Tuberculous endometritis: its association with infertility and other ACCEPTED

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1 JCM Accepts, published online ahead of print on 8 October 2008 J. Clin. Microbiol. doi: /jcm Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Tuberculous endometritis: its association with infertility and other gynaecological complaints in Indian women P. Kumar 1, N.P. Shah 1 A. Singhal* 1, D.S Chauhan 4, V.M.Katoch 4, S.Mittal 2, S. Kumar 2, M.K. Singh 3, S. Datta Gupta 3 and H.K. Prasad 1 1 Departments of Biotechnology, 2 Obstetrics and Gynaecology, 3 Pathology, All India Institute of Medical Sciences, New Delhi , India. 4 National JALMA Institute of Leprosy and other Mycobacterial Diseases Tajganj, Agra , *Present address: Scientist, Lab. of Molecular Pathology of Tuberculosis, Pasteur Institute 642 rue Engeland Brussels B-1180 Belgium Author for correspondence: H. Krishna Prasad, Tel: ; Fax: , ; hk_prasad@hotmail.com Key Words: Female Infertility, M.tb., M.bo., nested PCR, hupb Running Title: Detection of M.tb. and M.bo. in endometrial biopsies 1

2 Abstract Endometrial biopsies derived from 393 patients with assorted gynecological complaints were investigated for mycobacterial infection. Employing 4 different techniques mycobacterial pathogens were detected irrespective of the nature / type of clinical complaint. Mycobacterium tuberculosis was the predominant pathogen detected among the samples investigated. 2

3 Tuberculosis occurs worldwide and causes widespread morbidity and mortality. Pulmonary and extra-pulmonary sites are known to be associated with Mycobacterium tuberculosis (M.tb.) infection. In fact it is well known that pulmonary tuberculosis patients go on to develop extra-pulmonary tuberculosis. One such manifestation has been the occurrence of female genital tuberculosis (FGTB). The spread of the pathogen to fallopian tubes, endometrium and ovaries, leading to a variety of clinical conditions has been described (1, 8, 15). The present study was undertaken to detect mycobacterial infection in endometrial biopsies (EB) collected from patients registered in the gynaecological OPD. 393 patients attending the Obstetrics and Gynaecology OPD of AIIMS, New Delhi were included in the study. 285 of them were infertility patients, 80 had menstrual dysfunction complaints, 17 had chronic lower abdominal or pelvic pain & the remaining 11 were patients with ovarian cyst, fibroid, prolapse uterus, post recanalization etc. EB were processed as described by Chakravorty and Tyagi (3). Four methods were used to detect mycobacteria in the EB. The processed EB extracts were microscopically examined for AFB, isolation of mycobacteria by inoculation on Lowenstein Jensen media and processed for extraction of target DNA for N-PCR, (12). Culture results at the time of writing were available for 262 samples. 295 EB were processed for histopathological examination by H&E staining. The N-PCR for hupb DNA target was carried out as described previously (10). The N-PCR products were electrophoresed on 10% polyacrylamide gel and stained with ethidium bromide. The amplicon size for M.tb. and M. bovis (M.bo.) were ~116 bp and 89 bp respectively. Species level identification 3

4 of isolates obtained was done by spoligotyping (9) & by standard biochemical tests (16). Randomly selected EB samples showing dual bands (116 and 89 bp) were cloned into the pgemt vector using TA cloning kit (Promega, USA). The clones were sequenced at the DNA sequencing facility, South Campus Delhi University, New Delhi, India. The detection and identification of M.tb. and M.bo. in representative EB specimens have been depicted in the Figure. N-PCR amplified products equivalent to 116 bp were obtained in 5 of the 7 samples (Lanes 1-3, 7-8). These samples were considered to be infected with M.tb. A representative sample depicting mixed infection with M.tb. and M.bo. has been shown in lane 14 (Fig.). Samples with dual bands were eluted and sequenced. The sequence of the dual bands corresponding to 116 and 89 bp matched with that of the C-terminal part of the hupb gene of M.tb. and M.bo. respectively as described previously (14). In EB investigated differences in the detection of AFB / histopathological evidence of tuberculosis infection / isolation by culture and detection of M.tb. & / M.bo. by N-PCR was observed, (Table). Of the 393 EB collected, AFB was detected in twenty extracts (20 / 393, 5.1%) they included patients with chronic lower abdominal or pelvic pain (2/17, 11.8%) infertility (16 / 285, 5.6%) & menstrual dysfunction complaints (2 / 80, 2.5%). Granulomatous tissue reaction compatible with tuberculosis was observed exclusively in seven EB (7 / 220, 3.2%) derived from infertile patients. Mycobacteria were isolated from eleven samples by culture (11/262, 4.2%). Nine strains were lost on 4

5 subculture. Eight of these isolates were derived from patients with complaints of infertility (8/174, 4.6%), two were from patients with complaints of menstrual dysfunction (2/71, 2.8%) & one isolate was obtained from (1/11, 9.0%) a patient with ovarian cyst. Ten isolates have been identified as M.tb. by spoligotyping and standard biochemical criteria. One isolate was characterized as M. chelonae by biochemical criteria. However in comparison to isolation by culture, M.tb. / M.bo. / mixed infection was detected in 123 (123 / 393, 31.3%) samples by N-PCR. Of these 109 were associated with M.tb. (27.7%) and 31 (7.8%) with M.bo. infection. One hundred & eleven of these EB extracts (111 / 285, 39%) were from infertile patients, nine were from (9 / 80, 11.3%) patients with complaints of menstrual dysfunction, one was from a patient with pain in the lower abdomen (1 / 17, 5.9%) the remaining two were from (2 / 11, 18.2%) patients with complaints of post recanalization & fibroid (miscellaneous category, Table). The comparative % of sensitivity of detection of mycobacteria by the 4 methods showed that N-PCR assay has the highest sensitivity of 31.3%. AFB detection by microscopy showed a sensitivity of 5.1% and isolation of these pathogens by the culture technique (2.4%). was found to be 4.2%. However, the least sensitive technique was the histopathological examination for granulomatous tissue reaction compatible with tuberculosis infection 5

6 Comparing the various clinical conditions with the techniques used in the study for detection of mycobacteria in the samples, mycobacteria were detected by all the methods used in infertility cases. In cases of patients with menstrual dysfunction mycobacteria could be detected only by 3 methods, namely AFB microscopy, isolation by culture and N-PCR. In these individuals no evidence of granulamtous reaction of an ongoing mycobacterial infection was detected. Taking into consideration that isolation of mycobacteria is the gold standard for diagnosis of tuberculosis, eight isolates were obtained from infertility cases compared to 2 from patients with menstrual disorder and one from other conditions. Similarly comparing the N-PCR results, all categories of patients were positive. The highest percentage of positivity was in infertility cases, (39 %, 111 / 285). These results show that infertility with mycobacterial infection is a significant clinical problem in India. The prevalence of FGTB in infertility clinics has been reported to range from 1-19% (2, 11, 13). Beside M.tb., M.bo. infection has been reported in FGTB (4, 7).The failure to isolate M.bo. in the present study may be due to use of inappropriate media, (5,6). Non-specific clinical presentation; inefficacy of laboratory diagnostic tests, accessibility of reproductive clinics, has resulted in under reporting of FGTB. Hence patients with complaints of infertility and other gynaecological complaints must necessarily be investigated for tuberculosis of the genital tract. The N- PCR, histopathology and culture results confirm that infertility is a common clinical condition associated with FGTB. 6

7 Senior Research Fellowships to PK and AS from Indian Council of Medical Research and University Grants Commission are acknowledged. The financial support of DBT, Government of India is acknowledged. Mycobacterium tuberculosis H37Rv DNA was obtained from TB research material, NIAD, NIH, USA. 7

8 References: 1. Abebe, A., M. Lakew, D. Kidane, Z. Lakew, K. Kiros, and M. Harboe Female genital tuberculosis in Ethiopia. Int. J. Gynecol. Obstet. 84: Bhanu, N.V., U.B. Singh, M. Charaborty, N.Suresh, J.Arora, T.Ranu, D.Takkar, and P.Seth Improved diagnostic value of PCR in the diagnosis of female genital tuberculosis leading to infertility. J. of Med. Microbiol. 54: Chakravorty S., M. Dudeja, M. Hanif and J.S Tyagi Utility of universal sample processing methodology, combining smear microscopy, culture, and PCR, for diagnosis of pulmonary tuberculosis. J. Clin. Microbiol. 43: Chowdhury, N.N.R Overview of tuberculosis of the female genital tract. J. Ind. Med. Assoc. 94: Corner, L.A., C.Nicolacopoulos Comparison of media used for the primary isolation of Mycobacterium bovis by veterinary and medical diagnostic laboratories. Aust Vet J.65(7): Cousins, D.V., B.R. Francis, and B.L.Gow Advantages of a new agar medium in the primary isolation of Mycobacterium bovis. Vet Microbiol. 20(1): Gatongi D.K., G. Gitau, V. Kay, S. Ngwenya, C. Lafong, and A. Hasan Female genital tuberculosis. The Obstetrician & Gynaecologist. 7: Gupta N., J.B.Sharma, S. Mittal, N. Singh, R. Mishra, and M. Kukreja Genital tuberculosis in Indian infertility patients. Int. J. Gynaecol. Obstet. 97: Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, and S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, J. van Embden.1997.Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.j. Clin. Microbiol. 35: Mishra A., A. Singhal, D.S.Chauhan, V.M.Katoch, K.Srivasatava, S.S.Thakral, S.S.Bharadwaj, V. Sreenivas and H.K.Prasad Direct detection and identification of Mycobacterium tuberculosis and Mycobacterium bovis in bovine samples by a novel nested PCR assay: correlation with conventional techniques. J. Clin. Microbiol. 43:

9 11.Parikh F.R., N.Naik, S.G.Nadkarni, S.B Soonawala, S.A Kamat, and R.M.Parikh.1997.Genital tuberculosis a major pelvic factor causing infertility in Indian woman. Fertil. Steril. 67: Prabhakar S, A. Mishra, A. Singhal, V.M.Katoch, S.S.Thakral, J.S.Tyagi, and H.K.Prasad.2004.Use of the hupb gene encoding a Histone-like protein of Mycobacterium tuberculosis as a target for detection & differentiation of Mycobacterium tuberculosis & M. bovis. 42(6): Roy A., S. Mukherjee, S. Bhattacharya, S. Adhya, and P. Chakraborty Tuberculous endometritis in hills of Darjeeling: a clinicopathological and bacteriological study. Indian J. Pathol. Microbiol. 36(4): Shah, NP., A. Singhal, A. Jain, P. Kumar, S. S. Uppal, M. V. P. Srivatsava, and H.K.Prasad Occurrence of Overlooked Zoonotic Tuberculosis: Detection of Mycobacterium bovis in Human Cerebrospinal Fluid. J. Clin. Microbiol. 44(4): Verma TR Genital tuberculosis and subsequent fertility. Int. J. Gynecol. Obstet.35: Vestal, A.L Procedures of isolation and identification of mycobacteria. U.S. Department of Health, Education, and Welfare publication no. (CDC) , p Centers for Disease Control, Atlanta, Ga. 9

10 TABLE: Comparative analysis of smear microscopy for AFB, histopathology, Culture & N-PCR, of 393 patients investigated with various complaints. Clinical category a AFB b Histopathology c Culture d PCR e Infertility f (285) Menstrual dysfunction g (80) Chronic lower abdominal or pelvic pain h (17) Miscellaneous i (11) Total (393) a- Patients were categorised as complain of infertility, Menstrual dysfunction, Chronic lower abdominal or pelvic pain & miscellaneous etc b- Detection of AFB by Auramine O staining in biopsy extracts c- H&E stained EB sections examined for tissue reaction compatible with tuberculosis d- Growth on Lowenstein-Jensen with pyruvate on solid media, at 37 0 C e- 116 bp & 89 bp amplicon generated by N-PCR for M. tuberculosis & M. bovis respectively. f- EB from patients who have never been able to conceive a pregnancy after a minimum of 1 year of attempting to do so, through unprotected intercourse g- Menstrual dysfunction like Polymenorrhagia, Amenorrhea (Primary Amenorrhea, Secondary Amenorrhea), Oligomenorrhea, Menorrhagia, Irregular, post menstrual, continuous & Dysfunctional uterine Bleeding etc. h- Chronic lower abdominal or pelvic pain i- Miscellaneous complain like ovarian cyst, fibroid, prolapse uterus, endometosis, post Recanalization etc. 5.6 (16/285) 2.5 (2/80) 11.8 (2/17) * Results available at the time of analysis 3.2 (7/220)* 4.6 (8/174)* (2/71)* 38.9 (111/285) 11.3 (9/80) (1/17) (1/11) 5.1 (20/393) 2.4 (7/295)* 4.2 (11/262)* 18.1 (2/11) 31.3 (123/393) 10

11 Figure Legend: 100 bp 80 bp Fig. Nested PCR for detecting and differentiating Mtb and Mbo in Endometrial biopsy extracts. The ethidium bromide stained amplification products of Mtb and Mbo generated by using F and R primers were electrophoresed on nondenaturing 10% polyacrylamide gels. The 116 and 89 bp products obtained in Mtb and Mbo, respectively are indicated. Sample showing dual infection has been shown (Lane 14). Lane: 1, EB7; 2, EB17; 3, EB26; 4 & 13, Mtb positive control; 5 & 12, 100 bp molecular weight marker; 6 & 11, Mbo positive control, 7, EB40; 8, EB83; 9, EB55; 10, negative control; 14, EB bp Fig. 116 bp 89 bp 11