In a recent study posted to medRxiv*, researchers validated monkeypox virus (MPXV) quantitative polymerase chain reaction (qPCR) assays.
Human monkeypox (MPX) cases have been rarely detected outside MPXV-endemic countries since its discovery in the 1970s. This zoonotic disease has been neglected until the ongoing outbreak, despite warnings of global spread and the evidence of increased human-to-human transmission. More than 77,000 MPX cases have been reported from over 100 countries in the ongoing MPX outbreak.
MPX cases may present with fever, lymphadenopathy, and a papillary rash. Immunocompromised subjects are at risk of severe MPX manifestations, including death. The antiviral drug, tecovirimat (Tpoxx), and the JYNNEOS vaccine developed originally for smallpox are the only countermeasures against MPX. Since early and rapid virus detection is necessary for MPX prevention and treatment, developing qPCR assays is vital to disrupt transmission networks.
The study and findings
In the present study, researchers evaluated two MPXV-specific qPCR assays targeting the G2R and F3L loci and compared their sensitivity, specificity, and accuracy with the Centers for Disease Control and Prevention’s (CDC’s) pan-orthopoxvirus assay (OPXV-E9L). The authors commercially obtained synthetic MPXV DNA (VR-3270SD) due to the short supply of MPXV clinical samples and genomic DNA in Spring 2022.
The purchased template (VR-3270SD) had binding sites for primers and probes for numerous previously-developed assays. The researchers estimated the precise copy number of this standard by droplet digital PCR (ddPCR) to be 1.29 x 108 copies/ml for the MPXV-F3L and OPXV-E9L loci and 1.3 x 108 copies/ml for the MPXV-G2R locus.
The authors tested 15 herpes simplex virus (HSV)-positive, 17 varicella-zoster virus (VZV)-positive, and 52 HSV/VZV-negative skin swab samples using the OPXV-E9L assay and verified they were negative for MPXV. The F3L assay did not detect MPXV DNA in these specimens, whereas in the G2R assay, one VZV-positive sample was found positive for G2R but tested G2R-negative on repeat.
The G2R and F3L assays had negative percent agreement of 98.8% and 100%, respectively, across 84 samples. Next, the team tested for cross-reactivity of the MPXV assays with camelpox (CMLV), cowpox (CPXV), and vaccinia (VACV) viruses, which are close phylogenetic relatives to MPXV. The MPXV-F3L and -G2R assays did not detect the DNAs of CMLV, CPXV, and VACV, whereas the OPXV-E9L assay showed average cycle threshold (Ct) values of 19.8, 25.8, and 23.8, respectively.
The ability to detect MPXV DNA was assessed using contrived positives. The positive percent agreement was 100% for the OPXV-E9L assay and 99.1% for MPXV-G2R and -F3L assays. Further, they tested whether G2R and F3L assays would accurately detect MPXV in (clinical) samples and secured seven MPXV-positive skin swab specimens from a public health laboratory (PHL).
The PHL specimens were diluted 1:40 before extraction due to volume constraints. Twenty MPXV clinical specimens testing positive on the F3L assay were concomitantly tested with OPXV-E9L and MPXV-G2R assays. The MPXV-specific assays had, on average, lower Ct values than the OPXV-E9L assay for each specimen. Moreover, the G2R assay had a lower Ct than the F3L assay in each sample, consistent with two G2R copies in the MPXV genome.
The researchers estimated the limit of detection (LoD) to be 330 copies/ml, equivalent to 3.3 copies per (PCR) reaction using standard extraction methods for MPXV-F3L and -G2R assays. They also validated the MPXV-F3L assay using different specimen types, such as cerebrospinal fluid, urine, serum, plasma, whole blood, and oral/rectal/nasopharyngeal/vaginal swabs.
The negative percent agreement was 100% for all specimen types. The positive percent agreement was 100% for nasopharyngeal/recta/vaginal swabs, plasma, cerebrospinal fluid, whole blood, and breast milk, 95.7% for urine samples, and 95.5% for serum samples. The LoD for these samples was estimated at 1000 copies/ml (10 copies/ml per reaction).
Using MPXV clinical specimens, the negative percent agreement was 100% for saliva, semen, and rectal/oral/dry swabs. The positive percent agreement was 100% for saliva, rectal swabs, and semen and 95% for oral/dry swabs. The LoD for the F3L assay was 260 copies/ml for semen, 780 copies/ml for saliva, rectal and oral swabs, and 810 copies/swab for dry swabs.
In summary, the present study assessed the sensitivity and specificity of MPXV-specific primer and probe sets along with a pan-orthopoxvirus assay. The LoD for these assays was as low as 3.3 copies per (PCR) reaction by extracting from up to a 250 μl specimen. Neither assay showed cross-reactivity with VZV or HSV, indicating high specificity.
Overall, the performance of the three assays for MPXV detection was comparable, with each being highly sensitive for MPXV DNA. The MPXV-F3L assay had high specificity for MPXV and did not cross-react with HSV or other orthopoxviruses and will be a critical tool for decreasing MPXV transmission in the ongoing outbreak.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.