The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) Chikungunya (CHIKV), O'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group is investigating the natural history, epidemiology and medical management of infection by these viruses, to identify knowledge gaps and to propose recommendations for direct future investigations and rectification measures. Here, we present the first report dedicated to diagnostic aspects of CHIKV, ONNV and MAYV. Regarding diagnosis of the disease at the acute phase, molecular assays previously described for the three viruses require further evaluation, standardized protocols and the availability of international standards representing the genetic diversity of the viruses. Detection of specific IgM would benefit from further investigations to clarify the extent of cross-reactivity among the three viruses, the sensitivity of the assays, and the possible interfering role of cryoglobulinaemia. Implementation of reference panels and external quality assessments for both molecular and serological assays is necessary. Regarding sero-epidemiological studies, there is no reported high-throughput assay that can distinguish among these different viruses in areas of potential co-circulation. New specific tools and/or improved standardized protocols are needed to enable large-scale epidemiological studies of public health relevance to be performed. Considering the high risk of future CHIKV, MAYV and ONNV outbreaks, the Working Group recommends that a major investigation should be initiated to fill the existing diagnostic gaps.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to investigate natural history, epidemiology and clinical aspects of infection by these viruses. Here, we present a report dedicated to entomological aspects of CHIKV, ONNV and MAYV. Recent global expansion of chikungunya virus has been possible because CHIKV established a transmission cycle in urban settings using anthropophilic vectors such as Aedes albopictus and Aedes aegypti. MAYV and ONNV have a more limited geographic distribution, being confined to Africa (ONNV) and central-southern America (MAYV). ONNV is probably maintained through an enzootic cycle that has not been characterized yet, with Anopheles species as main vectors and humans as amplification hosts during epidemics. MAYV is transmitted by Haemagogus species in an enzootic cycle using non-human primates as the main amplification and maintenance hosts, and humans becoming sporadically infected when venturing in or nearby forest habitats. Here, we focused on the transmission cycle and natural vectors that sustain circulation of these viruses in their respective locations. The knowledge of the natural ecology of transmission and the capacity of different vectors to transmit these viruses is crucial to understand CHIKV emergence, and to assess the risk that MAYV and ONNV will expand on wide scale using anthropophilic mosquito species not normally considered primary vectors. Finally, the experts identified knowledge gaps and provided adapted recommendations, in order to address future entomological investigations in the right direction.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to identify gaps of knowledge about the natural history, epidemiology and medical management of infection by these viruses, and to provide adapted recommendations for future investigations. Here, we present a report dedicated to ONNV epidemiological distribution. Two large-scale ONNV outbreaks have been identified in Africa in the last 60 years, interspersed with sporadic serosurveys and case reports of returning travelers. The assessment of the real scale of ONNV circulation in Africa remains a difficult task and surveillance studies are necessary to fill this gap. The identification of ONNV etiology is made complicated by the absence of multiplex tools in co-circulation areas and that of reference standards, as well as the high cross-reactivity with related pathogens observed in serological tests, in particular with CHIKV. This is a specific obstacle for seroprevalence studies, that necessitate an improvement of serological tools to provide robust results. The scarcity of existent genetic data currently limits molecular epidemiology studies. ONNV epidemiology would also benefit from reinforced entomological and environmental surveillance. Finally, the natural history of the disease deserves to be further investigated, with a specific attention paid to long-term complications. Considering our incomplete knowledge on ONNV distribution, GloPID-R CHIKV, ONNV and MAYV experts recommend that a major effort should be done to fill existing gaps.

Serological surveys are essential to quantify immunity in a population but serological cross-reactivity often impairs estimates of the seroprevalence. Here, we show that modeling helps addressing this key challenge by considering the important cross-reactivity between Chikungunya (CHIKV) and O’nyong-nyong virus (ONNV) as a case study. We develop a statistical model to assess the epidemiology of these viruses in Mali. We additionally calibrate the model with paired virus neutralization titers in the French West Indies, a region with known CHIKV circulation but no ONNV. In Mali, the model estimate of ONNV and CHIKV prevalence is 30% and 13%, respectively, versus 27% and 2% in non-adjusted estimates. While a CHIKV infection induces an ONNV response in 80% of cases, an ONNV infection leads to a cross-reactive CHIKV response in only 22% of cases. Our study shows the importance of conducting serological assays on multiple cross-reactive pathogens to estimate levels of virus circulation.

Real-time molecular techniques have become the reference methods for direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process of preparation of oligonucleotide primers and hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared versus the classic protocol using extemporaneously prepared liquid reagents using (i) sensitivity study, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C mimicking transportation without cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO as to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). Results of our study demonstrate that (i) Lyoph-P&P is stable for at least 4 days at 37 °C supporting shipping without the need of cold chain, (ii) Lyoph-P&P rehydrated solution is stable at +4 °C for at least two weeks, (iii) sensitivity observed with Lyoph-P&P is at least equal to, often better than, that observed with liquid formulation, (iv) validation of results observed with low-copy specimens is rendered easier by higher fluorescence level. In conclusion, Lyoph-P&P holds several advantages over extemporaneously preparer liquid formulation that merit to be considered when a novel real-time molecular assay is implemented in a laboratory in charge of routine diagnostic activity.