This document summarizes diagnostic methods for female genital tuberculosis (FGTB). It discusses the following: 1. Smear examination is the first choice method under RNTCP due to its low cost and simplicity, but has low sensitivity around 22-78% compared to culture. Sensitivity can be improved to around 10% higher using fluorescent microscopy. 2. Culture is the gold standard but is slow, taking weeks, and has low sensitivity. Methods to improve detection include using carbon dioxide production, oxygen consumption, and radiometric or fluorescent techniques to decrease turnaround time by half. 3. Gene Xpert provides results in 2 hours and is sensitive and specific for detecting Mycobacterium tuberculosis, including in

2. Laboratory Diagnosis of
Female Genital Tuberculosis
Prof. Ashok Rattan,
MD, MAMS, INSA DFG, WHO Lab Director
Academics, Industry: Research, Diagnosis, Public Health, Academics

3. A disease of great antiquity
• Fossile bones 8000 BC show evidence
• Egyptian mummies: Spinal caries 2400 BC
• 1500 BC mention of consumption in India
• Hippocrates 460 – 370 BC recognized phthisis (felt it was heredity)
Tuberculosis, Phthisis

4. WHO declares TB a Public Health Emergency 1993:
Recommends DOTS
• Directly
• Observed
• Therapy
• Short Term
• Detect 70% of cases
• Treat 85% of those detected
• RNTCP (2010) reported in 2014
• 100% geographical coverage
• 71% detection rate
• 87% treatment success
• 7 x decrease in death rate ( from 29 to 4%)

5. Tuberculosis now
There is a gathering storm of drug resistant tuberculosis and the strategy we has
Been using has failed to control the disease or prevent emergence of MDR/XDR TB
- Doctors without borders MSF Alert Fall 2006
500,000
MDR TB
2 million death
10 million cases
DOTS detects 70%
Cures 85%
2 billion persons
With latent TB

7. Commonest method for diagnosis of TB
1885
2017

8. Burden of Tuberculosis 2017
(in 1000 )
country population TB mortality
in HIV -ve
TB mortality
in HIV +ve
Incidence HIV TB
incidence
Global 7,440,000 1,300 374 10,400 1,030
India 1,324,000 423 12 2,790 87
China 1,404,000 50 1.8 895 11
Pakistan 193,000 44 2.1 518 6.9
Bangladesh 163,000 66 0.1 360 0.5

10. Sites of infection in FGTB
90 – 100 % 
50 – 80 % 
20 – 30 % 
5 – 15 % 
1 – 2 % 

11. Epidemiology
Prevalence & Incidence of FGTB
• USA : 1% in women attending infertility clinics
• South Africa: 6 – 21 %
• India: 1 – 19%
• In infertility patients FGTB incidence varies from 3 to 16%
• In AIIMS, New Delhi [Suneeta Mittal, JB Sharma] 26 %
• Incidence of infertility in FGTB [AIIMS] 42.5%
• Women seeking assisted reproduction [AIIMS] 24 %
• Tubal factor infertility [AIIMS] 48.5%

12. Investigations
• Common Investigations:
• Blood & Urine Examination
• Chest X Ray
• Mycobacterium based
• TST or IGRA
• Smear examination
• Culture
• Histopathology
• Gene Xpert
• Additional Investigations:
• Imaging
• Endoscopy
• PET/CT
• Biomarkers [LTBI vs Active TB]

14. Acceptable specimens
• Endometrial biopsy / curettage / aspiration
• Menstrual blood within 12 hours of onset
• Biopsy from TO mass
• Peritoneal fluid / biopsy
• Cx or Vaginal secretion
• Other supportive samples
• Sputum for AFB/culture/Gene Xpert
• Blood

15. The First Choice Diagnostic Laboratory
Smear examination
[revised national TB control program RNTCP]
• ZN method
• Kinyoun cold stain
• Auromine O
• Rapid, economical, simple
• Adequate sensitivity in symptomatics
• Overall Low sensitivity (22 – 78% of culture)
• Limit of detection 1 x 104 vs 102 bacilli/ml
• Species cannot be identified

16. How to improve microscopy
LED Microscopy
• Fluorescent microscope
• Pro:
• High power rather than oil immersion
• Black background, yellow bacilli
• Increased sensitivity by 10%
• Con:
• High cost, maintenance
• FluoLED Microscopy

17. The First Choice Diagnostic Laboratory
Loop mediated Isothermal Amplification (LAMP)

18. Culture based detection
Gold standard
Slow : weeks
Low sensitivity

19. Improving detection
• CO2 production
• O2 consumption
– Radiometric
– Fluorescent
– Colourimetric
– Pressure sensor
– Decrease TAT by half
•14 C Palmitate  14 CO2

20. Speed Of Growth Of M.tuberculosis (MGIT 960 vs L J Media)
1st wk 2nd wk 3rd wk 4th wk 5th wk 6th wk
166
0
132
23
55
46
23
63
13
48
2
24
391 204

21. Gene expert
Gene Xpert

24. 533 150 [S+ 92, S- 58] 129 4
533
150
92
58
129
4
250
MDR 50; HIV 16

27. Gene Xpert MTB/RIF assay in the diagnosis of urinary
tuberculosis from urine specimen
Yu Pang et al. Scientific Reports, Nature: 2017; 7: 6181

28. Diagnostic usefulness of Xpert MTB/RIF assay for detection of
tuberculous meningitis using CSF
Syed Beenish Rufai et al. J Infect 2017; 75 (2): 125
• 267 CSF samples
• Smear by ZN -ve
• MGIT 52 (19.5%) 5 RIF resistant
• Xpert 38 (14.2%) 4 Rif R, 3 RIF I, 31 RIF S
• Xpert has sensitivity of 55.1%
• specificity of 94.8%

31. Genotype Mycobacterium test [Hain’s Test]
• DNA Strip technology: Three steps
• 1. DNA isolated from culture
• 2. Multiplex amplification with biotinylated primers  230 bp product
• 3. Reverse hybridization:
• Chemical denaturation of amplified product
• Hybridization of SS biotin labeled amplicon to membrane bound probes
• Stringent washing
• Addition of Steptavidin/alkaline phosphatase
• AP mediated staining reaction

32. The First Choice Diagnostic Laboratory

33. Genotype MTBDR assay

34. M. tuberculosis
Drug susceptibile
XDR
M. tuberculosis
Drug susceptible
INH + RIF Resistant
= MDR
FQ
Resistant
Injectable
Resistant
MDR [multiple drug resistant] TB XDR [extensively drug resistant] TB

35. 1st line drugs
2nd line drugs
INH INH
RIF RIF
PZA PZA
Etham Etham
FQ FQ
Injectable Injectable
Ethio Ethio
Cyclo Cyclo
PAS PAS
MDR TB XDR TB
2 most important
2 most important

36. Antibodies tests
• From Agglutination to SAFA all techniques have been tried
• Cannot differentiate between infection and disease
• Positive even in healthy individuals
• WHO and GOI have banned the use of Antibody tests

37. In Vitro Diagnostic Tests
Antigen
presenting
cell
Memory
T-cell
Presentation of
mycobacterial antigens
IFN-
IFN-
IL-8, etc.
IL-8, etc.
TNF-
TNF-
Andersen P, et al. Lancet 2000;356:1099

38. ESAT 6 & CFP 10
Specific antigens of M. tuberculosis
(RD 1 region)

39. 3rd Generation
Quantiferon TB Gold IT (in Tube method)
• Grey cap tube (Nil control)
• Red cap tube (TB Ag)
• Purple cap tube (Mitogen control)
• Collect 1 ml blood into each tube and mix well
• Transfer to Lab ASAP

40. RESULT INTERPRETATION
POSITIVE ESAT-6 and/or CFP-10
responsiveness detected
M. tuberculosis infection likely
NEGATIVE No ESAT-6 or CFP-10
responsiveness detected
M. tuberculosis unlikely
INDETERMINATE MTB infection status cannot be
determined as a result of
impaired immunity and/or
incorrect performance of the
test

41. Genital TB Algorithm
21st century approach
Tests Results TAT
Smear &/or
culture
+ -- -- + -- 24 hrs
6 wks
Gene Xpert + + -- -- -- 4 hrs
Quantiferon
Gold IT or
Platinum
+ + + -- -- 48 hrs
Diagnosis Genital TB Genital TB Latent TB MOTT
contamination
No evidence
of TB
infection
10 days

42. Carry Home Messages : Diagnostic Methods
[Genital TB is nearly always secondary to TB elsewhere]
1. Smear is inexpensive, rapid but insensitive (104 AFB/ml), can be
improved by using Fluorescent Microscopy (103 AFB/ml)
2. Liquid culture is sensitive & specific [102 M tb/ml], faster than solid
culture but still slow, isolate available for additional AST
3. IGRA is better than TST: both detect LTBI
4. Serological tests, have been banned, as they had no diagnostic value
5. Gene Xpert is sensitive & specific for M tb: both pulmonary & extra
6. Diagnostic tests : culture & non culture tests should be used
simultaneously in an algorithm to improve definitive diagnosis