This document provides information on laboratory diagnosis of tuberculosis. It discusses how mycobacteria are acid-fast bacilli with lipid-rich cell walls. Sputum and saliva samples are examined microscopically using Ziehl-Neelsen staining. Culture methods like Lowenstein-Jensen medium and Middlebrook 7H10 agar are used to grow mycobacteria. Newer methods like Xpert MTB/RIF assay and line probe assays allow for rapid detection of tuberculosis and drug resistance directly from samples.

1. LAB DIAGNOSIS OF
TUBERCULOSIS
Dr Emil Mohan
RD-III ,Dept of respiratory medicine

2. • obligate aerobes growing most successfully in tissues with high oxygen
content
• They are facultative intracellular pathogens usually infecting mononuclear
phagocytes (e.g. macrophages), slow-growing with a generation time of 12
to 18 hours hydrophobic with a high lipid content in the cell wall. Because
the cells are hydrophobic and tend to clump together, they are
impermeable to Gram's stain.
• Mycobacteria are "acid-fast bacilli" because of their lipid-rich cell walls,
which are relatively impermeable to various basic dyes unless the dyes are
combined with phenol.
• Once stained, the cells resist decolorization with acidified organic solvents
and are therefore called "acidfast"

3. Sputum and saliva
• SPUTUM
• Specimens appear mucoid even
blood stained
• Contains many polymopho
neutrophils
• SALIVA
• Appears clear, watery and frothy
• Contains manysquamous
epithelial cells
• Absence of poly morpho
neutrophils

4. Ziehl–Neelsen stain
• Paul Ehrlich developed a stain for mycobacterium tuberculosis, called the alum
hematoxylin stain
• It is named for two German doctors who modified the stain: the bacteriologist
Franz Ziehl and the pathologist Friederich Neelson
• at least requires 5000 bacilli/mm3 of sputum
• Flurscent staining offer advantage of screening at alow power.
• Influenced by type of specimens,thickness of smear,extent of decolourisation,type of
counter stain used and training and experience of examiner
• Concentration by cytocentrifugation enhance sensitivity to 100%
• Liquefaction of sputum by Na hypochlorite enhances sensitivity upto 70%
• Treatment with zwiteronic detergent interfere with innate buoyancy of bacilli and
enhances results of microscopy

5. • Place the slides on staining rack
• Cover each slide with 1% carbol fuchsin-primary stain
• Heat each slide from below,do not boil
• Leave for 10 min..longer time will improve staining
• Rinse the slide with water and drain off excess water
• Add 25% H2SO4 and leave for 3 min
• Rinse each slide with water
• Cover with 0.1%methylene blue for 1’.
• Rinse and drain off excess water

6. Grades of smear microscopy
exam results grade No.of fields
>10 AFB /oil immersion field positive 3+ 20
1–10 AFB per oil immersion
field
positive 2+ 50
10–99 AFB per 100 oil
immersion fields
positive 1+ 100
1–9 AFB per 100 oil
immersion fields
positive Scanty 100
No AFB per 100 oil
immersion fields
negative - 100

7. Processing direct smear negative specimens
• Sputum microscopy can be improved with liquefaction, concentration
and gravity sedimentation
• Solvents
• Sodium hypochlorite
• Sodium hydroxide
• Ammonium sulphate
• N –acetyl L-cysteine NaOH

8. Benefits of liquefaction and Concentration
• Major studies showed processing with chemicals and centrifugation
improved sensitivity upto 18%.
• Incremental yield upto 9%
• Treating specimens with sodium hypochlorite is mycobactericidal and
kills HIV and improves safte.y

9. Modifications
• 1% sulfuric acid alcohol for actinomycetes, nocardia.
• 0.5–1% sulfuric acid alcohol for oocysts of isospora, cyclospora.
• 0.25–0.5% sulfuric acid alcohol for bacterial endospores.
• Differential staining – glacial acetic acid used, no heat applied,
secondary stain is Loeffler's methylene blue.
• Kinyoun modification (or cold Ziehl–Neelsen technique) is also
available.
• A protocol in which a detergent is substituted for the highly
toxic phenol in the fuchsin staining solution

10. Fluorescent microscopy
• Light emitting diode based
• Stain 0.1% auramine,rhodamine,acridine orange
• Decolourizer 0.5%acid alcohol
• slides can be examined at a lower magnification, thus allowing the
examination of a much larger area per unit of time
• In fluorescence microscopy, the same area that needs examination for
10 minutes with a light microscope can be examined in 2 minutes

12. Culture
Conventional egg based solid medium
• Lowenstein –jensen medium
• Egg-based – Petragnani medium and Dorset medium
• Middlebrook 7H10 agar
• Middlebrook 7H11 agar
• Blood-based – Tarshis medium
• Serum-based – Loeffler medium
• Potato-based – Pawlowsky medium
Liquid based medium
• Dubos' medium
• Middlebrook 7H9 broth
• Proskauer and Beck's medium
• Sula's medium
• Sauton's medium

13. Lowenstein –jensen medium
• The medium is named after the Austrian pathologist Ernst
Löwenstein and the Danish medical doctor Kai Adolf Jensen
• Cultures should be read within 5–
to 7 days after inoculation and once
a week thereafter for up to 8 weeks.
• The typical colonies are non pigmented, rough, dry on LJ medium.
• The green color of the medium is due to the presence of malachite
green which is one of the selective agents to prevent growth of most
other contaminants.
• penicillin and nalidixic acid are also present in LJ medium to inhibit
growth of Gram-positive and Gram-negative bacteria

14. Lowenstein –jensen medium

15. Middlebrook medium
• Since this medium is only partially selective, bacteria other than
mycobacteria may grow if specimens are not appropriately
pretreated for decontamination.
• Mycobacteria are strict aerobes and growth is stimulated by
increased levels of CO2. Screw caps on tubes or bottles should be
handled as directed for exchange of CO2.
• Middlebrook 7H10 Agar requires incubation in a 5-10%
CO 2 atmosphere in order to recover mycobacteria.
• Inoculated media should be kept away from light or excessive heat,
as exposure results in the release of formaldehyde in the media
which may inhibit or kill mycobacteria.

16. Middlebrook medium

17. radiometric BACTEC 460 TB method
• C14 labelled palmitic acid used in 7H12 medium.
• When C14 labelled substrate in medium is metabolizd,14CO2 is
produced and is measured by BACTEC system and reported as growth
index value
• Mycobacteria in clinical samples can be detected in half time as
compared to conventional culture methods.

18. MGIT 960 mycobacteria detection system
• Automated system for growth and detection on MTB
• Incubate and monitor mycobacteria indicator tube every 60 min for
incearse in fluorescence
• Based on AFB metabolic O2 utilization
• Rapid ,accurate and cost effective method for high volume
mycobacteria laboratory

19. Limitations
• Assay is not indicated for
• Patients being treated with ATT
• Monitor bacteriologis cure
• Monitor response to therapy
• Negative test does not exclude the possibility of isolating MTB
complex from sputum sample.
• Positive test does not necessarily indicate the presence of viable
organisms.

20. • Xpert MTB /RIF assay does not diifertiate between the species of the
MTB complex

21. MB/BacT system
• Non-radiometric continuous monitoring system with computerized
data management system.
• Based on colorimetric detection of CO2
• Acceptable alternative for BACTEC system
• Increased contamination
• Longer detection time

22. Genotypic methods-PCR
• PCR allows sequences of DNA present in onlya few copies of
mycobacteria to be amplified in vitro such that the amount of DNA
can be visualized and identified.
• CBNAAT is aqualitative ,nested real time polymerase chain reaction in
vitro diagnostic test that can detect mycobaterium tuberculosis and
rifampicin resistance in 2 hours of starting the assay
• Most common target gene is IS6110

23. Advantages
• Unlike conventional NAAT ,in Xpert MTB sample processing ,PCR
amplification and detection are integrated into single self enclosed unit-
catridge
• Assay also detects the rifampicin resistance associated mutations of rpo B
gene. sequence of the rpo B gene is probed with five molecular beacons
• Sample reagent is tuberculocidal which largely eliminates concerns of bio
safety during procedure
• So, the technology can be taken out of central Lab nearer to patients.
• Require uninterrupted stable power supply ,temperature control, yearly
calibration

24. • As an initial test
• Pooled sensitivity=88%
• Pooled specificity=99%(22 studies ,9008 participants)
• As an add on test
• Pooled sensitivity=65%
• Pooled specificity=99%
(Source:Xpert TB for diagnosis of tuberculosis Policy Update
,WHO,2013)

25. Limitations
• Assay is not indicated for
– Patients being treated with ATT
– Monitor bacteriologic cure
– monitor response to therapy
• Negative test does not exclude the possibility of isolating
MTB-complex from the sputum sample
• Positive test does not necessarily indicate the presence of
viable organisms

26. Limitations
• Xpert MTB/RIF Assay does not differentiate between the
species of the MTB-complex
• Culture must also be performed to determine if
mycobacteria other than tuberculosis complex (MOTT) is
present in addition to MTB-complex.
• M. scrofulaceum when tested at concentration of 108
CFU/mL produced a false positive Xpert MTB/RIF Assay result.

27. • Unlike conventional NAAT ,in Xpert MTB sample processing ,PCR
amplification and detection are integrated into single selif enclosed
unit-catridge
• Assay also detects the rifampicin resistance associated mutations of
rpo B gene.sequence of the rpo B gene is probed with five molecular
beacons
• Sample reagent is tacberiocidal which largely elimates concers of
biosafety during procedure
• So,the technology can be taken out of central Lab nearer to patients.

28. Xpert MTB/RIF to diagnose EPTB and R Resist
• lymph node TB in samples from biopsy or fine- needle
aspiration – Sen=84.9%, Spe=92.5%
• pleural TB in pleural fluid – Sen=43.7%, Spe=98%.
• “Pleural fluid is not regarded as a suitable specimen for the
microbiological diagnosis of pleural TB. Pleural biopsy is the
preferred sample type for diagnosing pleural TB. ”

30. Xpert MTB/RIF to diagnose EPTB and R Resist
• TB in samples of CSF – Sen= 79.5%, Spe=98.6%
• TB in gastric fluid(lavage) – Sen= 83.8%, Spe=98.1%
• TB in tissue samples – Sen= 81.2%, Spe=98.1%

31. • CBNAAT should be used rather than conventional microscopy ,culture
as initial diagnmostic test in adults suspected of MDR TB/PLHA
patients(strong recommendation)
• CBNAAT should be used rather than conventional microscopy ,culture
as initial diagnmostic test in children suspected of MDR TB/PLHA
patients.
• CBNAAT can be used as afollow on test to microscopy in adults
suspected of having TB but not at the risk of MDRTB especially when
further testing of muicroscopy is needed

32. Principle of LPA
• Line probe assays are family of DNA strip based tests that determine
drug resistance profile a MTB strain
• WHO approved tests for rapid detection of resistance to first line and
second line drugs
Detected by
• binding of amplicons to probes targeting most common mutations
• Lack of hybridisation of amplicons to WT probes

33. Limitations
• Resistance cannot be completely excluded even in tghe presence of
all WT probes.Additional Phenotypic DST may be reqiured
• Less efficient in samples having both drug susceptible and resistance
bacteria (hetero resistance)
• LPA defined as indeterminate if corresponding locus control for the
specific drug is missing while test is valid
Sensitivity of FLLPA=96.7%
Specificity=98.8%(Natavithara et al,Eur Res. J.2017,49)

34. • Isoniazid
• Inh A prmoter mutation-low level resistance
• Kat G –high level resistance
• Rifampicin –rpo B gene
• Moxifloxacin-gyr A or B gene
• Amikacin,Kanamycin ,capremycin-rrs gene