多细胞株微量培养法在病毒性脑炎、脑膜炎患儿病原学检测的应用研究

Microculture of multiple cell lines to detect pathogens responsible for viral encephalitis and meningitis

目的探讨多细胞株微量培养法在病毒性脑炎、脑膜炎病原学检测中的应用价值。方法将脑脊液(CSF)标本过滤除菌后分别接种于含有青霉素和链霉素微量细胞培养板单层细胞上,每份标本平行接种8株细胞:MA104、MDCK、HEL、HEK293、Vero、Hep-2、Hela和BHK-21。静置培养,获得稳定的病毒株。以肠道病毒(EV)组合血清鉴定引起病毒性脑炎、脑膜炎的常见病毒类型。结果 126份病毒性脑炎、脑膜炎患儿CSF标本82份分离到病毒,阳性率63.0%,其中MA104阳性率为57.1%,MDCK阳性率为19.8%,HEL阳性率为11.9%,HEK阳性率为7.1%,Vero阳性率为6.3%,Hep-2阳性率为4.8%,Hela阳性率为4.0%,BHK-21阳性率为2.4%。病变细胞经血清学鉴定,78例鉴定出病毒株,鉴定率为92.8%,这些病毒中EV所占比例较高,为69.2%;ADV占11.5%,HSV-Ⅰ占5.1%,HSV-Ⅱ占6.4%;CMV占5.1%,INF占2.6%。结论 MA104细胞是分离CSF中病毒较敏感的细胞,可以作CSF中病原分离的首选细胞;联合多株细胞可提高细胞病变检出率。多株细胞微孔板培养法分离CSF病毒具有推广应用价值。

Objective To explore the use of microculture of multiple cell lines to detect pathogens responsible for viral encephalitis and meningitis.Methods Cerebrospinal fluid（CSF） specimens were aseptically filtered and plated on microporous plates in single cell layers containing penicillin and streptomycin.Each plate was seeded in parallel with eight types of cells： MA104,MDCK,HEL,HEK293,Vero,Hep-2,Hela,and BHK-21.After static culturing,stable strains were obtained.Serum containing Enterovirus（EV） was used to identify the common pathogens responsible for viral encephalitis and meningitis.Results Viruses were isolated in specimens from 82（63.0%） of 126 children with viral encephalitis or meningitis.Of the children,57.1% tested positive for MA104,19.8% tested positive for MDCK,tested positive for HEL was 11.9%,7.1% tested positive for HEK,6.3% tested positive for Vero,4.8% tested positive for Hep-2,4.0% tested positive for Hela,and 2.4% tested positive for BHK21.Pathological cells were then identified by serology in 78 patients.The rate of detection was 92.8%.The most prevalent virus was EV,found in 69.2%,followed by ADV in 11.5%,HSV-Ⅰin 5.1%,HSV-Ⅱin 6.4%,CMV in 5.1%,and INF in 2.6%.Conclusion MA104 cells were most susceptible to viruses isolated from CSF.These cells are first attacked by the pathogens in CSF.Combining these cells with other cell lines can improve the detection of cytopathic changes.Microtiter plate culture of multiple cell lines is a worthwhile way to isolate viruses in CSF.