Use of endo-ovarian tissue biopsy and pelvic aspirated fluid for the diagnosis of female genital tuberculosis by conventional versus molecular methods.

Til date, none of the diagnostic techniques available for the detection of female genital tuberculosis (FGTB) are 100% accurate. We therefore, proposed to use the endometrial tissue biopsies (ETBs), ovarian tissue biopsies (OTBs) and pelvic aspirated fluids (PAFs) for the diagnosis of FGTB among infertile women by conventional versus molecular methods.A total of 302 specimens were collected both from 202 infertile women highly suspected of having FGTB on laparoscopy examination and 100 control women of reproductive age. Out of 302 specimens, 150 (49.67%) were ETBs, 95 (31.46%) were OTBs and 57 (18.87%) were PAFs. All specimens were tested by conventional techniques, later compared with multi-gene PCR for the detection of Mycobacterium tuberculosis (MTB) and correlated with laparoscopic findings. The presence of MTB DNA was observed in 49.5% of ETBs, 33.17% of OTBs and 5.44% of PAF specimens collected from highly suspected FGTB patients. All women of control group were confirmed as negative for tuberculosis. The conventional methods showed 99% to 100% specificity with a low sensitivity, ranging from 21.78% to 42.08% while hematoxylin and eosin staining showed a sensitivity of 51.48%. Multi-gene PCR was found to have much higher sensitivity of 70.29% with MTB64 gene, 86.63% with 19 kDa antigen gene at species and TRC4 element at regional MTB complex and 88.12% with 32 kDa protein gene at genus level. The specificity of multi-gene PCR was 100%. Compared with culturing and Ziehl-Neelsen's staining, multi-gene PCR demonstrated improvement in the detection of FGTB (χ2 = 214.612, 1 df, McNemar's test value <0.0001).We suggest site specific sampling, irrespective of sample type and amplification of the 19 kDa antigen gene in combination with TRC4 element as a successful multi-gene PCR for the diagnosis of FGTB and differentiation of mycobacterial infection among endo-ovarian tissue biopsies and PAFs taken from infertile women

Agreement between culture (L-J egg media) and multi gene PCR results using four set of primers.

<p><b>Abbreviations</b>: n: number of patients; %: percentage; H & E staining: Hematoxylin and Eosin; L-J egg medium: Lowenstein Jensen; AFB+Ve: Acid fast bacilli positive; Z-N staining: Ziehl-Neelsen's; PCR: Polymerase Chain Reaction; 19 kDa (131 bp):19 kDa antigen gene; TRC4 (173 bp): TRC4 element; MPB64 (240 bp): MPB64 antigen gene; 32 kDa (506 bp): 32 kDa protein (MTP-59 α-antigen) gene.</p

2.5% Agarose Gel Electrophoresis was carried out with Multi-gene/multi-primer PCR products.

<p>Lanes in the first row (118 to 136) were loaded with PCR products of FGTB patients; Lanes in the second row (C7 to C20) were loaded with PCR products of control patients; Lane +Ve Ctrl was loaded with positive Reference Strain (<i>M. tuberculosis</i>, ATCC 35836); Lane –Ve Ctrl was loaded with negative control (H₂O); Lane 50 bp marker was loaded with 50 base pair (bp) molecular weight ladder (50 bp size of product starts from the bottom side of gel and ends with 650 bp product on the top/upper side of the gel), The band corresponding to 131 bp was noted as 19 kDa antigen gene, 173 bp was noted as TRC4 element, 240 bp was noted as MPB64 gene, 506 bp was noted as 32 kDa protein/MPT59 α- antigen gene. Primer dimers were also noted at the bottom during the end of sample run.</p

Laparoscopic/Hysteroscopic findings of infertile women suspected with female genital tuberculosis (FGTB) cases and control groups. Note

<p>: Some patients had more than one abnormal finding.</p

Statistical significance tests between conventional and molecular methods (n = 302).

<p><b>Note</b>: “a” denotes binomial distribution used. Statistical analysis showed 0 cells (0.0%) have expected count less than 5 for all methods. Variation is observed for different methods in terms of the minimum expected counts (MEC), i.e. MEC for “b” is 34.77; “c” is 14.57; “d” is 28.48; “e” is 27.15; “f” is 42.05; “g” is 42.05; “h” is 47.02; and “i” is 41.06. Degree of freedom (df) is one (1) for all calculations. Data is considered statistically significant if p value is less than 0.05.</p><p><b>Abbreviations</b>: n: number of patients; %: percentage; H & E staining: Hematoxylin and Eosin; L-J egg medium: Lowenstein Jensen; AFB+Ve: Acid fast bacilli positive; Z-N staining: Ziehl-Neelsen's; PCR: Polymerase Chain Reaction; 19 kDa (131 bp):19 kDa antigen gene; TRC4 (173 bp): TRC4 element; MPB64 (240 bp): MPB64 antigen gene; 32 kDa (506 bp): 32 kDa protein (MTP-59 α-antigen) gene.</p

Details of genes of <i>Mycobacterium tuberculosis</i> Complex.

<p>T: thymine; A: adenine; G: guanine; C: cytosine.</p

Z-N staining of endo-ovarian tissue biopsy and cultures in the detection of acid fast bacilli (AFB).

<p>a) Red/pink colour rod like beaded structures were observed in the tissue biopsy; b) Red/pink colour rod like structures were observed on pale blue background in the cultures.</p

Histopathological examination of endo-ovarian tissue biopsy.

<p>a) Endometrial tissue biopsies from the surface resemble those of cervical microglandular hyperplasia with beaded spindle-cells; b) Beaded and malignant lymphoid cells were observed in ovarian tissue biopsies; c) Appearance of spindle-cells with superficial strips of positive endometrial glands and stroma; d) Lymphocytic infiltrations and initial stages of granulomatous were observed. <b>Note</b>: The microscopic studies were carried out with tissue biopsies and aspirated fluids containing tissue pieces. Thereafter, the slides were viewed under bright field (40X), Inverted microscope. The contrasts of the photographs are changed to improve the visibility.</p

Demographic and clinical findings of FGTB cases and control groups (n = 302).

<p><b>Note</b>: Some patients had more than one abnormal finding; Data are presented as mean ± Standard Deviation (SD); n: number of patients; %: percentage.</p