|Year : 2012 | Volume
| Issue : 2 | Page : 182-186
Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis
AK Maurya1, S Kant1, VL Nag2, RAS Kushwaha1, TN Dhole2
1 Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University, Lucknow 226 003, (Erstwhile King George Medical College), India
2 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, Uttar Pradesh, India
|Date of Submission||22-Aug-2011|
|Date of Acceptance||22-Oct-2011|
|Date of Web Publication||28-May-2012|
T N Dhole
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Purpose: Extrapulmonary tuberculosis (EPTB) is emerging problem in developing and developed countries. The diagnosis of EPTB in its different clinical presentations remains a true challenge. IS6110-based polymerase chain reaction (PCR) is used for rapid identification and positivity rate of the Mycobacterium tuberculosis complex in clinical isolates of different sites of EPTB. The present study was carried out to study the prevalence of M. tuberculosis complex in clinical isolates of EPTB at tertiary care centres in Lucknow. Materials and Methods: Seven hundred fifty-six specimens were collected from the suspected cases of EPTB which were processed for Mycobacteria by Ziehl Neelson (ZN) staining and BACTEC culture. All the specimens were also processed for IS6110-based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of the M. tuberculosis complex. Results: Of these 756 specimens, 71(9.3%) were positive for acid fast bacilli (AFB) by ZN staining, 227(30.1%) were positive for mycobacteria by BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 165 (20.7%) isolates. We found a significant difference in sensitivities of different tests (P<0.05). Conclusions: This study reveals the positivity of M. tuberculosis complex in clinical isolates of EPTB case in tertiary care hospitals in Northern India. 72.7% of M. tuberculosis complex was confirmed by IS6110-PCR in culture isolates from different sites of EPTB. The high prevalence of the M. tuberculosis complex was seen in lymph node aspirate and synovial fluid. However, utility of PCR may play a potentially significant role in strengthening the diagnosis of EPTB especially targeting IS6110.
Keywords: Extrapulmonary tuberculosis, IS6110, Mycobacterium tuberculosis complex, polymerase chain reaction
|How to cite this article:|
Maurya A K, Kant S, Nag V L, Kushwaha R, Dhole T N. Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis. Indian J Med Microbiol 2012;30:182-6
|How to cite this URL:|
Maurya A K, Kant S, Nag V L, Kushwaha R, Dhole T N. Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis. Indian J Med Microbiol [serial online] 2012 [cited 2020 Jan 23];30:182-6. Available from: http://www.ijmm.org/text.asp?2012/30/2/182/96688
| ~ Introduction|| |
Tuberculosis (TB) is an increasing public health problem in developing countries. India is the highest TB burden country accounting for one fifth of the global incidence. Reported global annual incidence of TB cases estimate is 9.4 million cases, out of which it is estimated that 1.8 million cases are from India.  Extrapulmonary TB (EPTB) is a significant health problem in both developing and developed countries.  The reported prevalence of EPTB in India varies between 8.3% and 13.1% in different districts according to cohort analysis by Central TB Division, Ministry of Health and Family Welfare in 2002.  The reported proportions of EPTB among all TB cases in other developed countries ranged from 12% to 28.5%. , The diagnosis of EPTB in its different clinical presentations remains a true challenge. The lack of a sensitive, specific and rapid method for the early diagnosis of EPTB poses a difficulty in initiating early therapy.  Conventional diagnostic tests like smear examination for acid fast bacilli (AFB) and culture identification have serious limitations of sensitivity and the long time taken for growth of the bacilli (6-8 weeks). In the cases of EPTB like in the CNS (central nervous system), genitourinary tract and abdomen, the bacterial load is much less as compared to the pulmonary tuberculosis (PTB).  At present, nucleic acid amplification (NAA) technologies such as PCR are revolutionizing the detection of infectious pathogens such as M. tuberculosis. They offer new prospective for the diagnosis of EPTB in a few hours with a high degree of sensitivity and specificity. The insertion sequence IS6110 is a mobile genetic element typical for the Mycobacterium tuberculosis complex. IS6110-based PCR amplification of insertion sequences is most commonly used for the detection of M. tuberculosis complex.  IS6110-PCR is a more rapid, sensitive and specific in comparison to other methods routinely used in clinical laboratories. The aim of this study was to know the prevalence of M. tuberculosis complex in clinical isolates of different cases of EPTB in patients attending at two tertiary care centres in Lucknow.
| ~ Materials and Methods|| |
The study was performed prospectively in a blinded manner.
Clinical specimens and data collection
The entire patients were attending indoor (IPD) and outdoor (OPD) wards from January 2007 and December 2010 at two tertiary care centres in Lucknow. 2-10 ml of specimens was collected from 756 specimens, non-repeated specimens from suspected cases of EPTB. The specimens were included as lymph node aspirate and cold abscesses, pleural fluid, cerebrospinal fluid (CSF), synovial fluid, ascitic fluid, urine, gastric aspirate, pus, bone marrow aspirates, wound swabs and biopsy materials. All the patients signed due consent for sample collection. The clinical history regarding present and past history of antitubercular treatment (ATT), family history of tuberculosis and any other associated disease were taken in prescribed Performa.
Smear examination, culture and identification of M. tuberculosis complex
Specimens was divided into two part one part was kept at -20°C for PCR till processing and another part was processed for mycobacterial smear preparation and BACTEC culture. Smears were stained with Ziehl Neelsen (ZN) stain and examined for acid-fast bacilli (AFB).  BACTEC vials were incubated and interpreted as per Becton Dickinson (BD, Sparks, MD, USA) manual instructions. 
The mycobacterial isolates obtained in BACTEC culture were subjected to biochemical testing for species characterization by carrying out the Niacin test (commercially supplied Niacin strips by Sigma were used) and identification of isolates belonging to M. tuberculosis complex was performed by the BACTEC NAP test  (Becton Dickinson Diagnostic Instrument System, USA) where NAP (p-nitro-a-acetylamino-b-hydroxypropiophenone) was added to the culture to inhibit the growth of bacteria belonging to the M. tuberculosis complex. Standard strain M. tuberculosis complex, H37Rv ATCC™ No. 27294 was used as positive control.
Extraction of DNA
Extraction of DNA was done by the CTAB (cetyl-tri-methyl-ammonium bromide)-phenol chloroform extraction method.  Specimens were centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and the pellet suspended in 567 μl of TE (Tris EDTA, pH 7.4) buffer, 30 μl 10% SDS (sodium dodecyl sulfate) and 3 μl proteinase K (20 mg/ml), mixed and incubated at 20°C for 1 h. After incubation, 100 μl of 5 M NaCl and 80 μl of high-salt CTAB buffer (containing 4 M NaCl, 1.8% CTAB) were added and mixed followed by incubation at 65°C for 10 min. An approximate equal volume (0.7-0.8 μl) of chloroform-isoamyl alcohol (24:1) was added, mixed thoroughly and centrifuged for 4-5 min in a microcentrifuge at 12,000 rpm. The aqueous viscous supernatant was carefully decanted and transferred to a new tube. An equal volume of phenol: Chloroform-isoamyl alcohol (1:1) was added followed by a 5 min spin at 12,000 rpm. The supernatant was separated and then mixed with 0.6 volume of isopropanol to get a precipitate. The precipitated nucleic acids were washed with 75% ethanol, dried and re-suspended in 100 μl of TE buffer.
IS6110 is a 1191-bp repetitive insertion sequence that is usually present 6-20 times in the M. tuberculosis complex genome although as few as one copy has been observed.  The oligonucleotide primers used were IS1 and IS2: 5'-CCT GCG AGC GTA GGC GTC GG 3' and 5' CTC GTC CAG CGC CGC TTC GG 3' (SBS Gentech Co. Ltd). The IS6110 repetitive insertion sequence was designed for specific pair of primers to amplify 123 bp as reported earlier. 
DNA amplification by polymerase chain reaction
The amplification reaction was performed in a final volume of 20 μl. The reaction mixture contained 10 μl 5X Phire Reaction Buffer (1.5 mM MgCl 2 ), 1 μl of 10 mM dNTPs 0.4 μl of Phire Host Start II DNA polymerase (Finnzymes Oy, Espoo, Finland), 1 μl (10 pmole) of each primer, 5 μl of water (nuclease free) and 2 μl of extracted DNA. The PCR amplification was done in thermal cycler (MJ Research, PTC-100, GMI, Inc, USA), temperature cycling conditions included 98°C for 3 min, followed by 18 cycles at 98°C for 5 s, annealing of primers at 60°C for 5 s, and primer extension at 72°C for 10 s, and final extension at 72°C for 2 min. The amplified products were separated on 2% agarose gels, visualized on a UV-light transilluminator (Bangalore Genei, Bangalore, India). The presence of 123 bp fragment indicates the positive test for M. tuberculosis complex. The positive controls included the DNA of H37Rv strain. Negative control included PCR grade water [Figure 1].
|Figure 1: PCR-based detection of the M. tuberculosis complex targeting IS6110. Electrophoresis separation of the amplicon into 2% agarose gel is documented across Lanes 1-8. The presence of a 123 bp amplicon in the Lanes 1,3,4,5 indicated the presence of the target while the absence of the amplicon in the Lane 2 pointed towards the absence of the target. Lane NC was negative control and Lane PC was positive control (H37Rv). Ladder 100 bp was well shown in Lane L|
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Data were analysed using SPSS 15.0 (Statistical Package for the Social Sciences, Chicago, IL, USA) for Windows. The difference was significance when P<0.05.
| ~ Results|| |
Demographical characteristics of extrapulmonary tuberculosis cases
In the evaluation of the demographic characteristics of the EPTB patients, a total of 756 specimens from 756 patients with presumptive clinical diagnosis of EPTB were evaluated for the presence of extrapulmonary M. tuberculosis infection by molecular, biochemical and conventional methods. The specimens included are shown in [Table 1]. Out of 756 specimens, 71(9.3%) were positive for AFB by ZN staining and 227(30.1%) were positive for mycobacteria by BACTEC culture. 165 (20.6%) were confirmed as M. tuberculosis complex by the biochemical and molecular test. Of these 165 patients, the mean age of all patients was 32.12 + 5.23. In the study, 94 (56.9%) were males and 71 (43.1%) were females. Patients of 25-44 years of age group accounted for 43% of the total cases. 120 (72.7%) were new cases (they had not taken ATT in the past or were less than 1 month on ATT) and 45 (27.3%) were cases as previous treated cases (they had taken ATT in the past or were more than 1 month on ATT) (P<0.05). The history of contact with TB patients was determined in 45 cases (27.3%), 19 (11.5%) were having the history of diabetic mellitus, family history of TB were present in 23 (13.9%) and 3 (1.8%) cases were HIV positive.
|Table 1: Comparison of positivity rate of M. tuberculosis complex by conventional, biochemical and molecular methods|
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Positivity of M. tuberculosis complex among EPTB specimens by conventional and molecular methods
A total of 227 (30.1%) patients were positive for mycobacteria by BACTEC culture and positivity of specimens is summarized in [Table 1]. We performed the NAP test for differentiation of M. tuberculosis complex and Mycobacterium other than tuberculosis on 227(30.1%) culture positive isolates. We have identified M. tuberculosis complex on the basis of biochemical characterization of 165(72.7%) isolates. After that we performed the molecular test for the final confirmation of M. tuberculosis complex by the IS6110-based PCR test. 165/227 (72.7%) were found to be positive for M. tuberculosis complex by the NAP test and IS6110 PCR [results as shown in [Table 1]].
Prevalence of M. tuberculosis complex in EPTB cases
The prevalence of M. tuberculosis complex was finally identified by the IS6110 PCR test. We found 165 (72.7%) isolates as M. tuberculosis complex by the IS6110 PCR test. Of 165 M. tuberculosis complex isolates, 55(83.4%) isolates were in lymph node aspirate and cold abscesses, 5(83.4%) were in synovial fluid, 8(80%) were in ascitic fluid, 4(80%) isolates were in pericardial fluid, 10(71.4%) were in urine, 12 (70.5%) were in gastric aspirate, 7 (70%) were in bone marrow aspirates, 13 (68.4%) were in pus, 20 (68.9%) isolates were in pleural fluid, 14 (66.7%) were in C.S.F, 6 (66.7%) isolates were in wound swabs and 11 (61.1%) isolates were in biopsy materials [results as shown in [Table 2]].
| ~ Discussion|| |
India has about 1.8 million new cases of TB annually, accounting for one fifth of new cases in the world - a greater number than in any other country.  EPTB is burning health dilemma in both developed and developing countries. , Still diagnosis of EPTB in its different clinical presentations remains a true major challenge. EPTB is underestimated by clinicians and the uses of less sensitive conventional methods have contributed to the difficulties in managing patients with EPTB.  Problems can arise when clinical specimens contain very few mycobacteria and their slow growth rate limit detection by the conventional method such as acid-fast staining and bacterial culture. In recent years, rapid diagnostic tests based on NAA tests have been developed. , NAA tests have appeared with the goal of enabling clinicians to make a rapid and accurate diagnosis. PCR is the most excellent known NAA test. It amplifies target nucleic acid regions that uniquely identify the M. tuberculosis complex. The only repetitive target useful for a NAA in tuberculosis, which is so far available, is an insertion sequence designated IS6110. , IS6110 specific for the M. tuberculosis complex (M. tuberculosis, M. africanum, M. bovis, M. microti and M. canetti) generally occurs in 1-20 copies per cell, making it an ideal target for amplification. ,, The sequence has been used for detection and for molecular epidemiological studies of the M. tuberculosis complex group. , In this study, we have confirmed that IS6110-PCR is a useful tool for the diagnosis of EPTB. In our study, smear examination for AFB was done in 71 (9.4%) and conventional bacteriological techniques were positive for Mycobacteria in 227 (30.1%), whereas IS6110-PCR showed positivity in 165 (72.6%) for the M. tuberculosis complex in EPTB cases. This difference was found to be statistically significant (P<0.05).
Earlier studies reported ,,, the increased positivity by PCR targeting the IS6110 element in specimens of EPTB and 63%, 51%, 77% and 62% as positivity among EPTB specimens. In comparison, we found 72.6% positivity by PCR targeting the IS6110 element in specimens of EPTB. Earlier studies reported the positivity of the M. tuberculosis complex by PCR targeting the IS6110 element in specimen-wise distribution of the M. tuberculosis complex in EPTB. Sekar et al.  showed that the positivity of the M. tuberculosis complex was seen by IS6110 PCR on EPTB specimens, 61.5% in lymph node aspirates, 73.9% in CSF, 60.9% in ascitic fluid, 45.8% in pleural fluid and 100% of M. tuberculosis complex were in synovial fluid. Our study showed that the higher positivity of the M. tuberculosis complex among lymph node aspirates and synovial fluid was 83.4%, and 83.4%, respectively. Another studied by Gomez et al.  showed that very good sensitivity results for the detection of M. tuberculosis complex using IS6110 PCR, 95% were in CSF, 94% in pleural fluid and 93% in ascitic fluids. They were showing very good sensitivity results for the detection of the M. tuberculosis complex using IS6110 PCR but our sensitivity showed that less in CSF, pleural fluid and ascitic fluids. Less sensitivity may arise when specimens contain very few mycobacteria. GarcÍa et al.,  Kolk et al.,  Haron et al.,  Pietrzak et al.,  Singh et al.  and Shukla et al.  showed the detection of M. tuberculosis by PCR results as summarized in [Table 2]. Reported studies showed data of the positivity of the M. tuberculosis complex by IS6110 PCR on 50 to 250 sample size of EPTB case. In comparison, we showed data of the positivity of M. tuberculosis complex by IS6110 PCR in 756 cases of EPTB. Our results showed the positivity of the M. tuberculosis complex by IS6110 PCR from various sites of EPTB and its comparisons of prevalence of the M. tuberculosis complex are summarized in [Table 2].
However, the absence or presence of fewer copies of target sequence IS6110 in some more strains of M. tuberculosis has been reported. , Some of earlier studies reported that IS6110 PCR assays targeting IS6110 were more sensitive. ,,, On the basis of our PCR results, we can confirm that IS6110-based PCR methodology for M. tuberculosis can be highly specific and sensitive for detecting EPTB from variety of clinical specimens. The rapidity, high sensitivity and simplicity of PCR targeting IS6110 gene sequence are used in the diagnosis of EPTB.
| ~ Conclusion|| |
This study reveals the positivity of the M. tuberculosis complex in clinical isolates of EPTB cases in tertiary care hospitals. We found maximum presences of the M. tuberculosis complex in lymph node aspirate and synovial fluid. However, utility of PCR may play a potentially significant role in strengthening the diagnosis of EPTB especially targeting IS6110. This will ensure early treatment to patients and prevent further transmission of disease.
| ~ Acknowledgment|| |
This work was supported by grant from Indian Council of Medical Research, New Delhi (Extramural ICMR Project Sanction No. 5/8/5/4/2007-ECD-I). Authors would like to thank the Technical Member of Mycobacteriology Laboratory, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Science, Lucknow, India, for their technical support during research work.
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[Table 1], [Table 2]
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