Phthisiology

1. LABORATORY DIAGNOSIS
OF TUBERCULOSIS
Dr. D.P. Rajani
Medical Microbiologist
Microcare Laboratory & TRC
Surat

2. CLASSIFICATION OF MYCOBACTERIA
Mycobacterium tuberculosis complex refers to a genetically related
groups of Mycobacterium species that can cause TUBERCULOSIS
[TB] in humans. It includes;
Mycobacterium tuberculosis,
Mycobacterium bovis [M.bovis, subsp bovis, M.bovis subsp.caprae and M.bovis BCG]
Mycobacterium africanum,
Mycobacterium microti,
Mycobacterium canetti,
Mycobacterium mungi,
Mycobacterium orygis and
Mycobacterium pinnipedii

3. SPECIMEN COLLECTION
In every clinical Microbiology sample collection
“Results are as good as Specimen” Good quality sample
is very important.
Sputum samples of good quality collected in wide mouth
sterile containers.
Quantity sufficient
Extra pulmonary samples collected in sterile containers
/syringes.

4. Mucoid Sample
Purulent Sample
Bloody Sample
Salivary Sample

5. DIAGNOSIS OF TUBERCULOSIS
Smear Microscopy
AFB Culture
Manual & Liquid Sensitivity
Molecular Diagnostic

6. In low income and high tuberculosis prevalence
countries, sputum smear microscopy is the only cost-
effective tool for diagnosing patients with infectious
tuberculosis and to monitor their progress in treatment.
Sputum smear microscopy is a simple, inexpensive,
appropriate technology method which is relatively easy to
perform and to read.
Classical Ziehl-Neelsen stain used-AFB
SMEAR MICROSCOPY

7. Smear prepared from thick purulent parts of samples.
Size of smear should be 3cmx2cm
At least 300 fields examined
Smear is positive in samples which contain 5000- 10000
bacteria /ml
Sensitivity ranges from 25%-65%
 Sensitivity increases by examination of more than one
smear
SMEAR MICROSCOPY

8. SIZE OF SMEAR
2 x 3
1 x 2 Uniform Smear

9. Good Evenness Smear
Uneven Smear
Good Thickness Smear
Too Thick Smear
Too Thin Smear

10. Smears reported as Positive or
Negative
Quantity of AFB observed
should be noted
Factors influencing smear
sensitivity are type of specimen,
staining technique, experience
of reader
Laboratory Quality Control
important
ZN STAINING

11. FLUORESCENCE MICROSCOPY
Fluorochrome stain used
Can be examined at lower magnification(40 X)
Rapid but more false positive
LED fluorescence microscopy has been evaluated- rapid
and good results, lower cost
LED attachment to microscope Primo Star iLED from Carl
Zeiss

12. FLUORESCENCE
MICROSCOPY

13. DISADVANTAGES OF SMEAR
MICROSCOPY
Needs a large no of bacilli per ml of specimen to be
detected positive
Cannot differentiate between dead and live bacilli
Cannot differentiate between Mtb and NTM
No idea of drug resistance

14. AFB CULTURE
GOLD STANDARD
Provides definitive diagnosis of TB
Pure growth of mycobacteria to do speciation and drug
sensitivity.
Technically demanding and complex
High level of Biosafety needed

15. AFB CULTURE BY L.J. MEDIA [SOLID]
Detection of 10-100 viable
bacilli/ml of specimen
Specimens have to be
decontaminated before
inoculation to remove the
normal bacterial flora.
Solid culture – Conventional LJ
method.
Mycobacteria slow growing
and hence take 2-8 weeks to
grow
LOWENSTEIN JENSEN
[L.J. ] MEDIA

16. LIQUID CULTURE
Many Commercial systems available- BACTEC systems
MGIT960, BacT/ALERT 3D system
Liquid culture yield significantly rapid results than solid
media and isolation rates for mycobacteria are higher
Liquid media- Middle brook 7H9 media used
MGIT system( Mycobacterial growth indicator tubes)
contains a modified Middle brook 7H9 broth with a
fluorescence quenching based oxygen sensor. Growth of
mycobacteria leads to oxygen depletion and indicator
fluoresces brightly
Cultures positive in 10-14 days

17. LIQUID CULTURE BY BACTEC

18. DRUG SENSITIVITY FOR AFB
Sensitivity to first and second line drugs available
Expensive
High degree of technical expertise and lab infrastructure
required
Rigorous quality control needed

19. NON COMMERCIAL METHOD
MODS [Microscopic observed drug susceptibility]
 A micro colony method in liquid culture , based on
inoculation of specimens into drug free and drug
containing media, followed by microscopic examination of
early growth .
Recommended as direct or indirect tests for rapid
screening of patients suspected of having MDR TB

20. CRI
( Colorimetric redox indicator)
Indirect testing methods based on the reduction of a
coloured indicator added to liquid culture medium on a
microtitre plate after exposure of M. tb strains to anti TB
drugs in vitro

21. NRA
(Nitrate reductase assay)
 A direct or indirect method on solid culture based on the
ability of M. tuberculosis to reduce nitrate, which is
detected by a colour reaction

22. SEROLOGICAL TESTS
NEGATIVE RECOMMENDATION from WHO in 2011
SHOULD NOT BE ORDERED

23. MOLECULAR TEST
Genotypic methods have considerable advantage of
speed, standardization of testing and reduced requirement
for Biosafety
1. LINE PROBE ASSAY
( HAINS TEST)
2. GENEXPERT

24. 1. LINE PROBE ASSAY ( HAINS TEST)
Simultaneous identification for M.tuberculosis complex
Molecular assay for the detection of resistance to INH &
RIF of M.tuberculosis complex
By detection of most significant mutations to – inhA,
RpoB and the katG genes
Based on DNA strip technology
Can be done from positive cultures (from MGIT,
BacT/ALERT bottles or LJ)
Pulmonary samples which are smear +ve can be done
directly

25. Detection of multiple genes responsible for the antibiotic
resistance &
Simultaneous recognition of missing wild type gene
Also Available for Second secondline
and identification of some strain of NTM
Limitations of Genotype MTBDRplus
Needs preprocessing of samples.
Needs a PCR set up
Technically demanding
Panic of contamination
Special infrastructure required
Needs dedicated staff and space.

26. 2. GENEXPERT [CBNAAT]
The Xpert MTB/RIF is a cartridge based nucleic acid
amplification test , automated diagnostic test that can
identify Mycobacterium tuberculosis (MTB) DNA and
resistance to Rifampicin (RIF) by Nucleic Acid
Amplification Test(NAAT).
SAMPLES;
Pulmonary samples( Sputum, BAL )
Extra pulmonary samples [Lymph node tissue and
aspirates, CSF, Pus , Gastric lavage and aspirates ( in
children) & Other Tissues]

27. Pulmonary samples - Xpert MTB/ Rif Sensitivity
Status Sensitivity %
Smear +ve culture +ve 98
Smear –ve culture +ve 68
People with HIV 79
People without HIV 86
Extra pulmonary samples Xpert MTB/Rif - sensitivity and specificity
Samples Sensitivity % Specificity %
Lymphnode tissue and
aspirate
84.9 92.5
CSF 79.5 98.6
Pleural fluid 43.7 98.1
Gastric lavage and
aspirations
83.8 98.1
Other tissue 81.2 98.1

28. ABOUT MICROCARE LABAORATORY & TRC
Microcare Laboratory was Certified by ISO 9001:2000 in the year
of 2007, First in south Gujarat.
Microcare Laboratory was accredited by NABL in the year of March
2011, first laboratory in south Gujarat to get NABL Accreditation in
the field of Microbiology.
With the fully & favorable support of STO, STATE TB CELL and
whole team of RNTCP Gujarat government, Microcare laboratory
accredited by National Mycobacteriology Accreditation system of
Central TB Division, Govt. of India. 1st and only one lab In Gujarat in
private sector for solid C&DST.

29. C&DST BY L.J. C&DST BY
Bactec MGIT
MDR [TB]
Detection by
Genotype [Line
Probe Assay].
Detection of
MTB &
Rifampicin By
GeneXpert.
& Routine Culture and sensitivity testing of all clinical samples.
FLUORESCENCE
MICROSCOPY

30. From May 2013 to Dec. 2015 More than 200
patients were Notified by Microcare
laboratory through Nikshay

32. YEAR 2015
DRUG SENSITIVITY [%]
Streptomycin 91
Isoniazid 100
Rifampicin 100
Ethambutol 92

33. Thank you
Dr.D.P.Rajani
& Microcare laboratory Team