|Year : 2013 | Volume
| Issue : 1 | Page : 24-28
Multiplex polymerase chain reaction using insertion sequence 6110 (IS6110) and mycobacterial protein fraction from BCG of Rm 0.64 in electrophoresis target genes for diagnosis of tuberculous lymphadenitis
K Sharma1, N Gupta2, A Sharma3, G Singh1, PK Gupta1, A Rajwanshi2, SC Varma3, M Sharma1
1 Department of Medical Microbiology, PGIMER, Chandigarh, India
2 Department of Cytology and Gynaeco Pathology, PGIMER, Chandigarh, India
3 Department of Internal Medicine , PGIMER, Chandigarh, India
|Date of Submission||21-Aug-2012|
|Date of Acceptance||02-Dec-2012|
|Date of Web Publication||15-Mar-2013|
Department of Medical Microbiology, PGIMER, Chandigarh
Source of Support: None, Conflict of Interest: None
Purpose: Tubercular lymphadenitis (TBLA) is a common manifestations of extrapulmonary tuberculosis (EPTB) accounting for 30-40% of cases. Prompt diagnosis and timely initiation of anti-tubercular therapy (ATT) is the key for successful clinical outcome. This study was carried out to evaluate multiplex polymerase chain reaction (MPCR) using MPB64 and IS6110, and compare with the conventional methods for rapid diagnosis of TBLA. Materials and Methods: In our study, lymph node fine-needle aspirates of patients were evaluated for TBLA. They were classified as Group I: TBLA group, divided into (a) Confirmed TBLA cases (n0 = 80): Culture/smear-positive or cytological examination showing presence of epithelioid cell granuloma with or without multinucleate giant cell and caseation necrosis with presence of AFB, and (b) suspected TBLA cases ( n = 30): Culture/smear-negative and cytological examination showing presence of epithelioid cell granuloma and response to ATT and Group II (Control) (n = 25): Patients of lymphadenopathy confirmed to be caused by other diseases such as sarcoidosis, lymphoma, etc., All samples were subjected to conventional tests and MPCR. For MPCR we used Mycobacterium tuberculosis-specific deoxyribonucleic acid sequences specific for the MPB64 and IS6110 region. Results: In the confirmed TBLA group, Ziehl-Neelsen (ZN) smear, cytology, culture, and MPCR positivity was 30%, 70%, 26.3%, and 91.3% respectively. In the suspected TBLA group, smear and culture were negative, and sensitivity of cytology and MPCR was 73.3% and 86.6%, respectively. In the control group all tests were found to be negative, thus giving a specificity of 100% to all the tests in the study. Conclusion: In conclusion, techniques like MPCR with high sensitivity and specificity can play an important role in rapid diagnosis of TBLA.
Keywords: Insertion sequence6110, mycobacterial protein fraction from BCG of Rm 0.64 in electrophoresis, multiplex polymerase chain reaction, tubercular lymphadenitis
|How to cite this article:|
Sharma K, Gupta N, Sharma A, Singh G, Gupta P K, Rajwanshi A, Varma S C, Sharma M. Multiplex polymerase chain reaction using insertion sequence 6110 (IS6110) and mycobacterial protein fraction from BCG of Rm 0.64 in electrophoresis target genes for diagnosis of tuberculous lymphadenitis. Indian J Med Microbiol 2013;31:24-8
|How to cite this URL:|
Sharma K, Gupta N, Sharma A, Singh G, Gupta P K, Rajwanshi A, Varma S C, Sharma M. Multiplex polymerase chain reaction using insertion sequence 6110 (IS6110) and mycobacterial protein fraction from BCG of Rm 0.64 in electrophoresis target genes for diagnosis of tuberculous lymphadenitis. Indian J Med Microbiol [serial online] 2013 [cited 2019 Dec 11];31:24-8. Available from: http://www.ijmm.org/text.asp?2013/31/1/24/108714
| ~ Introduction|| |
Among the extrapulmonary tuberculoses (EPTBs), tubercular lymphadenitis (TBLA) is one of the most common manifestations, accounting to 30-40% of all cases.  Prompt diagnosis and timely initiation of anti-tubercular therapy (ATT) is the key for successful clinical outcome and better patient management.  Fine-needle aspiration (FNA) of the lymph nodes, with cytological and microbiological smear examination, along with culture, has simplified its diagnosis to a great extent. The cytological criteria for diagnosis of TBLA have been defined as "epitheloid cell granuloma with or without multinucleate giant cells and caseation necrosis".  However, in cases presenting with cold abscess, well-formed granuloma may not be seen. In addition, there are a number of non-tubercular causes for granulomatous reaction on histopathological examination, such as, atypical mycobacterial lymphadenitis; viral, bacterial, fungal lymphadenitis; sarcoidosis; toxoplasmosis; cat scratch fever; collagen vascular diseases; and disease of the reticuloendothelial system.  Conventional microbiological methods such as microscopy and culture are inadequate for rapid diagnosis of TBLA because of limited sensitivity. , Although culture is considered as the "gold standard" in the diagnosis of tuberculosis, it takes 6-8 weeks for culture to be positive, causing delay in initiation of treatment.
Nucleic acid amplification techniques (NAATs) such as polymerase chain reaction (PCR) play an important role in rapid diagnosis of TBLA.  Most studies have used only a single target gene, that is, IS6110; however, it is well known that IS6110 is missing or in low copy numbers in 10-40% of M. tuberculosis isolates from India.  So, an alternative approach is to use multiplex PCR (MPCR) where a numbers of targets are amplified simultaneously. This method is cost-effective and chances of contamination are also less as shown in our previous experience with osteoarticular and meningeal tuberculosis. , We have not been able to ascertain if others have used MPCR for TBLA. Therefore, this study was carried out to evaluate MPCR using MPB64 and IS6110, and to compare it with conventional methods such as microscopy, culture, and cytological examination for rapid diagnosis of TBLA.
| ~ Materials and Methods|| |
A total of 135 FNA from various lymph nodes, with relevant history, were received at the Mycobacteriology Laboratory at the Department of Medical Microbiology and Department of Cytology at Post Graduate Institute of Medical Education and Research, Chandigarh, India. The samples received at the Mycobacteriology Laboratory were coded and processed in a biosafety level-II cabinet. They were processed for (a) smear examination by Ziehl-Neelsen (ZN) method, (b) culture on Lowenstein-Jensen (LJ) medium, and (c) NAA by MPCR as per standard protocol. ,,, The portion for NAA tests was kept at −20°C until further use. The cytological findings were recorded independently by pathologist. On the basis of results of ZN staining, culture, cytological examinations, and response to treatment, patients were grouped as follows:
Group I: TBLA group
Confirmed TBLA cases
Culture/smear-positive or cytological examination showing presence of epithelioid cell granuloma with or without multinucleate giant cell and caseation necrosis with presence of acid-fast bacilli (AFB).
Suspected TBLA cases
Culture/smear-negative and cytological examination showing presence of epithelioid cell granuloma and response to ATT.
Group II (Control)
Patients with lymphadenopathy (LAP) confirmed to be caused by other diseases such as sarcoidosis, lymphoma, etc.
Deoxyribonucleic acid extraction
Two hundred microliters of stored sample was used for DNA extraction according to the cetyltrimethyl ammonium bromide (CTAB)-phenol-chloroform extraction method, with minor modifications as described earlier. ,,
Multiplex polymerase chain reaction
MPCR was performed using primers (Sigma, Bangalore, India) for the IS6110 (IS1: 5'- CCTGCGAGCGTAGGCGT-3' and IS2: 5'-CTCGTCCA GCGCCGCTTCGG-3') and MPB64 (MPB1: 5'- ACC AGGGAGCGGTTCGCCTGG-3' and MPB2: 5'- GATCT GGGGGTCGTCGGAGCT-3') region of the M. tuberculosis complex; amplification conditions have been described earlier.  The PCR product was run on 1.5% agarose gel stained with ethidium bromide. Specific bands of 123 bp for IS6110 and 240 bp for MPB64 were visualized using a digital gel documentation system (Alpha Innotech, San Leandro, CA, USA) as shown in [Figure 1]. The sample was considered positive for MPCR if a specific band was seen in any or both of the two gene targets chosen. In every MPCR assay, results were compared with one positive (DNA of H 37 Rv strain) and a negative (PCR-grade water) control. A 100-bp ladder was used as a molecular marker. At the time of analysis on the basis of results, the subjects were grouped as confirmed TBLA (n0 = 80), suspected TBLA (n = 30), and control group ( n = 25) [Table 1].
|Figure 1: LI: 100-bp molecular marker, L2: Positive control, L3,4: Patient sample DNA positive for both the bands, L5: Clinical sample positive for IS6110, L6: Clinical sample positive for MPB64 band, L7: Negative control, L8: Negative patient sample|
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|Table 1: Comparison of smear, culture, cytological examination and multiplex polymerase chain reaction|
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Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using SPSS version 14.0 software.
| ~ Results|| |
In the confirmed TBLA group, conventional microscopy (ZN stain) was positive in 24/80 (30%) cases whereas culture was positive in 21/80 (26.3%) cases. Cytology showed epithelioid cell granuloma with or without multinucleated giant cell, caseation necrosis, and AFB in 56/80 (70%) samples. Among this group, MPCR was positive in 73/80 (91.3%) cases, out of which MPB64 band appeared in 70 (87.5%) and IS6110 in 68 (85%) cases [Table 1]. Only MPB64 band was seen in 4 (5%) and only IS6110 in 3 (3.75%) cases. [Figure 1] shows the gel photograph of MPCR.
All suspected TBLA cases were found to be negative on smear and culture examination. Cytological examination was suggestive of TBLA in 22/30 (73.33%) cases. This figure increased to 26/30 (86.66%) using MPCR. All MPCR-positive cases were MPB64-positive whereas the IS6110 band could be demonstrated in 23 cases only. All cases of the suspected and confirmed group responded to treatment.
In the control group, all tests were found to be negative, thus giving a specificity of 100% for all the tests in the study, that is, AFB smear, culture, cytological examination, and MPCR. A final diagnosis of TBLA was made for 110 patients based on the results of smear examination, culture, cytological examination, and response to ATT. MPCR was positive in 99 of 110 cases, culture in 21, smear in 24, and cytology in 78 cases. Thus sensitivity of MPCR, smear, culture, and cytology was 90%, 21.8%, 19.09%, and 70.9%, respectively [Table 2].
|Table 2: Sensitivity and specifi city of fi ne needle aspiration cytology, multiplex polymerase chain reaction, smear culture in tuberculous lymphadenitis|
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| ~ Discussion|| |
In developing countries tuberculosis is the most common cause of LAP.  Diagnosis is based on clinical criteria, conventional microbiological techniques (smear examination and culture), cytology, and histopathology. Conventional microbiological methods have poor sensitivity, , so these are often supplemented with additional investigations such as cytology and biopsy. In the last few decades, FNA has been used as a less invasive procedure than excision biopsy. But granuloma, with or without necrosis, of possible TBLA may be seen in several infectious and non-infectious conditions. ,,,
Culture is taken as the gold standard for diagnosis of TB. In the present study, smear and culture had a sensitivity of 21.8% and 19.1%, respectively. The most important reason for the low sensitivity of culture is unrepresentative or inadequate sample volume.  Other reasons as stated by other studies could be paucibacillary nature,  presence of bacteriostatic substances,  partially treated case,  harsh decontaminations,  and presence of necrosis.  It has been reported that lymph nodes are immunologically competent factories, therefore the organisms in them are, most of the time, in a state of metabolic inhibition and therefore growth is not always possible.  The sensitivity of conventional methods is known to be very low, which varies from 10% to 50% for smear examination and 28% to 65% for culture. , There were few cases, which were smear-negative but culture-positive. All cultures were identified as M. tuberculosis and no non tubercular mycobacteria were isolated in this study.
In our study, sensitivity of cytology examination was 70.9%, which is similar to previously reported studies, and varies between 25% and 77%. , The discrepancy in the results of AFB positivity among the confirmed group as reported by the microbiologist (30%) and pathologist (70%) may be due to paucibacillary nature, inadequate volume received, or inappropriate apportioning of the sample for various diagnostic tests resulting in non-uniform distribution of organisms.
For rapid diagnosis of TBLA, NAA techniques can play an important role. PCR as highlighted by previous studies has shown good sensitivity and specificity, which varies between 50% and 91%, and 60% and 100%, respectively.  The repetitive nature of the IS6110 insertion sequence in M. tuberculosis makes it an attractive target for PCR amplification and most of the studies have evaluated IS6110 for diagnosis of tuberculosis.  However, presence of a few copies or complete absence of this sequence might miss the diagnosis. Study from India has also reported that a large number of clinical isolates (40%) had either a single copy or no copy of the insertion sequence.  To overcome this problem, simultaneous use of multiple targets increases sensitivity and decreases the chances of false-negative results. We had also reported previously that multiplex targets increase the sensitivity of the tests especially in smear-negative extrapulmonary conditions such as tubercular meningitis and osteoarticular tuberculosis. , MPCR has many advantages such as less chances of contamination as all reaction takes place in same tube and cost-effective, as stated earlier. , Therefore, in the present study we evaluated MPCR targeting IS6110 and MPB64 together for diagnosis of the M. tuberculosis complex in clinically suspected TBLA cases.
MPCR was positive in 91.3% of confirmed and 86.6% of suspected TBLA cases. Similar to our previous reports , higher sensitivity was obtained for MPB64 (87.27%) compared with IS6110 (82.72%). Singh et al. also reported that by using an additional target such as MPB64 they could pick up an additional seven (8.6%) patients who would have been missed by using only IS6110 as the target gene. In the present study, we could have missed 7.3% (detected by MPB64) or 2.7% (detected by IS6110) cases using a single target for amplification. An increase in sensitivity was also seen in earlier studies, , by using MPCR of the above two genes. By using an additional target, an increase in sensitivity for diagnosis of TB was observed as stated by earlier studies also. , However, most of the earlier studies had used separate PCRs for different targets, which increases cost and chances of contamination. In the present study, we used MPCR.
There were cases, which were detected only by MPCR but missed by conventional methods. This might represent disease in the early part of incubation before appearance of cytological lesion or a scanty number below the detection limit of microscopy or culture. In the confirmed TBLA group, MPCR was positive in 73/80 confirmed TBLA patients; there were seven cases, which were missed by MPCR (1 culture-positive, 1 smear-positive, and 5 cytology-positive) and various reasons for negative PCR have been highlighted below. The sensitivity of MPCR was 90% in the total number of TBLA cases and there were 10% cases, which were missed by MPCR. It may be due to the presence of low number of bacteria, insufficient or uneven distribution of sample, insufficient lysis of the tough cell wall of the M. tuberculosis complex, loss of DNA during purification, and presence of inhibitors (such as blood, host proteins, eukaryotic DNA, etc.) in high concentration interfering with amplification, as reported by earlier studies. ,
| ~ Conclusion|| |
Techniques like MPCR with high sensitivity and specificity can play an important role in rapid diagnosis of TBLA. Use of such a diagnostic technique especially in infections with paucibacillary conditions where a small quantity of sample is obtained from invasive procedures is justified. To the best of knowledge, we for the first time in India have evaluated MPCR using these two targets for rapid diagnosis of TBLA.
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[Table 1], [Table 2]