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.
[email protected].
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 amplifycation 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.
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Table 1. Applied program for amplification of M. tuberculosis PCR.
Temperature (°°C) 94 94 64 72 95 64 67 10
Time (s)
Number of repeats
180 50 50 30 30 50 20 Storage
1 5
40
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 sterile conditions before being processed. For all specimens, half of the sediment or the tissue biopsy specimen was stored at 20°C for the target amplification procedure, and the other half was 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 manifestation. 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).
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Table 2. Frequency of pulmonary and extrapulmonary TB in Men and Women.
Men 21 (18.59%) 38 (33.63%) 59 (52.22%)
Pulmonary Extrapulmonary Total
Women 27 (23.89%) 27 (23.89%) 54 (47.78)
Total 48 (42.48%) 65 (57.52%) 113
Table 3. Comparison of three applied methods.
Pulmonary Extrapulmonary Total
Total positive 48 65 113
Smear 12 (25%) 14(21.53%) 26
Culture 23 (48.92%) 26(40%) 49
PCR 41 (85.42%) 46(70.79%) 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. Sensitivity and Specificity of the PCR The lowest detection limit of the test was 500 microorganisms 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
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). 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-pulmonary 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 extrapulmonary tuberculosis (Iscman, 2000; Fanning, 1999). However we are not able to evaluate the role of HIV infection on the increasing of extrapulmonary tuberculosis
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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.
Number tested Pulmonary Extrapulmonary Total
46 65 113
Culture positive PCR positive PCR Negative 23 0 26 0 49 0
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
Culture negative PCR positive PCR Negative 18 5 20 19 38 26
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
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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. REFERENCES Beek LAMT, Werf MJVD, Richter C, Borgdorff MW (2006). Extrapulmonary tuberculosis by nationality, the Netherlands, 19932001. Emerg. Infect. Dis., 12 (9). Centers for Disease Control and Prevention (2000). Surveillance Report. Reported Tuberculosis in the United States. Atlanta, GA: CDC. Ersoz C, Polat A, Serin MS, Soylu L, Demircan O (1998). Fine needle aspiration (FNA) cytology in tuberculous lymphadenitis. Cytopathol., 9: 201–207. Chakravorty S, Sen MK, Tyagi JS (2005). Diagnosis of Extrapulmonary Tuberculosis by Smear, Culture, and PCR Using Universal Sample Processing Technology. J. Clin. Microbiol., 43(9): 4357-62. Fanning A (1999). Tuberculosis: 6. Extrapulmonary disease. CMAJ., 160: 1597-603.
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Gopal R, Padmavathy BK, Vasanthi S, Jayashree K (2001). Extrapulmonary Tuberculosis – a retrospective study. Indian J. 'Tuber., 48(4): 225-6. Garg SK, Tiwari RP, Tiwari D, Singh R, Malhotra D, Ramnani VK, Prasad GBKS, Chandra R, Fraziano M, Colizzi V, Bisen PS (2003). Diagnosis of tuberculosis: Available technologies, limitations, and possibilities. J. Clin. Lab. Anal., 17(5): 155–163. Hajia M, Rahbar M, Alkhani Y (2005). Efficiency of PCR Method for Screening Pulmonary Tuberculosis Patients under DOTs Protocol Therapy. Iranian J. Public Health, 34(2): 9-13. Iscman MD (2000). Tuberculosis in relation to human immunodeficiency virus and acquired immunodeficiency syndrome. In: Iseman MD, editor. A clinician’s guide to tuberculosis. Philadelphia: Lippincott Williams and Wilkins. pp. 199-252. Masjedi MR, Fadaizadeh L, Taghizadeh AR (2007). Notification of patients with tuberculosis detected in the private sector, Tehran, Iran. The International J. Tuber. Lung Dis., 11(8): 882-886. Masjedi MR, Farnia P, Sorooch S, Pooramiri MV, Mansoori SD, Zarifi AZ (2006). Extensively drug-resistant tuberculosis: 2 years of surveillance in Iran. Clin. Infect. Dis. 43: 841–7. Ministry of Health, Iran CDC, Frequency and Incidence of Tuberculosis in 1386 (April 2007- March 2008). Mirsaeidi SM, Tabarsi P, Khoshnood K (2005). Treatment of multidrugresistant tuberculosis (MDR-TB) in Iran (preliminary report). Int. J. Infect. Dis., 6: 317–22. Moon JW, Chang YS, Kim SK, Kim YS, Lee HM, Kim SK, Chang J (2005). The Clinical Utility of Polymerase Chain Reaction for the Diagnosis of Pleural Tuberculosis. Clin. Infect. Dis., 41: 660–666. Nagesh B, Sehgal SS, Jindal SK, Arora SK (2001). Evaluation of polymerase chain reaction for the detection of Mycobacterium tuberculosis in pleural fluid. Chest, 119: 1737-1741. Parandaman V, Narayanan S, Narayanan PR (2000). Utility of polymerase chain reaction using two probes for rapid diagnosis of tubercular pleuritis in comparison to conventional methods. Indian J. Med. Res., 112: 47-51. Rahbar M, Hajia M (2007). Value of gastric lavage for diagnosis of pulmonary tuberculosis. Pakistan J. Med. Sci., 23(1): 51-53. Woods GL (2001). Molecular techniques in mycobacterial detection. Arch. Pathol. Lab. Med., 125:122–126. World Health Organization (WHO) (2001). TB Manual National Tuberculosis Program Guidelines. Warsaw. World Health Organization (WHO) (2004). Laboratory Biosafety Manual (Third Edition). Geneva.
