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Role of Laboratory Services in TB Control Part - II Role of Culture, PCR & Serology C.N. PARAMASIVAN Tuberculosis Research Centre Indian Council of Medical Research Chennai Indications for Culture in DOTS Failures of re-treatment cases Seriously ill cases; extra-pulmonary cases

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role of laboratory services in tb control part ii role of culture pcr serology
Role of Laboratory Servicesin TB ControlPart - IIRole of Culture, PCR & Serology

C.N. PARAMASIVAN

Tuberculosis Research Centre

Indian Council of Medical Research

Chennai

indications for culture in dots
Indications for Culture in DOTS
  • Failures of re-treatment cases
  • Seriously ill cases;
    • extra-pulmonary cases
    • smear negative cases
    • childhood TB & HIV-TB
  • For DRS
  • Not for New Smear Positive Cases
mycobacterial culture
MYCOBACTERIAL CULTURE

Advantages:

  • Increases number of cases found
  • Detects cases among smear negative patients
  • Establishes viability of organisms
  • Distinguishing between Mycobacterial species
  • Helps in performing DST
  • Helps in diagnosing cases of failure

Limitations:

  • Expensive
  • Require enriched media
  • Require considerable expertise
  • Time consuming
decontamination procedures
Decontamination Procedures
  • 1915 – Petroff’s NaOH
  • 1946 – Trisodium Phosphate
  • 1955 – Pancreatin Desogen
  • 1958 – Pancreatin + 1% cetrimide
  • 1962 – Zephiran Trisodium PO4
  • 1963 – N-acetyl L-cysteine + 2%NaOH
  • 1969 – Swab culture technique + 1% cetrimide
  • 1975 – CPC + NaCl2
culture media solid
Culture Media : Solid
  • LJ
  • LJ with Na pyruvate
  • LJ with out asparagine
  • Middlebrook’s 7H10 & 7H11
  • Selective 7H10 & 11
  • Ogawa
  • Tarshi’s Blood Agar
petroff s method
PETROFF’S METHOD

Advantages:

  • Simple, inexpensive & control the growth of contaminants
  • Twenty samples can be processed in 2 Hrs, with centrifuge capacity being the limiting factor
  • Sterilized NaOH can be kept for several weeks

Limitations:

  • The specimen exposure times must be strictly followed to prevent over kill of tubercle bacilli. The initial kill is independent of additional contributory factors such as heat build-up in the centrifuge and centrifugal efficiency
processing of sputum with cpc method
Processing of sputum with CPC Method
  • If delay of more than 48 hours between collection and processing is anticipated, the sputum should be collected with 1%CPC and 2%NaCl2
  • CPC acts as homogenizing and decontaminating agent
  • It helps in retaining viability of Tubercle bacilli up to 7 days
  • These specimens should not be treated with NaOH ( Petroff’s)
colony morphology of m tuberculosis
Colony Morphology of M.tuberculosis
  • Dry wrinkled warty growth.
  • Eugonic
reading and reporting
Reading and Reporting

Characteristics of Tubercle bacilli

  • Growth of Primary culture takes 2 – 4 weeks to obtain visible colonies
  • Colonies are buff colored and rough, having the appearance of bread crumbs or cauliflower
  • Not easily emulsified but give a granular suspension
  • Microscopically frequently arranged in serpentine cords of varying length or show linear clumping
other culture methods
Other Culture Methods
  • Septi-check AFB
  • MGIT 960
  • Backtec/MB/Bact
  • ESP Culture ii
  • Microscopic Observation of Broth Culture
  • MODS: Micro Colony Detection System
nucleic acid amplification for mycobact diagnosis
Nucleic acid amplification for mycobact. diagnosis

Genus specific protocols

Targeting genes code for 16S rRNA

65KDa hsp

M.TB Complex specific is 6110

Other targets:

Genes encoding 38 KDa

MPB 64

mtp 40

PMT 64

Methods:

Target amplification - PCR

(TMA, LCR, SDA or signal amplification EG: QB amplification)

Current status: adjunct to standard procedure

What is new in the diagnosis of TB

PFYFFER G.E. J.INF. 1999, 39, 21-26.

TRC/ICMR

30

diagnostic performance of naa for direct detection of mtb complex
Diagnostic performance of NAA for Direct detection of MTB complex

What is new in the diagnosis of TB

31

Pfyffer . G.E. J.Inf 1999 39 21-26

TRC/ICMR

evaluation of in house pcr for the detection of m tb
Evaluation of in-house PCR for the detection of M.TB.
  • PCR Results from 6 labs
  • Samples reconstituted with defined amount of

M.TB cells

  • Each lab used specific conditions of
      • Sample processing
      • NA Amplification
      • Amplicon detection
  • Large differences observed in sensitivity & specificity

Conclusion: in house PCR can not be used as a single diagnostic tool

What is new in the diagnosis of TB

TRC/ICMR

32

Suffs. P. et al Int. J. Tuberc. Lung Dis 2000, 4(2) 179-183.

serological diagnosis of tb
Serological diagnosis of TB
  • Advantages
      • Low turn around time
      • High NPV
      • Useful as a screening test
  • Limitation
      • Low sensitivity in Smear Negative
      • In HIV positive

- Low NPV

- Low sensitivity

      • Disease Endemic Countries

 Latency - Low PPV

      • High Cost
      • Extensive Personnel Training
      • Difficulty in distinguishing MTB / NTM

What is new in the diagnosis of TB

Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000,89,131-140

TRC/ICMR

34

antigens used in serological diagnosis of tb
Antigens used in serological diagnosis of TB
  • Mycobacterial sonicates
  • Extracted glycolipids
  • PPD
  • Ag5 (38KDa Ag)
  • A60
  • 45 / 47 – KDa Ag
  • Ag Kp 90
  • 30 KDa Ag
  • P32 Ag
  • Cord Factor (trehalase dimycolate)
  • LAM

TRC/ICMR

What is new in the diagnosis of TB

33

Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000, 89, 131-140

antigens used in serological diagnosis of tb16
Antigens used in serological diagnosis of TB
  • Mycobacterial sonicates
  • Extracted glycolipids
  • PPD
  • Ag5 (38KDa Ag)
  • A60
  • 45 / 47 – KDa Ag
  • Ag Kp 90
  • 30 KDa Ag
  • P32 Ag
  • Cord Factor (trehalase dimycolate)
  • LAM

TRC/ICMR

What is new in the diagnosis of TB

33

Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000, 89, 131-140

sensitivity of smear negative vs smear positive tb
Sensitivity(%) of smear-negative vs smear-positive TB

What is new in the diagnosis of TB

Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000,89,131-140

TRC/ICMR

35

summary role of culture in dots
SummaryRole of culture in DOTS
  • Culture has no role in the diagnosis of TB in DEDC.
  • Indicated in;
    • Failures of re-treatment cases
    • Seriously ill cases;
      • extra-pulmonary cases
      • smear negative cases
      • childhood TB & HIV-TB
    • For DRS