A multiplex-NGS approach to identifying respiratory RNA viruses during the COVID-19 pandemic.

A methodological approach based on reverse transcription (RT)-multiplex PCR followed by next-generation sequencing (NGS) was implemented to identify multiple respiratory RNA viruses simultaneously. A convenience sampling from respiratory surveillance and SARS-CoV-2 diagnosis in 2020 and 2021 in Montevideo, Uruguay, was analyzed. The results revealed the cocirculation of SARS-CoV-2 with human rhinovirus (hRV) A, B and C, human respiratory syncytial virus (hRSV) B, influenza A virus, and metapneumovirus B1. SARS-CoV-2 coinfections with hRV or hRSV B and influenza A virus coinfections with hRV C were identified in adults and/or children. This methodology combines the benefits of multiplex genomic amplification with the sensitivity and information provided by NGS. An advantage is that additional viral targets can be incorporated, making it a helpful tool to investigate the cocirculation and coinfections of respiratory viruses in pandemic and post-pandemic contexts.

Transmission cluster of COVID-19 cases from Uruguay: emergence and spreading of a novel SARS-CoV-2 ORF6 deletion.

BACKGROUND: Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES: We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS: Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS: We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS: Our findings provide evidence for the origin and spread of deletion variants and emphasise indels' importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

Incidence of Guillain-Barré syndrome in an Uruguayan population. A prospective cohort study.

Guillain-Barre syndrome (GBS) is an acute autoimmune peripheral inflammatory neuropathy and the most frequent cause of non-poliovirus acute flaccid paralysis worldwide. Background annual GBS incidence rates (IRs) in Latin America (LA) varies from 0.40 to 2.12/100000 persons per year. We performed a prospective population-based epidemiological study to determine the incidence and clinical profile of GBS in the most densely populated regions in Uruguay. The incidence of GBS in the population living in Montevideo and Canelones was studied in the period between June 01, 2018 and May 31, 2020. Patients older than 16 years of age diagnosed with GBS were prospectively enrolled. The mean global annual IR in the Uruguayan population was 1.7/100000 persons (95% CI 1.25-2.25). The highest rate was observed in the 65 to 74 age group among men (5.25/100000 per year) and in the 55 to 64 age group among women (2/100.000 per year). The mean age was 53.9 ± 19.5, years, without difference by sex (53.5 women, 54.5 men). The in-hospital mortality rate was 5.8%. A total of 51 patients were diagnosed with GBS: 42 (82%) had typical GBS, 5 (10%) Miller-Fisher syndrome (MFS), 3 (7%) a bilateral facial nerve palsy, 1 patient had a GBS-MFS overlap (2.3%). This is the first population-based GBS incidence study in LA using a prospective design. Our IR can be a useful tool in establishing the background rate to examine future disease trends caused by the introduction of new viruses or vaccines in Uruguay.

Genomic and eco-epidemiological investigations in Uruguay reveal local Chikungunya virus transmission dynamics during its expansion across the Americas in 2023.

Uruguay experienced its first Chikungunya virus outbreak in 2023, resulting in a significant burden to its healthcare system. We conducted analysis based on real-time genomic surveillance (30 novel whole genomes) to offer timely insights into recent local transmission dynamics and eco-epidemiological factors behind its emergence and spread in the country.

HIV Drug Resistance in Adults Initiating or Reinitiating Antiretroviral Therapy in Uruguay-Results of a Nationally Representative Survey, 2018-2019.

The first nationally representative cross-sectional HIV drug resistance (HIVDR) survey was conducted in Uruguay in 2018-2019 among adults diagnosed with HIV and initiating or reinitiating antiretroviral therapy (ART). Protease, reverse transcriptase, and integrase genes of HIV-1 were sequenced. A total of 206 participants were enrolled in the survey; 63.2% were men, 85.7% were >25 years of age, and 35.6% reported previous exposure to antiretroviral (ARV) drugs. The prevalence of HIVDR to efavirenz or nevirapine was significantly higher (OR: 1.82, p < 0.001) in adults with previous ARV drug exposure (20.3%, 95% CI: 18.7-22.0%) compared to adults without previous ARV drug exposure (12.3%, 11.0-13.8%). HIVDR to any nucleoside reverse transcriptase inhibitors was 10.3% (9.4-11.2%). HIVDR to ritonavir-boosted protease inhibitors was 1.5% (1.1-2.1%); resistance to ritonavir-boosted darunavir was 0.9% (0.4-2.1%) among adults without previous ARV drug exposure and it was not observed among adults with previous ARV drug exposure. Resistance to integrase inhibitors was 12.7% (11.7-13.8%), yet HIVDR to dolutegravir, bictegravir, and cabotegravir was not observed. The high level (>10%) of HIVDR to efavirenz highlights the need to accelerate the transition to the WHO-recommended dolutegravir-based ART. Access to dolutegravir-based ART should be prioritised for people reporting previous ARV drug exposure.

Genomic and eco-epidemiological investigations in Uruguay reveal local Chikungunya virus transmission dynamics during its expansion across the Americas in 2023.

Uruguay experienced its first Chikungunya virus outbreak in 2023, resulting in a significant burden to its healthcare system. We conducted analysis based on real-time genomic surveillance (30 novel whole genomes) to offer timely insights into recent local transmission dynamics and eco-epidemiological factors behind its emergence and spread in the country.

Genomics-based timely detection of Dengue Virus type I genotypes I and V in Uruguay.

We report the first whole-genome sequences of Dengue Virus type I genotypes I and V from Uruguay, including the first cases ever reported in the country. Through timely genomic analysis, identification of these genotypes was possible, aiding in timely public health responses and intervention strategies to mitigate the impact of dengue outbreaks.

End-of-season influenza vaccine effectiveness during the Southern Hemisphere 2022 influenza season - Chile, Paraguay, and Uruguay.

OBJECTIVES: This study estimated the 2022 end-of-season influenza vaccine effectiveness (VE) against severe acute respiratory illness (SARI) hospitalization in Chile, Paraguay, and Uruguay. METHODS: We pooled surveillance data from SARI cases in 18 sentinel surveillance hospitals in Chile (n = 9), Paraguay (n = 2), and Uruguay (n = 7) from March 16-November 30, 2022. VE was estimated using a test-negative design and logistic regression models adjusted for country, age, sex, presence of ≥1 comorbidity, and week of illness onset. VE estimates were stratified by influenza virus type and subtype (when available) and influenza vaccine target population, categorized as children, individuals with comorbidities, and older adults, defined per countries' national immunization policies. RESULTS: Among the 3147 SARI cases, there were 382 (12.1%) influenza test-positive case patients; 328 (85.9%) influenza case patients were in Chile, 33 (8.6%) were in Paraguay, and 21 (5.5%) were in Uruguay. In all countries, the predominant subtype was influenza A(H3N2) (92.6% of influenza cases). Adjusted VE against any influenza-associated SARI hospitalization was 33.8% (95% confidence interval: 15.3%, 48.2%); VE against influenza A(H3N2)-associated SARI hospitalization was 30.4% (95% confidence interval: 10.1%, 46.0%). VE estimates were similar across target populations. CONCLUSION: During the 2022 influenza season, influenza vaccination reduced the odds of hospitalization among those vaccinated by one-third. Health officials should encourage influenza vaccination in accordance with national recommendations.

A Multiplex Nanopore Sequencing Approach for the Detection of Multiple Arboviral Species.

The emergence and continued geographic expansion of arboviruses and the growing number of infected people have highlighted the need to develop and improve multiplex methods for rapid and specific detection of pathogens. Sequencing technologies are promising tools that can help in the laboratory diagnosis of conditions that share common symptoms, such as pathologies caused by emerging arboviruses. In this study, we integrated nanopore sequencing and the advantages of reverse transcription polymerase chain reaction (RT-PCR) to develop a multiplex RT-PCR protocol for the detection of Chikungunya virus (CHIKV) and several orthoflaviviruses (such as dengue (Orthoflavivirus dengue), Zika (Orthoflavivirus zikaense), yellow fever (Orthoflavivirus flavi), and West Nile (Orthoflavivirus nilense) viruses) in a single reaction, which provides data for sequence-based differentiation of arbovirus lineages.

Emergence and spreading of the largest SARS-CoV-2 deletion in the Delta AY.20 lineage from Uruguay.

The genetic variability of SARS-CoV-2 (genus Betacoronavirus, family Coronaviridae) has been scrutinized since its first detection in December 2019. Although the role of structural variants, particularly deletions, in virus evolution is little explored, these genome changes are extremely frequent. They are associated with relevant processes, including immune escape and attenuation. Deletions commonly occur in accessory ORFs and might even lead to the complete loss of one or more ORFs. This scenario poses an interesting question about the origin and spreading of extreme structural rearrangements that persist without compromising virus viability. Here, we analyze the genome of SARS-CoV-2 in late 2021 in Uruguay and identify a Delta lineage (AY.20) that experienced a large deletion (872 nucleotides according to the reference Wuhan strain) that removes the 7a, 7b, and 8 ORFs. Deleted viruses coexist with wild-type (without deletion) AY.20 and AY.43 strains. The Uruguayan deletion is like those identified in Delta strains from Poland and Japan but occurs in a different Delta clade. Besides providing proof of the circulation of this large deletion in America, we infer that the 872-deletion arises by the consecutive occurrence of a 6-nucleotide deletion, characteristic of delta strains, and an 866-nucleotide deletion that arose independently in the AY.20 Uruguayan lineage. The largest deletion occurs adjacent to transcription regulatory sequences needed to synthesize the nested set of subgenomic mRNAs that serve as templates for transcription. Our findings support the role of transcription sequences as a hotspot for copy-choice recombination and highlight the remarkable dynamic of SARS-CoV-2 genomes.

Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genes.

Deletions frequently occur in the six accessory genes of SARS-CoV-2, but most genomes with deletions are sporadic and have limited spreading capability. Here, we analyze deletions in the ORF7a of the N.7 lineage, a unique Uruguayan clade from the Brazilian B.1.1.33 lineage. Thirteen samples collected during the early SARS-CoV-2 wave in Uruguay had deletions in the ORF7a. Complete genomes were obtained by Illumina next-generation sequencing, and deletions were confirmed by Sanger sequencing and capillary electrophoresis. The N.7 lineage includes several individuals with a 12-nucleotide deletion that removes four amino acids of the ORF7a. Notably, four individuals underwent an additional 68-nucleotide novel deletion that locates 44 nucleotides downstream in the terminal region of the same ORF7a. The simultaneous occurrence of the 12 and 68-nucleotide deletions fuses the ORF7a and ORF7b, two contiguous accessory genes that encode transmembrane proteins with immune-modulation activity. The fused ORF retains the signal peptide and the complete Ig-like fold of the 7a protein and the transmembrane domain of the 7b protein, suggesting that the fused protein plays similar functions to original proteins in a single format. Our findings evidence the remarkable dynamics of SARS-CoV-2 and the possibility that single and consecutive deletions occur in accessory genes and promote changes in the genomic organization that help the virus explore genetic variations and select for new, higher fit changes.

Phylogenetic analysis of pandemic 2009 influenza A virus circulating in the South American region: genetic relationships and vaccine strain match.

The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 influenza A virus strain (H1N1pdm). Understanding the evolution of H1N1pdm strains within the South American region is essential for studying global diversification, emergence and resistance, as well as determining vaccine efficacy. In order to gain insight into these matters, phylogenetic analysis was performed using 29 hemagglutinin (HA) gene sequences from H1N1pdm strains isolated in South America. The results of these studies revealed that clade 7 was the dominant H1N1pdm lineage in South America. None of the strains isolated in South America clustered together with the 2010 H1 vaccine strain. Amino acid substitutions P100S, S220T and I338V were found in almost all HAs of South American H1N1pdm strains.

A deletion in SARS-CoV-2 ORF7 identified in COVID-19 outbreak in Uruguay.

The analysis of genetic diversity in SARS-CoV-2 is the focus of several studies, providing insights into how the virus emerged and evolves. Most common changes in SARS-CoV-2 are single or point nucleotide substitutions; meanwhile, insertions and deletions (indels) have been identified as a less frequent source of viral genetic variability. Here, we report the emergence of a 12-nucleotide deletion in ORF7a, resulting in a 4-amino acid in-frame deletion. The Δ12 variant was identified in viruses from patients of a single outbreak and represents the first report of this deletion in South American isolates. Phylogenetic analysis revealed that Δ12 strains belong to the lineage B.1.1 and clustered separated from the remaining Uruguayan strains. The ∆12 variant was detected in 14 patients of this outbreak by NGS sequencing and/or two rapid and economic methodologies: Sanger amplicon sequencing and capillary electrophoresis. The presence of strong molecular markers as the deletion described here are useful for tracking outbreaks and reveal a significant aspect of the SARS-CoV-2 evolution on the robustness of the virus to keep its functionality regardless loss of genetic material.

Genome Sequences of SARS-CoV-2 P.1 (Variant of Concern) and P.2 (Variant of Interest) Identified in Uruguay.

Two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants associated with increased transmission and immune evasion, P.1 and P.2, emerged in Brazil and spread throughout South America. Here, we report genomes corresponding to these variants that were recently detected in Uruguay. These P.1 and P.2 genomes share all substitutions that are characteristic of these variants.

Phylodynamics of HIV-1 circulating recombinant forms 12_BF and 38_BF in Argentina and Uruguay.

BACKGROUND: Although HIV-1 CRF12_BF and CRF38_BF are two epidemiologically important recombinant lineages circulating in Argentina and Uruguay, little is known about their population dynamics. METHODS: A total of 120 "CRF12_BF-like" and 20 "CRF38_BF-like" pol recombinant sequences collected in Argentina and Uruguay from 1997 to 2009 were subjected to phylogenetic and Bayesian coalescent-based analyses to estimate evolutionary and demographic parameters. RESULTS: Phylogenetic analyses revealed that CRF12_BF viruses from Argentina and Uruguay constitute a single epidemic with multiple genetic exchanges among countries; whereas circulation of the CRF38_BF seems to be confined to Uruguay. The mean estimated substitution rate of CRF12_BF at pol gene (2.5 x 10-3 substitutions/site/year) was similar to that previously described for subtype B. According to our estimates, CRF12_BF and CRF38_BF originated at 1983 (1978-1988) and 1986 (1981-1990), respectively. After their emergence, the CRF12_BF and CRF38_BF epidemics seem to have experienced a period of rapid expansion with initial growth rates of around 1.2 year-1 and 0.9 year-1, respectively. Later, the rate of spread of these CRFs_BF seems to have slowed down since the mid-1990s. CONCLUSIONS: Our results suggest that CRF12_BF and CRF38_BF viruses were generated during the 1980s, shortly after the estimated introduction of subtype F1 in South America (~1975-1980). After an initial phase of fast exponential expansion, the rate of spread of both CRFs_BF epidemics seems to have slowed down, thereby following a demographic pattern that resembles those previously reported for the HIV-1 epidemics in Brazil, USA, and Western Europe.

Sexual behavior and HBV infection among noninjecting cocaine users (NICUs).

The aim is to estimate HBV prevalence and the associated risks among noninjecting cocaine users (NICUs). In 2002-2003, a total of 824 NICUs from Buenos Aires (Argentina) and Montevideo (Uruguay) were interviewed using a structured questionnaire. Serologic tests were carried out for Human Immunodeficiency Virus (HIV), hepatitis B (HBV), syphilis, and others. The population was divided into two serologic groups: HBV-infected and seronegative group. Univariate and binary logistic model were developed. The results seem to indicate that, among NICUs, HBV is transmitted through sexual contact. Prevention measures, including vaccine, are needed in order to control and minimize risks. The study's limitations are noted.

Epidemiology and Genetic Variability of Circulating Influenza B Viruses in Uruguay, 2012-2019.

Influenza B viruses (IBV) are an important cause of morbidity and mortality during interpandemic periods in the human population. Two phylogenetically distinct IBV lineages, B/Yamagata and B/Victoria, co-circulate worldwide and they present challenges for vaccine strain selection. Until the present study, there was little information regarding the pattern of the circulating strains of IBV in Uruguay. A subset of positive influenza B samples from influenza-like illness (ILI) outpatients and severe acute respiratory illness (SARI) inpatients detected in sentinel hospitals in Uruguay during 2012-2019 were selected. The sequencing of the hemagglutinin (HA) and neuraminidase (NA) genes showed substitutions at the amino acid level. Phylogenetic analysis reveals the co-circulation of both lineages in almost all seasonal epidemics in Uruguay, and allows recognizing a lineage-level vaccine mismatch in approximately one-third of the seasons studied. The epidemiological results show that the proportion of IBV found in ILI was significantly higher than the observed in SARI cases across different groups of age (9.7% ILI, 3.2% SARI) and patients between 5-14 years constituted the majority (33%) of all influenza B infection (p < 0.05). Interestingly, we found that individuals >25 years were particularly vulnerable to Yamagata lineage infections.

Transmission cluster of COVID-19 cases from Uruguay: emergence and spreading of a novel SARS-CoV-2 ORF6 deletion

BACKGROUND Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS Our findings provide evidence for the origin and spread of deletion variants and emphasise indels' importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

Evaluation of a new NASBA assay for the qualitative detection of hepatitis C virus based on the NucliSens Basic Kit reagents.

BACKGROUND: Direct detection of HCV RNA by nucleic acid amplification methods is an essential tool in the diagnosis of HCV infections. In-house developed methods based on reverse transcribed polymerase chain reaction (RT-PCR) are widely used but they are laborious and usually lack the standardization required by clinical laboratories. OBJECTIVES: To evaluate the sensitivity and the clinical performance of an HCV specific nucleic acid sequence based amplification (NASBA) assay based on the commercially available, NucliSens Basic Kit (bioMérieux) reagents. STUDY DESIGN: The analytical sensitivity of the Basic Kit-based HCV assay (BK-HCV) was determined using dilutions of the First World Health Organization International Standard for HCV RNA. The performance of the BK-HCV was evaluated at two study sites in comparison with in-house RT-nested PCR (RT-nPCR) by testing a total of 77 plasma specimens. Additional HCV laboratory tests such as Amplicor HCV v2.0 (Roche Diagnostics) and genotype were also included in the comparative analysis. RESULTS: The sensitivity of the BK-HCV was 100-150 IU/ml HCV RNA (85-100% hit rate). When evaluating the clinical performance, we found 96-100% correlation between BK-HCV and RT-nPCR, and 85-91% correlation between BK-HCV and Amplicor. The level of efficiency of the BK-HCV for detecting prevalent HCV genotypes was equal to in house RT-nPCR and Amplicor. CONCLUSIONS: The BK-HCV offers adequate sensitivity for diagnostic purposes and equivalent clinical performance to in-house RT-nPCR assays. The BK-HCV could become a suitable alternative to the in-house amplification methods, providing standardized reagents and procedures, plus rapid results to clinical laboratories.