Analytical and clinical evaluation of a novel real-time PCR-based detection kit for Mpox virus

Abstract

Outbreaks of emerging diseases, like Mpox in 2022, pose unprecedented challenges to global healthcare systems. Although Mpox cases globally decreased since the end of 2022, numbers are still significant in the African Region, European Region, Region of the Americas, and Western Pacific Region. Rapid and efficient detection of infected individuals by precise screening assays is crucial for successful containment. In these assays, analytical and clinical performance must be assessed to ensure high quality. However, clinical studies evaluating Mpox virus (MPXV) detection kits using patient-derived samples are scarce. This study evaluated the analytical and clinical performance of a new diagnostic MPXV real-time PCR detection kit (Sansure Monkeypox Virus Nucleic Acid Diagnostic Kit) using patient-derived samples collected in Germany during the MPXV clade IIb outbreak in 2022. Our experimental approach determined the Limit of Detection (LoD) to less than 200 cp/mL using whole blood samples and samples derived from vesicles or pustules. Furthermore, we tested potentially inhibiting substances and pathogens with homologous nucleic acid sequences or similar clinical presentation and detected no cross-reactivity or interference. Following this, the assay was compared to a CE-marked test in a clinical performance study and achieved a diagnostic sensitivity of 100.00% and diagnostic specificity of 96.97%. In summary, the investigated real-time PCR assay demonstrates high analytical performance and concurs with the competitor device with high specificity and sensitivity.

Keywords: Clinical diagnostics; MPXV; Mpox; Mpox detection assay.

Fig. 1

Precision testing in different matrices. MPXV reference DNA was diluted and spiked into MPXV-negative samples of (A) whole blood, (B) vesicles, and (C) pustules and tested over 21 days

Fig. 2

Determination of Limit of Detection (LoD). MPXV-negative samples of whole blood (A) or swabs obtained from vesicles (B) or pustules (C) were inoculated with MPXV DNA. The cycle threshold (Ct) values are presented for varying DNA concentrations spiked into the different matrices. (D) The positive detection rate of the MPXV DNA in the different sample types. The dotted line indicates the 95%-positive detection rate

Fig. 3

Competitive interference and cross-reactivity testing. Endogenous and exogenous substances were introduced into samples containing a defined MPXV DNA concentration at the LoD. The samples without any additions are labeled as the control (grey). For pathogens, three samples were tested, and for substances, nine samples were tested. The dotted line indicates the mean value of the control. The lines represent the mean, and the whiskers represent the standard deviation (SD)