Indian Journal of Medical Microbiology

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Indian Journal of. Medical Microbiology. Volume 33 Number 2 April 2015. Publication of Indian Association of Medical Microbiologists www.ijmm.org.
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ISSN 0255-0857

Volume 33 Number 2 April 2015

Indian Journal of Medical Microbiology

Indian Journal of Medical Microbiology

• Volume 33 • Issue 2 • April-June 2015 • Pages 203-***

Publication of Indian Association of Medical Microbiologists www.ijmm.org

Indian Journal of Medical Microbiology, (2015) 33(2): 274-276

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Brief Communication

Poor performance characteristics of conventional PCR in detection of respiratory syncytial virus-experience of a tertiary care centre in Southern India G Nandhini, *S Sujatha, N Jain, R Dhodapkar, T Kadhiravan, S Krishnamurthy

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. Key words: Acute respiratory infections, conventional PCR, diagnostic accuracy, real‑time PCR, respiratory syncytial virus

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.

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).

Materials and Methods

Conventional PCR

During the study period from January 2012 - March 2013, patients (both children and adults) attending the outpatient care facilities of our hospital with symptoms

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.

*Corresponding author (email: ) Department of Microbiology (NG, NJ, RD, SS), Medicine (TK), Pediatrics (SK), Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry - 605 006, India Received: 21‑02‑2014 Accepted: 09-10-2014 Access this article online Quick Response Code:

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.

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Sequencing

DOI: 10.4103/0255-0857.154875

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).

April-June 2015

Ganesh, et al.: Performance of conventional PCR in RSV detection

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