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ORIGINAL ARTICLE  
Year : 2015  |  Volume : 58  |  Issue : 1  |  Page : 27-30
Role of multiplex polymerase chain reaction using IS6110 and Protein b for the diagnosis of extra-pulmonary tuberculosis: North India


1 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Neurology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication11-Feb-2015
 

   Abstract 

Background: Prompt and accurate diagnosis of extra-pulmonary tuberculosis (TB) is highly challenging. Current conventional techniques lack sensitivity and are time-consuming. Here, we report our experience with multiplex polymerase chain reaction (MPCR) using two targets namely IS6110 and protein antigen b in the diagnosis of extra-pulmonary TB. Materials and Methods: A total of 150 patients of extra-pulmonary TB visiting tertiary care center in north India between September 2008 and December 2009 were included in the study. Sixty-six biopsy samples and 84 were body fluids from these patients were subjected for microscopy (Ziehl-Neelsen), culture on LJ medium and for Multiplex PCR using IS6110 and Protein b antigen. Results: Smear positivity was noted in 11 samples (7.33%), and LJ culture yielded Mycobacterium tuberculosis in 8 biopsies and 9 body fluids with overall positivity of 11.3%. The multi-targeted PCR could detect M. tuberculosis in a total of 112 samples. Of 112 positive samples, only Protein b band was detected in 7 samples and only IS6110 was detected in 5 samples. Overall Protein b, PCR could detect 71.33% of the cases, whereas IS6110 was positive in 66.6% of the cases. Overall the sensitivities of microscopy, culture, IS6110 PCR, Protein b PCR and MPCR were 7.33%, 11.3%, 66.67%, 71.3% and 74.6%, respectively. Thus by using more than two targets the sensitivity increased from 66.67% of IS6110 to 74.6% in MPCR. Conclusion: Multiplex polymerase chain reaction using IS6110 and Protein b antigen is a highly sensitive and specific tool in the diagnosis of pauci-bacillary conditions like extra-pulmonary TB.

Keywords: Extra-pulmonary tuberculosis, multiplex polymerase chain reaction, Protein b antigen, IS6110

How to cite this article:
Sharma K, Appannanavar SB, Modi M, Singh M, Sharma A, Varma S. Role of multiplex polymerase chain reaction using IS6110 and Protein b for the diagnosis of extra-pulmonary tuberculosis: North India. Indian J Pathol Microbiol 2015;58:27-30

How to cite this URL:
Sharma K, Appannanavar SB, Modi M, Singh M, Sharma A, Varma S. Role of multiplex polymerase chain reaction using IS6110 and Protein b for the diagnosis of extra-pulmonary tuberculosis: North India. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Jul 5];58:27-30. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/27/151164



   Introduction Top


Extra-pulmonary tuberculosis (TB) accounts for around 20-50% of all TB cases. [1] Prompt and accurate diagnosis is essential for better management of this group of pauci-bacillary patients. Currently, the diagnosis of extra-pulmonary TB relies on clinical features, imaging findings and conventional microbiological tools like microscopy and culture. Microscopy is quite insensitive especially because of the pauci-bacillary nature of extra-pulmonary TB. Although culture is a gold standard, it has a slow turnaround time of 6-8 weeks. Thus, there is a need for rapid, sensitive and specific tests for the diagnosis of extra-pulmonary TB. Nucleic acid based tests have proven beneficial in the diagnosis of TB. The polymerase chain reaction is the most common nucleic acid amplification test. [2] A large number of sequences of mycobacterial genome like IS6110, IS986, 65 kDa and 38 kDa antigens have been used as targets in polymerase chain reaction (PCR) test. [3],[4] Most of the PCR-based studies have shown single target, out of which IS6110 is the most commonly used target because it has multiple copies (16-20). [5] However, in the Indian subcontinent, Chauhan et al. have shown that 10-40% of the Mycobacterial strains either have low copies or no copy of IS6110. [6] More reliable report may be obtained by using more than one target. Another important target is protein antigen b (38 kDa protein), which is specific for Mycobacterium tuberculosis complex and has shown good clinical utility in certain studies. [7],[8] Studies regarding evaluation of Protein b in the diagnosis of extra-pulmonary TB are scanty from our region. [7],[8] Thus in the current study, we report our experience in multiplex PCR using IS6110 and Protein b in the diagnosis of extra-pulmonary TB, and compare the multiplex polymerase chain reaction (MPCR) results with ZN smear examination and conventional culture using LJ in patients suspected of TB visiting tertiary care center in north India.


   Materials and Methods Top


Study population

The study was approved by institute ethics committee. Cases visiting our tertiary care center in North India, for mycobacterial culture, along with appropriate information were included in the study. A total of 150 clinical samples obtained from patients of extra-pulmonary TB with a strong clinical/radiological/histopathological evidence of TB visiting our center between September 2008 and December 2009 were included in the study. Out of 150 samples, 66 samples were biopsy samples and remaining 84 were body fluids. The cases were divided into three groups; first group included confirmed cases (n-17), the second group included suspected cases of TB (n = 133), which were further confirmed by their response to anti-tubercular therapy (ATT). Response to ATT during the follow-up period (feeling of wellbeing/weight gain/disappearance of fever) was taken as the gold standard in the study. The third group including 60 non TB cases served as a control group.

Sample processing

Clinical samples received from suspected cases of TB were processed in the mycobacteriology division of our tertiary care center. Body fluids like synovial fluid, pleural fluid and ascitic fluid which were collected aseptically and were expected to have no other contaminating bacteria were inoculated without decontamination procedure. Tissue samples collected aseptically were ground in a preautoclaved mortar and pestle and inoculated as such. Slides were stained by Ziehl-Neelsen method. The mycobacterial isolates obtained were subjected to limited biochemical testing for species' characterization by carrying out Niacin test and p-nitro-a-acetyl amino-b-hydroxypropriophenone sensitivity test.

Sample preparation for DNA extraction

Commercially available Qiagen DNA extraction kit was used for DNA extraction from samples like; body fluids (pleural fluid, ascitic fluid, synovial fluid) and tissue (skin, lymph node and other biopsy material). The DNA from all the samples were subjected to multi-targeted PCR using both sets of primers; IS6110 and Protein b in the same reaction.

Multiplex polymerase chain reaction protocol

Multiplex PCR was standardized and was found to have quantitative sensitivity to detect the DNA equivalent to 2-3 organisms. It tested positive with standard strain of M. tuberculosis, H37RV. In each independent MPCR assay, test results were compared with the results for one positive and one negative control. The positive control included was the DNA of H37Rv and negative control included was the PCR grade water. Identification of M. tuberculosis was done using a specific pair of primers designed to amplify IS6110 and Protein b in the M. tuberculosis complex and the expected band size was about 123 bp for IS6110, and 419 bp for Protein b [Figure 1]. The sequences of primers used for IS6110 were; ISI: 5'-CCTGCGAGCGTAGGCGT 3 and IS2: 5'-CTCGTCCAGCGCCGCTTCGG 3'. Primers used for Protein b were; Pb1:5'-TCCGCTGCCAGTCGTCTTCC-3', Pb2:5'-GTCCTCGCGAGT CTAGGCCA-3'. The MPCR mixture (for 50 μl reaction) included; PCR buffer 10X, dNTPs (Mix) 10 mM, Primer IS1 (10 pm/μl), IS2 (10 pm/μl), Pb1 (10 pm/μl) and Pb2 (10 pm/μl), Taq polymerase 5 U/μl, DNA template and water. DNA amplification was performed for 35 cycles following an initial denaturation step at 95°C for 5 min in thermo cycler by using the following program: Denaturation at 94°C for 1 min, annealing at 65°C for 1.5 min, extension at 72°C for 1.5 min and final extension: 72°C for 10 min.
Figure 1: L1-100bp MM, L2: Positive control with 123bp (IS6110 band) and 419bp (Protein b band), L3, L4 : Positive clinical samples, L5: Clinical sample positive with IS6110 band, L6: Clinical sample positive with Protein b band only, L7: Negative control, L8: Negative clinical sample


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Statistical analysis

Sensitivity, specificity positive predictive value and negative predictive values of the tests in the extra-pulmonary were calculated and compared.


   Results Top


Out of 150 extra-pulmonary samples, smear examination was positive in a total of 11 samples (7.33%) including six biopsy samples and five body fluids. Using LJ culture, M. tuberculosis was positive in eight biopsies and nine body fluids with overall positivity of 11.3%. The multi-targeted protocol could detect M. tuberculosis in a total of 112 samples. Out of 112 positive samples, only Protein b band was detected in seven samples and only IS6110 was detected in five samples. Protein b PCR was positive in 72.7% of the biopsies and 70.2% of the body fluids. Overall Protein b PCR could detect 71.33% of the cases, whereas IS6110 was positive only in 66.6% of the cases [Table 1]. Among the 17 confirmed cases, MPCR was positive in all with a sensitivity of 100%. Out of the 133 suspected cases, MPCR showed positivity in 71.42% (95/133).
Table 1: Comparison of PCR results with smear and culture in the diagnosis of extra-pulmonary TB

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Statistical analysis showed that the positive predictive value and specificity of smear, culture and PCR were 100%. The negative predictive value (NPV) of IS6110 (32.6%) was comparable to smear and culture techniques. However, NPV was found to be higher when Protein b target was used in the MPCR (61.22%). Moreover, the mean detection time for M. tuberculosis was less than 1 day by PCR test. Overall the sensitivities of microscopy, culture, IS6110 PCR, Protein b PCR and MPCR were 7.33%, 11.3%, 66.67%, 71.3% and 74.6% respectively. Thus by using more than two targets the sensitivity increased from 66.67% of IS6110 to 74.6% of MPCR [Table 2].
Table 2: Sensitivity and specificity of Protein b PCR test for diagnosis of extra-pulmonary TB


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   Discussion Top


Molecular techniques are being increasingly used in the diagnosis of TB in recent decades. The advent of PCR has opened new vistas and provided a rapid, sensitive and specific diagnosis of TB. IS6110 although a commonly used target in PCR had disadvantage of having lower or no copies in a large number of strains in our region. [6] Protein b antigen is restricted to M. tuberculosis complex, thus making it suitable for use in diagnostic tests. Previous studies have shown that the sensitivity of Protein b PCR is 10fg which is equivalent to two M. tuberculosis. [7],[8] In the current study, we found that MPCR using IS6110 and Protein b PCR had a significantly higher sensitivity when compared to microscopy and culture in the diagnosis of extra-pulmonary TB. The higher sensitivity of PCR may be attributed to the ability of DNA amplification tests to detect even the nonviable organisms. It is likely that the samples obtained from cases being treated with ATT may have nonviable mycobacteria, and the DNA amplification method would be a better test when compared to culture technique. The sensitivity was increased when the samples were subjected for MPCR using Protein b antigen and IS6110 primers. We noted that the positivity based on Protein b PCR alone was comparable to results by Negi et al. [8] However, using multiple targets (Protein b and IS6110) the sensitivity to diagnose extra-pulmonary TB increased. In the extra-pulmonary samples various targets like IS6110, IS1081 and devR, have shown varied sensitivity. [8],[9],[10] [Table 3] Recent reports suggested a high incidence of M. tuberculosis isolates with missing IS6110 element in their genome. [6] However, we noted that in five samples that were negative by Protein b PCR were found to be positive for IS6110 PCR. This is mainly due to the fact that IS6110 occurs in multiple copy numbers and hence when present it gives a higher sensitivity as compared to a single copy genes like Protein b. Thus in the current study, we were able to detect a higher number of positive cases by incorporating dual targets (a single copy gene and a multi-copy gene) in PCR when compared to using single target. In our previous experience with the multi-targeted PCR in the diagnosis of tubercular uveitis and tubercular meningitis, we noted a higher sensitivity when compared to conventional techniques and uniplex PCR. [11],[12] In the current study, we noted a sensitivity of 74.6% for MPCR in the diagnosis of extra-pulmonary TB. Thus by using more than two targets the sensitivity increased from 66.67% of IS6110 to 74.6% in MPCR. The major limitation in extra-pulmonary samples is lower tissue burden, lower yield of DNA which affects the sensitivity to a greater extent. Thus, by using more than one target in the PCR, the sensitivity increased and aided in the prompt diagnosis of extra-pulmonary TB.
Table 3: Comparison of multiplex PCR using Protein b and IS6110 with other studies using PCR in the diagnosis of extra-pulmonary TB


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Our study indicates that MPCR is highly sensitive and specific method in the diagnosis of extra-pulmonary TB which is often missed by conventional tests due to its pauci-bacillary nature, resulting in an unacceptable delay in initiation of therapy. In conclusion, MPCR is a cost effective method for the diagnosis of extra-pulmonary TB. In future, MPCR with more than 2-3 target genes may have an important role in strengthening the diagnosis of extra-pulmonary TB.

 
   References Top

1.
Sharma SK, Mohan A. Extrapulmonary tuberculosis. Indian J Med Res 2004;120:316-53.  Back to cited text no. 1
    
2.
Hermans PW, Schuitema AR, Van Soolingen D, Verstynen CP, Bik EM, Thole JE, et al. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J Clin Microbiol 1990;28:1204-13.  Back to cited text no. 2
    
3.
Brisson-Noel A, Aznar C, Chureau C, Nguyen S, Pierre C, Bartoli M, et al. Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet 1991;338:364-6.  Back to cited text no. 3
    
4.
Hermans PW, van Soolingen D, Dale JW, Schuitema AR, McAdam RA, Catty D, et al. Insertion element IS986 from Mycobacterium tuberculosis: A useful tool for diagnosis and epidemiology of tuberculosis. J Clin Microbiol 1990;28:2051-8.  Back to cited text no. 4
    
5.
Flores LL, Pai M, Colford JM Jr, Riley LW. In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: Meta-analysis and meta-regression. BMC Microbiol 2005;5:55.  Back to cited text no. 5
    
6.
Chauhan DS, Sharma VD, Parashar D, Chauhan A, Singh D, Singh HB, et al. Molecular typing of Mycobacterium tuberculosis isolates from different parts of India based on IS6110 element polymorphism using RFLP analysis. Indian J Med Res 2007;125:577-81.  Back to cited text no. 6
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7.
Sharma K, Sharma A, Singh M, Ray P, Dandora R, Sharma SK, et al. Evaluation of polymerase chain reaction using protein b primers for rapid diagnosis of tuberculous meningitis. Neurol India 2010;58:727-31.  Back to cited text no. 7
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8.
Negi SS, Anand R, Basir SF, Pasha ST, Gupta S, Khare S, et al. Protein antigen b (Pab) based PCR test in diagnosis of pulmonary and extra-pulmonary tuberculosis. Indian J Med Res 2006;124:81-8.  Back to cited text no. 8
[PUBMED]  Medknow Journal  
9.
Tiwari V, Jain A, Verma RK. Application of enzyme amplified mycobacterial DNA detection in the diagnosis of pulmonary & extra-pulmonary tuberculosis. Indian J Med Res 2003;118:224-8.  Back to cited text no. 9
    
10.
Singh KK, Muralidhar M, Kumar A, Chattopadhyaya TK, Kapila K, Singh MK, et al. Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy. J Clin Pathol 2000;53:355-61.  Back to cited text no. 10
    
11.
Sharma K, Gupta V, Bansal R, Sharma A, Sharma M, Gupta A. Novel multi-targeted polymerase chain reaction for diagnosis of presumed tubercular uveitis. J Ophthalmic Inflamm Infect 2013;3:25.  Back to cited text no. 11
    
12.
Kusum S, Aman S, Pallab R, Kumar SS, Manish M, Sudesh P, et al. Multiplex PCR for rapid diagnosis of tuberculous meningitis. J Neurol 2011;258:1781-7.  Back to cited text no. 12
    

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Correspondence Address:
Dr. Kusum Sharma
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.151164

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