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Not recommended for testing serum and plasma on patients older than 30 days unless viremia is suspected. Mycobacterium tuberculosis (MTB) DNA in a single tube-single step reaction and correlated with laparoscopic findings.
Female genital tuberculosis (FGTB) is a symptomless disease that evidences itself only when it is investigated for infertility. Out of 85 cultures positive cases, 81 (26.82%) FGTB cases were found AFB positive by Ziehl-Neelson’s (Z-N) staining. Most STDs are treatable, some are even curable, but you have to get tested first before they cause something more serious. Other testing services provide the same tests, use the same labs, and offer the same turnaround, but often charge you $100 or more than what we charge. About My Health has locations across the country, so testing is always close and easier than ever. For routine immune status screening for exposure, order HSVGB for HSV 1 and 2 IgG antibodies. Demonstration of the etiologic agent by H & E staining or Z-N staining for acid fast bacilli, smear microscopy, culture of menstrual blood, urine and sputum were often unsuccessful.
Statistical AnalysisThe sensitivity, specificity, positive predictive values and negatives predictive values were calculated using standard formulae [29] for diagnostic accuracy.
Ethical ApprovalThe study protocol was in compliance with the Declaration of Helsinki, approved by the ethic committee of MHRT Hospital and Research Centre, Hyderabad in India. Materials and MethodsA prospective case-control study was set in the Zoology Modular Lab, CFRD, Osmania University, Hyderabad, India. Case GroupA total of 302 specimens were taken for study which included 202 infertile women highly suspected of having genital tuberculosis on laparoscopic examination and 100 control women (without TB) of reproductive age. Control GroupA total of 100 specimens were collected control women of reproductive age (18-40 years).
Processing of Tissue BiopsyThe specimens (like ETBs, OTBs and PAFs) collected from the lesions over the endometrium, ovaries and pelvic were mixed with sterile normal saline, transported in sterile vials to the laboratory and processed as per standard protocols [17, 18, 47, 48, 49, 50].
Decontamination and Concentration (DC)All specimens were decontaminated and concentrated by modified HS–SH procedure [51]. Histopathological ExaminationThin slices of the processed tissue biopsies and PAFs were placed onto the slides and kept for air drying at room temperature. Ziehl-Neelson’s (Z-N) Staining of Tissue Sediments for AFBFor about 50?l of the decontaminated and concentrated endo-ovarian tissue sediments were spread onto the slides, kept air drying for 10 minutes at 60°C and heat-fixed for 10 minutes at 90°C. Culture of Tissue SedimentsAbout 50?l inoculum of the decontaminated and concentrated endo-ovarian tissue biopsy sediments were taken and applied rapidly on L-J egg medium slants using sterile micropipette. Laparoscopy usually detects macroscopic changes such as peritubal adhesions, tubercles on the tubes and small tubo-ovarian masses that are commonly seen in chronic cases. All control women (n = 100) were found negative for tuberculosis with endometrium, ovaries and pelvics. Subsequently, 67 (33.17%) were positive for 19kDa antigen (131bp) gene and 32kDa protein (506bp) gene by multi-gene PCR. The STD and HIV test experts at About My Health aim to provide knowledgeable, compassionate customer service and the highest quality lab testing, in the fastest, most affordable way.
We therefore, proposed to use the endo-ovarian tissue biopsies and pelvic aspirated fluids for the detection of FGTB among infertile women by conventional versus genotypic methods. Comparison of quantitative variables (age, age at menarche age, body mass index and duration of infertility among infertile women highly suspected with FGTB and control women) was performed by means of ANOVA analysis with the F-test using SPSS v 20 for Windows (IBM® SPSS® Statistics 20, Chicago, USA). Informed consent was taken by our Institution through Ethical Committee because patient’s samples were obtained by Operative Laparoscopy. During the period of our study (2006–2014), the samples from infertile women visiting the gynaecology clinics at two collaborating centres in Hyderabad were analysed.
Statistical analysis of menstrual problems among infertile women highly suspected with FGTB and control women were described in Table 3.
Tissue specimens were fixed with buffered formalin (10%), cleaned with xylene and dehydrated with absolute alcohol (100%).
The slides were then stained with carbolfuchsin, decolorized by acid alcohol and counterstained by methylene blue and rinsed with water to remove excess methylene blue. Slant and bottled culture media were incubated in a horizontal plane until the inoculum was absorbed.
However, one should use varied combinations of tests to overcome the limitations such as the false positivity by way of contaminations, false negativity and dead bacilli or past infection or asymptomatic TB at another site. FGTB also presents distinctive diagnostic challenges including subtle clinical manifestations that were over looked in laparoscopy during early stages of infection [8].
Disparity among detection of FGTB by Z-N staining for AFB positive, histopathological evidence of tuberculosis infection, isolation by culture, and detection of M.


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This is the most common form of extrapulmonary tuberculosis (TB), accounting for about 27% (range, 14 to 41%) worldwide [6]. All patients were negative for chest X-ray and 42 (20.79%) patients were positive for Mantoux test.
Women who attended the same clinic for other gyaecological disorders, tubal sterilization and laparoscopy for menorrhagia were selected as controls.
The culture tubes were then incubated at 37°C for 6-8 weeks until heavy growth was obtained. Reagent preparation, DNA extraction, DNA amplification and detection were performed in separate rooms to avoid cross-contamination of amplicons. Even technical considerations, such as the use of suitable controls, standard strains and the retesting of doubtful positive samples, considerably influence the sensitivity and specificity.
11 (5.44%) samples were positive for 19kDa antigen (131bp) gene and 32kDa protein (506bp) gene by multi-gene PCR (Table 7). Genital and dermal swabs from lesions, ocular fluid, semen, and tissue specimens in viral transport media (Remel M4, M4RT, M5, M6, and BD Universal Transport Media).
A total of 302 specimens were collected from 202 infertile women highly suspected of having FGTB on laparoscopic examination and from 100 control women of reproductive age. It generates worrying effects by causing irreversible damage to the fallopian tube consequential in infertility which is hard to treat both by medical and surgical methods [15, 16]. Comparison among qualitative variables (menstrual irregularities, laparoscopic findings, types of biopsies and findings of conventional versus molecular methods among infertile women highly suspected with FGTB and control women) was performed by Pearson Chi-square test or Fisher’s exact test or McNemar’s test, as needed.
18-40 years of age having irregular periods with laparoscopic finding indicating beaded, blocked and tubal adhesions, infertility, pelvic pain, scanty menstruation, amenorrhoea and frozen pelvic and histopathological evidence in biopsy of premenstrual endometrial tissues or demonstration of tubercle bacilli in culture of menstrual blood or pelvic aspirated fluids or endometrial curetting.
Each specimen (from TBS) was divided into two portions-one kept for decontamination and concentration for subsequent studies such as Z-N staining, culturing on L-J media and extraction of DNA for multi-gene PCR method, and the other portion kept was used for histopathology, RNA and protein extraction. Then, the slides were stained with Weigert’s iron hematoxylin, washed with water, differentiated with acid alcohol (1%) and alcohol (95%) and washed with water. A positive control was included in each test and distilled water was included as a negative test control. Therefore, this investigation has been conducted meticulously with appropriate controls, replications, large sample size and with different combination of experiments. Amongst, the 202 specimens collected from highly suspected cases of FGTB, 175 (86.63%) specimens were positive for 19kDa antigen (131bp) gene (MTB species-specific gene) and TRC4 (173bp) repetitive element (south Indian MTB complex specific).
Your results will not be added to your permanent medical record without your express permission. In 80-90% cases, it affects women with menstrual irregularities accounting for about 27% of manifestations of FGTB [11], even this rate can be higher among patients with tubal factor infertility (39- 41%) [12]. It is estimated that at least 11% of the patients lack symptoms and FGTB is often detected in diagnostic workup of women attending infertility clinics [17].
All the patients were asymptomatic, resistant to all therapeutic treatments other than anti TB therapy. Menstrual irregularities such as oligomenorrhoea (3%) and mild menorrhagia (7%) were seen in the control women. The mixture was mixed for 5 seconds and centrifuged at 12,000 rpm for 15 min at 4°C using aerosol proof shields. Followed by, the slides were counterstained with eosin [30, 31, 32], dehydrated with increasing gradients of alcohol, cleaned with xylene and mounted.
The Clinical and Laboratory Standards Institute (CLSI, 2001 [36]) recommendations and regulations were followed for quality control and standards.
The PCR amplification was done using master cycler gradient PCR system (Eppendorf, Hamburg, Germany). The present study assesses the role of multi-gene PCR in the diagnosis of FGTB using ETBs, OTBs and PAF samples and correlated with laparoscopic findings.
The difference was due to over representations of clinical symptoms in infertile women with FGTB (Pearson ?2 = 127.492, 1df, McNemar’s test value = 0. Thangappah et al, revealed that 57% of infertile women in whom the presence of TB was suspected on clinical grounds had a positive endo-TB-PCR test, whereas only 9.5% had a positive test with no clinical ground for suspicion [18]. Information on the general, obstetric and gynaecological details including family history, marital status, age at menarche, length of menstrual cycle, associated symptoms, duration and amount of blood loss, duration of infertility, and socio demographic details like social status, occupation, lifestyle, age, body mass index (BMI), limited information on diet were obtained.
The supernatant was discarded into a splash-proof container with a tuberculocidal solution and the pellet was resuspended in 200µl of sterile TBS buffer and mixed for 5 seconds. Mounted slides were viewed under bright field (40x), Inverted Biological Microscope (BLM-290, BestScope, China). Contaminations were removed by subsequent washing with two different buffers according to manufacturer instructions.


Primer selection of present study was done based on local characteristics of strains and their importance in the diagnosis of the disease (tabulated in Table 4). Therefore, a high degree of suspicion assisted by intensive investigation is significant in the diagnosis of the disease.
Apart from routine examinations, laparoscopy and hysteroscopy was performed for infertility workup as and when needed. All patients were fertile, negative for chest X-ray and laparoscopically confirmed to be without FGTB.
Homogenised tissue sediments were used for culturing on L-J medium, Z-N staining and for the isolation of mycobacterial DNA.
The presence of caseating granulomas surrounded by epitheloid cells, malignant lymphocytic infiltrations, beaded plasma cells and giant polymorphornuclear cells were diagnostic of FGTB (Figure 1).
Pure genomic DNA was finally eluted under low ionic strength conditions in a slightly alkaline elution buffer. The nucleotide sequences of primers used for the detection of Mycobacterium in this study were described and validated as diagnostic markers in the past [17,18,29,37-46].
All subjects were HIV negative and negative for pulmonary TB on the basis of complete history, physical examinations; chest X-ray, lung plain X-ray and by appropriate tests such as tuberculin test [19]. The purity of DNA was checked on 0.8% agarose gel electrophoresis, incorporated with ethidium bromide. The bands in the gel were photographed under a Gel Doc-XRT with image lab software (Molecular Image, Bio-Rad, Hercules, CA, United States of America).
A standard protocol of investigations revealed a number of causes for fertility deprivation.
A positive chest X-ray for healed or active pulmonary tuberculosis, contact history, elevated erythrocyte sedimentation rate (ESR), positive tuberculin test and sampling by laparoscopy may specify the need for further investigations [19, 20, 21]. Details of hystero-laparoscopy findings like unilateral or bilateral tubal block with hydrosalphinx, omental adhesions, frozen pelvics, tubo-ovarian masses, tubercular salphingitis, beaded tubes and tubercles were noted. The bands in the gel were photographed under a Bio-Rad gel documentation system and quantified. Studies such as serial sections of tissues need to be studied, because the lesions are frequently erratic and that a positive endometrial culture for TB, is found only in about 25% of cases of tuberculous endometritis as the endometrium is often focal and the functionalis layer is shed every four weeks (granulomas take two weeks to develop) [22, 23]. Beaded appearance of tubes, frank tubercules on uterus and pelvic mass in variable combination aroused a suspicion. Moreover, due to the cyclical shedding of the endometrium, granulomas do not have enough time to form, so the granulomatous endometrium may not show evidence of tuberculosis in all the cycles. Constitutional symptoms such as sweating, increase in temperature and weight loss were not major complaints while local organ dysfunction manifested in amenorrhea, omental adhesions and bilateral tubal blockage are seen on hysterosalpingographic study.
Oophoritis is an inflammation of the ovaries, often seen in combination with salpingitis (inflammation of the fallopian tubes).
It is a relatively rare condition, usually follows haematogenous spread and causes infertility.
Since, there is no way to take the fallopian tubes out, sampling from the ovaries and endometrium was suggested for the detection of FGTB [24]. Use of menstrual blood for bacteriologic or molecular diagnosis has been recommended [25] but was reported to show low sensitivity [26]. Sampled sites may not represent the infected area and the infected site can be simply missed due to sparse number of paucibacillary nature of mycobacteria [18].
In addition, tissue reaction in those having tuberculosis may at times be atypical and bacteriologically mute [17]. These methods have poor sensitivity and specificity for the diagnosis of FGTB throughout the sub-clinical stages. On the other side, a range of PCR techniques have been mechanized for the detection of specific nucleic acid sequences of MTB and other mycobacteria [27]. Identi?cation of genes encoding the virulence determinants, available targets in genome and highly expressing factors are current important markers in the detection of FGTB.
Biological, molecular and immunological studies have resulted in identification of more than 33 different useful proteins, some of which are specific to MTB or MTB complex [28]. Out of which, four genes has been selected for our study considering their importance in the analysis of ethiopathogenesis and diagnosis of FGTB.



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