A spatial case-control study on symptomatic and inapparent primary dengue infections in an endemic city in Brazil.

We conducted a spatial case-control study nested in a dengue incidence cohort to explore the role of the spatial and socioeconomic factors in the proportion of symptomatic (cases) and inapparent primary dengue virus infections (controls). Cohort participants were children and adolescents (2 to 16 years of age) at the beginning of the follow-up. Case definitions were, for symptomatic cases, fever plus a positive lab result for acute dengue (NS1, RT-PCR, ELISA IgM/IgG), and for inapparent infection a positive result for dengue IgG (ELISA) in subjects without symptoms and with a previously negative result at baseline. The covariates included sociodemographic factors, residential location, and socioeconomic context variables of the census tracts of residence of cases and controls. We used principal component analysis to reduce the contextual covariates, with the component values assigned to each one based on their residences. The data were modeled in a Bayesian context, considering the spatial dependence. The final sample consisted of 692 children, 274 cases and 418 controls, from the first year of follow-up (2014-2015). Being male, older age, higher educational level of the head of the family and having a larger number of rooms in the household were associated with a greater chance of presenting dengue symptomatic infection at the individual level. The contextual covariates were not associated with the outcome. Inapparent dengue infection has extensive epidemiological consequences. Relying solely on notifications of symptomatic dengue infections underestimates the number of cases, preserves a silent source of the disease, potentially spreading the virus to unaffected areas.

Phylogenetics, Epidemiology and Temporal Patterns of Dengue Virus in Araraquara, São Paulo State.

Dengue virus (DENV) is a prominent arbovirus with global spread, causing approximately 390 million infections each year. In Brazil, yearly epidemics follow a well-documented pattern of serotype replacement every three to four years on average. Araraquara, located in the state of São Paulo, has faced significant impacts from DENV epidemics since the emergence of DENV-1 in 2010. The municipality then transitioned from low to moderate endemicity in less than 10 years. Yet, there remains an insufficient understanding of virus circulation dynamics, particularly concerning DENV-1, in the region, as well as the genetic characteristics of the virus. To address this, we sequenced 37 complete or partial DENV-1 genomes sampled from 2015 to 2022 in Araraquara. Then, using also Brazilian and worldwide DENV-1 sequences we reconstructed the evolutionary history of DENV-1 in Araraquara and estimated the time to the most recent common ancestor (tMRCA) for serotype 1, for genotype V and its main lineages. Within the last ten years, there have been at least three introductions of genotype V in Araraquara, distributed in two main lineages (L Ia and L Ib, and L II). The tMRCA for the first sampled lineage (2015/2016 epidemics) was approximately 15 years ago (in 2008). Crucially, our analysis challenges existing assumptions regarding the emergence time of the DENV-1 genotypes, suggesting that genotype V might have diverged more recently than previously described. The presence of the two lineages of genotype V in the municipality might have contributed to the extended persistence of DENV-1 in the region.

A spatial case-control study on symptomatic and inapparent primary dengue infections in an endemic city in Brazil

ABSTRACT We conducted a spatial case-control study nested in a dengue incidence cohort to explore the role of the spatial and socioeconomic factors in the proportion of symptomatic (cases) and inapparent primary dengue virus infections (controls). Cohort participants were children and adolescents (2 to 16 years of age) at the beginning of the follow-up. Case definitions were, for symptomatic cases, fever plus a positive lab result for acute dengue (NS1, RT-PCR, ELISA IgM/IgG), and for inapparent infection a positive result for dengue IgG (ELISA) in subjects without symptoms and with a previously negative result at baseline. The covariates included sociodemographic factors, residential location, and socioeconomic context variables of the census tracts of residence of cases and controls. We used principal component analysis to reduce the contextual covariates, with the component values assigned to each one based on their residences. The data were modeled in a Bayesian context, considering the spatial dependence. The final sample consisted of 692 children, 274 cases and 418 controls, from the first year of follow-up (2014-2015). Being male, older age, higher educational level of the head of the family and having a larger number of rooms in the household were associated with a greater chance of presenting dengue symptomatic infection at the individual level. The contextual covariates were not associated with the outcome. Inapparent dengue infection has extensive epidemiological consequences. Relying solely on notifications of symptomatic dengue infections underestimates the number of cases, preserves a silent source of the disease, potentially spreading the virus to unaffected areas.

Levels of angiopoietin-2 are predictive for mortality in yellow fever virus infected patients.

In 2018 there was a large yellow fever outbreak in São Paulo, Brazil, with a high fatality rate. Yellow fever virus can cause, among other symptoms, haemorrhage and disseminated intravascular coagulation, indicating a role for endothelial cells in the disease pathogenesis. Here, we conducted a case-control study and measured markers related to endothelial damage in plasma and its association with mortality. We found that angiopoietin-2 is strongly associated with a fatal outcome and could serve as a predictive marker for mortality. This could be used to monitor severe patients and provide care to improve disease outcome.

Viral Kinetics in Sylvatic Yellow Fever Cases.

BACKGROUND: Yellow fever is a mosquito-borne zoonotic disease caused by yellow fever virus (YFV). Between 2017 and 2019, more than 504 human cases and 176 deaths were confirmed in the outskirts of São Paulo city. Throughout this outbreak, studies suggested a potential association between YFV viremia and mortality. METHODS: Viral ribonucleic acid was measured using reverse-transcription quantitative polymerase chain reaction in plasma samples collected at up to 5 time points, between 3 and 120 days after symptoms onset. RESULTS: Eighty-four patients with confirmed YFV infection were included. Most were males, median age was 42, and 30 (36%) died. Deceased patients were older than survivors (P = .003) and had a higher viremia across all time points (P = .0006). Mean values of viremia had a positive, statistically significant correlation with peak values of neutrophils, indirect bilirubin, aspartate transaminase, international normalized ratio, and creatinine. Finally, a Cox proportional hazards model adjusted for age and laboratory variables showed that viremia is independently associated with death, with a mean 1.84-fold increase (84%) in the hazard of death (P < .001) for each unit increase in mean log10 viremia. CONCLUSIONS: Our results raise the importance of monitoring YFV viremia and suggest a potential benefit of antiviral drugs or neutralizing monoclonal antibodies early in the course of this infection to improve disease outcomes.

Use of saliva and RT-PCR screening for SARS-CoV-2 variants of concern: Surveillance and monitoring.

Genomic surveillance has been applied since the beginning of the COVID-19 pandemic to track the spread of the virus, leading to the characterization of multiple SARS-CoV-2 variants, including variants of concern (VOC). Although sequencing is the standard method, a rapid molecular test for screening and surveillance of VOC is considered for detection. Furthermore, using alternative saliva as specimen collection facilitates the implementation of a less invasive, self-collected sample. In this study, we applied a combinatory strategy of saliva collection and reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 VOC detection. Saliva samples from patients attending a tertiary hospital with suspected COVID-19 were collected and SARS-CoV-2 RNA was detected using SARS-CoV-2 RT-qPCR reagent kit (PerkinElmer). Positive saliva samples were screened for SARS-CoV-2 VOC with previously described RT-PCR for Alpha, Beta, and Gamma variants. Saliva samples were positive in 171 (53%) of 324 tested. A total of 108 (74%) from positive samples were also positive for VOC by RT-PCR screening. Those samples were found between January and August 2021. This approach allowed us to successfully use an alternative and complementary tool to genomic surveillance to monitor the circulation of SARS-CoV-2 VOC in the studied population.

NS1-based ELISA test efficiently detects dengue infections without cross-reactivity with Zika virus.

OBJECTIVES: The aim of this study was to achieve greater specificity of dengue virus (DENV) serological tests based on a recombinant antigen derived from non-structural protein 1 (ΔNS1) with regard to cross-reactive Zika virus (ZIKV) anti-NS1 antibody responses. This is of relevance in endemic regions for the serological discrimination of both DENV and ZIKV, such as Brazil and other tropical countries. METHODS: The ΔNS1 proteins were obtained as recombinant antigens and were evaluated as solid-phase-bound antigens in the ELISA test to detect anti-NS1 IgG antibodies. The performance of the ∆NS1-based DENV IgG ELISA was assessed with both mouse and human serum samples previously exposed to DENV or ZIKV. RESULTS: The ∆NS1-based DENV IgG ELISA detected anti-DENV NS1 IgG without cross-reactivity with ZIKV-positive serum samples. The sensitivity and specificity of the assay determined using samples previously characterized by real-time PCR (qRT-PCR) or plaque reduction neutralization assay (PRNT) were 82% and 93%, respectively. CONCLUSION: The ∆NS1-based DENV IgG ELISA conferred enhanced diagnostic specificity for anti-DENV serological tests and may be particularly useful for serological analyses in endemic regions for both DENV and ZIKV transmission.

Subacute Cognitive Impairment in Individuals With Mild and Moderate COVID-19: A Case Series.

Background: Previous reported neurologic sequelae associated with SARS-CoV-2 infection have mainly been confined to hospital-based patients in which viral detection was restricted to nasal/throat swabs or to IgM/IgG peripheral blood serology. Here we describe seven cases from Brazil of outpatients with previous mild or moderate COVID-19 who developed subacute cognitive disturbances. Methods: From June 1 to August 15, 2020, seven individuals 18 to 60 years old, with confirmed mild/moderate COVID-19 and findings consistent with encephalopathy who were observed >7 days after respiratory symptom initiation, were screened for cognitive dysfunction. Paired sera and CSF were tested for SARS-CoV-2 (IgA, IgG ELISA, and RT-PCR). Serum and intrathecal antibody dynamics were evaluated with oligoclonal bands and IgG index. Cognitive dysfunction was assessed by the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and the Clock Drawing Test (CDT). Results: All but one of our patients were female, and the mean age was 42.6 years. Neurologic symptoms were first reported a median of 16 days (IQR 15-33) after initial COVID-19 symptoms. All patients had headache and altered behavior. Cognitive dysfunction was observed mainly in phonemic verbal fluency (MoCA) with a median of six words/min (IQR 5.25-10.75) and altered visuospatial construction with a median of four points (IQR 4-9) (CDT). CSF pleocytosis was not detected, and only one patient was positive for SARS-Co Conclusions: A subacute cognitive syndrome suggestive of SARS-CoV-2-initiated damage to cortico-subcortical associative pathways that could not be attributed solely to inflammation and hypoxia was present in seven individuals with mild/moderate COVID-19.

Torquetenovirus in saliva: A potential biomarker for SARS-CoV-2 infection?

Torquetenovirus (TTV) is present in biological fluids from healthy individuals and measurement of its titer is used to assess immune status in individuals with chronic infections and after transplants. We assessed if the titer of TTV in saliva varied with the presence of SARS-CoV-2 in the nasopharynx and could be a marker of COVID-19 status. Saliva from 91 individuals positive for SARS-CoV-2 in nasal-oropharyngeal samples, and from 126 individuals who were SARS-CoV-2-negative, all with mild respiratory symptoms, were analyzed. Both groups were similar in age, gender, symptom duration and time after symptom initiation when saliva was collected. Titers of TTV and SARS-CoV-2 were assessed by gene amplification. Loss of smell (p = 0.0001) and fever (p = 0.0186) were more prevalent in SARS-CoV-2-positive individuals, while sore throat (p = 0.0001), fatigue (p = 0.0037) and diarrhea (p = 0.0475) were more frequent in the SARS-CoV-2 negative group. The saliva TTV and nasal-oropharyngeal SARS-CoV-2 titers were correlated (p = 0.0085). The TTV level decreased as symptoms resolved in the SARS-CoV-2 infected group (p = 0.0285) but remained unchanged in the SARS-CoV-2 negative controls. In SARS-CoV-2 positive subjects who provided 2-4 saliva samples and in which TTV was initially present, the TTV titer always decreased over time as symptoms resolved. We propose that sequential TTV measurement in saliva is potentially useful to assess the likelihood of symptom resolution in SARS-CoV-2-positive individuals and to predict prognosis.

SARS-CoV-2 reinfection caused by the P.1 lineage in Araraquara city, Sao Paulo State, Brazil.

Reinfection by the severe acute respiratory syndrome coronavirus type 2 (SARS-COV-2) has been reported in many countries, suggesting that the virus may continue to circulate among humans despite the possibility of local herd immunity due to massive previous infections. The emergence of variants of concern (VOC) that are more transmissible than the previous circulating ones has raised particular concerns on the vaccines effectiveness and reinfection rates. The P.1 lineage was first identified in December 2020 in Manaus city and is now globally spread. We report the first case of reinfection of SARS-CoV-2 caused by the P.1 variant outside of Manaus. The potential of these new variants to escape naturally and vaccine- induced immunity highlights the need for a global vigilance.

Dengue Virus Serotype 2 Intrahost Diversity in Patients with Different Clinical Outcomes.

Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it might also be linked to viral pathogenesis. Dengue virus serotype 2 (DENV-2) has circulated in Brazil since 1990 and is associated with severe disease and explosive outbreaks. Intending to shed light on the viral determinants for severe dengue pathogenesis, we sought to analyze the DENV-2 intrahost genetic diversity in 68 patient cases clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18). Unlike previous DENV intrahost diversity studies whose approaches employed PCR, here we performed viral whole-genome deep sequencing from clinical samples with an amplicon-free approach, representing the real intrahost diversity scenario. Striking differences were detected in the viral population structure between the three clinical categories, which appear to be driven mainly by different infection times and selection pressures, rather than being linked with the clinical outcome itself. Diversity in the NS2B gene, however, showed to be constrained, irrespective of clinical outcome and infection time. Finally, 385 non-synonymous intrahost single-nucleotide variants located along the viral polyprotein, plus variants located in the untranslated regions, were consistently identified among the samples. Of them, 124 were exclusively or highly detected among cases with warning signs and among severe cases. However, there was no variant that by itself appeared to characterize the cases of greater severity, either due to its low intrahost frequency or the conservative effect on amino acid substitution. Although further studies are necessary to determine their real effect on viral proteins, this heightens the possibility of epistatic interactions. The present analysis represents an initial effort to correlate DENV-2 genetic diversity to its pathogenic potential and thus contribute to understanding the virus's dynamics within its human host.

Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing.

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

Plaque Reduction Neutralization Test (PRNT) in the Congenital Zika Syndrome: Positivity and Associations with Laboratory, Clinical, and Imaging Characteristics.

The short duration of viremia, low blood viral load, inaccessibility to timely specific diagnostic tests, and cross-reactions with other flaviviruses have hindered laboratory confirmation of Congenital Zika Syndrome (CZS). This study analyzes the positivity of the plaque reduction neutralization test (PRNT) in children with clinical or imaging characteristics of CZS and its association with laboratory, clinical, and imaging characteristics. The 94 clinical cases of CZS submitted to the ZIKV PRNT90 test were followed from 2016 to 2018. The mean age of children at PRNT90 collection was 22 ± 6 months Standard Deviation. The ZIKV PRNT90 was positive (titer ≥ 10) in 40 (42.5%) children. ZIKV PRNT90 positivity was associated with severe microcephaly in newborns (p = 0.016), lower head circumference z-score at birth (p = 0.043) and 24 months of age (p = 0.031), and severe reduction of the cerebral parenchyma volume (p = 0.021), expressing greater disease severity. Negative PRNT90 in children with characteristic signs of CZS may be due to false-negative results, indicating that the diagnosis of CZS should be primarily syndromic.

Genomic detection of a virus lineage replacement event of dengue virus serotype 2 in Brazil, 2019.

BACKGROUND Despite efforts to mitigate the impact of dengue virus (DENV) epidemics, the virus remains a public health problem in tropical and subtropical regions around the world. Most DENV cases in the Americas between January and July 2019 were reported in Brazil. São Paulo State in the southeast of Brazil has reported nearly half of all DENV infections in the country. OBJECTIVES To understand the origin and dynamics of the 2019 DENV outbreak. METHODS Here using portable nanopore sequencing we generated20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities of São Paulo State, Araraquara and São José do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. FINDINGS The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Our analysis shows that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Dating phylogeographic analysis suggests that DENV2-III BR-4 was introduced to Brazil in or around early 2014, possibly from the Caribbean region. MAIN CONCLUSIONS Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.

Specificity of NS1-based immunochromatographic tests for dengue virus with regard to the Zika virus protein.

OBJECTIVES: This study was performed to determine whether Dengue virus (DENV) immunochromatographic tests can detect and differentiate nonstructural protein 1 (NS1) from each of the four DENV serotypes and do not cross-react with the Zika virus (ZIKV) NS1 protein. METHODS: We compared the specificity of six NS1-based DENV immunochromatographic tests (point of care) in the detection of NS1 proteins from each of the four DENV serotypes and ZIKV. The tests were performed with NS1 proteins produced in mammalian cells. Cross-reactivity was confirmed with a purified recombinant ZIKV NS1 protein and DENV+ or ZIKV+ human serum samples. RESULTS: Cross-reaction was observed in 2 out of the 6 evaluated tests using cell culture supernatants containing NS1 protein of each tested virus. Cross-reactivity with ZIKV was confirmed with purified recombinant ZIKV NS1 produced in Escherichia coli. Further analyses with serum samples collected from DENV+ or ZIKV+ patients confirmed the cross-reactivity with ZIKV protein in 2 tests. CONCLUSIONS: The detection of the NS1 protein is the basis for several commercially available serological DENV diagnostic tests. The present results emphasize the relevance of testing specificity of presently available NS1-based DENV serological tests and the need of adjustments of tests that cross-react with the ZIKV protein. Our results are particularly relevant for regions where both viruses are endemically found, as in the case of Brazil.

First report of Aedes albopictus infected by Dengue and Zika virus in a rural outbreak in Brazil.

In Brazil, Dengue (DENV) and Zika (ZIKV) viruses are reported as being transmitted exclusively by Aedes aegypti in urban settings. This study established the vectors and viruses involved in an arbovirus outbreak that occurred in 2019 in a rural area of Espírito Santo state, Brazil. Mosquitoes collected were morphologically identified, sorted in samples, and submitted to molecular analysis for arboviruses detection. Phylogenetic reconstruction was performed for the viral sequence obtained. All 393 mosquitoes were identified as Aedes albopictus. DENV-1 genotype V was present in one sample and another sample was positive for ZIKV. The DENV-1 clustered with viruses that have circulated in previous years in large urban centers of different regions in Brazil. This is the first report of A. albopictus infected by DENV and ZIKV during an outbreak in a rural area in Brazil, indicating its involvement in arboviral transmission. The DENV-1 strain found in the A. albopictus was not new in Brazil, being involved previously in epidemics related to A. aegypti, suggesting the potential to A. albopictus in transmitting viruses already circulating in the Brazilian population. This finding also indicates the possibility of these viruses to disperse across urban and rural settings, imposing additional challenges for the control of the diseases.

Data on dengue incidence in South-eastern Brazil, 2014-2018.

Data from the routine surveillance systems have been extensively used to estimate the incidence of dengue. However, routine surveillance data frequently underestimate the diseases' incidence. Underreporting of dengue cases is related to the varying spectrum of its clinical presentation, with a large proportion of mild and asymptomatic infections, to its unspecific signs and symptoms, to the limitations of access to health care, and to the performance of the surveillance system itself [1-3]. In order to obtain accurate figures on dengue incidence, a cohort of children and adolescents was set up and followed during four years. The incidence of reported cases was used as a reference for the sample size calculation, which was stratified by age groups. A two-stage procedure was used to select the participants: census tracts were randomly selected, and within each one, a pre-determined number of children of each age group was randomly selected. The parents or legal guardians of the participating children and adolescents provided a written informed consent. In the first home visit, they responded to a questionnaire containing data on socio-demographic characteristics, housing, access to water, sewage, and garbage collection. Also, during the first visit a blood sample of the participating child/adolescent was collected for dengue baseline serology. Beginning in the week after the enrolment, the parent or legal guardian that was designated in the first visit received weekly phone calls for fever surveillance. If the child/adolescent had fever during the week, a nurse was dispatched to the family's home to collect more detailed data on the fever episode and collect a blood sample for dengue diagnosis (IgG, IgM, NS1 and PCR). If the dengue diagnosis was confirmed, a medical appointment was scheduled, and another blood sample for confirmatory tests was collected. It was also agreed that in every anniversary of their participation, they would receive another visit for a blood collection for dengue serology, regardless if they had a fever episode or a confirmed dengue diagnosis during the previous year. This article contains the description of the cohort's dataset. It is associated with the article published in Acta Tropica, under the title "A cohort study to assess the incidence of dengue, Brazil, 2014-2018" [4]. The associated article focused on the seroprevalence and incidence of dengue, and explored some associations between both outcomes and some explanatory variables.