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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 21  |  Issue : 1  |  Page : 35-39

Suitability of IS6110 based polymerase chain reaction for the detection of Mycobacterium tuberculosis in sputum of new pulmonary tuberculosis cases


1 Department of Microbiology, Agartala Government Medical College, Tripura, India
2 Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India

Date of Web Publication4-Mar-2016

Correspondence Address:
Niladri Sekhar Das
Research Scientist VDL-ICMR, Department of Microbiology, Agartala Government Medical College, Tripura
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-9903.178103

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  Abstract 

Background and Objectives: Early diagnosis of pulmonary tuberculosis (PTB) is one of the primary challenges in curtailing its spread. Nucleic acid amplification methods targeting Mycobacterium tuberculosis (MTB) sequences in clinical specimens are increasingly in use as a tool for early tuberculosis (TB) diagnosis. Insertion sequence 6110, specific for MTB complex was targeted in sputum of new pulmonary TB patients in the present study, to determine its suitability for rapid diagnosis. Materials and Methods: A total of 100 new symptomatic for PTB attending a teaching hospital between January 2008 and December 2010 were enrolled in this study. Satisfactory sputum sample from all symptomatic was processed for microscopy by Ziehl-Neelsen (ZN) method, culture on Lowenstein-Jensen medium and polymerase chain reaction (PCR) for the IS6110 element. Results: Concordance among three methods was 87% and discordance in 13%. Positivity by ZN, culture, and PCR was 29%, 35%, and 35%, respectively. PCR picked up additional five cases, which were negative by both smear and culture. Excluding samples which grew MTB isolates lacking IS6110 the sensitivity of target IS6110 was found to be 100% with respect to ZN microscopy and culture. Interpretation and Conclusions: PCR targeting IS6110 in sputum was found to be very sensitive and specific in the rapid diagnosis of new PTB cases.

Keywords: IS6110 Mycobacterium tuberculosis, polymerase chain reaction, pulmonary tuberculosis


How to cite this article:
Das NS, Mendiratta DK, Narang R, Thamke DC, Narang P. Suitability of IS6110 based polymerase chain reaction for the detection of Mycobacterium tuberculosis in sputum of new pulmonary tuberculosis cases. J Mahatma Gandhi Inst Med Sci 2016;21:35-9

How to cite this URL:
Das NS, Mendiratta DK, Narang R, Thamke DC, Narang P. Suitability of IS6110 based polymerase chain reaction for the detection of Mycobacterium tuberculosis in sputum of new pulmonary tuberculosis cases. J Mahatma Gandhi Inst Med Sci [serial online] 2016 [cited 2020 Jul 14];21:35-9. Available from: http://www.jmgims.co.in/text.asp?2016/21/1/35/178103


  Introduction Top


Tuberculosis (TB) remains a major global health problem and causes ill-health among millions of people each year and ranks as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus. [1] It is estimated that about 40% of Indian population is infected with TB bacillus and the prevalence of TB in India was estimated to be 249/100,000 populations with mortality of 23/100,000. [2] Hence, early diagnosis of TB is crucial for prompt treatment and for the control of disease transmission.

Though sputum microscopy is simple and cheap, it has low sensitivity and specificity, and culture using Lowenstein-Jensen (LJ) though more sensitive than microscopy and also highly specific delays diagnosis by 3-8 weeks. Liquid culture systems, though rapid and more sensitive and specific than LJ, are very expensive. Since conventional techniques have limitations in early and rapid TB diagnosis, nucleic acid amplification methods targeting Mycobacterium tuberculosis (MTB) sequences in clinical specimens are increasingly in use as a tool for early TB diagnosis. [3] Polymerase chain reaction (PCR) targeting IS6110 sequence specific for M. tuberculosis complex (MTBC) [4] (MTB, M. africanum, M. bovis, M. microti, and M. canetti) and present in multiple copies (up to 20), [5] has attracted enormous attention for the diagnosis of MTB in sputum for over two decades now. The primary impetus worldwide responsible for this has been (i) the lack of sensitivity [6] (detection limit: 10,000 bacilli/ml) and specificity [6] (identifies only acid fast bacilli) of the preferred rapid method, that is, Ziehl-Neelsen (ZN) microscopy, (ii) ability of PCR to diagnose smear negative cases (sensitivity: 46-63%), [6] and (iii) long waiting period (6-8 weeks) for the availability of results by the sensitive (10-100 viable bacilli required) LJ culture method. The usefulness of IS6110 in the detection and identification of MTBC in clinical samples has been demonstrated in many studies. [7],[8],[9] As many as 138 papers between 1991 and 2011 showed how IS6110 could be used as a useful tool in the diagnosis of TB when compared to other conventional diagnostic methods. IS 6110 targeted PCR sensitivity has been reported to range between 70% and 90%. [3] Sensitivity is reported to be higher in smear-positive samples (95-100%) than in smear-negative samples (46-63%). [6] PCR targeting IS6110 in sputum when compared to culture has shown a sensitivity and specificity of 83.5% and 99.0%. [10] The rapid detection of MTB by PCR targeting IS6110, therefore becomes meaningful provided its utility is demonstrated convincingly.

In view of this, the present study was undertaken to determine the suitability of IS6110 element detection in sputum by PCR for the early diagnosis of new TB cases.


  Materials and Methods Top


Specimens

Two sputum samples (spot and deposited sputum throughout the night) were collected from 100 new pulmonary TB cases (not having or received anti tubercular treatment for >4 weeks) of age 15 years or more, attending a rural teaching hospital in central India, from January 2008 to December 2010.

Processing of the samples

All sputum samples were processed in TB Laboratory of the Department of Microbiology. Sputum microscopy was done by ZN method. All samples were decontaminated and concentrated using the N-acetyl-l-cysteine-sodium hydroxide. [11] The concentrated sputum sediments were inoculated on two LJ medium slopes and incubated at 37°C in 5% CO 2 up to 8 weeks. Before inoculation on the LJ medium 1 ml of the sediment was aliquoted for the DNA extraction. Culture results were monitored weekly. All isolates were confirmed to be MTB by niacin, nitrate, and P-nitro-alpha acetylamino-beta-hydroxy-propiophenone test. [12]

DNA extraction

A volume of 400 μl of Tris-ethylenediaminetetraacetic acid buffer was added to 1 ml of decontaminated centrifuged sediment of sputum sample and DNA was extracted according to method described by van Soolingen et al. 1991. [13] PCR [14] extracted DNA was amplified using readymade primers from Raogene Company (Details not disclosed by the company) and concentration used was as per company directive. The amplification was carried out using 50 μl reaction mix containing ×10 PCR buffer, 10 mM deoxynucleotide triphosphates, 3 μg/μl taq polymerase, 20 μl of the two primers, 37.5 μl of distilled water and 5 μl of template DNA. The thermal profile using thermo cycler TC312 (Barloworld Scientific Ltd.) involves 35 cycles: Initial denaturation 94°C for 5 min, denaturation 94°C for 1 min, annealing 68°C for 1.30 min, extension 72°C for 1.30 min, final extension 72°C for 10 min. H37Rv was used as a positive control whereas M. fortuitum was used as a negative control.

Detection of polymerase chain reaction products

Amplified products were electrophoresed on 1.5% agarose gel stained with ethidium bromide and examined under UV light for 123 bp product [Figure 1].

For standardization PCR was performed on 10 known smear positives (included all types of smear grades) and 10 known smear negative sputum samples along with, H37Rv and M. fortuitum. All smear positives and the H37Rv showed a band at 123 bp by PCR. Among the smear negatives PCR was positive in four and in all these samples grew MTB on LJ culture. M. fortuitum did not show a band at 123 bp.
Figure 1: Polymerase chain reaction for IS6110 element at 123 bp. Lane-1 and Lane-2 showing positive sputum sample for Mycobacterium tuberculosis (MTB), Lane-3 negative sputum sample for MTB, Lane-4 positive control of H37RV, Lane-5 negative control of nontubercular Mycobacteria, Lane-6 molecular marker

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The study was initiated after due approval by Institutional Ethical Committee and samples collected after informed written consent.


  Results Top


Among the 100 sputum samples examined by ZN microscopy, culture on LJ, and PCR for IS6110 target, 40 samples were positive by one or more methods: 29 (29%) by ZN staining, 35 (35%) by culture (all MTB isolates) and 35 (35%) by PCR [Table 1]. All PCR positives showed a band at 123 bp, specific for the target gene (IS6110) [Figure 1]. A concordance of 87% was seen in all the three methods (27 positive and 60 negative by all methods) and discordance in 13% samples [Table 1] (two in S+C+PCR−; three in S−C+PCR+; three in S−C+PCR−, and five in S−C−PCR+). Concordances of PCR with only smear and that of PCR with only culture was 90%, whereas discordance was 10% with either respectively [Table 1].
Table 1: PCR status with respect to smear, culture and IS6110 status

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About 85.7% (30/35) culture positive samples and 7.7% (5/65) culture negatives were positive by PCR [Table 1]. Similar figures in smear positive and smear negatives were 93.1% (27/29) and 11.3% (8/71) respectively [Table 1]. PCR was negative in five cases positive by other methods (three smear negative culture positive and two smear and culture positive) [Table 1]. However, all these five cases of MTB isolates were also negative by repeat PCR. PCR picked up additional five cases negative by smear and culture [Table 1].

Excluding the five samples whose culture isolates were also PCR negative (IS6110 lacking or present in fewer copies), the sensitivity of IS6110 as a target rose from 93.1% to 100% with respect to ZN microscopy and from 85.7% to 100% with respect to culture [Table 2]. There was however, not much difference in PCR specificity with respect to either smear or culture.
Table 2: Sensitivity and specificity of PCR with respect to ZN microscopy and culture on LJ

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Results for PCR in sputum were available in <48 h, whereas those for culture in 4-8 weeks.


  Discussion Top


The present study showed the sensitivity and specificity of IS6110 targeted PCR against culture, the gold standard, to be 85.7% and 92.3% respectively [Table 2]. This sensitivity is almost similar to 83.5% and specificity quite close to 99.0% reported by Ramachandran and Paramasivan. [10] Negi et al. [15] from Delhi evaluated four different MTB specific DNA targets (IS6110, 65 kDa, 38 kDa and mRNA coding for 85B protein) and reported that target IS6110 gave the highest positivity due to presence of multiple copies against single copy for other targets. Five MTB isolates in our study were PCR negative, though they were smear and/or culture positive [Table 1]. This is the limitation of targeting IS6110, when it is either absent or present in fewer copies in some strains of MTB. [5] Studies in India documented that 41% of MTB isolates harbored a single copy of IS6110 and 1% no copy. [16] Use of other targets for PCR in addition to IS6110 for the detection of TB can be of help in such a situation. [17],[18],[19],[20] Barani et al. [21] have advocated use of target TRC4 in addition to IS6110 for improved detection of South Indian IS6110 deficient MTB strains. Singh et al. [22] also showed the usefulness of using two targets, MPBC64 and IS6110, by demonstrating combined sensitivity of 77.8% as against 69.1% by IS6110 alone. However, the sensitivity of target IS6110 detection in our study rose to 100% [Table 2] against culture as also ZN microscopy, when isolates lacking IS6110 were excluded.

The discordance between PCR and ZN microscopy was seen in 10 cases. In two of these cases (smear positive PCR negative) the MTB isolates lacked/had fewer copies of IS6110 element. However, PCR has shown clear advantage over smear by detecting eight cases which were smear negative [Table 1]. The sensitivity of PCR over smear has been well documented and has been reported to range between 46% and 63% in smear negative cases. [6],[10] Further IS6110 is an insertion element found exclusively within the members of the MTBC, whereas ZN microscopy cannot differentiate between MTBC and nontubercular Mycobacteria's, thus giving an advantage to IS6110 targeted PCR over microscopy and making it an important diagnostic tool in the identification of MTBC species.

Polymerase chain reaction had a clear advantage over culture also as it could detect five cases negative by culture and smear [Table 1]. The sensitivity of PCR has been reported to be higher (1-10 bacilli/ml) than that of culture (100 bacilli/ml by culture on LJ). [23] Besides, culture allows the growth of only viable bacteria, while PCR can be positive in the presence of nonviable bacteria due to treatment. Different studies have reported false-positive PCR ranging between 6% and 12% [24],[25] due to crossover contamination or contamination from previous amplification. [6],[24],[25] However, in our study the contamination aspect was taken care of very thoroughly and only new cases were included. Further, in the absence of an alternate gold standard, it is not possible to clarify whether samples with false negative culture actually contained noncultivable bacteria. [26] Hence, low specificity of PCR in these five smear and culture negative cases, might not be true false-positives, but rather represent the apparent low sensitivity of the culture and smear. Routine diagnosis of pulmonary tuberculosis (PTB) by ZN microscopy and/or culture would have missed these five symptomatic cases had PCR not been resorted too.

The mean detection time for MTB was 24 days by LJ culture and less than 48 h by smear examination and PCR. Considering the limitations of conventional methods and the high sensitivity as also ability of PCR to detect MTBC only directly from clinical specimens, [27] PCR in clinically suspected PTB cases is currently considered the most sensitive, rapid diagnostic laboratory method. [28]


  Conclusion Top


Diagnosis of PTB by IS6110 directed PCR on sputum samples of clinically suspected cases has a great potential to improve the clinicians' ability for early and rapid diagnosis of MTBC, thus ensuring early treatment and preventing further transmission of disease.

Acknowledgments


We are grateful to the Kasturba Health Society, Sevagram for funding the study and also want to acknowledge the immense help received from the staff of the Department of Microbiology.

Financial support and sponsorship


Kasturba Health Society, Sevagram.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
World Health Organization (WHO). Global Tuberculosis Report 2013. Geneva, Switzerland: WHO; 2013.  Back to cited text no. 1
    
2.
Annual Status Report. Central TB Division. TB India 2012. Revised National TB Control Programme.   Back to cited text no. 2
    
3.
Roth A, Schaberg T, Mauch H. Molecular diagnosis of tuberculosis: Current clinical validity and future perspectives. Eur Respir J 1997;10:1877-91.  Back to cited text no. 3
    
4.
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.  Back to cited text no. 4
    
5.
Butcher PD, Hutchinson NA, Doran TJ, Dale JW. The application of molecular techniques to the diagnosis and epidemiology of mycobacterial diseases. Soc Appl Bacteriol Symp Ser 1996;25:53S-71.  Back to cited text no. 5
    
6.
Noordhoek GT, van Embden JD, Kolk AH. Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: An international collaborative quality control study among 30 laboratories. J Clin Microbiol 1996;34:2522-5.  Back to cited text no. 6
    
7.
Sankar S, Balakrishnan B, Nandagopal B, Thangaraju K, Natarajan S. Comparative evaluation of nested PCR and conventional smear methods for the detection of Mycobacterium tuberculosis in sputum samples. Mol Diagn Ther 2010;14:223-7.  Back to cited text no. 7
    
8.
Gupta N, Sharma K, Barwad A, Sharma M, Rajwanshi A, Dutta P, et al. Thyroid tuberculosis - Role of PCR in diagnosis of a rare entity. Cytopathology 2011;22:392-6.  Back to cited text no. 8
    
9.
Inoue M, Tang WY, Wee SY, Barkham T. Audit and improve! Evaluation of a real-time probe-based PCR assay with internal control for the direct detection of Mycobacterium tuberculosis complex. Eur J Clin Microbiol Infect Dis 2011;30:131-5.  Back to cited text no. 9
    
10.
Ramachandran R, Paramasivan CN. What is new in the diagnosis of tuberculosis? Part 1: Techniques for diagnosis of tuberculosis. Indian J Tuberc 2003;50:133.  Back to cited text no. 10
    
11.
Vestal AL. Procedure for the Isolation and Identification of Mycobacteria. Atlanta (GA): Department of Health, Education and Welfare, Public Health Service, CDC; 1975.  Back to cited text no. 11
    
12.
Koneman EW, Allen SD, Willam MJ, Schreckenberger PC, Winn WC Jr. Color Atlas and Text Book of Diagnostic Microbiology. 5 th ed.: Lippincott Williams and Wilkins; 1997. p. 893-938.  Back to cited text no. 12
    
13.
van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: Evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 1991;29:2578-86.  Back to cited text no. 13
    
14.
Eisenach KD, Sifford MD, Cave MD, Bates JH, Crawford JT. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis 1991;144:1160-3.  Back to cited text no. 14
    
15.
Negi SS, Khan SF, Gupta S, Pasha ST, Khare S, Lal S. Comparison of the conventional diagnostic modalities, bactec culture and polymerase chain reaction test for diagnosis of tuberculosis. Indian J Med Microbiol 2005;23:29-33.  Back to cited text no. 15
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16.
Narayanan S, Das S, Garg R, Hari L, Rao VB, Frieden TR, et al. Molecular epidemiology of tuberculosis in a rural area of high prevalence in South India: Implications for disease control and prevention. J Clin Microbiol 2002;40:4785-8.  Back to cited text no. 16
    
17.
Narayanan S, Parandaman V, Narayanan PR, Venkatesan P, Girish C, Mahadevan S, et al. Evaluation of PCR using TRC(4) and IS6110 primers in detection of tuberculous meningitis. J Clin Microbiol 2001;39:2006-8.  Back to cited text no. 17
    
18.
Das S, Paramasivan CN, Lowrie DB, Prabhakar R, Narayanan PR. IS6110 restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, south India. Tuber Lung Dis 1995;76:550-4.  Back to cited text no. 18
    
19.
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. 19
[PUBMED]  Medknow Journal  
20.
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. 20
    
21.
Barani R, Sarangan G, Antony T, Periyasamy S, Kindo AJ, Srikanth P. Improved detection of Mycobacterium tuberculosis using two independent PCR targets in a tertiary care centre in South India. J Infect Dev Ctries 2012;6:46-52.  Back to cited text no. 21
    
22.
Singh HB, Singh P, Jadaun GP, Srivastava K, Sharma VD, Chauhan DS, et al. Simultaneous use of two PCR systems targeting IS6110 and MPB64 for confirmation of diagnosis of tuberculous lymphadenitis. J Commun Dis 2006;38: 274-9.  Back to cited text no. 22
    
23.
Forbes BA, Hicks KE. Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction. J Clin Microbiol 1993;31: 1688-94.  Back to cited text no. 23
    
24.
Pierre C, Lecossier D, Boussougant Y, Bocart D, Joly V, Yeni P, et al. Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol 1991;29:712-7.  Back to cited text no. 24
    
25.
Shankar P, Manjunath N, Mohan KK, Prasad K, Behari M, Shriniwas, et al. Rapid diagnosis of tuberculous meningitis by polymerase chain reaction. Lancet 1991;337:5-7.  Back to cited text no. 25
    
26.
Parvez MA, Hasan KN, Rumi MA, Ahmed S, Salimullah M, Tahera Y, et al. PCR can help early diagnosis of pulmonary tuberculosis. Southeast Asian J Trop Med Public Health 2003;34:147-53.  Back to cited text no. 26
    
27.
Soini H, Musser JM. Molecular diagnosis of mycobacteria. Clin Chem 2001;47:809-14.  Back to cited text no. 27
    
28.
Del Portillo P, Murillo LA, Patarroyo ME. Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis. J Clin Microbiol 1991;29:2163-8.  Back to cited text no. 28
    


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