LABORATORY DIAGNOSIS OF TUBERCULOSIS Dr. D.P. Rajani Medical MicrobiologistMicrocare Laboratory & TRC Surat

CLASSIFICATION OF MYCOBACTERIA Mycobacterium tuberculosis complex refers to a genetically related groups of Mycobacterium species that can cause TUBERCULOSIS [TB] in humans. It includes; Mycobacterium tuberculosis,Mycobacterium bovis [M.bovis, subsp bovis, M.bovis subsp.caprae and M.bovis BCG]Mycobacterium africanum,Mycobacterium microti,Mycobacterium canetti,Mycobacterium mungi,Mycobacterium orygis and Mycobacterium pinnipedii

SPECIMEN COLLECTION In every clinical Microbiology sample collection “Results are as good as Specimen” Good quality sample is very important. Sputum samples of good quality collected in wide mouth sterile containers. Quantity sufficientExtra pulmonary samples collected in sterile containers /syringes.

Mucoid Sample Purulent Sample Bloody Sample

Salivary Sample

DIAGNOSIS OF TUBERCULOSIS Smear MicroscopyAFB CultureManual & Liquid SensitivityMolecular Diagnostic

In low income and high tuberculosis prevalence countries, sputum smear microscopy is the only cost-effective tool for diagnosing patients with infectious tuberculosis and to monitor their progress in treatment. Sputum smear microscopy is a simple, inexpensive, appropriate technology method which is relatively easy to perform and to read. Classical Ziehl-Neelsen stain used-AFB

SMEAR MICROSCOPY

Smear prepared from thick purulent parts of samples. Size of smear should be 3cmx2cm At least 300 fields examinedSmear is positive in samples which contain 5000- 10000 bacteria /mlSensitivity ranges from 25%-65% Sensitivity increases by examination of morethan one smear

SMEAR MICROSCOPY

SIZE OF SMEAR 2 x 31 x 2 Uniform Smear

Good Evenness Smear Uneven SmearGood Thickness Smear Too Thick Smear

Too Thin Smear

Smears reported as Positive or NegativeQuantity of AFB observed should be notedFactors influencing smear sensitivity are type of specimen, staining technique, experience of readerLaboratory Quality Control important

ZN STAINING

FLUORESCENCE MICROSCOPYFluorochrome stain usedCan be examined at lower magnification(40 X)Rapid but more false positiveLED fluorescence microscopy has been evaluated- rapid and good results, lower costLED attachment to microscope Primo Star iLED from Carl Zeiss

FLUORESCENCE MICROSCOPY

DISADVANTAGES OF SMEAR MICROSCOPYNeeds a large no of bacilli per ml of specimen to be detected positiveCannot differentiate between dead and live bacilliCannot differentiate between Mtb and NTMNo idea of drug resistance

AFB CULTUREGOLD STANDARDProvides definitive diagnosis of TBPure growth of mycobacteria to do speciation and drug sensitivity.Technically demanding and complexHigh level of Biosafety needed

AFB CULTURE BY L.J. MEDIA [SOLID] Detection of 10-100 viable bacilli/ml of specimenSpecimens have to be decontaminated before inoculation to remove the normal bacterial flora.Solid culture – Conventional LJ method.Mycobacteria slow growing and hence take 2-8 weeks to grow

LOWENSTEIN JENSEN [L.J. ] MEDIA

LIQUID CULTUREMany Commercial systems available- BACTEC systems MGIT960, BacT/ALERT 3D systemLiquid culture yield significantly rapid results than solid media and isolation rates for mycobacteria are higherLiquid media- Middle brook 7H9 media usedMGIT system( Mycobacterial growth indicator tubes) contains a modified Middle brook 7H9 broth with a fluorescence quenching based oxygen sensor. Growth of mycobacteria leads to oxygen depletion and indicator fluoresces brightlyCultures positive in 10-14 days

LIQUID CULTURE BY BACTEC

DRUG SENSITIVITY FOR AFBSensitivity to first and second line drugsavailableExpensiveHigh degree of technical expertise and lab infrastructure requiredRigorous quality control needed

NON COMMERCIAL METHODMODS [Microscopic observed drug susceptibility] A micro colony method in liquid culture , based on inoculation of specimens into drug free and drug containing media, followed by microscopic examination of early growth .Recommended as direct or indirect tests for rapid screening of patients suspected of having MDR TB

  CRI ( Colorimetric redox indicator)Indirect testing methods based on the reduction of a coloured indicator added to liquid culture medium on a microtitre plate after exposure of M. tb strains to anti TB drugs in vitro

 NRA(Nitrate reductase assay) A direct or indirect method on solid culture based on the ability of M. tuberculosis to reduce nitrate, which is detected by a colour reaction

SEROLOGICAL TESTSNEGATIVE RECOMMENDATION from WHO in 2011   SHOULD NOT BE ORDERED

MOLECULAR TESTGenotypic methods have considerable advantage of speed, standardization of testing and reduced requirement for Biosafety

1. LINE PROBE ASSAY ( HAINS TEST)2. GENEXPERT

1. LINE PROBE ASSAY ( HAINS TEST)Simultaneous identification for M.tuberculosis complexMolecular assay for the detection of resistance to INH & RIF of M.tuberculosis complex By detection of most significant mutations to – inhA, RpoB and the katG genes Based on DNA strip technologyCan be done from positive cultures (from MGIT, BacT/ALERT bottles or LJ)Pulmonary samples which are smear +ve can be done directly

Detection of multiple genes responsible for the antibiotic resistance &Simultaneous recognition of missing wild type geneAlso Available for Second secondline and identification of some strain of NTM Limitations of Genotype MTBDRplusNeeds preprocessing of samples.Needs a PCR set upTechnically demandingPanic of contaminationSpecial infrastructure requiredNeeds dedicated staff and space.

2. GENEXPERT [CBNAAT] The Xpert MTB/RIF is a cartridge based nucleic acid amplification test , automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) DNA and resistance to Rifampicin (RIF) by Nucleic Acid Amplification Test(NAAT). SAMPLES;Pulmonary samples( Sputum, BAL )Extra pulmonary samples [Lymph node tissue and aspirates, CSF, Pus , Gastric lavage and aspirates ( in children) & Other Tissues]

Pulmonary samples - Xpert MTB/ Rif SensitivityStatus Sensitivity %Smear +ve culture +ve 98Smear –ve culture +ve 68People with HIV 79People without HIV 86Extra pulmonary samples Xpert MTB/Rif - sensitivity and specificity Samples Sensitivity % Specificity %Lymphnode tissue and aspirate 84.9 92.5

CSF 79.5 98.6Pleural fluid 43.7 98.1Gastric lavage and aspirations 83.8 98.1Other tissue 81.2 98.1

ABOUT MICROCARE LABAORATORY & TRCLABORATORY ACHIEVEMENTMicrocare Laboratory was Certified by ISO 9001:2000 in the year of 2007, First in south Gujarat. Microcare Laboratory was accredited by NABL in the year of March 2011, first laboratory in south Gujarat to get NABL Accreditation in the field of Microbiology.With the fully & favorable support of STO, STATE TB CELL and whole team of RNTCP Gujarat government, Microcare laboratory accredited by National Mycobacteriology Accreditation system of Central TB Division, Govt. of India. 1st and only one lab In Gujarat in private sector for solid C&DST.

MICROCARE LABORATORY FACILITIES C&DST BY L.J. C&DST BY

Bactec MGITMDR [TB] Detection by Genotype [Line Probe Assay].

Detection of MTB & Rifampicin By GeneXpert.& Routine Culture and sensitivity testing of all clinical samples.

FLUORESCENCE MICROSCOPY

TB NOTIFICATIONFrom May 2013 to Dec. 2015 More than 200

patients were Notified by Microcare laboratory through Nikshay

AWARDS

MICROCARE LABORATORY AWARDED BY SMC ON WORLD TB DAY 2015 FOR BEST PERFORMANCE & SUPPORT IN TB DIAGNOSIS

PROFICIENCY TESTING RESULTS 2015 EVALUATE BY NIRT [National Institute for

Research In Tuberculosis] CHENNAI

YEAR 2015 DRUG SENSITIVITY [%]

Streptomycin 91

Isoniazid 100

Rifampicin 100

Ethambutol 92

Thank youDr.D.P.Rajani & Microcare laboratory Team