Journal of Cell and Molecular Biology 2: 99-103, 2003.
Haliç University, Printed in Turkey.
Polymerase chain reaction is a good diagnostic tool for Mycobacterium
tuberculosis in urine samples
Serdar Arisan, N. Cem Sönmez*, Ömer Onur Çak›r, Erbil Ergenekon
fiiflli Etfal Research and Training Hospital, 1 st Urology Clinics, fiiflli, Istanbul, Turkey (*author for
Received 3 June 2003; Accepted 27 June 2003
Tuberculosis is a major public health problem in developing countries. Tuberculosis of the urinary tract is not
uncommon in all over world, and it continues to be an important clinical problem, mainly because of its nonspecific
clinical presentation and variable radiographic appearance, which often mimic other pathologic lesions. Detection of
suspects bacterial population; direct smears can be used but they are often negative and do not differentiate
tuberculosis from nontuberculous mycobacterium. Culture, which is more sensitive, takes 6 to 8 weeks because of
the slow growth rate of mycobacterium. Polymerase chain reaction (PCR) is a technique used to amplify extremely
small amounts of a specific genomic sequence rapidly. The presence of an extremely small number of bacteria can
thus be detected within 24 to 48 hours. Therefore PCR is a promising tool for rapid detection of urinary tract
tuberculosis in urine samples.
Key words: PCR, Mycobacterium tuberculosis, diagnosis
‹drar örneklerinde Mycobacterium tuberculosis tan›s› için polimeraz zincir
Tuberküloz geliflmekte olan ülkelerde oldukça önemli bir sa¤l›k sorunudur. Uriner sistemde meydana gelen
tuberküloz ise pek yayg›n de¤ildir. Ancak spesifik olmayan klinik bulgular› yüzünden ve de¤iflken radyolojik
görünümü ve di¤er patolojik lezyonlar› taklit etti¤i için önemli bir klinik problem halini alm›flt›r. fiüpheli bakterial
populasyonlar›n tan›s› do¤rudan smear alarak gerçeklefltirilebilmektedir. Ancak al›nan sonuçlar s›kl›kla negatif veya
tüberküloz tipi olmayan mikobakterileri tan›mlamaktad›r. Polimeraz zincir reaksiyonu (PCR) küçük miktarlarda
genomlar› spesifik genom dizilerinde ço¤altmay› hedefleyen bir araç olarak kullan›lmaktad›r. ‹drarda çok az
miktarda bulunan bakteriler 24-48 saat içerisinde bu yolla teflhis edilebilmektedir. Bundan ötürü, PCR uriner sistem
tuberküloz tan›s›nda oldukça umut vadeden bir araçt›r.
Anahtar sözcükler: PCR, Mycobacterium tuberculosis, tan›
Tuberculosis is a major public health problem in
developing countries. Despite the decline in incidence
seen in the 1980s, resurgence has occurred and 8% to
15% of patients with pulmonary tuberculosis go on to
develop urinary tract tuberculosis (UTB). The
estimated involvement is 400 per 100,000 populations.
100 Serdar Arisan et al.
(Styblo, 1980; Peterson, 1994). Tuberculosis of the
urinary tract is not uncommon in all over world, and it
continues to be an important clinical problem, mainly
because of its nonspecific clinical presentation and
variable radiographic appearance, which often mimic
other pathologic lesions (Figure 1).
The earliest urographic change occurs in the minor
calices, and caliceal dilation is the first sign. However,
it is often so minimal that the diagnosis can be
extremely difficult (Ney and Friendenberg, 1981). A
slight loss of sharpness of the calix, which represents
mucosal edema, is another subtle initial sign (Elkin,
1990). As the disease progresses, the calix appears
moth-eaten or feathery (Taylor, 1939). An instance of
calcification in renal tuberculosis was found in 7% of
cases by Crenshaw (1930) and in 14.4% of cases by
Gow (1965). Urethral strictures have been reported in
about 10% to 56% of patients, and bladder
involvement is found in one third of cases of UTB
(Royalance et al., 1970).
Early diagnosis of the disease allows
administration of antitubercular treatment at a stage at
which it may be curative. However, more often than
not, the diagnosis is delayed because of a delay in
presentation and in making a definitive diagnosis, with
the consequence that a number of patients present with
nonfunctioning kidneys, ureteral stricture, and
shrunken bladders. These changes can be avoided if
the diagnosis is made early and treated effectively.
The diagnostic criterion for UTB is the isolation of
Mycobacterium tuberculosis from urine. This is not
easy to achieve, as the discharge of organisms into the
urine is sporadic and, more importantly, involves few
organisms (Gow et al., 1984; Gow, 1992). Direct
smears are often negative and do not differentiate
tuberculosis from nontuberculous mycobacterium.
Culture, which is more sensitive, takes 6 to 8 weeks
because of the slow growth rate of mycobacterium
(Manjunanth et al., 1991). This study conducted by the
fiiflli Etfal Research and Training Hospital in 29
patients suspected of having urinary tract tuberculosis,
the incidence of a positive urine culture for
Mycobacterium tuberculosis among histological
positive cases was 15% (Colabawalla, 1990). In the
literature, the majority of molecular TB detection
protocols is based on the PCR and have been
developed "in house". PCR is a technique used to
amplify extremely small amounts of a specific
genomic sequence rapidly. The presence of an
extremely small number of bacteria can thus be
detected within 24 to 48 hours (Noel-Brisson et al.,
1989; 1991). The high sensitivity of PCR is
particularly useful in paucibacillary situations such as
non-pulmonary tuberculosis. The aims of this study
were to reveal the importance of PCR analysis for
rapid and sharp results for detection of Mycobacterium
tuberculosis in urine samples.
Figure 1: A Ziehl-Neelsen acid-fast stain of some sputum
positive for tuberculosis. Arrows indicate mycobacteria,
which appear as red rods.
Materials and methods
In the first step of study, urine samples were collected
from 29 patients with suspected urinary tract
tuberculosis between 2000-2002 years in fiiflli Etfal
Research and Training Hospital 1 st Urology Clinics.
These urine specimens used for both routine culture
and PCR techniques.
100 ml urine samples were used in 2 hours after taken
and concentrated to 2 ml. Bacterial DNA extraction
and PCR for amplification of the Mycobacterium
tuberculosis complex (Yamaguchi et al., 1989) were
carried out according to a previously published
protocol (Seth et al., 1996). DNA was extracted with
QIAamp DNA Mini Kit (Qiagen).
The urine specimens were prepared for PCR
amplification according to the following protocol.
Sediments were washed three times with an equal
volume of Tris-EDTA buffer (10 mM Tris-HCL, 1 mM
EDTA; pH: 8.0) at 5000-x g for 5 min. The resulting
pellet was resuspended in 0.25 mL of Tris-EDTA
buffer and then boiled for 20 min. After centrifuged at
5000-x g for 5 min, 5 µl of the supernatant was
analyzed by PCR in a 50 µl reaction mixture. The PCR
reaction mixture contained 50 mM KCL, 10 mM
Tris-HCl, pH: 8.3, 1.5 mM MgCl2, 200 µM
deoxynucleoside triphosphates (dNTP), 2.5 U Taq
polymerase (Boehringer Mannheim, Germany) and
0.5 µM (each) of the primers. The primer sets used to
amplify the 123-bp IS 6110 gene fragment consisted of
TBC1 (CCT GCG AGC GTA GGC GTC GG) and
TBC2 (CTC GTC CAG GGC CGC TTC GG)
(Eisenach et al., 1991; Bennedsen et al., 1996 and
Desjardin et al., 1998-Qiagen). The reaction mixture
was subjected to 30 cycles of amplification (95°C, 30
sec; 68°C, 30 sec; 72°C, 30 sec) followed by a 5 min
extension at 72°C (Thermocycler; Techne). 15 µl of
the amplification products were analyzed by
electrophoresis in an ethidium bromide stained 2%
agarose gel (Sambrock et al., 1989).
Total of 29 urine specimens were collected from 15
women and 14 men. Their average ages for women 35
and for men were 42. 9 patient were positive for
tuberculosis by both test, while remain of the study
group were negative. 4 urine samples could not
evaluated by PCR due to the presence of inhibitory
substances of nature. The bladder biopsy was
diagnostic of tuberculosis in 5 (20%) of the 24 patients
biopsied. Fine needle aspiration cytological
examination was done in 1 patient with epididymal
swelling and was suggestive of tuberculosis. The
urinary PCR was falsely positive in 1 (4%) patients
Mycobacterium tuberculosis organisms were found
to be excreted intermittently in the urine of infected
patients. 7 men patients were showing testicular
Table 1: The comparision of PCR and routine culture results
Culture Staining PCR + PCR -
11 positive 11 positive 10 1
14 negative 14 negative 2 14
swelling, perianal sinus or genital ulcer. 2 women and
3 men patients had secondary coliform infection. 1
woman and 5 men had gross hematuria. Microscopic
hematuria was present 9 men and 11 women. All
patients had frequent urination, initially during the
day; later, at night. Dysuria, frank pain, suprapubic
pain, blood or pus in the urine presented in all patients.
Cystourethroscopy was performed in 29 patients. The
bladder revealed evidence of chronic cystitis in 24
patients. After detailed consultation other fungal
infection for 2 patient, pyonephrosis for 1 patient,
calyceal diverticula for 2 patients and bladder cancer
for 1 patient were detected. Complete blood cell count,
sedimentation rate, serum chemistry were investigated
for all suspect patients. The erythrocyte sedimentation
rate was elevated in 16 patients. Chest and spine
radiographs showed negative results for all patients.
Two patients had abnormal renal parameters. Kidney,
urethra and bladder radiographs reveal calcifications in
the kidney for 4 patients. Calcifications were
intraluminal. Bladder calcification was not obtained
from radiographic results. There was no HIV positive
patient. All positive patients were consulted by
infection consultants and treated with INH, Rifampin,
pyrazinamide and ethambutol for first 2 months and
rifampin and INH treatment were continued for
resistance to either agent exists.
Mycobacterium diagnosis by PCR 101
Tuberculosis is still rampant in underprivileged
societies and continues to take a heavy toll. It can
affect any organ system in the body and produce
protean manifestations. UTB occurs in 8% to 20% of
patients with pulmonary tuberculosis, with a
prevalence of 400 per 100,000 population (Styblo,
1980; Peterson, 1994). The most common presenting
symptoms in patients with UTB are irritative voiding
symptoms and hematuria, with the reported incidence
in world literature at around 60% and 50%,
respectively (Narayan, 1982). The speed and
simplicity of amplification techniques can greatly
assist TB diagnosis, therapy and epidemiology. This
improvement will continue with the rapid advances in
the tools available in molecular biology. No long series
has been reported on the sensitivity and specificity of
urinary PCR in UTB. Although, PCR test has been
extensively studied and proved highly sensitive,
specific and rapid. In various studies, data show
sensitivity ranging from 87-100% (usually >90%) and
specificity from 92,2-99,8 % (usually >95%). This
102 Serdar Arisan et al.
compares to cultures (37%), bladder biopsies (47%)
and intravenous pyelogram (IVP) examinations. Along
an accurate clinical assessment, PCR is the best
available tool to avoid delay in starting treatment
because it takes only 6 hours (Davies et al., 1999).
False-negative findings may result from the presence
of inhibitors; non-homogeneous distribution of
bacteria in the specimen so that the fraction tested does
not contain mycobacterium; or low numbers of
mycobacterium in the specimen, which decreases the
probability of the presence of organisms in the fraction
analyzed by PCR (Missirliu et al., 1996; Kolk et al.,
1992). False-positive results with PCR may be caused
by contamination due to the presence of amplicons or
Mycobacterium tuberculosis complex bacilli or DNA
(van Vollenhoven et al., 1996).
Most PCR assays employed for the diagnosis of
tuberculosis do not distinguish between infections
caused by Mycobacterium bovis and Mycobacterium
tuberculosis. The species-specific PCR assays
developed for the differentiation of Mycobacterium
tuberculosis and Mycobacterium bovis have been
invalidated due to false negative results owing to
absence of specific target sequences such as mtp40 in
some Mycobacterium tuberculosis strains (Gillespie
and McHugh, 1997 and Cousins, 1992).
In conclusion, in this study PCR results were same
with routine culture results. Therefore, PCR is a rapid
tool to detect Mycobacterium tuberculosis in urine
samples for UTB patients.
We thank to E. Damla Büyüktunçer for editorial
comments and advisory critics.
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