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

Local 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.A 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.Our 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.Hand, foot and mouth disease (HFMD) is an unremarkable illness that commonly occurs in young children. This condition is caused by human species A enteroviruses [1] most commonly Coxsackievirus A16 (CVA16) but has more recently been associated with human enterovirus 71 (HEV71) which can also cause neurological complications [2]. In the most common manifestation which gives the syndrome its name, children typically present with vesicular exanthema on the soles of their feet, the palms of their hands and in their mouths, causing discomfort and feeding difficulties. CVA16 and HEV71 are also associated with herpangina, but this is often also caused by other species A enteroviruses such as Coxsackievirus A8 (CVA8) and Coxsackievirus A10 (CVA10). Etiological diagnosis of HFMD and herpangina has classically been dependent upon isolation of viruses and the identification of these either by neutralization tests using Lim-Benyesh-Melnick (LBM) pools or monospecific a