Establishment And Application Of Multiplex PCR Assay To Study Early Multiple Herpesviruses Infection During HSCT

PARTⅠ Establishment of multiplex PCR assay for eight herpesviruses【Objective】To establish a multiplex PCR assay to detect eight herpesviruses at the same time.【Methods】The conserved sequence of the 8 viruses were identified from NCBI.Proper probes and primers were designed by software and the feasibility of the primers and probes were checked.Two hundred blood samples from patients after hematopoietic stem cell transplantation were collected randomly to test the sensitivity and specificity of the multiplex PCR assay.In the experiment,for HSV-1,HSV-2,EBV,and CMV,the specificity with multiplex PCR assay were compared with their standard kits.For those viruses without standard kits,the 1st generation sequencing was done.【Results】(1)In the study,we designed probes and primers of the multiplex PCR assay,successfully.It could detect eight herpesviruses at the same time.After the improvement of the reaction conditions and system,the amplification efficiency of multiplex PCR was feasible.(2)There were no difference of specificity between multiplex PCR assay and HSV-1,HSV-2,EBV,CMV kit.For VZV,HHV-6,HHV-7 and HHV-8 positive samples,the results of 1st generation sequencing showed a correct sequence of those viruses.The specificity of the assay was strong.(3)The multiplex PCR assay could detect the samples with under 1×103copies.It showed that the sensitivity of the assay was high.【Conclusion】The established multiplex PCR assay is sensitive and specific to detect eight humanherpes viruses at the same time.PARTⅡ Clinical study of early Multipleherpesviruses infection duringhematopoietic stem cell transplantation using multiplex PCR assay【Objective】To study the prevalence of eight herpesviruses infection within 90 days after HSCT with the multiplex PCR assay and to explore the association between multiple herpesviruses infection and clinical characteristics in HSCT patients and its impact on post-transplant complications and prognosis.【Methods】Ninety consecutive patients undergoing HSCT at the First Affiliated Hospital of Soochow University between February 2017 and August 2017 were enrolled in the study,734 peripheral blood specimens were obtained before and within 90 days after transplantation at different time points,Lab-Aid824 Nucleic Acid Extraction Mini Reagent was used to extract DNA and multiplex PCR assay was used to detect eight human herpes viruses at once.We analyzed the prevalence of multiple herpesviruses infection within 90 days after HSCT and explored the correlation between multiple herpesviruses infection and clinical characteristics in HSCT patients and its impact on post-transplant complications and prognosis by various statistical methods.【Results】1.There were 53 male and 37 female patients with a median age of 29.5years old(range:9-51).The median follow-up time was 192 days(range: 35-308).Thirty-two(35.6%)patients suffered from grade II-IV a GVHD.The median time of neutrophil engraftment was 12days(range:8-23).The median time of platelet recovery was 14days(range:9-36).The cumulative incidence of relapse and NRM(non-relapse mortality)were 11.7% and 5.6%,respectively.The overall survival(OS)and progression free survival(PFS)were 90.6% and 88.3%,respectively.2.Multiplex PCR detected HSV-1 in 18(20%),HSV-2 in 4(4.4%),VZV in 3(3.3%),EBV in 24(26.7%),CMV in 5(5.6%),HHV-6 in 4(4.4%),HHV-7 in 2(2.2%),HHV-8 in3(3.3%)samples of 90 blood samples before HSCT.Multiple detection of ≥ 2 kinds of viruses was observed in 21(23.3%)samples.Multiplex PCR detected HSV-1 in 80(12.4%),HSV-2 in 10(1.6%),VZVin11(1.7%),EBV in 132(20.5%),CMV in 112(17.4%),HHV-6 in37(5.7%),HHV-7 in 14(2.2%),and HHV-8 in 9(1.4%)samples of 644 blood samples from all patients within 90 days after HSCT.Meanwhile,two viruses infection was detected simultaneously in 8.3%(62/644)samples,and 3 kinds of viruses simultaneously was observed in 1.7%(11/644)samples.There were 77.8%(70/90)patients suffering from herpesviruses infection.The infection rates of HSV-1,HSV-2,VZV,EBV,CMV,HHV-6,HHV-7 and HHV-8 after HSCT were 28.9%(26/90),5.6%(5/90),5.6%(5/90),50%(45/90),46.7%(42/90),14.4%(13/90),5.6%(5/90),6.7%(6/90),respectively.Multiple detection of 3viruses was observed in 34.6%(18/52)patients.The most frequent coinfection in all patients was a combination of HSV-1,EBV and CMV(38.9%,7/18).The coinfection of 4kinds of viruses was observed in 4 cases.3.34 patients in our study experienced clinical symptoms associated with viral infection.The main manifestations were hemorrhagic cystitis(HC)in 27 patients interstitial pneumonia in 3 patients,enteritis in 2 patients,viral encephalitis in 3 patients and unexplained fever in 3 patients.Herpes virus infection was detected in 20 of 27 HC patients,coinfection with EBV and CMV was the most common in patients with HC.Multiple detection of ≥2 kinds of viruses was observed in all patients with interstitial pneumonia.Coinfection of HSV-1,CMV,HHV-6,and HHV-7 and dual infection of HSV-1and HHV-6 were detected in 2 patients with enteritis.Among the patients of viral encephalitis,dual infection of HSV-1 and EBV and mixed infection of HSV-2,EBV and HHV-8 were observed in 2 cases.HSV-1 and EBV were detected in 2 patients of unexplained fever,respectively.4.There was a correlation between HSV-1 and HSV-2 infection.The correlation between HHV-6 and HHV-7 infection was also observed.The correlation between the herpesvirus infection and clinical characteristics was analyzed.The age of patients was correlated with the incidence of HSV-1 infection before transplantation.The results showed that ≥ 2 kinds of viruses coinfection was associated with HLA mismatch(P=0.003),grade II-IV a GVHD(P=0.022)and the use of ATG(P=0.048).Multivariate analysis showed that HLA mismatch and grade II-IV a GVHD were independent risk factors for ≥ 2 kinds of viruses coinfection.5.We analyzed the impact of herpesvirus infection on engraftment,the incidence of grade II-IV a GVHD,relapse,NRM,PFS and OS after transplantation.The results showed that with HHV-6 infection and ≥ 2 herpesviruses infection increased the incidence of grade II-IV a GVHD.【Conclusion】The infection of single and multiple herpesviruses exists before and after transplantion.There is a correlation between HSV-1 and HSV-2 infection.HHV-6 and HHV-7 infection have the correlation too.HLA mismatch and grade II-IV a GVHD can increase the incidence of ≥ 2 kinds of viruses coinfection.With HHV-6 infection and ≥ 2herpesviruses infection,which both increase the incidence of grade II-IV a GVHD.