicion is required for early diagnosis andrecognition of the capillary leak associated withDHF. Though dengue diagnosis has improvedover years, still a definite test for earlyrecognition of vascular leak which is subtle atthe onset, and the need for fluids is lacking.Rising hematocrit with falling platelet count is afeature unique to DHF and serial estimation ofthese parameters along with repeated clinicalassessments form the cornerstone of diagnosisand management of DHF. Imaging studies likexray chest and ultrasonography of chest andabdomen detect capillary leak before it isclinically evident and before the changes inhematocrit. Tourniquet test is a very usefulclinical tool for early recognition of increasedvascular permeability and can be used as an alertin a suspected case. Dengue serology, thoughuseful in the diagnosis does not differentiatebetween DF and DHF at presentation and hencean intelligent use is required with caution.PCR and viral isolations are research tools and

researchers are also looking at estimation ofheparin sulfate in the urine as an early marker ofincreased capillary permeability.

Points to Remember

• Differentiation between dengue fever anddengue hemorrhagic fever is essential forproper patient management.

• Diagnosis of dengue and denguehemorrhagic fever is based mainly onserial clinical assessment of the patient.

• Tourniquet test is a simple and usefulclinical tool to predict increased capillaryfragility in DHF

• Simple tools like serial complete bloodcounts with hematocrit and platelet counthelp differentiate DF from DHF. Risinghematocrit and falling platelet count arefeatures unique to DHF

• Dengue serology, though useful in thediagnosis does not differentiate betweenDF and DHF.

References

1. Anonymous. Clinical Diagnosis. In: Denguehemorrhagi fever, diagnosis, treatment,prevention and control; 2

nd Edn,Geneva, World

Health Organization 1997; pp12-23.

2. Halstead SB. Dengue and Dengue HemorrhagicFever. In: Text book of Pediatric InfectiousDiseases, 5

th Edn, Feigin, Cherry, Demmler,

Kaplan, Eds, 2004; pp2178-2200.

3. Dengue haemorrhagic fever: Diagnosis,treatment, prevention and control (ed 2), WorldHealth Organization, Geneva, Switzerland(1997).

4. José G Rigau-Pérez. Severe dengue: the needfor new case definitions The Lancet InfectiousDiseases 2006; 6:297-302 DOI:10.1016/S1473-3099(06)70465-0.

5. Teeratkul A, Limpakarnjanarat K, Nisalak A,Nimmanitya S. Predictive value of clinical and

88

2009; 11 (3) : 293

laboratory findings for early diagnosis of dengueand dengue haemorrhagic fever (Abstract).Southeast Asian J Trop Med and Pub Health.1990; 21: 696-697.

6. Nanthakorn Eu-Ahsunthornwattanaa, Jakris Eu-

ahsunthornwattanab and Usa Thisyakorn

a .

Peripheral blood count for dengue severityprediction. A prospective study in Thai children.Pediatr Vol. 121 Supplement January 2008, pp.S127-S128 (doi:10.1542/peds.2007-2022KKKK)

7. Lall R, Dhandha V. Dengue hemorrhagic feverand dengue shock syndrome in India. Natl MedJ India 1996: 9: 20-23.

89

8. Srikiatkhachom, Anon MD, Krantrachue.Natural History of Plasma Leakage in DengueHemorrhagic Fever: A serial untrasonographicstudy. Pediatr Infect Dis J 2007;26:283-290.

9. Colbert, James A, Gordon, et al. Ultrasoundmeasurement of gall bladder wall thickening asa diagnostic test and prognostic indicator forsevere dengue in pediataric patients. PediatrInfect Dis J 2007;26:850-852.

10. Chuansumrit, Ampaiwan; Chaiyaratana. TheUse of Dengue Nonstructural Protein 1 Antigenfor the Early Diagnosis During the Febrile Stagein Patients with Dengue Infection. Pediatr InfectDis J 2008;27:43-48.

SOUTH PEDICON 2009XXII South Zone Conference of Indian Academy of Pediatrics

34th

Annual Conference of IAP Tamil Nadu State Chapter

Hosted by

Indian Academy of Pediatrics

Kancheepuram District Branch

On 14th, 15

th & 16

th of August 2009

at GRT Temple Bay, Mahabalipuram, Tamilnadu.

Delegate Fee

Particulars 15/4Rs. 31/5Rs. 31/7Rs. SpotRs.

IAP 2000 2500 3000 3500

Non IAP 2500 3000 3500 4000

Post Graduate 1500 1800 2500 3000

Accompanying Person 1750 2000 2500 3000

DD in favour of “Kanchi South Pedicon 2009” Payable at Sriperumbudur.

For details contactDr. Major K. Nagaraju Dr. S. YoganandanV.N. Hospital, No.1, Kattabomman Street, 39, Thiruvalluvar Street,Shanmugam Road, Tambaram-West, Chennai - 600 045. Kancheepuram.Modbile : 0984030199 E-mail: [email protected] Mobile : 0944443824

NEWS AND NOTES


TINEA CAPITIS

* Madhu R

Abstract:Tinea capitis, a fungal infection of thescalp and scalp hair is the most commondermatophytosis of childhood and is caused bydermatophytes belonging to the generaMicrosporum and Trichophyton. It is contagiousand communicable and is also a notifiabledisease in the United States of America andUnited Kingdom. This infection may betransmitted via infected persons, select animalvectors and by fomites. The clinical presentationdepends on the type of hair invasion and the levelof host resistance. Griseofulvin has been the drugof choice for the past five decades, but the newerantifungal agents such as fluconazole,itraconazole and terbinafine have also beenfound to be safe and effective in the treatment oftinea capitis.

Key Words: Tinea capitis, Children, Partial hairloss, Broken hair, Fomites

Tinea capitis, the most commondermatophytosis of childhood is a fungalinfection of the scalp and scalp hair that is causedby dermatophytes belonging to the generaMicrosporum and Trichophyton.1 It is alsoreferred to as ring worm of the scalp or Tineatonsurans.2 Cassius Felix in 400 AD first coinedthe term “tinea” to mean “ring worm” in latin.3

Dermatophytes causing tinea capitis may beanthropophilic, zoophilic or geophilic.

DERMATOLOGY

Predominant organisms vary with geographicarea and from time to time. For instance, in NorthAmerica, initially Microsporum canis andMicrosporum audouinii were the dominantorganisms, which have now been replaced byT. tonsurans. M.canis is the most commoncausuative organism in most parts of Europe.In the urban areas of United kingdom, a rise inthe prevalence of T.tonsurans has been recorded.4

Trichophyton violaceum, Trichiphytontonsurans, Trichophyton rubrum, Trichophytonmentagraphytes, Trichophyton simii are some ofthe dermatophytes causing tinea capitis in India,with T.violaceum being the most common agent.5

Tinea capitis occurs predominantly inprepubertal children, especially those betweenthe ages of 4 and 14 years.1 In generalapproximately 30% of the infections occur inchildren below 5 years of age and in all about96% in those below 10 years.5 Infants and adultsare occasionally affected. In the adults, it is seenmore in the adult females who fondle children.6

Decreased incidence of tinea capitis after pubertymay be due to certain unsaturated fatty acidspresent in the sebum of post pubertal individualsthat are inhibitory to dermatophytes.2

Dermatophytes have a short incubationperiod of 1- 3 weeks. Boys are more commonlyaffected than girls. Tinea capitis is transmittedvia infected persons, fallen infected hairs, selectanimal vectors and by fomites such ascontaminated combs, hair brushes, barber shopinstruments, caps, towels, bed sheet, pillow cases,bedding, clothing, furniture in the houses, backsof seats in the theatre, shared seats7 and even toys.Trauma is considered an important prerequisite

* Assistant Professor,Dept. of Dermatology and Leprosy,Govt. Stanley Hospital, Chennai.

90

2009; 11 (3) : 295

for infection in tinea capitis.8,9 Many a times,infection occurs after a scalp shave. Poor personalhygiene, over-crowding, large family size andlow socioeconomic status act as commonpredisposing factors for this infection.7 Incidenceof tinea capitis is high in the siblings of infectedchildren.5 Carriers (asymptomatic carriage)constitute a major reservoir for transmission ofthe organisms causing the disease. Carriers donot have any signs or symptoms of the infection.Adults in the family, siblings, care takers andplaymates are the usual carriers. Asymptomaticcarrier state is most common with theanthropophilic organisms. Hair care practices(use of oils or greases, styling, frequency of hairwash etc) do not appear to play a major role inthe acquisition of infection.10

Pathogenesis

Trauma assists inoculation which isfollowed after approximately 3 weeks by clinicalevidence of hair shaft infection. Infection usuallybegins in the perifollicular stratum corneum andthe hyphae generally spread into and around thehair shaft. They descend along the follicle andpenetrate the midportion of the hair.Subsequently, the intrapilary hyphae descend tothe border of the keratogenous zone where thehyphae continue to grow in equilibrium with thekeratinisation process, never entering thenucleated zone. The terminal tuft of hyphae atthis zone is termed Adamson’s fringe. There arethree types of hair invasion namely the ectothrixtype, the endothrix type and the favus type. Inthe ectothrix type, the intrapilary hyphaeproliferate and divide into arthrospores that reachthe cortex of the hair and are transported upwardson the surface of the hair. Arthrospores are seenon the surface of the hair shaft rather than withinit. In Endothrix infection, the hyphae proliferateand divide into arthroconidia inside the hair, withthe cortex being intact. Affected hair is fragileand breaks off close to the scalp surface. In favic

type, broad regularly septate hyphae and airspaces are seen in the hair shaft withdisarticulated arthroconidia being absent.1,2,5

Clinical types

The clinical presentation of tinea capitis isindeed variable, depending on the type ofhair invasion, the level of host resistance andthe degree of inflammatory host response.The appearance, therefore may vary from a fewdull grey, broken off hairs with a little scaling, toa severe painful inflammatory mass on the scalp.In all types, the cardinal features are partialhair loss with variable degree of inflammation.There are non- inflammatory and inflammatorytypes of infection. Non-inflammatory types oftinea capitis are the grey patch (Fig.1), black dot(Fig.2), the seborrhoeic type, smooth patch(Fig.3) of baldness and the adult type (glabroustype).1,2 Inflammatory types of tinea capitis arekerion (Fig.4), abscess, favus and pustulartypes.1,7 Mixed type may comprise of either twotypes of non-inflammatory or may be acombination of non-inflammatory andinflammatory types.11 Non-inflammatory typessuch as the grey patch, black dot and theseborrhoeic types are commoner than theglabrous and the inflammatory types such askerion, abscess. Non inflammatory types aremore common in older children and adults.3

Silent transmission of the disease from them tochildren and other contacts seem to be the mainfactor for a high incidence and endemicity of thedisease in South India.3 Lymphadenopathy,especially cervical or suboccipital, is verycommon in symptomatic patients with tineacapitis.

Grey patch or Epidemic type is characterizedby patches of partial hair loss with fine scaling.2

These patches are often circular in shape andshow numerous broken off hairs that are dull greyand lusterless. Itching may be present in somepatients.

91


Seborrhoeic type is more commonly caused byT.tonsurans and is characterised by diffusescaling.7

Black dot pattern is most often caused byendothrix organisms. Because of thearthroconidia inside the hair shaft, it is extremelybrittle and breaks at the level of the scalp. Theremnant of hair left behind in the infected follicleappears as a black dot on clinical examination.5

Smooth patch of baldness mimics alopeciaareata with almost total loss of hair. There isminimal scaling.1

Glabrous type is the common type seen in adults.Lesion usually extends from the nape of neck to

occiput or from the face, forehead and cheek tothe temporal or frontal areas.

Inflammatory types are caused most commonlyby zoophilic organisms. Kerion is a painful,inflammatory boggy mass studded with brokenhairs, oozing purulent material from follicularorifices. It represents a vigorous host immuneresponse to the dermatophyte. Regional lymphnode enlargement and Id reactions are commonin kerion and abscess type.3

Agminate folliculitis is characterised bypustules, papules with folliculitis without scaling.

Favus, an inflammatory type of tinea capitis thatbegins in early life and commonly extends into

92

Fig. 1. Grey patch

Fig. 2. Black dot

Fig. 4. Smooth bald patch

Fig. 3. Kerion

2009; 11 (3) : 297

93

adulthood is caused by T.schoenleinii and isendemic in Kashmir.5 It is characterised by thepresence of yellowish, cup shaped crusts knownas scutula. This infection frequently results inscarring and permanent alopecia.2

Differential diagnosis of tinea capitis

Differential diagnosis would includeseborrhoeic dermatitis (dandruff), atopicdermatitis, and psoriasis, when there is minimalinflammation and diffuse scaling.1 Seborrhoeicdermatitis is usually more diffuse and there areno broken hairs. In psoriasis, the scales on thescalp are thicker, but the hair is not broken off.When alopecia is present, conditions likealopecia areata, trichotillomania are to beconsidered. Smooth patches of hair loss inalopecia areata are non scaly. In trichotillomania,hair is broken off at varying lengths. Tractionalopecia is characterized by oval or linear hairloss in the periphery. In bacterial pyodermas(folliculitis, impetigo), loosening of the hair isnot normally present.1 If scarring has occurred,non infectious conditions like discoid lupuserythematosis. lichen planopilaris, pseudopeladeand radiation dermatitis should be considered.1

Diagnosis

Microscopic examination of the scalp scalesand the hair root in 10% and 40% KOH solutionreveals branched septate hyphae, arthrosporesand endothrix or ectothrix invasion respectively.5

Wood’s lamp examination may be of value ininfection due to Microsporum species andT.schoenleinii. Isolation of the fungus by cultureis the gold standard for diagnosis of tinea capitis.

Treatment

Topical antimycotic agents are ineffectiveas sole therapy for tinea capitis, as an oralantimycotic agent is required to penetrate the hairfollicles. Griseofulvin has been the drug of choice

for the past five decades by virtue of the efficacy,safety profile and cost.5 In fact, it was the onlydrug approved by the FDA for the treatment oftinea capitis. Ketoconazole, a broad spectrumazole agent is effective against dermatophytes.However, the risk of hepatotoxicity, lack of aliquid preparation and availability of newerantifungal agents have precluded the use of thisdrug in the treatment of tinea capitis. The newerantifungals - Fluconazole, Itraconazole, andTerbinafine have been found to be useful in thetreatment of tinea capitis.7,12,13 In 2007, theUS FDA has approved the use of terbinafine HCloral granules for the treatment of tinea capitis inpatients aged 4 years and older.

Drug dosage is as follows:

Drug Dosage/Day Duration

Tab Griseofulvin 15mg/kg after 12 weeks125mg food in

divided doses

Tab Fluconazole 6mg/kg 20 days150mg

Cap Itraconazole 5mg/kg 4 weeks100mg

Tab Terbinafine < 20 kg 4 weeks250mg 62.5 mg od

20 – 40 kg125 mg od> 40 kg250 mg od

Pulse regimen may be administered withItraconazole or Terbinafine as given below:

Pulse regimen - 5 mg/kg/d for 1 wk – 1 pulse

Second pulse after 2 weeks

Third pulse after 3 weeks

With regard to the safety profile of thesystemic antifungal agents in children,


fluconazole is considered safe for newborn, whilegriseofulvin and itraconazole can be administeredfrom 6 months of age. Ketoconazole andterbinafine can be given from 2 years of age.Shampoos like ketoconazole, selenium sulphide,zinc pyrithione etc used twice weekly decreasesthe spore load and therefore reduces transmissionof infections. Some experts recommend that allthe family members and contacts use antifungalshampoo twice weekly to eliminate the carrierstate. Use of oral corticosteroids in kerion iscontroversial. Steroids may be used in severecases of kerion associated with id reaction.

Spread of the infection by fomites is animportant concern. Therefore children should bewarned not to share combs or other hair careitems and to throughly cleanse or avoid possiblecontaminated items. Patients who are infected byzoophilic organisms need to have the infectedanimal appropriately treated.

Last, but not the least, it is indeed veryimportant to counsel the child and to reassureregarding the regrowth of the hair, as childrenwith tinea capitis frequently are ridiculed,isolated, and bullied by classmates or playmates.In some cases, the disease can cause severeemotional impairment in vulnerable children andhence counselling the child and parents is ofutmost importance.

Points to Remember

• Tinea capitis is a fungal infection of thescalp and trichophylon species are thecommon causative dermatophytes of tineacapitis in children.

• Children below 10 years arepredominantly affected.

• Infection spreads through contacts,fomites, sharing of combs and selectanimal vectors.

• Partial hair loss, itching, pain andlymphadenopathy are the commonfeatures.

• KOH wet mount of scalp scales and hairroot is a simple way to demonstrate thefungus.

• Oral griseofulvin is the drug of choice:the newer antifungals like fluconazole,itraconazole and terbinafine are alsoeffective.

References

1. Martin AG, Kobayashi GS. Superficial Fungalinfection. In : Freedberg IM, Eisen AZ, WolffK, Austen KF, Goldsmith LK, Katz SI,Fitzpatrick TB, Eds. Fitzpatrick’s Dermatologyin General Medicine. 5

th Edn, Mc Graw Hill.

Health Profession & Division, 1999; pp2337-2357.

2. Hay RJ, Moore M, Mycology tinea capitis. In :Champion RH, Burton JL, Burns DA.Brcathnach SM, Eds. Textbook of Dermatology.6

th Edn, Oxford: Blackwell Scientific

Publications, 1998; pp1277-1376.

3. Kamalam, A. Observations on the Gamut /Galaxy of the fungal infections in TamilnaduState. Kavaka 1984; 12 : 15-36.

4. Elewski B. Tinea capitis. Dermatol clin 1996;14(1) : 23-31.

5. Kanwar AJ, Manta, Chander J. Superficialfungal infections, Tinea capitis. In : Valia RG,Valia AR. Eds. Textbook and Atlas ofDermatology. Vol.I, 2

nd Edn, Bhalani Publishing

House, 2001; pp215-258.

6. Kamalam A, Thambiah AS. Tinea capitis inSouth Indian families. Mykosen, 1979; 22(7):251-254.

7. Elewski, BE. Tinea capitis. A currentperspective. J Am Acad Dermatol 2000;42:1-20.

8. Kamalam A, Chetty GN, Balasubramanian N,Chandrasekaran N, Thambiah AS. Tinea capitisin a Moslem school. Indian J Medi Res 1979;70 : 41-43.

94

2009; 11 (3) : 299

9. Kamalam A, Thambiah AS. Tinea capitis,endemic disease in Madras. Mycopathologica1980; 71 : 45-51.

10. Paller AS, Mancini AJ. Eds. Skin disorders dueto fungi. In: Hurwitz Clinical PediatricDermatology. 3

rd Edn, Elsevier Saunders, 2006;

451- 453.11. Jahangir M, Hussain I, Khurshid K, Haroon TS.

A clinic etiological correlation in tinea capitis.Int J Dermatol 1999;38: 275-278.

95

12. Friedlander SF, Fungal infections. In :SchachnerLA , Hansen RC, Eds. Pediatric Dermatology.3

rd Edn, Mosby. 2003;pp1093 -1100.

13. Gupta AK, Adam P, et al. Therapeutic optionfor the treatment of tinea capitis caused byTrichophyton species: Griseofulvin versus theNew oral antifungal agents, Terbinafine,Itraconazole and Fluconazole. Pediatr Dermatol2001; 18: 433- 438.

Meningococcal infections

Meningococcal disease is a contagious bacterial disease caused by Neisseria meningitidis(N. meningitidis). People who have had close contact with someone who has a meningococcalinfection and populations with known high carriage rates are offered antibiotics in order toeradicate the bacteria and thus prevent disease. Rifampin (also known as rifampicin),ciprofloxacin and ceftriaxone are effective agents for eradicating carriage of N. meningitidis.However, the use of rifampin may have a disadvantage as development of resistance to theantibiotic has been noted following treatment.

Fraser A, Gafter-Gvili A, Paul M, Leibovici L. Antibiotics for preventingmeningococcal infections. Cochrane Database of Systematic Reviews 2006, Issue 4. Art.No.: CD004785. DOI: 10.1002/14651858.CD004785.pub3. This version first publishedonline: January 24. 2005 Last assessed as up-to-date: November 11. 2008

CLIPPINGS

Adenotonsillectomy for obstructive sleep apnoea in children

The current treatment of choice for surgical treatment of obstructive sleep apnoea (reductionof airflow at the nose and mouth during sleep) in children is adenotonsillectomy (the removalof the adenoids and tonsils), due to its perceived efficacy, cost effectiveness and the relativesize of adenoid and tonsil tissue in children. There is a lack of strong evidence to support theuse of adenotonsillectomy in children with sleep apnoea, although there are some data to indicatethat a procedure which removes part of the tonsils (temperature controlled radiofrequencytonsillectomy and adenoidectomy) leads to quicker return to normal diet in the post-surgeryphase than complete tonsillectomy and adenoidectomy. There is some debate as to the diagnosisof obstructive sleep apnoea in children and further research would need to undertake extensivediagnostic tests to make an appropriate diagnosis.

Lim J, McKean MC. Adenotonsillectomy for obstructive sleep apnoea in children.Cochrane Database of Systematic Reviews 2001, Issue 3. Art. No.: CD003136. DOI:10.1002/14651858.CD00313. This version first published online: January 20. 2003Lastassessed as up-to-date: November 11. 2008


RADIOLOGIST TALKS TO YOU

* Associate Professor** Asst. Professor

*** ProfessorDepartment of Radiology,Chenglepet Medical College Hospital,Chenglepet, Tamilnadu.

AQUEDUCTAL STENOSIS AND

HYDROCEPHALUS

* Vijayalakshmi G** Elavarasu E

*** Venkatesan MD

We now pass on to the next two stages ofneural development which result in actualformation of neurons. These are the stages ofneuronal proliferation, histogenesis,differentiation and the stage of cellular migration.These two stages run concurrently from thesecond to the fifth month of intrauterine life.They result in the formation and organizationof neurons.

Disorders belonging to the group ofneuronal proliferation, histogenesis anddifferentiation are the commonest. This groupincludes arachnoid cysts, obstructive hydro-cephalus and vein of Galen aneurysm. Of these,the commonest is aqueductal stenosis. Thisresults in dilatation of the lateral and thirdventricles. The fourth ventricle is normal. (Fig.1)Obstructive hydrocephalus due to any cause canalso occur due to obstruction at the fourthventricle outlet foramina. This is termed as non-communicating tetraventricular hydrocephalusand the cisterna magna is not dilated. When theobstruction is distal to the foramina of Lushka

and Magendie, it is called communicatinghydrocephalus as in Fig 2. Here there is dilatationof all four ventricles and the cisterna magna isalso dilated. Compare with the normal midsagitalsection in Fig. 3. where the cisterna magna is athin black line below the cerebellum(C). Themidsagital section in Fig. 2 shows the distendedblack cisterna magna inferior to the cerebellum.So, this is communicating hydrocephalus wherethe level of obstruction is at the arachnoid villiand basal cisterns. The etiology in this type areinfections and hemorrhage. Whatever theetiology or level of obstruction, it is essential tomake a diagnosis of obstructive hydrocephalusbecause of the need to refer the patient for a VPshunt. Ultrasound is the initial modality in theevaluation of macrocrania or suspectedhydrocephalus. A simple and quick scan throughthe open fontanelle will suffice to make adiagnosis. CT is done pre-operatively. Rememberthat cerebral masses can also causehydrocephalus. CT will of course reveal thepresence of any mass. Ultrasound is also usefulfor post-shunt follow-up.

Dilated ventricles do not always signifyhydrocephalus. Fig. 4 shows dilated lateral andthird ventricles. However, look at the largequantity of extra-axial fluid. This is due to lossof cerebral volume because of atrophy. Since thecranium is a closed box there is a passivedilatation of ventricles and secondary dilatationof subarachnoid cisterns to make up for thecerebral parenchymal volume loss. The sulci andsylvian fissure appear wide for the same reason.

Sometimes there is dilatation of the occipitalhorns alone as in Fig. 5. The other horns of the

96

2009; 11 (3) : 301

97

Fig.1 Aqueduct stenosis. Dilatedlateral and third(T) ventricles

Fig.2 Communicating hydrocephalus

Fig.3 Normal sagital section. C isthe cerebellum

Fig.4 CT picture of cerebralatrophy

Fig.5 ColpocephalyFig.6 Benign external hydrocephalus.F is the fluid in the cisterns


lateral ventricles and the other ventricles are notdilated. This is called colpocephaly and isassociated with mental retardation, microcephalyand seizures. It is a passive dilatation due toneuronal loss in the occipital lobes. Colpocephalymay be diagnosed in a late antenatal scan andmaybe misdiagnosed as hydrocephalus. Postnatalscans are more accurate. It may accompany manyother congenital anomalies. In Fig 5 there isagenesis of the corpus callosum also.

One commonly encountered problem is aninfant with macrocephaly. These children mayhave benign external hydrocephalus. Ultrasoundcan be used as a screening investigation to ruleout obstructive hydrocephalus. Externalhydrocephalus is a condition that is characterizedby enlargement of the subarachnoid spaces in aninfant with macrocephaly. The subarachnoidspaces are enlarged and typically seen in thefrontal areas and interhemispheric

fissures.(Fig.6) Doppler will confirm thesubarachnoid location of fluid if veins can be seencoursing through the fluid collection. This is todifferentiate it from subdural effusions which willnot have any veins running through . Incommunicating hydrocephalus also, there areprominent subarachnoid spaces but the ventriclesare much more dilated. Cerebral atrophy is alsoaccompanied by enlarged subarachnoid spacesbut there is no macrocephaly. Ventricles cansometimes be dilated mildly in benign externalhydrocephalus, as in Fig 6. It is usually a self-limited condition that presents between ages of3 and 12 months and resolves spontaneously byabout two years. Psychomotor development mayat times be delayed, though usually normal.Ultrasound is good as a screening procedure.Parents can be reassured., and resolution canalso be followed with serial ultrasound withoutany radiation risk.

98

CLIPPINGS

Hand washing for preventing diarrhoea

Strategies to encourage hand washing can reduce the incidence of diarrhoea by about onethird.

Diarrhoea is a serious global public health problem, particularly in low-income and middle-income countries. The World Health Organization estimates that over three million episodesoccur each year, with many people dying, especially children aged less than five years in low-and middle-income countries. Persistent diarrhoea can also contribute to malnutrition, reducedresistance to infections, and sometimes impaired growth and development. The organismscausing diarrhoea can be transmitted from infected faeces to people through food and water,person-to-person contact, or direct contact. Hand washing after defecation and handling faeces,and before preparing and eating food can reduce the risk of diarrhoea. This review looked attrials of interventions to increase the use of hand washing in institutions in high-income countriesand in communities in low- or middle-income countries, and found many of the interventionslike educational programmes, leaflets, and discussions to be effective.

Ejemot RI, Ehiri JE, Meremikwu MM, Critchley JA. Hand washing for preventingdiarrhoea. Cochrane Database of Systematic Reviews 2008, Issue 1. Art. No.: CD004265.DOI: 10.1002/14651858.CD004265.pub2. This version first published online: January 23.2008.

2009; 11 (3) : 303

ESOPHAGOGASTRIC JUNCTIONAL

ADENOCARCINOMA IN A CHILD

* Radhika** Malathi S*** Janani S

**** Kalaiselvi

Malignancies of the gastrointestinal tract(GI) are rare in children constituting not morethan 5 % of all pediatric malignancies and usuallyhave a poor outcome. Gastric carcinomasrepresent 0.05% of all the GI malignancies1.Adenocarcinoma of the esophageal gastricjunction in children though rare has beendocumented in literature. The reason for theirrarity is probably because epidemiologicalstudies have documentted that oesophagealmalignancies are predominantly environmentrelated requiring long latent period forcarciongenesis. We report a 12-year-old boy withadenocarcinoma of the esophago gastric junction(EGJAC).

Case Report

A 12-year-old overweight boy presentedwith recurrent non-bilious, post-cibal vomitingassociated with vague upper abdominaldiscomfort and sitophobia (aversion to food) of

one and a half months duration. There was nohistory of dysphagia, odynophagia, retrosternalburn, abdominal distension, GI bleed, recurrentcough, wheeze, aspiration, fever, headache,diarrhea or jaundice. He was overweight(56kg, BMI 26.7) prior to the illness and had lost8 Kgs during this period. There was no familyhistory of tuberculosis or GI malignancies.

He was a well nourished young boy(wt 48 kg) with no significant lymphadenopathy.Abdominal examination was normal except forepigastric tenderness. The other systems werenormal. The haematological and biochemicalparameters were normal, but stool for occultblood was positive. The X-ray chest was normal.Esophagogastric duodenoscopy revealed anencircling friable ulcero proliferative lesion atthe lower end of the esophagus narrowing thelumen and involving the esophago gastricjunction. A smooth extraneous impression wasseen in the fundus and the rest of the stomachbeing normal. Biopsy of the lesion revealed amoderate to poorly differentiated grade III to IVjunctional adenocarcinoma. There was noevidence of intestinal metaplasia, Barrett’sepithelium or H. pylori.

Contrast enhancing computerisedtomography (CECT) of the abdomen showed amass lesion involving the esophago gastricjunction extending along the lesser curvature ofthe stomach without any liver metastases. CECTthorax showed a small nodule at the base of theright lung. A diagnosis of EGJAC with extensioninto the lesser curvature with probable metastasesto the right lung was considered. Since the lesion

* Resident in Pediatrics,** Senior Consultant Pediatric

Gastroenterologist*** Senior Consultant Pediatrician

**** Senior Consultant Oncologist

Kanchi Kamakoti Childs Trust Hospital,Chennai - 34.

CASE STUDY

99


was wide spread, it was decided to manage thechild with preoperative chemotherapy followedby surgery if feasible. The child received 8 cyclesof chemotherapy consisting of 5 flurouracil andcisplatin. After the completion of third cycle, hehad an episode of febrile neutropenia, which wastreated with antibiotics and colony stimulatingfactors. He had temporary improvement in hissymptoms but soon developed progressivedysphagia. A repeat upper GI endoscopy showeda stricturous lesion which was confirmative ofadenocarcinoma on biopsy. Parents were notwilling for surgery or stenting. The child wassupported with tube feeding and parenteralnutrition. He succumbed to the illness 9 monthsafter the diagnosis.

Discussion

EGJAC is very rare in pediatrics consideringthe fact that gastric carcinomas constitute 0.05%of all GI cancers which by itself is only 5% ofthe childhood malignancies1. There have beenfew reports of EGJAC in children over the lasttwo decades which has been identified mainly inadolescents. Case reports of esophagealsquamous cell carcinoma and esophagealadenocarcinoma in Indian children have alsobeen documented2. This is probably the youngestchild with EGJAC reported from India.

In adults over the past two decades theepideminology of esophageal adenocarcinoma ischanging with a dramatic rise in the incidence ofadenocarconomas of the lower esophagus,esophagogastric junction and adenocarcinoma ofthe gastric cardia especially in North Americaand Western Europe. This has been linked to themetap las ia -dysp las ia -adenocarc inomaconsequence. Studies have reported an increaseamong white men ranging from 1.5% to 17% peryear. In US the reported annual increase hs beenclose to 10% which exceeds that of any othermalignancy in that population.

The strongest risk for esophagealadenocarcinoma is Barrett’s esophagus;secondary to long-standing gastroesophagealreflux disease which is associated with a 30 – 40fold risk of adenocarcinoma oesophagus.Barrett’s epithelium in childhood usually presentswith symptoms of reflux disease but may be silentand present with adeoncarcinoma in childhoodor early adulthood3. GERD and its associationwith adenocarcinoma is well recognized. Hencechildren with long-standing reflux disease, hiatushernia, post tracheo-esophageal atresia repair andthose with family history of GERD should befollowed up regularly for cancer surveillance.

Obesity (BMI >30) is a true and significantrisk factor for the development of esophagealadenocarcioma independent of reflux3. This childwas overweight and was taunted for the same byhis peers prior to his presentation. The gene forsevere pediatric GERD has also been mapped to13q144. An autosomal dominant pattern with highpenetrance of severe GERD has beendocumented in some families. Several geneticvariants such as mutations of p53 tumoursuppressor and reduced expression of E-cadheringene have been reported in these malignancies.

Dysphagia is the most common presentationfor EGJAC. This child presented with vomitingand significant weight loss whereas dysphagiawas a late manifestation. Vomiting with weightloss overt or occult bleed or the presence of otherred flag signs ae indicators for an earlyendoscopy. Diagnosis is based on a high indexof suspicion in children who present withsymptoms mimickig acid peptic disease5.

Adenocarcinomas of the GE junction areclassified into three well recognized types andform the basis for surgical treatment, predictingprognosis. This child probably had type II lesion(true lesion of the cardia arising at the GEjunction), since there was no evidence of

100

2009; 11 (3) : 305

intestinal metaplasia or Barrett’s oesophagus(BE) on histopathology as seen6. Endoscopicallyit is not easy to differentiate adenocarcinomaassociated with BE and adenocarcinoma of thecardia involving the OG junction except for adetailed histopathology which will help indifferentiating the types of EGJAC. The surgeryof choice is usually oesophagectomy withresection of the proximal stomach or extendedtotal gastrectomy with transhiatal / transthoracicresection of the distal oesophagus dependingupon the grading6. Neoadjuvant chemotherapy isrecommended in advanced cases when surgeryis not feasible. Epirubicin, Cisplatin withcontinuous 5 flurouracil has been reported to havea good clinical outcome and is currently the“Goldstandard chemotherapy” for this disease7.Despite good symptomatic response, this regimengiven preoperatively in unresectable or lymphnode positive gastroesophageal adenocarcinomasdoes not seem to improve the final outcome.Other regimens include Epirubicin, Doxorubicin,Cisplatin (EAP); Cisplatin, Etoposide and 5 – FU(PEF); Irinotecan and cisplatin8,2.

The prognosis of adenocarcinoma involvingOG junction is poor because of the widespreadlymphatic metastases present even at the time ofdiagnosis making curative resection difficult.Even in patients with potentially curable disease,the 5-year survival rate is approximately only20%8. Since the important risk factors are GERD,Barrett’s oesophagus and obesity which arepreventable, this malignant lesion hasbeen labeled as a preventable disease.These malignancies are rare in children butthe risk factors may be present in childhood.Life style modifications such as avoiding fastfood, smoking and alcohol, increased intake ofgreen leafy vegetables and regular exercise, may

help in decreasing the incidence in adults andalso prevent the sporadic occurrences inadolescents.

This case report highlights a very rare andfatal malignancy in a child where an earlyendoscopy as the first line of investigation helpedin diagnosis.

References

1. Goldthorn JE, Canizaro PC. Gastrointestinalmalignancies in infancy, childhood andadolescence. Surg Clin North Am 1986;66:845-861.

2. Gangopadhyay AN, Mohanty PK, Gopal SC,et al. Adenocarcinoma of the esophagus in an8 year old boy. J Pediatr Surg 1997;32:1259-1263.

3. Spechler SJ, Robbins AH, Rubins HB, et al.Adenocarcinoma and Barett’s oesophagus. Anover-rated risk. Gastrenterology. 1984;87:927-933.

4. Hu FZ, Preston RA, Post JC, et al. Mapping ofa gene for severe pediatric gastroesophagealreflux to chr 13q14. JAMA 2000;284:325-334.

5. McGill TW, Downey EC, Westbrook J, et al.Gastric carcinoma in children. J Ped Surg1993;28:1620-1621.

6. Monig SP, Schroder W, Beckurts KT, HolscherAH. Classification, diagnosis and surgicaltreatment of the gastroesophageal junction.Hepatogastroenterology 2001;48:1231-1237.

7. Geh JI, Glynne-Jones R, Kwok QSK, et al.Pre-operative ECF chemotherapy in gastro-esophageal adenocarcinoma. Clin Oncol2000;12:182-187.

8. Ilson DH. Epirubicin, cisplatin and fluorouracilin gastric and esophageal cancer : a step ahead?.J Clin Oncol 2002; 20:1996-2004.

101


DUODENAL ANGIODYSPLASIA –

A RARE CASE OF UPPER

GASTROINTESTINAL BLEED IN A

YOUNG CHILD

* Sumathi B* Nirmala D

** Bhaskar Raju B*** Rajalakshmi V

Gastrointestinal (GI) bleeding can be a lifethreatening problem in children. The mostcommon cause of large recurrent GI bleeds inchildren is variceal bleed secondary to portal veinthrombosis. Vascular lesions are an uncommon,but important cause of obscure upper GI bleedingand the most common vascular lesions arevascular ectasias. Arteriovenous malformation orangiodysplasia are names often usedsynonymously for vascular ectasias. However,arteriovenous malformations are developmentalanomalies in contrast to vascular ectasias, whichare acquired lesions. There has been littleexperience with GI angiodysplasia in pediatricpopulation. Their natural history is unknown anddiagnosis is often missed or delayed.

Angiodysplasia is a distinct clinical entity,characterized by abnormal dilatation ofsubmucosal veins, the clinical presentation ofwhich may vary from iron deficiency anemia dueto chronic GI blood loss to massive GI bleed

which can be a potentially life threateningcondition. These lesions are recognized as asignificant cause of lower gastrointestinal bleedin elderly patients. Vascular degeneration due toageing causes the lesions and hence uncommonin the young. Seventy percent of these lesions inadults are located in caecum and ascending colon,fifteen percent in jejunum and ileum, and theremaining are seen in descending and sigmoidcolon.1

Diagnosis of angiodysplasia can be difficult.Sometimes conventional endoscopy can missthem and Capsule Endoscopy, Intra-OperativeEnteroscopy, selective visceral angiography andother imaging modalities including Isotopestudies may be required to diagnose thecondition. Here we report a rare case of recurrentGI bleed in a very young child due to duodenalangiodysplasia. Endoscopy was the diagnostictool that identified the cause of massive bleedin our patient.

Case Report

A three year old female child born to non-consanguineous parents was admitted forGI bleed the second time, with history of fivediscrete episodes of painless and massivehemetemesis within the previous 48 hours,associated with hemodynamic instability.The child was resuscitated with six units of bloodtransfusion. Endoscopy that was done for a minorGI bleed during the previous admission a weekearlier, with 7mm fibreoptic Olympus GIF N30modified gastroscope fitted with a video adapterwas negative. There was no history of umbilical

CASE STUDY

* Assistant Professor,** Professor & Head

Department of Pediatric Gastroenterology*** Professor & Head

Dept. of Pathology, ICH & HC, Chennai.

102

2009; 11 (3) : 307

sepsis or umbilical vein catheterization in the newborn period, drug intake or bleeding diathesis.Clinical examination was unremarkable exceptfor pallor and shock. No cutaneous or mucosalangiomatous lesions were seen. Bleeding time,clotting time, platelet count, coagulation profile,renal function tests, chest X ray and ultrasoundabdomen were normal. After hemodynamicstabilization, endoscopy was repeated with thesame gastroscope. A flat red spot with smoothsurface in the anterior inferior wall of duodenum,proximal to the ampulla, suggestive of a vascularlesion was seen (Fig.1). A diagnosis ofangiodysplasia of the duodenum was made andthe child referred for surgical management, sinceprovisions for management of such lesionsendoscopically were not available in our institute.Surgical excision of the affected segment wasdone and specimen was sent for histopathology.Macroscopic examination of the excised segmentshowed multiple areas of focal serosalhemorrhages. Luminal surface showed focallyedematous mucosa with small dilated andcongested vessels. Microscopic examination

showed thin walled dilated vascular channelsfilled with blood, extending into mucosa andsubmucosa, suggestive of angiodysplasia ofduodenum (Fig.2). Postoperative period wasuneventful with no further recurrence of bleed.She has been followed up for one year and isasymptomatic.

Discussion

Angiodysplasia is increasingly reported in elderlyindividuals and often due to ageing where as inchildren it is usually congenital. Angiodysplasiais an important vascular lesion of the gut,probably responsible for about 1.2 to 8.0% casesof hemorrhage from the upper GI tract.2 Gastricangiodysplasia is rare in infants. In older children,they may be rarely seen in conditions likeBernard-Soulier syndrome.3 Small bowelangiodysplasia should be considered in all casesof gastrointestinal bleed of obscure origin. Thevascular morphology in angiodysplasia variesfrom small focal lesions to large dilated tortuoussubmucosal veins. Bleeding occurs when theseabnormal vessels rupture.

Lesion inDuodenum

Fig. 1. Endoscopic view of thevascular lesion

Fig. 2. Photo micrograph of excised ofduodenal mucosa with submucosashowing thin walled dilated vascularchannels filled with blood extendinginto mucosa suggestive ofangiodysplasia of duodenum

103


Very few cases of angiodysplasia in childrenare reported in literature.4 The age ranged fromnewborn to 15 years, males being morecommonly affected. A variety of conditions likepseudoxanthoma elasticum, von Willebrand’ssyndrome, Ehlers-Danlos syndrome,Osler-Weber-Rendu Disease, Klippel-Trenaunay-Weber syndrome, progressive systemic sclerosiscan be associated with vascular ectasias. As perclassification described by Moore and co-workers, Type 1 represents colonic lesions in theelderly, predominantly on right side, oftensolitary and acquired. Type 2 is extracoloniclesions occurring in the age group less than50 years, commonly in small intestine.The lesions are frequently multiple and largerthan Type1. They are congenital as different fromtype 1 lesions which are caused by senescence.Type 3 is punctate angiomas seen in HereditaryHemorrhagic Telangiectasia. Our patientprobably had type 2 lesion. Relative deficiencyof collagen type IV in mucosal vessels maybe responsible for development of ectasia.5

Various diagnostic modalities have been used todiagnose cases of angiodysplasia includingendoscopy, selective visceral angiography andoperative angiography, with variable successrates. The most important investigation fordiagnosis of angiodysplasia is angiography orarteriography, which locates and delineates thelesion.6 Surgical resection is advocated as thefirst-line therapeutic measure in bleedingectasias. However, other measures like bipolarelectrocoagulation, vasopressin infusion and gelfoam embolization can been tried to controlbleeding, wherever available.7 Use of metalhemoclips for control of non variceal bleedingin children has also been reported.8

Conclusion

Angiodysplasia of gastrointestinal tractthough rare is an important cause of recurrentobscure GI bleeding in children. Early diagnosisand prompt treatment is essential to avoid apossible fatal outcome.

References

1. Richardson JD. Vascular lesion of intestine. AmJ Surg 1991;161:284.

2. Foutch PG. Angiodysplasia of thegastrointestinal tract. Am J Gastroenterol1993;88: 807-818.

3. Kaya Z, Gursel T, Dalgic B, Aslan D. Gastricangiodysplasia in a child with Bernard – Souliersyndrome: Efficacy of Octreotide in Long-TermManagement: Pediatr Hematol Oncol 2005;22:223-227.

4. Torre Mondragon DL, Vargas Gomez MA,Mora Tiscarreno MA, Mayans RJ.Angiodysplasia of the colon in children.J Pediatr Surg 1995;30:72-75.

5. Roskell DE, Biddolph SC, Warren BF.Apparent deficiency of mucosal vascularcollegen type IV associated with angiodysplasiaof the colon. J Clin Pathol 1998;5:18-20.

6. Boley SJ, DiBiase A, Brandt LJ, SammartanoRJ. Lower intestinal bleeding in the elderly.Am J Surg 1979;137:57-64.

7. DeDiego JA, Molina LM, Diez M, Delgado I,Moreno A, Solana M, et al. Intestinalangiodysplasia: Retrospective study of 18 cases.Hepatogastroenterology 1988;35:255-259.

8. Lee YJ, Oh JM, Park SE, Park JK. Successfultreatment of a gastric Dieulafoy’s lesion with ahemoclip in a newborn infant. GastrointestEndosc 2003;57:435-436.

104

INDIAN JOURNAL OF PRACTICAL PEDIATRICSijpp.in/admin/uploadimage/jul-sep-09.pdf2009; 11 (3) : 205...

Documents

Transcript of INDIAN JOURNAL OF PRACTICAL PEDIATRICSijpp.in/admin/uploadimage/jul-sep-09.pdf2009; 11 (3) : 205...