Fatal Oropouche Virus Infections in Nonendemic Region, Brazil, 2024.

We report acute Oropouche virus infections in 2 previously healthy women from a nonendemic region of Brazil outside the Amazon Basin. Infections rapidly progressed to hemorrhagic manifestations and fatal outcomes in 4-5 days. These cases highlight the critical need for enhanced surveillance to clarify epidemiology of this neglected disease.

Differential diagnosis of exanthematous viruses during the 2022 Mpox outbreak in Minas Gerais, Brazil.

The monkeypox virus (MPXV) outbreak, primarily endemic to Africa, has spread globally, with Brazil reporting the second-highest number of cases. The emergence of MPXV in non-endemic areas has raised concerns, particularly due to the co-circulation of other exanthematous viruses such as varicella-zoster virus (VZV) and molluscum contagiosum virus (MOCV). To perform an accurate differential diagnosis of MPXV during the ongoing outbreak in Minas Gerais, Brazil, a 5PLEX qPCR assay targeting orthopoxviruses (OPV), VZV, and MOCV was used to retrospectively analyze all clinical samples that tested negative for MPXV in the initial screening conducted at Funed. In summary, our study analyzed 1,175 clinical samples received from patients suspected of MPXV infection and found a positivity rate of 33.8% (397 samples) for MPXV using the non-variola qPCR assay. Testing the 778 MPXV-negative clinical samples using the 5PLEX qPCR assay revealed that 174 clinical samples (22.36%) tested positive for VZV. MOCV DNA was detected in 13 and other OPV in 3 clinical samples. The sequencing of randomly selected amplified clinical samples confirmed the initial molecular diagnosis. Analysis of patient profiles revealed a significant difference in the median age between groups testing positive for MPXV and VZV and a male predominance in MPXV cases. The geographic distribution of positive cases was concentrated in the most populous mesoregions of Minas Gerais state. This study highlights the challenges posed by emerging infectious diseases. It emphasizes the importance of epidemiological surveillance and accurate diagnosis in enabling timely responses for public health policies and appropriate medical care. IMPORTANCE: Brazil ranks second in the number of cases during the global monkeypox epidemic. The study, conducted in Minas Gerais, the second most populous state in Brazil with over 20 million inhabitants, utilized differential diagnostics, revealing a significant number of positive cases for other exanthematous viruses and emphasizing the need for accurate diagnoses. During the study, we were able to assess the co-circulation of other viruses alongside monkeypox, including varicella-zoster virus, molluscum contagiosum virus, and other orthopoxviruses. The significance of the research is underscored by the concentration of positive cases in populous areas, highlighting the challenges posed by emerging infectious diseases. This demographic context further amplifies the importance of the research in guiding public health policies and medical interventions, given the substantial population at risk. The study not only addresses a global concern but also holds critical implications for a state with such a large population and geographic expanse within Brazil. Overall, the study emphasizes the pivotal role of surveillance and precise diagnosis in guiding effective public health responses and ensuring appropriate medical interventions.

Orthohantavirus infections in South America: a systematic review and meta-analysis.

Orthohantaviruses are zoonotic pathogens that cause acute and severe syndromes in humans. This review was performed to estimate the occurrence of human orthohantaviruses in South America between 2010 and 2022. A careful evaluation of the eligibility and quality of the articles was carried out after a systematic bibliographic search of four databases. The pooled frequency of human orthohantaviruses was calculated using a random effects model meta-analysis. The heterogeneity of estimates (resulting from the chi2 test and I2 statistics) was investigated by subgroup analysis and meta-regression. 1,962 confirmed cases of orthohantavirus infections were diagnosed among 35,548 individuals from seven South American countries. The general occurrence of orthohantaviruses was estimated to be 4.4% (95% confidence interval: 2.9-6.2%) based on general pooling of human cases from 32 studies. In a subgroup analysis considering the study design and method of diagnosis, the percentages of diagnosed orthohantavirus infections differed substantially (I2 = 97.8%, p = 0.00) among South American countries. Four genetic variants of orthohantavirus have been identified circulating in Argentina, Brazil, Bolivia, Chile, Colombia, and Peru. Although laboratory diagnosis of orthohantaviruses is not performed in many countries in South America, there is evidence that four different orthohantaviruses are circulating in the region. The pooled occurrence of viral infection was approximately 4.0% in more than half of the South American countries. Updated information on the occurrence of human infections is essential for monitoring the territorial spread and determining the frequency of this zoonosis.

SARS-CoV-2 Spillback to Wild Coatis in Sylvatic-Urban Hotspot, Brazil.

We tested coatis (Nasua nasua) living in an urban park near a densely populated area of Brazil and found natural SARS-CoV-2 Zeta variant infections by using quantitative reverse transcription PCR, genomic sequencing, and serologic surveillance. We recommend a One Health strategy to improve surveillance of and response to COVID-19.

Genomic Surveillance of Monkeypox Virus, Minas Gerais, Brazil, 2022.

Phylogenetic analysis of 34 monkeypox virus genome sequences isolated from patients in Minas Gerais, Brazil, revealed initial importation events in early June 2022, then community transmission within the state. All generated genomes belonged to the B.1 lineage responsible for a global mpox outbreak. These findings can inform public health measures.

Rapid Epidemic Expansion of Chikungunya Virus East/Central/South African Lineage, Paraguay.

The spread of Chikungunya virus is a major public health concern in the Americas. There were >120,000 cases and 51 deaths in 2023, of which 46 occurred in Paraguay. Using a suite of genomic, phylodynamic, and epidemiologic techniques, we characterized the ongoing large chikungunya epidemic in Paraguay.

Ultrastructural analysis of monkeypox virus replication in Vero cells.

In early May 2022, the first worldwide monkeypox virus (MPXV) outbreak was reported, with different clinical aspects from previously studied human monkeypox infections. Despite monkeypox medical importance, much of its biological aspects remain to be further investigated. In the present work, we evaluated ultrastructural aspects of MPXV asynchronous infections in Vero cells by transmission electron microscopy (TEM). The viral strain was isolated from a male patient infected during the 2022 outbreak. TEM analysis showed: (i) adhered intracellular mature virus particles before entry of the host cell; (ii) a reorganization of the rough endoplasmic reticulum cisternae into the so-called "mini-nuclei" structure associated with genome replication; and (iii) noticeably different sites within the viral factory presenting granular or fibrillar aspects. We also observed viral crescents, different MPXV particle morphotypes, and cellular alterations induced by infection, such as changes in the cytoskeleton structure and multimembrane vesicles abundance. Taken together, to the best of our knowledge, these results revealed for the first-time ultrastructural aspects of different steps of the MPXV cycle.

Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed.

BACKGROUND: The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES: To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS: Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS: Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS: Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

Rickettsia amblyommii infecting Amblyomma sculptum in endemic spotted fever area from southeastern Brazil.

The Rickettsia bacteria include the aetiological agents for the human spotted fever (SF) disease. In the present study, a SF group Rickettsia amblyommii related bacterium was detected in a field collected Amblyomma sculptum (Amblyomma cajennense species complex) tick from a Brazilian SF endemic site in southeastern Brazil, in the municipality of Juiz de Fora, state of Minas Gerais. Genetic analysis based on genes ompA,ompB and htrA showed that the detected strain, named R. amblyommii str. JF, is related to the species R. amblyommii.

Resurgence of Dengue Virus Serotype 3 in Minas Gerais, Brazil: A Case Report.

This report provides a detailed overview of the resurgence of DENV-3 in the state of Minas Gerais, Brazil, which is a concerning scenario in the context of dengue, a mosquito-borne viral disease. Historically, Brazil has grappled with dengue epidemics caused primarily by the DENV-1 and DENV-2 serotypes. However, in 2023, a significant shift in this pattern was observed as DENV-3 made a notable resurgence. This resurgence was characterized by the increase in DENV-3 cases within the country and the region of the Americas. Given the absence of sustained DENV-3 circulation in Brazil in previous years, this situation poses a significant risk, making the population highly susceptible to a potential novel epidemic. In November 2023, a 31-year-old male patient in Belo Horizonte exhibited symptoms of acute febrile syndrome. Multiplex RT-qPCR using the Kit Molecular ZC D-Tipagem confirmed DENV-3 infection, suggesting a likely autochthonous case, as the patient reported no travel history. To promptly assess this resurgence, we applied the nanopore sequencing technology. This allowed for the rapid characterization of the initial DENV-3 case isolated in Minas Gerais in 2023, representing a 13-year interval since the serotype's previous documented circulation in that state. This case report underscores the critical importance of proactive monitoring and the swift implementation of targeted control strategies to address the evolving dynamics of dengue, with a specific emphasis on the resurgence of DENV-3 in the state.

Metagenomic Analysis for Diagnosis of Hemorrhagic Fever in Minas Gerais, Brazil.

Viral hemorrhagic fever poses a significant public health challenge due to its severe clinical presentation and high mortality rate. The diagnostic process is hindered by similarity of symptoms across different diseases and the broad spectrum of pathogens that can cause hemorrhagic fever. In this study, we applied viral metagenomic analysis to 43 serum samples collected by the Public Health Laboratory (Fundação Ezequiel Dias, FUNED) in Minas Gerais State, Brazil, from patients diagnosed with hemorrhagic fever who had tested negative for the standard local hemorrhagic disease testing panel. This panel includes tests for Dengue virus (DENV) IgM, Zika virus IgM, Chikungunya virus IgM, yellow fever IgM, Hantavirus IgM, Rickettsia rickettsii IgM/IgG, and Leptospira interrogans IgM, in addition to respective molecular tests for these infectious agents. The samples were grouped into 18 pools according to geographic origin and analyzed through next-generation sequencing on the NextSeq 2000 platform. Bioinformatic analysis revealed a prevalent occurrence of commensal viruses across all pools, but, notably, a significant number of reads corresponding to the DENV serotype 2 were identified in one specific pool. Further verification via real-time PCR confirmed the presence of DENV-2 RNA in an index case involving an oncology patient with hemorrhagic fever who had initially tested negative for anti-DENV IgM antibodies, thereby excluding this sample from initial molecular testing. The complete DENV-2 genome isolated from this patient was taxonomically classified within the cosmopolitan genotype that was recently introduced into Brazil. These findings highlight the critical role of considering the patient's clinical condition when deciding upon the most appropriate testing procedures. Additionally, this study showcases the potential of viral metagenomics in pinpointing the viral agents behind hemorrhagic diseases. Future research is needed to assess the practicality of incorporating metagenomics into standard viral diagnostic protocols.

Exploring Microorganisms Associated to Acute Febrile Illness and Severe Neurological Disorders of Unknown Origin: A Nanopore Metagenomics Approach.

Acute febrile illness (AFI) and severe neurological disorders (SNDs) often present diagnostic challenges due to their potential origins from a wide range of infectious agents. Nanopore metagenomics is emerging as a powerful tool for identifying the microorganisms potentially responsible for these undiagnosed clinical cases. In this study, we aim to shed light on the etiological agents underlying AFI and SND cases that conventional diagnostic methods have not been able to fully elucidate. Our approach involved analyzing samples from fourteen hospitalized patients using a comprehensive nanopore metagenomic approach. This process included RNA extraction and enrichment using the SMART-9N protocol, followed by nanopore sequencing. Subsequent steps involved quality control, host DNA/cDNA removal, de novo genome assembly, and taxonomic classification. Our findings in AFI cases revealed a spectrum of disease-associated microbes, including Escherichia coli, Streptococcus sp., Human Immunodeficiency Virus 1 (Subtype B), and Human Pegivirus. Similarly, SND cases revealed the presence of pathogens such as Escherichia coli, Clostridium sp., and Dengue virus type 2 (Genotype-II lineage). This study employed a metagenomic analysis method, demonstrating its efficiency and adaptability in pathogen identification. Our investigation successfully identified pathogens likely associated with AFI and SNDs, underscoring the feasibility of retrieving near-complete genomes from RNA viruses. These findings offer promising prospects for advancing our understanding and control of infectious diseases, by facilitating detailed genomic analysis which is critical for developing targeted interventions and therapeutic strategies.

Detection of Multiple Human Viruses, including Mpox, Using a Wastewater Surveillance Approach in Brazil.

Sewage surveillance can be used as an effective complementary tool for detecting pathogens in local communities, providing insights into emerging threats and aiding in the monitoring of outbreaks. In this study using qPCR and whole genomic sewage surveillance, we detected the Mpox virus along with other viruses, in municipal and hospital wastewaters in Belo Horizonte, Brazil over a 9-month period (from July 2022 until March 2023). MPXV DNA detection rates varied in our study, with 19.6% (11 out of 56 samples) detected through the hybrid capture method of whole-genome sequencing and 20% (12 out of 60 samples) through qPCR. In hospital wastewaters, the detection rate was higher, at 40% (12 out of 30 samples) compared to 13.3% (4 out of 30 samples) in municipal wastewaters. This variation could be attributed to the relatively low number of MPXV cases reported in the city, which ranged from 106 to 341 cases during the study period, and the dilution effects, given that each of the two wastewater treatment plants (WWTP) investigated serves approximately 1.1 million inhabitants. Additionally, nine other virus families were identified in both hospitals and municipal wastewaters, including Adenoviridade, Astroviridae, Caliciviridae, Picornaviridade, Polyomaviridae, Coronaviridae (which includes SARS-CoV-2), Herspesviridae, Papillomaviridae and Flaviviridae (notably including Dengue). These findings underscore the potential of genomic sewage surveillance as a robust public health tool for monitoring a wide range of viruses circulating in both community and hospitals environments, including MPXV.

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.

Exploring Dengue Infection in a Vaccinated Individual: Preliminary Molecular Diagnosis and Sequencing Insights.

This study examines a case involving a 7-year-old child who developed dengue symptoms following Qdenga vaccination. Despite initial negative diagnostic results, molecular analysis confirmed an infection with DENV4. Next-generation sequencing detected viral RNA from both DENV2 and DENV4 serotypes, which were identified as vaccine-derived strains using specific primers. Phylogenetic analysis further confirmed that these sequences belonged to the Qdenga vaccine rather than circulating wild-type viruses. This case underscores the critical need for precise diagnostic interpretation in vaccinated individuals to avoid misdiagnosis and to strengthen public health surveillance. A comprehensive understanding of vaccine-induced viremia is essential for refining dengue surveillance, improving diagnostic accuracy, and informing public health strategies in endemic regions.

High Frequencies of kdr Mutation and Chikungunya Infection in Aedes aegypti Population from Minas Gerais, Brazil.

The Chikungunya virus (CHIKV) presents global health challenges, with Brazil experiencing outbreaks since its introduction in 2014. In 2023, following a CHIKV outbreak in Minas Gerais (MG), social media was used to optimize an entomological survey aimed at identifying vectors and viral lineages and assessing insecticide resistance. Following Instagram posts, residents with suspected CHIKV infection were able to schedule mosquito aspirations. In total, 421 mosquitoes (165 Aedes aegypti and 256 Culex quinquefasciatus) were captured from 40 households in Salinas city (MG) and tested for the Dengue, Zika, and Chikungunya viruses through RT-qPCR. Twelve of 57 pools (10 Ae. aegypti and two Cx. quinquefasciatus) tested positive for CHIKV RNA. Viral RNA was also detected in the heads of nine Ae. aegypti, indicating viral dissemination but not in Cx. quinquefasciatus. Genome sequencing yielded the first near-complete genome from the 2023 outbreak, unveiling that the CHIKV strain belonged to the East/Central/South African (ECSA) genotype. Additionally, genetic analyses revealed high frequencies of kdr alleles, including in CHIKV-infected mosquitoes, suggesting resistance to pyrethroid insecticides in this Ae. aegypti population. Social media was important for guiding mosquito-capture efforts in CHIKV transmission hotspots, thus optimizing the opportunity for viral detection. These findings emphasize the urgent need for innovative vector studies and control strategies, as well as interdisciplinary approaches in public health interventions.

Corrigendum: Genomic characterization of SARS-CoV-2 from an indigenous reserve in Mato Grosso do Sul, Brazil.

[This corrects the article DOI: 10.3389/fpubh.2023.1195779.].

Loxosceles amazonica Brown Spider venom: Insights into enzymatic activities, immunorecognition, and novel phospholipase D isoforms.

The Loxosceles genus represents one of the main arachnid genera of medical importance in Brazil. Despite the gravity of Loxosceles-related accidents, just a handful of species are deemed medically important and only a few have undergone comprehensive venom characterization. Loxosceles amazonica is a notable example of a potentially dangerous yet understudied Loxosceles species. While there have been limited reports of accidents involving L. amazonica to date, accidents related to Loxosceles are increasing in the North and Northeast regions of Brazil, where L. amazonica has been reported. In this work, we provide a complementary biochemical and immunological characterization of L. amazonica venom, considering its most relevant enzymatic activities and its immunorecognition and neutralization by current therapeutic antivenoms. Additionally, a cDNA library enriched with phospholipase D (PLD) sequences from L. amazonica venom glands was built and subsequently sequenced. The results showed that L. amazonica venom is well immunorecognised by all the tested antibodies. Its venom also displayed proteolytic, hyaluronidase, and sphingomyelinase activities. These activities were at least partially inhibited by available antivenoms. With cDNA sequencing of PLDs, seven new putative isoforms were identified in the venom of L. amazonica. These results contribute to a better knowledge of the venom content and activities of a synanthropic, yet understudied, Loxosceles species. In vivo assays are essential to confirm the medical relevance of L. amazonica, as well as to assess its true toxic potential and elucidate its related pathophysiology.

Exploring the Genomic Dynamics of the Monkeypox Epidemic in Paraguay.

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.

Retrospective Analysis of Omicron in Minas Gerais, Brazil: Emergence, Dissemination, and Diversification.

Brazil is one of the countries most affected by COVID-19, with the highest number of deaths recorded. Brazilian Health Institutions have reported four main peaks of positive COVID-19 cases. The last two waves were characterized by the emergence of the VOC Omicron and its sublineages. This study aimed to conduct a retrospective surveillance study illustrating the emergence, dissemination, and diversification of the VOC Omicron in 15 regional health units (RHUs) in MG, the second most populous state in Brazil, by combining epidemiological and genomic data. A total of 5643 confirmed positive COVID-19 samples were genotyped using the panels TaqMan SARS-CoV-2 Mutation and 4Plex SC2/VOC Bio-Manguinhos to define mutations classifying the BA.1, BA.2, BA.4, and BA.5 sublineages. While sublineages BA.1 and BA.2 were more prevalent during the third wave, BA.4 and BA.5 dominated the fourth wave in the state. Epidemiological and viral genome data suggest that age and vaccination with booster doses were the main factors related to clinical outcomes, reducing the number of deaths, irrespective of the Omicron sublineages. Complete genome sequencing of 253 positive samples confirmed the circulation of the BA.1, BA.2, BA.4, and BA.5 subvariants, and phylogenomic analysis demonstrated that the VOC Omicron was introduced through multiple international events, followed by transmission within the state of MG. In addition to the four subvariants, other lineages have been identified at low frequency, including BQ.1.1 and XAG. This integrative study reinforces that the evolution of Omicron sublineages was the most significant factor driving the highest peaks of positive COVID-19 cases without an increase in more severe cases, prevented by vaccination boosters.