The GeneXpert HSV PCR test will be validated against HSV viral cultures and routine quantitative PCR. Validation will occur in two populations: 1) nonpregnant women in STI clinics with clinically-apparent HSV lesions (Group 1, n=300), and 2) pregnant women in active labor with no visible evidence of HSV infection (Group 2, n=12,500). All testing of samples on the GeneXpert platform, routine quantitative PCR, and viral culture will be done at the UAB Central Laboratory. Women in each group will have specimens obtained from genital lesions (Group 1) or vaginal swabs (Group 2). Specimens from all women in Group 1 will be evaluated by HSV culture, routine HSV PCR, and GeneXpert HSV PCR. Approximately half of the women in Group 2 will be tested by routine HSV PCR and GeneXpert HSV PCR; specimens from the rest of the women in Group 2 will be stored for possible testing in the future by routine HSV PCR and GeneXpert HSV PCR. In this manner, we will maximize the data from which to compare GeneXpert PCR results with routine PCR, while maintaining flexibility to ensure an adequate number of specimens are positive for HSV DNA by routine PCR.
Swabs from pregnant women in labor will be placed in viral transport media, frozen at -20°C, and batch-shipped to the UAB Central Laboratory for analysis on the GeneXpert instrument and by routine HSV quantitative PCR. Swabs from nonpregnant women in STI clinics will be placed in viral transport media, refrigerated at 4°C, and shipped to the UAB Central Laboratory for real time analysis on the GeneXpert instrument and by HSV culture and routine quantitative HSV PCR. Specimens from the first 300 women enrolled in Group 2 will be run as individual routine PCRs and in batches of 5 samples per PCR run. In this manner, we will validate that the level of detection from batching of samples for routine PCR is acceptable. Once this validation occurs, specimens from the approximately half of remaining Group 2 women will be batched for real-time routine PCR analysis. If a batch run of 5 specimens is negative, no further testing will be performed. If a batch run of 5 specimens is positive, all of the specimens will be separated out for re-running as individual PCRs. All specimens evaluated by routine PCR will also be evaluated by GeneXpert PCR; in this manner, we will have individual routine PCR results results for comparison against GeneXpert PCR results.
A blood specimen will be obtained from each nonpregnant (Group 1) and pregnant (Group 2) woman at the time of enrollment, and if she is determined to be shedding HSV by routine PCR, GeneXpert PCR, or culture then type-specific serologic testing will be performed. Correlation of viral typing from the virologic sampling with HSV-1 and HSV-2 serostatus will allow for categorization of infection (first-episode primary, first-episode nonprimary, or recurrent infection).
Those women in Group 1 who have a positive HSV culture will be contacted directly when the result is known. Women in Group 2 with a positive HSV PCR result will not be contacted because routine HSV PCR and GeneXpert PCR are not FDA-cleared tests in this population; thus, we will not know the test performance characteristics (e.g., sensitivity, specificity, etc.) until completion of the trial. All pregnant women in Group 2 will receive written materials at the time of enrollment educating them on signs and symptoms of neonatal HSV disease. All postpartum women will be contacted by telephone 60-90 days post-delivery and an inquiry will be made to determine if their babies developed neonatal HSV disease.
Data on the incidence of neonatal HSV disease among babies delivered to women in Group 2 will be compared with the incidence data from Brown et al.1 In their study of almost 60,000 women conducted over a 20 year period, this group of researchers has reported an incidence rate for neonatal HSV disease of 1 in 3,200 live births.
