Background:
In May 2022 an increasing number of Monkeypox virus (Mpox) cases in non-endemic countries, including the United States, was noted. This outbreak of Mpox has primarily affected men who have sex with men who have reported recent sex with new or multiple partners. This study describes the performance of a single-well dual-target real-time PCR (RT-PCR) test for the detection of Mpox without the need for tiered testing.
Methods:
Lesion swabs collected in viral transport media (VTM) were extracted and tested by RT-PCR using a non-variola Orthopoxvirus target (E9L-NVAR) and an Mpox virus clade II (West African) target (MPXV-WA). We assessed analytical sensitivity, specificity, precision, accuracy and specimen stability during storage. RT-PCR cycle threshold (Ct) distributions were determined for specimens submitted for clinical testing.
Results:
Analytical sensitivity was 100 copies/mL from swabs in VTM. Intra- and inter-assay precision had a %CV of < 4.9 across 3 target levels. Analytical specificity was 100% when tested among unrelated pathogens including herpes simplex virus (HSV) and varicella zoster virus (VZV), which can also cause lesions. An Mpox-positive clinical specimen diluted in negative swab media was stable for 24 hours at room temperature, 7 days refrigerated and 30 days frozen. Most (96.8%) Mpox-positives were obtained from males with a median age of 35 [IQR: 30-43; range: 3 - 81]. Although Ct values for both RT-PCR targets were highly correlated (Spearman rho 0.995), 12 specimens had undetectable Cts for MPXV-WA in spite of detectable Cts for E9L-NVAR. Six specimens submitted for sequencing harbored the recently described Mpox crmB gene deletion. While a single swab was typically collected, multiple swabs were collected from 26.3% (6,577/24,981) of the patients. Ct values from patients with multiple swabs that demonstrated both Mpox-positive and negative results had values that were 4.2 Cts (95% CI: 3.4-5.5) higher than Ct values for patients with exclusively Mpox-positive swabs (Table).
Conclusions:
A sensitive, dual-target, RT-PCR assay to enable Mpox diagnosis without tiered testing demonstrated excellent performance in clinical specimens. The inclusion of 2 targets in a single well improved throughput relative to multi-well tests and reduced the risk of false negatives resulting from genomic deletions. Higher Ct values noted for patients with discordant positive and negative swabs may be associated with variability in lesion stage and swabbing efficiencies.
Demographics for patients having positive, mixed and negative Mpox tests