Quantification of saxitoxin in human blood by ELISA.

Saxitoxin (STX) is a potent marine toxin that causes paralytic shellfish poisoning (PSP) which can result in significant morbidity and mortality in humans. Low lethal doses, rapid onset of PSP symptoms, and brief STX half-life in vivo require sensitive and rapid diagnostic techniques to monitor human exposures. Our laboratory has validated an enzyme-linked immunosorbent assay (ELISA) for quantitative detection of STX from 0.020 to 0.80 ng/mL in human whole blood and from 0.06 to 2.0 ng/mL in dried human blood which is simple, sensitive, rapid, and cost-effective. To our knowledge, this is the first validated method for the quantitation of saxitoxin in whole blood. Microsampling devices were used in sample collection which allows for standardized collection of blood, stable storage, and cost-efficient shipping. Quality control precision and accuracy were evaluated over the course of validation and were within 20% of theoretical concentrations. No detectable background concentrations of STX were found among fifty whole blood and dried blood convenience samples. Additionally, ten spiked individual whole blood and dried blood samples were tested for accuracy and precision and were within 20% of theoretical concentrations. Gonyautoxins 2&3 (GTX2&3) cross-reacted with this ELISA by 21%, but all other structurally related PSP toxins tested cross-reacted less than two percent. While clinical diagnosis or treatment of PSP would be unaffected by GTX2&3 cross-reactivity by ELISA, to accurately quantify individual PSP toxins, these results should be coupled with high performance liquid chromatography mass spectrometry measurements.

Comparison of vector competence of Aedes mediovittatus and Aedes aegypti for dengue virus: implications for dengue control in the Caribbean.

BACKGROUND: Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes. METHODS: To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1-4 per os at viral titers of 5-6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution. RESULTS: Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus. CONCLUSIONS: This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.

Discovery and characterization of potential prognostic biomarkers for dengue hemorrhagic fever.

Half a million patients are hospitalized with severe dengue every year, many of whom would die without timely, appropriate clinical intervention. The majority of dengue cases are uncomplicated; however, 2-5% progress to severe dengue. Severe dengue cases have been reported with increasing frequency over the last 30 years. To discover biomarkers for severe dengue, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to analyze dengue virus positive serum samples from the acute phase of infection. Using this method, 16 proteins were identified as candidate biomarkers for severe dengue. From these 16 biomarkers, three candidates were selected for confirmation by enzyme-linked immunosorbent assay and Western blot: vitronectin (Vtn, 55.1 kDa), hemopexin (Hx, 52.4 kDa), and serotransferrin (Tf, 79.2 kDa). Vitronectin, Hx, and Tf best differentiated between dengue and severe dengue.