Abstract Background The WHO declared the current Monkeypox (Mpox) outbreak a public health emergency of international concern. PCR test is the preferred diagnostic assay. External Quality Assessment Programs (EQAP) provide an independent assessment of the effectiveness of analytical systems, especially with emerging pathogens. There are a few references about EQAP for Mpox. Here, we report the results of the first Mpox EQAP conducted by a Brazilian EQAP provider, which follows the criteria of ABNT NBR ISO/IEC 17043:2011. Methods The quality control samples were inactivated lyophilized suspension of Vero cells (BCRJ 0245/ATCC CCL-81) infected with viable Mpox virus particles and cultured under BSL-3 conditions. The EQAP surveys were conducted in September and October/2022. In the first round, two samples were sent—one negative and one positive [cycle threshold (CT) of 23]. In the second round, 5 samples with different viral loads were sent (CT of 24.9/negative/26.5/34.8/34.6). The percentage of correct results (%CR), sensitivity (SE), specificity (SP), false positive (FP), and false negative (FN) were calculated for each sample, and the performance of laboratory-developed test (LDT) or in vitro diagnosis (IVD) was compared. Results A total of 49 laboratories (38 from Brazil) submitted 319 datasets with qualitative results. In the first round, 30 laboratories used LDT methods and 11 IVD methods, and in the second round, it was 32 and 14, respectively. The %CR ranged from 81.4%–100%, SE from 83.3%–100%, SP from 95.45%–97.7%, FP from 2.3%–4.55%, and FN from 2.3%–16.3%. Samples with higher FN have lower viral loads (P = 0.00044).The overall % of CR, SE, SP, FP, and FN for LDT methods were 76.7%–100%, 79.3%–100%, 93.3%–100%, 0%–6.7%, and 0%–20%, and for IVD methods were, 90.9%–100%, 90.9%–100%, 90.9%–100%, 0%–3.1%, and 0%–7.7%, respectively. No statistical difference was found between methods regarding these parameters. Conclusion This is the first report of EQAP of Mpox molecular testing from Brazil. The EQAP showed that Mpox PCR had an overall good accuracy and performance. FN rates ranged from 2.3% to 16.3%, suggesting there is room for assay improvement, especially with samples with low viral loads. LDT and IVD tests presented similar performance.
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