Rapid Diagnosis of Genital Tuberculosis by Real-time Polymerase Chain Reaction

Objective: Rapid diagnosis of genital tuberculosis (GTB) is essential as it is an important cause of infertility among women. Diagnosis can be done by imaging techniques, direct visualization by endoscopy, serology, histopathology, culture and polymerase chain reaction (PCR) tests. Laparoscopy detects macroscopic changes only; histology is only suggestive and not confirmatory unless acid fast bacilli (AFB) are demonstrated in the lesion but sensitivity is poor in paucibacillary disease. Culture methods are the gold standard but slow growth of most pathogenic mycobacteria delays the diagnosis. PCR can rapidly detect even few copies of DNA with high sensitivity and specificity. Aim: Comparative evaluation of AFB smear examination, culture in Middlebrook 7H9 media and IS6110 based real-time PCR assay for the detection of mycobacteria in various female genital samples. Materials and methods: A total of 555 female genital samples like endometrial and fallopian tube biopsies, menstrual blood and vaginal discharge were processed by modified Petroff’s method. The deposit was used for detection of mycobacteria by AFB smear, culture on Middlebrook 7H9 media and IS6110 based real-time PCR. Results: Out of 555 samples, 25.22 % (140/555) were positive by the combination of all the methods used. Overall positivity by real-time PCR alone was 23.78% (132/555), by culture 8.28% (46/555) and 2.70% (15/555) by AFB smear examination. Out of total positives, 94.28% (132/140) were positive by PCR alone, 32.85% (46/140) by culture and 10.71% (15/140) by AFB smear. Eight (5.71%) culture positive samples were negative by smear and PCR, six of these were nontubercular mycobacteria (NTM) and two samples had PCR inhibitors as confirmed by spiking with positive DNA. Contamination was observed in 25/555 (4.5%) which were reported negative by culture but three of these were PCR positive. AFB smear results were available in 1 hour, PCR in 1 day and culture in 4 to 6 weeks. Conclusion: PCR was found to be the most rapid and sensitive (94.28%) method, 9-fold more sensitive than smear examination and 3-fold than the culture for detection of mycobacteria. Results were available in 4 to 6 weeks time for culture but in only 1 day by PCR. IS6110 PCR can detect only MTB and not the NTM. Use of multiplex PCR with genus and MTB specific primers will increase the sensitivity of test but care needs to be taken to prevent false positivity due to cross contamination and false negative due to PCR inhibitors.