LABORATORY DIAGNOSIS OF TUBERCULOSIS

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LABORATORY DIAGNOSISOF TUBERCULOSIS
Present and Future

• Angkana Chaiprasert, Dr.rer.nat.
• Division of Mycology and Mycobacteriology
• Department of Microbiology
• Faculty of Medicine Siriraj Hospital, Mahidol
  University

16 July 2002
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MYCOBACTERIA

• Actinomycetales
• Mycobacteriaceae
• Acid-fast bacilli (AFB)
• Long generation time

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• Long generation time

• 2-4 hrs Rapid grower
• M. chelonae
• M. fortuitum

• 1-7 day Slow grower
• M. avium complex (MAC)
• M. leprae
• M. tuberculosis complex
• M. ulcerans

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Diseases Etiologic agents

• Tuberculosis M. tuberculosis cpx.
• Leprosy M. leprae
• Avian tuberculosis MAC
• Swimming pool M. marinum
• granuloma
• Buruli ulcer M. ulcerans
• Multiple subcu- M. fortuitum
• taneous granuloma M. chelonae cpx.

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lab. ????????????
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Pulmonary
Sputum 5 - 10 ml ????? ????? 4C
(3x) ???????? ??????? Aerosol-induce BAL 10-50
ml sterile PF 50 ml Gastric lavage 10-50
ml ???? 100 mg Na-carbonate
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?????????????????
????????
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Extrapulmonary
Urine 50 ml (3x) sterile ????? 4C CSF 2
ml Tissue biopsies 0.5 - 1 cm3 Joint fluids
2-10 ml Pus 2-10 ml Blood 5-10 ml EDTA, 4C
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LABORATORY METHODS

• 1. Direct detection
• Mycobacterial cells Acid fast staining

• Smear diameter 1.5 cm
• Safety cabinet
• Expose UV 2 hr
• Kinyoun stain

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LABORATORY METHODS

• Acid fast staining Report

Negative No AFB in ? 100 fields Actual count 1
- 9 AFB/100 fields 1 10 - 99 AFB/100
fields 2 1 - 10 AFB/field in ? 50
fields 3 gt10 AFB/field in gt20 fields
WHO / IUATLD version (from NIH)
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LABORATORY METHODS

• 1. Direct detection
• Mycobacterial cells
• Acid fast staining

Sensitivity 104 cells/ml Specificity AFB
Mycobacteria Turnaround Time within 24 hr
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• Nucleic acid detection by PCR, SDA

• 16S rRNA gene target
• PCR product 306 bp (one-tube nested)
• M. tuberculosis complex
• (Q.C. for each run with negative
• and positive control)
• Report Negative or Positive for
• M. tuberculosis complex DNA
• Turnaround time 7 days
• Cost 650 Baht

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In House PCR for Rapid Detectionof Mycobacterium
tuberculosis complex

• PRINCIPLE

1. dNTP 2. PCR buffer 3. Taq Pol. 4. Primers
Bacterial cell
Denaturation step Annealing step Extension step
1 cycle
DNA Thermal-cycler
2n
(n number of cycle)
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DETECTION LIMIT

• Molecular detection test

In House PCR 16S rRNA 1 wk

• SAMPLE SENSITIVITY SPECIFICITY
• Sputum 91.9 95.0
• Bronchoalveolar lavage 70.0 84.0
• Pleural fluid lt10.0 gt95.0
• Cerebrospinal fluid 20.0 gt85.0

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M. tuberculosis
M. bovis
5 M. bovis and 11 M. tuberculosis isolates
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• Fast/Plaque

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ICT Tuberculosis
BIOLINE TB
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LiPA
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Microarray
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• 2. Isolation of mycobacteria by culture

• Conventional culture system

• Löwenstein-Jensen Medium

• Report AFB positive or negative
• Turnaround time 6 weeks
• Cost 150 Baht

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Dark
Light
Dark
Light
PHOTOCHROMOGENS
SCOTOCHROMOGENS
Dark
Light
NONCHROMOGENS
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• Automated culture system

• Hemoculture BACTEC 9240
• 5 ml blood Myco /F Lytic
• Other specimen MB/BacT Med, MGIT 960
• Report AFB positive or negative
• Turnaround time 4 weeks
• Cost 300 Baht

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MB/BacT Med
BACTEC 9240
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BACTEC MGIT 960
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• 3. Identification to species from culture

• Conventional biochemical test

• 10 tests
• Turnaround time 4 weeks
• Cost 200 Baht

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IN HOUSE PCR, PCR/REA, or SEQUENCING

• Molecular identification test

• 16S rRNA gene
• KS4
• mtp40
• 16S-23S spacer region
• hsp65
• rpoB

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Using In-house PCR with specific primer

• Specific primers from
• 16S rRNA gene, KS4, and mtp40
• Able to identify
• M. tuberculosis complex
• M. avium-intracellulare complex
• M. fortuitum complex
• M. marinum
• Detect DNA band by electrophoresis
• Q.C.with known species (/- control) Time 1-2
  d
• Cost 200 Baht

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M. nonchromogenicum
M. nonchromogenicum
PCR-REA
M. tuberculosis
M. tuberculosis
M. africanum
M. bovis BCG
M. avium
M. terrae
M. africanum
M. bovis BCG
M. avium
M. terrae
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Using nucleotide sequencing from 16S rRNA gene

• Unclassify and important isolates VIP case
• Able to identify
• all mycobacterium species
• Turnaround time 7 days
• Cost 2,000 Baht

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Fluorescent DNA sequencing
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• 4. Susceptibility test for mycobacteria

• Conventional Test

• Direct Susceptibility Test

- Directly from clinical sample Smear
positive - M7H10 agar - Isoniazid and
Rifampicin - Turnaround Time 2-4 wks - Cost 120
Baht per test

• Indirect Susceptibility Test

- From pure culture - Turnaround Time 4 wks -
M7H10 agar or LJ - Cost 250 Baht per test
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MIC determination

• E-Test

• M7H11 media
• MIC for each drug
• Turnaround Time 1-2 wks
• Cost
• 500 Baht/drug

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• Automated AST with MGIT 960

• Liquid media drug
• - Isoniazid
• - Rifampicin
• - Streptomycin
• - Ethambutol
• Turnaround Time 2-3 wks
• after culture positive
• Cost 250 Baht/drug

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THANK YOU
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THANK YOU
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THANK YOU
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• 5. M. tuberculosis strain typing (research
  purpose)

• To prove
• Institutional Outbreak of TB
• Community Transmission of TB
• Cross-Contamination in Laboratory

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• Standard strain typing technique for M.
  tuberculosis

• Isolation of DNA using lysozyme
• Digest genomic DNA with PvuII
• DNA probe IS6110
• Reference strain
• M. tuberculosis Mt14323

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Completely digested DNA
Genomic DNA of M. tuberculosis
Separated restriction DNA fragment
Immobilized-DNA on membrane
Hybridized DNA
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BJ
NB
H
FB
SB
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• Other typing technique

• Spoligotyping
• PCR (VNTR)