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1
Department of Obstetric and Gynecology; 2Oncology and Stem Cell Working Group;
3
Department of Clinical Pathology, 4Limijati Maternal and Child Hospital, Faculty of
Medicine, Universitas Padjadjaran-Dr. Hasan Sadikin Hospital, Bandung 40161, Indonesia
Clinical Features
Symptoms appears on each patient can be
differently found based on severity sites
and stage of the disease. Some of the
patients may not develop any symptoms.
Several symptoms discovered on patients Magnification 200x
are:
Formation of a large pelvic mass
Chronic pelvic inflammatory
disease symptoms
Menstrual abnormalities, eg.
Amenorroea, menorrhagia,
hypomenorroea, polymenorhoea,
postmenopausal bleeding and
oligomenorrhoea.
Excessive vaginal discharge Magnification 400x
General symptoms of typical
tuberculosis such as weight loss,
anorexia, pyrexia.
Infertility problems which may be
primary or secondary.
Histological Examination
For histopathological studies, a
portion of the endometrial and falopian- Magnification 400x
tube tissue from the lesion was fixed in ten Figure 2. Presence of caseating granulomas
percent formalin; routine processing was surrounded by epitheloid cells,
done and the stained with haemotoxylin
lymphocytes, plasma cells and giant cells
and eosin (H&E). Presence of caseating
granulomas surrounded by epitheloid
cells, lymphocytes, plasma cells and giant
cells were diagnostic of genital
tuberculosis (TB).
Magnification 100x
Magnification 100x
centrifuged at 11200 g for 20 min. The
supernatant was discarded and the pellet
was processed to extract DNA.
(ii) Isolation of DNA - Pellets were
resuspended in 500l of TE buffer by
repeated pipetting. Then 50l of 10 mg/ml
of lysozyme was added, mixed well and
incubated for one hour at 37oC. To this,
70l of 10 percent SDS (sodium dodecyl
Magnification 200x sulphate) and 6l of 10 mg/ml of
proteinase K were mixed and incubated for
10 min at 65oC. After incubation 100l of 5
M NaCl was added and mixed thoroughly.
The samples were further incubated with
80l of CTAB/NaCl (Cetyl trimethyl
ammonium bromide in sodium chloride)
solution for 10 min at 65oC. To this
prepared sample approximately equal
volume (700-800l) of chloroform/
Magnification 400x isoamyl alcohol were added, mixed
thoroughly and centrifuged for 10 min. All
these chemicals were purchased from
sigma chemical (St. Louis MO), USA. To the
supernatant, 0.6 volume isopropanol was
added to precipitate the nucleic acids and
placed at -20oC for 60 min. The resultant
sample was spun at 16128 g for 20 min at
6oC. The resulting DNA pellet was washed
with 70 percent ethanol to remove
Magnification 400x
residual CTAB. The supernatant was
Figure 3. Presence of caseating granuloma
carefully removed and the pellet was
surrounded by epitheloid cells, dried. The prepared pellet was re-
lymphocytes, plasma cells and giant cells dissolved in 25l of TE buffer (910 mM TRIS
and 1 mM EDTA) and stored at 4oC for
future use.
Polymerase chain reaction
examination
To diagnose tuberculosis of fallopian
tubes and endometrial tuberculosis,
polymerase chain reaction procedure was
done as described below:
(i) Processing of samples - The
endometrial and falopian-tube tissue was
finely chopped using a sterile scalpel and
homogenized in TE buffer (TRIS-EDTA-
Figure 4. PolyChain Reaction (PCR)
10mM Tris. Cl. pH 8.0; 1 mM EDTA pH 8.0)
until the solution became turbid. This was
(iii) Amplification of mycobacterial DNA initial denaturation at 95oC for 5 min,
PCR was performed using Gene followed by denaturation at 94oC for 30
amplification 9700 Thermal cycler with sec, annealing at 58oC for 30 sec, extension
standard 25l working volume (Gene at 72oC for 30 sec with 25 cycles and a final
Amplification PCR System 9700-Applied extension at 72oC for 5 min. Detection of
Biosystems USA). Precautions were taken amplified products was done by agarose
to avoid false positivity. Preparation of PCR gel electrophoresis (2%) at 80 volts for 45
reagents, addition of template DNA and min. Gel was stained with ethidium
analysis of amplified products were done bromide and viewed under UV
in three different rooms to avoid carryover transilluminator (VILBER-LOURMAT,
contamination. Reagents were aliquoted France, TCP-20.M).
and each aliquot was used only once. Wax (v) Evaluation of specific diagnostics - For
beads were added to minimize nonspecific the diagnosis of genital TB there is no
amplification. DNAs from the samples absolute gold standard test available.
were amplified using the following Therefore, based on the clinical profile
primers. and laparoscopic evaluation of patients, a
IS6110 forward (5 CCT GCG AGC GTA diagnostic criteria were derived to suspect
GGC GTC GG 3) TB. A woman was said to be suspected of
IS6110 reverse (5 CTC GTC CAG CGC having genital TB if she has had findings
CGC TTC GG 3) suggestive of TB at laparoscopy with one
The IS6110 primers amplify a fragment or more of the following findings: A
with a length of 123 base pair (bp). DNA definite past history of TB, in the presence
extraction chemicals and PCR chemicals of active extra-genital TB, characteristic
were obtained from USB, Amersham features on histerosalphyngograpgy
Bioscience. (HSG), elevated erytocyte sedimen rate
(ESR), positive Mantoux test, evidence of
calcification/complex adnexal mass by
scan.
Nucleic Acid extraction
PCR amplification
Conclusion
We report three cases of female
with genital tuberculosis diagnosed during
laparoscopic surgery and confirmed with
pathology anatomy result. All of these
three cases were managed distinctively
according to its clinical presentation and
course. Most of the patients came with
clinical symptoms of irregular menstrual
cycle and infertility that mimicked other
disease. Our case series is a good
educational lesson that can be used by
obstetrician for better diagnosing and
handling of patients with similar problems.
12. Malhotra M. Genital tuberculosis.
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