The recent outbreaks of West Nile (WN) encephalitis and St. Louis encephalitis (SLE) in the United States have highlighted the need for rapid and specific methods of detecting arboviral antigens in mosquitoes. We evaluated rapid, field-usable assays for detecting and differentiating WN and SLE viruses in mosquito pools, based on a patent-pending, immunochromatographic technology (VecTest) formatted on a dipstick. The device provides results in less than 20 min and can be used in laboratories with adequate containment facilities. In laboratory assessments, both the SLE and WN virus tests demonstrated sensitivity comparable with that of an antigen capture ELISA, but less than can be achieved with Vero cell plaque or reverse-transcriptase polymerase chain reaction assays. There was no evidence of cross-reaction when tested with high concentrations of heterologous flavivirus antigens or with Eastern equine encephalitis or Western equine encephalitis viruses. Both the WN and SLE dipstick tests delivered a clear positive result with a single positive specimen in a pool of 50 mosquitoes. This virus assay technology reduces the time required to obtain test results and will allow rapid medical threat assessment and effective targeting of vector control measures.
Abstract. To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest TM Malaria) is a rapid, one-step procedure using a`dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The`VecTest TM Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for >15 weeks in dry conditions up to 45 C and in humid conditions up to 37 C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.
The determination of the presence or absence of malaria sporozoites in wild-caught Anopheles mosquitoes remains an integral component to the understanding of the transmission dynamics in endemic areas. To improve that capability, there has been on-going development of a new device using dipstick immunochromatographic technology for simplifying the testing procedure and reducing the time required to obtain results. As part of a larger multi-center effort, we evaluated the sensitivity and specificity of a prototype malaria sporozoite antigen panel assay (Medical Analysis Systems, Camarillo, CA) against three human Plasmodium species/polymorphs. The wicking (dipstick) assay was compared against a standard parasite antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of human circumsporozoite protein (CSP) in wild-caught mosquitoes. Over 6,800 Anopheles mosquitoes, representing 20 species collected from malaria endemic areas of Indonesia were tested either individually or in pools of up to 10 mosquitoes each. From 1,442 pooled test strip assays and ELISA formats, nine mosquito pools were found reactive for P.falciparum, P. vivax 210, or P. vivax 247 CSP. There was complete concordance between test strip results and ELISA results. Sensitivity was 100% and given some minor problems with false positives or negatives, specificity (n = 488) was 97%. Most strips judged as false positive produced very weak signals compared with negative control blank strips and paired ELISA-negative samples. The dipstick test proved technically simpler to perform and interpret than the ELISA and results were obtained within 15 min of exposure to mosquito suspension. This qualitative assay appears an attractive alternative to the CSP ELISA for detection of sporozoites in fresh or dried mosquitoes.
Vector-borne diseases such as malaria, dengue, and leishmaniasis are a threat to military forces deployed outside of the United States. The availability of specific information on the vector-borne disease threat (e.g., presence or absence of a specific disease agent, temporal and geographic distribution of competent vectors, and vector infection rates) allows for effective implementation of appropriate measures to protect our deployed military forces. Vector diagnostics can provide critical, real-time information crucial to establishing effective vector prevention/control programs. In this article we provide an overview of current vector diagnostic capabilities, evaluate the use of vector diagnostics in Operation Enduring Freedom and Operation Iraqi Freedom, and discuss the concept of operations under which vector diagnostics are employed.
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