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Occurrence of female genital tuberculosis among infertile women: a study from a tertiary maternal health care research centre in South India V. Bhanothu, J. P. Theophilus, P. K. Reddy & R. Rozati

European Journal of Clinical Microbiology & Infectious Diseases ISSN 0934-9723 Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-014-2164-1

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Author's personal copy Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-014-2164-1

ARTICLE

Occurrence of female genital tuberculosis among infertile women: a study from a tertiary maternal health care research centre in South India V. Bhanothu & J. P. Theophilus & P. K. Reddy & R. Rozati

Received: 17 March 2014 / Accepted: 14 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The purpose of this investigation was to estimate the proportion of female genital tuberculosis (FGTB) among infertile women, along with the mean cost of diagnosis by different methods. The study was carried out on 211,335 women, of which 31,755 (15.02 %) were infertile. 202 women were highly suspected of FGTB on laparoscopy, which was later ascertained by multi-gene polymerase chain reaction (PCR), and the cost was estimated. The majority of the patients were infertile (77.23 %), with menstrual disturbances (61.88 %). Many of them were having beaded tubes (68.81 %), tubal block with hydrosalpinx (58.91 %) and tubercular salpingitis (48.01 %). Out of 302 case-controls, 105 infertile women were positive by haematoxylin and eosin staining, 14.57 % were acid-fast bacilli-positive and 86 infertile women were positive on culture. 178 (58.94 %) endoovarian tissue biopsies and pelvic aspirated fluid specimens were positive for a 32-kDa protein gene as amplified using multi-gene PCR. The proportion of proven FGTB cases was very high (58.94 %) among infertile women highly suspected of FGTB. The estimated cost in rupees (Rs) of FGTB diagnosis by the conventional method ranges from Rs 3.36 to 38.11, while multi-gene PCR was established as being very expensive (Rs 254.21). The expected time of FGTB diagnosis by the conventional method ranges from 0.5 to 1.83 h, whereas culture took 4–8 weeks. The logical time of FGTB diagnosis by multi-gene PCR was V. Bhanothu (*) : J. P. Theophilus Department of Zoology, UCS, Osmania University, Hyderabad 500007, AP, India e-mail: [email protected] P. K. Reddy Department of Tribal Welfare, Andhra Pradesh, India R. Rozati MHRT Hospital & Research Centre, Hyderabad, AP, India

6.48 h. Compared to Ziehl–Neelsen’s staining and culturing, multi-gene PCR improved the detection rate of suspected FGTB. Therefore, FGTB can be diagnosed if multi-gene PCR is considered in the evaluation of infertile patients in areas where tuberculosis is endemic.

Introduction Female genital tuberculosis (FGTB) is an uncommon form of extra-pulmonary tuberculosis, of which infertility is the most common symptom [1, 2]. Genital tuberculosis (GTB) represents 15–20 % of extra-pulmonary tuberculosis [3] and remains a major health problem in developing countries [4]. The incidence of infertility in GTB worldwide varies from 10 to 85 % [5, 6]; it is endemic in India, with an incidence of 58 % [7] and the majority of patients are in the same reproductive age group (15–45 years) [8]. Mycobacterium tuberculosis (MTB) is the most common cause of human tuberculosis (TB), accounting for more than 99 % of the cases [9], with the others being caused by M. bovis mainly [10]. In 80–90 % cases, it affects women with menstrual irregularities, accounting for about 27 % of manifestations of FGTB [11]; this rate is observed to 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 the diagnostic workup of women attending infertility clinics [13]. 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 polymerase chain reaction (PCR) test, whereas only 9.5 % had a positive test with no clinical grounds for suspicion [14]. The actual incidence may be under-reported due to asymptomatic, varied clinical presentations, diverse imaging, transforming laparoscopic results and a mixed bag of bacteriological and serological tests [5, 15]. Though absolute diagnosis cannot be made from characteristic

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features in hysterosalpingogram (HSG) or laparoscopy, laparoscopy is a valuable procedure for obtaining tissue for culture, identification of acid-fast bacilli (AFB) in smears stained by the Ziehl–Neelsen (Z–N) technique and histopathological examination [16–19]. On the other hand, these methods have poor sensitivity and specificity for the diagnosis of FGTB throughout the sub-clinical stages, due to the lack of a gold standard method. With the overall increasing time trend of extra-pulmonary TB worldwide, the occurrence of FGTB becomes a significant topic in many countries. In consequence, a range of PCR techniques have been mechanised for the detection of specific nucleic acid sequences of MTB and other mycobacteria [20]. Unfortunately, there are insufficient studies to assess the costto-benefit ratio of conventional and molecular methods in the detection of FGTB. Therefore, the present study was proposed to expand our previous research work [21], by taking all possible quality control measures into consideration. Further, we attempted to report the proportion of FGTB among infertile women with diverse clinical presentations, and later compared the costs associated with different methods used for the diagnosis of FGTB. Statistical analysis The sensitivity, specificity, positive predictive value and negatives predictive value were calculated using standard formulae [22] for diagnostic accuracy. The association between various quantitative variables (age, age at menarche, body mass index and duration of infertility among infertile women highly suspected of FGTB and control women) was analysed by using SPSS v20 for Windows (IBM® SPSS® Statistics 20, Chicago, IL, USA). The data were presented as the mean ± standard deviation (SD). Association among qualitative variables (findings of conventional and molecular methods among infertile women highly suspected of FGTB and control women) were statistically analysed by Pearson’s Chi-square test or Fisher’s exact test or McNemar’s test, as needed. Significant differences in the positive rates of the different methods were analysed. Data were considered statistically significant if the p-value was less than 0.05.

Materials and methods Ethical statement The study protocol was in compliance with the Declaration of Helsinki, approved by the ethics committee of MHRT Hospital & Research Centre, Hyderabad, India. Informed consent from the patients were obtained by our institution through the

ethical committee, since patients’ samples were obtained by operative laparoscopy. Study design A prospective case–control study was set in the Zoology Modular Lab, CFRD, Osmania University, Hyderabad, India. During the period of our study (September 2006–February 2014), the samples from infertile women visiting the gynaecology clinics at two collaborating maternal health care centres in Hyderabad were analysed. All patients met the definitive/confirmed inclusion criterion for selecting FGTBsuspected cases showing clinical symptoms such as infertility among the reproductive age group (18–40 years), pelvic pain, scanty menstruation with irregular periods, dysmenorrhoea, oligomenorrhoea, amenorrhoea, general malaise and menorrhagia leading to the abortions. Radiological findings may or may not be indicative of TB and laparoscopic findings indicated caseation, granuloma/tubercles and/or beaded/thickened tubes, hydrosalpinx, peritubal/periovarian adhesions and tubo-ovarian mass without frank tubercles. Histopathological findings indicated chronic inflammation or lesions such as proliferative solid epithelioid granulomas, dense polymorphonuclear cells, lymphocytic infiltrations, giant or beaded cells, enlarged lymphoid cells and accumulation of plasma and spindle cells. Demonstration of tubercle bacilli in culture, Z– N staining of menstrual blood fluids, pelvic aspirated fluids (PAFs), endometrial curetting, endometrial tissue biopsies (ETBs) and ovarian tissue biopsies (OTBs) are indicative of TB. Exclusion criteria were as follows: women above 40 years of age, symptoms suggesting pulmonary TB/extra-pulmonary TB except infertility, with normal abdominal and vaginal examinations, pregnant and nursing women, women with autoimmune disorders, pulmonary infections, human immunodeficiency virus (HIV) co-infection, women with diabetes, malnutrition and other medical disorders like hypertension, peritoneal adhesions due to previous abdominal surgery, infertility due to male factors and abnormality in ovulations. 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 sociodemographic details like social status, occupation, lifestyle, age, body mass index (BMI), limited information on dietary and nutritional factors were obtained. All subjects were HIVnegative and negative for pulmonary TB on the basis of complete history and physical examinations (chest X-ray, lung plain X-ray and by appropriate tests, such as a tuberculin skin test) [23]. Apart from routine examinations, laparoscopy and hysteroscopy were performed at infertility workup as and when needed. Details of hystero-laparoscopy findings were noted. Beaded appearance of tubes, frank tubercles on the uterus and pelvic mass in variable combination aroused a

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suspicion. Constitutional symptoms such as sweating, increase in temperature and weight loss were not major complaints, while local organ dysfunction manifested in amenorrhoea, omental adhesions and bilateral tubal blockage are seen on hysterosalpingographic study. All samples collected were examined by haematoxylin and eosin (H&E) staining [24], Z–N staining for AFB [25], as well as culture on Löwenstein–Jensen (L–J) egg media [26] and the MTBspecific multi-gene/multi-primer PCR method [21] by which FGTB was confirmed. Primer selections for the multi-gene PCR study were done based on local characteristics of the strains and their importance in the diagnosis of the disease. The nucleotide sequences of primers used for the detection of Mycobacterium in this study were described and validated as diagnostic markers in the past [21]. MTB (ATCC 35836) reference stain isolates provided by the Department of Microbiology, Nizam’s Institute of Medical Sciences (NIMS), Hyderabad, India, were used as controls in each assay. The diagnosis was made based on morphological [23] and molecular investigations.

Fig. 1 Histopathological examination of endo-ovarian tissue biopsy: a polymorphonuclear cells were observed in endometrial tissue biopsies; b beaded lymphocytic infiltrations and initial stages of granulomatous were observed

pale blue background was noted as Mycobacterium [21, 25, 26] (shown in Fig. 2). Culture of tissue sediments

Processing of tissue biopsy The specimens (such as ETBs, OTBs and PAFs) collected from the lesions over the endometrium, ovaries and pelvis were mixed with sterile normal saline, transported in sterile vials to the laboratory and processed as per standard protocols [13, 14, 21]. Decontamination and concentration (D&C) All specimens were decontaminated and concentrated by a modified hypertonic saline–sodium hydroxide (HS–SH) procedure [21, 27]. Histopathological examination Thin slices of the processed tissue biopsies and PAFs were placed onto the slides and kept for air drying at room temperature. Specimens were fixed, dehydrated and stained with Weigert’s iron H&E [21, 24, 25]. Then, the slides were cleaned and mounted. Mounted slides were viewed under a bright field (40×), Inverted Biological Microscope (BLM290, BestScope, China). The presence of caseating granulomas surrounded by epithelioid cells, malignant lymphocytic infiltrations, plasma cells and giant polymorphonuclear cells were diagnostic of FGTB (Fig. 1). Z–N staining of tissue sediments for AFB Stained slides were viewed under an Inverted Biological Microscope. The portion of smear that stained pink/red on a

Inoculated culture tubes were incubated at 37 °C for 6– 8 weeks until heavy growth was obtained [21]. Cultures were stained by Z–N staining for confirmation of AFB growth [26]. “Standard Precautions” [28] and institutional guidelines have been followed in handling all items contaminated with blood and other body fluids. Quality control A positive control was included in each test and distilled water was included as a negative test control. All these investigations have been conducted meticulously with appropriate controls, replications, large sample size and with different combinations of parameters and experiments. The recommendations and regulations of the Clinical and Laboratory Standards Institute (CLSI/NCCLS, 2001 [28]) were followed for quality control and standards. DNA extraction and purification Pure genomic DNA was extracted by using the DNASure Tissue mini kit from Genetix Biotech Asia Pvt. Ltd., New Delhi, India. The protocol was modified according to the manufacturer’s instructions [21, 29]. The purity of DNA was checked on 0.8 % agarose (Amresco, Amresco LLC, USA) gel electrophoresis, incorporated with ethidium bromide. The electrophoresis was carried out with 1X tris-acetic acid-EDTA buffer at a constant voltage (110 V) for one hour. The bands in the gel were visualized and quantified under ultraviolet illumination (Gel Doc-XRT system, Bio-Rad, Hercules, CA, United States of America).

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Fig. 2 Ziehl–Neelsen (Z–N) staining of endo-ovarian tissue biopsy and cultures in the detection of acid-fast bacilli (AFB): a red/pink colour rodlike beaded structures were observed in the tissue biopsy; b red/pink colour rod-like structures were observed on a pale blue background in the cultures

Multi-gene/multi-primer PCR method [21] Two looped touchdown multi-gene PCR programmes, each with 25 cycles, was followed. In the first loop, the template DNA was initially denatured at 95ºC for 5 min and then denatured at 94ºC for 45 s, annealed at 68ºC for 45 s, extended at 72ºC for 45 s and continued for a total of 25 cycles. In the second loop, DNA was denatured at 94ºC for 45 s, annealed at 58ºC for 45 s, extended at 72ºC for 45 s and continued for a total of 25 cycles with a final extension at 72ºC for 15 min. The PCR amplification was done using the Mastercycler gradient PCR system (Eppendorf, Hamburg, Germany). PCR products were subjected to electrophoresis on a 2.5 % agarose gel incorporated with ethidium bromide, along with Gene Rule 50 bp DNA ladder/molecular weight marker. The electrophoresis was carried out with 1X tris-acetic acid-EDTA buffer, at a constant voltage (110 V) for one hour. The bands in the gel were visualized under ultraviolet illumination (Gel Doc-XRT with molecular image lab software, Bio-Rad, Hercules, CA, United States of America). As, we found clear and accurate banding patterns by agarose gel electrophoresis, sequencing of the PCR product was not suggested. The results of the case–control groups were compared with the reference strain (shown in Fig. 3). Estimation of costs All the patients admitted during the study period were diagnosed by physical examinations, HIV test, chest X-ray, lung plain X-ray, tuberculin skin test, hystero-laparoscopy, conventional methods and MTB-specific multi-gene PCR method, by which FGTB was confirmed [21]. In this context, we intended to estimate the cost of diagnostic laboratory tests and time associated with different methods. According to our understanding, the cost of diagnostic laboratory tests includes the cost of the laboratory building, instrumentation, manpower,

furniture, chemicals and consumables, kits and reagents, glasswares and miscellaneous supplies, pipettes and dispensers, overheads and stationary. Of the different methods of diagnosis, the cost of the HIV test, cost of chest X-ray and lung plain X-ray, cost of hystero-laparoscopy, cost of tuberculin skin test and physician fee (100 %) were borne by the patients themselves. The estimated costs of the laboratory building, instrumentation, manpower, vehicles, furniture, pipettes, dispensers, transportations, overheads and stationary were excluded because of the difficulty in estimating these kinds of costs, due to collaborations. At present, we estimated the direct cost of laboratory investigations including costs of chemicals, consumables, kits, reagents, glasswares and miscellaneous supplies required by different methods. The mean cost of each test per patient was calculated by taking the total cost of the test divided by the total number of patients. The time taken for reporting of the initial sample was calculated and considered as the time spent for each test.

Results The study was carried out on 211,335 women examined for different problems over a period of eight years; 31,755 (15.02 %) women were found to be infertile. A standard protocol of investigations revealed a number of causes for fertility deprivation, of which 12,384 (39 %) patients had a lower socioeconomic status, 65 % were from urban areas and 46 % patients were below undergraduate level of education (Fig. 4). On diagnostic laparoscopy, 9,272 (29.2 %) were found to have endometriosis, 5,713 (17.99 %) had polycystic ovaries (PCOs), 4,919 (15.49 %) were struggling with ovulatory failure, 1,810 (5.7 %) had sexual dysfunction, 1,406 (4.43 %) had idiopathic infertility and there were 1,363 (4.3 %) infertile women who fulfilled the eligibility criteria by which diagnostic laparoscopy was carried out based on subtle physico-clinicopathological manifestations excluding the symptoms of PCOs and endometriosis [30, 31] and by which the diagnostic criteria were derived to suspect FGTB (Fig. 5). Physico-clinicopathological symptoms found during our investigation were mild and local, such as infertility, duration of infertility, abdominal pain and menstrual irregularities, while laparoscopy generally detects most common macroscopic changes, such as tubal blockage, beaded tubes, tubercular salpingitis, tubercles on tubes and omental adhesions that were seen in chronic cases. Further, a total of 302 specimens were taken for study, which included samples from 202 infertile women highly suspected of having GTB on laparoscopic examination and samples from 100 control women (without TB) of reproductive age. Out of 202 FGTB suspected patients, 42 (20.79 %) were positive for the Mantoux test. All of them were negative for chest X-ray and the erythrocyte sedimentation rate (ESR)

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Fig. 3 2.5 % agarose gel electrophoresis was carried out with multi-gene/ multi-primer polymerase chain reaction (PCR) products. Lanes 65 to 72 were loaded with PCR products of female genital tuberculosis (FGTB) patients. Lanes C41 to C49 were loaded with PCR products of control patients. Lane +Ve Ctrl was loaded with positive reference strain (Mycobacterium tuberculosis, ATCC 35836). Lane –Ve Ctrl was loaded with negative control (H2O). Lane Mrkr was loaded with 50 base pair (bp)

molecular weight ladder (the 50-bp size of product starts from the bottom side of the gel and ends with 650 bp of product on the top/upper side of the gel). The band corresponding to 131 bp was noted as the 19-kDa antigen gene, 173 bp was noted as TRC4 element, 240 bp was noted as the MPB64 gene, 506 bp was noted as the 32-kDa protein/MPT59 αantigen gene. Primer dimers were also noted at the bottom during the end of the sample run

was elevated among these patients. The mean age of these subjects was 28.54 ± 4.46 years, the mean duration of

infertility was 3.92±3.03 years and the mean BMI was 24.36±1.47. Statistical association was observed in relation No. of cases (n=31755)

Percentage (%)

20641

14607 12384

11750

11114 9844 7304

Resident

Occupaons

9

Educaon Qualificaon

6352 Above PGs

46

PG any branches

37

UG any branches

23

2858

10th -12th standard

39

Government employees

31

House holders with business

7 Agriculture

Living in Rural

35

Soware employees

65

1905 6 Below 10th standard

2223

Living in urban

Fig. 4 Social status of the infertile population

Author's personal copy Eur J Clin Microbiol Infect Dis Fig. 5 Varied clinical presentations of the infertile population in South India. Note that some patients had symptoms pertaining to two or more systems

No. of cases (n=31755) Tubo-ovarian masses

160

Tubercular salphingis

294

Tubal block with Hydrosalphinx

386

Beaded Tubes

523 5713

Polycysc Ovary Syndrome (PCOS)

9272

Endometriosis Pelvic inflammatory disease Idiopathic inferlity Sexual dysfuncon Ovulatory failure Male factor

with different parameters among case–control groups (F-test = 151.653, 1 df, p