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
of the body. While pulmonary tuberculosis has its most
common presentation, extrapulmonary tuberculosis is
also an important clinical problem (Centers for Disease
Control and Prevention 2000; Gopal et al., 2001).
Differentiation between pulmonary and extrapulmonary
tuberculosis depends on the location of the infection that
can be either inside or outside the lungs. The most
frequent forms of extrapulmonary reported types are:
lymphatic, pleural, bone and/or joint, central nervous
system, and abdominal. Other types such as miliary,
pericarditis and genitourinary forms have also been
frequently reported.
Applied diagnostic methods are observation of AFB
(Acid Fast Bacillai) and culture in most of clinical
laboratories. However, the sensitivity of AFB smear and
culture are low (Ersoz et al., 1998; Garg et al., 2003;
Chakravorty et al., 2005) especially in extrapulmonary
cases. Thus, there remain samples that are both AFB
and culture negative. Culture provides definitive
identification but it is time-consuming, requiring 6 – 8 weeks
*Corresponding author. rahbar@health.gov.ir.
weeks. Its efficacy in extrapulmonary tuberculosis is
rather low BACTEC system is capable of reducing the
required time for isolation but has not been applied in
most laboratories of our country.
Amplification techniques for the diagnosis of
tuberculosis have attracted considerable interest in
diagnosis, particularly with the hope of shortening the
time required to detect and identify Mycobacterium
tuberculosis in respiratory and non-respiratory specimens
(Hajia et al., 2005; Nagesh et al., 2001; Parandaman et
al. 2000). However, despite numerous reports in the
literature (Woods, 2001; Moon et al., 2005), amplification
techniques do not yet have an established role in the
laboratory for tuberculosis diagnosis, nor have they
replaced traditional techniques. The most important pitfall
is false negative results. Weak extraction procedures
result false negative due to the presence of inhibitors,
especially in suspected TB samples, that several clinical
specimens are received in diagnostic laboratories.
Availability of various commercial extraction kits has
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
increasing the efficiency and reproducibility of the results.
878 Afr. J. Microbiol. Res
Table 1. Applied program for amplification of M. tuberculosis PCR.
Temperature (°°C) Time (s) Number of repeats
94 180 1 sterile conditions before being processed. For all specimens, half of
94 50
64 50 5 the target amplification procedure, and the other half was
72 30
95 30
64 50 40
67 20
10 Storage
Although numerous studies have contributed to our
understanding of PCR performance for diagnosis
pulmonary and extra-pulmonary tuberculosis, PCR has
not provided suitable contentment. Here, we have
analyzed an extended number of various clinical samples
to estimate the rate of various extrapulmonary forms and
to compare its sensitivity with microscopic examination
and culture.
MATERIALS AND METHODS
Definitions
Extra-pulmonary TB may be characterized by swelling of the
particular infected site (lymph node), mobility impairment (spine), or
severe headache and neurological dysfunction (TB meningitis) etc.
Clinical presentation is characterized by chronic pain and swelling
as the principal features. The criteria for a positive diagnosis of
tuberculosis in this study were considered as follows: documented
tuberculosis with positive culture and/or positive microscopic
examination or probable tuberculosis with compatible clinical and
radiographic evidence. In cases of extrapulmonary tuberculosis,
which remained without bacteriologic or histological confirmation,
diagnosis was based on recognition of signs and symptoms raised
from involvement of particular organ or system (WHO, 2001).
Patients and specimens
Two thousand one hundred and twenty three pulmonary and
extrapulmonary specimens were analyzed by microscopic
examination, culture and PCR technique. These specimens were
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
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
obtained and analyzed by Microsoft Excel during their
hospitalization (Version, 2007).
Specimen processing
Upon receipt, the specimens were stored at 4°C prior to being
processed. Fluid samples were first centrifuged at 3,000 g for 20
min. Bone marrow aspirates were received either in Isolators or in
Vacutainers (Becton Dickinson, Le Pont de Claix, France). All
specimens were divided after being processed in the laboratory,
with approximately two-thirds used for culture and one-third used
for DNA extraction or preparation.
Tissue specimens were cut and homogenized in a mortar under
the sediment or the tissue biopsy specimen was stored at 20°C for
inoculated onto culture medium and used for acid-fast staining.
Pettrof decontamination method was applied for non-sterile
specimens (such as sputum and so on). Smears were directly
prepared for staining by Zeihl-Nelsen method for each specimen
(Rahbar et al., 2007). In addition, all specimens were inoculated
onto 2 slops of Lowenstein–Jensen and incubated at 35°C. Slops
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).
DNA extraction and PCR protocols
DNA extraction was performed in an identical manner for all
patients’ samples using High Pure PCR Template preparation kit
(provided from Roche Co.). The kit is designed to purify nucleic
acids from different requested specimens for PCR test. It contains a
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
guanidium-HCl, 10 mM urea, 20% Triton X -100 and Tris-HCl) on
samples, then use of inhibitor removal, washing and elution buffers
respectively. At each step reagents will be added to the filter tube.
Supernatant will be passed through collection tube after
centrifugation. This procedure will highly reduce the contaminations
and increase the efficiency of the recovery rate of the nucleic acids
as much as possible.
PCR carried out on the prepared purified nucleic acid use of M.
tuberculosis PCR kit (DNA Technology Co.). It contained specific
primers to target transposable element (IS6110) for amplification
330 base pair of template. 5 µl of template, 10 µl PCR buffer, 10 µl
mixture (containing specific primers and dNTP, 2.5U taq
polymerase) were mixed and amplified with the recommended
program (Table 1). The applied PCR kit was constructed in a format
of competitive PCR with internal control. Provided specific primers
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
extraction procedure. The kit was also contained specific labeled
probes for specific and internal products to enable us for detection
the amplified products by the fluorescence detector (DNA
Technology Co.), called Fluorescent Amplification-based Specific
Hybridization method.
RESULTS
Between January, 2007 till December, 2008, 113 TB
positive cases (5.18%) were reported from a total 2123
suspected cases. Tuberculosis was confirmed in 87
patients by laboratory results while the remained 26
patients were diagnosed based of just their clinical mani-
festation. Pulmonary and extra-pulmonary tuberculosis
were proved in 48 (42.48%) and 65 (57.52%) cases with
the mean age of 34.95, 42.19 years old for them
respectively. Male patients had lower pulmonary and
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
The most frequent type of extrapulmonary forms was
central nervous system 22 (33.84%), followed by pleural
18 (27.70%), skeletal 9 (13.84%), abdominal and
lymphatic forms 6 for each (9.23%) (Table 3 and 4). Two
out of six lymphatic specimens were excised biopsy of
the lymph nodes with positive results for AFB, culture and
PCR.
tuberculosis patients in this study. The sensitivity of these
three tests was reduced in extrapulmonary forms. It was
70.79, 40 and 21.53% respectively (Table 3). The highest
rate of microscopic examination was determined in
pleural, but culture and PCR had the highest rate in CSF
specimens (Table 4). All culture positive had PCR
positive results too, while 38 of culture negative had also
positive results by PCR method (Figure 1 and Table 5).

Sensitivity and Specificity of the PCR
The lowest detection limit of the test was 500 micro-
organisms in 1 ml of the specimens. The test has
negative results with a wide range of commensal and
pathogenic organisms encountered in the respiratory
tract.
Results of clinical specimens
Sensitivity of the PCR, culture and staining methods were
76.99, 43.36 and 23.43% in all confirmed positive
DISCUSSION
In our study, extrapulmonary TB was determined in
57.52% of cases which is very high in comparison with
other reports (Beek et al., 2006). Distribution of Extra-pul-
monary TB is in a wide range in different provinces, with
the average of 27.63% of all proved TB cases that were
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
reported that co-infection with HIV raised the extra-
pulmonary tuberculosis (Iscman, 2000; Fanning, 1999).
However we are not able to evaluate the role of HIV
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
studied patients. It seems other parameters should have
also influenced on the increased rate of this study.
According to the Iranian National Tuberculosis Program
(NTP) guidelines, physicians-in-charge and laboratories
are obliged to report TB patients after confirmation of the
diagnosis to the district health centers of the MOH for
notification. Thereafter, the rule is to treat patients under
direct supervision of the health centers. But obviously
there is currently no active supervision on private sector,
despite the fact that a large number of patients are
treated in this sector specially in urban population, more
attention should be paid to strengthening
communications between the private and public sectors
(Masjedi et al., 2007).
Improper anti-tuberculosis therapy is another affecting
parameter that should be considered. Masjedi (Masjedi et
al., 2006) reported 54.1% of 695 culture positive
specimens were susceptible to all 4 anti-tuberculosis
drugs tested. Mirsaeidi et al. (2005) reported 76% of
Iranian patients with MDR TB responded well to the
second-line treatment. The best way to stop MDR TB is
to detect and treat drug-susceptible or drug-resistant TB
before it evolves into MDR TB and before it spreads.
Therefore, it is essential to perform drug-susceptibility
testing on the first-line drugs, to identify patients with
drug-resistant and MDR TB, and, for patients receiving
the relevant second-line agents, to optimize the treatment
of drug-resistant disease. At the present time there are
only a few centers which are carrying out the
susceptibility test for mycobacterium in Iran this needs to
be more developed.
However it seems main affecting parameter can be
laboratory diagnosis of the TB. Sensitivity of the culture
was 40% in extrapulmonary tuberculosis in this study
.Therefore if more sensitive method could be applied the
diagnosis rate of extrapulmonary TB will be increased.
The poor performance of conventional microbiological
techniques in extrapulmonary specimens has stimulated
the increased use of PCR tests in the laboratory
diagnosis of tuberculosis. The exact diagnostic role of
PCR assay for M. tuberculosis in high-prevalence areas
for tuberculosis has to be assessed, particularly in the
case of extrapulmonary tuberculosis.
There are numerous examples in the literature of
amplification-based test performances being marred by
inhibitory substances present in clinical specimens,
notably biopsies and proteinaceous pleural effusions
which could include blood, host proteins, and even
eukaryotic DNA that can inhibit amplification when
present in a high concentration.
Sensitivity of the PCR will be dropped in those
improper taken specimens and sent samples as well as
those inappropriate extracted specimens. In contrast to
pulmonary specimens, the major problem of
extrapulmonary specimens is insufficient volume of
specimens. The second major inconvenience of PCR in
extrapulmonary specimens is the presence of inhibitors,
which interfere with amplification-based techniques. A
multistep process is often required to eliminate inhibitors

and to obtain highly purified DNA. Therefore, the major
difficulty with mycobacterium is achieving best recovery
rate of nucleic acid from specimens. Commercial applied
extraction kits and use of signal based technique caused
increasing sensitivity and accuracy of the test in the
present study. The Roche extraction kit provides suitable
preparation guideline for various clinical specimens
(Sputum, BAL, Ascitic fluid, blood, pleural fluid, tissue
biopsy specimens, bone marrow aspirates, urine and so
on).
Conclusion
Apply pretreatment protocols for various samples before
extraction and use of internal control achieved enabled
us to have high performance of the PCR technique.
Necessity of Dots performance for urban population the
same as rural population, and controlling the MDR
tuberculosis and HIV infection seems to be the affecting
parameters to reduce extrapulmonary TB.
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