Diagnosis of Female Genital TB
Anuj Sharma
17 May 2007
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TB
One third of world population infected Life time risk of TB following infection ~5-10% Global emergency
10 million new cases per year 3 million deaths every year
India 14 million people 5-16% cases of infertility
Drug resistant TB HIV co-infection
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GUTB
Common site of extrapulmonary TB 15-20% of extrapulmonary cases of TB
(developing countries); M:F = 5:3 Kidneys, ureter, bladder, or genital organs Clinical symptoms develop 10-15 yrs after
primary infection ~25% GUTB patients have known h/o TB;
about half of these patients have normal CXR
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GUTB
Mycobacterium sp M. tuberculosis complex
M. tuberculosis – most common M. bovis
(M. microti, M. africanum, M. canetti)
MOTT/NTM Mycobacterium kansasii Mycobacterium fortuitum Mycobacterium avium-intracellulare Mycobacterium xenopi Mycobacterium celatum
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M. tuberculosis
Aerobic bacillus Non-spore forming Non-motile Generation time: 12-20 hours Culture
3-6 weeks 1-2 weeks
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GTB
Still rampant in India Genital TB used to be the commonest
cause of tubal infertility in the past Today genital TB much less common But, often misdiagnosed in infertile
women, leading to a lot of heartbreak and distress
Infection / Disease
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Pathogenesis
FGTB is usually secondary to pulmonary TB; although in some cases, cervical TB - primary infection
Begins with a focus in the endosalpinx Fallopian tubes - 100% Endometrium - 50% Ovaries - 20% Cervix - 5% Vagina and vulva - <1%
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Diagnostic criteria Genital tuberculosis
Endometrial adhesions with deformity, and obliteration of the endometrial cavity,
Obstruction of the fallopian tubes with multiple areas of constriction, and calcified lymph nodes in the adnexal region
Advanced tuberculous endometritis may mimic severe uterine adhesions as seen in Asherman syndrome
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HSG - GTB
Rigid pipe-stem tubes A clubbed ampula with retort-shaped
hydrosalpingx Vascular or lymphatic intravasation of contrast Small shrunken uterine cavity with filling
defects Long and dilated cervical canal & dye in
cervical crypts Bilateral cornual block Punctate opacification of crypts and diverticulae
in lumen of tubes
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Investigations
CBC, ESR, KFT, CRP
CXR
Pelvic ultrasound / hystero-salpingography
Laparoscopy
Histopathology
Microbiology Mantoux test
QTG-T
Serology
AFB microscopy / culture
EA / EB / EC / menstrual blood
Urine – 3 consecutive days (smear vs culture - St: 52% / 65%; Sp: 89-96 / 100%)
Molecular tests
HIV
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Genital tuberculosisA diagnostic dilemma*
Varied clinical presentations Diverse results on imaging and laparoscopy Mixed lab tests Pelvic ultrasound - initial screening test
Ascites / loculated fluid (100%) Adnexal mass (93%) Peritoneal thickening(69%) Omental thickening(61%) Endometrial involvement (83%) Peritoneal tubercles and adhesions
MRI, hysterosalpingography, and endoscopy Diagnosed on the collective evidence from imaging
techniques, endoscopy, histopathology and microbiology
*J Obstet Gynecol India 2006; Vol. 56, No. 3: 203-204
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GTB
Chest radiographs, urine, and sputum cultures are not specific for FGTB
But, helpful to rule out dissemination to other organs
Infertile women with a positive PPD skin test - early laparoscopy direct visualisation of fallopian tubes collection of specimens for histopathology
and microbiology
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New approaches
LTBI – QFTG, T spot TB NAA
Amplicor MTB test (Roche, PCR) Amplified MTD (Gen-Probe, TMA) ProbeTec ET (BD, SDA)
High specificity / PPV; low sensitivity / NPV To be used in conjunction with conventional tests
and clinical data Rapid detection of drug resistance
Molecular beacons – Rif / INH Line probe assays – INNO-LiPA Rif TB kit Phage based assays – FASTPlaque TB,
FASTPlaque TB MDRi kit, FASTPlaque TB-Response
Proc Am Thorac Soc 2006; Vol 3: 103-10
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Outline
Mantoux QuantiFERON-TB Gold Microscopy Culture Molecular tests – Gen-probe / PCR Identification by Accuprobe FASTPlaque TB
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Mantoux
Diagnostic role of a positive Mantoux (PPD) is controversial
Almost 45% of infertile women with strong indirect evidence of pelvic TB, such as laparoscopic findings (thickened tubes, areas of caseation, etc) - negative Mantoux
In 27 infertile women with a positive Mantoux, only 11 had clear laparoscopic findings suggestive of FGTB
Mantoux test in women with laparoscopically diagnosed tuberculosis sensitivity - 55% specificity - 80%
* Raut VS, Mahashur AA, Sheth SS: The Mantoux test in the diagnosis of genital tuberculosis in women. Int J Gynaecol Obstet 2001, 72:165-169
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QuantiFERON-TB GoldQFTG
In vitro laboratory diagnostic test (May ’05) Indirect test for M. tuberculosis complex
M. tuberculosis M. bovis, M. africanum, M. microti, M. canetti
infection Tuberculosis disease OR latent tuberculosis
infection (LTBI)- cannot distinguish between them
Intended for use in conjunction with risk assessment, radiography, and other medical and diagnostic evaluations
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QFTG
IFN-g release assay (IGRA)Fresh heparinised whole blood from sensitised persons incubated with mixtures of synthetic peptides (two proteins present in M. tuberculosis) ESAT-6 (early secretory antigenic target-6) CFP-10 (culture filtrate protein-10)
Lymphocytes in blood of TB patients recognize these mycobacterial antigens - generation and secretion of interferon-γ (IFN-γ)
Detection and subsequent quantification of IFN-γ by ELISA
These proteins are absent in BCG strains and from most NTMs (except M. kansasii, M. szulgai and M. marinum)
Higher specificity than with PPD (Mantoux)
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QFTG
Single patient visit - whole blood sample - 4 ml of heparinised whole blood
Must be transported to lab to allow initiation of testing within 12 hours (viable lymphocytes)
Rapid results (within 24 hours) No booster response (measured by
subsequent tests - which can happen with Mantoux)
No reader bias (cf Mantoux) Not affected by prior BCG vaccination Impaired or altered immune function ST: 80-95% (Mantoux 75-90%) SP: 95-100% (Mantoux 70-95%)
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QFTG
Result InterpretationPositive(ESAT-6 and/or CFP-10responsiveness detected)
M. tuberculosis infection likely
Negative(No ESAT-6 or CFP-10responsiveness detected)
M. tuberculosis infection unlikely, but cannot be excluded in immunocompromised patients, or highly probable cases
Indeterminate Test not interpretable
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Microscopy
Ziehl-Neelsen, Kinyoun Fluorochrome - Auramine-rhodamine (direct
fluorescence) Higher sensitivity; faster screening
ST: 22-78% (cf culture) MC Detection limit in sputum: 5000-10000
orgs/mlCulture: 100 orgs/ml
Presumptive identification; confirmation by culture / NAA test
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Culture
Decisive step for diagnosis, treatment & control of TB
Combination of solid & liquid media- “gold standard” for primary isolation
Recommended turn around time (CDC) 14 days (culture)
21-30 days (identification & susceptibility)
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BacT/ALERT 3D MB
Fully automated
Non-invasive
Continuously monitored non-radiometric system
Revised antibiotic supplement kit
Medium - modified Middlebrook 7H9 broth with supplements
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BacT/ALERT 3D
The first BacT/ALERT 3D 960 in India
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BacT/ALERT MP
10 ml Middlebrook 7H9 Broth BSA, Catalase
Decontaminated clinical specimen and sterile body fluid specimen (other than blood)
MB/BacT Antibiotic Supplement Kit Ampho B, Azlocillin, NA, Polymyxin
B, Trimethoprim and Va
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BacT/ALERT MB
30 ml Middlebrook 7H9 Broth
SPS, Glycerol
For blood and sterile body fluids
Direct inoculation
No processing
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BacT/ALERT 3D MB
CO2 released by mycobacteria detected by sensor
Colour changes - increase in reflectance units
Positive broth - 106-107 orgs/ml Higher biomass - direct inoculation of
identification panels & susceptibility tests
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Mean detection time
BacT/ALERT-3D (days)
LJ(days)
Italy2001
MTB• Smear +ve 11.5 20.6• Smear –ve 19.9 32.1
NTM 19.6 27.8
Italy1999
MTB• Smear +ve 11.8 28.5• Smear –ve 21 36.2
NTM 12.7 36.2
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SGRH Experience
BacT/Alert 3D
LJ medium
Mean detection time (days)
11.95 22
AFB S/T 10.45 21
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Molecular diagnosis of TB
DNA probes From cultures Direct samples > 10,000 organisms
rRNA probes Gene amplification
PCR Isothermal amplification
Gen-probe AMTD, NASBA, SDA (IS6110), QB replicase
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PCRAMPLICOR M. tb test
Uses Rapid diagnosis in smear negative samples
65 kDA protein encoding gene mpt64 gene
Differentiate M. tb / NTM Species specific IS6110
Genetic markers for drug resistance Rifampicin – rpoB INH – codon 315 of katG
False positives & false negatives (inhibitors) Negative result cannot rule out TB & positive
result is not always confirmatory
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Gen-Probe – MTD testAmplified M. tb direct (MTD) test
TMA - isothermal amplification of M. tb complex 16s rRNA
>1 billion copies of RNA amplicons; Hybridisation Protection assay (HPA); Single tube
Detection of amplicons with acridinium ester-labelled DNA probe
St – 91-95% Sp - 99-100% PPV - 84-100% NPV - 98.4-99.6%
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34Evaluation of women with infertility and genital TB
Biopsy or curettage samples from 65 women clinically suspected to have genital tuberculosis were investigated with smear microscopy, histopathology, culture, and PCR for mycobacteria
Of the 65 clinically suspected patients investigated 8 were acid fast bacilli (AFB) smear positive 12 were culture positive 17 were histology positive 28 were positive by PCR
A combination of PCR with the other available techniques is the best method of achieving sufficient sensitivity and specificity for the diagnosis of female genital tuberculosis
J Obstet Gynecol India 2006, Vol. 56, No. 5: 423-426
17 May 2007
35Improved diagnostic value of PCR in the diagnosis of FGTB leading to infertility
Double-blind study; 25 women suffering from infertility 61 samples, consisting of EAs, EBs and fluid from POD PCR - mpt64 gene of Mycobacterium tuberculosis
14 out of 25 patients (56.0 %) compared to 1 smear with acid-fast bacilli (1.6%) and2 culture-positive samples (3.2 %)
53.3%of EBs, 47.6% of EAs and 16% of POD fluid samples All patients with laparoscopy suggestive of tuberculosis
60% of those with a probable diagnosis and 33% of those with incidental findings, were positive by PCR1 EA sample from an infertile patient with normal laparoscopy was also positive
Multiple sampling from different sites and amplification of the mpt64 gene segment by PCR offered increased sensitivity in determining tuberculous aetiology in female infertility
Journal of Medical Microbiology (2005), 54, 927–931
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36Meta-analyses / systematic reviewAccuracy of NAA test for TB
> 40 studies; Pulm & extrapul TB Commercial tests High Specificity; St lower / variable NAA to be done in conjunction with smears /
cultures Clinical value depends on pretest probability
J Clin Microbiol 2003; 41: 5355-65
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Accuprobe
Gen-probe’s culture identification tests Definitive identification of common
mycobacteria Specificity of DNA probe /
convenience/speed of HPAMycobacterial Identification Sensitivity Specificity
Mycobacterium avium 99.3% 100%
Mycobacterium intracellulare 100% 100%
Mycobacterium avium complex 99.9% 100%
Mycobacterium gordonae 98.8% 99.7%
Mycobacterium kansasii 92.8% 100%
Mycobacterium tuberculosis complex 99.2% 99.0%
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AccuProbe tests
Based on hybridisation of nucleic acids 4 steps
Sample preparation Hybridisation Selection of the hybrid Detection of the hybrid
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FASTPlaque TB
Mycobacteriophage detection system M. smegmatis lytic cycle: 90 mins Not expensive; safe Viable bacilli, intact phage receptors Affected by effective ATT – monitor trt
success Phage inhibitory substances Analytical ST: 100-300 bacilli/ml Mixed results
Good sp (96-99%) Less st (70-87%)
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Diagnosis of TBSGRH Microbiology
Microscopy SGRH charges (NH/PvtOPD)
ZN 170 DF (Auramine-Rhodamine) 500
Culture, Identification & Sensitivity AFB Culture (manual)/ST 900 AFB Culture (automated - BacT/ALERT-3D) MB3DNAI Accuprobe nil
NAA NASBA - Gen-probe – TMA 2000
Serology / Mantoux / QFT-G 1200 / 80 / 2500
3DNAI – DF, rapid culture, NA Id and ST 2200
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