No evidence of Zika, dengue, or chikungunya virus infection in field-caught mosquitoes from the Recife Metropolitan Region, Brazil, 2015.

Background: The Recife Metropolitan Region (RMR), north-eastern Brazil, was the epicentre of the 2015 Zika virus (ZIKV) epidemic, which was followed by a 2016 chikungunya virus (CHIKV) epidemic. It historically has amongst the highest incidence of dengue virus (DENV) infections and is the only remaining focus of lymphatic filariasis (LF) in Brazil. In early 2015, a molecular xenomonitoring surveillance project focused on Culex (Cx.) quinquefasciatus commenced to inform LF elimination activities. Aedes (Ae.) aegypti mosquitoes were also collected, concurrent with the first microcephaly cases detected in the RMR. In terms of the 2015 ZIKV epidemic, these are the earliest known field-collected mosquitoes, preserved for potential RNA virus detection, when ZIKV was known to be circulating locally. Methods:   Adult mosquitoes were collected in two sites (0.4 km 2) of Sítio Novo, Olinda, RMR, from July 22 to August 21, 2015. Mosquitoes were morphologically identified, sorted by physiological status, and pooled (up to 10 mosquitoes per house per day or week). RNA was extracted, reverse transcribed and the cDNA tested by real-time PCR. Results: A total of 10,139 adult female Cx. quinquefasciatus and 939 adult female Ae. aegypti were captured. All female Ae. aegypti specimens were included within 156 pools and screened for ZIKV, DENV and CHIKV. In addition, a sub-set of 1,556 Cx. quinquefasciatus adult females in 182 pools were screened for ZIKV. No evidence of infection with any of the three arboviruses was found. Conclusions: The absence of arbovirus detection may have been expected given the extremely restricted geographic area and collection of mosquitoes during a very short time period of peak mosquito abundance (July-September), but low arbovirus circulation (November-March).  However, this study demonstrates the potential to retrospectively screen for additional unexpected pathogens in situations of rapid emergence, such as occurred during the outbreak of ZIKV in the RMR

Development of an urban molecular xenomonitoring system for lymphatic filariasis in the Recife Metropolitan Region, Brazil.

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

Results of a Zika Virus (ZIKV) Immunoglobulin M-Specific Diagnostic Assay Are Highly Correlated With Detection of Neutralizing Anti-ZIKV Antibodies in Neonates With Congenital Disease.

BACKGROUND:  Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates. METHODS:  We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated. RESULTS:  Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P < .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P < .0001). CONCLUSIONS:  Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs in equivocal cases

Association between Zika virus infection and microcephaly in Brazil, January to May, 2016: preliminary report of a case-control study.

BACKGROUND: The microcephaly epidemic, which started in Brazil in 2015, was declared a Public Health Emergency of International Concern by WHO in 2016. We report the preliminary results of a case-control study investigating the association between microcephaly and Zika virus infection during pregnancy. METHODS: We did this case-control study in eight public hospitals in Recife, Brazil. Cases were neonates with microcephaly. Two controls (neonates without microcephaly), matched by expected date of delivery and area of residence, were selected for each case. Serum samples of cases and controls and cerebrospinal fluid samples of cases were tested for Zika virus-specific IgM and by quantitative RT-PCR. Laboratory-confirmed Zika virus infection during pregnancy was defined as detection of Zika virus-specific IgM or a positive RT-PCR result in neonates. Maternal serum samples were tested by plaque reduction neutralisation assay for Zika virus and dengue virus. We estimated crude odds ratios (ORs) and 95% CIs using a median unbiased estimator for binary data in an unconditional logistic regression model. We estimated ORs separately for cases with and without radiological evidence of brain abnormalities. FINDINGS: Between Jan 15, 2016, and May 2, 2016, we prospectively recruited 32 cases and 62 controls. 24 (80%) of 30 mothers of cases had Zika virus infection compared with 39 (64%) of 61 mothers of controls (p=0·12). 13 (41%) of 32 cases and none of 62 controls had laboratory-confirmed Zika virus infection; crude overall OR 55·5 (95% CI 8·6-∞); OR 113·3 (95% CI 14·5-∞) for seven cases with brain abnormalities; and OR 24·7 (95% CI 2·9-∞) for four cases without brain abnormalities. INTERPRETATION: Our data suggest that the microcephaly epidemic is a result of congenital Zika virus infection. We await further data from this ongoing study to assess other potential risk factors and to confirm the strength of association in a larger sample size. FUNDING: Brazilian Ministry of Health, Pan American Health Organization, and Enhancing Research Activity in Epidemic Situations

Zika virus infection in pregnancy: Establishing a case definition for clinical research on pregnant women with rash in an active transmission setting.

Defining cases of Zika virus (ZIKV) infection is a critical challenge for epidemiological research. Due to ZIKV's overlapping clinical features and potential immunologic cross-reactivity with other flaviviruses and the current lack of an optimal ZIKV-specific diagnostic assay, varying approaches for identifying ZIKV infections have been employed to date. This paper presents the laboratory results and diagnostic criteria developed by the Microcephaly Epidemic Research Group for defining cases of maternal ZIKV infection in a cohort of pregnant women with rash (N = 694) recruited during the declining 2015-2017 epidemic in northeast Brazil. For this investigation, we tested maternal sera for ZIKV by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Immunoglobulin (Ig) M and IgG3 enzyme-linked immunosorbent assays (ELISAs), and Plaque Reduction Neutralization Test (PRNT50). Overall, 23.8% of participants tested positive by qRT-PCR during pregnancy (range of detection: 0-72 days after rash onset). However, the inter-assay concordance was lower than expected. Among women with qRT-PCR-confirmed ZIKV and further testing, only 10.1% had positive IgM tests within 90 days of rash, and only 48.5% had ZIKV-specific PRNT50 titers ≥20 within 1 year of rash. Given the complexity of these data, we convened a panel of experts to propose an algorithm for identifying ZIKV infections in pregnancy based on all available lines of evidence. When the diagnostic algorithm was applied to the cohort, 26.9% of participants were classified as having robust evidence of a ZIKV infection during pregnancy, 4.0% as having moderate evidence, 13.3% as having limited evidence of a ZIKV infection but with uncertain timing, and 19.5% as having evidence of an unspecified flavivirus infection before or during pregnancy. Our findings suggest that integrating longitudinal data from nucleic acid and serologic testing may enhance diagnostic sensitivity and underscore the need for an on-going dialogue regarding the optimization of strategies for defining cases of ZIKV in research

SARS-CoV-2 Viremia is Associated with COVID-19 Severity and Predicts Clinical Outcomes

Background: SARS-CoV-2 viral RNA (vRNA) is detected in the bloodstream of some patients with COVID-19 (“RNAemia”) but it is not clear whether this RNAemia reflects viremia (i.e., virus particles) and how RNAemia/viremia is related to host immune responses and outcomes. Methods: SARS-CoV-2 vRNA was quantified by ultra-sensitive RT-PCR in plasma samples (0.5-1.0 ml) from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-ICU), and 23 ICU patients, and vRNA levels compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in pelleted plasma. Results: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6% and 11.1% of ICU, non-ICU, and outpatients respectively. Virions were detected in plasma pellets by electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (p6,000 copies/ml was strongly associated with mortality (HR: 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (p<0.01) but not with plasma neutralizing antibody titers (p=0.8). Conclusions: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia quantified by ultrasensitive RT-PCR correlate strongly with disease severity, patient outcome and specific inflammatory biomarkers but not neutralizing antibody titers

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

Zika-related adverse outcomes in a cohort of pregnant women with rash in Pernambuco, Brazil.

BACKGROUND: While Zika virus (ZIKV) is now widely recognized as a teratogen, the frequency and full spectrum of adverse outcomes of congenital ZIKV infection remains incompletely understood. METHODS: Participants in the MERG cohort of pregnant women with rash, recruited from the surveillance system from December/2015-June/2017. Exposure definition was based on a combination of longitudinal data from molecular, serologic (IgM and IgG3) and plaque reduction neutralization tests for ZIKV. Children were evaluated by a team of clinical specialists and by transfontanelle ultrasound and were classified as having microcephaly and/or other signs/symptoms consistent with congenital Zika syndrome (CZS). Risks of adverse outcomes were quantified according to the relative evidence of a ZIKV infection in pregnancy. FINDINGS: 376 women had confirmed and suspected exposure to ZIKV. Among evaluable children born to these mothers, 20% presented with an adverse outcome compatible with exposure to ZIKV during pregnancy. The absolute risk of microcephaly was 2.9% (11/376), of calcifications and/or ventriculomegaly was 7.2% (13/180), of additional neurologic alterations was 5.3% (13/245), of ophthalmologic abnormalities was 7% (15/214), and of dysphagia was 1.8% (4/226). Less than 1% of the children experienced abnormalities across all of the domains simultaneously. Interpretation: Although approximately one-fifth of children with confirmed and suspected exposure to ZIKV in pregnancy presented with at least one abnormality compatible with CZS, the manifestations presented more frequently in isolation than in combination. Due to the rare nature of some outcomes and the possibility of later manifestations, large scale individual participant data meta-analysis and the long-term evaluation of children are imperative to identify the full spectrum of this syndrome and to plan actions to reduce damages

Placental transfer and kineticis of maternal transferred dengue-specific antibodies in a cohort of children during the first year.

Submitted by Stephany Silva ([email protected]) on 2016-09-23T14:03:16Z No. of bitstreams: 1 2016castanha-pms.pdf: 1456214 bytes, checksum: 8fc7b4fe1efd0a8f729a4d6d9904da42 (MD5)Approved for entry into archive by Adagilson Silva ([email protected]) on 2016-09-23T19:45:38Z (GMT) No. of bitstreams: 1 2016castanha-pms.pdf: 1456214 bytes, checksum: 8fc7b4fe1efd0a8f729a4d6d9904da42 (MD5)Made available in DSpace on 2016-09-23T19:45:38Z (GMT). No. of bitstreams: 1 2016castanha-pms.pdf: 1456214 bytes, checksum: 8fc7b4fe1efd0a8f729a4d6d9904da42 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilAnticorpos antidengue materno transferidos têm sido implicados na imunopatogênese da dengue grave em lactentes. Postula-se que esses anticorpos possuam um papel distinto durante os primeiros anos de vida: ao nascimento, anticorpos antidengue materno adquiridos conferem proteção, e em seguida declinam a níveis subneutralizantes capazes de facilitar a infecção pelo vírus dengue (DENV) mediante o mecanismo de ADE (Antibody dependent enhancement), aumentando o risco de ocorrência das formas graves da dengue. Estudos prospectivos conduzidos em lactentes Asiáticos têm mostrado que o pico de anticorpos materno adquiridos com a capacidade de mediar aumento da infecção pelo DENV ocorre entre o sexto e o nono mês de vida, o que correlaciona com a epidemiologia da dengue grave em lactentes dessa região. Esta tese descreve o perfil de imunidade materna antidengue e a transferência placentária de anticorpos dengue-específicos em pares mãe-cordão recrutados em um estudo de coorte prospectivo conduzido na cidade do Recife, Nordeste do Brasil. Adicionalmente, esse trabalho analisa o papel dos níveis de IgG total maternos e da imunidade antidengue materna na transferência de anticorpos ao feto. Na coorte de lactentes, a tese avalia a cinética de declínio dos anticorpos antidengue materno-transferidos e sua capacidade de mediar ADE durante os primeiros anos de vida. Níveis de IgG DENV-especifico e de anticorpos neutralizantes sorotipo específicos (DENV1-4) foram determinados em 376 pares mãe-cordão. A cinética de anticorpos materno transferidos neutralizantes e/ou mediadores de ADE foi investigada em uma subamostra das crianças inseridas na coorte. A maior parte das gestantes apresentava imunidade ao sorotipo DENV-3 (53,7 por cento) ou a combinação DENV-3/ DENV-4 (30,6 por cento). Níveis de IgG DENV-específico e de anticorpos neutralizantes para os sorotipos DENV-3 e DENV-4 foram significativamente maiores nas amostras do cordão umbilical do que nas respectivas amostras maternas. Mães imunes a apenas um sorotipo do vírus transferiram maiores níveis de IgG DENV-especifico (p=0,02) e de anticorpos neutralizantes para o DENV-3 (p=0,04) quando comparado a mães imunes a múltiplos sorotipos do DENV. Níveis de anticorpos materno transferidos para o DENV-3 foram indetectáveis em 90 por cento das crianças aos 4 meses de idade. O pico de atividade ADE foi detectado no segundo mês de vida (p0,0001) e rapidamente declinou no quarto mês de vida (p=0,0035). Diferente do observado em lactentes asiáticas, o pico de atividade ADE mediado pelos anticorpos maternos transferidos ocorreu mais nos lactentes Brasileiros. Esses resultados possuem importantes implicações para a imunopatogênese da dengue durante a infância e para o estabelecimento de futuros esquemas vacinais antidengue nos primeiros anos de vida (AU