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 degrees C, and (iii) long-term stability at 37 degrees 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 degrees C supporting shipping without the need of cold chain, (ii) Lyoph-P&P rehydrated solution is stable at +4 degrees 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.

The Republic of Congo (RoC) declared a chikungunya (CHIK) outbreak on 9 February 2019. We conducted a ONE-Human-Animal HEALTH epidemiological, virological and entomological investigation. Methods: We collected national surveillance and epidemiological data. CHIK diagnosis was based on RT-PCR and CHIKV-specific antibodies. Full CHIKV genome sequences were obtained by Sanger and MinION approaches and Bayesian tree phylogenetic analysis was performed. Mosquito larvae and 215 adult mosquitoes were collected in different villages of Kouilou and Pointe-Noire districts and estimates ofAedes (Ae.)mosquitos' CHIKV-infectious bites obtained. We found two new CHIKV sequences of the East/Central/South African (ECSA) lineage, clustering with the recent enzootic sub-clade 2, showing the A226V mutation. The RoC 2019 CHIKV strain has two novel mutations, E2-T126M and E2-H351N. Phylogenetic suggests a common origin from 2016 Angola strain, from which it diverged around 1989 (95% HPD 1985-1994). The infectious bite pattern was similar for 2017, 2018 and early 2019. OneAe. albopictuspool was RT-PCR positive. The 2019 RoC CHIKV strain seems to be recently introduced or be endemic in sylvatic cycle. Distinct from the contemporary Indian CHIKV isolates and in contrast to the original Central-African strains (transmitted byAe. aegypti), it carries the A226V mutation, indicating an independent adaptive mutation in response to vector replacement (Ae. albopictusvsAe. aegypti).