Detection Of Enterovirus 71 And Coxsackievirus A16 From Children With Hand, Foot And Mouth Disease In Kunming, 2008

Objective Human enterovirus 71(EV71) and coxsakievirus A16(CoxA16) are two major causes of hand-foot-and-mouth disease(HFMD).Since poliovirus has been eradicated,EV71 has come to medical attention as the most important enterovirus related to severe complications and sequelae.This study was conducted to investigate the casual causes of HFMD in children during an epidemic from April to June of 2008 in Kunming.Methods 58 specimens which include vesicle fluid,urine and throat swabs were collected from 25 patients who got HFMD.Those suspected patients were diagnosed through real time fluorescence-probing RT-PCR.The positive amplified products of EV71 were sequenced and then compared with references viruses from GenBank.The phylogenetic analysis was based on VP1 gene of enterovirus. Meanwhile,epidemiological data were collected and analyzed with software SPSS11.5.Results1.Totally 58 specimens include throat swab,vesical fluid and urine were collected from 25 patients.Vesical fluid was a sensitive specimen to detect enterovirus.The viral load of throat swab is(1.07±2.05)×10~5,and(4.21±1.10)×10~6 in vesical samples.The virus yield of throat swab samples and vesical fluid samples has no significant difference in statistics.2.13 patients were identified as positive for CoxA16 and 5 patients were identified as positive for EV71,the rate of CoxA16 and EV71 was 52%and 20% respectively.3.The positive rate of real time fluorescence-probing RT-PCR in detecting clinical samples of HFMD is 72%.The mean time of sample collection is 2.17d.Viral load of enterovirus in clinical sample is 2.99×102～3.70×107 per milliliter.4.Two isolated strains of EV71 were compared with existent virus strain in GenBank which share high homology with Genogroup C.5.The partial VP1 sequences of the PCR products was analyzed phylogenetically, by using VP1-based genetic classification,the isolated were classified as subgenogroup C4 which shows a close genetic relationship to recent subgenogroup C4 strains in mainland China.6.Phylogenetic tree was prepared by the neighbor-joining method and newly identified subgenogroup C4 strains.The results demonstrate that the viruses isolated in mainland China were belong to the same group but different from the original virus in America.7.All of the admitted patients were slight and had rashes on hand,foot and mouth.Some of the patients got fever or weakness.The clinical manifestations of CoxA 16 and EV71 were alike.8.Most of the HFMD patients were two to four years old,and third of those patients had contactant history.It has a high number of cases occurred in kindergarten or has a family assembling tendency in the HFMD epidemic.Conclusions1.The major causes of HFMD in this epidemic were CoxA16 and EV71.2.Two isolated EV71 viruses were analyzed based on partial VP1 sequences and the result shows that the isolated strains were belong to a new lineage(C4) within genogroup C.3.All of the HFMD patients got rashes on hand,foot and mouth.Clinicians can hardly distinguish these two viruses through clinical manifestations.4.Vesicle swabs give a high diagnostic yield in this study,but it has no significant difference of viral load from throat swabs.5.All of the urine samples were detected as negative for CoxA 16 and EV71.