| BackgroundHand,foot,and mouth disease(HFMD)is a common infectious disease occurring in children caused by enteroviruses,which has caused many outbreaks worldwide and brings seriously threat to the children’s life and health.Enterovirus A71(EV-A71)and Coxsackievirus A16(CVA16)are identified as the predominant pathogens of HFMD.In recent years,Coxsackievirus A6(CVA6)and Coxsackievirus A10(CVA10)have played more and more important role in a series of HFMD outbreaks.It indicates that the pathogenic spectrum of HFMD has been changing.Guangzhou city is one of the most serious cities in HFMD with high incidence,and the recent relevant research on HFMD in Guangzhou is rare.Therefore,it is urgent to establish a comprehensive surveillance on the pathogenic spectrums of HFMD in Guangzhou.In addition,CVA6 has caused many outbreaks in recent years and the atypical clinical manifestations of HFMD caused by CVA6 may increase the difficulty in HFMD diagnosis and treatment.Although vaccines against EV-A71 have been successfully developed,they cannot protect children from infection with CVA6.No effective antiviral treatments have been developed for HFMD.Therefore,there is an urgent need to develop a simple,rapid,and accurate detection method for the early diagnosis of CVA6 infection to improve clinical outcomes and prevent transmission.In this study,we not only aim to understand the epidemic characteristics associated with HFMD outbreak in Guangzhou,2018,but also establish a one-step reverse-transcription recombinase polymerase amplification(RTRPA)combined with a disposable lateral flow strip(LFS)assay to detect CVA6.Methods1.Epidemiological investigation of HFMD in Guangzhou in 2018The clinical and laboratory data of 1220 enterovirus-associated HFMD patients in Guangdong Women and Children Hospital in 2018 were analysed in this study.Molecular diagnostic methods were performed to identify its serotypes.Phylogenetic analyses were depicted based on the complete VP1 gene of CVA6,CVA10,and CVA16.2.Establishment of a rapid detection method for the detection of CVA6RPA primers and probe were designed to target the highly conserved regions of CVA6 VP1 gene.Subsequently,the RT-RPA-LFS assay was established and optimized for the detection of CVA6.To determine the sensitivity of the RT-RPA-LFS assay,tenfold serial dilutions of a CVA6 RNA standard were used as reaction templates,and all the samples were also tested using a commercial q RT-PCR diagnostic kit for comparison.The RT-RPA-LFS assay specificity was assessed using non-CVA6enteroviruses(CVA2,4,5,9,10,16,EV-A71,CVB2,3,and Echovirus 3,11,18).To validate the clinical performance of the RT-RPA-LFS assay,142 clinical samples confirmed by commercial enterovirus kits was analysed using a commercial real-time quantitative RT-PCR(q RT-PCR)diagnostic kit and reverse transcription semi-nested PCR(RT-sn PCR)in parallel with the RT-RPA-LFS assay.Results1.Epidemiological investigation of HFMD in Guangzhou in 2018There were 21 enterovirus serotypes detected in Guangzhou in 2018.Three serotypes of enterovirus,CVA6(364/1220,29.8%),CVA10(305/1220,25.0%),and CVA16(397/1220,32.5%),were identified as the causative pathogens and accounted for 87.3% among all 1220 HFMD patients.In different seasons,CVA6 was the predominant pathogen of HFMD during autumn,and CVA10 as well as CVA16 were more prevalent in summer.Patients infected by CVA6,CVA10 or CVA16 showed similar clinical features and laboratory characteristics,and the ratios of severe HFMD were 5.8%,5.9%,and 1.5% in the three serotypes.Phylogenetic analyses of VP1 sequences showed that the CVA6,CVA10,and CVA16 sequences belonged to the subgenogroup E2,genogroup E,and genogroup B1,respectively.2.Establishment of a rapid detection method for the detection of CVA6In this study,a simple,rapid and accurate one-step RT-RPA-LFS assay was developed to detect CVA6.This assay can be performed in less than 35 min at 37℃without expensive instruments,and the result can be observed directly with the naked eye.The sensitivity of the RT-RPA-LFS was 10 copies per reaction,which was comparable to that of the conventional q RT-PCR assays.Moreover,the assay specificity was 100%.The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples,and the coincidence rate between RT-RPA-LFS,q PCR,and RT-sn PCR was 100%.ConclusionsCVA6,CVA10,and CVA16 were the predominant and co-circulated serotypes in Guangzhou China,2018.Moreover,a significant seasonal change in the occurrences of enteroviruses was observed in this study.CVA6 was the predominant pathogen of HFMD during autumn,and the co-circulation of CVA10 and CVA16 was more prevalent in summer.In addition,we developed a highly sensitive and specific one-step RT-RPA-LFS assay that can easily,rapidly,and visually detect CVA6 without any expensive instruments,which will be hopeful for the prevention and control of HFMD. |
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