Rapid Identification and Typing of Herpes Simplex Virus in Clinical Specimens by a Direct Microneutralization Test

Abstract

Now that various drugs for treating patients suffering from herpes simplex virus (HSV) infections are available (I, 3, 5, 6, 11), it is desirable to give a quick and accurate diagnosis by a method feasible for clinical laboratories. It is also necessary to determine the type of HSV to trace the route of infection as well as to accumulate data for epidemiological analysis. Isolation of the virus from patients followed by a microneutralization test for viral identification and typing (8,12), if performed within a short period of time, would serve these purposes. The isolation rate seems to be nearly 100% when a suitable transport medium and adequate cells are used (9). However, in this procedure, we have to spend days for preparation of monolayer cultures of an appropriate cell age, observation of cytopathic effect (CPE) and the final neutralization test. To shorten the whole process, we used the clinical specimens directly in a microneutralization test in parallel with conventional cell inoculation. The results were satisfactory in that more than 90% of the isolation-positive specimens could be identified within 3 days by means of the direct assay.Outpatients visiting the Department of Dermatology, Tokyo Women's Medical College, suspected of having herpetic infections were examined by applying a skin swab to the lesion. Just before use, the swab was soaked in divalent cation-free phosphate buffered saline (2) containing 1,000 p,g of streptomycin and 5,000 units of penicillin per ml. After rubbing the lesion, the swab was quickly squeezed into 2 ml of a transport medium which consisted of either Earle's saline plus 0.5% lactalbumin hydrolysate and 0.1 % yeast extract or Eagle's minimum essential medium (MEM) supplemented with 0.11 % NaHCO a and 20% inactivated calf serum. The tube was rubber-stoppered and kept frozen until tested. The suspension was thawed only once and passed through a Millipore membrane of 0.45-p, porosity. No special pretreatment to prevent viral adsorption to the membrane was necessary in this case (8).In most cases, the direct assay was performed simultaneously with inoculation of cells according to the conventional procedure. In the direct assay, the clinical 541