Fig 2 -

Analysis of Ct/Cq values for (A) monkeypox virus PCR results and (B) for orthopoxvirus PCR results for different test systems. The grey boxes display all results for the respective sample, and the distributions of specific manufacturer-based collectives are illustrated as smaller, colored box plots in overlay with the total results. For all boxes, the whiskers stretch from the 1st quartile—1.5*(interquartile range) to the 3rd quartile + 1.5*(interquartile range).</p

Fig 1 -

Distribution of the qualitative PCR results for the four samples of the monkeypox EQA survey for A) monkeypox virus (MPXV) and B) orthopoxvirus (OPXV). Numbers in the columns represent the actual number of results for the corresponding category.</p

S4 Table -

Results of Levene’s test of equality of variance of the reported assay-specific Ct/Cq values per MPX positive sample for A) MPXV detection and B) OPXV detection. When equality of variance was not found (labeled in red colour), one collective (marked with *) was identified and excluded before rerun the test to be able to show the equality of the variances for the other collectives (labeled in green colour). (XLSX)</p

INSTAND Monkeypox EQA September 2022—all results.

This table contains the raw results of the EQA participants without any correction. Results that were excluded from evaluation are highlighted in orange. (XLSX)</p

Passing Bablok fit of laboratory-based Ct/Cq values (black dots) for all pairs of EQA samples, differing in concentration by a power of ten.

The Passing Bablok regression line (green) is shown with the lower and upper 95% confidence limits (red).</p

Tables with OPXV qualitative results submitted by EQA participants.

Samples 418001, 418002 and 418004 are positive for MPXV DNA. Test kits that showed less than 5 results were aggregated into "other (NS1 Table. The correct result for the corresponding samples is highlighted in light grey. The quota is defined as percentage of correct results in relation to all results for both the individual test kit (horizontally) and over all test kits for the corresponding sample (sum). (XLSX)</p

Distribution of the quantitative PCR results for monkeypox virus (blue) and orthopoxvirus (red) for the digital PCR (squares) and PCR/NAT (dots) methods.

Seven participants reported results in copies/mL. The colored lines indicate the mean values of the quantitative results for MPXV (blue) and OPXV (red) respectively, and the blue area indicates the 95% confidence interval of the mean value for the quantitative MPXV PCR results. A confidence interval for OPXV was not feasible.</p

Sample properties.

BackgroundIn May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests.MethodsWe analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level.Results141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each.ConclusionThe EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.</div