Optimising the surveillance of Aedes aegypti in Brazil by selecting smaller representative areas within an endemic city.

OBJECTIVES: Arboviruses, such as dengue (DENV), zika (ZIKV), and chikungunya (CHIKV), constitute a growing urban public health threat. Focusing on Aedes aegypti mosquitoes, their primary vectors, is crucial for mitigation. While traditional immature-stage mosquito surveillance has limitations, capturing adult mosquitoes through traps yields more accurate data on disease transmission. However, deploying traps presents logistical and financial challenges, demonstrating effective temporal predictions but lacking spatial accuracy. Our goal is to identify smaller representative areas within cities to enhance the early warning system for DENV outbreaks. METHODS: We created Sentinel Geographic Units (SGUs), smaller areas of 1 km2 within each stratum, larger areas, with the aim of aligning the Trap Positivity Index (TPI) and Adult Density Index (ADI) with their respective strata. We conducted a two-step evaluation of SGUs. First, we examined the equivalence of TPI and ADI between SGUs and strata from January 2017 to July 2022. Second, we assessed the ability of SGU's TPI and ADI to predict DENV outbreaks in comparison to Foz do Iguaçu's Early-Warning System, which forecasts outbreaks up to 4 weeks ahead. Spatial and temporal analyses were carried out, including data interpolation and model selection based on Akaike information criteria (AIC). RESULTS: Entomological indicators produced in small SGUs can effectively replace larger sentinel areas to access dengue outbreaks. Based on historical data, the best predictive capability is achieved 2 weeks after infestation verification. Implementing the SGU strategy with more frequent sampling can provide more precise space-time estimates and enhance dengue control. CONCLUSIONS: The implementation of SGUs offers an efficient way to monitor mosquito populations, reducing the need for extensive resources. This approach has the potential to improve dengue transmission management and enhance the public health response in endemic cities.

LaSap vaccine: Immunotherapy and immunochemotherapy associated with allopurinol in dogs naturally infected with Leishmania infantum.

Canine visceral leishmaniasis is a parasitic zoonosis that has a profound impact on public health in countries where it is endemic. Chemotherapeutic treatments cannot keep dogs stable for long periods, and the risk of generating parasitic resistance must be considered. Forty-four symptomatic and naturally infected dogs with Leishmania infantum were tested with two treatment protocols (i) immunotherapy with LaSap vaccine and (ii) immunochemotherapy with LaSap vaccine plus allopurinol. At 90 days after the end of the treatment, it was verified that, although both protocols had generated significant clinical improvements with a greater production of IFN-γ/IL-10, in relation to the parasite load, mainly in the skin, the dogs treated only with immunotherapy maintained the same profile. These results indicate that LaSap is a good strategy to control dog parasitism.

Bivalent Vaccine against Streptococcus agalactiae and Aeromonas hydrophila in Nile Tilapia (Oreochromis niloticus): A Laboratory-Phase and Large-Scale Study.

One of the main factors limiting tilapia's production is the occurrence of infections caused by Aeromonas and Streptococcus species. This work intended to evaluate a bivalent vaccine against A. hydrophila and S. agalactiae by intraperitoneal (i.p) administration in Nile tilapia (Oreochromis niloticus) in Brazil. The study was carried out in two phases: one in the laboratory, on a small scale, and from the results obtained, the study was expanded to a large scale in a production system in cages. The vaccine proved to be safe and effective in laboratory tests, with a vaccine efficacy (VE) of 93.66%. However, in large-scale tests with 12,000 tilapias, the VE was 59.14%, with a better food conversion ratio (1.54 kg) in the vaccinated group compared to the control group (1.27 kg). These results corroborate the efficiency of this tested vaccine; however, they indicate the need for field tests to attest to real protection.

Cross-sectional spatial and epidemiological analysis of canine visceral leishmaniasis cases in the triple border region, Brazil, Argentina and Paraguay, between 2015 and 2020.

Visceral Leishmaniasis is one of the most important vector-borne zoonoses in the world. In 2013, the first autochthonous canine case of the disease in the state of Paraná, southern region of Brazil, was reported in Foz do Iguaçu, on the triple border between Brazil, Argentina and Paraguay. In 2015, the first human case was related. Once the endemic was confirmed, the Zoonoses Control Center (ZCC), an agency of the Municipal Health Department, started actions to implement the Human Visceral Leishmaniasis (HVL) Surveillance and Control Program (VLSCP), of the Ministry of Health. Between 2015 and 2020, 12,205 dog samples were analyzed for the diagnosis of the disease. A prevalence of 37.94% (4,630 samples) was found: 2016 had the highest prevalence, with 46.25%, and the year with the lowest prevalence was 2020, with 25.98%. Possible risk factors for dogs were analyzed, and the results obtained were: whether the request for the exam was performed by the ZCC was a significant protective factor, with a lower prevalence (37.5%) than dogs coming from private clinics (OR = 0.89, p-value = 0.016). Males were significantly more infected than females, with 41.1% and 35.7% positivity, respectively (OR = 1.24, p < 0.0001). Companion dogs and mixed breed dogs were significantly less affected than the other groups tested (OR = 0.44, p < 0.001; OR = 0.79, p = 0.012, respectively). The dogs' dark coat color was a significant risk factor with respect to the other color categories. Short and medium coat sizes were significantly considered risk factors, with 41.3% and 31.3% positivity. Long-haired dogs had only 22.7% positivity. In univariate analyses, giant, large and medium dogs were significantly more affected than small dogs. Dogs up to four years of age were significantly less affected than those in other age groups. There was a coincidence of human and canine cases in the spatial distribution. However, according to the literature, a higher incidence would be expected in humans, due to the high prevalence found in dogs. Therefore, further studies should be carried out to understand the dynamics of the disease in this region.

Adaptation of a Human Diagnostic Kit to Detect Dengue, Zika, and Chikungunya Viruses in Mosquito Samples (Aedes aegypti and Aedes albopictus): A Contribution to Public Health in the International Triple Border (Brazil, Paraguay, and Argentina).

Objective: The objective of this work was to adapt a diagnostic kit developed for humans to identify Dengue (DENV1, DENV2, DENV3, DENV4), Zika (ZIKV) and Chikungunya virus (CHIKV) in females of Aedes aegypti and Aedes albopictus and to verify if the occurrence of mosquitoes infected with these three arboviruses are being found in regions with high occurrence of these diseases in humans. Materials and Methods: For this purpose, live mosquitoes were captured between January and June 2020 using 3,476 traps permanently installed in the field were used. After capture, the species were identified, then the females were placed in a pool of 2 to 10 specimens and sent to the laboratory for detection of DENV1, DENV2, DENV3, DENV4, ZIKV and CHIKV by RT-PCR using a commercial human kit for arboviruses. Results: Of the 76 mosquito pools collected, six (7.9%) pools tested positive for the DENV2 virus. The DENV-positive mosquitoes were collected in regions with a high incidence of reported cases of Dengue or in adjacent areas. Conclusion: The absence of kits for the detection of these arboviruses in Aedes is a limiting factor and the adequacy of commercial kits, already used for the diagnosis of arboviruses in humans, the results presented demonstrate that it is possible to identify the presence of DENV2 in mosquitoes with the respective kit, reinforcing the use of RT-qPCR as a robust diagnostic tool for epidemiological surveillance allowing managers to receive timely results for decision-making regarding prevention and control actions.

Infection in asymptomatic carriers of SARS-CoV-2 can interfere with the achievement of robust immunity on a population scale.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide as a severe pandemic, and a significant portion of the infected population may remain asymptomatic. Given this, five surveys were carried out between May and September 2020 with a total of 3585 volunteers in the municipality of Foz do Iguaçu, State of Paraná, a triple border region between Brazil/Argentina/Paraguay. Five months after the first infection, volunteers were re-analysed for the production of IgG anti-Spike and anti-RBD-Spike, in addition to analyses of cellular immunity. Seroconversion rates ranged from 4.4 % to a peak of 37.21 % followed by a reduction in seroconversion to 21.1 % in September, indicating that 25 % of the population lost their circulating anti-SARS-CoV-2 antibodies 3 months after infection. Analyses after 5 months of infection showed that only 17.2 % of people still had anti-RBD-Spike antibodies, however, most volunteers had some degree of cellular immune response. The strategy of letting people become naturally infected with SARS-CoV-2 to achieve herd immunity is flawed, and the first contact with the virus may not generate enough immunogenic stimulus to prevent a possible second infection.

Estudo da conservação de material biológico com a tecnologia Fta-card® / Study on the conservation of biological material with Fta-card® technology / Estudio de conservación de material biológico con la tecnología Fta-card®

O Whatman FTA-Card® é um papel-filtro quimicamente tratado, destinado à coleta, transporte, armazenamento de amostras para posterior extração de ácidos nucléicos. A tecnologia FTA-Card® é utilizada para manter estável DNA e RNA em temperatura ambiente, podendo ser utilizados para fixação de uma ampla variedade de material orgânico ou tecidos. Foram realizados testes para certificar sua eficiência na conservação do material a ser analisado com o intuito de eliminar a cadeia fria de conservação, agilizando o processo e diminuindo os custos da execução de exames moleculares associados ao diagnóstico de patologias. Foram testadas oito amostras de felinos na forma de sangue total e soro, para a extração utilizou-se o kit Magazorb RNA Total mini-prep kit (Promega®, EUA), para o diagnóstico foi utilizada a técnica de PCR em tempo real para amplificar o gene CI2 de mamíferos, a fim de visualizar a eficácia na conservação de ácidos nucleicos. A utilização desse método torna possível que o material biológico seja enviado por serviços de transporte postais, reduzindo os custos e viabilizando diagnósticos provenientes de áreas mais remotas.(AU)

Whatman FTA-Card® is a chemically treated filter paper intended for the collection, transport, and storage of samples for later extraction of nucleic acids. FTA-Card® technology is used to keep DNA and RNA stable at room temperature and can be used to fix a wide variety of organic material or tissues. Tests were carried out to certify its efficiency in the conservation of the material to be analyzed in order to eliminate the cold conservation chain, speeding up the process and decreasing the costs of performing molecular tests associated with the diagnosis of pathologies. By using this method, biological material can be sent by postal transport services, reducing costs and making diagnoses from more remote areas feasible. Samples of feline specimens were tested in the form of whole blood and serum, using the Magazorb RNA Total mini-prep kit (Promega®, USA) for the extraction. Diagnosis was performed using real-time PCR technique to amplify the mammalian CI2 gene in order to visualize the effectiveness in conserving nucleic acids.(AU)

Whatman FTA-Card® es un papel de filtro tratado químicamente, destinado a la recogida, transporte, almacenamiento de muestras para su posterior extracción de ácidos nucleicos. La tecnología FTA-Card® se usa para mantener el ADN y el ARN estables a la temperatura ambiente y se puede usar para la fijación de una amplia variedad de materiales o tejidos orgánicos. Se realizaron pruebas para certificar su eficiencia en la conservación del material a analizar con el fin de eliminar la cadena de frío de conservación, agilizando el proceso y reduciendo los costos de realización de pruebas moleculares asociadas al diagnóstico de patologías. Se analizaron ocho muestras felinas en forma de sangre total y suero, para la extracción se utilizó el mini-prep kit Magazorb RNA Total (Promega®, USA), para el diagnóstico se utilizó la técnica de PCR en tiempo real para amplificar el CI2 de mamífero gen, con el fin de visualizar la efectividad en la conservación de ácidos nucleicos. El uso de ese método permite el envío de material biológico por los servicios de transporte postal, lo que reduce los costes y permite realizar diagnósticos desde zonas más remotas.(AU)

The adoption of the One Health approach to improve surveillance of venomous animal injury, vector-borne and zoonotic diseases in Foz do Iguaçu, Brazil.

Public health institutions with sectorized structure and low integration among field teams, old-fashioned practices such as paper-based storage system, and poorly qualified health agents have limited ability to conduct accurate surveillance and design effective timely interventions. Herein, we describe the steps taken by the Zoonosis Control Center of Foz do Iguaçu (CCZ-Foz) in the last 23 years to move from an archaic and sectorized structure to a modern and timely surveillance program embracing zoonotic diseases, venomous animal injuries, and vector-borne diseases epidemiology under the One Health approach. The full implementation of the One Health approach was based on 5 axes: (1) merging sectorized field teams; (2) adoption of digital solutions; (3) health agents empowerment and permanent capacitation; (4) social mobilization; and (5) active surveys. By doing so, notifications related to zoonotic diseases and venomous animals increased 10 and 21 times, respectively, with no impairment on arbovirus surveillance (major concern in the city). Open sources database (PostgreSQL) and software (QGis) are daily updated and create real-time maps to support timely decisions. The adoption of One Health approach increased preparedness for endemic diseases and reemerging and emerging threats such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Immunogenicity of HLA-DR1 and HLA-A2 peptides derived from Leishmania major Gp63 in golden hamsters.

AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.

Malathion insecticide resistance in Aedes aegypti: laboratory conditions and in situ experimental approach through adult entomological surveillance.

OBJECTIVE: In Brazil, the most common method of controlling outbreaks of arbovirus is by the use of chemical sprays, which kill the insect vector, Aedes aegypti. The main objective of this study was to evaluate the resistance of Ae. aegypti to the insecticide, malathion, in situ. The location of this study was the municipality of Foz do Iguaçu, in the state of Paraná, Brazil. METHODS: Ultra-low-volume (ULV) fogging equipment was used, by vehicle, to apply the insecticide in situ, and mosquito populations after treatment were compared with those of control areas. The resistance of strains collected from the municipality was compared to the Rockefeller strain under laboratory conditions. RESULTS: We found 220 adult female specimens and 7423 eggs of Ae. aegypti in the areas subjected to UBV treatment, whereas 245 adult females and 10 557 eggs were found in the control areas. The UBV treatment area showed no significant difference compared to the control area, for all the indices. Mortality of the Rockefeller colony varied more quickly when there were slight variations in malathion concentration than the Foz do Iguaçu population.

OBJECTIF: Au Brésil, la méthode la plus courante de lutte contre les épidémies d'arbovirus consiste à utiliser des pulvérisations chimiques qui tuent l'insecte vecteur, Aedes aegypti. L'objectif principal de cette étude était d'évaluer la résistance de Ae. aegypti à l'insecticide, le malathion, in situ. Le lieu de cette étude était la municipalité de Foz do Iguaçu, dans l'état du Paraná, au Brésil. MÉTHODES: Un équipement de brumisation à très faible volume (ULV) a été utilisé, par véhicule, pour appliquer l'insecticide in situ et les populations de moustiques après le traitement ont été comparées à celles des zones témoins. La résistance des souches collectées dans la municipalité a été comparée à la souche Rockefeller dans des conditions de laboratoire. RÉSULTATS: Nous avons trouvé 220 spécimens femelles adultes et 7.423 œufs d'Ae. aegypti dans les zones soumises au traitement ULV, alors que 245 femelles adultes et 10.557 œufs ont été trouvés dans les zones témoins. La zone de traitement ULV n'a montré aucune différence significative par rapport à la zone témoin, pour tous les indices. La mortalité de la colonie de Rockefeller variait plus rapidement lorsqu'il y avait de légères variations dans la concentration de malathion que la population de Foz do Iguaçu.

Molecular Detection of Dengue Virus in Mosquitoes as an Early Indicator to Aid in the Prevention of Human Infection in Endemic Areas.

Human cases of dengue virus based on the National Dengue Control Plan were compared with the molecular detection of the dengue virus in trapped mosquitoes, verifying the prediction and efficacy potentials of vector control between the two methodologies in a city with three endemic frontiers. Molecular detection of dengue virus in trapped mosquitoes was significantly higher than in human cases (p = 0.0435). Thus, molecular detection could be used as an early indicator to help prevent more human cases of dengue.

False-negative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors / Resultado falso negativo no diagnóstico molecular de SARS-CoV-2 em amostras com inibidores de amplificação

ABSTRACT Introduction: Although reverse transcription-polymerase chain reaction (rRT-PCR) is the gold standard method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), some factors, such as the presence of amplification inhibitors, lead to false-negative results. Objective: Here we describe the differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to the presence of amplification inhibitors. Material and method: Viral ribonucleic acid (RNA) from samples of nasopharyngeal swabs from 20 patients previously detected as "Negative" and 21 patients detected as "Positive" for SARS-CoV-2 was performed with the EasyExtract DNA-RNA kit (Interprise®). The rRT-PCR was performed with the OneStep/COVID-19 kit (IBMP), with normal and diluted (80 µl of H2O RNAse free) samples, totaling 82 tests. Results: The results indicate that there is an average variation (a < 0.05) delaying the Cq between the results of amplification of the internal control (IC), N gene (NG), and ORF1ab (OF), 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. Discussion: The extraction kit does not completely purify the inhibitor compounds; therefore, no amplified product result may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution; this process reduces the efficiency of rRT-PCR to 29.8% in detecting SARS-CoV-2. Conclusion: Knowing the rRT-PCR standards of diluted samples can assist in the identification of false-negative cases and, consequently, avoid incorrect diagnosis.

RESUMEN Introducción: Aunque la reacción en cadena de la polimerasa con transcriptasa reversa en tiempo real (rRT-PCR) sea el método de referencia para detección del coronavirus tipo 2 del síndrome respiratorio agudo grave (Sars-CoV-2), algunos factores como la presencia de inhibidores de amplificación conducen a resultados falsos negativos. Objetivo: Describimos las diferencias entre los resultados de rRT-PCR para infección por Sars-CoV-2 en muestras normales y diluidas, simulando la necesidad de dilución debido a la presencia de inhibidores de amplificación. Material y método: La extracción de ácido ribonucleico (ARN) viral de muestras de hisopos nasofaríngeos de 20 pacientes previamente detectados como "negativos" y 21 pacientes detectados como "positivos" para Sars-CoV-2 se realizó con el kit Easy Extract DNA-RNA (Interprise®). La rRT-PCR se realizó con el kit OneStep/Covid-19 (IBMP), con muestras normales y diluidas (80 µl de H2O libre de ARNasa), totalizando 82 pruebas. Resultados: Los resultados indican que hay una variación media (a < 0,05) retrasando el ciclo de cuantificación (Cq) entre los resultados de amplificación del control interno (CI), gen N (GN) y ORF1ab (OF) de 1,811 Cq, 3,840 Cq y 3,842 Cq. Discusión: El kit de extracción no purifica completamente los compuestos inhibidores; por lo tanto, puede ocurrir no amplificación. Obtuvimos un diagnóstico falso negativo de 19,04% después de la dilución de la muestra; ese proceso reduce la eficiencia de la rRT-PCR hacia 29,8% en la detección de Sars-CoV-2. Conclusión: Conocer los patrones de la rRT-PCR de muestras diluidas puede ayudar en la identificación de casos falsos negativos y, por consiguiente, evitar un diagnóstico equivocado.

RESUMO Introdução: Embora a reação em cadeia da polimerase de transcrição reversa (rRT-PCR) seja o método padrão-ouro para detecção de coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2), alguns fatores como a presença de inibidores de amplificação levam a resultados falso negativos. Objetivo: Descrevemos as diferenças entre os resultados de rRT-PCR para infecção por SARS-CoV-2 em amostras normais e diluídas, simulando a necessidade de diluição devido à presença de inibidores de amplificação. Material e método: A extração de ácido ribonucleico (RNA) viral de amostras de suabes nasofaríngeos de 20 pacientes previamente detectados como "negativos" e 21 pacientes detectados como "positivos" para SARS-CoV-2 foi realizada com kit o EasyExtract DNA-RNA (Interprise®). A rRT-PCR foi realizada com o kit OneStep/COVID-19 (IBMP), com amostras normais e diluídas (80 µl de H2O RNAse-free), totalizando 82 testes. Resultados: Os resultados indicam que existe uma variação média (a < 0,05) atrasando o Cq entre os resultados de amplificação do controle interno (CI), gene N (GN) e ORF1ab (OF) de 1,811 Cq, 3,840 Cq e 3,842 Cq, respectivamente. Discussão: O kit de extração não purifica completamente os compostos inibidores, portanto, pode ocorrer não amplificação. Obtivemos um diagnóstico falso negativo de 19,04% após a diluição da amostra; esse processo reduz a eficiência da rRT-PCR para 29,8% na detecção de SARS-CoV-2. Conclusão: Conhecer os padrões da rRT-PCR de amostras diluídas pode auxiliar na identificação de casos falso negativos e, consequentemente, evitar um diagnóstico incorreto.

Building Infestation Index for Aedes aegypti and occurrence of dengue fever in the municipality of Foz do Iguaçu, Paraná, Brazil, from 2001 to 2016.

INTRODUCTION: the Building Infestation Index (BII) uses the Rapid Assay of the Larval Index for Aedes aegypti (LIRAa) to express the relationship between positive and surveyed properties. We evaluated LIRAa and the relationship between the BII and climate variables for dengue cases in Foz do Iguaçu municipality, Paraná. METHODS: Spearman's correlations for mean precipitation, mean temperature, BII, and dengue cases (time lag). RESULTS: positive correlations between BII and cases, and mean temperature and cases at two months. Weak correlation between precipitation and cases at three months. CONCLUSIONS: LIRAa and climate variables correlate with dengue cases.

Building Infestation Index for Aedes aegypti and occurrence of dengue fever in the municipality of Foz do Iguaçu, Paraná, Brazil, from 2001 to 2016

Abstract INTRODUCTION: the Building Infestation Index (BII) uses the Rapid Assay of the Larval Index for Aedes aegypti (LIRAa) to express the relationship between positive and surveyed properties. We evaluated LIRAa and the relationship between the BII and climate variables for dengue cases in Foz do Iguaçu municipality, Paraná. METHODS: Spearman's correlations for mean precipitation, mean temperature, BII, and dengue cases (time lag). RESULTS: positive correlations between BII and cases, and mean temperature and cases at two months. Weak correlation between precipitation and cases at three months. CONCLUSIONS: LIRAa and climate variables correlate with dengue cases.