African Journal of Microbiology Research Vol. 3(12) pp.

877-881 October, 2009

Available online http://www.academicjournals.org/ajmr
ISSN 1996-0808 ©2009 Academic Journals

Full Length Research Paper

Is PCR assay reliable for diagnosis of extrapulmonary

tuberculosis?
Massoud Hajia1, Mohammad Rahbar1, 2* and Rana Amini1
1
Research Center and Health Reference Laboratory, Iran.
2
Milad Hospital, Tehran, Iran.
Accepted 8 October, 2009

Extrapulmonary tuberculosis is one of the major problems in clinical practice. The aim of this study was
to evaluate the rate of all diagnosed tuberculosis cases among those urban patients referred to private
clinical laboratories, and comparing the results of PCR technique with traditional methods. In the
present study, we examined 2123 specimens who were referred from private clinical laboratories
between 2006 till 2008. All specimens were stained for acid fast bacilli, cultured with standard
procedures and tested with PCR using signal based method after ensuring of nucleic acid purification.
One hundred and thirteen patients were positive for TB, pulmonary tuberculosis were proved in 48
patients whilst the remaining 65 cases were classified as extrapulmonary tuberculosis. Positive rates
for PCR, culture and staining were 41, 23 and 12 respectively. The respective figures for extrapulmonary
tuberculosis were 46, 26 and 14. This study demonstrates that PCR has a high sensitivity in diagnosis
of TB than traditional method.

Key words: Extrapulmonary tuberculosis, PCR methods.

INTRODUCTION

Tuberculosis can potentially involve any system or organ weeks. Its efficacy in extrapulmonary tuberculosis is
of the body. While pulmonary tuberculosis has its most rather low BACTEC system is capable of reducing the
common presentation, extrapulmonary tuberculosis is required time for isolation but has not been applied in
also an important clinical problem (Centers for Disease most laboratories of our country.
Control and Prevention 2000; Gopal et al., 2001). Amplification techniques for the diagnosis of
Differentiation between pulmonary and extrapulmonary tuberculosis have attracted considerable interest in
tuberculosis depends on the location of the infection that diagnosis, particularly with the hope of shortening the
can be either inside or outside the lungs. The most time required to detect and identify Mycobacterium
frequent forms of extrapulmonary reported types are: tuberculosis in respiratory and non-respiratory specimens
lymphatic, pleural, bone and/or joint, central nervous (Hajia et al., 2005; Nagesh et al., 2001; Parandaman et
system, and abdominal. Other types such as miliary, al. 2000). However, despite numerous reports in the
pericarditis and genitourinary forms have also been literature (Woods, 2001; Moon et al., 2005), amplification
frequently reported. techniques do not yet have an established role in the
Applied diagnostic methods are observation of AFB laboratory for tuberculosis diagnosis, nor have they
(Acid Fast Bacillai) and culture in most of clinical replaced traditional techniques. The most important pitfall
laboratories. However, the sensitivity of AFB smear and is false negative results. Weak extraction procedures
culture are low (Ersoz et al., 1998; Garg et al., 2003; result false negative due to the presence of inhibitors,
Chakravorty et al., 2005) especially in extrapulmonary especially in suspected TB samples, that several clinical
cases. Thus, there remain samples that are both AFB specimens are received in diagnostic laboratories.
and culture negative. Culture provides definitive Availability of various commercial extraction kits has
identification but it is time-consuming, requiring 6 – 8 weeks allowed the diagnostic laboratories to achieve amplify-
cation techniques with higher accuracy. Introducing
commercial PCR kits especially signal based procedures
has also caused in reduction of PCR errors and
*Corresponding author. rahbar@health.gov.ir. increasing the efficiency and reproducibility of the results.
878 Afr. J. Microbiol. Res

Table 1. Applied program for amplification of M. tuberculosis PCR. specimens were divided after being processed in the laboratory,
with approximately two-thirds used for culture and one-third used
Temperature (°°C) Time (s) Number of repeats for DNA extraction or preparation.
Tissue specimens were cut and homogenized in a mortar under
94 180 1 sterile conditions before being processed. For all specimens, half of
94 50 the sediment or the tissue biopsy specimen was stored at 20°C for
64 50 5 the target amplification procedure, and the other half was
inoculated onto culture medium and used for acid-fast staining.
72 30
Pettrof decontamination method was applied for non-sterile
95 30 specimens (such as sputum and so on). Smears were directly
64 50 40 prepared for staining by Zeihl-Nelsen method for each specimen
67 20 (Rahbar et al., 2007). In addition, all specimens were inoculated
onto 2 slops of Lowenstein–Jensen and incubated at 35°C. Slops
10 Storage were examined for growth of M. tuberculosis. Suspected colonies
were examined for Acid Fast bacilli with Zeihl-Nelsen methods and
confirmed by biochemical tests. All biosafety rules were considered
in accordance with WHO biosafety manual (WHO, 2004).
Although numerous studies have contributed to our
understanding of PCR performance for diagnosis
pulmonary and extra-pulmonary tuberculosis, PCR has DNA extraction and PCR protocols
not provided suitable contentment. Here, we have
analyzed an extended number of various clinical samples DNA extraction was performed in an identical manner for all
patients’ samples using High Pure PCR Template preparation kit
to estimate the rate of various extrapulmonary forms and (provided from Roche Co.). The kit is designed to purify nucleic
to compare its sensitivity with microscopic examination acids from different requested specimens for PCR test. It contains a
and culture. primary step as a pre-lysis for some specific specimens such as
tissue or even embedded tissue. The main steps are started of
applying proteinase K and binding buffer (containing 6 M
MATERIALS AND METHODS guanidium-HCl, 10 mM urea, 20% Triton X -100 and Tris-HCl) on
samples, then use of inhibitor removal, washing and elution buffers
Definitions respectively. At each step reagents will be added to the filter tube.
Supernatant will be passed through collection tube after
Extra-pulmonary TB may be characterized by swelling of the centrifugation. This procedure will highly reduce the contaminations
particular infected site (lymph node), mobility impairment (spine), or and increase the efficiency of the recovery rate of the nucleic acids
severe headache and neurological dysfunction (TB meningitis) etc. as much as possible.
Clinical presentation is characterized by chronic pain and swelling PCR carried out on the prepared purified nucleic acid use of M.
as the principal features. The criteria for a positive diagnosis of tuberculosis PCR kit (DNA Technology Co.). It contained specific
tuberculosis in this study were considered as follows: documented primers to target transposable element (IS6110) for amplification
tuberculosis with positive culture and/or positive microscopic 330 base pair of template. 5 µl of template, 10 µl PCR buffer, 10 µl
examination or probable tuberculosis with compatible clinical and mixture (containing specific primers and dNTP, 2.5U taq
radiographic evidence. In cases of extrapulmonary tuberculosis, polymerase) were mixed and amplified with the recommended
which remained without bacteriologic or histological confirmation, program (Table 1). The applied PCR kit was constructed in a format
diagnosis was based on recognition of signs and symptoms raised of competitive PCR with internal control. Provided specific primers
from involvement of particular organ or system (WHO, 2001). could also amplify a product from fragment encoding 900 base pair
as internal control to ensure of proper extraction and removal of any
expected inhibitors. This fragment was added before commencing
Patients and specimens extraction procedure. The kit was also contained specific labeled
probes for specific and internal products to enable us for detection
Two thousand one hundred and twenty three pulmonary and the amplified products by the fluorescence detector (DNA
extrapulmonary specimens were analyzed by microscopic Technology Co.), called Fluorescent Amplification-based Specific
examination, culture and PCR technique. These specimens were Hybridization method.
collected from various clinical laboratories to investigate for
tuberculosis infections in Tehran during two years from January,
2006 till December, 2008. All of the specimens were collected prior RESULTS
to the commencement of antituberculosis chemotherapy. Clinical
information of patients (fever, weight loss, night sweats, chest x-ray,
previous history of tuberculosis, place of birth, and life style) were Between January, 2007 till December, 2008, 113 TB
obtained and analyzed by Microsoft Excel during their positive cases (5.18%) were reported from a total 2123
hospitalization (Version, 2007). suspected cases. Tuberculosis was confirmed in 87
patients by laboratory results while the remained 26
patients were diagnosed based of just their clinical mani-
Specimen processing
festation. Pulmonary and extra-pulmonary tuberculosis
Upon receipt, the specimens were stored at 4°C prior to being were proved in 48 (42.48%) and 65 (57.52%) cases with
processed. Fluid samples were first centrifuged at 3,000 g for 20 the mean age of 34.95, 42.19 years old for them
min. Bone marrow aspirates were received either in Isolators or in respectively. Male patients had lower pulmonary and
Vacutainers (Becton Dickinson, Le Pont de Claix, France). All higher extrapulmonary rate than female (Table 2).
 Hajia et al. 879

Table 2. Frequency of pulmonary and extrapulmonary TB in Men and Women.

Men Women Total

Pulmonary 21 (18.59%) 27 (23.89%) 48 (42.48%)
Extrapulmonary 38 (33.63%) 27 (23.89%) 65 (57.52%)
Total 59 (52.22%) 54 (47.78) 113

Table 3. Comparison of three applied methods.

Total positive Smear Culture PCR

Pulmonary 48 12 (25%) 23 (48.92%) 41 (85.42%)
Extrapulmonary 65 14(21.53%) 26(40%) 46(70.79%)
Total 113 26 49 87

Table 4. Comparison of three diagnosis methods applied in various tested samples.

Specimens Total positive Smear Culture PCR

1 CSF 22 - 9 16
2 Pleural 18 6 7 13
3 Synovial and bone marrow aspiration 9 2 3 7
4 Asitic fluid 6 - - 5
5 Urine 2 - - 1
6 Lymph node 6 - 2 4
7 Pericarditis 2 - - -
Total 65 14 26 46

Frequency of various tuberculosis forms tuberculosis patients in this study. The sensitivity of these
three tests was reduced in extrapulmonary forms. It was
The most frequent type of extrapulmonary forms was 70.79, 40 and 21.53% respectively (Table 3). The highest
central nervous system 22 (33.84%), followed by pleural rate of microscopic examination was determined in
18 (27.70%), skeletal 9 (13.84%), abdominal and pleural, but culture and PCR had the highest rate in CSF
lymphatic forms 6 for each (9.23%) (Table 3 and 4). Two specimens (Table 4). All culture positive had PCR
out of six lymphatic specimens were excised biopsy of positive results too, while 38 of culture negative had also
the lymph nodes with positive results for AFB, culture and positive results by PCR method (Figure 1 and Table 5).
PCR.

DISCUSSION
Sensitivity and Specificity of the PCR
In our study, extrapulmonary TB was determined in
The lowest detection limit of the test was 500 micro- 57.52% of cases which is very high in comparison with
organisms in 1 ml of the specimens. The test has other reports (Beek et al., 2006). Distribution of Extra-pul-
negative results with a wide range of commensal and monary TB is in a wide range in different provinces, with
pathogenic organisms encountered in the respiratory the average of 27.63% of all proved TB cases that were
tract. under-supervision of Ministry of Health (MOH) (MOH,
2008). All these suspected patients referred to the MOH
services are diagnosed just by routine procedures. It is
Results of clinical specimens reported that co-infection with HIV raised the extra-
pulmonary tuberculosis (Iscman, 2000; Fanning, 1999).
Sensitivity of the PCR, culture and staining methods were However we are not able to evaluate the role of HIV
76.99, 43.36 and 23.43% in all confirmed positive infection on the increasing of extrapulmonary tuberculosis
880 Afr. J. Microbiol. Res

Figure 1. Analysis of PCR Result in fluorescent detector. No.1: negative control, No.2: Positive control, No. 3 is positive TB
samples. No. 4 and 5 is back ground to calibrate the applied labeled probes.

Table 5. Comparison the results of PCR with culture in examined specimens.

Culture positive Culture negative

Number tested PCR positive PCR Negative PCR positive PCR Negative
Pulmonary 46 23 0 18 5
Extrapulmonary 65 26 0 20 19
Total 113 49 0 38 26

tuberculosis, since no HIV test has been applied for these susceptibility test for mycobacterium in Iran this needs to
studied patients. It seems other parameters should have be more developed.
also influenced on the increased rate of this study. However it seems main affecting parameter can be
According to the Iranian National Tuberculosis Program laboratory diagnosis of the TB. Sensitivity of the culture
(NTP) guidelines, physicians-in-charge and laboratories was 40% in extrapulmonary tuberculosis in this study
are obliged to report TB patients after confirmation of the .Therefore if more sensitive method could be applied the
diagnosis to the district health centers of the MOH for diagnosis rate of extrapulmonary TB will be increased.
notification. Thereafter, the rule is to treat patients under The poor performance of conventional microbiological
direct supervision of the health centers. But obviously techniques in extrapulmonary specimens has stimulated
there is currently no active supervision on private sector, the increased use of PCR tests in the laboratory
despite the fact that a large number of patients are diagnosis of tuberculosis. The exact diagnostic role of
treated in this sector specially in urban population, more PCR assay for M. tuberculosis in high-prevalence areas
attention should be paid to strengthening for tuberculosis has to be assessed, particularly in the
communications between the private and public sectors case of extrapulmonary tuberculosis.
(Masjedi et al., 2007). There are numerous examples in the literature of
Improper anti-tuberculosis therapy is another affecting amplification-based test performances being marred by
parameter that should be considered. Masjedi (Masjedi et inhibitory substances present in clinical specimens,
al., 2006) reported 54.1% of 695 culture positive notably biopsies and proteinaceous pleural effusions
specimens were susceptible to all 4 anti-tuberculosis which could include blood, host proteins, and even
drugs tested. Mirsaeidi et al. (2005) reported 76% of eukaryotic DNA that can inhibit amplification when
Iranian patients with MDR TB responded well to the present in a high concentration.
second-line treatment. The best way to stop MDR TB is Sensitivity of the PCR will be dropped in those
to detect and treat drug-susceptible or drug-resistant TB improper taken specimens and sent samples as well as
before it evolves into MDR TB and before it spreads. those inappropriate extracted specimens. In contrast to
Therefore, it is essential to perform drug-susceptibility pulmonary specimens, the major problem of
testing on the first-line drugs, to identify patients with extrapulmonary specimens is insufficient volume of
drug-resistant and MDR TB, and, for patients receiving specimens. The second major inconvenience of PCR in
the relevant second-line agents, to optimize the treatment extrapulmonary specimens is the presence of inhibitors,
of drug-resistant disease. At the present time there are which interfere with amplification-based techniques. A
only a few centers which are carrying out the multistep process is often required to eliminate inhibitors
 Hajia et al. 881

and to obtain highly purified DNA. Therefore, the major Gopal R, Padmavathy BK, Vasanthi S, Jayashree K (2001). Extra-
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