Sinval Pinto Brandão Filho

Natural Leishmania infantum infection in Migonemyia migonei (França, 1920) (Diptera:Psychodidae:Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil

A study of the natural infection of phlebotomine sand flies by Leishmania (Leishmania) infantum was conducted in an area of visceral leishmaniasis in São Vicente Férrer, located in the northern part of the Atlantic rain forest region in the State of Pernambuco, Brazil. In a previous study, Migonemyia migonei have been found predominantly in peridomiciles and houses in this endemic area. The analysis of M. migonei, collected by CDC light trap, by multiplex PCR assay coupled to non-isotopic hybridization showed that 2 females out of 50 were infected by L. infantum. This is the first finding of natural infection of M. migonei by L. infantum suggesting that M. migonei may be the vector of L. infantum in areas of visceral leishmaniasis where Lutzomyia longipalpis, the usual vector, is absent.

Characterization of Leishmania spp. causing cutaneous leishmaniasis in Manaus, Amazonas, Brazil

In the State of Amazonas, American tegumentary leishmaniasis is endemic and presents a wide spectrum of clinical variability due to the large diversity of circulating species in the region. Isolates from patients in Manaus and its metropolitan region were characterized using monoclonal antibodies and isoenzymes belonging to four species of the parasite: Leishmania (Viannia) guyanensis, 73% (153/209); Leishmania (Viannia) braziliensis, 14% (30/209); Leishmania (Leishmania) amazonensis, 8% (17/209); and Leishmania (Viannia) naiffii, 4% (9/209). The most prevalent species was L. (V.) guyanensis. The principal finding of this study was the important quantity of infections involving more than one parasite species, representing 14% (29/209) of the total. The findings obtained in this work regarding the parasite are further highlighted by the fact that these isolates were obtained from clinical samples collected from single lesions.

Association between microcephaly, Zika virus infection, and other risk factors in Brazil: final report of a case-control study

BACKGROUND A Zika virus epidemic emerged in northeast Brazil in 2015 and was followed by a striking increase in congenital microcephaly cases, triggering a declaration of an international public health emergency. This is the final report of the first case-control study evaluating the potential causes of microcephaly: congenital Zika virus infection, vaccines, and larvicides. The published preliminary report suggested a strong association between microcephaly and congenital Zika virus infection. METHODS We did a case-control study in eight public maternity hospitals in Recife, Brazil. Cases were neonates born with microcephaly, defined as a head circumference of 2 SD below the mean. Two controls without microcephaly were matched to each case by expected date of delivery and area of residence. We tested the serum of cases and controls and the CSF of cases for detection of Zika virus genomes with quantitative RT-PCR and for detection of IgM antibodies with capture-IgM ELISA. We also tested maternal serum with plaque reduction neutralisation assays for Zika and dengue viruses. We estimated matched crude and adjusted odds ratios with exact conditional logistic regression to determine the association between microcephaly and Zika virus infection. FINDINGS We screened neonates born between Jan 15 and Nov 30, 2016, and prospectively recruited 91 cases and 173 controls. In 32 (35%) cases, congenital Zika virus infection was confirmed by laboratory tests and no controls had confirmed Zika virus infections. 69 (83%) of 83 cases with known birthweight were small for gestational age, compared with eight (5%) of 173 controls. The overall matched odds ratio was 73·1 (95% CI 13·0-∞) for microcephaly and Zika virus infection after adjustments. Neither vaccination during pregnancy or use of the larvicide pyriproxyfen was associated with microcephaly. Results of laboratory tests for Zika virus and brain imaging results were available for 79 (87%) cases; within these cases, ten were positive for Zika virus and had cerebral abnormalities, 13 were positive for Zika infection but had no cerebral abnormalities, and 11 were negative for Zika virus but had cerebral abnormalities. INTERPRETATION The association between microcephaly and congenital Zika virus infection was confirmed. We provide evidence of the absence of an effect of other potential factors, such as exposure to pyriproxyfen or vaccines (tetanus, diphtheria, and acellular pertussis, measles and rubella, or measles, mumps, and rubella) during pregnancy, confirming the findings of an ecological study of pyriproxyfen in Pernambuco and previous studies on the safety of Tdap vaccine administration during pregnancy. FUNDING Brazilian Ministry of Health, Pan American Health Organization, and Enhancing Research Activity in Epidemic Situations.

First International Workshop on Zika Virus Held by Oswaldo Cruz Foundation FIOCRUZ in Northeast Brazil March 2016 – A Meeting Report

In the first two days of March 2016, an International Workshop on Zika Virus was held in the city of Recife, Brazil, hosted by the Oswaldo Cruz Foundation (Fiocruz). The workshop took place at the Research Institute Aggeu Magalhaes (IAM), the state of Pernambuco research branch of Fiocruz, responsible for hosting the meeting and its organization. Representatives from several institutions working on Zika virus science attended, including researchers and academics from several other Fiocruz research entities (based in Rio de Janeiro, Belo Horizonte and Bahia), from major Brazilian Universities (University of Sao Paulo, Federal University of Rio de Janeiro and others) and from other Brazilian research centers (such as the Evandro Chagas Institute—IEC). Foreign participants included representatives from the National Institute of Allergy and Infectious Diseases NIAID-USA and the University of Glasgow, among others. The Workshop brought together more than 600 participants and was accessible, live, through the web (over four thousand accessions from 26 countries). The purpose of the meeting was to provide an updated view on Zika virus in Brazil and to discuss recent advances and challenges on its research. With the recent Zika virus outbreak in Northeast Brazil and especially the remarkable increase in newborns suffering a congenital disorder termed microcephaly—a rare neurological condition in which an infants head is significantly smaller than the heads of other children of the same age—recently linked to Zika virus infection during pregnancy [1–5], the Brazilian public health authorities declared a National Public Health Emergency on Nov 11, 2015. This was followed by the recognition by the World Health Organization—WHO, on February 1, 2016, of the cluster of microcephaly cases and other neurological disorders as a health emergency, declaring a Global Emergency and the spread of the Zika virus an “extraordinary event” with a public health threat to other parts of the world (source: http://www.who.int/emergencies/zika-virus/en/). During the meeting, special sessions were organized by experts in the following research fields: Arboviruses, Zika and vaccines–discussed data regarding different circulating arthropod borne viruses in Brazil (such as chikungunya and dengue), new data about Zika virus biology, in vitro models of infection and also strategies for vaccine development; Biology of the virus vector interaction–focused on new findings on virus vector studies, including an evaluation of potential vectors for Zika virus and countermeasures to mosquito control; Clinical–with the intention of discussing the last clinical findings on Zika virus infection and major birth congenital malformations; Diagnosis—plotted new strategies to improve laboratory tests, development of technological products and also a historical update of Zika virus identification in Brazil; Epidemiology—this session was designed to provide an update on the clinical epidemiological studies under development in Brazil, with a presentation of the data on a case-control investigation of microcephaly cases and cohort studies. The first day the meeting opened with a talk from the IEC team on Zika virus infection and the global challenge to control its spread. Subsequent talks covered the clinical manifestations presented by Zika infected individuals, from a medical perspective. Medical doctors from Northeast Brazil reported that the major clinical findings on microcephaly confirmed cases were calcifications (approximately 98%), ventriculomegalia and disorder of cortical development, including simplification of gyrus and lissencephaly with a lower proportion of other abnormalities such as pachygyria. Official data from the Brazilian Ministry of Health confirmed that currently Zika virus is circulating in 22 of the 27 states in Brazil, with two confirmed deaths (Fig 1). By March 1, a total of 5.909 cases of microcephaly were reported and are currently under investigation. From 641 confirmed microcephaly cases, the number of newborn deaths reached 139 (stillbirths and/or newborn deaths) and from the total number of microcephaly cases investigated, 82 were confirmed to be Zika by laboratory tests (RT-qPCR and/or serology). Most of the cases were reported from the state of Pernambuco (1.672 cases) (source: Brazilian Ministry of Health). Fig 1 Brazilian federative units with Zika documented autochthonous cases (triangles) and number of microcephaly confirmed cases in each federal state (color shading of the affected Brazilian states) (source: Brazilian Ministry of Health and Secretary of Vigilance ... On the Diagnosis panel, the discussions were mainly on the development of new laboratory tools. Given the extensive cross reactivity with other circulating arboviruses in Brazil, the lack of reliable and specific serologic tests for Zika virus represents a major challenge. Currently, only two serological tests are licensed for Zika virus diagnosis in Brazil (Anti-Zika Virus ELISA IgG/IgM and IIFT Arboviral Fever Mosaic 2 IgG/IgM, both from EUROIMMUN). In addition to serological tests, Fiocruz is providing a recently licensed multiplex Dengue/Zika and Chikungunya assay based on NAT technology (nucleic acid test or nucleic acid amplification test). Discussions about development and characterization of monoclonal antibodies and recombinant proteins, as biotechnological products to be applied on different platforms for Zika virus diagnostic, occurred during the event, however no new kits are being licensed at the moment, which could be explained, in part, by the lack of reliable serum panels for kits validation. Researchers from IAM investigating the microcephaly cases reported the detection of Zika virus specific IgM in roughly 90% of the samples of cerebrospinal fluid (CSF) tested from microcephalic newborns, which strongly correlates Zika virus infection with brain malformation. The team from the Oswaldo Cruz Institute (IOC—a Fiocruz branch from Rio de Janeiro) provided results on Zika virus identification from several body fluids such as urine, semen (up to 2 months after symptoms), saliva and CSF. The data showed that Zika virus found in unusual samples (urine, saliva, semen) can persist for longer time periods than in the blood. So far, however, only sexual, perinatal and vector transmissions have been documented for Zika virus. Therefore, is concerning the fact that Zika virus detection on semen for long periods after the initial infection has serious implications on sexual transmission. Although organ transplantation and blood transfusion are probable transmission routes, there are no reports yet in the literature [6–11]. Despite the fact that new Zika candidates vaccines may not enter clinical trials until the next 2 or 3 years, scientific efforts to achieve a safe and efficient Zika vaccine are crucial to counteract this new emerged viral disease. Vaccine development is still very preliminary and to the moment only pre clinical studies are in course. On vaccine discussion topic, scientists presented different strategies for vaccine development, based on several immunization platforms, such as viral chimeras using the vaccinal Yellow Fever 17D strain, virus attenuation by reverse genetics (infectious clones) and subunit vaccines (DNA and/or recombinant proteins). Mosquitoes from the genus Aedes (Ae. albopictus and Ae. Aegypti) are the main species responsible for Zika virus transmission in Brazil [12,13]. During the Biology of the Virus Vector Interaction session, researchers from IAM reviewed the evidence regarding Zika virus transmission and presented results derived from laboratory experiments that implicate other mosquito species as potential vectors involved in the Zika virus life cycle. Using mosquitos artificially fed with Zika virus infected blood, they were able to detect the virus in the salivary glands of Culex quinquefasciatus 7 and 15 days post-feeding, confirming a high infection rate of 100% and 67%, respectively. In addition, advances and new approaches for mosquito control were presented in the session and challenging aspects for the implementation of control measures discussed, taking into account the socio-economical and environmental conditions of urban areas in Brazil. Since its introduction in Brazil, Zika virus infection has spread rapidly through the tropical Americas and we hypothesize that sequential flaviviruses exposures can represent a complicating factor for its spread, especially in Brazil where rates for prior dengue infection are extremely high. Currently, a number of independent groups are developing projects on Zika virus investigation in Brazil. Recently, Fiocruz (the largest, health related, research institute in Latin America, with a total of 18 units scattered throughout different regions in Brazil) established a National Network Zika Committee through the integration of efforts carried out by several of its research branches. These efforts are focusing on the development of better diagnostic tools, vaccine research, virus genetic studies, immunopathologic studies on Zika patients and research on vector populations and control. Currently, Zika virus is still expanding within the Brazilian territory and in other countries from Central and South America, probably due to the availability/abundance of vectors and the challenges in achieving efficient levels of vector reduction with a consequent impact on virus transmission. It is currently very difficult to determine how and to where will the virus spread over time. Assumptions by field specialists testify that when herd immunity reaches a level of ~80%, Zika virus transmission could then drop, however this is only speculative and based on previous data from recent outbreaks of dengue and chikungunya [14]. Serious funding and science investments are thus critical in order to face this newly emerged disease.

Phlebotomine sandflies (Diptera: Psychodidae) in São Vicente Férrer, a sympatric area to cutaneous and visceral leishmaniasis in the state of Pernambuco, Brazil.

INTRODUCTION In the last decades, a considerable geographic expansion of the leishmaniases in all regions of Brazil has been observed. The present study was carried out to identify the composition of the phlebotomine sandfly fauna and verify the seasonal variation of the main species after environmental changes occurred in São Vicente Férrer Municipality, State of Pernambuco, Brazil. METHODS Captures were carried out during four consecutive nights of each month using Centers for Disease Control and Prevention light traps from September 2009 to September 2010. The correlation between the number of phlebotomine sandflies captured and climatic factors (temperature and rainfall) was evaluated. RESULTS A total of 13,872 specimens belonging to 20 species were captured, of which, 6,247 (45%) were females, and 7,625 (55%) were males. Lutzomyia migonei was the most abundant species with 9,964 (71.8%) specimens, being predominant in the intradomicile and peridomicile areas with 108 (86.4%) and 9,746 (97%), respectively. In the forest remnants, Lutzomyia complexa 2,395 (65%) and Lutzomyia sordellii 770 (20.8%) predominated. The correlation analysis between the total number of sandflies captured and climatic factors did not show a significant influence on population density. CONCLUSIONS The high abundance of Lutzomyia migonei and Lutzomyia complexa indicates the possibility of new cases of cutaneous leishmaniasis (CL).

Sandfly fauna in an area endemic for visceral leishmaniasis in Aracaju, State of Sergipe, Northeast Brazil

INTRODUCTION In recent years, visceral leishmaniasis, a major public health problem, has been spreading from the rural to urban areas in many areas of Brazil, including Aracaju, the capital of the State of Sergipe. However, there are no studies of the sandfly fauna in this municipality or its variation over the year. METHODS Phlebotomine sandflies were collected from a rural area of Aracaju from September 2007 to July 2009. Modified CDC ultra-violet (UV) light traps were used to evaluate sandfly monthly distribution and their presence in the domestic and peridomestic environments. RESULTS The most abundant species was Lutzomyia longipalpis (90.4%) followed by Evandromyia lenti (9.6%). A chicken shed trap site had the highest proportion of L. longipalpis (51.1%) and large numbers of L. longipalpis were also collected in the houses closest to the chicken shed. There was a positive correlation between monthly rainfall and L. longipalpis abundance. CONCLUSIONS Lutzomyia longipalpis is the most abundant species and is probably the main vector of the visceral leishmaniasis agent in the rural area of Aracaju. An increase in L. longipalpis frequency was observed during the rainy season. The peridomicile-intradomicile observations corroborate the importance of chicken sheds for the presence of L. longipalpis in the peridomestic environment. The great numbers of L. longipalpis inside the houses confirm the endophilic behaviour of this species and the possibility of visceral transmission in the intradomicile.

Molecular detection of Leishmania in phlebotomine sand flies in a cutaneous and visceral leishmaniasis endemic area in northeastern Brazil

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

Bilateral anomaly in Evandromyia evandroi (Diptera: Psychodidae: Phlebotominae) captured in Vicência municipality, Northern Rainforest region of Pernambuco State, Brazil.

Abstract Approximately 800 species of phlebotomine sand flies, many of which are vectors of Leishmania, have been described. Besides morphological similarities within groups, the occurrence of anomalies within a species may lead to an erroneous description of new species. This paper describes one phlebotomine sand fly, Evandromyia evandroi, with a symmetrical bilateral anomaly in the number of spines on the gonostyle. In this specimen, the anomalous spine is located in the external region of gonostyle, inserted between the upper external and the lower external spines. It is important to document morphological anomalies, so as to avoid erroneous sand fly identifications.

First Internal Workshop of the Reference Services at Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz

1. Departamento de Parasitologia Centro de Pesquisas Aggeu Magalhaes, Fundacao Oswaldo Cruz, Recife, PE. 2. Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhaes, Fundacao Oswaldo Cruz, Recife, PE. 3. Departamento de Entomologia, Centro de Pesquisas Aggeu Magalhaes, Fundacao Oswaldo Cruz, Recife, PE. 4. Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhaes, Fundacao Oswaldo Cruz, Recife, PE. 5. Servico de Referencia Nacional em Filarioses, Recife, PE. 6. Servico de Referencia em Esquistossomose, Recife, PE. 7. Servico de Referencia em Leishmaniose, Recife, PE. 8. Servico de Referencia em Culicideos Vetores. Recife, PE. 9. Servico de Referencia Nacional em Peste, Recife, PE. 10. Servico de Referencia em Doencas de Chagas, Recife, PE.

Lutzomyia naftalekatzi, a new species of phlebotomine sand fly (Diptera: Psychodidae) from Zona da Mata region, Pernambuco, Brazil.

Lutzomyia naftalekatzi, a new phlebotomine sand fly belonging to the L. aragaoi species group, is described from specimens collected in Zona da Mata region of Pernambuco, Brazil.