A combined ELISA for infection-induced and vaccine-induced mpox antibodies during the clade Ib outbreak in Rwanda: an observational, cross-sectional, clinical validation study
Clarke, Jenny Semukunzi, Herve Faustini, Sian E Heaney, Jennifer L J Kwok, Hin Fai Uwimana, Jean Marie Vianney Gokani, Karishma Rukundo, Gilbert Musabyimana, Jean Pierre Ingabire, Prosper
Clarke, Jenny
Semukunzi, Herve
Faustini, Sian E
Heaney, Jennifer L J
Kwok, Hin Fai
Uwimana, Jean Marie Vianney
Gokani, Karishma
Rukundo, Gilbert
Musabyimana, Jean Pierre
Ingabire, Prosper
Abstract
Background: Validated serological assays for mpox are scarce, particularly in outbreak regions where they are most urgently needed. We aimed to conduct an in-country validation of a serum-based mpox IgG ELISA during an outbreak of clade Ib mpox in Rwanda, and report immunological observations after infection and vaccination and on background seropositivity.
Methods: This observational, cross-sectional, clinical validation study of an mpox-specific IgG ELISA was conducted at the Rwanda National Reference Laboratory, within the Rwanda Biomedical Centre, Kigali, Rwanda. Adults (aged ≥18 years) were recruited into one of three groups of equal size: those who had previously had a microbiologically confirmed mpox infection (the infection cohort), those who had previously received an mpox vaccination (the vaccination cohort), and individuals with no history of mpox infection or vaccination (the unexposed cohort). Venous blood and dried blood spot (DBS) samples were collected from the participants. The IgG assay, containing four immunodominant mpox antigens, was developed into plate-based ELISA kits and used to analyse the serum and DBS samples, and an IgG ratio was obtained from the optical density readout. The performance of the assay was assessed by analysis of the receiver operating characteristic curve, intra-assay and interassay precision, and linearity, and the agreement between serum and DBS concentrations was tested with Spearman's correlation and Bland-Altman analysis.
Findings: Between Aug 1 and Aug 12, 2025, 194 individuals were invited to participate in the study, of whom 150 were enrolled, 50 in each of the three cohorts. Median IgG ratios were 3·24 (IQR 1·33-8·01) in the infection cohort, 1·77 (0·90-2·86) in the vaccination cohort, and 0·68 (0·39-1·16) in the unexposed cohort. IgG ratios in the infection and vaccination cohorts overlapped, but were both significantly higher than in the unexposed cohort (p<0·0001 for the infection cohort; p=0·0002 for the vaccination cohort). Intra-assay (coefficient of variation ≤13%) and interassay (≤10%) precision was high, and ratios were linear across dilutions. DBS and serum measurements were strongly correlated (R2=0·84), but DBS measurements showed a small underestimation. In the vaccination cohort, IgG ratios were higher in female participants (median 2·70 [IQR 0·83-3·77], n=21) than in male participants (1·35 [0·90-2·42], n=29; p=0·0030).
Interpretation: We show that an mpox ELISA can be implemented within a national reference laboratory in a resource-limited setting as part of outbreak-response measures. The assay is suitable for population-level surveys and can analyse both DBS and serum samples. These findings provide a platform for larger population studies and further work to explore immune correlates of protection in mpox.
MIDER Authors
Date
2026-03-09
Type
Article
Subject
Communicable diseases, Vaccination, Antigens, Africa, Disease outbreaks
Collections
Citation
Clarke J, Semukunzi H, Faustini SE, Heaney JLJ, Kwok HF, Uwimana JMV, Gokani K, Rukundo G, Musabyimana JP, Ingabire P, Bitunguhari L, de Dieu Harelimana J, Richter AG, Muvunyi CM, Green CA; MpoxCARE Study Team. A combined ELISA for infection-induced and vaccine-induced mpox antibodies during the clade Ib outbreak in Rwanda: an observational, cross-sectional, clinical validation study. Lancet Infect Dis. 2026 Mar 9:S1473-3099(26)00006-X. doi: 10.1016/S1473-3099(26)00006-X. Epub ahead of print.
Journal / Source Title
The Lancet. Infectious Diseases
DOI
10.1016/S1473-3099(26)00006-X
PMID
41819115
Publisher
Elsevier Science
Lancet Publishing Group
Lancet Publishing Group
Publisher’s URL
https://www.sciencedirect.com/journal/the-lancet-infectious-diseases