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
– smear negative cases
– childhood TB & HIV-TB
• For DRS

• Not for New Smear Positive Cases

 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
 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
 LJ
 LJ with Na pyruvate
 LJ with out asparagine
 Middlebrook’s 7H10 & 7H11
 Selective 7H10 & 11
 Ogawa
 Tarshi’s Blood Agar
 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

 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

 Dry wrinkled warty

growth.
 Eugonic
 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

• 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
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,
Current SDA
status: or signal
adjunct amplification
to standard procedureEG: QB amplification)
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
Technique Target Sensitivity % Specificity %
Overall Sm-ve

Home-brew protocols IS6110 85.0 73.0 88.0

PCR IS6110(ne) 77.1 57.9 -
Kit-based test formats 16S rRNA 74.0 53.0 93.0
PCR(Amplicor;Roche) 97.8 - 98.9
PCR (Cobas Amplicor;Roche)16S rRNA 84.3 57.9 100
92.4 59.6 -
TMA (MTD;Gen-Probe) rRNA 83.0 68.0 99.0
100 - 99.3
LCR (LCx;Abbott) 38-kDa protein 77.0 57.0 99.0
77.1 36.8 -
90.8 53.0 100
90.2 70.0 98.4
SDA (BDProbeTec;Becton IS6110 plus 97.9 92.3 96.5
Dickinson) 16S rRNA
Qß (Galileo;Gene-Trak) 23S rRNA 84.0 69.2 97.0
What is new in the diagnosis of TB Pfyffer . G.E. J.Inf 1999 39 21-26 TRC/ICMR 31
 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 Suffs. P. et al Int. J. Tuberc. Lung Dis 2000, 4(2) 179-183. TRC/ICMR 32
 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
 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 Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000, 89, 131-140 33
 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 Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000, 89, 131-140 33
 Sensitivity(%) of smear-negative
vs smear-positive TB
Antibody detection to Age ‘x’ Sm+ Cult+ Sm- Cult+
Glycolipid 96 97
19KDa 57 55
LAM (Myco Dot) 26 7
LAM (Elisa) 88 67
P32Ag of M. bovis IgA 33 IgA 29
BCG IgG 52 IgG 35
38KDa 45- 89 16-82
P90 75 68
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
• 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