Paridade de Ochlerotatus scapularis em condições de laboratório e campo

OBJECTIVE: To identify the parity status of Ochlerotatus scapularis females in laboratory and field conditions. METHODS: Field collections were carried out fortnightly using a hand-held aspirator in Vale do Ribeira, Southeastern Brazil, from April 2003 to March 2004. In laboratory, 100 Ochlerotatus scapularis F1 females were obtained and followed up individually, reporting the number of blood meals, the length of gonotrophic cycle, survival and number of eggs laid. The parity status and ovarian development were observed through ovarian dissection of 90 field females for each collection and all females reared in laboratory. RESULTS: The parity status diagnoses of the 100 females reared in laboratory and dissected agreed with 55% of the oviposition data, and it was underestimated in 37% and overestimated in 2%. Also in the laboratory, 106 gonotrophic cycles were completed, around 55% of females needed more than one blood meal before laying eggs. In laboratory conditions the species survival was 26 days. A total of 1,180 field females of Ochlerotatus scapularis were dissected: 418 (35.4%) nulliparous, 655 (55.5%) uniparous, 46 (3.9%) biparous and 61 (5.2%) could not be evaluated. Ninety field females were found in Christophers and Mer's phase III-V. CONCLUSIONS: The gonotrophic discordance hypothesis could be confirmed based on field and laboratory observations of Ochlerotarus scapularis females.OBJETIVO: Identificar o estado de paridade em fêmeas de Ochlerotatus scapularis de campo e obtidas em condições de laboratório. MÉTODOS: Para obtenção das fêmeas de campo, foram realizadas coletas quinzenais no Vale do Ribeira utilizando-se a técnica da aspiração manual, de abril de 2003 a março de 2004. Em laboratório, 100 fêmeas (F1) de Ochlerotatus scapularis foram mantidas e observadas individualmente anotando-se o número de repastos realizados, duração do ciclo gonotrófico, tempo de sobrevivência e número de ovos colocados. A observação do estado de paridade e desenvolvimento ovariano foi feita pela dissecção dos ovários de 90 fêmeas por coleta, e de todas as fêmeas mantidas em laboratório. RESULTADOS: Das 100 fêmeas mantidas em laboratório e dissecadas, o diagnóstico de condição de paridade conferiu com os resultados constatados em 55% dos casos, sendo subestimados em 37% e superestimados em 2%. Ainda em laboratório, de 106 ciclos gonotróficos completados, cerca de 55% das fêmeas necessitaram de mais de um repasto sangüíneo antes de ovipor. A sobrevivência observada em laboratório foi de até 26 dias para a espécie. Foram dissecadas 1.180 fêmeas de Ochlerotatus scapularis do campo: 418 (35,4%) foram consideradas nulíparas, 655 (55,5%) uníparas, 46 (3,9%) como bíparas e 61 (5,2%) não puderam ser avaliadas. Noventa fêmeas apresentavam-se na fase III-V de Christophers e Mer. CONCLUSÕES: Pode-se confirmar a hipótese de discordância gonotrófica, com base nas observações de fêmeas de Ochlerotatus scapularis no campo e laboratório

Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.</p

Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 – 2018

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Parity and ovarian development of Ochlerotatus scapularis (Diptera: Culicidae) in laboratory and field conditions in Vale do Ribeira region, São Paulo, Brazil

Objetivo. Identificar o estado de paridade em fêmeas de Ochlerotatus scapularis em condições de laboratório e campo. Das fêmeas coletadas em campo, determinar o grau de paridade considerando sua importância epidemiológica como possível transmissor de patógenos. Métodos. O trabalho foi dividido em duas partes: no campo foram realizadas coletas quinzenais no Vale do Ribeira de abril de 2003 a março de 2004, utilizando-se a técnica da aspiração manual. No laboratório, 100 fêmeas (FI) de Ochlerotatus scapularis foram criadas e observadas individualmente anotando-se o número de repastos por elas realizados, duração do ciclo gonotrófico e número de ovos colocados. A observação do estado de paridade e desenvolvimento ovariano foi feita através da dissecção dos ovários de 90 fêmeas, por coleta, e de todas as fêmeas criadas. Resultados. Das 100 fêmeas mantidas em laboratório e dissecadas, o diagnóstico de condição de paridade conferiu com os resultados constatados em 55% dos casos, sendo subestimados em 37% e superestimados em 2%. Ainda em laboratório, de 106 ciclos gonotróficos completados, cerca de 55% das fêmeas necessitaram de mais de um repasto sangüíneo antes de ovipor, indicando uma possível discordância gonotrófica para a espécie. Sob condições laboratoriais observou-se uma sobrevivência de até 26 dias para a espécie. Foram dissecadas 1180 fêmeas de Oco scapularis do campo: 418 (35,4%) foram consideradas nulíparas, 655 (55,5%) uníparas, 46 (3,9%) como bíparas e 61 (5,2%) não puderam ser avaliadas. Foi observado que 90 fêmeas apresentavam-se na fase ill-V de Christopher e Mer, reforçando a hipótese de discordância gonotrófica. A variável estado de paridade variou ao longo do ano. Conclusões: Observando fêmeas de Oco scapularis no campo e laboratório, confirmamos a hipótese de discordância gonotrófica.Objective. Identify the parity status of Ochlerotatus scapularis females in laboratory and field conditions. Regarding the field females, to determine the parity degree, considering this species epidemiologic importance as a possible pathogens vector. Methods. The research has two parts: field collections every 15 days were carried out in Vale do Ribeira from April 2003 to March 2004, using a hand-held aspirator. In laboratory, 100 Ochlerotatus scapularis F1, females were obtained and followed up individually, reporting the number of blood meals, the lentgh of gonotrophic cycle and survival. The parity status and the ovarian development were observed through ovarium dissection of 90 field females for each collection and all females reared in laboratory. Results. The parity status diagnoses of the 100 females reared in laboratory and dissecated were according to 55% of the oviposition data, were underestimated in 37% and overestimated in 2%. Also in the laboratory, 106 gonotrophic cycles were completed, around 55% of females needed more than one blood meal before laying eggs. Under laboratory conditions, the females survived for up to 26 days. 1180 field females of Ochlerotatus scapularis were dissecated: 418 (35,4%) nulliparous, 655 (55,5%) uniparous, 46 (3,9%) biparous and 61 (5,2%) could not be evaluated. We observed that 90 field females were found in Christopher and Mer phase Ill-V. Conclusions. Based on field and laboratory observations on Ochlerotarus scapularis females, we confirm the gonotrophic discordance hypothesis. The qualitative variable of parity status differed significantly throughout the year

Feeding habits of Aedes aegypti and Culex quinquefasciatus and its implications on the reproductive capacit

O estudo de hábito alimentar de espécies vetaras de doenças, ou potencialmente vetoras, indica os organismos envolvidos na transmissão e manutenção das doenças. Com a expansão de áreas urbanas e consequente aglomeração humana, como acontece no município de Marília, criou-se ambiente propício para a criação de mosquitos adaptados à essa situação. Esse trabalho teve por objetivo caracterizar o hábito alimentar de culicídeos capturados em área urbana do município de Marília e relacionar com o nível socioeconômico. A técnica utilizada para a identificação do hábito alimentar foi o ELISA (Enzyme Linked lmmunosorbent Assay) considerando quatro hospedeiros (humano, cão, ave e roedor). Entre as espécies coletadas com maior frequência estavam os Aedes aegypti e Culex quinquefasciatus. Em laboratório foram testados os aspectos da capacidade reprodutiva para fêmeas dessas espécies, alimentadas artificialmente com sangue dos mesmos hospedeiros observados em campo. As espécies coexistem em toda a área mesmo em diferentes níveis socioeconômicos. A não concorrência pode ser explicada por apresentarem perfis hematofágicos distintos. Diferentemente do Ae. aegypti, o Cx. quinquefasciatus é espécie eclética quanto as fontes de hematofagia, no entanto apresentou maior frequência de repastas sanguíneos em roedores. Os Ae. aegypti se mostraram essencialmente antropofílicos.The study of the feeding habits of vectors diseases species, or potentially vectors species, indicates the organisms involved in the maintenance and transmission of diseases. With the expansion of urban areas and consequent overcrowding, as occurs in Marília city, an adapted environment for breeding mosquitoes was created. This study aimed to characterize the feeding habits of mosquitoes captured in urban region of Marília and correlate with socioeconomic status. ELISA (Enzyme Linked lmmunosorbent Assay) was the technique used to identify the mosquitoes feeding habits, considering four hosts (human, dog, bird and rodent). The most frequently collected species were Aedes aegypti and Culex quinquefasciatus. Aspects of reproductive capacity in females of these species were tested in laboratory through artificially feeding, with blood of the field observed hosts. The mosquitoes species coexist in the whole area, even in different socioeconomic levels. The absence of competition can be explained by differences in hematophagous profiles. Although Cx. quinquefasciatus is an eclectic specie in blood sources, in opposite of Ae. aegypti, a high frequency of biting in rodents was observed. Ae. aegypti showed a mainly anthropophilic biting activity

Parity of Ochlerotatus scapularis in laboratory and field conditions

OBJECTIVE: To identify the parity status of Ochlerotatus scapularis females in laboratory and field conditions. METHODS: Field collections were carried out fortnightly using a hand-held aspirator in Vale do Ribeira, Southeastern Brazil, from April 2003 to March 2004. In laboratory, 100 Ochlerotatus scapularis F1 females were obtained and followed up individually, reporting the number of blood meals, the length of gonotrophic cycle, survival and number of eggs laid. The parity status and ovarian development were observed through ovarian dissection of 90 field females for each collection and all females reared in laboratory. RESULTS: The parity status diagnoses of the 100 females reared in laboratory and dissected agreed with 55% of the oviposition data, and it was underestimated in 37% and overestimated in 2%. Also in the laboratory, 106 gonotrophic cycles were completed, around 55% of females needed more than one blood meal before laying eggs. In laboratory conditions the species survival was 26 days. A total of 1,180 field females of Ochlerotatus scapularis were dissected: 418 (35.4%) nulliparous, 655 (55.5%) uniparous, 46 (3.9%) biparous and 61 (5.2%) could not be evaluated. Ninety field females were found in Christophers and Mer's phase III-V. CONCLUSIONS: The gonotrophic discordance hypothesis could be confirmed based on field and laboratory observations of Ochlerotarus scapularis females

Evaluation of Methods for Collecting Mosquitoes (Culicidae: Diptera) in Canopy and Ground Strata in the Brazilian Savanna

The hand-net is the standard method for capturing mosquitoes with sylvatic diurnal activity in disease outbreaks in Brazil. However, occupational risks and biases related to the collectors&rsquo; abilities and attractiveness are important limitations. In this study, we compared hand-nets with automatic traps (CDC) associated to CO2 and BG-Lure&reg; in the Vassununga State Park, a Brazilian Savanna protection area. The collections carried out over 27 days on the ground and the forest canopy. A total of 1555 mosquitoes were obtained in 20 taxa. The diversity index ranged between 1.12 and 1.79 and the dominance index from 0.22 to 0.40. The dominant species on the ground was Aedes scapularis (46.0%), and in the canopy, Hg. janthinomys/capricornii (31.9%). Haemagogus leucocelaenus was rare (n = 2). The hand-net resulted in the greatest diversity and abundance of species in both strata, followed by the traps associated with CO2. A low degree of similarity was observed between the hand-net on the ground compared to the other capture methods. The use of BG-Lure&reg; alone resulted in a low number of specimens. In conclusion, the hand-net is still the method of choice for collecting arbovirus vectors in the diurnal period, especially yellow fever vectors

Evaluation of Methods to Collect Diurnal Culicidae (Diptera) at Canopy and Ground Strata, in the Atlantic Forest Biome

Hand-held insect nets are the standard method for capturing vector mosquitoes of sylvatic arboviruses; however, occupational risks and biases due to individual skill and attractiveness are important limitations. The use of chemical attractants and automatic traps could be an alternative to resolve these limitations. This study compares the yields achieved using nets with those employing electrical traps with CO2 and BG-Lure&reg;, near the ground and in the canopy strata (6.0 and 8.0 m high). The study was conducted at the Cantareira State Park, which is in the Brazilian Atlantic Forest biome. In the 18 collections performed, 3570 specimens of 52 taxa were obtained. The most frequent species captured near the ground were Wyeomyia confusa and Limatus durhamii, whereas Sabethes albiprivus, Sabethes purpureus, and Haemagogus leucocelaenus were the most frequent in the canopy. The nets resulted in greater species richness and abundance, followed by the trap employing CO2. The combination of CO2 traps with BG-Lure&reg; did not improve performance. The use of BG-Lure&reg; alone resulted in low abundance and a low number of species. Our results demonstrate that the use of traps with CO2 can be complementary to collections with nets; however, for species of epidemiological interest such as those of the genera Haemagogus and Sabethes, especially in the canopy, the net remains the method of choice