Monitoramento das Produções

by Dipu T. Sathyapalan, Athira Padmanabhan, Merlin Moni, Binny P-Prabhu, Preetha Prasanna, Sabarish Balachandran, Sreekrishnan P. Trikkur, Soumya Jose, Fabia Edathadathil, Jagan O. Anilkumar, Rekha Jayaprasad, Gireeshkumar Koramparambil, Ravindra C. Kamath, Veena Menon, Vidya Menon

Severe thrombocytopenia in dengue often prompts platelet transfusion primarily to reduce bleeding risk. In India, about 11–43% of dengue patients report receiving platelet transfusions which is considered scarce and expensive especially in resource limited settings. Herein, we evaluated the efficacy and safety of Carica papaya leaf extract (CPLE) in the management of severe thrombocytopenia (≤30,000/μL) in dengue infection. 51 laboratory confirmed adult dengue patients with platelet counts ≤30,000/μL were randomly assigned to either treatment (n = 26) or placebo (n = 24) group. By day 3, CPLE treated patients reported significantly (p = 0.007) increased platelet counts (482%± 284) compared to placebo (331%±370) group. In the treatment group, fewer patients received platelet transfusions (1/26 v/s 2/24) and their median time for platelets to recover to ≥ 50,000/μL was 2 days (IQR 2–3) compared to 3 days (IQR 2–4) in placebo. Overall, CPLE was safe and well tolerated with no significant decrease in mean hospitalization days. Plasma cytokine profiling revealed that by day 3, mean percent increase in TNFα and IFNγ levels in treatment group was less compared to that observed in placebos; (TNFα: 58.6% v/s 127.5%; p = 0.25 and IFNγ: 1.93% v/s 62.6% for; p = 0.12). While a mean percent increase in IL-6 levels occurred in placebos (15.92%±29.93%) by day 3, a decrease was noted in CPLE group (12.95%±21.75%; p = 0.0232). Inversely, CPLE treated patients reported a mean percent increase compared to placebo by day 3 (143% ±115.7% v/s 12.03%± 48.4%; p = 0.006). Further, by day 3, a faster clearance kinetics of viral NS1 antigenemia occurred–mean NS1 titers in treatment group decreased to 97.3% compared to 88% in placebos (p = 0.023). This study demonstrates safety and efficacy of CPLE in increasing platelet counts in severe thrombocytopenia in dengue infections. A possible immunomodulatory and antiviral activity may be attributed to CPLE treatment. These findings merit validation in larger prospective studies.
Trial registration
Name of the registry: Clinical Trials Registry—India (CTRI)
Registration No.: CTRI-REF/2017/02/013314

by Izaskun Alejo-Cancho, Jessica Navero-Castillejos, Aida Peiró-Mestres, Rosa Albarracín, Josep Barrachina, Alexander Navarro, Verónica Gonzalo, Víctor Pastor, José Muñoz, Miguel J. Martínez
Background Dengue virus (DENV) is the most important arbovirus worldwide, causing infections in endemic countries and returning travellers from these areas. Rapid diagnostic tests are needed to improve patient management and monitor local transmission. The detection of DENV non-structural protein 1 (NS1) is a useful tool for the diagnosis, but the currently available methods can be time consuming or lack sensitivity. The objective of our study was to evaluate a new rapid and semi-quantitative microfluidic DENV NS1 immuno-magnetic agglutination assay based on aggregation of magnetic nanoparticles detected by an electronic reader (Virotrack Dengue Acute and Blubox, Blusense diagnostics, Copenhagen, Denmark). Methodology/Principal findings A panel of 135 serum samples from travelers returning from dengue endemic countries was analyzed (74 DENV positive samples including the four DENV serotypes, 26 Zika virus positive samples, 25 chikungunya virus positive samples, 5 malaria positive samples and 5 negative samples). Samples were tested by three different antigen detection methods: SD Dengue NS1 Ag ELISA, SD BIOLINE Dengue Duo and ViroTrack Dengue Acute. The sensitivity observed for SD Dengue NS1 Ag ELISA, ViroTrack Dengue Acute and SD BIOLINE Dengue Duo was 97.2%, 91.1% and 68.1%, respectively. All methods showed high specificity (98.4% for ViroTrack Dengue Acute and 100% for both SD Dengue NS1 Ag ELISA and SD BIOLINE Dengue Duo). SD Dengue NS1 Ag ELISA and ViroTrack Dengue Acute only failed to detect samples positive for DENV-2. Conclusions/Significance ViroTrack Dengue Acute is a sensitive and specific assay for DENV NS1 detection. It provides faster results than the ELISA method and a better performance than the rapid immunochromatographic tests. ViroTrack Dengue Acute could represent a valuable tool for rapid diagnosis of DENV infections in returning travellers from endemic countries.

by Rachel Sippy, Daniel F. Farrell, Daniel A. Lichtenstein, Ryan Nightingale, Megan A. Harris, Joseph Toth, Paris Hantztidiamantis, Nicholas Usher, Cinthya Cueva Aponte, Julio Barzallo Aguilar, Anthony Puthumana, Christina D. Lupone, Timothy Endy, Sadie J. Ryan, Anna M. Stewart Ibarra
Background Dengue, chikungunya, and Zika are arboviruses of major global health concern. Decisions regarding the clinical management of suspected arboviral infection are challenging in resource-limited settings, particularly when deciding on patient hospitalization. The objective of this study was to determine if hospitalization of individuals with suspected arboviral infections could be predicted using subject intake data. Methodology/Principal findings Two prediction models were developed using data from a surveillance study in Machala, a city in southern coastal Ecuador with a high burden of arboviral infections. Data were obtained from subjects who presented at sentinel medical centers with suspected arboviral infection (November 2013 to September 2017). The first prediction model—called the Severity Index for Suspected Arbovirus (SISA)—used only demographic and symptom data. The second prediction model—called the Severity Index for Suspected Arbovirus with Laboratory (SISAL)—incorporated laboratory data. These models were selected by comparing the prediction ability of seven machine learning algorithms; the area under the receiver operating characteristic curve from the prediction of a test dataset was used to select the final algorithm for each model. After eliminating those with missing data, the SISA dataset had 534 subjects, and the SISAL dataset had 98 subjects. For SISA, the best prediction algorithm was the generalized boosting model, with an AUC of 0.91. For SISAL, the best prediction algorithm was the elastic net with an AUC of 0.94. A sensitivity analysis revealed that SISA and SISAL are not directly comparable to one another. Conclusions/Significance Both SISA and SISAL were able to predict arbovirus hospitalization with a high degree of accuracy in our dataset. These algorithms will need to be tested and validated on new data from future patients. Machine learning is a powerful prediction tool and provides an excellent option for new management tools and clinical assessment of arboviral infection.

by Shengxue Luo, Wei Zhao, Xiaorui Ma, Panli Zhang, Bochao Liu, Ling Zhang, Wenjing Wang, Yuanzhan Wang, Yongshui Fu, Jean-Pierre Allain, Tingting Li, Chengyao Li

Zika virus (ZIKV) has spread in many countries or territories causing severe neurologic complications with potential fatal outcomes. The small primate common marmosets are susceptible to ZIKV, mimicking key features of human infection. Here, a novel simian adenovirus type 23 vector-based vaccine expressing ZIKV pre-membrane-envelope proteins (Sad23L-prM-E) was produced in high infectious titer. Due to determination of immunogenicity in mice, a single-dose of 3×108 PFU Sad23L-prM-E vaccine was intramuscularly inoculated to marmosets. This vaccine raised antibody titers of 104.07 E-specific and 103.13 neutralizing antibody (NAb), as well as robust specific IFN-γ secreting T-cell response (1,219 SFCs/106 cells) to E peptides. The vaccinated marmosets, upon challenge with a high dose of ZIKV (105 PFU) six weeks post prime immunization, reduced viremia by more than 100 folds, and the low level of detectable viral RNA (103.66) and T-cell response (>726 SFCs/106 PBMCs) were acquired 1–2 weeks post exposure to ZIKV, while non-vaccinated control marmosets developed long-term high titer of ZIKV (105.73 copies/ml) (P

Title: External control viral-like particle construction for detection of emergent Arboviruses by real-time reverse-transcription PCR
Authors: Borghetti, Ivo Alberto; Zambenedetti, Miriam Ribas; Requião, Luciana; Vieira, Deusilene Souza; Krieger, Marco Aurélio; Rampazzo, Rita de Cássia Pontello

by Krijn Paaijmans, Marco Brustolin, Carles Aranda, Roger Eritja, Sandra Talavera, Nonito Pagès, Silvie Huijben

by Stefan Malafa, Iris Medits, Judith H. Aberle, Stephan W. Aberle, Denise Haslwanter, Georgios Tsouchnikas, Silke Wölfel, Kristina L. Huber, Elena Percivalle, Pascal Cherpillod, Melissa Thaler, Lena Roßbacher, Michael Kundi, Franz X. Heinz, Karin Stiasny
Background Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals. Methodology Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments. Principal findings Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses. Conclusions Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.

Title: Vertical Transmission of Zika Virus (Flaviviridae, Flavivirus) in Amazonian Aedes aegypti (Diptera: Culicidae) Delays Egg Hatching and Larval Development of Progeny
Authors: Chaves, Barbara Aparecida; Vieira Junior, Ademir Bentes; Silveira, Karine Renata Dias; Paz, Andreia da Costa; Vaz, Evelyn Beatriz da Costa; Araujo, Raphaela Guedes Pereira; Rodrigues, Nilton Barnabe; Campolina, Thais Bonifacio; Orfano, Alessandra da Silva; Pimenta, Rafael Nacif; Villegas, Luis Eduardo Martinez; Melo, Fabrício Freire de; Silva, Breno de Mello; Monteiro, Wuelton Marcelo; Guerra, Maria das Graças Vale Barbosa; Lacerda, Marcus Vinicius Guimarães de; Norris, Douglas Eric; Secundino, Nagila Francinete Costa; Pimenta, Paulo Filemon Paolucci

by Juliane F. Oliveira, Moreno S. Rodrigues, Lacita M. Skalinski, Aline E. S. Santos, Larissa C. Costa, Luciana L. Cardim, Enny S. Paixão, Maria da Conceição N. Costa, Wanderson K. Oliveira, Maurício L. Barreto, Maria Glória Teixeira, Roberto F. S. Andrade

The co-circulation of different arboviruses in the same time and space poses a significant threat to public health given their rapid geographic dispersion and serious health, social, and economic impact. Therefore, it is crucial to have high quality of case registration to estimate the real impact of each arboviruses in the population. In this work, a Vector Autoregressive (VAR) model was developed to investigate the interrelationships between discarded and confirmed cases of dengue, chikungunya, and Zika in Brazil. We used data from the Brazilian National Notifiable Diseases Information System (SINAN) from 2010 to 2017. There were three peaks in the series of dengue notification in this period occurring in 2013, 2015 and in 2016. The series of reported cases of both Zika and chikungunya reached their peak in late 2015 and early 2016. The VAR model shows that the Zika series have a significant impact on the dengue series and vice versa, suggesting that several discarded and confirmed cases of dengue could actually have been cases of Zika. The model also suggests that the series of confirmed and discarded chikungunya cases are almost independent of the cases of Zika, however, affecting the series of dengue. In conclusion, co-circulation of arboviruses with similar symptoms could have lead to misdiagnosed diseases in the surveillance system. We argue that the routinely use of mathematical and statistical models in association with traditional symptom-surveillance could help to decrease such errors and to provide early indication of possible future outbreaks. These findings address the challenges regarding notification biases and shed new light on how to handle reported cases based only in clinical-epidemiological criteria when multiples arboviruses co-circulate in the same population.

by Elba Caraballo, B. Katherine Poole-Smith, Kay M. Tomashek, Brenda Torres-Velasquez, Luisa I. Alvarado, Olga D. Lorenzi, Carmen Ramos, Jessica Carrión, Elizabeth Hunsperger
Background Dengue is an important arboviral disease with about 100 million dengue cases per year, of which, ~5% result in severe disease. Clinical differentiation of dengue from other acute febrile illnesses (AFI) is difficult, and diagnostic blood tests are costly. We evaluated the utility of anti-DENV IgM in urine to identify dengue cases among AFI patients enrolled in a clinical study. Methods Between May 2012-March 2013, 1538 study participants with fever for ≤7 days were enrolled, a medical history was obtained, and serum and urine specimens were collected. Serum was tested for DENV RNA and anti-DENV IgM. Urine was tested for anti-DENV IgM, and its sensitivity and specificity to detect sera laboratory-positive dengue cases were calculated. We evaluated if urine anti-DENV IgM positivity early (≤5 days post-illness onset [DPO]) and late (6–14 DPO) in the clinical course was associated with dengue severity. Results Urine anti-DENV IgM sensitivity and specificity were 47.4% and 98.5%, respectively, when compared with serum anti-DENV IgM ELISA results, and 29.7% and 91.1% when compared with serum rRT-PCR results. There was no correlation between urine anti-DENV IgM positivity and patient sex or pre-existing chronic disease. Early in the clinical course, a significantly higher proportion of those who developed dengue with warning signs had anti-DENV IgM in their urine when compared to those without warning signs (20.4% vs. 4.3%). There was no difference in the proportion with urine anti-DENV IgM positivity between severity groups late in the clinical course. Conclusion While detection of urine anti-DENV IgM lacked adequate diagnostic sensitivity, it is a highly specific marker for laboratory-positive dengue, and its presence early in the clinical course may distinguish those with more severe disease. Further assessment of urine anti-DENV IgM by DPO is warranted to determine its utility as an early diagnostic (and possibly prognostic) marker for dengue.

Title: Avaliação de larvicidas experimentais à Base de Bacillus thuringiensis svar. israelensis, desenvolvidos no Estado de Pernambuco, para o controle de Aedes aegypti
Authors: Revorêdo, Amanda Maria Cabral
Abstract: Instituições brasileiras de pesquisa estão empenhadas no desenvolvimento biotecnológico de larvicidas à base de Bacillus thuringiensis israelenses (Bti), para o controle de Aedes aegypti. O objetivo deste estudo foi avaliar a atividade tóxica de caldos fermentados (CF), biomassas concentradas (BC), pó primário (PP) e duas formulações em grânulos dispersíveis em água (FMA e FMB), produzidas com a cepa IPS82, no Centro Acadêmico de Vitória/UFPE. Foram realizados em laboratório bioensaios in vivo com larvas (L4) de Ae. aegypti para estimar os valores das concentrações letais para 50% e 90% as larvas (CL50 e CL90). Para avaliar o desempenho dos formulados experimentais foram realizados testes sob condições simuladas de campo (TCS). A eficácia inicial foi mensurada pela mortalidade de L4, em recipientes-teste de 50 L, 24 horas após o tratamento e a persistência, pela adição semanal de L1 e contagem de pupas sobreviventes. Foram avaliadas 16 bateladas de produção com 32 amostras cujas CL50 variaram de 1,03 a 4,08 mg/L para os CF e de 0,34 a 0,84 mg/L para suas respectivas BC. Para três destas bateladas o princípio ativo (cristais proteicos e esporos), recuperado do CF por floculação, foi também submetido à secagem para obtenção do PP, para o qual foi observado um aumento da atividade biológica na ordem de  34 vezes, passando de 1,03 mg/L para 0,03 mg/L ao longo dos dois processos de concentração. Os formulados contendo 33% do PP, revelaram potências similares, 2.742 UTI/mg (FMA) e 2.965 UTI/mg (FMB). Nos recipientes-teste tratados com 3,4 mg/L dos formulados a eficácia inicial de controle foi de 100%, já a persistência, eliminando ≥ 80% das larvas, foi de 104 dias para o FMA e de 90 dias para o FMB. Os resultados revelaram a reprodutibilidade das condições de produção dos formulados e a similaridade entre a atividade larvicida residual de ambos, embora possuíssem substâncias adjuvantes diferentes, sugerindo o potencial de uso deles em criadouros de campo de Ae. aegypti.

by Perran A. Ross, Jason K. Axford, Qiong Yang, Kyran M. Staunton, Scott A. Ritchie, Kelly M. Richardson, Ary A. Hoffmann

Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia are being released into natural mosquito populations in the tropics as a way of reducing dengue transmission. High temperatures adversely affect wMel, reducing Wolbachia density and cytoplasmic incompatibility in some larval habitats that experience large temperature fluctuations. We monitored the impact of a 43.6°C heatwave on the wMel infection in a natural population in Cairns, Australia, where wMel was first released in 2011 and has persisted at a high frequency. Wolbachia infection frequencies in the month following the heatwave were reduced to 83% in larvae sampled directly from field habitats and 88% in eggs collected from ovitraps, but recovered to be near 100% four months later. Effects of the heatwave on wMel appeared to be stage-specific and delayed, with reduced frequencies and densities in field-collected larvae and adults reared from ovitraps but higher frequencies in field-collected adults. Laboratory experiments showed that the effects of heatwaves on cytoplasmic incompatibility and density are life stage-specific, with first instar larvae being the most vulnerable to temperature effects. Our results indicate that heatwaves in wMel-infected populations will have only temporary effects on Wolbachia frequencies and density once the infection has established in the population. Our results are relevant to ongoing releases of wMel-infected Ae. aegypti in several tropical countries.

Title: Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium
Authors: Mattos, Diogo A.; Silva, Marlon V.; Gaspar, Luciane P.; Castilho, Leda R.
Abstract: Neste trabalho, alterações nos métodos de cultivo de células Vero foram empregadas para melhorar as condições de crescimento celular, a fim de obter maiores densidades celulares viáveis e aumentar os títulos virais. A propagação do vírus da febre amarela 17DD (YFV) em células Vero cultivadas em microtransportadores Cytodex I foi avaliada em vasos de 3 L de biorreator. Antes das mudanças atuais, as células Vero exibiam repetidamente colonização insuficiente por microtransportador. Um processo de cultivo modificado com quatro alterações resultou em densidades celulares mais altas e títulos de vírus mais altos do que o observado anteriormente para o 17DD YFV.

by Jian Cheng, Hilary Bambrick, Laith Yakob, Gregor Devine, Francesca D. Frentiu, Do Thi Thanh Toan, Pham Quang Thai, Zhiwei Xu, Wenbiao Hu
Background Many studies have shown associations between rising temperatures, El Niño events and dengue incidence, but the effect of sustained periods of extreme high temperatures (i.e., heatwaves) on dengue outbreaks has not yet been investigated. This study aimed to compare the short-term temperature-dengue associations during different dengue outbreak periods, estimate the dengue cases attributable to temperature, and ascertain if there was an association between heatwaves and dengue outbreaks in Hanoi, Vietnam. Methodology/Principal findings Dengue outbreaks were assigned to one of three categories (small, medium and large) based on the 50th, 75th, and 90th percentiles of distribution of weekly dengue cases during 2008–2016. Using a generalised linear regression model with a negative binomial link that controlled for temporal trends, temperature variation, rainfall and population size over time, we examined and compared associations between weekly average temperature and weekly dengue incidence for different outbreak categories. The same model using weeks with or without heatwaves as binary variables was applied to examine the potential effects of extreme heatwaves, defined as seven or more days with temperatures above the 95th percentile of daily temperature distribution during the study period. This study included 55,801 dengue cases, with an average of 119 (range: 0 to 1454) cases per week. The exposure-response relationship between temperature and dengue risk was non-linear and differed with dengue category. After considering the delayed effects of temperature (one week lag), we estimated that 4.6%, 11.6%, and 21.9% of incident cases during small, medium, and large outbreaks were attributable to temperature. We found evidence of an association between heatwaves and dengue outbreaks, with longer delayed effects on large outbreaks (around 14 weeks later) than small and medium outbreaks (4 to 9 weeks later). Compared with non-heatwave years, dengue outbreaks (i.e., small, moderate and large outbreaks combined) in heatwave years had higher weekly number of dengue cases (p

by Carolina Mendoza, Gloria-Isabel Jaramillo, Thomas H. Ant, Grace M. Power, Robert T. Jones, Juliana Quintero, Neal Alexander, Jayne Webster, Lyda Osorio, James G. Logan
Background Arboviruses transmitted by day-biting Aedes mosquitoes are a major public health concern. With the challenges inherent in arbovirus vaccine and therapeutics development, vector control and bite prevention strategies are among the limited options available for immediate intervention. Bite prevention through personal protective technologies (PPT), such as topical mosquito repellents or repellent-impregnated clothing, may help to decrease biting rates and, therefore, the risk of disease in groups most susceptible to adverse outcomes from Zika virus. However, achieving high uptake and compliance with PPT can be challenging. Methodology/Principal findings To gain an insight into the knowledge and concerns of pregnant women surrounding Zika and their opinions regarding PPT, particularly repellent clothing, a focus group study was carried out with pregnant women, women of reproductive age, and semi-structured interviews with their male partners in two cities in Colombia. The discussions revealed shortfalls in basic knowledge of Zika virus, with several pregnant participants reporting being unaware of the potential for Zika-related congenital malformations. Although participants generally considered Zika to be a significant personal threat, most rated it as less of a concern than dengue or diarrheal diseases. Overall, repellent clothing and other forms of PPT were viewed as effective, although some participants expressed concerns over the high costs of repellents, and safety fears of regular contact with repellent chemicals, which they perceived as potentially harmful. Plant-derived repellents were considered to be safer than synthetic chemical repellents. Discussions also highlighted that health centers were the preferred source of information on bite-reduction. Conclusions/Significance Achieving high uptake and compliance with PPT in populations most at risk of adverse outcomes from Zika infection requires engaging key users in open dialogue to identify and address any practical issues regarding PPT use, and concerns over safety. The findings presented here suggest that educational campaigns should strongly emphasize the risks associated with Zika during pregnancy, and discuss safety profiles of approved synthetic repellents and the availability of EPA-approved plant-based repellents. In addition, the economic and political context should be a major consideration when evaluating personal mosquito-repellent strategies.

by Crystal Garae, Kalkoa Kalo, George Junior Pakoa, Rohan Baker, Phill Isaacs, Douglas Spencer Millar
Background The family flaviviridae and alphaviridae contain a diverse group of pathogens that cause significant morbidity and mortality worldwide. Diagnosis of the virus responsible for disease is essential to ensure patients receive appropriate clinical management. Very few real-time RT-PCR based assays are able to detect the presence of all members of these families using a single primer and probe set. We have developed a novel chemistry, 3base, which simplifies the viral nucleic acids allowing the design of RT-PCR assays capable of pan-family identification. Methodology/Principal finding Synthetic constructs, viral nucleic acids, intact viral particles and characterised reference materials were used to determine the specificity and sensitivity of the assays. Synthetic constructs demonstrated the sensitivities of the pan-flavivirus detection component were in the range of 13 copies per PCR. The pan-alphavirus assay had a sensitivity range of 10–25 copies per reaction depending on the viral strain. Lower limit of detection studies using whole virus particles demonstrated that sensitivity for assays was in the range of 1–2 copies per reaction. No cross reactivity was observed with a number of commonly encountered viral strains. Proficiency panels showed 100% concordance with the expected results and the assays performed as well as, if not better than, other assays used in laboratories worldwide. After initial assay validation the pan-viral assays were then tested during the 2016–2017 Vanuatu dengue-2 outbreak. Positive results were detected in 116 positives from a total of 187 suspected dengue samples. Conclusions/Significance The pan-viral screening assays described here utilise a novel 3base technology and are shown to provide a sensitive and specific method to screen and thereafter speciate flavi- and/or alpha- viruses in clinical samples. The assays performed well in an outbreak situation and can be used to detect positive clinical samples containing any flavivirus or alphavirus in approximately 3 hours 30 minutes.

Title: Estudo da aplicabilidade do teste de ativação de monócitos na detecção de pirogênios na vacina contra febre amarela
Authors: Navega, Elaine Cristina Azevedo
Abstract: A necessidade de alternativas ao uso de animais no teste de pirogênio é guiada pelo principio dos 3R’s, culminando na aceitação pela Farmacopeia Europeia (FE) do método alternativo in vitro, o Teste de Ativação de Monócitos (MAT). Respeitando esta premissa, o MAT utiliza como matriz, fontes de monócitos humanos sendo considerado um método promissor e eficiente por excluir riscos inerentes à extrapolação inter-espécies, detectar amplo espectro de pirogênios e suprir limitações dos testes atuais, i.e o Teste de Endotoxina Bacteriana (LAL) e o Teste de Pirogênio em coelhos (RPT). Apesar das vantagens abordadas, alguns obstáculos técnico-científico-regulatórios devem ser transpostos para a implantação efetiva do MAT na rotina industrial, em especial, de produtos biológicos. Neste contexto, o Laboratório de Controle de Qualidade de Bio-Manguinhos (Seção de Controle Biológico) em colaboração com o Instituto Nacional de Controle de Qualidade em Saúde (INCQS), visando reforçar a melhoria nos ensaios de controle de qualidade e fortalecer cientificamente o uso dos testes alternativos seguindo os pré-requisitos regulamentados pelas farmacopeias, selecionou de seu portifólio como alvo de estudo, a vacina contra a febre amarela (VFA) como primeiro modelo de aplicabilidade do MAT em vacinas virais por: (1) ser o carro-chefe de Bio-Manguinhos, (2) apresentar processo de produção complexo, com fatores críticos de interferência como uso de vírus vivo e resíduos de componentes de processos (ovos embrionados) e (3) possibilitar potencial investigativo de outras fontes pirogênicas não detectadas pelo método descrito na monografia da vacina. Os métodos farmacopeicos descritos para o MAT utilizando IL-1/sangue criopreservado, IL-6/ sangue fresco e a proposta de método para rotina industrial utilizando IL-6/ sangue criopreservado, foram validados conforme requisitos da FE. A verificação produto-específica foi realizada com a VFA/10 doses e fatores interferentes avaliados através da máxima diluição válida. Após determinação da diluição, o método quantitativo foi aplicado aos lotes vacinais, com interpretação entre os sistemas de leitura e matrizes do MAT e LAL. Os dados mostram reprodutibilidade do teste utilizando diferentes doadores e medidores de pirogenicidade (IL-6/IL-1) frente ao estímulo da endotoxina. Após verificação da aplicabilidade do MAT à VFA com diluição 1:10, demonstramos que os sistemas utilizando sangue criopreservado (IL-1β/IL-6) respeitam os limites de recuperação da endotoxina e se mostraram mais adequados do que o sistema IL-6/sangue fresco. Os resultados quantitativos da resposta pirogênica no produto - determinada pelo MAT - correspondem àqueles observados pelo LAL. Os dados demonstram a aplicabilidade do MAT em vacinas, sendo o modelo VFA compatível com a utilização.

by Rosemary J. Steinbach, Nicole N. Haese, Jessica L. Smith, Lois M. A. Colgin, Rhonda P. MacAllister, Justin M. Greene, Christopher J. Parkins, J. Beth Kempton, Edward Porsov, Xiaojie Wang, Lauren M. Renner, Trevor J. McGill, Brandy L. Dozier, Craig N. Kreklywich, Takeshi F. Andoh, Marjorie R. Grafe, Heidi L. Pecoraro, Travis Hodge, Robert M. Friedman, Lisa A. Houser, Terry K. Morgan, Peter Stenzel, Jonathan R. Lindner, Robert L. Schelonka, Jonah B. Sacha, Victoria H. J. Roberts, Martha Neuringer, John V. Brigande, Christopher D. Kroenke, Antonio E. Frias, Anne D. Lewis, Meredith A. Kelleher, Alec J. Hirsch, Daniel Neal Streblow

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

Title: Dengue virus infection of the central nervous system (CNS): a case report from Brazil
Authors: Nogueira, R. M. R.; Filippis, A. M. B.; Coelho, J. M. O.; Sequeira, P. C.; Schatzmayr, H. G.; Paiva, F. G.; Ramos, A. M. O.; Miagostovich, M. P.

Title: Infants with microcephaly due to ZIKA virus exposure: nutritional status and food practices
Authors: Santos, Samira Fernandes Morais dos; Soares, Fernanda Valente Mendes; Abranches, Andrea Dunshee de; Costa, Ana Carolina Carioca da; Moreira, Maria Elisabeth Lopes; Matos Fonseca, Vania de

by Shih-Chao Lin, Brian D. Carey, Victoria Callahan, Ji-Hyun Lee, Nicole Bracci, Anurag Patnaik, Amy K. Smith, Aarthi Narayanan, Benjamin Lepene, Kylene Kehn-Hall

Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.

Os coletivos da Covid-19
Carvalheiro, José da Rocha
Ao estudar um fenômeno que atinge a população de diversos continentes, cuidamos de coletivo de pessoas. A pandemia que assola a humanidade é pouco conhecida. Buscando entendê-la, aparecem outros coletivos no discurso: coletivo do pensamento, aglomeração, rede, teste, UTI, rebanho, comboio. Na análise desses coletivos, buscamos elucidar controvérsias. Na formulação de políticas inovadoras em saúde, aproveitamos textos próprios, de análise de epidemias anteriores. Pelo limitado conhecimento da História Natural da Covid-19, têm relevância os “procedimentos não farmacológicos”, ligados ao comportamento das pessoas. Não bastasse o rigor da invasão epidêmica do
vírus, sofremos uma invasão léxica inédita: achatamento de curvas, distanciamento social e etiqueta respiratória. Principais controvérsias: a disputa pela cloroquina, com populistas em ação e o equivocado uso da Herd Immunity como objetivo a alcançar, e milhões de óbitos.

Manual sobre biossegurança para reabertura de escolas no contexto da Covid-19
Pereira, Ingrid D’avilla Freire; Corbo, Anamaria D’Andrea; Paula, Tainah Silva Galdino de; Mendonça, Flávia Coelho Ribeiro; Carvalho, Paulo Roberto de; Bottino, Fernanda de Oliveira; Stephanelli, Lásaro Linhares; Valle, Silvio
Manual tem como proposta disponibilizar informações facilmente acessíveis para escolas públicas, destacando a comunicação sobre os mecanismos de transmissão da Covid-19 e a implementação de boas práticas que possam contribuir para a promoção da saúde e a prevenção dessa doença nas escolas. (AU)
2. ed. rev.

Historia, coronavirus y la crisis de liderazgo mundial
Cueto, Marcos

by Basile Kamgang, Marie Vazeille, Armel N. Tedjou, Theodel A. Wilson-Bahun, Aurélie P. Yougang, Laurence Mousson, Charles S. Wondji, Anna-Bella Failloux
Introduction Dengue is the most important mosquito-borne diseases worldwide but was considered scarce in West-Central Africa. During the last decade, dengue outbreaks have increasingly been reported in urban foci in this region suggesting major epidemiological changes. However, in Central Africa where both vectors, Aedes aegypti and Aedes albopictus are well established, the role of each species in dengue transmission remains poorly investigated. Methodology/Principal findings Field-collected strains of Ae. aegypti and Ae. albopictus from different ecological settings in Central Africa were experimentally challenged with dengue 2 virus (DENV-2). Mosquitoes were analysed at 14- and 21-days post-infection. Analysis provide evidence that both Ae. aegypti and Ae. albopictus in Central Africa were able to transmit dengue virus with Ae. aegypti exhibiting a higher transmission rate. Unexpectedly, two Ae. aegypti populations from Bénoué and Maroua, in northern Cameroon, were not able to transmit DENV-2. Conclusions/Significance We conclude that both Ae. aegypti and Ae. albopictus are susceptible to DENV-2 and may intervene as active dengue vectors. These findings highlight the urgent need to plan a vector surveillance program and control methods against dengue vectors in Central Africa in order to prevent future outbreaks.

by Zachary J. Madewell, Ryan R. Hemme, Laura Adams, Roberto Barrera, Stephen H. Waterman, Michael A. Johansson
Background Detecting and monitoring the transmission of arboviruses such as Zika virus (ZIKV), dengue virus, and chikungunya virus is critical for prevention and control activities. Previous work has compared the ability of different human-focused surveillance strategies to detect ZIKV transmission in U.S. counties where no known transmission had occurred, but whether virological surveillance in mosquitoes could represent an effective surveillance system is unclear. Objectives We leveraged a unique set of data from human and virological surveillance in Ae. aegypti during the 2016 ZIKV epidemic in Caguas, Puerto Rico, to compare alternative strategies for detecting and monitoring ZIKV activity. Methods We developed a simulation model for mosquito and human surveillance strategies and simulated different transmission scenarios with varying infection rates and mosquito trap densities. We then calculated the expected weekly number of detected infections, the probability of detecting transmission, and the number of tests needed and compared the simulations with observed data from Caguas. Results In simulated high transmission scenarios (1 infection per 1,000 people per week), the models demonstrated that both approaches had estimated probabilities of detection of greater than 78%. In simulated low incidence scenarios, vector surveillance had higher sensitivity than human surveillance and sensitivity increased with more traps, more trapping effort, and testing. In contrast, the actual data from Caguas indicated that human virological surveillance was more sensitive than vector virological surveillance during periods of both high and low transmission. Conclusion In scenarios where human surveillance is not possible or when transmission intensity is very low, virological surveillance in Ae. aegypti may be able to detect and monitor ZIKV epidemic activity. However, surveillance for humans seeking care for Zika-like symptoms likely provides an equivalent or more sensitive indicator of transmission intensity in most circumstances.

by Michel J. Counotte, Christian L. Althaus, Nicola Low, Julien Riou

The 2015–2017 epidemics of Zika virus (ZIKV) in the Americas caused widespread infection, followed by protective immunity. The timing and burden of the next Zika virus outbreak remains unclear. We used an agent-based model to simulate the dynamics of age-specific immunity to ZIKV, and predict the future age-specific risk using data from Managua, Nicaragua. We also investigated the potential impact of a ZIKV vaccine. Assuming lifelong immunity, the risk of a ZIKV outbreak will remain low until 2035 and rise above 50% in 2047. The imbalance in age-specific immunity implies that people in the 15–29 age range will be at highest risk of infection during the next ZIKV outbreak, increasing the expected number of congenital abnormalities. ZIKV vaccine development and licensure are urgent to attain the maximum benefit in reducing the population-level risk of infection and the risk of adverse congenital outcomes. This urgency increases if immunity is not lifelong.

by Chih-Cheng Hsieh, Ming-Yuan Hong, Tzong-Shiann Ho, Ching-Chuan Liu, Guey-Chuen Perng, Chia-Chang Chuang
Background Severe dengue virus (DENV) infection involves plasma leakage and vascular collapse, and leads to significant morbidity and death. Serum soluble ST2 (sST2 [interleukin (IL)-1 receptor like-1 protein: IL-1-RL-1]) levels are high in pediatric cases of DENV infection, and the disease progresses. However, the correlation between serum sST2 levels and the outcomes of DENV infection in the elderly (≥65 years) is unclear. We thus explored the mechanisms of serial sST2 level changes involved in the coagulopathy and bloodstream infections of elderly patients in Taiwan’s 2015 DENV outbreak. Methods This retrospective study was done in a tertiary medical center in southern Taiwan during the outbreak. All DENV-infected patients who, between July 1, 2015, and December 31, 2015, provided a written informed consent for at least two blood sample analyses were enrolled and reviewed. The serum levels of sST2 were quantified. ΔsST2 is defined as the “changes of sST2 levels in serially paired samples”. Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) analyses were used to evaluate the prognostic ability of ΔsST2. Results Forty-three patients with DENV infection were enrolled. Mean patient age was 75.0 ± 12.2 years and the case fatality rate was 44.2% (19/43). Significantly more non-survivors than survivors had increased ST2 level (78.9% vs. 12.5%, p 0.001). The AUC value for serum ΔsST2 level was 0.857 for predicting DENV fatality. Moreover, patients given frozen fresh plasma (FFP) transfusions were significantly (p = 0.025) more likely to have higher serum ST2 level changes than were those who had not. DENV-infected patients with early bloodstream infections (BSIs) seemed to have higher ST2 levels than those who did not have BSIs. Conclusions Serum ST2 levels increased in the elderly (≥ 65 years of age) with DENV infection. The changes in serum sST2 levels might be a critical indicator of DENV infection severity for the elderly; sST2 is an important modulator of coagulopathy in severe DENV infections.

by Romrawin Chumpu, Nirattaya Khamsemanan, Cholwich Nattee

Dengue and dengue hemorrhagic pose significant burdens in many tropical countries. Dengue incidences have perpetually increased, leading to an annual (uncertain) peak. Dengue cases cause an enormous public health problem in Thailand because there is no anti-viral drug against the dengue virus. Searching for means to reduce the dengue incidences is a challenging and appropriate strategy for primary prevention in a dengue outbreak. This study constructs the best predictive model from past statistical dengue incidences at the provincial level and studies the relationships among dengue incidences and weather variables. We conducted experiments for 65 provinces (out of 77 provinces) in Thailand since there is no dengue information for the remaining provinces. Predictive models were constructed using weekly data during 2001-2014. The training set are data during 2001-2013, and the test set is the data from 2014. Collected data were separated into two parts: current dengue cases as the dependent variable, and weather variables and previous dengue cases as the independent variables. Eight weather variables are used in our models: average pressure, maximum temperature, minimum temperature, average humidity, precipitation, vaporization, wind direction, wind power. Each weather variable includes the current week and one to three weeks of lag time. A total of 32 independent weather variables are used for each province. The previous one to three weeks of dengue cases are also used as independent variables. There is a total of 35 independent variables. Predictive models were constructed using five methods: Poisson regression, negative binomial regression, quasi-likelihood regression, ARIMA(3,1,4) and SARIMA(2,0,1)(0,2,0). The best model is determined by combinations of 1–12 variables, which are 232,989,800 models for each province. We construct a total of 15,144,337,000 models. The best model is selected by the average from high to low of the coefficient of determination (R2) and the lowest root mean square error (RMSE). From our results, the one-week lag previous case variable is the most frequent in 55 provinces out of a total of 65 provinces (coefficient of determinations with a minimum of 0.257 and a maximum of 0.954, average of 0.6383, 95% CI: 0.57313 to 0.70355). The most influential weather variable is precipitation, which is used in most of the provinces, followed by wind direction, wind power, and barometric pressure. The results confirm the common knowledge that dengue incidences occur most often during the rainy season. It also shows that wind direction, wind power, and barometric pressure also have influences on the number of dengue cases. These three weather variables may help adult mosquitos to survive longer and spread dengue. In conclusion, The most influential factor for further cases is the number of dengue cases. However, weather variables are also needed to obtain better results. Predictions of the number of dengue cases should be done locally, not at the national level. The best models of different provinces use different sets of weather variables. Our model has an accuracy that is sufficient for the real prediction of future dengue incidences, to prepare for and protect against severe dengue outbreaks.

Title: The citrus flavonoid naringenin impairs the in vitro infection of human cells by Zika virus
Authors: Cataneo, Allan Henrique Depieri; Kuczera, Diogo; Koishi, Andrea Cristine; Zanluca, Camila; Silveira, Guilherme Ferreira; Arruda, Thais Bonato de; Suzukawa, Andréia Akemi; Bortot, Leandro Oliveira; Dias-Baruffi, Marcelo; Verri, Waldiceu Aparecido; Robert, Anny Waloski; Stimamiglio, Marco Augusto; Santos, Claudia Nunes Duarte dos; Wowk, Pryscilla Fanini; Bordignon, Juliano