Citywide Integrated Aedes aegypti Mosquito Surveillance as Early Warning System for Arbovirus Transmission, Brazil.

Arbovirus epidemiology lacks efficient and timely surveillance systems with accurate outbreak alert signals. We devised a citywide integrated surveillance system combining entomologic, epidemiologic, and entomo-virologic data gathered during 2017-2020 in Foz do Iguaçu, Brazil. We installed 3,476 adult mosquito traps across the city and inspected traps every 2 months. We compared 5 entomologic indices: traditional house and Breteau indices for larval surveys and trap positivity, adult density, and mosquitoes per inhabitant indices for adult trapping. We screened for dengue, Zika, and chikungunya viruses in live adult Aedes aegypti mosquitoes collected from traps. Indices based on adult mosquito sampling had higher outbreak predictive values than larval indices, and we were able to build choropleth maps of infestation levels <36 h after each round of trap inspection. Locating naturally infected vectors provides a timely support tool for local public health managers to prioritize areas for intervention response to prevent virus outbreaks.

'Candidatus Mycoplasma haematonasua' and tick-borne pathogens in ring-tailed coatis (Nasua nasua Linnaeus, 1976) from the Iguaçu National Park, Paraná State, southern Brazil.

The Iguaçu National Park (INP) is the largest remnant of Atlantic Forest in southern Brazil, representing an ecological continuum with Argentina. The INP harbours a diverse fauna, with ring-tailed coatis (Nasua nasua Linnaeus, 1976, Carnivora: Procyonidae) in close contact with tourists either begging and/or snatching food from visitors. A potentially novel haemotropic Mycoplasma sp. has been previously detected in the ring-tailed coatis from central-western and southern Brazil. Therefore, the aims of this study were to investigate the occurrence of haemotropic Mycoplasma sp. and tick-borne pathogens in wild ring-tailed coatis from the INP, Foz do Iguaçu municipality, Paraná State, southern Brazil. Blood samples were collected from 18 wild ring-tailed coatis and evaluated by conventional PCR (cPCR) assays for haemotropic Mycoplasma spp. (16S and 23S rRNA), Theileria/Babesia spp. (18S rRNA) and Ehrlichia/Anaplasma spp. (16S rRNA, sodB, dsb and groEL). Eight out of 18 (44.44%; 95% CI: 24.56%-66.28%) animals were positive for haemotropic Mycoplasma spp. All ring-tailed coatis tested negative for Theileria/Babesia spp. and only one out of 18 (5.56%; 95% CI: 0.99%-25.76%) animals tested positive for Ehrlichia/Anaplasma spp. by the 16S rRNA cPCR. Unfortunately, multiple attempts to sequence the 16S rRNA gene of the Ehrlichia/Anaplasma-positive sample have failed. Phylogenetic and network analysis of the hemoplasma 16S and 23S rRNA gene fragments confirmed that animals were infected by a potentially novel haemotropic Mycoplasma sp. previously reported in ring-tailed coatis from Brazil. The name 'Candidatus Mycoplasma haematonasua' is proposed for this novel organism.

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.