Diagnostic performance of a field-deployable iiPCR platform and viral load dynamics of MPXV Clade IIb in a cohort with high prevalence of HIV

Abstract

BACKGROUND: Since 2022, the global outbreak of Mpox caused by the Mpox virus (MPXV) Clade IIb has underscored the need for timely and decentralized molecular diagnostics. Although quantitative real-time PCR (qPCR) remains the diagnostic gold standard, its infrastructure requirements may limit accessibility in resource-constrained settings. Insulated isothermal PCR (iiPCR) represents a potential field-deployable alternative; however, clinical validation data for MPXV remains limited. METHODS: We prospectively enrolled 24 qPCR-confirmed Mpox patients between 2023 and 2024. Multiple specimen types were tested using the POCKIT Central MPXV iiPCR system and reference qPCR. Analytical sensitivity was assessed using serial viral dilutions. Viral load dynamics were evaluated using cycle threshold (Ct) values across specimen types and days post-symptom onset. Phylogenetic characterization was performed using a four-gene Sanger sequencing approach. RESULTS: PFU/mL and complete qualitative concordance in cultured samples. Lesion-derived specimens showed the highest detection rates (100%) and consistently lower Ct values, whereas non-lesion specimens exhibited lower and more variable positivity. No clear differences in viral load-based diagnostic performance were observed between people living with HIV receiving antiretroviral therapy (ART) and people without HIV. Phylogenetic analysis confirmed exclusive circulation of MPXV Clade IIb. CONCLUSIONS: Under the conditions evaluated, the POCKIT Central iiPCR system achieved diagnostic performance comparable to qPCR, particularly for lesion-derived specimens. In this ART-treated HIV-prevalent cohort with preserved immune function, HIV co-infection was not associated with differences in diagnostic performance, supporting the potential utility of iiPCR for decentralized Mpox testing.