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Immunocompromised Host:LABORATORY APPROACH
Immunocompromised Host:LABORATORY APPROACH
Prof. Dr.Özay Arıkan Akan
Ankara University Medical School Ibni Sina Hospital Central Microbiology Laboratories
Lower respiratory tract microbiology
Diagnosis (mimicking clinical presentations)
Establishing the agent (Clinical/radiological
findings are nonspecific) Success of the therapy (atypical microorganisms,
antimicrobial resistance) Collecting the epidemiological data
Laboratory diagnosis
Microbiological approach Microscopy Culture (blood, sputum
and respiratory tract secretions)
Serologic tests Nucleic asit amplification
tests (eg: PCR)
Microbiology Laboratory Rapid Reliable Reproducible results Continuous care
QUALIFIED SERVICE
Right specimen
Right area
Right technique
Right time
Right amount
Right transportation
Limits of diagnosis
There is lung involvement in at least 75% of febrile neutropenic patients, but etiological agents can be microbiologically documented in only 50% of them.
Diagnostic problems in immunosuppressed patients
Specimen collection is difficult PMNLs are absent in neutropenic patients Normal flora Bacteria can cause infection Fastidious and resistant bacteria can be observed Serological antibody response is not sufficient
Etiological agents Bacteria I.group S.pneumoniaeH.influenzaeM.catharralis Gram negative bacilli EnterobactericeaeAcinetobacterPseudomonas aeruginosaStenotrophomonas maltophiliaS.aureusNonfermenter bacteria
Bacteria II group. Nocardia,Legionella, M.pneumoniae Chlamydophila pneumoniae, Mycobacterium tuberculosisMycobacteria other than M.tbc
Fungi: Candida, Aspergillus, Cryptococcus, Zygomycetes grubu ,Pneumocystis jiroveciiDematiaceous molds Fusarium- Scedosporium-Geothricum candidum.-Trichosporon spp.
Viruses: RSV- influenza-parainfluenzaAdenovirusCoronavirus, Metapneumovirus, Herpes viruses
Parasites: Strongloides stercoralis Toxoplasma gondiiEnterocytozoon bieneusi
Causes of pneumonia
Agents of infectious pneumoniae
Conventional bacteria 37 %
Fungi 14 %
Viruses 15 %
Pneumocystis jirovecii 8 %
Nocardia asteroides 7 %
Mycobacterium tuberculosis 1 %
Mixed infections 20 %
ONE WHO DOES NOT KNOW
WHAT HE IS LOOKING FOR,
CANNOT UNDERSTAND
WHAT HE HAS FOUND
Blood cultures 15-30 % positive in pneumonia of immunosupressed
patients
Especially conditions in which bacteria predominates; Febrile neutropenia, Early phases of Heart and Lung transplants.
CollinBA, Clin Infect Dis N Am 1998
Saleh, A.F. et al. 8th ECCMID 1997
Comparison of Manuel and Automated blood culture results
Clinical study, 1442 blood culture sets. 16,14 % specimen positivity
Time (hours)
14Cockerill FR et al. CID 38: 1724-30, 2004
Urinary antigen L.pneumophila serogroup 1 Sensitivity 70-90 %, Specificity 99-100 %
S.pneumoniae Sensitivity 72%, Specificity %90
Histoplasmosis and blastomycosis by ELISA
Respiratory tract specimens Nasopharyngeal swab, aspirats
Sputum
In the diagnosis of viral infections, M.pneumoniae and Chlamydophilia
Standard sputum evaluationGram stain an Cultura
Advantages Easy Rapid Cheap
Disadvantages Nonproduction of sputum Upper Respiatory tract
contamination (20-25%) Insufficient with some
organisms Experts for evaluation
Appropriate specimen and transport Rejection criteria Induced sputum for P. jirovecii , M. tbc and various
bacteria Endotracheal aspirates can replace sputum in
immunsupressed patients.
Baughman RP et al In:Pulmonary infections in immunocompromised patients, 2009
Invazive techniques in the diagnosis of pneumonia
Bronchoscopic Standard bronchial lavage BAL PSB (protected specimen brush)
Nonbronchoscopic Tracheal /transtranscheal aspiration Blind protected brush Blind protected bronchial lavage lavaji Distal lung aspiration with teleschopic catheter Biopsi (plevral, transthoracic, toracoschopic ve torachotomic)
Advantages Reaches distal airways High sensitivity and specificity? Diagnostic standardization with quantitative cultures
Disadvantages Hard to perform High cost Antibacterial effect of local anestetics Indications Reproducibility ? (Point to borderline results) complications
Diagnosis of pneumonia in immunocompromised patient
Baughman RP et al In:Pulmonary infections in immunocompromised patients, 2009
Specimen cost Bacteria P.jirovecii M.tbc CMV Aspergillus
Blood Low +1 0 0 +1 0
Sputum Low +3 +2 +3 0 +1
Tracheal aspirat
Low +2 +2 +3 0 1
PSB High +4 0 0 0 0
Non Bronchoscopic BAL
Medium +4 +2 +3 0 +2
BAL Medium +4 +4 +4 +3 +2
TBB High 0 + +3 +3 +3
SLB Very High
0 +4 +4 +4 +4
Specimen Bronchitis CAP- outpatient
HAP VAP Immunsuppression
URT (+) (+) _ _ (+)
Sputum + + + _ +
TA (+) (+) _
Blood culture
_ _ + + +
Urine _ (+) (+) _ (+)
Br wash/ Br brush
(+) _ _ _ _ / (+)
PSB _ _ + + (+)
BAL _ _ + + +
TBB/TBNA _ _ _ _ (+)
TTA _ _ (+) _ _
TTNA _ _ _ _ (+)
OLB _ _ _ _ (+)
Value of bronchoscopic specimens for various diseases
Specimen type Disease Laboratory approach
Bronchial wash Specific pathogens ; M.Tbc , Legionella and endemic fungi
Special media and stains, medium,stain boya DFA for legionella and Pneumocystis
PSB Bacterial pnömonia Gram stain and quantitative culture
BAL For opportunistic pathogens
TBB Malignancy,sarcoidosis,Limited use in pneumonia
Histopathologic evaluation, Mycobacterium and Pneumocystis aranabilir.
procedure Agent technique property
Stain Bacteria, Nocardia sppMycobacteria, Fungi,Pneumocystis jirovecii
Gram ARB Calcoflor white
Giemsa Toluidine blue
TA: epithelial cell > 10 (10X) No mo on Gram stai
BAL: Epithelial cell>1% .contaminationPNL containing bacteria >5% pathognomonic for pneumoniae
Culture Bacteria , Nocardia, Mycobacteria, viruses
M.pneumoniae. Legionella pneumophila, fungi
Quantitative cultures,
special media
Tracheal aspirate >10 5
BAL >10 4
Cytology Hücre differansiasyonu Intrasellüler patojen
Sitopatojenik etki
Antigen tests (latex or IF)
Bacteria legionellaPneumocystis jirovecii
Fungi , Chlamydia,Virüses
GalactomannanBeta glucan
Nükleic acit Amplification
tests
C. pneumoniaeM.tbc, P.jirovecii,
Legionella, M.pneumoniae,viruses
High cost hard to standardize Colonization-infection
Mycobacterium tuberculosis Direct smear (AFB) Culture: classical (4-8 weeks), BACTEC Serology ? PCR
With 2 consecutive sputumspecimens
Diagnosis rate: 95%
Value of examining three acid-fast bacillus sputum smears for diagnosis
of pulmonary tuberculosis. J Clin Microbiol 2000; 38:4285
365 specimens124 mycobacterial isolates 10 nontuberculous mycobacteria 114 M.tbc
Conventional culture (LJ agar)
BACTEC MGIT 960
Recovery rate 94% 75.8%
Contamination rate
4.1% 5.5%
TTD Mycobacteria 30.6 days 10.7 days
Smear +Smear -
10.1 days12.6 daysp:0.06
Chien HP et al. INt J Tuberc Lung Dis 200
Classical method TAT New method TAT
EZN 15 min Fluorescent stain 3 min sens 10%
Solid medium 1-8 wks Liquid medium 1-3 wks (7-14 days less)
Phenotypical identification
3-8 wks DNA probes HPLC 1-3 days
- Nucleic asit amplification for
M.tbc
2-6 hours
Drug susceptibilitiy with solid media
3 wks Drug susceptibilitiy with liquid media
5-7 days
Diagnosis in invazive aspergillozis
Wheat LJ, Walsh T. Eur J Clin Microbiol Inf Dis 2008Klont R CID 2004
Galactomannan antigen (serum and BAL) (1-3)-B-D-glucan PCR : sensitivity for aspergillozis 45-93 % specificity 72-100 %Galactomannan GMI >0.5 sensitivity 100 % specificity 97.5% meta analysis: sensitivity % 79 specificity % 86 frequency: 2 times /week Patient population :neutropenia in hematological
malignancy and allogeneic SCT Sensitivity is higher in BAL compared to serum (85 -
100% vs %73-83)
Diagnosis of invasive pulmonary aspergillosis using Galactomannan antigenemia EIA on BAL specimens
Population Cut off 0.5 Cut off 1.0 reference
Sensitivity- % Specificity- % Sensitivity- % Specificity- %
Hematology Not stated Not stated 98.1 100(CO 1.1) 27
Hematology Not stated Not stated 100 100 28
Bone marrow transplant
76 94 61 98 29
Solid organ transplant
100 84 100 91 30
Solid organ transplant
67 95 67 98 31
Intensive care unit
88 87 Not stated Not stated 33
nonimmunocompromised
100 78 100 88 32
27: Li-Yang Hsu et al. BM C Infect Dİs 2010 28:Becker MJ et al.Br J haematol 2003, 29:Musher B et al. J Clin Microbiol 2004, 30:Clancy CJ et al. J Clin Microbiol 2007, 31:Hussain S et al. Transplantatiaon 2007 32:Nguyen MH et al. J Clin Microbiol 2007 33: Meersseman W et al. Am J Respir Crit Care 2008
Beta glucan
Panfungal? -Zygomycosis and Cryptococcosis-
Cannot differentiate between Aspergillus -Candida
ECIL 2010 High cost False positive reactions
Diagnosis and management is a teamwork