BackgroundIn 2005–2006 a major epidemics of Chikungunya disease occurred in South-West Indian Ocean islands. In Reunion Island, the magnitude of Chikungunya infection related symptoms was high and with over 38% of serological prevalence in the population. This epidemics illustrated the potential threat of emerging arboviral diseases for inhabitants of Reunion Island and elsewhere since vectors are worldwide distributed. A sentinel surveillance network was set-up to detect emerging pathogens associated with fever over 38 °C and in the absence of known etiologic causes. Leptospirosis is caused by a pathogenic spirochete of the Leptospira genus and is an endemic and recurrent seasonal disease of great concern in Reunion Island. To accurately diagnose potentially infected patients and to advise Health authorities on the presence of emerging pathogens, a rapid diagnostic test was needed that could differentiate between these 3 pathogens.MethodsA one-step multiplex real-time PCR assay was developed that can simultaneously detect RNA of Chikungunya and Dengue viruses and leptospiral DNA with good performance for a routine diagnostic use.ResultsSimplex protocols already published were used with key modifications to implement a triplex assay which was set-up with a small reaction volume to improve cost efficiency.ConclusionsThis approach has enabled greater diagnostic capacity in our laboratory. We established a multiplex approach validated and valuable for cost savings, and with the concurrent detection of 3 pathogens of public health concern.

BackgroundAlphaviruses are arthropod borne RNA viruses of medical importance. Geographical expansion of mosquitoes of the Aedes genus in the past decades has been associated with major Alphavirus-associated outbreaks. Climate changes and intensification of air travels have favored vector expansion and virus dissemination in new territories leading to virus emergence not only in tropical areas but also in temperate regions. The detection of emergence is based upon surveillance networks with epidemiological and laboratory investigation.MethodA specific, sensitive and rapid screening test for genus-specific Alphavirus is critically required. To address this issue, we developed a new molecular assay targeting nsP4 gene and using a TaqMan® real time RT-PCR method for the specific detection of all major Alphavirus genus members.ResultsThis assay was tested for specificity using several Alphavirus species. We also tested successfully clinical sensitivity using patient’s samples collected during the Chikungunya outbreak of 2005–2006 in the Indian Ocean.ConclusionsThis new pan-Alphavirus molecular diagnostic tool offers great potential for exclusion diagnosis and emergence detection given its broad specificity restricted to Alphavirus genus.

BackgroundAlphaviruses are arthropod borne RNA viruses of medical importance. Geographical expansion of mosquitoes of the Aedes genus in the past decades has been associated with major Alphavirus-associated outbreaks. Climate changes and intensification of air travels have favored vector expansion and virus dissemination in new territories leading to virus emergence not only in tropical areas but also in temperate regions. The detection of emergence is based upon surveillance networks with epidemiological and laboratory investigation.MethodA specific, sensitive and rapid screening test for genus-specific Alphavirus is critically required. To address this issue, we developed a new molecular assay targeting nsP4 gene and using a TaqMan® real time RT-PCR method for the specific detection of all major Alphavirus genus members.ResultsThis assay was tested for specificity using several Alphavirus species. We also tested successfully clinical sensitivity using patient’s samples collected during the Chikungunya outbreak of 2005–2006 in the Indian Ocean.ConclusionsThis new pan-Alphavirus molecular diagnostic tool offers great potential for exclusion diagnosis and emergence detection given its broad specificity restricted to Alphavirus genus.

BackgroundIn 2005–2006 a major epidemics of Chikungunya disease occurred in South-West Indian Ocean islands. In Reunion Island, the magnitude of Chikungunya infection related symptoms was high and with over 38% of serological prevalence in the population. This epidemics illustrated the potential threat of emerging arboviral diseases for inhabitants of Reunion Island and elsewhere since vectors are worldwide distributed. A sentinel surveillance network was set-up to detect emerging pathogens associated with fever over 38 °C and in the absence of known etiologic causes. Leptospirosis is caused by a pathogenic spirochete of the Leptospira genus and is an endemic and recurrent seasonal disease of great concern in Reunion Island. To accurately diagnose potentially infected patients and to advise Health authorities on the presence of emerging pathogens, a rapid diagnostic test was needed that could differentiate between these 3 pathogens.MethodsA one-step multiplex real-time PCR assay was developed that can simultaneously detect RNA of Chikungunya and Dengue viruses and leptospiral DNA with good performance for a routine diagnostic use.ResultsSimplex protocols already published were used with key modifications to implement a triplex assay which was set-up with a small reaction volume to improve cost efficiency.ConclusionsThis approach has enabled greater diagnostic capacity in our laboratory. We established a multiplex approach validated and valuable for cost savings, and with the concurrent detection of 3 pathogens of public health concern.