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Polymerase chain reaction is a good diagnostic tool - Journal of Cell ...

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

correspondence)

Received 3 June 2003; Accepted 27 June 2003

Abstract

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

reaksiyonunun kullan›m›

Özet

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›

Introduction

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.

99


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

Patients

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.

DNA extraction

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).

PCR analysis

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).

Results

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

(Table 1).

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.

Discussion

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.

Acknowledgement

We thank to E. Damla Büyüktunçer for editorial

comments and advisory critics.

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