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RESULTS

Megha Singhal Molecular diagnostics of genital tuberculosis

PhD Thesis 95 University of Delhi, Delhi

Results

On the basis of detailed history, the study subjects (400 infertile females fulfilling

inclusion criteria) were categorized with regard to their demographic profile, type of

infertility and duration of married life. Patients were also categorized according to their

gynecological symptoms, menstrual disturbance, history of contact or past history of

TB and history suggestive of active disease. Findings of molecular tests (DNA PCR,

mRNA-based RT-PCR, real-time and gene fingerprinting for mutation monitoring in

the drug resistant genes) were correlated with a cascade of clinical profile/laparoscopic

findings and pregnancy outcome following diagnosis and anti-tubercular treatment.

Demographic profile of study subjects

Socioeconomic status

When the educational and professional/economical background of the patients was

compared, it was found that maximum patients (43%) belonged to middle strata of the

society while 37% and 20% patients belonged to lower and higher strata of the society

as shown in fig 15 (110).

Fig 15: Pie diagram depicting socioeconomic status of the study subjects.

State-wise geographic distribution

Maximum patients visiting the infertility clinic were from Delhi (276/400) followed by

Uttar Pradesh (68/400), Haryana (48/400) and other states (8/400). The geographic

distribution of patients among various states is shown in fig 16.

37%

43%

20%

Socioeconomic Status

lower middle high



Fig 16: Graph depicting the state distribution among the study subjects

Details of Study subjects & healthy fertile Controls

The patients in the study group were between the age group 18 and 40 years. The

mean age of the patients was 27.8 years with a standard deviation of 4.7 years.

Table 19: Age distribution of study subjects.

Age (years) No. of cases Percentage

18-25 98 24.5%

26-30 160 40%

31-35 107 26.75%

36-40 39 9.75%

Total 400

Fig 17: Graph showing age distribution among the study subjects.

0%

20%

40%

60%

80%

Delhi Haryana Uttar Pradesh

Others

69%

12% 17%2%

Geographic distribution

24.5%

40%

26.8%

9.8%

0

10

20

30

40

50

18-25 yrs 26-30 yrs 31-35 yrs 36-40 yrs

Age Distribution



Table 20: Age distribution among fertile controls.

On analysis, we found that maximum number of Study subjects (40%) belonged to the

age group 26-30 years suggesting that female genital tuberculosis affects females in

their early reproductive age.

Duration of marital life

The duration of marital life ranged between 1.5 years and 17 years. The mean duration

of marital life was 7.2 years with a standard deviation of 3.5 years.

Type of infertility

Primary infertility was present in 275 (68.75%) women, while 129 (32.25%) women

had secondary infertility. Among the patients with secondary infertility, 14.7% had

previous living issue, 44.9% had previous abortions, and 14.7% had ectopic pregnancy.

Fig 18: Pie chat depicting the type of infertility among the patients.

1°infertility,

68.75%

2°infertility,

32.25%

Type of infertility

Distribution of age group among the Fertile controls (n=100)

Age (years) 30-35 36-40 41-45 46-50

Percentage (%) 21 43 28 8



Gynecological symptoms

In the present study, ~52% patients only had infertility and did not have any

gynecological symptoms. Maximum patients (64%) had normal menstrual cycles while

menstrual disturbances in the form of amenorrhea, oligomenorrhea and menorrhagia

were seen in 5.25%, 23.75% and 8% patients, respectively.

Fig 19: Graph depicting menstrual history of the study subjects.

Oligomenorrhea was the pre dominant menstrual disturbance seen in the subjects.

Dysmenorrhea and dysparenuia were seen in 3.25% (13/400) and 0.5% (2/400) cases,

respectively. Urinary symptoms like increased frequency and burning micturation were

seen in 5% cases. Chronic pelvic pain not responding to treatment was seen in 6%

cases.

One patient presented with active genital tuberculosis with granulomas in the uterus.

In this woman, ESR was 35, Mantoux was positive with an induration of 19 mm.

Laparoscopy findings showed tubercles with dense peritubal and periovarian adhesions.

While correlating the lab results of this patient later during the study, pre-menstrual

endometrial sample had tested positive for AFB smear, culture, histopathology, PCR

and RT-PCR suggestive of active tubercular disease.

5.25%

23.8%

8%

64%

0

50

100

150

200

250

300

Amenorrhea Oligomenorrhea Menorrhagia Normal

Menstrual History



Table 21: Gynecological symptoms observed among 400 study subjects.

Symptoms Number % age

No symptom other than infertility 208 52%

Menstrual disturbance 146 36%

Dysmenorrhea 13 3.25%

Dysparenuia 3 0.75

Chronic pelvic pain 24 6%

Vaginal Discharge 32 8%

Urinary Symptom 20 5%

Previous history of tuberculosis

Nearly 30% (121/400) patients had a past history of tuberculosis. This included

patients affected with pulmonary as well as extra pulmonary cases (such as lymph

node, recurrent uveitis, cervical lymphadenopathy, abdominal, spine and bone

tuberculosis). All these patients adequately received anti-tubercular therapy (ATT) for 9

months to 1.5 years as per the Revised National Tuberculosis Control Programme

(RNTCP).

Among the patients with past history of tuberculosis, 18.2% (22/121) were found be

positive in DNA PCR, 14.8%, (18/121) had suggestive laparoscopic findings while

2.48% (3/121), 5.79% (7/121) and 1.65% (2/121) were positive in smear, culture and

histopathology respectively.

Fig 20: Pie chart showing the past history of tuberculosis among the patients.

Present30%

Absent70%

Past history of Tuberculosis



Table 22: Positivity by each parameter among the patients (121) with past history of tuberculosis.

AFB smear AFB culture HPE DNA PCR Laparoscopy

Positive 3 7 2 22 18

Negative 118 114 119 99 103

Findings of Mantoux and HIV Test Results

The total leucocyte count (TLC) ranged between 7-11 109/l and the differential

leucocyte count (DLC) was within normal limits for all the subjects including the cases

which tested positive for genital tuberculosis.

Mantoux test (Mx)

Result of Mantoux test was available on 336 study subjects. A positive tuberculin

induration was defined as induration size equal to or greater than 10 mm. In our study,

tuberculin >10 mm was seen 21.4% (72/336) subjects. Out of these 72 Mantoux

positive cases however, only 29 (40.2%) showed corroborative evidence of tuberculosis

from other tests of our study; whereas the remaining 43 cases were not positive in any

other test carried-out in this study.

HIV status

Tuberculosis and HIV have a synergistic relationship, as tuberculosis being an

opportunistic infection may suppress the immune status in AIDS patients. Therefore,

all subjects and their partners were tested for the presence of HIV infection; but all

were negative for HIV.

Findings of Chest X-ray

In the present study, chest radiography (poster anterior view) was done in 330 cases.

The findings were normal except in 7 cases with findings of opacified cavities,

heterogenous cavities and ill defined opacities suggesting healed lesions with fibrous

scars. As none of the cases had symptoms pertaining to respiratory system, sputum for

AFB was not done in these cases.



Findings of Laparoscopy

Results from diagnostic endoscopy (Laparoscopy) were obtained from all the subjects.

A systemic and thorough evaluation of pelvis and abdominal cavity was carried out for

evidence of TB and findings such as granulomas, caseation, calcification, tubercles and

loculated ascites were looked for. The fallopian tubes were also evaluated for the

presence of proximal and distal blocks and hydrosalpinx. Presence of adhesions was

also noted. Pelvis and peritoneal cavity were also evaluated for presence of other

pathology. Presence of fluid in Pouch of Douglas (POD) was also noted. Pelvic

pathology like fibroid uterus, endometriosis, and polycystic ovaries was seen as

incidental findings. Table 23 shows the laparoscopic findings among the study subjects.

The laparoscopic findings were subdivided into:

Group A: This group included findings suggestive of tuberculosis.

Suggestive findings, which included:

Affirmative findings (presence of tubercles, peritubal and/or periovarian

adhesions, tubo-ovarian mass, beaded tubes, cornual blockage)

Suspicious findings (hydrosalpinx, sacculated tubes, signs of chronic inflammation,

pelvic inflammatory disease, and mild adhesions)

*More than one positive finding (tubercle/adhesions/beaded tubes/blockage)

was considered as being suggestive for tuberculosis in laparoscopy.

Group B: This included findings other than tuberculosis or normal findings.

Normal findings: This included findings other than tuberculosis like normal pelvic

findings, endometriosis, fibroid uterus etc.

Suggestive of Tuberculosis: 38/400

- Affirmative: 26/38 - Suspicious: 12/38

Not suggestive: 362/400

Fig 21: Laparoscopic findings of the patients



i) Dilated tube

ii) Adhesions



iii) Adhesions with hydrosalpinx



iv) Hydrosalpinx with fluid in POD

v) Inflammation

vi) Tubercles on fallopian tube



Table 23: Laparoscopy findings among study subjects

Laparoscopic findings No. of patients

Adhesions (Peritoneal/Pelvic/POD)

36

Beaded tube appearance 4 Tubo-ovarian mass 8 Tubal block (uni) 49 Tubal block (bi) 32

Tube absent 8 Hydrosalpinx 10 Frozen Pelvis 7 Tubercles 4 Fluid in POD 3 Caseous granuloma 5 Endometriosis 11 Calcification 1 PID 5

PCOS 21

While correlating the results, it was observed that all the 38 laparoscopy positives

were also DNA PCR positive during the present study.

Findings of Conventional Laboratory Tests

Findings of histopathological examination (HPE)

Pre-menstrual endometrial biopsy samples were taken and sent for histopathology.

Out of the 400 women, 1.25% (5/400) samples showed positive for tuberculosis. In

these cases, the histology showed sub epithelial tissue shows multiple caseating

granulomas with langerhans giant cells and diffuse and dense inflammatory infiltrate

comprising lymphocytes, plasma cells and neutrophils. The impression was said to be

as granulomatous highly suggestive of tuberculosis. Another 14 patients showed signs

of non tubercular endometritis (fig 23).

Secretory endometrium was seen in 81.2% (325/400) patients while proliferative

endometrium was seen in 13.2% (53/400) patients. Hyperplastic endometrium was

observed in 3 (0.75%) women (table 24). In one case which presented as active genital

tuberculosis, the histology showed tubercular salpingitis and clear evidence of

tuberculosis were also seen in laparoscopy. Among the patients in control group, no



sample was found to be positive for tuberculosis in histopathology. All patients

positive in histopathology were also positive in Laparoscopy and DNA PCR.

Fig 22: Histopathological findings among the infertile patients

Table 24: Various results in histopathology in the study group

Result of HPE Number Percentage

Study group (n=400) Tubercular endometritis Chronic endometritis Proliferative phase Secretory phase Hyperplastic endometrium Control group (n=100) Tubercular etiology

5 14 53 325 3 0

1.25% 3.5%

13.25% 81.2% 0.75%

0

Smear and Culture

Smears were prepared from all the study and control subjects and examined by Ziehl-

Neelsen method. Out of the 400 samples in the study group, AFB Smear detected

1.75% (7/400) cases. In the control group however, none of the samples was found to

be positive.

1.25% 3.5%

13.25%

81.20%

Histopathological findings

Tubercular endometritis

Chronic endometritis

Proliferative phase

Secretory phase



Fig 23: Results of AFB culture in the study group.

Culture by BACTEC 460 TB systems was done in all the samples. Since these extra

pulmonary samples are paucibacillary in nature their processing included milder

decontamination. The reading on the BACTEC machine was taken every alternate day.

Table 25: Positivity in AFB smear/culture among the study and control groups.

Endometrial biopsy Smear* %% tage Culture* % tage

Study subjects (n=400) 7 1.75% 11 2.75%

Controls (n=100) 0 0 0 0

*All AFB smear and culture positive were also positive in DNA PCR.

Culture for acid-fast bacilli using BACTEC 460 TB systems detected 2.75% (11/400)

cases (fig 23). The cultures were identified as positive within 12 days and were

identified as MTB on the basis of NAP test. Apart from this, 6 cultures got

contaminated. However none of the contaminated sample was positive in AFB smear

or PCR.

2.75%

95.75%

1.50%

Results of AFB culture in study group

Positive

Negative

Contaminated



Molecular Diagnostic Tests

Findings of Objective 1:

DNA PCR for 65 kDa gene for diagnosis of genital tuberculosis

PCR was carried-out to amplify -globin gene using specific primers to ascertain the

quality of extracted DNA suitable for PCR amplification, showed amplification bands

at 110 bp (fig 24).

Fig 24: PCR result of -globin gene region (110 bp), lane L: 100 bp ladder, lane1-10: clinical

samples & lane12: negative control

Results DNA PCR on Controls & Study subjects

Out of the first set of controls i.e. 100 fertile females, 4 tested positive in DNA PCR.

Among the second set of controls i.e. 25 known positive AFB cultures for

ascertaining the detection efficacy of DNA PCR tests, all the 25 samples (which were

expected to be positive) actually Out of 400 clinical samples in the study group,

tested positive in DNA PCR for 65 kDa gene. 12.75% (51/400) tested positive by

DNA PCR of 65kDa antigen coding gene (fig 25)

Fig 25: PCR result of 65 kDa HSP gene of M. Tuberculosis (165 bp), lane L: 100 bp

ladder, lane 1: negative control, lane 2-8: clinical samples and lane 9: positive control

1 2 3 4 5 6 7 L 9 10 L 12

110 bp

1 2 3 4 5 6 7 8 9 L

165 bp



Comparative analysis of DNA PCR result with Other tests and Laparoscopy

Comparative analysis of DNA PCR results with other tests is tabulated was carried out

with previous tests. The results are shown in table 26 and fig 26.

Table 26: Comparative analysis of DNA PCR with conventional tests and laparoscopy

Tests Histopath Smear Culture Laparo DNA PCR

Positivity

5/400 7/400 11/400 38*/400 51*^/400

*Of 38 laparoscopy positives, 4 which initially tested negative or doubtful in DNA PCR turned positive in repeat PCR after spiking; later confirmed with DNA sequence homology for 65 kDa gene. ^All these 51 PCR positive samples were confirmed to be true positives by gene sequence analysis of the PCR products.

Fig 26: Positivity by different parameters in the diagnosis of female genital tuberculosis.

Confirmation of DNA PCR positivity by Gene sequencing/BLAST search

Gene signatures of 165 bp region of 65kDa gene of M. tuberculosis

The samples that tested positive for 65 kDa gene were analyzed by partial nucleotide

sequencing (fig 27). The sequences were subjected to BLAST search to confirm DNA

sequence homology with 65 kDa gene of M. tuberculosis. The blast results showed 100%

identity with the 65 kDa gene of M. tuberculosis suggesting it to be M. tuberculosis and

thus no false amplification was seen. Following is the 165 bp sequence of 65kDa gene

of M. tuberculosis.

0.00%

2.00%

4.00%

6.00%

8.00%

10.00%

12.00%

14.00%

AFB Smear AFB Culture Histopathology Laparoscopy DNA PCR

1.75%2.50%

1.25%

9.50%

12.75%



Fig 27: Blast results of the sequences obtained from positive samples.



> M.tuberculosis - 65 kDa antigen (cell wall protein a) gene

Homology of reference sequences of 65kDa gene with gene sequences of our study subjects. All the 51 DNA-positives had identical sequences.


Utility of mRNA RT-PCR to detect active cases of genital TB

mRNA-based RT-PCR for detection of 85B antigen gene for viability of M. tuberculosis

Nested RT-PCR carried out to amplify 216 bp region of 85B antigen gene of M.

tuberculosis using specific primers. Gel picture of the standardized PCR is shown below

(fig 28):

Fig 28: Nested RT-PCR result of 85B antigen gene region (216 bp), lane L: 100 bp

ladder, lane 8: positive control, lane1-4,6,7: clinical samples and lane5: negative control

Out of the 51 DNA PCR positive patients in the study group, 31.3% (16/51) patients tested positive for mRNA-based RT-PCR.

1 2 3 4 5 6 7 8 L

216 bp



Results of mRNA-based RT-PCR on Controls

Out of the first set of controls i.e. 100 fertile females, NONE tested positive in mRNA-

RT-PCR. Among the second type of controls i.e. 25 known positive AFB cultures

used as second set of controls for ascertaining the detection efficacy of DNA PCR tests,

all the 25 samples (which were expected to be positives) actually tested positive m-

RNA RT-PCR for 85B antigen gene of M. tuberculosis (table 27). Of 51 DNA PCR

positives in this study, ~30% (16/51) tested positive in m-RNA PCR for 85B

antigen gene of M. tuberculosis.

Table 27. Active cases of genital tuberculosis among PCR-positive patients

Positivity (out of 51 DNA positives)

DNA PCR mRNA RT-PCR 51 16*

*mRNA-based RT-PCR of the 85B antigen gene of M. tuberculosis was detected in ~30% DNA PCR positive patients.

Correlation of Laparoscopic findings with different Laboratory tests:

The patients positive in different tests were grouped accordingly to their laparoscopic findings

viz. affirmative findings, suspicious findings and normal findings. It was observed

that among the 38 patients showing suggestive findings for tuberculosis in

laparoscopy, DNA PCR confirmed positivity in 100% (38/38) laparoscopy positive

cases; while turned positive in while AFB smear, culture and histopathology were far

inferior as reflected in the table 28.

Table 28: Correlation of laparoscopic findings with different laboratory tests

Laparoscopy HPE (n=5)

Smear (n=7)

Culture (n=11)

DNA PCR

(n=51)

mRNA PCR (Useful for ascertaining

viability of tubercle bacilli) (n=16)

A Affirmative findings (n=26) 4 6 7 26 11

B Suspicious findings (n=12) 1 1 3 12 3

C Normal findings (n=362) 0 0 1 13 2

Fertile Controls (n=100) 0 0 0 4 0



Confirmation mRNA results by gene sequencing of 216bp product of 85B antigen gene

The samples that tested positive by 85B antigen gene amplification were analyzed by

partial nucleotide sequencing. The results of sequencing were blasted onto the global

genome data bank (NCBI website) to confirm the presence of 85B antigen gene of M.

tuberculosis.

Blast search result of 216 bp sequence of 85B antigen gene of M. tuberculosis showing 100%

identity of our sequences with the 216 bp 85B antigen gene sequence of the standard M.

tuberculosis strain Rv1886c (gene) >Rv1886c, suggesting that our RT-PCR products were

true positives (fig 29).


Homology of in-silico deduced amino acid reference sequences of 85B antigen gene of M. tuberculosis with amino acid sequences of our study subjects. All the 16 mRNA-positives had identical sequences.



Sequence relatedness between the 85B antigen gene sequences from study subjects and controls

Fig 30: Sequence diversity between the study and control group.



Structure of the active site: 85B antigen gene

Using m-fold web server, the structure of active site was drawn out of the sequences

of 85B antigen gene from our study and control groups, but no difference was found in

the binding site; yet again confirming that our mRNA PCR positives were true

positives (fig 31).

dG= -79.64 [initially -89.30] dG= -82.36 [initially -88.40]

Fig 31. Structure of active site of 85B antigen gene from Study Subjects and AFB culture controls

Deduced amino acid sequence from our 85 B antigen gene sequence

ERMTTQDVEAITPQTLINIRPVVAAIKEFFGTSQLSQFMDQNNPLSGLTHKR

RLSALGPGGLSRERAGLEVRDVHPSHYGRMCPIETPEGPNIGLIGSLSVYARV

NPFRVHRNAVP

Findings of Taqman real-time PCR using our ‘self in-silico designed’ novel target for mRNA-based Taqman Real-time PCR for mRNA

A total of 19 out of 51 DNA confirmed positives (i.e. 37.25%) were positive in our

developed TaqMan assay as against only 16 detected by conventional RT-PCR for

mRNA of 85B antigen gene of M. tuberculosis to differentiate between active and latent



cases of genital tuberculosis. Real-time PCR was carried-out in the ABI 7900 HT

machine using the novel targets depicted in table 29. The amplification plot is shown in

fig 32.

Fig 32. TaqMan Real-time PCR standardized using novel target. It depicts one positive control, one negative control

along with two samples that tested positive for 146 bp region of 85B antigen gene of M. tuberculosis.

Table 29. Primers and probe along with annealing temperatures for novel targets

Name Sequence Position Orientation No. (nt) Anneal temp C 85B_F (primer)

GTTCGTAGCAGCAACCTGAA

817 forward 20 59.08

85B_R (primer)

GAACTCTGCAGGTCACCCTT

943 reverse 20 59.3

Probe CCAGGATGCGTACAACGCCG

840 forward 20 69.06



Comparative efficacy of Conventional RT-PCR vs Novel Real-time RT-PCR:

Table 30 compares the detection rates by conventional RT-PCR and novel real-time PCR.

Table 30: Detection rates by conventional RT-PCR and novel real-time PCR

By conventional RT-PCR 16/51 DNA PCR positives

By Novel TaqMan Real-time PCR 19*/51 DNA PCR positives

*The three extra positives detected by real-time PCR could not be confirmed through sequence analysis. Hence it is only indicative of the marginal superiority of the technique.


Findings on drug-resistance genes (rpo B, kat G and inh A)

Detection of rpo B, kat G and inh A genes in 51 DNA PCR-positive infertile females

For mutation analysis, DNA from all 51 samples was subjected to amplification of

partial region of rpoB, katG and inhA genes associated with multidrug resistance. Table

31 shows the primer sequences used for the multidrug resistant genes along with their

annealing temperature, nucleotide position and amplicon size (bp). Primer sequences

and corresponding Gel pictures of PCR amplification of 350 bp rpoB, 620 bp katG and

248 bp inhA genes are shown in figures 33, 34 and 35:

Table 31: Primer sequences used for the multidrug resistant genes along with their annealing temperature, nucleotide position and amplicon size (bp)

Gene Sequence Anneal temp C Position(nt) Amplicon size

rpoB

F- GGGAGCGGATGACCACCC 60 2266 350 bp R- GCGGTACGGCGTTTCGATGAAC

2615

katG F- AGCTCGTATGGCACCGGAAC 60 904 620 bp

R- TTGACCTCCCACCCGACTTG 1523 inhA F- CCTCGCTGCCCAGAAAGGGA 45 Upstream

of inhA gene 248 bp

R- ATC CCC CGG TTT CCT CCG GT



Fig 33: PCR result of rpoB gene region (350 bp), lane L: 100bp ladder, lane 3:

negative control, lane 1, 2 and 4: clinical samples

Fig 34: PCR result of katG gene region (620 bp), lane L: 100bp ladder, lane 2-

6: clinical samples

Fig 35: PCR result of inhA gene region (248 bp), lane L: 100bp ladder, lane 3:

negative control, lane 1, 2 and 4: clinical samples

Analysis of DNA sequences to unveil new mutation(s), if any in the rpo B, kat G and inh A genes from 51 DNA PCR-positive cases for drug resistance to Rifampicin & Isoniazid

Sequencing was carried-our as per the protocol mentioned earlier for sequencing of

DNA PCR positive amplicons. The sequences were subjected to BLAST search base

(NCBI website); and subsequent mutation analysis by comparison with the previously

reported sequences.

350 bp

1 2 3 L 4

L 2 3 4 5 6

248 bp

1 2 3 4 L

620 bp



Gene signatures of 350 bp region of rpoB gene

Samples that amplified the 350 bp, 620 bp and 248 bp region of rpoB, katG and inhA

genes were analyzed by partial nucleotide sequencing. The results of sequencing were

blasted onto the global genome data bank (NCBI website) to search for the reference

sequence of M. tuberculosis.

After the blast results, reference sequence was selected and mutation analysis was

carried out using various softwares like Mega v4.1, BioEdit v7.0.5.3 and SeqScape® v2.7

(Applied Biosystems). All the sequences were submitted to global NCBI GenBank.

BLAST search results:

The following figure shows the blast results obtained for positive samples (fig 36).




Mutation analysis of drug resistance genes in DNA PCR positives

Mutation analysis was carried out in the drug resistance genes. The drug resistance

analysis results for rpoB gene are shown in table 32. Figure 37 depicts the point of

mutation on the rifampicin resistance determining region (RRDR).

Fig 37: Rifampicin resistance determining region (RRDR) showing mutations at two codon positions

Table 32: Mutations in the rpoB gene

Codon no. Mutation Position of Mutation

516 GACTAC (AspTyr) T replaces G at first position

531 TCGTTG (SerPhe) T replaces C at first position

TCGTGG (SerTrp) G replaces at second position

572 ATCTTC (IlePhe) T replaces A at first position

584 TTCTTT C (PhePhe) Insertion of T at the third position

Accession no.: JF268583 to JF268611 (NCBI, Gene Bank)

Fig 38: Multiple Sequence alignment depicting mutations in rpoB gene



Fig: 39: Amino acid alignment of the partial region of rpoB gene of M. tuberculosis depicting differences in amino acid at various positions.



Mutations in the katG gene

Only 2 mutations at codon position 315 and 463 already reported previously in other

forms of tuberculosis were also found in the amplicon sequences of katG gene from

our study subjects.

Table 33: Mutations in the katG gene of M. tuberculosis.

Codon no. Mutation Position of Mutation 315 AGCACC (Ser315Thr) C replaces G at second position

463 CGGCTG (Arg463Leu) T replaces G at second position

*Codon 463 is a polymorphic site. This substitution was seen in almost all samples. It has been shown to be associated not with resistance but with evolutionary genetics.

Mutations in the inh A gene

No mutation was seen in the promoter region of inhA gene of M. tuberculosis.

Profile of 51 DNA PCR confirmed cases of genital tuberculosis of our study

After compiling the results from various tests, it was found that a total of 51 samples

tested positive for genital tuberculosis by various parameters. The clinical profile of the

positive patients is shown in table 34:

Table 34: Showing profile of 51 DNA PCR confirmed cases of genital tuberculosis of our study

Age (years)

18-25 26-30 31-35 36-40

12 22 14 3

Type of Infertility

Primary (1) Secondary (2)

35 16

History of TB

Present Absent

22 29

Menstrual History

Amenorrhea Oligomenorrhea Menorrhagia Normal

2 21 4 24



Comparison of results between female genital TB positive and negative patients

Various parameters were compared between genital TB positive and negative patients.

Oligomennorhea was found to have a significant correlation between the two groups.

Table 35 shows the comparison of various characteristics between the two groups.

Table 35. Clinical characteristics of positive and negative patients.

Clinical feature Positive patients (%) Negative patients (%) p-value

Amenorrhea 3.9 5.4 0.6448

Oligomenorrhea 41.2 21.2 0.0017

Menorrhagia 7.8 8.02 0.9647

1 infertility 68.6 68.6 0.9838

H/O TB 43.1 28.4 0.319

Comparison of results between mRNA-positive and mRNA-negative patients

Comparison of various clinical characteristics between the patients who were DNA

positive-mRNA positive and those who were DNA positive-mRNA negative did

not reveal any significant difference in any parameter between the two groups (table

36).

Table 36: Comparison of clinical features between mRNA-positive & negative patients

Clinical Features mRNA-positive patients (16/51)

mRNA-negative patients (35/51)

p-value

Amenorrhea 0 2 0.329

Oligomenorrhea 4 10 0.79

Menorrhagia 2 3 0.661

1 infertility 27 8 0.052

H/o TB 9 13 0.201



Profile of 13 FGTB positive-laparoscopy negative patients

The clinical features of 13 female genital tuberculosis positive-laparoscopy negative

patients were analyzed. They are presented in table 37.

Table 37: Profile of 13 FGTB positive-laparoscopy negative patients

Parameter Findings

Type of infertility Seven (53.8%) patients had 1 infertility while six (46.1%) had

2 infertility.

Menstrual cycle 23.07% had Oligomenorrhea, 7.7% had Menorrhagia while 61.5% had normal cycles

H/o TB Three patients (30.7%) had a previous history of TB (pulmonary and abdominal tuberculosis).

Smear and Histopathology None of the patients were positive by smear and histopathology

Culture One patient (7.7%) had a positive culture

mRNA -based RT-PCR Two patients (15.3%) had a positive RT-PCR

Follow-up of Study Subjects and Pregnancy Outcome

Among the 400 subjects, a total of 51 subjects tested positive for M. tuberculosis by

different parameters. All the subjects were put on anti-tubercular therapy using the

standard regimen. Patients with multidrug resistant endometrial tuberculosis were put

on second line anti-tubercular drug regimen as per the revised tuberculosis control

program for multidrug-resistant tuberculosis.

Table 38: Showing the number of patients who conceived after treatment.

Number of patients put on ATT as per Laparoscopy and/or DNA PCR results of this study

Conceived=7

Not conceived even after treatment=6

Still under treatment

Lost interest

Lost to follow-up

IUI IVF Spontaneous IUI IVF

3 1 3 4 2 19 12 7



Seven (7/51; 13.72%) patients conceived post-ATT. These included one patient from

group A with positive DNA-PCR and RT-PCR; one patient from group B, with a

positive DNA-PCR and culture and five patients from group C, all with positive

DNA-PCR, one of which had a positive RT-PCR (table 38).

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