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

http://images.google.com.tr/imgres?imgurl=http://birool.googlepages.com/kedisyan.jpg&imgrefurl=http://pardus007.blogcu.com/hayata-bazen-farkli-acilarla-bakmak-lazim/227722&usg=__HR2jj3xdbJuQtrJuq5so-t7Madg=&h=400&w=400&sz=62&hl=tr&start=4&sig2=yBeWoqYADlj7wb3LNlstWg&itbs=1&tbnid=djXLfr3Hz6zOIM:&tbnh=124&tbnw=124&prev=/images?q=bakmak&hl=tr&sa=G&gbv=2&tbs=isch:1&ei=B0TZS6vTK471_AaLur3GDw