|Year : 2013 | Volume
| Issue : 4 | Page : 370-373
Evaluation of multiplex Polymerase chain reaction utilising multiple targets in Mycobacterium tuberculosis direct test negative but culture positive cases: A potential method for enhancing the diagnosis of tuberculosis
K Sharma1, D Ashkin2, P Fiorella3, D Willis3, S Dean3, A Sharma4, KK Singh5, Y Lee3, M Pedrosa3, G Singh1, M Sharma1, S Laal5
1 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 A.G. Holley State Hospital , Lantana, Florida, USA
3 Department of Health, Bureau of Laboratories, Jacksonville, Florida, USA
4 Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
5 Department of Pathology , New York University School of Medicine, New York, USA
|Date of Submission||09-Mar-2013|
|Date of Acceptance||11-Sep-2013|
|Date of Web Publication||25-Sep-2013|
Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
Purpose: To evaluate multiplex Polymerase Chain Reaction (MPCR) utilising multiple targets (IS6110, Protein b [Pab] and MPB64 genes) in Mycobacterium tuberculosis Direct Test (MTD) negative but culture positive cases and comparison of MPCR with Real-Time polymerase chain reaction (RT-PCR) for diagnosis of tuberculosis. Materials and Methods: MPCR was carried out on 28 culture positive sputum samples. Out of 28 culture positive samples, 17 were originally reported, as MTD test negative and 11 were MTD test positive, respectively. The results of MPCR were compared with RT-PCR. To check the specificity of the tests, MPCR and RT-PCR were also evaluated with 16 non-tuberculous mycobacterial (NTM) isolates. Results: Out of 28 culture positive sputum samples, MPCR was positive in all 28/28 samples, whereas RT-PCR was positive in 27/28 samples and MTD test was originally tested positive in six sputum samples and on repeating MTD testing, five more sputum samples were positive and thus total number of MTD positive were 11/28 sputum samples, respectively. All the tests were negative on evaluation with all the 16 NTMs, thus giving specificity of 100% to all the tests; sensitivity of MPCR, RT-PCR and MTD tests were 100%, 96.42% and 39.28%, respectively, in these specifically selected samples. Conclusions: MPCR may be an important tool in the rapid diagnosis of tuberculosis especially in disease endemic, resource limited countries.
Keywords: Diagnosis, multiplex Polymerase chain reaction, Mycobacterium tuberculosis direct test, real time Polymerase chain reaction, tuberculosis
|How to cite this article:|
Sharma K, Ashkin D, Fiorella P, Willis D, Dean S, Sharma A, Singh K K, Lee Y, Pedrosa M, Singh G, Sharma M, Laal S. Evaluation of multiplex Polymerase chain reaction utilising multiple targets in Mycobacterium tuberculosis direct test negative but culture positive cases: A potential method for enhancing the diagnosis of tuberculosis. Indian J Med Microbiol 2013;31:370-3
|How to cite this URL:|
Sharma K, Ashkin D, Fiorella P, Willis D, Dean S, Sharma A, Singh K K, Lee Y, Pedrosa M, Singh G, Sharma M, Laal S. Evaluation of multiplex Polymerase chain reaction utilising multiple targets in Mycobacterium tuberculosis direct test negative but culture positive cases: A potential method for enhancing the diagnosis of tuberculosis. Indian J Med Microbiol [serial online] 2013 [cited 2020 Jan 25];31:370-3. Available from: http://www.ijmm.org/text.asp?2013/31/4/370/118896
| ~ Introduction|| |
Tuberculosis (TB) is an important global public health problem. According to the World Health Organisation (WHO), approximately nine million new cases and two million deaths are reported worldwide annually. , Early and rapid diagnosis is crucial for successful disease management as well as effective control of the disease. Smear microscopy and culture are time consuming and can be insensitive in paucibacillary conditions. 
Nucleic acid-based amplification (NAA) tests have emerged as important tools for diagnosing TB. Most of the NAA based diagnostic methods have used a single target (e.g. IS6110) for the amplification and detection of Mycobacterium tuberculosis. However, IS6110 may be absent in 10-40% of M. tuberculosis isolates especially in geographically specific endemic areas. , An alternative approach may be to use multiplex Polymerase Chain Reaction (MPCR), in which several target genes for M. tuberculosis complex (MTBC) are amplified simultaneously to increase the sensitivity and specificity of the test. MPCR also has several strengths such as cost effectiveness and reduced possibility of PCR contamination.
The present study was undertaken to attempt to improve the diagnosis of TB by potentially increasing the diagnostic sensitivity of TB deoxyribonucleic acid (DNA) detection by using a MPCR method that targets IS6110, Protein b (Pab) and MPB64 genes. To the best of our knowledge, the use of a MPCR assay using these three targets has not been evaluated for the diagnosis of TB in sputum samples and never been compared before with Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe Inc., San Diego, CA) and Real-Time PCR (RT-PCR). In the United States, the MTD is the only Food and Drug Association (FDA)-approved test for both smear negative and positive specimens that has been widely evaluated and utilised for the detection of tuberculosis. This study focussed on respiratory specimens that were found in routine clinical practice to be MTD negative but subsequently culture positive.
| ~ Materials and Methods|| |
Specimens and bacterial strains
The Mycobacteriology section of the Florida Department of Health, Bureau of Laboratories (BOL) facility in Jacksonville, FL, functions as the referral laboratory for M. tuberculosis strains for the state of Florida. For this study, the Florida BOL provided a blinded sputum sample set consisting of 17 specimens that were initially found in clinical practice to be MTD negative and subsequently culture positive for MTBC as well as 11 specimens that were initially found to be MTD positive and subsequently culture positive for MTBC in clinical practice.
Parallel studies with the same sample set were also tested at the Veterans Administration Medical Centre, New York University School of Medicine (New York, NY) at a later date. To confirm the initial results for the purpose of this study, repeated MTD testing, including inhibition testing, was performed by The Florida BOL on 11 of the 17 initial MTD negative, culture positive samples (the other six samples did not have enough specimen left for repeat testing). To check the specificity, a set of 16 non-tuberculous mycobacterial (NTM) isolates were also provided by the FL BOL and subsequently evaluated by MTD, MPCR and RT-PCR. Ethical approval was not required for the study as it was a retrospective study.
Processed sputum samples and Mycobacteria Growth Indicator Tube (MGIT) (Becton Dickinson) cultures were extracted by method as described earlier.  Control DNA was prepared from solid cultures of a M. tuberculosis reference strain (H37RV) and M. kansasii laboratory-identified strain grown on Lowenstein Jensen (LJ) agar. DNA extraction was carried as described above.
The appropriate positive and negative controls, H37RV DNA and PCR grade water, respectively, were included in each MPCR run. In addition, PCR and amplicon analysis by gel electrophoresis of the blinded sample set was performed twice at each location (FL and NY) on different days to evaluate the reproducibility of the MPCR test. Identification of MTBC was done using specific primer pairs designed to amplify portions of the IS6110, Pab and MPB64 genes with resulting band sizes of 123 bp, 419 bp and 240 bp, respectively. The primer sequences, PCR mix recipe and cycle details were followed as described earlier. 
Real Time PCR (RT-PCR)
We used the RT-PCR protocol described by Halse et al.,  which targets the multi-copy IS6110 insertion element specific for MTBC DNA. To assess the PCR suitability of extracted DNA, an inhibition control plasmid (ICP)  provided by the Wadsworth Centre Laboratory (Albany, NY) was used. Positive and negative controls consisted of MTBC DNA and DNA grade water, respectively. Frozen aliquots of M. tuberculosis and M. kansasii cultures were used as controls for DNA extraction and were prepared concurrently with unknown samples.
| ~ Results|| |
Sensitivity and specificity of the MPCR assay
In the present study, sputum samples from 28 mycobacterial culture positive specimens were evaluated. We accepted culture as the gold standard and grouped these 28 patients as MTD negative/MTBC culture positive (n = 17), and MTD positive/MTBC culture positive (n = 11). In addition, to test for specificity of the tests, DNA was extracted from NTM isolates (n = 16). Of the 17 initial MTD negative/MTBC culture positive, repeated MTD testing was performed on 10 of these specimens for which specimen was still available. Of these, five tested positive on repeat MTD testing, two tested in the "gray zone" or indeterminate and only three were MTD negative on repeat testing. No inhibitors were found in any of these 10 specimens. By microscopic examination, only two of the selected sputum samples were acid-fast bacilli (AFB) smear positive; and were also positive by culture, as well as positive by MPCR and RT-PCR but tested negative by the MTD test (confirmatory repeat MTD testing could not be performed on these two specimens due to a lack of specimen availability). In the 17 culture positive/initial MTD negative sputum samples, MPCR was positive in all 17 (100%) samples [Figure 1], where as RT-PCR was positive in 16 of the 17 (94.11%) samples (the one specimen that tested negative on RT-PCR which was MTBC culture positive, tested MTD positive on repeat testing). In 11 culture positive/MTD positive sputum samples both MPCR and RT-PCR were positive in all 11 (100%) of these samples, respectively [Table 1]. We accepted culture as the gold standard, so the sensitivity of MPCR, RT-PCR, MTD test and AFB smear were 100%, 94.11%, 39.28% and 7%, respectively, in these specifically selected specimens. MPCR and RT-PCR were negative for the 16 NTM isolates, so the specificity was 100% [Table 2]. Sensitivity was estimated by serial dilutions of M. tuberculosis H37RV DNA. The MPCR assay detected 10 femto grams, which is equivalent to 2-3 cells.
|Table 2: Sensitivity, specificity, PPV and NPV of MPCR compared to RT PCR|
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|Figure 1: L1: 100bp molecular marker, L2: Positive control, L3, L4, L5, L6, L7: Positive clinical samples, L8: Negative control. IS6110: 123bp, Protein B : 419bp, MPB 64: 240 BP|
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| ~ Discussion|| |
In the present study, we evaluated MPCR using three different targets i.e., IS6110, MPB64 and Pab specific for MTBC. Most of the earlier studies have used single target, specifically IS6110, because of multiple copies (6-24).  In the present study, we accepted culture as the gold standard and selected 28 sputum specimens that were all culture positive, but of which 17 initially tested negative in routine clinical practice by MTD, an FDA-approved, commercially available test, for a sensitivity of the MTD test of 39.28% (11/28 cases). In this study, the MPCR test was positive in all 28 culture positive samples. Subsequently, of the 17 initial MTD negative/MTBC culture positive cases, 10 of these samples were available for confirmatory repeat MTD testing. Of these 10 samples, five were found to test positive after repeat MTD testing, three remained MTD negative and two tested in the "gray zone" or with indeterminate results. This is a disturbing finding from a clinical standpoint. It is unknown how many of specimens would have tested MTD positive on repeat confirmatory testing, but if the findings of this study (where 50% of the initial MTD negative subsequent culture positive specimens were MTD positive on confirmatory testing) is any indication, it may be clinically very significant. However, in day-to-day clinical practice, it probably would not be feasible to do confirmatory testing on all MTD negative specimens as it would be a significant work load increase that most laboratories cannot afford. A possible explanation for repeat testing of 10 samples by MTD showing five positive is that MTD is a technically demanding test and, if not performed diligently, can lead to errors. Thus, it is essential to have a test that has a higher sensitivity to try to avoid initially missing these very important cases.
When we compared the MPCR results with RT-PCR results, RT-PCR was positive in 27/28 (96.42%) cases. There was one culture positive sputum specimen that was negative by RT-PCR but MPCR as well as MTD positive (which targets the 16S rRNA). In that one culture positive sample, which was missed by RT-PCR, one reason postulated for the negative RT-PCR result was that MPCR targets three genes specific for MTBC, whereas RT-PCR only targets IS6110. However, in that sample, the MPCR was positive for IS6110 but negative by RT-PCR, which supports a lower detection limit of MPCR as compare to RT-PCR, as stated by another study.  The detection limit of RT-PCR is 10-12 organisms, whereas the detection limit for MPCR is two to three organisms. A number of earlier studies have used only IS6110 uniplex PCR on sputum samples and its sensitivity and specificity varies between 62-83% and 92-100%. , Similarly, Negi et al.,  evaluated Pab as a diagnostic target alone using a uniplex PCR in pulmonary samples and reported a sensitivity of 74% and specificity of 100%. Seth et al.,  evaluated a uniplex PCR using MPB64 as a target reported a sensitivity of 85%. In addition, a recently published review mentioned that targeting MPB64 is more sensitive than IS6110 in the diagnosis of TB. It is possible that using multiple targets to detect TB on a single PCR reaction may increase the sensitivity of NAA tests for the diagnosis of TB. 
MPCR in this study was shown to have good sensitivity and specificity. RT-PCR missed one culture positive sample in this study, which was detected by MPCR. This assay was as good as RT-PCR in the detection of MTBC in these initially MTD negative specimens. In agreement with Eisenach et al.,  MPCR was highly specific for MTBC. No amplification product (s) was produced with other mycobacterial species (NTMs) tested. MPCR could potentially be used for patient care in endemic recourse poor countries, where RT-PCR technologies is not feasible due to their cost and complexity. MPCR's potentially improved sensitivity and specificity to diagnose TB as well as its easier application in resource poor areas, may hold the potential to give clinicians the tool they need to better diagnose and ultimately manage this deadly disease. Smaller number of patient sample is one of the limitations of the study, which needs to be evaluated on larger number of samples as a prospective study in future. Another limitation of our study was that we did not use any primer to differentiate or to detect M. bovis.
| ~ Acknowledgment|| |
This work was funded by Fogarty NIH fellowship under the AIDS and TB Training and Research Program at the New York University School of Medicine, New York, NY, USA. We also acknowledge Dr. Max Salfinger for editing this paper.
| ~ References|| |
|1.||World Health Organization. The WHO's Report on Global TB. WHO; 2009. |
|2.||WHO Research for action, understanding and controlling tuberculosis in India: Regional office for Southeast-Asia, New Delhi; 2000. p. 70. |
|3.||Bonington A, Strang JI, Klapper PE, Hood SV, Parish A, Swift PJ, et al. TB PCR in early diagnosis of tuberculous meningitis: Evaluation of Roche semi-automated COBAS amplicor MTB test with reference to manual amplicor MTB PCR test. Tuber Lung Dis 2000;80:191-6. |
|4.||Daley P, Thomas S, Pai M. Nucleic acid amplification tests for diagnosis of tuberculous lymphadenitis: A systematic review. Int J Tuberc Lung Dis 2007;11:1166-76. |
|5.||Chauhan DS, Sharma VD, Parashar D, Chauhan A, Singh D, Singh HB, et al. Molecular typing of Mycobacterial tuberculosis isolates from different parts of India based on IS6110 element polymorphism using RFLP analysis. Indian J Med Res 2007;125:577-81. |
|6.||Anek-Vorapong R, Sinthuwattanawibool C, Podewils LJ, McCarthy K, Ngamlert K, Promsarin B, et al. Validation of GenoType MTB DR plus assay for detection of MDR-TB in a public health laboratory in Thailand. BMC Infect Dis 2010;10:123. |
|7.||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. |
|8.||Eisenach KD, Cave MD, Bates JH, Crawford JT. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis 1990;161:977-81. |
|9.||Drews SJ, Eshaghi A, Pyskir D, Chedore P, Lombos E, Broukhanski G, et al. The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens. Diagn Pathol 2008;3:37. |
|10.||Tiwari V, Jain A, Verma RK. Application of enzyme amplified mycobacterial DNA detection in the diagnosis of pulmonary and extra-pulmonary tuberculosis. Indian J Med Res 2003;118:224-8. |
|11.||Prasad R, Lath SK, Mukerji PK, et al. Clinical utility of PCR in Patients of pulmonary tuberculosis. Int J Tuberc 2001;48:135-8. |
|12.||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;24:81-8. |
|13.||Seth P, Ahuja GK, Bhanu NV, Behari M, Bhowmik S, Broor S, et al. Evaluation of polymerase chain reaction for rapid diagnosis of clinically suspected tuberculous meningitis. Tuber Lung Dis 1996;77:353-7. |
|14.||Cutrufello NJ, Karakousis PC, Fishler J, Albini TA. Intraocular tuberculosis. Ocul Immunol Inflamm 2010;18:281-91. |
|15.||Halse TA, Edwards J, Cunningham PL, Wolfgang WJ, Dumas NB, Escuyer VE, Musser KA. Combined real-time PCR and rpoB gene pyro-sequencing for rapid identification of Mycobacterium tuberculosis and determination of rifampin resistance directly in clinical specimens. J Clin Microbiol 2010;48:1182-8. |
[Table 1], [Table 2]