Development of a molecular xenomonitoring protocol to assess filariasis transmission.

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes.

Integrated Strategies for Aedes aegypti Control Applied to Individual Houses: An Approach to Mitigate Vectorial Arbovirus Transmission.

Aedes aegypti and Culex quinquefasciatus mosquitoes are vectors of different arboviruses that cause a large burden of disease in humans worldwide. A key step towards reducing the impact of arboviruses on humans can be achieved through integrated mosquito surveillance and control approaches. We carried out an integrated approach of mosquito surveillance and control actions to reduce populations of these insects along with a viral surveillance in a neighborhood of Recife (Northeastern Brazil) with high mosquito densities and arbovirus transmission. The actions were carried out in 40 different houses in the Nova Descoberta neighborhood. The area was divided into two groups, the control group using tools to monitor the mosquito density (1 OVT; 1 Double BR-ovt; monthly capture of alates) and the experimental group with control actions using surveillance tools in an intensified way (2 OVTs; 2 Double BR-ovts; fortnightly capture of alates; toxic baits). We evaluated the study's impact on the mosquito density via the Egg Density (ED) and Adult Density (AD) over a period of 12 cycles of 28 days each. The collected adult mosquitoes were processed via RT-qPCR for DENV, CHIKV and ZIKV and, subsequently, the Minimum Infection Rate (MIR) was calculated. After 12 cycles, we observed a 91% and 99% reduction in Aedes ED and AD in the monitored properties, as well as a 76% reduction in the AD of Cx. quinquefasciatus in the same properties. Moreover, we detected circulating arboviruses (DENV and ZIKV) in 19.52% of captured adult mosquitoes. We show that enhancing entomological surveillance tools can aid in the early detection of possible risk areas based on vector mosquito population numbers. Additionally, the detection of important arboviruses such as ZIKV and DENV raises awareness and allows for a better selection of risk areas and silent virus spread. It offers supplementary information for guiding emergency mosquito control measures in the target area. The goal is to minimize human-vector interactions and, subsequently, to lower the risk of transmitting circulating arboviruses.

Detection of Wuchereria bancrofti in the city of São Luís, state of Maranhão, Brazil: New incursion or persisting problem?

BACKGROUND: The elimination of lymphatic filariasis (LF) from Brazil by 2020 was not accomplished; however, this goal can be achieved in the upcoming years with the assistance of specific strategies. The surveillance of LF can be performed using molecular xenomonitoring (MX), a noninvasive method used to infer the presence of the parasite in the human population. Herein, São Luís (state of Maranhão) was the first city to be investigated to identify whether LF transmission in Brazil has been interrupted and if there were any new incursions. METHODOLOGY/PRINCIPAL FINDINGS: Mosquitoes were collected by aspiration at 901 points distributed among 11 neighborhoods in São Luís with records of patients with microfilaremia. Pools of engorged or gravid Culex quinquefasciatus females were evaluated by WbCx duplex PCR with endogenous control for mosquitoes and target for W. bancrofti for determining the vector infection rate. Among the 10,428 collected mosquitoes, the most abundant species were C. quinquefasciatus (85%) and Aedes aegypti (12%). Significantly larger numbers of mosquitoes were collected from the neighborhoods of Areinha and Coreia (p<0.05). MX performed using PCR validated 705 pools of engorged or gravid females, fifteen of which were positive for Wuchereria bancrofti in two neighborhoods. CONCLUSIONS: The high density of engorged C. quinquefasciatus females per home, inadequate sanitation, and detection of W. bancrofti-infected mosquitoes in the city of São Luís represent a warning of the possible upsurge of LF, a disease that is still neglected; this underscores the need for the ostensive monitoring of LF in Brazil.

Double BR-OVT: a new trap model for collecting eggs and adult mosquitoes from Culex quinquefasciatus and Aedes spp.

The circulation of arboviruses throughout the world and the maintenance of lymphatic filariasis endemicity in tropical countries, combined with the lack of vaccines and specific treatments, highlight the importance of reducing the populations of mosquitoes involved in the transmission of these pathogens, Aedes aegypti and Culex quinquefasciatus. To contribute to the development of new strategies for monitoring and controlling these culicids, we evaluated the performance of the Double BR-OVT trap individually and in pairs, in the field. After 18 months, the Double BR-OVT traps captured a mean of 3.5 ± 7.4 and 1.8 ± 3.2 of Culex and Aedes /residence/cycle, respectively, in addition to 410 ± 588.3 Aedes eggs/residence/cycle. When installed in pairs, the Double BR-OVT traps collected three times more adult mosquitoes of C. quinquefasciatus (9.4 ± 8.3 Culex /residence/bimester) and two times more Aedes spp. (3 ± 3.2 Aedes /residence/bimester) in comparison with the traps installed individually (2.6 ± 7.1 and 1.5 ± 3.2 Culex and Aedes /residence/bimester, respectively) (p < 0.05). The Double BR-OVT trap has an exceptional advantage: it aggregates different functionalities into a single instrument, as this type of trap can concomitantly collect eggs and adult mosquitoes of C. quinquefasciatus and A. aegypti , a feature that makes it a potentially useful tool among the strategies for monitoring and controlling these mosquitoes.

Avaliação do impacto de ações integradas de controle vetorial sobre a infestação de Aedes aegypti e Culex quinquefasciatus em Recife-PE

Aedes aegypti e Culex quinquefasciatus são vetores de patógenos que causam doenças no homem, sendo necessário investir em ações que reduzam suas populações, minimizando o contato mosquito-homem. Avaliamos o impacto do controle integrado de mosquitos (CIM) em dois bairros do Recife-PE. Ações realizadas: captura de alados, recolhimento massivo de ovos, iscas tóxicas para mosquitos e tratamento de criadouros. As ações foram realizadas em 80 imóveis, 40 no bairro da Várzea (Área 1), e 40 em Nova Descoberta (Área 2). Cada bairro com 2 extratos de 20 imóveis: ações simples (S) (1 OVT; 1 BR-ovt; captura de alados mensal) e ações intensificadas (I) (2 OVT; 2 BR-ovt; captura de alados quinzenal; iscas tóxicas). O Índice de Densidade de Ovos (IDO) e Densidade de Adultos (DA) foram utilizados para avaliar o impacto das ações de controle sob a densidade de mosquitos. Os mosquitos capturados foram processados por RT-qPCR e calculada a taxa de infecção mínima (MIR) para cada patógeno. Em A. aegypti foi investigada infecção vetorial DENV, CHIKV e ZIKV e em C. quinquefasciatus, a presença do parasita Wuchereria bancrofti e do vírus Zika. Após 12 ciclos de avaliação nas áreas houve redução de 88% no IDO da Área 1 e de 91% na Área 2. A DA de A. aegypti reduziu 89% na área 1 e 91% na área 2. A redução da DA de C. quinquefasciatus na área 1 foi de 92% e a área 2 de 76%. Em A. aegypti, capturados nos 12 ciclos de estudo, foram detectados pools infectados com ZIKV (Área 1 - MIR 84,14; Área 2 ­ MIR 153,43) e DENV (Área 2 ­ MIR 5,29). Já para C. quinquefasciatus, detectamos pools infectados com ZIKV (Área 1 ­ MIR 10,06; Área 2 ­ MIR 18,33) e Wuchereria bancrofti (Área 2 ­ MIR 4,23). Nossos resultados demonstram que o CIM, com ações simples ou intensificadas, reduzem a densidade local de culicídeos vetores em residências-alvo. Ressaltamos também a importância de inserir a vigilância de infecção vetorial em programas de controle de mosquitos para prever possibilidades de surtos de doenças e direcionar ações de controle emergenciais .