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Multiple epicenters of the SARS-CoV-2 pandemic have emerged since the first pneumonia cases in Wuhan, China, such as Italy, USA, and Brazil. Brazil is the third-most affected country worldwide, but genomic sequences of SARS-CoV-2 strains are mostly restricted to states from the Southeast region. Pernambuco state, located in the Northeast region, is the sixth most affected Brazilian state, but very few genomic sequences from the strains circulating in this region are available. We sequenced 101 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms that reside in Pernambuco. Phylogenetic reconstructions revealed that all genomes belong to the B lineage and most of the samples (88%) were classified as lineage B.1.1. We detected multiple viral introductions from abroad (likely from Europe) as well as six local B.1.1 clades composed by Pernambuco only strains. Local clades comprise sequences from the capital city (Recife) and other country-side cities, corroborating the community spread between different municipalities of the state. These findings demonstrate that different from Southeastern Brazilian states where the epidemics were majorly driven by one dominant lineage (B.1.1.28 or B.1.1.33), the early epidemic phase at the Pernambuco state was driven by multiple B.1.1 lineages seeded through both national and international traveling.

The emergence of SARS-CoV-2 in the human population has caused a huge pandemic that is still unfolding in many countries around the world. Multiple epicenters of the pandemic have emerged since the first pneumonia cases in Wuhan, first in Italy followed by the USA and Brazil. Up to now, Brazil is the second most affected country, however, genomic sequences of SARS-CoV-2 strains circulating in the country are restricted to some highly impacted states. Although the Pernambuco state, located in the Northeast Region, is the sixth most affected brazilian state and the second considering lethality rate, there is a lack of high quality genomic sequences from the strains circulating in this region. Here, we sequenced 38 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms. Phylogenetic reconstructions revealed that three lineages were circulating in the state and 36 samples belong to B1.1 lineage. We detected two introductions from European countries and five clades, corroborating the community spread of the virus between different municipalities of the state. Finally, we detected that all except one strain showed the D614G spike protein amino acid change that may impact virus infectivity in human cells. Our study brought new light to the spread of SARS-CoV-2 strains in one of the most heavily impacted states of Brazil.

Duas tecnologias alternativas para o controle de Aedes aegypti foram avaliadas: a aplicação espacial de larvicida biológico-Bti em potenciais criadouros peridomiciliares, e a liberação de machos estéreis para inviabilização reprodutiva das fêmeas do mosquito. As ações foram realizadas pelos Agentes dos Serviços de Saúde em 15 vilas da Ilha de Fernando de Noronha, e em uma área (900 imóveis) no bairro da Várzea/Recife/Pernambuco. A efetividade dos métodos foi avaliada por indicadores entomológicos, estimados pela presença, quantidade e viabilidade de ovos do mosquito, coletados em armadilhas, e por marcadores genéticos. A aplicação de Bti, com atomizador costal, ocorreu a cada 30 dias em ambas as áreas. Uma redução importante e sustentável da população de A. aegypti, por este método, foi alcançada em 2015/2016 na Várzea e, em 2016, na Ilha, onde a remoção de 18 toneladas de resíduos sólidos em 2015 contribuiu possivelmente para os resultados. Machos esterilizados com radiação gama foram produzidos em massa no laboratório e liberados em uma das vilas da Ilha. A análise espaço-temporal dos indicadores, de dez/2015 a ago/2016, revelou redução expressiva da densidade populacional do mosquito e da diversidade genética da população local. Ambas as abordagens parecem ter reduzido o contato homem-vetor e os riscos de transmissão de arboviroses na Ilha, apesar da elevada competência vetorial da população local do mosquito para os vírus Zika e Dengue. Os métodos testados se mostraram eficientes e passíveis de serem integradas às ações do SUS voltadas ao controle de A. aegypti.

Zika virus (ZIKV) is a negative sense RNA virus from the Flaviviridae family, which was relatively unknown until the first human epidemic in Micronesia, in 2007. Since then, it spread to French Polynesia and the Americas. Recife, the capital of Pernambuco state and epicenter of the Zika epidemic in Brazil, experienced a large number of microcephaly cases and other congenital abnormalities associated to the ZIKV infection from, 2015 to 16. Evidences suggest that both Aedes aegypti and Culex quinquefasciatus mosquitoes from Recife are capable of replicating and transmitting the virus. Here, we conducted high throughput sequencing of ZIKV genomes directly from Ae. aegypti and Cx. quinquefasciatus mosquitoes collected during the ZIKV epidemics in Recife, in order to investigate the variability and evolution of the virus. We obtained 11 draft ZIKV genomes derived from 5 pools from each Ae. aegypti and Cx. quinquefasciatus species. Genome coverage breadth ranged from 16 to 100% and average depth from 45 to 46,584×. Two of these genomes were obtained from pools of Cx. quinquefasciatus females with no sign of blood in the abdomen. Amino acid substitutions found here were not species-specific. In addition, molecular clock dating estimated that ZIKV draft genomes obtained here were co-circulating in other regions of the country during the epidemics. Overall results highlight that viral mutations and even minor variants can be detected in genomes directly sequenced from mosquito samples and insights about natural viral genomic variability and viral evolution can be useful when designing tools for mosquito control programs.

Background Zika virus (ZIKV) has been isolated from many mosquito species in nature, but it is believed that the main vectors in urban environments are species of the genus Aedes. Here, we detected and isolated ZIKV in samples from Aedes aegypti, Aedes taeniorhynchus and Culex quinquefasciatus, collected during the Zika epidemic in Vitória, southeast Brazil. Using quantitative real-time polymerase chain reaction, ZIKV detection was performed in mosquito samples collected from February to April 2016. Results Overall, six pools of mosquitoes were positive for ZIKV: four of Cx. quinquefasciatus, one of Ae. aegypti and one of Ae. taeniorhynchus. Their genomes were sequenced. Conclusions These results support and strengthen the hypothesis that other mosquito species can also be involved in ZIKV transmission.

Aedes aegypti (L.) has become an efficient vector of important arboviruses due to its anthropophilic and domiciliary behaviors. Since the 1980s, dengue affects thousands of people every year in Brazil; in Fernando de Noronha (FN), a touristic archipelago, dengue cases have occurred since 2001. Once Ae. aegypti populations are well established in the inhabited areas of FN, the threat of dengue or another arbovirus epidemic is continuously imminent. This study aimed to monitor the DENV serotypes in mosquito samples collected in FN, where at least one resident was clinically diagnosed as dengue patient. Entomological surveillance was conducted in 2011 and 2012. Mosquitoes were sorted by sex and location and were stored in pools. DENV detection was performed using polymerase chain reaction with reverse transcription (RT-PCR) and the Platelia Dengue NS1 Ag. RNA integrity was checked by RT-PCR using rpL8 primers, and the minimum infection rate (MIR) was calculated. In total, 339 pools were analyzed, and only one was positive (DENV-1) by Multiplex RT-PCR (MIR = 1.53). When considering only pools with RNA integrity, the MIR was 2.92. Using the Platelia kit, the MIR was 9.18 (considering all the pools) and 17.54 (only 140 pools with RNA integrity). Our results showed the importance of a constant entomological surveillance in that area, the need to improve storage and transportation protocols, and an endogenous control in the RT-PCR to avoid false-negative results. Finally, our study indicated that the NS1-Ag detection was the most sensitive method and should be used routinely for DENV surveillance in mosquitoes if the serotype identification is not required.

The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.

Introduction Molecular xenomonitoring (MX)—pathogen detection in the mosquito rather than human—is a promising tool for lymphatic filariasis (LF) surveillance. In the Recife Metropolitan Region (RMR), the last LF focus in Brazil, Culex quinquefasciatus mosquitoes have been implicated in transmitting Wuchereria bancrofti parasites. This paper presents findings on the ideal mosquito collection method, mosquito dispersion, W. bancrofti infection in mosquitoes and W. bancrofti antigen in humans to aid MX development. Methods Experiments occurred within two densely populated urban areas of Olinda, RMR, in July and August 2015. U.S. Centers for Disease Control and Prevention (CDC) light traps were compared to battery-powered aspirators as collection methods, and mosquito dispersion was measured by mosquito mark release recapture (MMRR). Female Cx. quinquefasciatus were tested by PCR for W. bancrofti infection, and study area residents were screened by rapid tests for W. bancrofti antigen. Results Aspirators caught 2.6 times more total Cx. quinquefasciatus, including 38 times more blood-fed and 5 times more gravid stages, than CDC light traps. They also collected 123 times more Aedes aegypti. Of the 9,644 marked mosquitoes released, only ten (0.01%) were recaptured, nine of which were < 50m (34.8m median, 85.4m maximum) from the release point. Of 9,169 unmarked mosquitoes captured in the MMR, 38.3% were unfed, 48.8% blood-fed, 5.5% semi-gravid, and 7.3% gravid. PCR on 182 pools (1,556 mosquitoes) found no evidence of W. bancrofti infection in Cx. quinquefasciatus. Rapid tests on 110 of 111 eligible residents were all negative for W. bancrofti antigen. Conclusions Aspirators were more effective than CDC light traps at capturing Ae. aegypti and all but unfed stages of Cx. quinquefasciatus. Female Cx. quinquefasciatus traveled short (< 86m) distances in this urban area. Lack of evidence for W. bancrofti infection in mosquitoes and antigen in humans in these fine-scale studies does not indicate that LF transmission has ceased in the RMR. A MX surveillance system should consider vector-specific collection methods, mosquito dispersion, and spatial scale but also local context, environmental factors such as sanitation, and host factors such as infection prevalence and treatment history., Author summary Lymphatic filariasis (LF) is a parasitic disease transmitted by mosquitoes, and can cause elephantiasis. It is the world’s leading cause of disability due to infectious diseases, affects over 120 million people globally, and is scheduled for global elimination via mass drug administration (MDA) and mosquito control. Molecular xenomonitoring (MX) is a process of screening mosquitoes—not humans—for parasites to estimate whether they are circulating in human populations. MX is especially useful during and following MDA, when new case detection becomes difficult, but is challenging to design and conduct in cities. Using two study sites in the Recife Metropolitan Region, Brazil, we investigated two crucial questions for urban MX development—“What is the best operationally feasible tool to catch adult mosquitoes?” and “How far do mosquitoes disperse in cities?”—in order to determine placement of future surveillance sites. We also screened a proportion of mosquitoes and all eligible residents from the study sites for LF infection. We determined that handheld battery powered aspirators were the best mosquito collection tool; that mosquitoes flew no more than about 85m; and—in this small sample of mosquitoes and very small sample of humans—there was no evidence of LF infection in mosquitoes or study area residents.

Arthropod-borne viruses, such as Dengue (DENV), Chikungunya (CHIKV), and Zika (ZIKV), pose a challenge to public health, due to their worldwide distribution and large-scale outbreaks. Dengue fever is currently one of the most important diseases and it is caused by four serotypes of DENV and is mainly transmitted by the mosquito Aedes aegypti. It is estimated that 50–100 million cases are reported every year worldwide. More recently, CHIKV and ZIKV, which are also transmitted by Ae. aegypti, have caused epidemics in countries in the Caribbean region, the Pacific region, and Americas. Cabo Verde faced its first dengue outbreak in 2009, with more than 21,000 reported cases and four registered deaths. The epidemic was caused by DENV-3 transmitted by Ae. aegypti mosquitoes. In addition, the country faced a Zika outbreak with more than 7,500 notified cases from October 2015 to May 2016. In the present study, we conducted a survey in mosquito samples to detect arboviruses circulating in the local vector population. Collections were performed from November 2014 to January 2015, in the City of Praia, the capital of Cabo Verde, using aspirators and BG-sentinel traps. Samples were examined by multiplex Reverse Transcription-polymerase chain reaction. A total of 161 Ae. aegypti adult females were analyzed (34 pools) and from these samples, eight pools were found positive for DENV-2 and DENV-4. Our results revealed a very high natural infection rate in the vector population and showed two different serotypes co-circulating in the island that differ from the one detected in the 2009 outbreak in Cabo Verde.

The World Health Organization (WHO) recommends that people travelling to or living in areas with Zika virus (ZIKV) outbreaks or epidemics adopt prophylactic measures to reduce or eliminate mosquito bites, including the use of insect repellents. It is, however, unknown whether repellents are effective against ZIKV-infected mosquitoes, in part because of the ethical concerns related to exposing a human subject’s arm to infected mosquitoes in the standard arm-in-cage assay. We used a previously developed, human subject-free behavioural assay, which mimics a human subject to evaluate the top two recommended insect repellents. Our measurements showed that DEET provided significantly higher protection than picaridin provided against noninfected, host-seeking females of the southern house mosquito, Culex quinquefasciatus, and the yellow fever mosquito, Aedes aegypti. When tested at lower doses, we observed a significant reduction in DEET-elicited protection against ZIKV-infected yellow fever mosquitoes from old and recent laboratory colonies. The reduction in protection is more likely associated with aging than the virus infection and could be compensated by applying a 5x higher dose of DEET. A substantial protection against ZIKV-infected and old noninfected mosquitoes was achieved with 5% DEET, which corresponds approximately to a 30% dose in the conventional arm-in-cage assays.

Zika virus (ZIKV) is a flavivirus that has recently been associated with an increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide; however, there is evidence that other mosquito species, including Culex quinquefasciatus, transmit the virus. To test the potential of Cx. quinquefasciatus to transmit ZIKV, we experimentally compared the vector competence of laboratory-reared Ae. aegypti and Cx. quinquefasciatus. Interestingly, we were able to detect the presence of ZIKV in the midgut, salivary glands and saliva of artificially fed Cx. quinquefasciatus. In addition, we collected ZIKV-infected Cx. quinquefasciatus from urban areas with high microcephaly incidence in Recife, Brazil. Corroborating our experimental data from artificially fed mosquitoes, ZIKV was isolated from field-caught Cx. quinquefasciatus, and its genome was partially sequenced. Collectively, these findings indicate that there may be a wider range of ZIKV vectors than anticipated.

Zika virus (ZIKV) is a flavivirus that has recently been associated with increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide, but there is evidence of other mosquito species, including Culex quinquefasciatus, playing a role in the Brazilian outbreak. To test this hypothesis, we experimentally compared the vectorial competence of laboratory-reared A. aegypti and C. quinquefasciatus. We found ZIKV in the midgut, salivary glands, and saliva of artificially fed C. quinquefasciatus. Additionally, we collected ZIKV-infected C. quinquefasciatus from urban areas of high microcephaly incidence in Recife, Brazil. Take into account; these findings indicate that there may be a wider range of vectors for ZIKV than anticipated.

The aim of this study was to determine the prevalence of the bla SHV gene in Klebsiella pneumoniae isolates from hospital and community infections and from the normal microbiota of healthy individuals in Recife, PE, Brazil. Fifty-two K. pneumoniae isolates were analyzed regarding the presence of the bla SHV gene, using PCR, and eight isolates were analyzed by DNA sequencing. This gene was detected in 16 isolates from hospital infections, four from community infections, and nine from the normal microbiota. This was the first study to find the bla SHV gene in K. pneumoniae isolates from the normal microbiota. Through DNA sequencing of eight K. pneumoniae isolates from hospital and community infections, with a resistance phenotype indicative of extended-spectrum β-lactamase production, a new SHV variant named SHV-122 was found. We also detected the presence of bla SHV-1, bla SHV-11, bla SHV-28, and bla SHV-108. The results show that in Recife, Brazil, K. pneumoniae isolates that presented resistance to oxyimino-β-lactams had high prevalence and diversity of the bla SHV gene. We also conclude that there was a high presence of the bla SHV gene among isolates from the normal microbiota of healthy individuals.

Emerging Microbes & Infections (2017) 6, e91; doi:10.1038/emi.2017.86; published online 18 October 2017.

The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RT-PCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain.