Indian Journal of Medical Microbiology Home 

BRIEF COMMUNICATION
[Download PDF]
Year : 2015  |  Volume : 33  |  Issue : 2  |  Page : 274--276

Poor performance characteristics of conventional PCR in detection of respiratory syncytial virus-experience of a tertiary care centre in Southern India

G Nandhini1, S Sujatha1, N Jain1, R Dhodapkar1, T Kadhiravan2, S Krishnamurthy3,  
1 Department of Microbiology, Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry - 605 006, India
2 Department of Medicine, Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry - 605 006, India
3 Department of Pediatrics, Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry - 605 006, India

Correspondence Address:
S Sujatha
Department of Microbiology, Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry - 605 006
India

Abstract

Respiratory syncytial virus (RSV) is a significant cause of contagious acute respiratory infections in children and older adults. Since there are contradictory reports regarding the efficacy of different methods to detect RSV, we evaluated the performance of the conventional PCR versus real-time PCR in 222 patients with acute respiratory infections (ARI) recruited between January 2012 and March 2013. Conventional PCR had a very poor sensitivity of 40% (95% CI: 19.2-63.9%) and failed to detect RSV in respiratory samples with low viral load. Thus, it may be prudent to replace it with real-time PCR to achieve precise diagnosis.

How to cite this article:
Nandhini G, Sujatha S, Jain N, Dhodapkar R, Kadhiravan T, Krishnamurthy S. Poor performance characteristics of conventional PCR in detection of respiratory syncytial virus-experience of a tertiary care centre in Southern India.Indian J Med Microbiol 2015;33:274-276

How to cite this URL:
Nandhini G, Sujatha S, Jain N, Dhodapkar R, Kadhiravan T, Krishnamurthy S. Poor performance characteristics of conventional PCR in detection of respiratory syncytial virus-experience of a tertiary care centre in Southern India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Sep 22 ];33:274-276
Available from: http://www.ijmm.org/text.asp?2015/33/2/274/154875

Full Text

 Introduction



Respiratory syncytial virus (RSV) is a leading cause of viral acute respiratory infections (ARI), particularly in children. The precise data concerning morbidity and mortality contributed by RSV is lacking due to non-availability of a standard protocol for RSV detection. Conventional PCR is considered to be a simple and an economical technique for any laboratory setting with the flexible ability of multiplexing. However, there is a discrepancy over the diagnostic accuracy of conventional PCR. [1],[2] Hence, this study was undertaken to assess the performance of conventional PCR in the detection of RSV in comparison with real-time PCR.

 Materials and Methods



During the study period from January 2012 - March 2013, patients (both children and adults) attending the outpatient care facilities of our hospital with symptoms of ARI (fever along with two or more symptoms of ARI i.e. cold/cough, sore throat, myalgia) were recruited after obtaining informed consent. In addition, specimens were also collected from all hospitalised patients who satisfied the case-definition of severe acute respiratory illness (SARI). [3] The study protocol was approved by the Institutional Human Ethics Committee.

Nasopharyngeal swabs were collected in Hi-viral transport medium (HiMedia, Mumbai, India) and immediately transported to the laboratory on ice. Samples were stored at −80°C until further use. Viral RNA was extracted using QIAamp viral RNA extraction kit (Qiagen, Germany).

Conventional PCR

Conventional PCR assay was done as one step reverse transcriptase PCR using primers (Sigma, U.S.A) specific for RSV N gene (approximately 705 bp.) [4] For all PCR amplifications, positive (RSV positive sample) and negative (nuclease free water) controls were included.

Real-time PCR

Real-time PCR reactions were carried out as one-step reverse transcriptase PCR using RSV real-time primers and Taqman probes specific to N gene (Applied Biosystems, U.S.A). [5] RNase P primer/probes were used as an internal control to check the quality of the specimen collected.

Sequencing

The conventional PCR product of representative samples was purified (Qiagen, Germany) and sequencing of RSV N gene was performed using the ABI 3730 Genetic analyzer (Applied Biosystems, U.S.A).

Statistical analysis

Sensitivity, specificity, negative likelihood ratio, positive predictive value and negative predictive value were calculated for evaluating the diagnostic performance of conventional PCR. A comparison of data obtained by conventional PCR and real-time data was analyzed by performing McNemar's and Student's t test. Statistical significance was concluded if the P < 0.05. Data were analyzed using QuickCalcs, GraphPad software.

 Results



Two hundred twenty-two patients with ARI were recruited for the study. Twenty samples (9%) were positive for RSV by conventional and/or real-time PCR. While real-time PCR detected all 20 (9%) positives, conventional PCR could detect only 8 (3.6%; P < 0.001) [Table 1].{Table 1}

Sensitivity of conventional PCR when compared to real-time PCR was 40% (95% CI: 19.2-63.9%). Specificity - 100% (95% CI: 98.2-100%); negative likelihood ratio -0.60 (95% CI: 0.42-0.86) and negative predictive value -94.4% (95% CI: 90.4-97.1%). The identity of amplicons obtained from positive samples was confirmed as human respiratory syncytial virus by sequencing.

The average threshold cycle (Ct) values of RSV samples positive by real-time PCR and negative by conventional PCR (n = 12) was 7.15 cycles higher than that of positive samples by conventional PCR (n = 8) which was found to be statistically significant (P value = 0.0009).

 Discussion



The burden of respiratory syncytial virus, a virus known to cause substantial morbidity globally, is underestimated in India and the data from the few reports available are difficult to compare due to various detection methods employed with diverse testing efficacy.

Our study, which aimed to evaluate the efficiency of conventional PCR in comparison with real-time PCR, showed that conventional PCR missed 60% of the positive RSV cases. False negatives can be associated with the ineffectiveness of gel electrophoresis detection system in conventional PCR as it may leave samples with lower viral load undetected. Similar findings were reported by Mentel et al., where real-time PCR was found to be 25% more sensitive than nested PCR for the detection of RSV. [6] However, our findings are contradictory to a recent report from India, where Choudhary et al., 2013 have reported multiplex conventional PCR to be as sensitive as real-time PCR in the detection of eighteen respiratory viruses. [2]

On analyzing the mean threshold cycle (Ct) values of real-time PCR and results of conventional PCR, we noticed that positive samples with lower Ct values (higher viral load) were detected by conventional PCR whereas those with higher Ct values (lower viral load) were not detected. These findings corroborate the observations of Van de Pol et al.[7] Thus, the decreased sensitivity of conventional PCR seen in the present study indicates the lower detection limit of this method, as it had failed to spot samples with low viral load.

In our study, we found that real-time PCR had a significantly improved turnaround time compared with conventional PCR and required minimal sample handling, which reduces the possibility of cross-contamination due to PCR products. In spite of being more expensive, the added costs of real-time PCR can be offset by more accurate diagnosis that in turn can help in better patient management, decreased hospital costs and length of stay.

Thus, to conclude, precise data regarding morbidity and mortality of respiratory syncytial viruses should be collected on a routine basis through a rapid and sensitive method. Conventional PCR showing significant false negatives and poor sensitivity cannot be considered as an accurate method to detect RSV, and it may be prudent to replace it with real-time PCR to achieve a specific diagnosis and to document true incidence of RSV in acute respiratory infections.

References

1Ginocchio CC, McAdam AJ. Current Best Practices for Respiratory Virus Testing. J Clin Microbiol 2011;49 (9 Suppl):S44-S48.
2Choudhary ML, Anand SP, Heydari M, Rane G, Potdar VA, Chadha MS, et al. Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real time RT-PCR. J Virol Methods 2013;189:15-9.
3WHO,WHO surveillance case definitions for ILI and SARI [Internet]. WHO. Available from: http://who.int/influenza/surveillance_monitoring/ili_sari_surveillance_case_definition/en/ [Last accessed on 2014 Jun 5].
4Bharaj P, Sullender WM, Kabra SK, Mani K, Cherian J, Tyagi V, et al. Respiratory viral infections detected by multiplex PCR among pediatric patients with lower respiratory tract infections seen at an urban hospital in Delhi from 2005 to 2007. Virol J 2009;26:6:89.
5Sanghavi SK, Bullotta A, Husain S, Rinaldo CR. Clinical evaluation of multiplex real-time PCR panels for rapid detection of respiratory viral infections. J Med Virol 2012;84:162-9.
6Mentel R, Wegner U, Bruns R, Gürtler L. Real-time PCR to improve the diagnosis of respiratory syncytial virus infection. J Med Microbiol 2003;52(Pt 10):893-6.
7Van de Pol AC, van Loon AM, Wolfs TF, Jansen NJ, Nijhuis M, Breteler EK, et al. Increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time PCR in samples from patients with respiratory symptoms. J Clin Microbiol 2007;45:2260-2.