Laboratory Diagnosis Culture Pcr

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 M.TB Complex specific is Other targets: Genes encoding

Methods: Target amplification

16S rRNA 65KDa hsp 6110 38 KDa MPB 64 mtp 40 PMT 64 - PCR

(TMA, LCR, SDA or signal amplification Current status: adjunct 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 % Overall Sm-ve

Specificity %

Home-brew protocols PCR

IS6110 IS6110(ne)

85.0 77.1

73.0 57.9

88.0 -

Kit-based test formats PCR(Amplicor;Roche)

16S rRNA

74.0 97.8

53.0 -

93.0 98.9

PCR (Cobas Amplicor;Roche)16S rRNA

84.3 92.4

57.9 59.6

100 -

TMA (MTD;Gen-Probe)

rRNA

83.0 100

68.0 -

99.0 99.3

LCR (LCx;Abbott)

38-kDa protein

77.0 77.1 90.8 90.2

57.0 36.8 53.0 70.0

99.0 100 98.4

SDA (BDProbeTec;Becton Dickinson) Qß (Galileo;Gene-Trak)

IS6110 plus 16S rRNA 23S rRNA

97.9

92.3

96.5

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

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 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

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 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

75

68

P90

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