|Year : 2016 | Volume
| 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
Niladri Sekhar Das1, Deepak Kumar Mendiratta2, Rahul Narang2, Dipak C Thamke2, Pratibha Narang2
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 Publication||4-Mar-2016|
Niladri Sekhar Das
Research Scientist VDL-ICMR, Department of Microbiology, Agartala Government Medical College, Tripura
Source of Support: None, Conflict of Interest: None
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|| |
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.  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.  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.  Polymerase chain reaction (PCR) targeting IS6110 sequence specific for M. tuberculosis complex (MTBC)  (MTB, M. africanum, M. bovis, M. microti, and M. canetti) and present in multiple copies (up to 20),  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  (detection limit: 10,000 bacilli/ml) and specificity  (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%),  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. ,, 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%.  Sensitivity is reported to be higher in smear-positive samples (95-100%) than in smear-negative samples (46-63%).  PCR targeting IS6110 in sputum when compared to culture has shown a sensitivity and specificity of 83.5% and 99.0%.  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|| |
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.  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. 
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.  PCR  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|| |
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].
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|| |
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.  Negi et al.  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.  Studies in India documented that 41% of MTB isolates harbored a single copy of IS6110 and 1% no copy.  Use of other targets for PCR in addition to IS6110 for the detection of TB can be of help in such a situation. ,,, Barani et al.  have advocated use of target TRC4 in addition to IS6110 for improved detection of South Indian IS6110 deficient MTB strains. Singh et al.  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. , 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).  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% , due to crossover contamination or contamination from previous amplification. ,, 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.  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,  PCR in clinically suspected PTB cases is currently considered the most sensitive, rapid diagnostic laboratory method. 
| Conclusion|| |
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.
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.
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