Background In 2018, Paraguay experienced a large dengue virus (DENV) outbreak. The primary objective of this study was to characterize dengue cases in the Central Department, where the majority of cases occur, and identify factors associated with DENV infection. Methods Patients were enrolled from January-May 2018 if they presented with a suspected arboviral illness. Acute-phase specimens (≤8 days after symptom onset) were tested using rRT-PCR, a rapid diagnostic test for DENV nonstructural protein 1 (NS1) and anti-DENV IgM and IgG, and ELISA for IgG against NS1 from Zika virus (ZIKV). Results A total of 231 patients were enrolled (95.2% adults) at two sites: emergency care and an outpatient clinical site. Patients included 119 (51.5%) dengue cases confirmed by rRT-PCR (n = 115, 96.6%) and/or the detection of NS1 and anti-DENV IgM (n = 4, 3.4%). DENV-1 was the predominant serotype (109/115, 94.8%). Epidemiologically, dengue cases and non-dengue cases were similar, though dengue cases were less likely to reside in a house/apartment or report a previous dengue case. Clinical and laboratory findings associated with dengue included red eyes, absence of sore throat, leucopenia and thrombocytopenia. At an emergency care site, 26% of dengue cases (26/100) required hospitalization. In univariate analysis, hospitalization was associated with increased viral load, anti-DENV IgG, and thrombocytopenia. Among dengue cases that tested positive for IgG against ZIKV NS1, the odds of DENV NS1 detection in the acute phase were decreased 10-fold (OR 0.1, 0.0–0.3). Conclusions Findings from a predominantly adult population demonstrate clinical and laboratory factors associated with DENV infections and the potential severity of dengue in this group. The combination of viral load and specific IgG antibodies warrant further study as a prognostic to identify patients at risk for severe disease.

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) 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.

SARS-CoV-2 variant detection relies on resource-intensive whole-genome sequencing methods. We sought to develop a scalable protocol for variant detection and surveillance in Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of 201 acute-phase nasopharyngeal samples were included. Samples were positive for the SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore) sequencing on a subset of samples with confirmed variant lineages. Samples had a mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%), which was consistent with the P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%), and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). The results were confirmed using Sanger sequencing in 181/181 samples, and variant calls were consistent with Nanopore sequencing in 29/29 samples. The Spike SNP assay could improve population-level surveillance for mutations associated with SARS-CoV-2 variants and inform the judicious use of sequencing resources.

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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.

RNA extraction is an essential step for detection and surveillance of common viral pathogens. Currently, sample processing and RNA extraction are costly and rely on proprietary materials that are difficult to acquire, maintain, and safely discard in low-resource settings. We developed an economical RNA extraction and storage protocol that eliminates the use of instrumentation, expensive materials, and cold chain requirements. Through an iterative process, we optimized viral lysis and RNA binding to and elution from glass fiber membranes. Protocol changes were evaluated by testing eluates in virus-specific real-time RT-PCRs (rRT-PCRs). Efficient, non-toxic viral lysis was achieved with a sucrose buffer including KCl, proteinase K and carrier RNA. Glass fiber membranes demonstrated concentration-dependent RNA binding of three arthropod-borne RNA viruses (arboviruses): dengue, chikungunya and Oropouche. Membrane binding was significantly increased in an acidic arginine binding buffer. For the clinical evaluation, 36 dengue virus (DENV)-positive serum samples were extracted in duplicate in the optimized protocol and results were compared to a commercial method. DENV RNA was successfully extracted from 71/72 replicates (98.6%) in the extraction packets, and rRT-PCR Ct values correlated between the techniques. Five clinical samples were selected to evaluate ambient-temperature storage up to 7 days on dried glass fiber membranes. DENV RNA was stable at 1, 3 and 7 days post extraction, with a mean difference in eluate RNA concentration of 0.14 log10 copies/uL. At a cost of $0.08 /sample, RNA extraction and storage packets address key limitations to available protocols and may increase capacity for molecular detection of RNA viruses. Keywords: ribonucleic acid, extraction, dengue virus, molecular testing

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.

El cáncer de cuello uterino es el segundo cáncer femenino más común a nivel mundial. El agente causal es el virus de papiloma humano (VPH). Se han identificado 13 tipos de virus de papiloma humano de alto riesgo oncogénico (VPH-AR), entre los cuales el VPH 16 y VPH 18 son los más frecuentemente detectados en cáncer de cuello uterino, siendo en Paraguay detectados en el 70% de casos de cáncer invasor. Por ello, el objetivo fue estandarizar y determinar el límite de detección de una técnica de PCR convencional para la detección de VPH 16 y 18. Para la detección de ADN de VPH 16 y 18, se observaron mejores resultados con 2mM de MgCl 2 y 60°C para la temperatura de alineamiento. El límite de detección para las PCR fue de 14,6x10-11 ng/µL para VPH 16 y 21,7x10-12 ng/µL para VPH 18. Este trabajo servirá de base a otros estudios de detección e identificación de estos tipos virales por PCR, con miras a identificar un grupo de mujeres positivas para VPH-AR que poseen mayor riesgo de desarrollo de lesión y cáncer de cuello uterino y precisan de un seguimiento más cercano.