Comparing Molecular Methods for Early Detection and Serotyping of Enteroviruses in Throat Swabs of Pediatric Patients

Abstract

BackgroundEnteroviruses include over 100 serotypes and usually cause self-limited infections with non-specific symptoms in children, with the exceptions of polioviruses and enterovirus 71 which frequently cause neurologic complications. Therefore, early detection and serotyping of enteroviruses are critical in clinical management and disease surveillance. Traditional methods for detection and serotyping of enteroviruses are virus isolation and immunofluorescence assay, which are time-consuming. In this study, we compare virus isolation and two molecular tests for detection and serotyping of enteroviruses in clinical samples.MethodsOne hundred and ten throat swabs were collected from pediatric outpatients with enterovirus-like illnesses (hand-foot-mouth disease, herpangina, and non-specific febrile illness). Virus isolation was conducted using multiple cell lines and isolated viruses were serotyped using immunofluorescent assay. In the molecular tests, a semi-nested RT-PCR and a novel CODEHOP platform were used to detect the 5′UTR and VP1 genes of enteroviruses, respectively. Amplified nucleotides were sequenced and genotyped.ResultsAmong the 110 cases, 39(35%), 52(47%), and 46(42%) were tested positive with these three tests, respectively. Using the consensus results of these three tests as the gold standard, agreement of the VP1 CODEHOP test was 96%, which is higher than those of the virus isolation (89%) and the 5′-UTR test (88%). The VP1 CODEHOP test also has the best performance on serotyping confirmed with serum neutralization tests.ConclusionsThe VP1 CODEHOP test performed well for detection and serotyping of enteroviruses in clinical specimens and could reduce unnecessary hospitalization cares during enterovirus seasons.

A Molecular Approach Applied to Enteroviruses Surveillance in Northern Taiwan, 2008-2012

“…As expected, detection rate of the VI-IFA was lower than that of the VP1-CODEHOP test (48% vs. 58%). After verification with the serotype-specific serum neutralization tests, we further confirmed high accuracy of the VP1-CODEHOP test, which is consistent with a previous study with a small sample size [8]. The previous study is a pilot study and included 110 cases for comparing two molecular methods (5'-UTR and VP1-CODEHOP) and the traditional method (virus isolation).…”

“…Based on the VP1-CODEHOP test, we found that multiple EV71 genotypes co-circulated in 2008–2012 but EV71 sub-genotypes B5b and B5c caused large-scale epidemics in 2008 and 2012, respectively, which are consistent with other studies analyzing full VP1 sequences and complete genomes [24, 27]. Although a degenerate primer set targeting conserved region of enteroviruses (5’-UTR) had been widely used on molecular diagnostic systems, the 5’-UTR-based molecular diagnosis cannot precisely identify serotypes of human enteroviruses [8, 28]. …”

“…Therefore, the enterovirus VP1 gene has been targeted to develop a CODEHOP-based reverse-transcriptase (RT) PCR (VP1-CODEHOP) for serotyping of enterovirus clinical isolates. Recently, accuracy of the VP1-CODEHOP test was further confirmed to be higher than that of the 5'-UTR RT-PCR and VI-IFA for detection and serotyping of enterovirus infections using throat swabs collected from pediatric outpatients [8, 11]. In this study, we further conducted a 5-year enterovirus surveillance comparing the VI-IFA and VP1-CODEHOP tests in northern Taiwan from 2008 to 2012.…”

“…Therefore, it is desirable to develop rapid laboratory diagnosis for class suspension and case management in the enterovirus surveillance system. Previous studies have documented that molecular diagnosis based on polymerase chain reaction (PCR) is more time-saving and sensitive than the VI-IFA for detection of EV71 infections in inpatients [5–7] and outpatients [8]. Besides, a novel molecular method using the consensus degenerate hybrid oligonucleotide primer (CODEHOP) has been developed to detect and identify distant-related pathogens with limited primers instead of a large number of primers.…”

Synthetic peptides for efficient discrimination of anti-enterovirus antibodies at the serotype level

“…Molecular methods such as polymerase chain reaction (e.g. nested PCR) are also used for the detection of viral ribonucleic acid [8]. Although these tests possess increased sensitivities in CSF and blood specimens, their utility is limited to the viremia phase of the infection (usually over a few days).…”