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

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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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