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.

Linear epitope mapping in the E and NS1 proteins of dengue and Zika viruses: Prospection of peptides for vaccines and diagnostics.

The arrival of the Zika virus (ZIKV) in dengue virus (DENV)-endemic areas has posed challenges for both differential diagnosis and vaccine development. Peptides have shown promise in addressing these issues. The aim of this study was to identify the linear epitope profile recognized by serum samples from dengue and Zika patients in the E and NS1 proteins of DENV and ZIKV. This cross-sectional study included individuals of all ages with laboratory-confirmed DENV and ZIKV infections, who were selected through convenience sampling. The serum samples from dengue and Zika patients detected epitopes evenly distributed across the viral proteins in a peptide microarray platform. However, several epitopes were located within "epitope hotspots", characterized by clusters of peptides recognized in more than 30% of the sub-arrays analyzed using individual or pooled serum samples. The serum samples from dengue and Zika patients showed a high level of cross-reactivity with peptides in the DENV and ZIKV proteins. Analysis using an additional peptide microarray platform, which contained peptides selected based on the results of the initial screening, revealed that two DENV and one ZIKV peptide, highly specific to their related viruses, were located within the epitope hotspots; however, they presented low detection rates (32.5, 35.0, and 28.6%, respectively). In addition, two DENV peptides detected at similarly high rates by both dengue and Zika patients were also found within the epitope hotspots. These hotspots contain several immunodominant epitopes that are recognized by a larger number of individuals when compared to 15-amino acid (aa) sequence peptides. Thus, epitope hotspots may have greater potential to serve as antigens in diagnostic tests and vaccine development than peptides composed of only 15 amino acids.

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.

Detection of neutralizing antibodies against flaviviruses in free-ranging birds, Paraguay (2016-2018).

Saint Louis encephalitis virus (SLEV), West Nile virus (WNV) and Ilheus virus (ILHV) are flaviviruses maintained by enzootic transmission networks between mosquitoes and birds. They have been detected in South America, with no records for Paraguay. We detected the presence of neutralizing antibodies for SLEV, WNV and ILHV in free-ranging birds collected in Paraguay (2016-2018). Four positive samples were detected in resident birds: one SLEV (rufous-bellied thrush), one WNV (barred antshrike) and two ILHV (white-tipped dove and shiny cowbird). These results bring new information about enzootic activity of flaviviruses in Paraguay.

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.

Implementación de un sistema de detección de flavivirus en mosquitos / Implementation of a flavivirus detection system in mosquitoes

Los Flavivirus constituyen virus transmitidos por artrópodos, principalmente mosquitos. Pueden producir enfermedades en humanos y animales, también incluyen virus específicos de insectos que solo infectan y se replican en los insectos, no así en vertebrados. En Paraguay los virus dengue, fiebre amarilla y Zika fueron detectados en infecciones humanas, pero los estudios de flavivirus en mosquitos son aún escasos. Por ello, el objetivo del presente estudio fue implementar un sistema de detección de flavivirus en mosquitos en el IICS-UNA. Primero, se organizaron capacitaciones en colecta, preparación de pools y procesamiento por técnicas de RT-PCRs convencionales realizadas por expertos internacionales a profesionales locales (bioquímicos y biólogos). Además, se implementaron planillas de registro de datos y de control de transporte de muestras de los lugares de colectas hasta el IICS-UNA. Se prepararon en total 201 pools de 1 a 35 mosquitos cada uno agrupados por especie, localidad, entre otros criterios. Para asegurar la integridad del RNA extraído se realizó la detección de un control interno (Actina-1), siendo todos los pools positivos para el mismo, 91/201 pools fueron positivos para flavivirus. Se realizó la secuenciación de 19/91 pools positivos para flavivirus identificándose flavivirus de insectos (detectándose principalmente Culex Flavivirus, cell fusing agents Flavivirus y Kamiti river virus), evidenciando la elevada distribución de estos virus. Estos resultados demuestran que fue factible implementar el sistema de detección de flavivirus en mosquitos, lo cual podría contribuir a fortalecer la vigilancia y control de estas virosis, así como el conocimiento sobre la importancia ecológica de flavivirus de insectos

Flaviviruses are viruses transmitted by arthropods, mainly mosquitoes. They can cause diseases in humans and animals, they also include specific insect viruses that only infect and replicate in insects, not in vertebrates. In Paraguay, dengue, yellow fever, and Zika viruses were detected in human infections, but studies of flaviviruses in mosquitoes are still scarce. Therefore, the objective of the present study was the implementation of a flavivirus detection system in mosquitoes at IICS-UNA. First, trainings on collection, pool preparation and processing by conventional RT-PCR techniques were organized by international experts for local professionals (biochemists and biologists). In addition, data log sheets and sample transport control forms from the collection sites to the IICS were implemented. A total of 201 pools of 1 to 35 mosquitoes were prepared, each grouped by species, locality, among others. To ensure the integrity of the extracted RNA, an internal control (Actin-1) detection was performed, all pools being positive for it; 91/201 pools were positive for flaviviruses. The sequencing of 19/91 pools positive for flavivirus was carried out, identifying flavivirus in all cases of insects (mainly detecting Culex Flavivirus, cell fusing agents Flavivirus and Kamiti river virus), evidencing the high distribution of these viruses. These results demonstrate that it was feasible to implement the flavivirus detection system in mosquitoes, which could contribute to strengthen the detection, surveillance and control of these viruses, as well as, the knowledge about the ecological importance of insect flaviviruses

Comparison of Anti-Dengue and Anti-Zika IgG on a Plasmonic Gold Platform with Neutralization Testing.

Antibody cross-reactivity confounds testing for dengue virus (DENV) and Zika virus (ZIKV). We evaluated anti-DENV and anti-ZIKV IgG detection using a multiplex serological platform (the pGOLD assay, Nirmidas, Palo Alto, CA) in patients from the Asunción metropolitan area in Paraguay, which experiences annual DENV outbreaks but has reported few autochthonous ZIKV infections. Acute-phase sera were tested from 77 patients who presented with a suspected arboviral illness from January to May 2018. Samples were tested for DENV and ZIKV RNA by real-time reverse transcription-PCR, and for DENV nonstructural protein 1 with a lateral-flow immunochromatographic test. Forty-one patients (51.2%) had acute dengue; no acute ZIKV infections were detected. Sixty-five patients (84.4%) had anti-DENV-neutralizing antibodies by focus reduction neutralization testing (FRNT50). Qualitative detection with the pGOLD assay demonstrated good agreement with FRNT50 (kappa = 0.74), and quantitative results were highly correlated between methods (P < 0.001). Only three patients had anti-ZIKV-neutralizing antibodies at titers of 1:55-1:80, and all three had corresponding DENV-neutralizing titers > 1:4,000. Hospitalized dengue cases had significantly higher anti-DENV IgG levels (P < 0.001). Anti-DENV IgG results from the pGOLD assay correlate well with FRNT, and quantitative results may inform patient risk stratification.

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.

Real-time RT-PCR for the detection and quantitation of Oropouche virus.

Oropouche virus (OROV) causes an acute, systemic febrile illness, and in certain regions of South America, this represents the second most common human arboviral infection after dengue virus. A new real-time RT-PCR was developed for OROV and reassortant species. The new OROV rRT-PCR proved linear across 6-7 orders of magnitude with a lower limit of 95% detection of 5.6-10.8 copies/µL. Upon testing dilutions of OROV and Iquitos virus reference genomic RNA, all dilutions with >10 copies/µL were detected in both the OROV rRT-PCR and a comparator molecular assay, but the OROV rRT-PCR detected more samples with ≤10 copies/µL (8/14 vs 0/13, respectively, P = 0.002). In a set of 100 acute-phase clinical samples from Paraguay patients with a suspected arboviral illness, no patients tested positive for OROV RNA using either assay. The OROV rRT-PCR provides a sensitive molecular assay for the study of this important yet neglected tropical arboviral infection.

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.

Alphaviruses: Serological Evidence of Human Infection in Paraguay (2012-2013).

INTRODUCTION: Alphaviruses can produce febrile illness and encephalitis in dead-end hosts such as horses and humans. Within this genus, the Venezuelan Equine Encephalitis virus (VEEV) complex includes pathogenic epizootic subtypes and enzootic subtypes that are not pathogenic in horses (except subtype IE, Mexican strains), although they can cause febrile symptoms in humans. The Rio Negro virus (RNV-VEEV subtype VI) circulates in Argentina, where it was associated with undifferentiated febrile illness. Mayaro (MAYV) and Una (UNAV) viruses belong to a different group, the Semliki Forest virus complex, with confirmed circulation. OBJECTIVE: The present study aimed to determine RNV, MAYV, and UNAV seroprevalences by plaque reduction neutralization test in 652 samples of Paraguayan individuals mainly from the Central Department, between years 2012 and 2013. METHODS: Samples with antibodies titer >1:20 against RNV were also tested for Mosso das Pedras-subtype IF, subtype IAB, and Pixuna (PIXV)-subtype IV viruses that belongs to VEEV antigenic complex. RESULTS: The overall seroprevalence of RNV was 3.83%, and for UNAV it was 0.46%, and no neutralizing antibodies were detected against MAYV in the studied population. Two of the twenty-seven heterotypic samples were positive for PIXV. The 50.1% of neutralizing antibody titers against RNV were high (equal to or greater than 1/640), suggesting recent infections. The effect of age on the prevalence of RNV was negligible. CONCLUSIONS: These results bring new information about neglected alphaviruses in South America, and these data will serve as the basis for future studies of seroprevalence of other VEEV, and studies to search potential hosts and vectors of these viruses in the region.

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.

Búsqueda de flavivirus en individuos con sospecha clínica de dengue y resultado negativo para el antígeno NS1 en Paraguay / Search of flavivirus in individuals with clinical suspicion of dengue and negative for the NS1 antigen in Paraguay

Los flavivirus son responsables de una considerable morbi-mortalidad a nivel mundial. Entre ellos, el virus del dengue (DENV) es causante de graves problemas de salud pública en Paraguay. El objetivo del estudio fue detectar infecciones por flavivirus a través de una reacción de RT-nested PCR genérica para flavivirus en 195 muestras de individuos con sospecha de dengue, negativos por el test inmunocromatográfico (antígeno NS1 ­ DENV), provenientes del área metropolitana de Asunción entre 2011 y 2013. Las muestras positivas para flavivirus fueron sometidas a dos reacciones de RT-nested PCRs específicas para DENV. El límite de detección (LD) para flavivirus fue de 0,2 UFP/reacción. En total 43/195 muestras fueron positivas para flavivirus. De estas, 38/43 (88,4%) correspondieron a DENV (6 DENV-1, 30 DENV-2 y 2 DENV-3). Además, 5/43 casos (11,6%) positivos para flavivirus fueron negativos para DENV por ambas reacciones específicas, pudiendo deberse a infecciones por otros flavivirus. Los resultados sugieren que la utilización de una reacción genérica seguida de otras reacciones específicas para DENV en casos febriles negativos para NS1 por el método inmunocromatográfico permitiría detectar más casos de infecciones por DENV y además, podría contribuir a la identificación de casos debido a infecciones por otros flavivirus.

Flaviviruses are responsible for considerable worldwide morbidity and mortality. Among them, the dengue virus (DENV) causes serious public health problems in Paraguay. The objective of the study was to detect flavivirus infections using a generic RT-nested -PCR in 195 samples of individuals with suspected dengue and negative for the inmunochromatographic test (NS1 antigen ­ DENV), from the metropolitan area of Asuncion between 2011 and 2013. The flavivirus-positive samples were subjected to two reactions of DENV-specific RT-nested PCRs. The detection limit (DL) for flavivirus was 0.2 PFU / reaction. In total, 43/195 samples were positive for flavivirus. Of them, 38/43 (88,4%) corresponded to DENV (6 DENV-1, 30 DENV-2 and 2 DENV-3). In addition, 5/43 cases (11.6%) positive for flavivirus were negative for DENV by both specific reactions, and may be infections caused by other flaviviruses. The results suggest that the use of a generic reaction followed by other DENV specific reactions in febrile negative cases for NS1 by the immunochromatographic method would allow the detection of more cases of DENV infections and could contribute to the identification of cases due to infections by others flaviviruses.

Desempeño del kit ELISA chagas IICS V.1 para el diagnóstico de la enfermedad de chagas / ELISA chagas test IICS V.1 evaluation for the diagnosis of chagas disease

En Paraguay, el tamizaje serológico para la enfermedad de Chagas en bancos de sangre es necesario, por lo cual es importante un método diagnóstico con alta sensibilidad. El ELISA Chagas test IICS V.1 es un ELISA indirecto sensibilizados con antígeno soluble de epimastigote de T.cruzide la cepa Ypsilon. El objetivo del presente trabajo fue evaluar el desempeño del ELISA Chagas test IICS V.1 en comparación con kits comerciales, ademásanalizar los resultados de la evaluación externa e interna de calidad del kit. En este estudio observacional de prueba diagnóstica se analizaron 56 muestras de suerospositivos y negativos para antígenos de Trypanosoma cruzi, testados por el ELISA BiosChile, obteniéndose una concordancia excelente entre el ELISA Chagas test IICS V.1y los kits comerciales: Chagatest ELISA-Wiener, con Índice kappa: 0,89 IC de 95% (0,76-1) y Test ELISA para Chagas III-Grupo BiosChile con Índice kappa: 0,92 IC 95% (0,82-1).En la evaluación externa de calidad realizada por la Fundação Pró-Sangue/Hemocentro de São Paulo, Brasil en el periodo 2001 al 2012 se analizaron 450 muestras: 372 negativas y 78 positivas para T. cruzi, obteniéndose en dicha evaluación la calificación "A" que indica ausencia de falsos positivos y negativos. Además, en el mismo periodo los valores del control interno se encontraron dentro del rango permitido de ±2DS. Los resultados obtenidos en el estudio demuestran la alta calidad de este test de producción nacional, que sumado al bajo costo del mismo, pueden ser utilizados en trabajos de campo, donde no necesita de instrumentación y las lecturas pueden realizarse a simple vista, constituyendo una herramienta válida y útil para el apoyo al diagnóstico de la enfermedad de Chagas.

In Paraguay, the serological screening for Chagas disease is mandatory in pregnant women and blood banks, therefore a high sensitivity diagnostic method is required. The aimof this study was to evaluate the ELISA Chagas test IICS V.1 by comparison with commercial kits and to analyze the external and internal quality evaluation results. In this descriptive observational study, 56 seropositive and seronegative to Trypanosoma cruzisamples were analyzed, obtaining an excellent concordance between the ELISA Chagas test IICS V.1 and these commercial kits: Chagatest ELISA-Wiener, Argentina (kappa index:0.89) and Test ELISA Chagas III-Grupo Bios, Chile (kappa index: 0.92).In the externalquality assessment carried out by the Fundação Pró-Sangue /Blood Center of São Paulo,Brazil in the period 2001 to 2012, 450 samples were analyzed: 372 seronegative and 78seropositive for T. cruzi. In this evaluation, an “A” score was obtained indicating theabsence of false positives and negatives. Additionally, in the same period of time theinternal control values were within the accepted range of ± 2SD, with a confidence intervalof 95%. The results obtained in the present study demonstrate the high quality of this locally produced test which added to its low cost, making it a valid and useful tool tosupport the diagnosis of Chagas disease.

Perfil clínico, hematológico y serológico en pacientes con sospecha de dengue del IICS-UNA, 2009-2013 / Clinical, hematological and serological profile in suspected dengue patients at IICS-UNA, 2009-2013

El dengue constituye una de las enfermedades transmitidas por mosquitos más importante a nivel mundial. La enfermedad puede cursar con un cuadro asintomático, presentarse con un amplio rango de manifestaciones clínicas inespecíficas o cierto porcentaje puede derivar en casos graves. Este estudio observacional descriptivo de corte transverso tuvo como objetivo determinar las características clínicas, parámetros hematológicos y presencia de IgM en 92 pacientes que acudieron al IICS-UNA con sospecha clínica de dengue en el periodo 2009 al 2013. Se utilizó el MAC-ELISA desarrollado en el IICS-UNA, se registraron los datos clínicos-epidemiológicos a través de una encuesta y se determinaron los parámetros hematológicos. Se obtuvieron resultados positivos para IgM en 51/92 (55%) pacientes y resultados negativos en 41/92 (45%). Las características clínicas más frecuentes fueron: fiebre, cefalea, mialgias y artralgias. Entre los pacientes con IgM positiva, 14/51 (27%) manifestaron dolores abdominales, 19/51 (37%) reportaron letargo o postración y 20/51 (39%) declararon tener náuseas y/o vómitos, 4/51 (8%) presentaron leucopenia, 10/51 (20%) valores de hematocrito disminuido y 6/51 (12%) plaquetopenia. Sólo 13/92 (14%) pacientes declararon haber cursado con la enfermedad anteriormente. Del total de pacientes, 4/92 (4%) manifestaron haber presentado algún tipo de hemorragia. Los resultados obtenidos en el estudio refuerzan la importancia de integrar todos los parámetros posibles: detección de IgM, perfil hematológico y la clínica del paciente con sospecha de dengue para brindar un mejor diagnóstico. Así también, es necesario resaltar en cuanto a los signos de alarma para una intervención rápida a fin de evitar complicaciones.

Dengue is one of the most important mosquito-borne diseases worldwide. The disease may present as asymptomatic, with a wide range of non-specific clinical manifestations or acertain percentage can result in severe cases. This observational, descriptive, crosssectional study aimed to determine the clinical features, hematological parameters and the presence of IgM in 92 patients who attended to IICS-UNA with clinical suspicion of denguefrom 2009 to 2013. The MAC-ELISA developed at IICS-UNA was used, clinical and epidemiological data were recorded through a survey and hematological parameters were determined. Positive results for IgM in 51/92 (55%) patients were obtained and negative results in 41/92 (45%). The most frequent clinical features were fever, headache, muscleand joint pains. Among patients with positive IgM, 14/51 (27%) reported abdominal pain,19/51 (37%) reported lethargy or prostration and 20/51 (39%) reported having nausea and/or vomiting, 4/51(8%) had leucopenia, 10/51 (20%) decreased hematocrit values and6/51 (12%) presented thrombocytopenia. Only 13/92 (14%) patients reported a previous DENV infection. Of the total, 4/92 (4%) presented some type of bleeding. The results of thestudy reinforce the importance of integrating all possible parameters: IgM detection, hematological and clinical profile of patients with suspected dengue to provide better diagnosis. It is also necessary to emphasize warning signs for a quick intervention to avoid complications.

[Clinical and epidemiological aspects of toxoplasmosis in patients with vision problems]. / Aspectos clínico-epidemiológicos de la toxoplasmosis en pacientes que consultan por problemas de visión.

INTRODUCTION: Toxoplasmosis is a worldwide disease; it can cause decreased vision or even blindness. The route of transmission in humans may vary according to the habits of the region; probably the ingestion of raw or undercooked meat is the main source of infection. OBJECTIVE: To determine the seroprevalence of toxoplasmosis in an eye clinic, the frequency of ocular toxoplasmosis (OT) and risk habits for acquiring the infection. MATERIALS AND METHODS: Adult patients consulting in the Retina Department of the Teaching Hospital of the National University of Asuncion, Paraguay between August and September, 2014 were included. Prior informed consent, socio-demographic and epidemiological data related to T. gondii infection were obtained. In addition a blood sample for the determination of anti T. gondii IgG antibodies by the ELISA method was taken and ophthalmologic evaluation for the diagnosis of OT was made. RESULTS: A total of 80 patients with mean ± SD age of 53 ± 20 years were studied, with slight predominance of women (55%). The seroprevalence of toxoplasmosis was 84% (67/80) and OT was detected in 8.9% of the 67 seropositive persons. The habit of not washing vegetables with sodium hypochlorite and eat meat from wild animals was related to higher risk of infection in this population. CONCLUSION: It is important to conduct research at the population level to establish the epidemiology of toxoplasmosis in our country. Information on prophylactic measures to prevent infection by T. gondii should be given to the population.