We used the following formula, where s is the shedding rate, ϕ is the specificity, and θk and pk are the sensitivity of PCR and the observed frequency of shedding in copy level range k, respectively (both s and the observed pk are estimated from the shedding studies of HSV-2-seropositive participants, θk was from the dilution study, and ϕ was from HSV-seronegative participants): misclassified = false positives + false negatives = (1 − ϕ) × (1 − s) + Σ (1 − θk) × pk × s.
Figure depicts the estimated false-positive, false-negative, and total misclassification rates, with shedding rates between 1% and 50%, using three cutoffs: ≥1, ≥5, and ≥10 copies per 20 μl. False-positive results comprised only a minority of misclassified results. At shedding rates greater than 8%, the lowest cutoff resulted in the lowest total misclassification rate.
Misclassification rates by shedding rate and cutoff for determination of positivity. Total bar height indicates overall misclassification, with shading distinguishing false negatives (neg.) and false positives (pos.).
We also examined the timing of low positives (below 10 copies per 20 μl) in relation to episodes of shedding, where an episode is any consecutive shedding period at copy levels greater than 10 copies per 20 μl. We tested whether low positives occurred randomly (as would be expected with contamination) or in close proximity to episodes. Of the 708 samples in which HSV DNA was detected at 1 to 10 copy numbers, 54% occurred during or within 2 days of a shedding episode, compared to 13% of 16,170 totally negative samples (P < 0.0001 by hypergeometric test), confirming that low positives are associated with confirmed HSV shedding episodes. A total of 15% of low positives also occurred coincidently with lesions, compared to 5% of completely negative samples (P < 0.0001).
Quantitative PCR for HSV DNA appears to be highly accurate. The test probably detects all shedding when the actual quantity is greater than 75 copies per 20 μl (3.75 × 103 copies/ml) (67% of received samples) and correctly identifies more than 99% of negative samples. Given the high specificity of the assay, and the occurrence of low positives in substantial numbers (3% of samples) and in temporal association with episodes and lesions, a detection of even low copy numbers of HSV DNA (1 copy/20 μl or 50 copies/ml) is almost invariably a true positive.
However, a cutoff determination also requires a consideration of the clinical laboratory context. For example, it may be deemed more risky to provide a false-positive diagnosis of genital HSV in an adult, and hence, we would prefer the cutoff with maximum specificity, whereas for detecting HSV encephalitis or neonatal herpes, the consequences of a false-negative assay are high.