The document discusses case finding and diagnostic strategies for tuberculosis. It describes the epidemiology of TB globally and in India. The key diagnostic methods discussed are microscopy, culture, and GeneXpert/CBNAAT. Microscopy requires 5000-10000 bacilli/ml but is simple and rapid. Culture is more sensitive, detecting 10-100 bacilli/ml, and allows for drug susceptibility testing. GeneXpert/CBNAAT detects TB and rifampin resistance within 2 hours and works directly from sputum, with sensitivity of 92% and specificity of 99%.

1. Case Finding and Diagnostic strategy
for Tuberculosis
• Dr. Yogita Mistry
• Microbiologist
• Intermediate Reference Laboratory
for Tuberculosis
• GMC,Surat

2. Objectives
• Introduction
• Epidemiology
• Programme
• Laboratory network
• Case finding and Diagnostic strategy
• Diagnostic methods in short

3. Introduction
• Mycobacterium tuberculosis, and rarely by other organisms of the
“tuberculosis complex”
• Transmitted by inhalation of infected droplet nuclei which are discharged in
the air when a patient with untreated sputum positive TB coughs or sneezes.
• If the bacillus succeeds in infecting a person, only about 5%–10% of such
infected persons (primary infection) develop active disease.
• In the remaining 90% to 95 % of infected persons, initial infection usually
goes unnoticed.

4. Extent of the Tuberculosis Problem
• One third of the global population
• The annual incidence of new cases of all forms of TB (pulmonary and extra-pulmonary) worldwide :8.8
million
• Globally, it is estimated that 1.8 million people die from TB each year—the majority of them in
developing countries.
• INDIA
• About 40% of the population in India is estimated to be infected with TB bacillus.
• Every year approximately 1.8 million people develop TB and nearly 400,000 die from it.
•In India, every day:
• • more than 5000 develop TB disease
• • more than 1000 people die of TB (i.e. 1 death every 1.5 minutes)

5. Programme
• The National Tuberculosis Programme of India (NTP) was
initiated in 1962, based on research by Tuberculosis research centre, Chennai and
National TB Institute, Bangalore.
• self administered standard drug regimens
• Despite the existence of the NTP, there was little impact on the TB burden till
1992.

6. RNTCP
• On the recommendations of an expert committee, a revised strategy to
control TB was pilot-tested in 1993 in a population of 2.35 million and
thereafter increased in phased manner.
• A full-fledged programme was started in 1997 and rapidly expanded with
excellent results.
• DOTS (Directly Observed Treatment, Short-course chemotherapy) strategy.

8. Organizational structure and functions
• The structure of RNTCP comprises of five levels:
• National level
• State level
• District level
• Sub-district level
• Peripheral health institution level

9. National Level
• Ministry of Health and Family Welfare (MoHFW)
• Directorate General Health Services
• Central TB Division
• The CTD is assisted by 3 national tuberculosis institutes, namely, National Tuberculosis Institute,
Bangalore, Tuberculosis Research Centre, Chennai, and Lala Ram Sarup Institute of Tuberculosis
and Respiratory Diseases, New Delhi.

10. Structure of RNTCP at the state level:

11. Structure of RNTCP Laboratory network:

12. Case Finding

24. Microscopy
• Light microscopy using ZN stain
• Florescence microscopy using fluorescence stains
• Needs atleast 5000-10000 bacillies/ml of sputum samples
• Extrapulmonary?
• Advantages
• Disadvantages

25. Factors which influence the sensitivity of
acid-fast smears include
• Amount of acid-fast bacilli present in the specimen( atleast 10000 bacilli/ml required)
• Experience of the reader,
• Stain used
• Number of fields examined
• Type of specimen (e.g., generally respiratory specimens have higher yield than non
respiratory)
• Volume of the specimen
• Smear pretreatment (direct vs. pre-treated, concentrated).

26. • Patient population being examined
• Immunocompromised individuals often present with lower bacterial loads making detection by
smear difficult.
• Up to 30% of persons (commonly children) are unable to produce sputum for a smear requiring
the use of more invasive methods (gastric washing, bronchoalveolar lavage, etc). So , a
concentration step provides increase sensitivity over direct smear microscopy. Extrapulmonary
tuberculosis, paediatric tuberculosis and in patients coinfected with HIV .

27. The advantages of smear microscopy are
• Most simplest
• Non invasive
• Rapid
• Inexpensive
• Very specific in high prevalence settings
• Results are obtained within hours of receipt of the sample
• Correlate with infectiousness
• Identify both patients at high risk of death from tuberculosis if untreated and
patients who require more drugs in the initial treatment regimen because of greater
bacterial load.
• Important role in follow up of TB treatment. Only when the smears are negative can
the intensive phase of the treatment be suspended.

28. Factors which influence the sensitivity of acid-
fast smears include
• Amount of acid-fast bacilli present in the specimen( atleast 10000 bacilli/ml required)
• Experience of the reader,
• Stain used
• Number of fields examined
• Type of specimen (e.g., generally respiratory specimens have higher yield than non
respiratory)
• Volume of the specimen
• Smear pretreatment (direct vs. pre-treated, concentrated).

29. • Patient population being examined
• Immunocompromised individuals often present with lower bacterial loads making detection by
smear difficult.
• Up to 30% of persons (commonly children) are unable to produce sputum for a smear requiring
the use of more invasive methods (gastric washing, bronchoalveolar lavage, etc). So , a
concentration step provides increase sensitivity over direct smear microscopy.
• Extrapulmonary tuberculosis, paediatric tuberculosis and in patients coinfected with HIV .
• Due to the requirement of serial sputum examinations, some patients who do not come back for
repeated sputum examinations become “diagnostic defaulters” . Some do not come back for
results.

31. Mercury bulb LED
1. Aspecific light (large spectrum) from UV to IR Light of specific wavelength
1. Bulbs of 50 to 100 Watt Only 3 watt
1. Use of excitation filter required No excitation filter as the light produced is specific
1. Expensive dichromatic beam splitter required in epi
fluorescence
No dichromatic beam splitter mirror required
1. Lifetime max. 300 hours=> counter required ( explosion
hazard); lamp must warm up before reading and cool before
switching on again
Life above 10,000 hours of use
No waiting time between switching on/off (i.e.
power cuts!!)
1. Bulb replacement complex and expensive++ Replacement of bulbs never needed?
1. Requires a stable power supply Low voltage and interruptions little disturbing
1. Heat emission LED=cold light, no heat
1. Photobleaching quiet fast during reading Photobleaching less likely to occur
1. Mercury harmful to the environment Bulbs contain silicium, irritating
1. Background sometimes too dark, makes focusing very
difficult or impossible
Background less dark,=> focusing easier
1. Needs dark room for better contrast Contrast OK in ordinary room

32. Culture
• Solid
• Liquid
• 10-100 bacilli/ml

33. Why is growth detection necessary?
• TB culture is more sensitive than acid-fast smear microscopy
• Microscopy requires 5000–10000 AFB per ml of sputum for detection
• Culture can detect as few as 10–100 viable bacteria per ml of sputum
• Viable mycobacterial cells may be necessary for final
identification
• Drug susceptibility tests require viable organisms
• Genotyping of mycobacteria requires large quantities of cells

34. Which media should be used?
Inoculation of both solid and
liquid media are considered
the “Gold Standard” for TB
culture

35. Growth detection: Lowenstein-Jensen (LJ) medium
• Can be less expensive than liquid
media (MGIT)
• Long shelf life: can be stored
refrigerated for several months
• A variety of decontamination
methods can be used
• Allows morphology to be
visualized, mixed cultures of
mycobacteria can be detected

36. Growth detection on LJ
• Supports growth of most mycobacteria
• Growth can be quantified
• Colony morphology and
pigmentation can be seen
• It is less likely to become contaminated
during preparation and inoculation
• Contamination usually involves the total
surface of the medium so it is easily seen

37. Growth detection in liquid media
• Benefits:
• Shorter turn-around time
• Greater sensitivity
• Manual, semi-automated and
fully automated formats
• Challenges:
• Higher isolation rate of NTM
• Higher contamination rate

38. Gene Xpert/ CBNAAT
• On average 100-103 bacilli/ml

39. CBNAAT/Gene Xpert
This is a heterogeneous group of tests that use either the polymerase chain reaction (PCR)
technique or Transcription mediated amplification (TMA) or other forms of nucleic acid
amplification methods to detect mycobacterial nucleic acid.
These test vary in which nucleic acid sequence they detect and vary in their accuracy.
sensitivity 92%
specificity 99%

41. GeneXpert is an automated molecular platform to detect
M. tuberculosis and rifampicin resistance testing by
targeting specific mutations in the rpoB gene.
It is approved for use directly on raw sputum and results
should be available within 2 hours in the laboratory but
available in health facilities within 48 hours.
• The test involves only three manual steps:
• The addition of sample treatment reagent to liquefy and
inactivate the sputum
• Transfer of 2ml of liquefied sputum to the cartridge
• Loading the cartridge into the device for the assay

42. Advantages of the test are :
It detects MTB and Rifampicin resistance from one specimen at the
same time.
Results are available in approx. 2 hours.
 It is specific for MTB complex; (it can differentiate MTB from other
mycobacteria).
 It can also be used on the following processed samples - CSF, aspirates
(gastric, lymph node) and tissue (i.e. pleural biospy)
The test for each specimen is carried out in a closed system (cartridge),
so there is a reduced risk of cross-contamination and human error.

43. • The limitations of this test are :
It cannot be used for monitoring treatment because it does not distinguish between live
and dead bacilli, its use is therefore limited to diagnosis
 A small proportion of Rifampicin resistance detected may not correlate with
physiological resistance (leading to discordance between Xpert and DST results or
clinical outcome)
 The assay is semi-quantitative and defines a positive test as “very low”, “low”,
“medium”, and “high”. This grading is not reported on the laboratory result.
There is no direct correlation between the Xpert semi-quantitative result and the smear
grading of scanty, +, ++ and +++. The rifampicin results can only be reported if MTB
complex is detected.
The test might be unsuccessful due to laboratory test errors, test failure or invalid results.
In these instances a second specimen must be collected for a repeat Xpert test.

44. LPA
DNA Extraction
Amplification
Hybridization

45. Line Probe Assay

46. laboratory layout
DNA-“dirty” area
DNA-free
area
Amplicon-
“dirty” area

47.  Advantages of the test are that:
 It detects MTB and resistance to RIF & INH at the same time from one specimen
 It reduces time to diagnosis of MDR-TB to 7 days
Cost-effective when compared with TB culture and DST
They also demonstrated significant patient benefits, including early targeted treatment
of MDR-TB and the potential interruption of transmission.

48. The limitations of the test are :
It cannot be used for monitoring patients on treatment because it does not distinguish
between live and dead bacilli, therefore its use is limited to diagnosis
It is dependent on smear results, can only be performed on smear positive or culture
positive sputum specimen
 labour intensive
Prone to contamination and human error
 Requires a lot of space - at least 3 separate rooms for the different steps ( National
Tuberculosis Management Guidelines 2014)
 A small proportion of resistance detected may not correlate with physiological resistance
(leading to discordance between LPA and conventional DST results or clinical outcome).
New:
Version 2 of the MTBDRplus which can be used on smear positive and negative sputum
specimens is available and currently being validated in the country.
 MTBDRsl is available for second line testing. This test may be used as a rule in test for
XDR-TB in high risk groups.

49. Summary
Molecular DST [e.g. cartridge-based automated nucleic acid
amplification test (CBNAAT) or line probe assay (LPA)]
First
Liquid culture isolation and LPA DST Second
Solid culture isolation and LPA DST Third
Liquid culture isolation and Liquid DST Fourth
Solid culture isolation and DST Fifth

50. •Thank You