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Journal of Clinical Microbiology, December 2008, p. 4068-4070, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01162-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Association of Tuberculous Endometritis with Infertility and Other Gynecological Complaints of Women in India

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*

Departments of Biotechnology,¹ Obstetrics and Gynaecology,² Pathology, All India Institute of Medical Sciences, New Delhi 110029, India,³ National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Tajganj, Agra 282001, India⁴

Received 19 June 2008/ Accepted 30 September 2008

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Endometrial biopsy samples derived from 393 patients with assorted gynecological complaints were investigated for mycobacterial infection. By employment of four 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.

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Tuberculosis occurs worldwide and causes widespread morbidity and mortality. Pulmonary and extrapulmonary sites are known to be associated with Mycobacterium tuberculosis infection. In fact, it is well known that pulmonary tuberculosis patients go on to develop extrapulmonary tuberculosis. One such manifestation is the occurrence of female genital tuberculosis (FGTB). The spread of the pathogen to fallopian tubes, endometria, and ovaries, leading to a variety of clinical conditions, has been described previously (1, 8, 15). The present study was undertaken to detect mycobacterial infection in endometrial biopsy (EB) samples collected from patients registered in the gynecological outpatient department of the All India Institute of Medical Sciences, New Delhi, India.

Three hundred ninety-three patients attending the obstetrics and gynecology outpatient department of the All India Institute of Medical Sciences were included in the study. Of these, 285 were infertility patients, 80 had menstrual dysfunction complaints, 17 had chronic lower abdominal or pelvic pain, and the remaining 11 were patients with complaints such as ovarian cyst, fibroid, prolapsed uterus, and postrecanalization. The EB samples were processed as described by Chakravorty et al. (3). Four methods were used to detect mycobacteria in the EB samples. The processed EB extracts were microscopically examined for acid-fast bacilli (AFB), processed for isolation of mycobacteria by inoculation on Lowenstein-Jensen medium, and processed for extraction of target DNA by nested PCR (N-PCR) (12). Culture results at the time of this writing were available for 262 samples. Two hundred ninety-five EB samples were processed for histopathological examination by hematoxylin and eosin staining. The N-PCR for the 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 sizes for M. tuberculosis and Mycobacterium bovis were 116 bp and 89 bp, respectively. Species-level identification of the isolates obtained was done by spoligotyping (9) and by standard biochemical tests (16). Randomly selected EB samples showing dual bands (116 and 89 bp) were cloned into the pGEMT vector by using a TA cloning kit (Promega). The clones were sequenced at the DNA sequencing facility, South Campus Delhi University, New Delhi, India.

The detection and identification of M. tuberculosis and M. bovis in representative EB specimens are depicted in Fig. 1. N-PCR-amplified products equivalent to 116 bp were obtained for five of the seven samples (lanes 1 to 3, 7, and 8). These samples were considered to be infected with M. tuberculosis. A representative sample depicting mixed infection with M. tuberculosis and M. bovis is shown in Fig. 1, lane 14. Samples with dual bands were eluted and sequenced. The sequences of the dual bands corresponding to 116 and 89 bp matched those of the C-terminal parts of the hupB genes of M. tuberculosis and M. bovis, respectively, as described previously (14).

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FIG. 1. N-PCR for detecting and differentiating M. tuberculosis and M. bovis in EB extracts. The ethidium bromide-stained amplification products of M. tuberculosis and M. bovis generated by using forward and reverse primers were electrophoresed on nondenaturing 10% polyacrylamide gels. The 116- and 89-bp products obtained from M. tuberculosis and M. bovis, respectively, are indicated. A sample with dual infection is shown (lane 14). Lanes: 1, sample EB7; 2, EB17; 3, EB26; 4 and 13, M. tuberculosis-positive controls; 5 and 12, 100-bp molecular weight markers; 6 and 11, M. bovis-positive controls, 7, EB40; 8, EB83; 9, EB55; 10, negative control; 14, EB34.

Among EB samples, differences between results for detection of AFB, histopathological evidence of tuberculosis infection, isolation by culture, and detection of M. tuberculosis and M. bovis by N-PCR were observed (Table 1). Of the 393 EB extracts collected, AFB were detected in 20 (20/393; 5.1%), including those from patients with chronic lower abdominal or pelvic pain (2/17; 11.8%), infertility (16/285; 5.6%), and menstrual dysfunction complaints (2/80; 2.5%). Granulomatous tissue reactions compatible with tuberculosis were observed exclusively in seven EB samples derived from infertile patients (7/220; 3.2%). Mycobacteria were isolated from 11 samples by culture (11/262; 4.2%). Nine strains were lost on 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%), and one was obtained from a patient with an ovarian cyst (1/11; 9.0%). Ten isolates were identified as M. tuberculosis by spoligotyping and standard biochemical criteria. One isolate was characterized as Mycobacterium chelonae by biochemical criteria. However, in comparison to what was found with isolation by culture, M. tuberculosis/M. bovis mixed infection was detected in 123 samples by N-PCR (123/393; 31.3%). Of these, 109 (27.7%) were associated with M. tuberculosis and 31 (7.8%) with M. bovis infection. One hundred eleven of these EB extracts were from infertile patients (111/285; 39%), nine were from patients with complaints of menstrual dysfunction (9/80; 11.3%), one was from a patient with pain in the lower abdomen (1/17; 5.9%), and the remaining two were from patients with complaints of postrecanalization and fibroid (2/11; 18.2%) (Table 1, miscellaneous category). Comparison of the percentages of sensitivity for detection of mycobacteria by the four methods showed that the N-PCR assay has the highest sensitivity (31.3%). AFB detection by microscopy showed a sensitivity of 5.1%, and that for detection by isolation of the pathogens by the culture technique was found to be 4.2%. The least sensitive technique was histopathological examination for granulomatous tissue reactions compatible with tuberculosis infection (2.4%).

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TABLE 1. Comparative analysis of smear microscopy results for AFB detection, histopathological examination, culture, and N-PCR for 393 patients investigated with various complaints

Results for the various clinical conditions and techniques used in the study for detection of mycobacteria in the samples revealed that mycobacteria were detected by all the methods used in infertility cases. In cases of patients with menstrual dysfunction, mycobacteria were detected by only three methods, namely, AFB microscopy, isolation by culture, and N-PCR. In these individuals, no evidence of granulomatous reactions compatible with ongoing mycobacterial infection was detected. With acknowledgment that isolation of mycobacteria is the gold standard for diagnosis of tuberculosis, eight isolates were obtained from infertility cases, two from patients with menstrual disorders, and one from a patient with an ovarian cyst. Similarly, N-PCR results for patients in all categories were positive. The highest percentage of positivity was for infertility cases (111/285; 39%). 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 to 19% (2, 11, 13). In addition to M. tuberculosis infection, M. bovis infection has been reported to occur in FGTB (4, 7).The failure to isolate M. bovis in the present study may be due to use of inappropriate media (5, 6). Nonspecific clinical presentation, inefficacy of laboratory diagnostic tests, and inaccessibility of reproductive clinics have resulted in underreporting of FGTB. Hence, patients with complaints of infertility and other gynecological 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.

 
Senior Research Fellowships to P.K. and A.S. from the Indian Council of Medical Research and the University Grants Commission and the technical assistance of Shailendra Kumar are acknowledged. The financial support of DBT, Government of India, is acknowledged.

Mycobacterium tuberculosis H37Rv DNA was obtained from the Tuberculosis Vaccine Testing and Research Materials program (NIAD/NIH grant AI-75320).

 
* Corresponding author. Mailing address: Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. Phone: 91 11 26594994. Fax: 91 11 26588663. E-mail: hk_prasad{at}hotmail.com

Published ahead of print on 8 October 2008.

Present address: Laboratory of Molecular Pathology of Tuberculosis, Pasteur Institute, 642 rue Engeland, Brussels B-1180, Belgium.

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Journal of Clinical Microbiology, December 2008, p. 4068-4070, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01162-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kumar, P. Articles by Prasad, H. K. Search for Related Content PubMed PubMed Citation Articles by Kumar, P. Articles by Prasad, H. K.