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.

Dengue virus (DENV) has been a major public health concern in Paraguay, with frequent outbreaks occurring since early 1988. Although control measures have been implemented, dengue remains a significant health threat in the country, and continued efforts are required for prevention and control. In response to that, in collaboration with the Central Public Health Laboratory in Asunción, we conducted a portable whole-genome sequencing and phylodynamic analysis to investigate DENV viral strains circulating in Paraguay over the past epidemics. Our genomic surveillance activities revealed the co-circulation of multiple DENV serotypes: DENV-1 genotype V, the emerging DENV-2 genotype III, BR4-L2 clade, and DENV-4 genotype II. Results additionally highlight the possible role of Brazil as a source for the international dispersion of different viral strains to other countries in the Americas emphasizing the need for increased surveillance across the borders, for the early detection and response to outbreaks. This, in turn, emphasizes the critical role of genomic surveillance in monitoring and understanding arbovirus transmission and persistence locally and over long distances.

In recent months, Paraguay has been grappled with a notable monkeypox outbreak, straining its healthcare infrastructure. The sudden spike in cases underlines the imperative need for a comprehensive understanding of the virus’s dynamics, enabling the formulation of robust containment measures. To address this challenge, our team joined forces with the Central Public Health Laboratory of Asunción and the Pan-American Health Organization. Through this collaboration, we employed portable whole-genome sequencing combined with phylodynamic analysis to examine the MPXV strains circulating in Paraguay. Our genomic monitoring approach has produced the first 30 whole-genome sequences from Paraguay, all of which were identified under lineage IIb. Interestingly, our data suggest that the origin of the monkeypox virus in Paraguay at the beginning of 2022 can be traced back to Brazil. This introduction subsequently catalyzed further community spread that was further exacerbated by several independent introduction events as time progressed. These findings not only shed light on the transmission patterns of the virus but also highlight the pivotal role such insights play in sculpting effective response strategies and driving impactful public health measures. Furthermore, our findings strongly advocate intensified surveillance at international borders, ensuring swift detection and proactive countermeasures against potential outbreaks in the future.

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.