Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: Improved primers for the specific detection of human enterovirus 71 by RT PCR

Abstract

BackgroundHuman enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.MethodsLocal strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens.ResultsA total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.ConclusionsOur new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

Genetic Characteristics of Human Enterovirus 71 and Coxsackievirus A16 Circulating from 1999 to 2004 in Shenzhen, People's Republic of China

“…Viruses of lineages B and C cocirculated in Japan beginning in 1995, with lineage C gradually becoming predominant. In Malaysian strains detected in 2000 and 2003, viruses of lineages B and C coexisted (with lineage C predominating) in 2000, while lineage C but not lineage B was identified in 2003 (19). These results indicate that three genetic lineages of CA16 (A, B, and C) have existed in Asia.…”

Combining Multiplex Reverse Transcription-PCR and a Diagnostic Microarray To Detect and Differentiate Enterovirus 71 and Coxsackievirus A16

“…In fact, reverse transcription-PCR (RT-PCR) has been used to diagnose enterovirus infections early (13,29), and DNA sequencing has been used for serotyping (7,14,35). However, genomic information for many enterovirus serotypes is limited, and there is a high genetic heterogeneity among different strains, so misdiagnosis might be frequent (27). Therefore, we are still left with a need for a rapid and accurate means of diagnosing HFMD.…”

Impact of Human Enterovirus 71 Genotypes in Meningoencephalitis in Iran

“…The VP1 protein contains numerous neutralization sites and corresponds to the virus serotyping; therefore, it was from this that most of the EV71 phylogenies were derived ( 15 - 17 , 31 , 36 ). It might be possible that two sub-genotypes, B3 and C4, are recombinants of EV-71 and prototype CV-A16/G10 ( 27 , 31 , 36 , 37 ).…”