Development of a fast and low-cost qPCR assay for diagnosis of acute gas pharyngitis.

Mustafa Kolukirik,Mesut Yılmaz,Orhan Ince,Ayşe Istanbullu Tosun,Canan Ketre,Bahar K Ince

doi:10.1186/s12941-016-0162-0

Abstract

BackgroundGroup A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15–30 % of cases of acute pharyngitis in children and 5–10 % of cases in adults. In this study, a real-time quantitative PCR (qPCR) based GAS detection assay in pharyngeal swab specimens was developed.MethodsThe qPCR assay was compared with the gold standard bacterial culture and a rapid antigen detection test (RADT) to evaluate its clinical performance in 687 patients. The analytical sensitivity of the assay was 240 cfu/swab. Forty-five different potential cross-reacting organisms did not react with the test. Four different laboratories for the reproducibility studies were in 100 % (60/60) agreement for the contrived GAS positive and negative swab samples.ResultsThe relative sensitivities of the RADT and the qPCR test were 55.9 and 100 %; and the relative specificities were 100 and 96.3 %, respectively. Duration of the total assay for 24 samples including pre-analytical processing and analysis changed between 42 and 55 min depending on the type of qPCR instrument used. A simple DNA extraction method and a low qPCR volume made the developed assay an economical alternative for the GAS detection.ConclusionWe showed that the developed qPCR test is rapid, cheap, sensitive and specific and therefore can be used to replace both antigen detection and culture for diagnosis of acute GAS pharyngitis.

Highlights

Group A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15–30 % of cases of acute pharyngitis in children and 5–10 % of cases in adults
As most pharyngitis is viral in origin, accurate diagnosis can reduce the unnecessary use of antibiotics and potential development of antibiotic resistance [6, 7]
We aimed to develop a quantitative Polymerase chain reaction (PCR) (qPCR) based GAS detection assay in pharyngeal swab specimens that is as rapid as the previous molecular tests, as sensitive and specific as the culture test and as economical as the lowcost antibiotic treatments

Summary

Introduction

Group A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15–30 % of cases of acute pharyngitis in children and 5–10 % of cases in adults. Acute pharyngitis is a nonspecific symptom that can result from a number of viral or bacterial infections. The cost per case of GAS pharyngitis was estimated to be approximately $205, with about half of the costs attributed to nonmedical costs such as missed days of work by parents for child care [2]. As most pharyngitis is viral in origin, accurate diagnosis can reduce the unnecessary use of antibiotics and potential development of antibiotic resistance [6, 7]. Accurate diagnosis of GAS pharyngitis is difficult for a number of reasons. Diagnosis of GAS pharyngitis using clinical signs alone is unreliable due to the broad overlap in symptoms between the viral and bacterial etiologies [4]; physicians miss up to 50 % of GAS pharyngitis cases and identify 20–40 % of non-GAS sore

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