METHODS: The fresh Azolla pinnata plant from Kuala Krai, Kelantan, Malaysia was used for crude extraction using Soxhlet and maceration methods. Then, the chemical composition of extracts and its structure were identified using GCMS-QP2010 Ultra (Shimadzu). Next, following the WHO procedures for larval bioassays, the extracts were used to evaluate the early 4th instar larvae of Aedes mosquito vectors.

RESULTS: The larvicidal activity of Azolla pinnata plant extracts evidently affected the early 4th instar larvae of Aedes aegypti mosquito vectors. The Soxhlet extraction method had the highest larvicidal effect against Ae. aegypti early 4th instar larvae, with LC50 and LC95 values of 1093 and 1343 mg/L, respectively. Meanwhile, the maceration extraction compounds were recorded with the LC50 and LC95 values of 1280 and 1520 mg/L, respectively. The larvae bioassay test for Ae. albopictus showed closely similar values in its Soxhlet extraction, with LC50 and LC95 values of 1035 and 1524 mg/L, compared with the maceration extraction LC50 and LC95 values of 1037 and 1579 mg/L, respectively. The non-target organism test on guppy fish, Poecilia reticulata, showed no mortalities and posed no toxic effects. The chemical composition of the Azolla pinnata plant extract has been found and characterized as having 18 active compounds for the Soxhlet method and 15 active compounds for the maceration method.

METHOD: A research-to-policy forum was convened by TDR, the Special Programme for Research and Training in Tropical Diseases, with researchers and representatives from ministries of health, in order to review research findings and discuss their implications for policy and research.

RESULTS: The participants reviewed findings of research supported by TDR and others. Surveillance and early outbreak warning. Systematic reviews and country studies identify the critical characteristics that an alert system should have to document trends reliably and trigger timely responses (i.e., early enough to prevent the epidemic spread of the virus) to dengue outbreaks. A range of variables that, according to the literature, either indicate risk of forthcoming dengue transmission or predict dengue outbreaks were tested and some of them could be successfully applied in an Early Warning and Response System (EWARS). Entomological surveillance and vector management. A summary of the published literature shows that controlling Aedes vectors requires complex interventions and points to the need for more rigorous, standardised study designs, with disease reduction as the primary outcome to be measured. House screening and targeted vector interventions are promising vector management approaches. Sampling vector populations, both for surveillance purposes and evaluation of control activities, is usually conducted in an unsystematic way, limiting the potentials of entomological surveillance for outbreak prediction. Combining outbreak alert and improved approaches of vector management will help to overcome the present uncertainties about major risk groups or areas where outbreak response should be initiated and where resources for vector management should be allocated during the interepidemic period.

METHODOLOGY/PRINCIPAL FINDINGS: We assessed oral susceptibility of Malaysian Ae. aegypti and Ae. albopictus by real-time PCR to an Australian RRV strain SW2089. Replication kinetics in midgut, head and saliva were determined at 3 and 10 days post-infection (dpi). With a 3 log10 PFU/ml blood meal, infection rate was higher in Ae. albopictus (60%) than Ae. aegypti (15%; p<0.05). Despite similar infection rates at 5 and 7 log10 PFU/ml blood meals, Ae. albopictus had significantly higher viral loads and required a significantly lower median oral infectious dose (2.7 log10 PFU/ml) than Ae. aegypti (4.2 log10 PFU/ml). Ae. albopictus showed higher vector competence, with higher viral loads in heads and saliva, and higher transmission rate (RRV present in saliva) of 100% at 10 dpi, than Ae. aegypti (41%). Ae. aegypti demonstrated greater barriers at either midgut escape or salivary gland infection, and salivary gland escape. We then assessed seropositivity against RRV among 240 Kuala Lumpur inpatients using plaque reduction neutralization, and found a low rate of 0.8%.