The airway microbial community may shape the variable outcomes of COVID-19, but its role is poorly understood. We profiled naso-oropharyngeal swab samples from 561 people using marker-gene amplicon sequencing, total-RNA metatranscriptomics, and shotgun DNA metagenomics, which together tracked SARS-CoV-2 lineages, detected respiratory co-infections, and recovered 190 microbial genomes including candidate previously undescribed species. Here we show that increasing COVID-19 severity is associated with a reproducible depletion of oral commensal bacteria — most consistently Gemella, with Rothia, Haemophilus and related genera — concordant across all three sequencing approaches and robust to sensitivity analyses, and accompanied by shifts in microbial metabolic and stress-response functions. In contrast, bacterial resistance-gene burden did not increase with antibiotic use or severity, host gene-expression changes were limited, and bacteria enriched in severely ill patients were largely environmental, consistent with low-biomass artifacts. These findings define a commensal-depletion signature of COVID-19 severity. High-resolution multi-omics of naso-oropharyngeal swabs defines a respiratory microbiome signature of COVID-19 severity: a reproducible, cross-platform depletion of oral commensal bacteria, most consistently Gemella.

Highly pathogenic avian influenza (HPAI) A(H5N1) viruses of clade 2.3.4.4b have recently spread across the Americas, prompting intensified surveillance efforts in Brazil aimed at early detection in wild birds. As part of these efforts, we identified a low pathogenic avian influenza A(H11N2) virus in a white-rumped sandpiper (Calidris fuscicollis) sampled at Lagoa do Peixe National Park (PNLP) in southern Brazil. Whole-genome sequencing revealed that seven of the eight gene segments shared high nucleotide similarity (approximately 98.8%) with viruses previously detected in shorebirds from Delaware Bay, North America. In contrast, the PB1 segment showed high nucleotide similarity (approximately 99%) to the PB1 lineage associated with clade 2.3.4.4b A(H5N1) genotype B3.2 viruses circulating in the Americas. Phylogenetic, nucleotide identity, and molecular clock analyses indicated that this lineage shares a recent common ancestor with North American LPAI viruses and was subsequently detected in distinct viral genetic backgrounds. Although no HPAI virus was identified in this study, the presence of a PB1 segment related to H5N1-associated lineages suggests that genetic components linked to these viruses were circulating among low pathogenic avian influenza viruses in South America. These findings highlight the importance of continued surveillance in migratory bird populations to improve understanding of avian influenza virus diversity and support epidemiological monitoring.

Objective:This study aimed to evaluate the Trueprep/Truelab platform with four Truenat assays (COVID-19, Mycobacterium tuberculosis - MTB, malaria, leptospirosis), focusing on analytical performance, repeat rates, and time-to-result. Methods:Remnant clinical specimens and commercial controls were analyzed under routine conditions. Performance metrics included accuracy, reproducibility, limit of detection (LOD), linearity, and specificity, when applicable. Each analyte was tested in replicates, and results were compared with established reference methods. Results:The Truenat COVID-19 assay achieved 100% agreement with the reference and LOD of 500 copies/mL. Truenat MTB showed 95% concordance with the comparator, though repeat runs were occasionally required (7.3%). Malaria assays demonstrated high reproducibility and linearity (R2 = 0.9996) with an observed LOD of 3000 copies/mL. Leptospira assays yielded 100% accuracy with a LOD of 750 copies/mL. Performance generally matched manufacturer specifications, though low-burden MTB samples were less consistently detected. Conclusion:Truenat assays showed reliable analytical performance, especially for COVID-19. While MTB detection remains more robust with Xpert Ultra in low-burden cases, Truenat provides a viable point-of-care alternative in resource-limited settings. For malaria and leptospirosis, broader clinical validation is needed before routine implementation in our service.

Introduction/Objective: Cytomegalovirus (CMV), a member of the Herpesviridae family, is widely distributed globally and usually causes asymptomatic infection or self-limited symptoms, followed by latency for an indeterminate time in immunocompetent individuals. However, in immunosuppressed patients, it can trigger severe clinical manifestations due to reactivation. The emergence of strains resistant to the antivirals ganciclovir, foscarnet, cidofovir, maribavir, and letermovir represents a growing challenge in treatment. In this context, early detection of mutations associated with viral resistance is essential to guide interventions and optimize therapeutic management. This study aimed to develop a molecular test based on next-generation sequencing (NGS) to identify mutations associated with resistance to the main antivirals in key CMV genes, using clinical samples. Methods: Specific primers were designed for the target regions of the UL27, UL51, UL54, UL56, and UL97 genes, where mutations previously associated with antiviral resistance occur. Viral DNA was extracted from clinical samples previously positive for CMV and then submitted to PCR, library preparation with the Microbial Amplicon Prep kit, and sequencing on an Illumina platform. Bioinformatic analysis was conducted with an in-house pipeline, and variants were compared with the HerpesDRG DB v03112024 database. Results: The assay demonstrated 98% sensitivity for detecting resistance variants in samples with viral load ≥1,500 IU/mL and 80% in samples with viral load of 1,000 IU/mL. A total of 39 clinical samples were analyzed, of which 5 showed clinically relevant mutations in the UL97 gene. Mutations in the UL54 and UL56 genes were validated using synthetic DNA. The main mutations identified included C603W, M460V, L595S, and A594V (UL97), I726T, T700A, and Q578H (UL54), and C325F and T244K (UL56), associated with resistance to different antivirals. The assay performance was validated in an international reference laboratory, with advantages such as broader genomic coverage, faster turnaround time, and simultaneous analysis of multiple genes. Conclusion: The developed test is robust, sensitive, and applicable to routine clinical practice for detecting CMV antiviral resistance. Its implementation contributes to personalized management of immunosuppressed patients, optimizing therapeutic choice and reducing the risk of treatment failure.

BACKGROUND:Diagnosing pediatric infectious diseases is challenging due to nonspecific presentations, small sample volumes, and the limited sensitivity of conventional microbiological tests (CMTs). Metagenomic next-generation sequencing (mNGS) enables broad, hypothesis-free pathogen detection, but its diagnostic performance in children remains insufficiently characterized. This study evaluates the diagnostic accuracy of mNGS in pediatric infectious diseases and compares its performance with CMTs. METHODS:This systematic review and meta-analysis was registered in PROSPERO (CRD42024542444). Searches were performed using multiple databases through August 2024. Eligible studies evaluated mNGS and CMTs in pediatric patients (≤21 years) with suspected infectious diseases and compared their respective results with clinical diagnosis. Pooled sensitivity, specificity, and diagnostic odds ratios (DORs) were calculated using a bivariate random-effects model. RESULTS:Thirty-three studies (n = 4,165) met inclusion criteria, and nine were eligible for meta-analysis. Pooled sensitivity and specificity of mNGS versus clinical diagnosis were 0.84 (95% CI: 0.82-0.86) and 0.71 (95% CI: 0.66-0.75), respectively, compared with 0.40 (95% CI: 0.37-0.43) and 0.82 (95% CI: 0.78-0.86) for CMTs. The pooled DOR favored mNGS (18.6 vs. 5.4). Respiratory infections were most frequently investigated, followed by bloodstream and mixed infections. Over two-thirds of studies reported changes in antimicrobial management following mNGS results. CONCLUSIONS:mNGS demonstrates superior sensitivity and diagnostic accuracy compared with CMTs, enabling comprehensive pathogen detection, including rare and co-infecting organisms, and informing targeted antimicrobial therapy. Despite limitations related to cost, complex interpretation, and methodological standardization, mNGS represents a promising complement to conventional diagnostics in pediatric infectious disease management.

Hepatitis A Virus (HAV) infects millions of individuals annually and is a major cause of acute viral hepatitis worldwide. This study aims to (1) assess HAV infection in suspected acute hepatitis patients at public healthcare institutions in Brazil; (2) evaluate the proportion of immunized individuals against HAV; (3) identify HAV genotypes; (4) examine the association between HAV infection and demographic data, as well as exposure to risk factors. This is a prospective, observational multicenter study conducted in primary health services in Brazil from October 2019 to May 2023, involving 1721 patients with suspected acute hepatitis. Acute HAV infection was identified in 108 (6.3%) patients, predominantly in young men (80%) and from South and Southeast regions of Brazil (97%). Anti-HAV IgG, indicating previous exposure or vaccination, was detected in 78.6% of individuals (74% in the South to 91% in the North). Genotype I.A was found in all cases and approximately 450 mutations were identified, most of them in the structural proteins VP1-3. Two viral groups were identified and related to two introductions of the virus: cosmopolitan sequences from North America, South America, and Europe, and a minor group of Brazilian sequences similar to Asian and South American ones. The high incidence of acute HAV infections highlights the need for targeted prevention and vaccination strategies. The characterization of HAV genetic diversity and molecular epidemiology contributes to monitoring and identifying emerging outbreaks.

Background and ObjectivesDengue virus (DENV) infection can cause acute encephalitis. Chronic encephalitis with progressive dementia is rarely reported.MethodsWe present a case of chronic encephalitis with rapidly progressive dementia, in which a potential DENV brain infection was identified with brain tissue metagenomic next-generation sequencing. Brain pathology and molecular diagnosis are also presented.ResultsA 20-year-old man from SP, Brazil, presented with rapidly progressive dementia, speech apraxia, and apathy in June 2022. By January 2023, cognitive testing showed severe global impairment (MMSE score of 18/30). MRI revealed white matter abnormalities and atrophy; CSF analysis disclosed a mild lymphocytic pleocytosis, mildly elevated protein levels, and positive CSF oligoclonal bands. Despite extensive testing ruling out common infectious and inflammatory causes, the patient's condition worsened with executive dysfunction, language impairment, tremors, and myoclonus. In August 2023, a brain biopsy and next-generation sequencing identified DENV-1 genotype V, linked to Brazilian sequences from 2000 to 2022.DiscussionThis case highlights a challenging instance of encephalitis with unknown etiology, where metagenomic analysis detected DENV-1 RNA in brain tissue, suggesting a possible cause.

PURPOSE:Cladophialophora bantiana is a rare cause of severe central nervous system phaeohyphomycosis. RESULTS:A 44-year-old woman from Northeastern Brazil with a history of scotomas, mental confusion, and impaired coordination presented Magnetic Resonance Imaging with vasogenic edema and nodular enhancement in the left occipital lobe. Brain biopsy showed neutrophilic exudate with eosinophils, macrophages, giant multinucleated cells, and septate hyphae with a brown-colored birefringent wall and acute angle branching. Metagenomic analysis of the biopsy's total RNA revealed the presence of RNA sequences highly similari to C. bantiana. Culture confirmed the presence of olivaceous-gray suede-like to floccose colonies, with septate dematiaceous hyphae and long conidia chains from undifferentiated conidiophores, consistent with C. bantiana. CONCLUSIONS:This case highlights the potential of metagenomic testing as a tool for early diagnosis of infections caused by uncommon fungal pathogens. To our knowledge, this is the first report of C. bantiana central nervous system infection in Brazil.

The outcomes of SARS-CoV-2 infection exhibit significant heterogeneity, and the role of the airway microbiota remains insufficiently understood. In a cohort comprising 561 participants, an integrated multi-omics approach was employed. This approach combined 16S/ITS amplicons (n = 542), total RNA metatranscriptomics (n = 495), and total DNA metagenomics (n = 113), with 101 samples analyzed using all three omics. Metatranscriptomes resolved lineage dynamics across major infection waves and identified microbial coinfections; shotgun metagenomes recovered 190 MAGs, including 13 putative novel species, expanding the catalog of respiratory taxa. Cross‑omics testing showed species‑level biomarkers and resistome burden were more linked to clinical outcomes than genus‑level signals, highlighting the value of high‑resolution profiling. Functional analyses revealed outcome‑based shifts in microbial processes and host metatranscriptomic signatures enriched for interferon programs and tissue remodeling pathways. This work delineates mechanisms associated with COVID‑19 severity, uncovers candidate biomarkers, and provides a multi‑omics framework for integrated pathogen–microbiome–host surveillance in future respiratory pandemics.

Background:We aimed to assess the accuracy, clinical efficacy, and limitations of metagenomic next-generation sequencing (mNGS) for diagnosing infectious meningoencephalitis. Methods:We performed a systematic literature review and meta-analysis of studies that evaluated the performance of mNGS to determine the cause of infectious meningoencephalitis. We explored PubMed, Cumulative Index to Nursing and Allied Health, Embase, Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, and Web of Science up to 12 November 2024. To perform a meta-analysis, we calculated the pooled diagnostic odds ratio (DOR) for mNGS and for conventional microbiological tests (CMTs) compared to the clinical diagnosis. Results:Thirty-four studies met the inclusion criteria, with mNGS-positive rates ranging from 43.5% to 93.5% for infectious meningoencephalitis. The meta-analysis included 23 studies with 1660 patients. The pooled sensitivity was 0.70 (95% confidence interval [CI], .67-.72), and its specificity was 0.93 (95% CI, .92-.94). The DOR for mNGS was 26.7 (95% CI, 10.4-68.8), compared to 12.2 (95% CI, 3.2-47.0) for CMTs. For tuberculosis meningoencephalitis, mNGS demonstrated a pooled sensitivity of 0.67 (95% CI, .61-.72) and specificity of 0.97 (95% CI, .95-.99), with a DOR of 43.5 (95% CI, 7.4-256.6). Conclusions:Our review indicates that mNGS can be a valuable diagnostic tool for infectious meningoencephalitis, offering high sensitivity and specificity. mNGS's superior DOR compared to that of CMTs highlights its potential for more accurate diagnoses and targeted interventions. Further research is needed to optimize which patients and at what point in the diagnostic process mNGS should be used.

BACKGROUND AND OBJECTIVES:Dengue virus (DENV) infection can cause acute encephalitis. Chronic encephalitis with progressive dementia is rarely reported. METHODS:We present a case of chronic encephalitis with rapidly progressive dementia, in which a potential DENV brain infection was identified with brain tissue metagenomic next-generation sequencing. Brain pathology and molecular diagnosis are also presented. RESULTS:A 20-year-old man from SP, Brazil, presented with rapidly progressive dementia, speech apraxia, and apathy in June 2022. By January 2023, cognitive testing showed severe global impairment (MMSE score of 18/30). MRI revealed white matter abnormalities and atrophy; CSF analysis disclosed a mild lymphocytic pleocytosis, mildly elevated protein levels, and positive CSF oligoclonal bands. Despite extensive testing ruling out common infectious and inflammatory causes, the patient's condition worsened with executive dysfunction, language impairment, tremors, and myoclonus. In August 2023, a brain biopsy and next-generation sequencing identified DENV-1 genotype V, linked to Brazilian sequences from 2000 to 2022. DISCUSSION:This case highlights a challenging instance of encephalitis with unknown etiology, where metagenomic analysis detected DENV-1 RNA in brain tissue, suggesting a possible cause.

Recent outbreaks of yellow fever in Brazil, with hundreds of cases despite available vaccination, have drawn attention to the pressing need for effective therapeutic interventions, with a special focus on the critically ill [...]

Background: Severe yellow fever (YF) can result in acute liver failure (ALF) and high mortality. The role of therapeutic plasma exchange (TPE) in managing YF-ALF remains unclear. This study evaluated the impact of TPE strategies in severe YF. Methods: This observational case-series study evaluated three groups of patients classified according to treatment: G1 (standard intensive care support [ICS]), G2 (ICS + high-volume-TPE [HV-TPE]), and G3 (ICS + intensive TPE). HV-TPE was performed during 3 consecutive days with extra sessions of one plasma-volume, if necessary, whereas intensive TPE consisted of one plasma volume/session performed twice daily, with additional fresh frozen plasma infusion. Hemostatic agents, including tranexamic acid, platelets, and cryoprecipitate, were administered as needed. TPE was de-escalated based on clinical and laboratory parameters. The primary outcome was mortality. Results: Sixty-six patients were included (G1: 41, G2: 11, G3: 14). Groups had similar baseline characteristics. Mortality was significantly lower in G3 (14%) compared to G2 (82%) and G1 (85%) (p < 0.001). Additionally, G3 patients showed a higher frequency of undetectable YF viral load. Conclusions: Intensive TPE is a feasible and effective intervention for severe YF, achieving an 84% reduction in mortality. The limitations of our results are the small sample size, observational and single-center study. Further studies are warranted to elucidate intensive TPE’s role in YF management.

Influenza A viruses of the H2 subtype represent a zoonotic and pandemic threat to humans due to a lack of widespread specific immunity. Although A(H2) viruses that circulate in wild bird reservoirs are distinct from the 1957 pandemic A(H2N2) viruses, there is concern that they could impact animal and public health. There is limited information on AIVs in Latin America, and next to nothing about H2 subtypes in Brazil. In the present study, we report the occurrence and genomic sequences of two influenza A viruses isolated from wild-caught white-rumped sandpipers (Calidris fuscicollis). One virus, identified as A(H2N1), was isolated from a bird captured in Restinga de Jurubatiba National Park (PNRJ, Rio de Janeiro), while the other, identified as A(H2N2), was isolated from a bird captured in Lagoa do Peixe National Park (PNLP, Rio Grande do Sul). DNA sequencing and phylogenetic analysis of the obtained sequences revealed that each virus belonged to distinct subtypes. Furthermore, the phylogenetic analysis indicated that the genomic sequence of the A(H2N1) virus isolated from PNRJ was most closely related to other A(H2N1) viruses isolated from North American birds. On the other hand, the A(H2N2) virus genome recovered from the PNLP-captured bird exhibited a more diverse origin, with some sequences closely related to viruses from Iceland and North America, and others showing similarity to virus sequences recovered from birds in South America. Viral genes of diverse origins were identified in one of the viruses, indicating local reassortment. This suggests that the extreme South of Brazil may serve as an environment conducive to reassortment between avian influenza virus lineages from North and South America, potentially contributing to an increase in overall viral diversity.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts.

We report 4 highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b viruses in samples collected during June 2023 from Royal terns and Cabot's terns in Brazil. Phylodynamic analysis revealed viral movement from Peru to Brazil, indicating a concerning spread of this clade along the Atlantic Americas migratory bird flyway.

The raising of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants led to the use of COVID-19 bivalent vaccines, which include antigens of the wild-type (WT) virus, and of the Omicron strain. In this study, we aimed to evaluate the impact of bivalent vaccination on the neutralizing antibody (NAb) response. We enrolled 93 volunteers who had received three or four doses of monovalent vaccines based on the original virus (n = 61), or a booster shot with the bivalent vaccine (n = 32). Serum samples collected from volunteers were subjected to neutralization assays using the WT SARS-CoV-2, and Omicron subvariants. In addition, immunoinformatics to quantify and localize highly conserved NAb epitopes were performed. As main result, we observed that the neutralization titers of samples from individuals vaccinated with the bivalent vaccine were higher for the original virus, in comparison to their capacity of neutralizing the Omicron variant and its subvariants. NAb that recognize epitopes mostly conserved in the WT SARS-CoV-2 were boosted, while those that recognize epitopes mostly present in the Omicron variant, and subvariants were primed. These results indicate that formulation of future vaccines shall consider to target present viruses, and not viruses that no longer circulate.

Introdução O vírus da Hepatite E (HEV) é responsável pelo desenvolvimento da hepatite E, e sua principal via de transmissão é fecal-oral. O genótipo 3 (HEV-3), zoonótico, está globalmente distribuído, sendo o consumo de carne suína mal cozida o principal fator de risco para infecção. No Brasil, o HEV está presente em suínos e produtos derivados, havendo ainda poucos relatos de infecção em humanos, embora os estudos sorológicos apontem para uma prevalência bem maior. A ausência de triagem rotineira dificulta a compreensão dessa infecção em nosso meio fazendo-se necessária a inclusão desse agente como hipótese diagnóstica nos casos de hepatite aguda sem etiologia definida. Resultados Paciente masculino, 67 anos, deu entrada no Hospital das Clínicas da FMUSP com quadro de hepatite aguda apresentando os seguintes sintomas: náusea, dor abdominal, urina escura e fezes claras. Os níveis de enzimas hepáticas estavam elevados (AST = 2.616 U/L; ALT = 2.654 U/L), fosfatase alcalina de 256 U/l, gama-glutamil transpeptidase de 210 U/l e bilirrubina total de 11,6 mg/dL. A tomografia de abdome apresentou fígado de dimensões um pouco aumentadas, de contornos regulares, sem evidência de lesões focais. A infecção pelos vírus das hepatites A, B ou C foi descartada por ausência de marcadores sorológicos dessas infecções. Foi realizada a pesquisa do HEV por PCR em tempo real sendo o RNA viral detectado com carga viral de 222,44 U/ul, além disso a sorologia para pesquisa de anticorpos anti-HEV IgM e IgG foi reagente. O genoma dessa cepa de HEV foi caracterizado e o genótipo classificado como 3 subtipo f (HEV-3f). Na análise filogenética essa sequência viral agrupou-se com cepas do HEV detectadas em suínos da região Nordeste do Brasil. Conclusão No Brasil até o presente momento apenas o genótipo 3 do HEV foi identificado infectando humanos e suínos. Curiosamente, o paciente infectado pelo HEV identificado neste estudo tinha um histórico de viagem recente para a cidade de Garanhuns - Pernambuco, onde foi relatada alta soroprevalência de HEV em suínos. A estreita relação filogenética do HEV isolado do paciente com cepas suínas isoladas no referido estado, em cidades próximas de onde o paciente esteve, sugere uma possível transmissão zoonótica do HEV nesta região. O estudo da diversidade genética do HEV é de grande relevância para o entendimento das vias de transmissão predominantes em nosso meio e para a avaliação da eficácia dos métodos moleculares utilizados para diagnóstico.

The hepatitis E virus (HEV) is an important causative agent of acute hepatitis (AH). Despite reports of human infection in Brazil, the investigation is not routinely conducted, even in cases of elevated liver enzymes. This study evaluated two groups: group 1-patients with acute hepatitis A (n = 44); group 2-patients with nonA-C AH (n = 47). They were tested by enzyme immunoassay for anti-HEV IgM/IgG and real-time PCR for HEV RNA detection. The positive sample for HEV RNA was submitted for sequencing. The seroprevalence of anti-HEV IgM and IgG in group 1 was 4% (2/44) and 14.5% (7/44), respectively. Viral RNA was not detected in any sample. In group 2, the anti-HEV IgM positivity was 4.3% (2/47), and IgG 14.9% (7/47). RNA was detectable in one case, which presented a viral load of 222.4 IU/μL and positive anti-HEV IgM/IgG. In the phylogenetic analysis, the genotype identified was HEV-3f. These results indicate that HEV infection should be considered a possible diagnosis in cases of non-A-C AH. The patient identified with acute hepatitis E had recently traveled to the Northeast region of Brazil (Garanhuns city in Pernambuco state), where there are reports of high HEV seroprevalence among pigs. The close phylogenetic relationship observed between the sequence characterized in this study and strains isolated from pigs in nearby cities where the patient went suggested a possible zoonotic transmission in this region. This study highlights the importance of expanding studies and improving surveillance to understand better and manage HEV infections nationwide.