Reemergence of human malaria in Atlantic Forest of Rio Grande do Sul, Brazil.

Unforeseen Plasmodium infections in the Atlantic Forest of Brazilian Extra-Amazonian region could jeopardise malaria elimination. A human malaria case was registered in Três Forquilhas, in the Atlantic Forest biome of Rio Grande do Sul, after a 45 years' time-lapsed without any malaria autochthonous notification in this southern Brazilian state. This finding represents the expansion of the malaria distribution areas in Brazil and the southernmost human malaria case record in South America in this decade. The coexistence of the bromeliad-breeding vector Anopheles (Kerteszia) cruzii and non-human primates in the Atlantic Forest regularly visited by the patient claimed for the zoonotic origin of this infection. The reemergence of Atlantic Forest human malaria in Rio Grande do Sul was also discussed.

Seasonal population dynamics of the primary yellow fever vector Haemagogus leucocelaenus (Dyar & Shannon) (Diptera: Culicidae) is mainly influenced by temperature in the Atlantic Forest, southeast Brazil.

BACKGROUND: Southeast Brazil has recently experienced a Yellow Fever virus (YFV) outbreak where the mosquito Haemagogus leucocelaenus was a primary vector. Climatic factors influence the abundance of mosquito vectors and arbovirus transmission. OBJECTIVES: We aimed at describing the population dynamics of Hg. leucocelaenus in a county touched by the recent YFV outbreak. METHODS: Fortnightly egg collections with ovitraps were performed from November 2012 to February 2017 in a forest in Nova Iguaçu, Rio de Janeiro, Brazil. The effects of mean temperature and rainfall on the Hg. leucocelaenus population dynamics were explored. FINDINGS: Hg. leucocelaenus eggs were continuously collected throughout the study, with a peak in the warmer months (December-March). The climatic variables had a time-lagged effect and four weeks before sampling was the best predictor for the positivity of ovitraps and total number of eggs collected. The probability of finding > 50% positive ovitraps increased when the mean temperature was above 24ºC. The number of Hg. leucocelaenus eggs expressively increase when the mean temperature and accumulated precipitation surpassed 27ºC and 100 mm, respectively, although the effect of rainfall was less pronounced. MAIN CONCLUSIONS: Monitoring population dynamics of Hg. leucocelaenus and climatic factors in YFV risk areas, especially mean temperature, may assist in developing climate-based surveillance procedures to timely strengthening prophylaxis and control.

Correction to: Frozen blood clots can be used for the diagnosis of distinct Plasmodium species in man and non-human primates from the Brazilian Atlantic Forest.

Following publication of the original article [1], it was flagged that one of the authors (Anielle de Pina Costa) is missing an affiliation in the article.

Capture of Alouatta guariba clamitans for the surveillance of sylvatic yellow fever and zoonotic malaria: Which is the best strategy in the tropical Atlantic Forest?

Howler monkey capture is an arduous and expensive task requiring trained and specialized professionals. We compared strategies and methods to most efficiently capture Alouatta guariba clamitans in remnants of the Atlantic Forest in Rio de Janeiro and its bordering states of Minas Gerais and São Paulo. We tested whether or not the success of expeditions in the forest with anesthetic darts, nets, and baited traps differed with and without the support of an information network, a contact chain built with key institutions and inhabitants to continuously monitor howler monkey presence. The influence of forest conditions (vegetation type and fragment size) upon darting success was also evaluated. We captured 24 free-living A. guariba clamitans. No howler monkey was caught with traps, probably due to the predominantly folivore feeding to high local plant diversity providing a great variety of food options. Captures based on an information network were significantly more efficient in terms of numbers of caught monkeys than without it. Captures with darts were considerably more efficient when performed in semideciduous forests and small forest fragments as opposed to ombrophilous forests or large woods. Although we walked great distances within the forest searching for howler monkeys, all but one animal were captured at the forest fringes. Hindrances to search and the darting method in the Atlantic Forest, for example, the steep terrain, high tree canopies, hunt pressure, and low A. guariba clamitans population density, were mitigated with the use of the information network in this monkey capture. Moreover, the information network enhanced the surveillance of zoonotic diseases, which howler monkeys and other nonhuman primates are reservoirs in Brazil, such as malaria and yellow fever.

Combination of surveillance tools reveals that Yellow Fever virus can remain in the same Atlantic Forest area at least for three transmission seasons.

BACKGROUND: In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES: To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS: We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS: Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS: Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

Culex quinquefasciatus mosquitoes do not support replication of Zika virus.

The rapid spread of Zika virus (ZIKV) in the Americas raised many questions about the role of Culex quinquefasciatus mosquitoes in transmission, in addition to the key role played by the vector Aedes aegypti. Here we analysed the competence of Cx. quinquefasciatus (with or without Wolbachia endosymbionts) for a ZIKV isolate. We also examined the induction of RNA interference pathways after viral challenge and the production of small virus-derived RNAs. We did not observe any infection nor such small virus-derived RNAs, regardless of the presence or absence of Wolbachia. Thus, Cx. quinquefasciatus does not support ZIKV replication and Wolbachia is not involved in producing this phenotype. In short, these mosquitoes are very unlikely to play a role in transmission of ZIKV.

Genomic and structural features of the yellow fever virus from the 2016-2017 Brazilian outbreak.

Southeastern Brazil has been suffering a rapid expansion of a severe sylvatic yellow fever virus (YFV) outbreak since late 2016, which has reached one of the most populated zones in Brazil and South America, heretofore a yellow fever-free zone for more than 70 years. In the current study, we describe the complete genome of 12 YFV samples from mosquitoes, humans and non-human primates from the Brazilian 2017 epidemic. All of the YFV sequences belong to the modern lineage (sub-lineage 1E) of South American genotype I, having been circulating for several months prior to the December 2016 detection. Our data confirm that viral strains associated with the most severe YF epidemic in South America in the last 70 years display unique amino acid substitutions that are mainly located in highly conserved positions in non-structural proteins. Our data also corroborate that YFV has spread southward into Rio de Janeiro state following two main sylvatic dispersion routes that converged at the border of the great metropolitan area comprising nearly 12 million unvaccinated inhabitants. Our original results can help public health authorities to guide the surveillance, prophylaxis and control measures required to face such a severe epidemiological problem. Finally, it will also inspire other workers to further investigate the epidemiological and biological significance of the amino acid polymorphisms detected in the Brazilian 2017 YFV strains.

Frozen blood clots can be used for the diagnosis of distinct Plasmodium species in man and non-human primates from the Brazilian Atlantic Forest.

BACKGROUND: Zoonotic infections with epidemic potential, as non-human primate malaria and yellow fever (YF), can overlap geographically. Optimizing a small blood sample for diagnosis and surveillance is of great importance. Blood are routinely collected for YF diagnosis and blood clots usually discarded after serum obtention. Aiming to take sample advantage, the sensitivity of a PCR using extracted DNA from long-term frozen clots from human and non-human primates for detection of Plasmodium spp. in low parasitaemia conditions was assayed. RESULTS: Malaria diagnosis with DNA extracted from blood clots generated results in agreement with samples obtained with whole blood, including mixed Plasmodium vivax/simium and Plasmodium malariae/brasilianum infections. CONCLUSION: Blood clots from human and non-human primates may be an important and low cost source of DNA for malaria surveillance in the Atlantic Forest.

Yellow fever outbreak in Brazil: the puzzle of rapid viral spread and challenges for immunisation.

We discuss the complex eco-social factors involved in the puzzle of the unexpected rapid viral spread in the ongoing Brazilian yellow fever (YF) outbreak, which has increased the reurbanisation risk of a disease without urban cases in Brazil since 1942. Indeed, this rapid spatial viral dissemination to the Southeast and South regions, now circulating in the Atlantic Forest fragments close to peri-urban areas of the main Brazilian megalopolises (São Paulo and Rio de Janeiro) has led to an exponential increase in the number of yellow fever cases. In less than 18 months, 1,833 confirmed cases and 578 deaths were recorded most of them reported in the Southeast region (99,9%). Large epizooties in monkeys and other non-human primates (NHPs) were communicated in the country with 732 YF virus (YFV) laboratory confirmed events only in the 2017/2018 monitoring period. We also discuss the peculiarities and similarities of the current outbreak when compared with previous great epidemics, examining several hypotheses to explain the recent unexpected acceleration of epizootic waves in the sylvatic cycle of the YFV together with the role of human, NHPs and mosquito mobility with respect to viral spread. We conclude that the most feasible hypothesis to explain this rapidity would be related to human behavior combined with ecological changes that promoted a significant increase in mosquito and NHP densities and their contacts with humans. We emphasize the urgent need for an adequate response to this outbreak such as extending immunisation coverage to the whole Brazilian population and developing novel strategies for immunisation of NHPs confined in selected reserve areas and zoos. Finally, we stress the urgent need to improve the quality of response in order to prevent future outbreaks and a catastrophic reurbanisation of the disease in Brazil and other South American countries. Continuous monitoring of YFV receptivity and vulnerability conditions with effective control of the urban vector Aedes aegypti and significant investments in YF vaccine production capacity and research and development for reduction of adverse effects are of the highest priority.

Anophelines species and the receptivity and vulnerability to malaria transmission in the Pantanal wetlands, Central Brazil.

BACKGROUND Studies on malaria vectors in the Pantanal biome, Central Brazil, were conducted more than half a century ago. OBJECTIVES To update anopheline records and assess receptivity and vulnerability to malaria transmission. METHODS Five-day anopheline collections were conducted bimonthly in Salobra, Mato Grosso do Sul state, for one year. Indoors, mosquitoes were collected from their resting places, while in open fields, they were captured using protected human-baited and horse-baited traps near the house and at the Miranda River margin, respectively. Hourly biting activity outdoors was also assessed. Secondary data were collected on the arrival of tourists, economic projects, and malaria cases. FINDINGS A total of 24,894 anophelines belonging to 13 species were caught. The main Brazilian malaria vector Anopheles darlingi was the predominant species, followed by An. triannulatus s.l. Hourly variation in anopheline biting showed three main peaks occurring at sunset, around midnight, and at sunrise, the first and last being the most prominent. The highest density of all species was recorded near the river margin and during the transition period between the rainy and early dry seasons. This coincides with the time of main influx of outsider workers and tourists, whose activities mostly occur in the open fields and frequently start before sunrise and last until sunset. Some of these individuals originate from neighbouring malaria-endemic countries and states, and are likely responsible for the recorded imported and introduced malaria cases. MAIN CONCLUSION Pantanal is a malaria-prone area in Brazil. Surveillance and anopheline control measures must be applied to avoid malaria re-emergence in the region.

Zika infection decreases Aedes aegypti locomotor activity but does not influence egg production or viability.

BACKGROUND: Zika has emerged as a new public health threat after the explosive epidemic in Brazil in 2015. It is an arbovirus transmitted mainly by Aedes aegypti mosquitoes. The knowledge of physiological, behavioural and biological features in virus-infected vectors may help the understanding of arbovirus transmission dynamics and elucidate their influence in vector capacity. OBJECTIVES: We aimed to investigate the effects of Zika virus (ZIKV) infection in the behaviour of Ae. aegypti females by analysing the locomotor activity, egg production and viability. METHODOLOGY: Ae. aegypti females were orally infected with ZIKV through an artificial feeder to access egg production, egg viability and locomotor activity. For egg production and viability assays, females were kept in cages containing an artificial site for oviposition and eggs were counted. Locomotor activity assays were performed in activity monitors and an average of 5th, 6th and 7th days after infective feeding was calculated. FINDINGS: No significant difference in the number of eggs laid per females neither in their viability were found between ZIKV infected and non-infected females, regardless the tested pair of mosquito population and virus strain and the gonotrophic cycles. Locomotor activity assays were performed regardless of the locomotor activity in ZIKV infected females was observed, in both LD and DD conditions. MAIN CONCLUSIONS: The lower locomotor activity may reduce the mobility of the mosquitoes and may explain case clustering within households reported during Zika outbreaks such as in Rio de Janeiro 2015. Nevertheless, the mosquitoes infected with ZIKV are still able to disseminate and to transmit the disease, especially in places where there are many oviposition sites.

Culex quinquefasciatus from areas with the highest incidence of microcephaly associated with Zika virus infections in the Northeast Region of Brazil are refractory to the virus.

Zika virus (ZIKV) is widely distributed in Brazil and the Northeast Region (NE) is the most affected zone, showing the highest incidence of microcephaly associated with ZIKV congenital infections worldwide. We report attempts to infect three populations of Culex quinquefasciatus from severely affected sites in the NE and Southeast Region (SE) of Brazil with three strains of ZIKV isolated from these localities. An Aedes aegypti population from the SE was used as a positive control. All tested Cx. quinquefasciatus populations were refractory to the ZIKV isolates. For these reasons, we believe Cx. quinquefasciatus should not be considered a potential vector of ZIKV in Brazil.

Zika puzzle in Brazil: peculiar conditions of viral introduction and dissemination - A Review.

This article discusses the peculiar conditions that favoured the unexpected introduction of Zika virus into the poorest northeastern region of Brazil in 2015, its speed of transmission to other Brazilian states, other Latin American countries and other regions, and the severity of related neurological disorders in newborns and adults. Contrasting with evidence that Zika had so far caused only mild cases in humans in the last six decades, the epidemiological scenario of this outbreak in Brazil indicates dramatic health effects: in 2015, an increase of 20-fold in notified cases of microcephaly and/or central nervous system (CNS) alterations suggestive of Zika congenital infection, followed by an exponential increase in 2016, with 2366 cumulative cases confirmed in the country by the end of December 2016. A significant increase in Guillain-Barré syndrome in adults has also been reported. Factors involved in viral dissemination, neural pathogenesis and routes of transmission in Brazil are examined, such as the role of social and environmental factors and the controversies involved in the hypothesis of antibody-dependent enhancement, to explain the incidence of congenital Zika syndrome in Brazil. Responses to the Zika outbreak and the development of new products are also discussed.

Genome analysis of yellow fever virus of the ongoing outbreak in Brazil reveals polymorphisms.

The current yellow fever outbreak in Brazil is the most severe one in the country in recent times. It has rapidly spread to areas where YF virus (YFV) activity has not been observed for more than 70 years and vaccine coverage is almost null. Here, we sequenced the whole YFV genome of two naturally infected howler-monkeys (Alouatta clamitans) obtained from the Municipality of Domingos Martins, state of Espírito Santo, Brazil. These two ongoing-outbreak genome sequences are identical. They clustered in the 1E sub-clade (South America genotype I) along with the Brazilian and Venezuelan strains recently characterised from infections in humans and non-human primates that have been described in the last 20 years. However, we detected eight unique amino acid changes in the viral proteins, including the structural capsid protein (one change), and the components of the viral replicase complex, the NS3 (two changes) and NS5 (five changes) proteins, that could impact the capacity of viral infection in vertebrate and/or invertebrate hosts and spreading of the ongoing outbreak.

First detection of natural infection of Aedes aegypti with Zika virus in Brazil and throughout South America.

Zika virus (ZIKV) has caused a major epidemic in Brazil and several other American countries. ZIKV is an arbovirus whose natural vectors during epidemics have been poorly determined. In this study, 1,683 mosquitoes collected in the vicinity of ZIKV suspected cases in Rio de Janeiro, Brazil, from June 2015 to May 2016 were screened for natural infection by using molecular methods. Three pools of Aedes aegypti were found with the ZIKV genome, one of which had only one male. This finding supports the occurrence of vertical and/or venereal transmission of ZIKV in Ae. aegypti in nature. None of the examined Ae. albopictus and Culex quinquefasciatus was positive. This is the first report of natural infection by ZIKV in mosquitoes in Brazil and other South American countries. So far, Ae. aegypti is the only confirmed vector of ZIKV during the ongoing Pan-American epidemics.

Culex mosquitoes are experimentally unable to transmit Zika virus.

We report that two laboratory colonies of Culex quinquefasciatus and Culex pipiens mosquitoes were experimentally unable to transmit ZIKV either up to 21 days post an infectious blood meal or up to 14 days post intrathoracic inoculation. Infectious viral particles were detected in bodies, heads or saliva by a plaque forming unit assay on Vero cells. We therefore consider it unlikely that Culex mosquitoes are involved in the rapid spread of ZIKV.

High level of vector competence of Aedes aegypti and Aedes albopictus from ten American countries as a crucial factor in the spread of Chikungunya virus.

UNLABELLED: Chikungunya virus (CHIKV) causes a major public health problem. In 2004, CHIKV began an unprecedented global expansion and has been responsible for epidemics in Africa, Asia, islands in the Indian Ocean region, and surprisingly, in temperate regions, such as Europe. Intriguingly, no local transmission of chikungunya virus (CHIKV) had been reported in the Americas until recently, despite the presence of vectors and annually reported imported cases. Here, we assessed the vector competence of 35 American Aedes aegypti and Aedes albopictus mosquito populations for three CHIKV genotypes. We also compared the number of viral particles of different CHIKV strains in mosquito saliva at two different times postinfection. Primarily, viral dissemination rates were high for all mosquito populations irrespective of the tested CHIKV isolate. In contrast, differences in transmission efficiency (TE) were underlined in populations of both species through the Americas, suggesting the role of salivary glands in selecting CHIKV for highly efficient transmission. Nonetheless, both mosquito species were capable of transmitting all three CHIKV genotypes, and TE reached alarming rates as high as 83.3% and 96.7% in A. aegypti and A. albopictus populations, respectively. A. albopictus better transmitted the epidemic mutant strain CHIKV_0621 of the East-Central-South African (ECSA) genotype than did A. aegypti, whereas the latter species was more capable of transmitting the original ECSA CHIKV_115 strain and also the Asian genotype CHIKV_NC. Therefore, a high risk of establishment and spread of CHIKV throughout the tropical, subtropical, and even temperate regions of the Americas is more real than ever. IMPORTANCE: Until recently, the Americas had never reported chikungunya (CHIK) autochthonous transmission despite its global expansion beginning in 2004. Large regions of the continent are highly infested with Aedes aegypti and Aedes albopictus mosquitoes, and millions of dengue (DEN) cases are annually recorded. Indeed, DEN virus and CHIK virus (CHIKV) share the same vectors. Due to a recent CHIK outbreak affecting Caribbean islands, the need for a Pan-American evaluation of vector competence was compelling as a key parameter in assessing the epidemic risk. We demonstrated for the first time that A. aegypti and A. albopictus populations throughout the continent are highly competent to transmit CHIK irrespective of the viral genotypes tested. The risk of CHIK spreading throughout the tropical, subtropical, and even temperate regions of the Americas is more than ever a reality. In light of our results, local authorities should immediately pursue and reinforce epidemiological and entomological surveillance to avoid a severe epidemic.

Simian malaria in the Brazilian Atlantic forest: first description of natural infection of capuchin monkeys (Cebinae subfamily) by Plasmodium simium.

BACKGROUND: In Brazil, two species of Plasmodium have been described infecting non-human primates, Plasmodium brasilianum and Plasmodium simium. These species are morphologically, genetically and immunologically indistinguishable from the human Plasmodium malariae and Plasmodium vivax parasites, respectively. Plasmodium simium has been observed naturally infecting monkeys of the genera Alouatta and Brachyteles in a restricted area of the Atlantic Forest in the south and southeast regions of Brazil. However, its reported geographical distribution and the diversity of its vertebrate hosts may be underestimated, since available data were largely based on analyses by microscopic examination of peripheral blood, a method with limited sensitivity, considering the potential sub-patent feature of these infections. The present study describes, for the first time, the natural infection of P. simium in capuchin monkeys from the Brazilian Atlantic Forest. METHODS: Blood samples from 30 non-human primates belonging to nine species kept in the Primate Centre of Rio de Janeiro were collected. Fragments of spleen and liver from one dead monkey found in the neighborhoods of the Primate Centre were also analysed. Molecular diagnosis was performed by nested PCR (18SSU rRNA) and the amplified fragment was sequenced. RESULTS: Thirty per cent of the captive animals were infected with P. simium and/or P. brasilianum. The dead monkey tested positive for DNA of P. simium. For the first time, Cebinae primates (two specimens of genus Cebus and two of genus Sapajos) were found naturally infected by P. simium. The infection was confirmed by sequencing a small fragment of 18SSU rRNA. CONCLUSION: The results highlight the possibility of infection by P. simium in other species of non-human primates whose impact could be significant for the malaria epidemiology among non-human primates and, if it becomes clear that this P. simium is able to infect monkeys and, eventually, man, also for the maintenance of transmission of human malaria in the context of a zoonosis in areas under influence of the Atlantic Forest.

Three-way interactions between mosquito population, viral strain and temperature underlying chikungunya virus transmission potential.

Interactions between pathogens and their insect vectors in nature are under the control of both genetic and non-genetic factors, yet most studies on mosquito vector competence for human pathogens are conducted in laboratory systems that do not consider genetic and/or environmental variability. Evaluating the risk of emergence of arthropod-borne viruses (arboviruses) of public health importance such as chikungunya virus (CHIKV) requires a more realistic appraisal of genetic and environmental contributions to vector competence. In particular, sources of variation do not necessarily act independently and may combine in the form of interactions. Here, we measured CHIKV transmission potential by the mosquito Aedes albopictus in all combinations of six worldwide vector populations, two virus strains and two ambient temperatures (20°C and 28°C). Overall, CHIKV transmission potential by Ae. albopictus strongly depended on the three-way combination of mosquito population, virus strain and temperature. Such genotype-by-genotype-by-environment (G × G × E) interactions question the relevance of vector competence studies conducted with a simpler set of conditions. Our results highlight the need to account for the complex interplay between vectors, pathogens and environmental factors to accurately assess the potential of vector-borne diseases to emerge.

Updating the geographical distribution and frequency of Aedes albopictus in Brazil with remarks regarding its range in the Americas.

The geographical distribution of Aedes albopictus in Brazil was updated according to the data recorded across the country over the last eight years. Countrywide house indexes (HI) for Ae. albopictus in urban and suburban areas were described for the first time using a sample of Brazilian municipalities. This mosquito is currently present in at least 59% of the Brazilian municipalities and in 24 of the 27 federal units (i.e., 26 states and the Federal District). In 34 Brazilian municipalities, the HI values for Ae. albopictus were higher than those recorded for Ae. aegypti, reaching figures as high as HI = 7.72 in the Southeast Region. Remarks regarding the current range of this mosquito species in the Americas are also presented. Nineteen American countries are currently infested and few mainland American countries have not confirmed the occurrence of Ae. albopictus. The large distribution and high frequency of Ae. albopictus in the Americas may become a critical factor in the spread of arboviruses like chikungunya in the new world.