Characterization of Dengue Virus 4 Cases in Paraguay, 2019-2020.

In 2019-2020, dengue virus (DENV) type 4 emerged to cause the largest DENV outbreak in Paraguay's history. This study sought to characterize dengue relative to other acute illness cases and use phylogenetic analysis to understand the outbreak's origin. Individuals with an acute illness (≤7 days) were enrolled and tested for DENV nonstructural protein 1 (NS1) and viral RNA by real-time RT-PCR. Near-complete genome sequences were obtained from 62 DENV-4 positive samples. From January 2019 to March 2020, 799 participants were enrolled: 253 dengue (14 severe dengue, 5.5%) and 546 other acute illness cases. DENV-4 was detected in 238 dengue cases (94.1%). NS1 detection by rapid test was 52.5% sensitive (53/101) and 96.5% specific (387/401) for dengue compared to rRT-PCR. DENV-4 sequences were grouped into two clades within genotype II. No clustering was observed based on dengue severity, location, or date. Sequences obtained here were most closely related to 2018 DENV-4 sequences from Paraguay, followed by a 2013 sequence from southern Brazil. DENV-4 can result in large outbreaks, including severe cases, and is poorly detected with available rapid diagnostics. Outbreak strains seem to have been circulating in Paraguay and Brazil prior to 2018, highlighting the importance of sustained DENV genomic surveillance.

Prevalencia de baja avidez de Inmunoglobulina G anti Toxoplasma gondii y comportamiento de riesgo para toxoplasmosis en embarazada

Introducción: la toxoplasmosis es una infección zoonótica producida por Toxoplasma gondii, protozoo intracelular que puede afectar al hijo de la mujer embarazada y causar severas secuelas por lo que el monitoreo serológico debe ser realizado. Objetivo: determinar la prevalencia de baja avidez IgG anti Toxoplasma gondii y el comportamiento de riesgo para la enfermedad de toxoplasmosis en mujeres que estuvieron embarazadas durante el período 2017-2019 que acudieron al Instituto de Investigaciones en Ciencias de la Salud de la Universidad Nacional de Asunción-Paraguay. Metodología: fueron analizadas 371 fichas de pacientes con serología IgG positiva para toxoplasmosis cuyas muestras fueron procesadas en el Departamento de Producción del Instituto de Investigaciones en Ciencias de la Salud entre los años 2017-2019. Posteriormente, en el año 2020, se realizó 149/371 encuestas digitales de en estas mismas mujeres sobre conocimiento y comportamiento de riesgos para Toxoplasmosis. Resultados: se observó una prevalencia de 18 % de baja avidez para toxoplasmosis. A partir de la encuesta se encontró el 98 % conoce la enfermedad, el 73 % adquirió información durante el embarazo y el 50,3 % recibió orientación de prevención, además, el 65 % refirió como formas de transmisión comer carnes mal cocidas y verduras crudas. En cuanto al comportamiento de riesgo 46 % consume de aguatería, 20 % consume carne a punto medio y 78 % vegetales crudos. El 54 % realiza actividad de cultivo, tienen mascotas como gatos 4,3 %, perros 82 %, además el 9 % refirió dormir con sus mascotas. Conclusión: la prevalencia de baja Avidez en la población estudiada fue del 18 %. Se evidenció algunos comportamientos de riesgo para la toxoplasmosis en las mujeres encuestadas, por lo que se demuestra la necesidad de aplicar programas de prevención primaria en nuestro país.

Introduction: toxoplasmosis is a zoonotic infection caused by Toxoplasma gondii, an intracellular protozoan that can affect children of pregnant women and cause severe sequelae; therefore, serological monitoring should be performed. Objective: to determine the prevalence of low avidity IgG anti-Toxoplasma gondii and the risk behavior for toxoplasmosis disease in pregnant women during the 2017-2019 time period, who attended the Health Sciences Research Institute of the Universidad Nacional de Asuncion - Paraguay. Methodology: a total of 371 patient records with positive IgG serology for toxoplasmosis, whose samples were processed in the Production Department of the Instituto de Investigaciones en Ciencias de la Salud between the years 2017-2019 were analyzed. Subsequently, in 2020, 149/371 digital surveys of the same women were conducted on their knowledge and risk behavior for toxoplasmosis. Results: a low avidity prevalence of 18 % for toxoplasmosis was observed. 98 % knew about the disease, 73 % acquired information during pregnancy, and 50.3 % received preventive orientation. 65 % reported that eating undercooked meat and raw vegetables is a form of disease transmission. Regarding risk behavior, 46 % of the participants consumed poultry, 20 % consumed medium-rare-cooked meat, and 78 % consumed raw vegetables. Fifty-four percent of the patients performed farming activities, 44.3 % had cats as pets, 82 % had dogs, and 9 % slept with their pets. Conclusion: some risk behaviors for toxoplasmosis were evident in the women surveyed, demonstrating the need to implement primary prevention programs in our country.

Prevalencia de baja avidez de Inmunoglobulina G anti Toxoplasma gondii y comportamiento de riesgo para toxoplasmosis en embarazada / Prevalence of low avidity of anti-Toxoplasma gondii immunoglobulin G and risk behavior for toxoplasmosis in pregnant women

Introducción: la toxoplasmosis es una infección zoonótica producida por Toxoplasma gondii, protozoo intracelular que puede afectar al hijo de la mujer embarazada y causar severas secuelas por lo que el monitoreo serológico debe ser realizado. Objetivo: determinar la prevalencia de baja avidez IgG anti Toxoplasma gondii y el comportamiento de riesgo para la enfermedad de toxoplasmosis en mujeres que estuvieron embarazadas durante el período 2017-2019 que acudieron al Instituto de Investigaciones en Ciencias de la Salud de la Universidad Nacional de Asunción-Paraguay. Metodología: fueron analizadas 371 fichas de pacientes con serología IgG positiva para toxoplasmosis cuyas muestras fueron procesadas en el Departamento de Producción del Instituto de Investigaciones en Ciencias de la Salud entre los años 2017-2019. Posteriormente, en el año 2020, se realizó 149/371 encuestas digitales de en estas mismas mujeres sobre conocimiento y comportamiento de riesgos para Toxoplasmosis. Resultados: se observó una prevalencia de 18 % de baja avidez para toxoplasmosis. A partir de la encuesta se encontró el 98 % conoce la enfermedad, el 73 % adquirió información durante el embarazo y el 50,3 % recibió orientación de prevención, además, el 65 % refirió como formas de transmisión comer carnes mal cocidas y verduras crudas. En cuanto al comportamiento de riesgo 46 % consume de aguatería, 20 % consume carne a punto medio y 78 % vegetales crudos. El 54 % realiza actividad de cultivo, tienen mascotas como gatos 4,3 %, perros 82 %, además el 9 % refirió dormir con sus mascotas. Conclusión: la prevalencia de baja Avidez en la población estudiada fue del 18 %. Se evidenció algunos comportamientos de riesgo para la toxoplasmosis en las mujeres encuestadas, por lo que se demuestra la necesidad de aplicar programas de prevención primaria en nuestro país.

Introduction: toxoplasmosis is a zoonotic infection caused by Toxoplasma gondii, an intracellular protozoan that can affect children of pregnant women and cause severe sequelae; therefore, serological monitoring should be performed. Objective: to determine the prevalence of low avidity IgG anti-Toxoplasma gondii and the risk behavior for toxoplasmosis disease in pregnant women during the 2017-2019 time period, who attended the Health Sciences Research Institute of the Universidad Nacional de Asuncion - Paraguay. Methodology: a total of 371 patient records with positive IgG serology for toxoplasmosis, whose samples were processed in the Production Department of the Instituto de Investigaciones en Ciencias de la Salud between the years 2017-2019 were analyzed. Subsequently, in 2020, 149/371 digital surveys of the same women were conducted on their knowledge and risk behavior for toxoplasmosis. Results: a low avidity prevalence of 18 % for toxoplasmosis was observed. 98 % knew about the disease, 73 % acquired information during pregnancy, and 50.3 % received preventive orientation. 65 % reported that eating undercooked meat and raw vegetables is a form of disease transmission. Regarding risk behavior, 46 % of the participants consumed poultry, 20 % consumed medium-rare-cooked meat, and 78 % consumed raw vegetables. Fifty-four percent of the patients performed farming activities, 44.3 % had cats as pets, 82 % had dogs, and 9 % slept with their pets. Conclusion: some risk behaviors for toxoplasmosis were evident in the women surveyed, demonstrating the need to implement primary prevention programs in our country.

Serum biomarkers and anti-flavivirus antibodies at presentation as indicators of severe dengue.

BACKGROUND: Dengue is the most common vector-borne viral disease worldwide. Most cases are mild, but some evolve into severe dengue (SD), with high lethality. Therefore, it is important to identify biomarkers of severe disease to improve outcomes and judiciously utilize resources. METHODS/PRINCIPAL FINDINGS: One hundred forty-five confirmed dengue cases (median age, 42; range <1-91 years), enrolled from February 2018 to March 2020, were selected from an ongoing study of suspected arboviral infections in metropolitan Asunción, Paraguay. Cases included dengue virus types 1, 2, and 4, and severity was categorized according to the 2009 World Health Organization guidelines. Testing for anti-dengue virus IgM and IgG and serum biomarkers (lipopolysaccharide binding protein and chymase) was performed on acute-phase sera in plate-based ELISAs; in addition, a multiplex ELISA platform was used to measure anti-dengue virus and anti-Zika virus IgM and IgG. Complete blood counts and chemistries were performed at the discretion of the care team. Age, gender, and pre-existing comorbidities were associated with SD vs. dengue with/without warning signs in logistic regression with odds ratios (ORs) of 1.07 (per year; 95% confidence interval, 1.03, 1.11), 0.20 (female; 0.05,0.77), and 2.09 (presence; 1.26, 3.48) respectively. In binary logistic regression, for every unit increase in anti-DENV IgG in the multiplex platform, odds of SD increased by 2.54 (1.19-5.42). Platelet count, lymphocyte percent, and elevated chymase were associated with SD in a combined logistic regression model with ORs of 0.99 (1,000/µL; 0.98,0.999), 0.92 (%; 0.86,0.98), and 1.17 (mg/mL; 1.03,1.33) respectively. CONCLUSIONS: Multiple, readily available factors were associated with SD in this population. These findings will aid in the early detection of potentially severe dengue cases and inform the development of new prognostics for use in acute-phase and serial samples from dengue cases.

Revisiting the dengue epidemic of 2011 in Paraguay: molecular epidemiology of dengue virus in the Asuncion metropolitan area.

BACKGROUND: Dengue is one of the most important re-emerging viral diseases and the most common human arthropod-borne viral infection worldwide. Any of the four Dengue virus serotypes (DENV-1 to 4) can cause asymptomatic infections or clinical manifestations that range in severity from a mild, self-limited illness, to a severe disease characterized by a shock syndrome that can lead to death. Paraguay suffers periodic epidemic outbreaks of dengue since 1988 when the DENV-1 was introduced in the country. Epidemics caused by all four serotypes have been reported and the country. Although dengue is endemic in Paraguay, few studies have described the molecular epidemiology of DENV in the country, which is important to understand the local and global spread, as well as the evolution of this pathogen. METHODS: This was a cross-sectional study of a convenience sample. Suspected dengue patients of any age were recruited from the Emergency Laboratory of the Central Hospital of the Institute of Social Welfare, Asuncion, Paraguay, from February to June of 2011. A DENV antigen test was used to confirm the infection. The protein E gene sequences of isolated viruses were sequenced for phylogenetic analysis. RESULTS: Dengue was confirmed in 55.1% of the participants (n = 98/178). The most frequent clinical findings were fever, headache, and myalgia. Identity analyses of the protein E gene sequence of 56 viruses isolated showed the circulation of DENV-1 (n = 45) and DENV-2 (n = 11) in the Asuncion metropolitan area in 2011. Molecular epidemiology analyses suggest that DENV-1 was introduced into Paraguay from Argentina, while the DENV-2 from Brazil, replacing previous virus lineages. CONCLUSIONS: We have analyzed the molecular epidemiology of DENV-1 and DENV-2 isolated in Paraguay in 2011. We found strong evidence that DENV-1 was introduced into Paraguay from Argentina, while the DENV-2 from Brazil, replacing previous virus lineages. Molecular epidemiology studies are of great interest to analyze the dynamic of DENV spread, which are useful for early implementation of containment measures to reduce the risk of explosive epidemics caused by this virus.

Implementation of a Multiplex rRT-PCR for Zika, Chikungunya, and Dengue Viruses: Improving Arboviral Detection in an Endemic Region.

Arboviral diagnosis has been complicated throughout the tropical and subtropical Americas by the recent co-circulation of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV). The aim of this study was to implement a multiplex real-time RT-PCR (rRT-PCR) for ZIKV, CHIKV, and DENV in Paraguay to test patients who were clinically suspected of having dengue. We tested 110 sera from patients who presented to the Hospital de Clínicas in 2016 and had testing for DENV nonstructural protein 1 (NS1; 40 positive and 70 negative). Using a composite reference standard, we confirmed 51 dengue cases (46.4%): 38/40 NS1 positive and 13/70 NS1 negative. Chikungunya virus and ZIKV were detected in one sample each, both were DENV NS1 negative. The NS1 test demonstrated good agreement with rRT-PCR for DENV. However, multiplex rRT-PCR identified a subset of dengue cases and additional arboviral infections that would not be detected if NS1 assays are relied upon for diagnosis.

Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection.

The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/µL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.

Real-time RT-PCR for Mayaro virus detection in plasma and urine.

BACKGROUND: Mayaro virus (MAYV) causes an acute febrile illness which can be difficult to differentiate from dengue or chikungunya. MAYV RNA can be detected in plasma during the first 3-5days of illness, but only a single rRT-PCR has been fully evaluated in the literature. OBJECTIVES: To develop an rRT-PCR for MAYV and evaluate assay performance using human plasma and urine samples spiked with different MAYV strains. STUDY DESIGN: A MAYV rRT-PCR targeting a region of the 5'UTR and nsp1 gene was designed from the alignment of all complete-genome MAYV sequences to be compatible with existing laboratory protocols. The assay was evaluated using human samples spiked with six MAYV strains, including strains from each of the three genotypes. RESULTS: The linear range of the MAYV rRT-PCR extended from 1.0 to 8.0 log10copies/µL, and the lower limit of 95% detection was 8.2copies/µL. No detection was observed when the MAYV rRT-PCR was tested with genomic RNA from related arboviruses. The assay demonstrated linear amplification of all 6 MAYV strains when spiked into human plasma samples as well as 2 strains spiked into urine. CONCLUSIONS: We report the design and evaluation of an rRT-PCR for MAYV. Given the concern for MAYV emergence in the Americas and the few molecular tests that have been evaluated in the literature, this assay should provide a useful diagnostic for patients with an acute febrile illness.