Towards an optimized inhibition of liver stage development assay (ILSDA) for Plasmodium falciparum.

BACKGROUND: Experimental vaccines targeting Plasmodium falciparum have had some success in recent years. These vaccines use attenuated parasites, recombinant sporozoite proteins, or DNA and virus combinations to induce cell-mediated immune responses and/or antibodies targeting sporozoite surface proteins. To capitalize on the success of these vaccines and understand the mechanisms by which these vaccines function, it is important to develop assays that measure correlates of protection in volunteers. The inhibition of liver stage development assay (ILSDA) tests antibodies for the ability to block sporozoite development in hepatocytes. As such the ILSDA is an excellent candidate assay to identify correlates of humoral protection, particularly against the liver stage of malaria infection. In addition, the ILSDA can be used as a tool to evaluate novel sporozoite antigens for future vaccine development. Historically the ILSDA has suffered from low sporozoite infection rates, absence of standardized reagents, and the subjectivity associated with the traditional primary outcome measures, which depend on microscopy of stained hepatocyte cultures. This study worked to significantly improve sporozoite infection rates in hepatocytes, modify key steps in the assay protocol to reduce experimental variability, and demonstrate the utility of the ILSDA in testing antibodies targeting the circumsporozoite protein. METHODS: Cryopreserved primary human hepatocytes, Plasmodium falciparum sporozoites, and circumsporozoite antibodies were used to optimize the ILSDA. RESULTS: Inoculation of cryopreserved primary human hepatocytes with Plasmodium falciparum sporozoites improved liver stage development in the ILSDA compared to HCO4 cells. In the ILSDA, circumsporozoite antibodies suppressed liver stage development in cryopreserved primary human hepatocytes in a concentration-dependent manner. Antibody-mediated suppression of parasite development in the ILSDA at a 96-hour endpoint was more robust than the 24-hour endpoint. CONCLUSIONS: ILSDA performance is improved by the use of cryopreserved primary human hepatocytes, expediting interactions between sporozoites and hepatocytes, and extending the assay endpoint.

Geographical distribution of Anopheles darlingi in the Amazon Basin region of Peru.

Malaria has reemerged as a significant public health disease threat in Peru, especially within the Amazon Basin region. This resurgence of human cases caused by infection with Plasmodium falciparum and Plasmodium vivax is thought to be associated with the spread of Anopheles darlingi, the principal South American malaria vector, into new areas of the Amazon Basin. However, comprehensive studies of the distribution for this species have not been conducted in Peru for several years, nor are historical accounts accurate enough to determine if An. darlingi was actually present and not collected or misidentified. Therefore, the objective of this study is to define the distribution of An. darlingi as well as obtain data on distribution and abundance of other Anopheles species in this region. Mosquitoes were collected during 2001 in the Departments of Loreto and Ucayali, the two largest Amazonian Departments of Peru. A total of 60,585 specimens representing 12 species of the subgenera Nyssorhynchus and Anopheles were collected at 82 (88.2%) of 93 collecting sites. The majority of mosquitoes obtained were identified as An. benarrochi, comprising 70.7% of mosquitoes collected, followed by An. darlingi (24.0%), Anopheles mattogrosensis (2.4%), and Anopheles triannulatus (1.5%). Anopheles darlingi was collected from 48.8% of sites, indicating that this species is established throughout central Loreto, including further west in the Amazon Basin than previously reported. These data suggest that this species is now found in areas of the Amazon Basin region where it has not been previously reported.

Seasonal abundance of Culex nigripalpus Theobald and Culex salinarius Coquillett in north Florida, USA.

North Florida is a transition zone between widespread Culex nigripalpus populations to the south and focal Culex salinarius populations to the north. Culex nigripalpus is a vector of St. Louis encephalitis (SLE) and eastern equine encephalitis (EEE) viruses in south Florida, while Cx. salinarius is a suspected New World vector of West Nile (WN) virus. Abundant vector populations are often a prerequisite for epidemic and epizootic transmission of arboviruses. Extensive SLE transmission has never been reported from north Florida, but sporadic WN transmission was reported there during the summer of 2001. The disparate flavivirus transmission patterns observed in north and south Florida may be due, in part, to the local geographical and seasonal distribution of Culex vectors. Here we report that from May 1991 to April 1994, Cx. salinarius was most commonly observed during the winter and spring in northeast Florida (Duval County), whereas Cx. nigripalpus was most abundant during the summer and autumn. An unusually mild spring in 1991 allowed Cx. nigripalpus to reproduce early in the year, resulting in a summer population that emerged more than 8 wks earlier than in 1992 and 1993. The 1991 Cx. nigripalpus population persisted through October, when SLE transmission was detected by sentinel chickens. Transmission of SLE was not detected in Duval County during 1992 or 1993. These data indicate that mild winter and spring conditions in north Florida may favor increased abundance and survival of Cx. nigripalpus in a region where this species is normally not abundant. A seasonal shift in population structure may increase the transmission risk of arboviruses for which Cx. nigripalpus is a competent vector, including SLE, WN, and EEE.