Screening for MPXV, VZV, HSV-1 and HSV-2, viruses responsible for mucocutaneous vesicular rashes, by multiplex HRM-PCR in the Democratic Republic of Congo and France

Introduction: Since May 2022, over 21,000 cases of monkeypox (MPOX) have been reported in Europe where the disease is not endemic. In 2023, the number of suspected cases of MPOX in the Democratic Republic of Congo (DRC) doubled compared to 2020 to 12,569 reported suspected cases, including 581 deaths. In 2024, the situation has worsened further with 15,664 potential cases and 548 deaths. Monkeypox virus (MPXV) represents a diagnostic challenge due to the diversity of clinical presentations. These symptoms are often like those encountered in infection caused by herpes simplex virus types 1 and 2 (HSV-1, HSV-2) and varicella-zoster virus (VZV). AFYIA Diagnostics has developed a qualitative screening test that allows the simultaneous identification of the viruses mentioned. Methods: The DIRASH kit is a multiplex HRM assay, based on the simultaneous detection of 4 viruses. The associated DISoft™ software was used to automatically analyze and interpret the HRM profiles obtained. Three studies were carried out to monitor cases of cutaneous-mucosal febrile vesicular eruptions from January 2021 to December 2022 during the clade II epidemic by CHU Montpellier and from December 2022 to June 2024 on clade 1 and 1b particularly these last months by INRB in Kinshasa. Each patient was screened by commercial or in-house PCRs. These samples were then retrospectively tested with the DIRASH kit in these laboratories. Results: 496 samples were retrospectively tested with the DIRASH kit, to compare its clinical sensitivity with that of partner laboratory kits. For Montpellier, sensitivity was 100% for MPXV, 94% for VZV, and 98% for HSV-1 and HSV-2. INRB reported 98% for MPXV and 93% for VZV. The DIRASH kit detected several unknown co-infections (MPXV/HSV-1, MPXV/HSV2 and MPXV/VZV). Results from the ongoing INRB clinical study will be presented at the conference. Discussion: Results of this study show that the clinical sensitivity of the PCRs from CHU Montpellier and INRB are comparable to those obtained with the DIRASH kit on the panel of samples tested. The DIRASH kit revealed the presence of several co-infections that would have never been detected with the other PCRs. The under-diagnosis of Monkeypox-positive samples partly explains these results, illustrating the advantage of syndromic diagnosis of febrile rashes. Conclusion: The DIRASH kit is rapid, reliable, transportable at room temperature, low-cost-multiplex test with automated result interpretation. The use of a multiplex kit for the simultaneous detection of viruses responsible for mucocutaneous vesicular eruptions meets the needs for differential diagnosis of MPXV, particularly in the DRC where the epidemic is the largest in Africa, but also in countries where the epidemic is not endemic. The kit is particularly well-suited to the crucial need to strengthen diagnostic capabilities in the DRC, to guide patient management and epidemic control.