Simultaneous detection of chikungunya virus, dengue virus and human pathogenic Leptospira genomes using a multiplex TaqMan® assay

Claude Giry, Bénédicte Roquebert, Ghislaine Li-Pat-Yuen, Philippe Gasque, Marie-Christine Jaffar-Bandjee

In 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. A 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. Simplex 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. This 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.