Rapid Adaptation of Established High-Throughput Molecular Testing Infrastructure for Monkeypox Virus Detection

Beginning in May 2022, a rising number of monkeypox cases were reported in non–monkeypox-endemic countries in the Northern Hemisphere. We adapted 2 published quantitative PCRs for use as a dual-target monkeypox virus test on widely used automated high-throughput PCR systems. We determined analytic performance by serial dilutions of monkeypox virus reference material, which we quantified by digital PCR. We found the lower limit of detection for the combined assays was 4.795 (95% CI 3.6–8.6) copies/mL. We compared clinical performance against a commercial manual orthopoxvirus research use only PCR kit by using clinical remnant swab samples. Our assay showed 100% positive (n = 11) and 100% negative (n = 56) agreement. Timely and scalable PCR tests are crucial for limiting further spread of monkeypox. The assay we provide streamlines high-throughput molecular testing for monkeypox virus on existing broadly established platforms used for SARS-CoV-2 diagnostic testing.

On the basis of diagnostic testing during the SARS-CoV-2 pandemic (8; C. Manohar et al., unpub. data, https://doi.org/10.1101/2021. 10.13.21264919), we chose a dual-target approach, in which 1 assay targets a conserved sequence of the Orthopoxvirus genus, not including variola major or minor viruses (9), and the other targets a MPXV-specific sequence (10) ( Table 1). The cobas 5800, 6800, and 8800 systems (Roche Diagnostics, https://diagnostics.roche.com) use a spike-in RNA full process control that is added automatically during extraction. The corresponding Beginning in May 2022, a rising number of monkeypox cases were reported in non-monkeypox-endemic countries in the Northern Hemisphere. We adapted 2 published quantitative PCRs for use as a dual-target monkeypox virus test on widely used automated highthroughput PCR systems. We determined analytic performance by serial dilutions of monkeypox virus reference material, which we quantified by digital PCR. We found the lower limit of detection for the combined assays was 4.795 (95% CI 3.6-8.6) copies/mL. We compared clinical performance against a commercial manual orthopoxvirus research use only PCR kit by using clinical remnant swab samples. Our assay showed 100% positive (n = 11) and 100% negative (n = 56) agreement. Timely and scalable PCR tests are crucial for limiting further spread of monkeypox. The assay we provide streamlines high-throughput molecular testing for monkeypox virus on existing broadly established platforms used for SARS-CoV-2 diagnostic testing.
internal control assay is preloaded in the open channel reagent for use with cobas omni Utility Channel (Roche Diagnostics) ( Table 2). We modified and optimized all assays for use on cobas 5800, 6800, and 8800 systems, including 2′O-methyl-RNA-modified primers and internal quenchers for TaqMan probes, as previously described (11).

In Silico Evaluation
As part of a support request for Utility Channel applications, we submitted all sequences of the duplex assay to Roche Diagnostics for evaluation of inclusivity and potential primer-probe interactions. The submitted sequences were aligned to currently available MPXV and orthopoxvirus sequences available in public databases.

Analytical Performance Evaluation
We We determined the lower limit of detection by serial 2-fold dilution of MPXV standard in universal transport medium from 100 to 0.78 copies/mL and 21 repeats per dilution step. Using MedCalc statistical software (https://www.medcalc.org), we determined the limit for 95% probability of detection. We assessed linearity by 10-fold serial dilution of MPXV standard (5 repeats per dilution step) at concentrations of ≈10 1 -10 7 copies/mL. We determined linearity and intra-assay variability by using Validation Manager software (Finbiosoft, https://finbiosoft. com). Concentrations represent copies per mL of specimen. For empirical inclusivity and exclusivity testing, we used the assay to test a set of 53 samples, including clinical samples, reference material, and external quality controls from a range of bloodborne and respiratory pathogens (Appendix Table  1, https://wwwnc.cdc.gov/EID/article/28/9/22-0917-App1.pdf). We used an experimental MVA vector-based SARS-CoV-2 vaccine as reference material for a non-MPXV orthopoxvirus.

Clinical Evaluation and Follow-Up Samples
For clinical validation, we used the RUO LightMix Modular Orthopoxvirus assay as reference test, 1766 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 28, No. 9, September 2022  which we performed according to manufacturer's recommendation by using the MagNA-pure96 system with 200-µL extraction volume. In total, we tested 67 clinical samples consisting of respiratory, skin, and genital swab samples with both assays. Of those samples, 11 were positive for MPXV DNA, which we obtained from 2 confirmed clinical cases in Hamburg, Germany. We analyzed 33 consecutive clinical samples from the same 2 patients and 2 additional cases by using the duplex assay (Appendix Table 2). The clinic provided globalized patient characteristics.

In Silico Analysis
We did not detect any concerning oligo interactions (Appendix Figure 1). Target-1: NVAR was still a 100% match for all but 1 MPXV sequence, which had 1 lowrisk mismatch. NVAR also had high sequence similarity with many other orthopoxviruses but might not be optimal for reliable detection of camelpox or cowpox (Appendix Figure 2). Target-2: MPOX is a perfect match for almost all Congo Basin strain MPXV sequences but has a known mismatch for West Africa strain sequences in the probe region. This mismatch is expected to slightly reduce relative fluorescence increase signals, as demonstrated in the clinical sample set. Other orthopoxviruses have extensive sequence mismatches with this assay and are not expected to produce detectable signals (Appendix Figure 3).

Analytical performance
We determined LoD was 9.697 (95% CI 7.424-15.327) copies/mL for the NVAR assay and 6.359 (95% CI 4.908-10.110) copies/mL for the MPOX assay by probability of detection analysis. Overall LoD for both targets combined was 4.795 (95% CI 3.598-8.633) copies/mL. We compiled hit rates (Table 3) and probability of detection plots (Appendix Figure 4) for the assay. The assay showed excellent linearity. Cycle threshold (Ct) values were 37-18, ≈10 1 -10 7 copies/ mL, and pooled SD and 95% CI were within linear range: Ct 0.194, SD 0.0662% for NVAR; Ct 0.175, SD 0.618% for MPXV (Figure 1). No false positives occurred within the inclusivity-exclusivity set. The MVA vector vaccine was correctly detected by the NVAR assay, and not by the MPXV assay (Appendix Table 1).

Clinical Evaluation
In total, we tested 67 clinical samples, consisting of respiratory, skin, and genital swab samples, with both assays. Of those, 11 samples obtained from 2 confirmed clinical monkeypox case-patients in Hamburg, Germany, were positive for MPXV DNA. We noted 100% positive (11/11) and 100% negative agreement (56/56) for the 2 assays ( Figure 2).

Results from Different Sample Types and Timepoints
Another 33 clinical samples were longitudinally col-  Table 2).

Discussion
The trajectory of the ongoing MPXV outbreak in Europe and North America has many uncertainties. However, the World Health Organization acknowledges that known clusters represent a change in transmission pattern and emphasizes the need to limit further spread (1). Broad availability of molecular testing with short turnaround times is a crucial prerequisite for reducing monkeypox spread.
We adapted 2 established nonvariola orthopoxvirus and MPXV qPCR assays (9,10) as a duplex test for the cobas 5800, 6800, and 8800 fully automated sample-to-result platforms, which are widely used for high-throughput SARS-CoV-2 diagnostic testing (13). Both assays have been validated extensively against other orthopoxvirus species in previous studies (9,10) and remain inclusive and highly specific for in silico analysis with currently available monkeypox sequences. We demonstrated excellent analytical performance of the duplex assay, showing single-digit detection limits and near-perfect PCR efficiency. A spike-in full-process control assay, similar to commercial in vitro diagnostic assays, already is included in the open channel reagents we used.
Our institution confirmed 4 clinical cases of monkeypox, and we used the initial clinical samples as our clinical positive set. Although the assay was only validated on swab samples, we also detected MPXV DNA in EDTA plasma, urine, and seminal fluid diluted in guanidine hydrochloride solution without any method adaptations. Among all tested clinical samples positive for MPXV DNA, swabs of skin   lesions consistently yielded early Ct values in the lowto mid-teens, indicating exceedingly high viral DNA loads, which might be a concern for both personnel safety and contamination risks. Viral DNA was readily detectable in oropharyngeal swab samples, as previously reported (2,14). Likewise, EDTA plasma samples were consistently positive but had later Ct values, mostly around 30. Further studies could evaluate the practical usefulness of plasma or urine for monkeypox diagnostic purposes or longitudinal viral load monitoring. Our data regarding MPXV DNA in different clinical specimen types are well in line with other published studies (2,14); overall, skin lesion swab samples appeared to be best suited for diagnostic purposes based on our sample set.
In conclusion, we provided technical performance evaluation for a laboratory-developed duplex qPCR assay for MPXV detection for use on the cobas 5800, 6800, and 8800 high-throughput systems. The assay we describe enables laboratories to adapt existing automated SARS-CoV-2 molecular testing infrastructure for a potential large-scale monkeypox outbreak. M.L. and D.N. received speaker honoraria and related travel expenses from Roche Diagnostics.