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Novel tests (BioPlex) for herpes simplex virus-1 (HSV-1) and HSV-2 IgG were compared with HerpeSelect HSV-1 and HSV-2 ELISAs for type-specific IgG. The sensitivity and specificity of BioPlex HSV-1 IgG were 94% (84/89) and 96% (119/124), respectively, with unselected sera, while the sensitivity and specificity of BioPlex HSV-2 IgG were 92% (109/118) and 98% (95/97), respectively. BioPlex IgM was compared with Diamedix IgM against sera from patients with culture-documented genital herpes. The test results were concordant in 81% of sera from HSV-1 patients and in 90% of sera from HSV-2 patients. Use of BioPlex IgM in addition to BioPlex IgG tests increased HSV-2 seroconversion detection from 47% of subjects to 70%. Use of Diamedix IgM in addition to Focus IgG ELISA increased HSV-2 detection from 40% of subjects to 70%. IgM was detected by BioPlex in 63% of sera from patients with early HSV-2 infection (< 30 days) and in 59% of sera by Diamedix. IgM was also detected in a large proportion of sera from subjects with established HSV-2 infection (33% by BioPlex and 29% by Diamedix). Addition of IgM testing substantially increased the ability to detect seroconversion early in infection. IgM is an indicator of recent infection only in subjects who lack detectable IgG.