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LIM/46 - Laboratório de Parasitologia Médica, Hospital das Clínicas, Faculdade de Medicina
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- Multiplex qPCR Discriminates Variants of Concern to Enhance Global Surveillance of SARS-CoV-2(2023) VOGELS, C. B. F.; BREBAN, M. I.; OTT, I. M.; ALPERT, T.; PETRONE, M. E.; WATKINS, A. E.; KALINICH, C. C.; EARNEST, R.; ROTHMAN, J. E.; JESUS, J. G. de; CLARO, I. M.; FERREIR, G. M.; CRISPIM, M. A. E.; SINGH, L.; TEGALLY, H.; ANYANEJI, U. J.; HODCROF, E. B.; MASON, C. E.; KHULLAR, G.; METTI, J.; DUDLEY, J. T.; MACKAY, M. J.; NASH, M.; WANG, J.; LIU, C.; HUI, P.; MURPHY, S.; NEAL, C.; LASZLO, E.; LANDRY, M. L.; MUYOMBWE, A.; DOWNING, R.; RAZEQ, J.; OLIVEIRA, T. de; FARIA, N. R.; SABINO, E. C.; NEHER, R. A.; FAUVER, J. R.; GRUBAUGH, N. D.Broadly accessible and inexpensive surveillance methods are needed to track Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOC) around the world. While sequencing is the gold standard to identify circulating SARS-CoV-2 variants, routine genomic surveillance is not available in many locations primarily due to a lack of resources and expertise. The Institutional Review Board from the Yale University Human Research Protection Program determined that the RT-qPCR testing and sequencing of de-identified remnant COVID-19 clinical samples conducted in this study is not research involving human patients. Multiplex-PCR products were purified by using AmpureXP beads, and quantification was carried out using the Qubit dsDNA High Sensitivity assay on the Qubit 3.0. © 2023 Jenny Stanford Publishing Pte. Ltd.
- Impact of COVID-19 RT-PCR testing of asymptomatic health care workers on absenteeism and hospital transmission during the pandemic(2023) MENDES, Elisa Teixeira; NETO, Danilo Glauco Pereira Villagelin; FERREIRA, Giulia Magalhaes; VALENCA, Ian Nunes; LIMA, Maria Patelli Juliani Souza; FREITAS, Maria Fernanda Marciano Barros de; DONALISIO, Maria Rita; MELO, Marcio Cristiano; LAZARI, Carolina; GOES, Jacqueline; MORALES, Ingra; JARDIM, Ana Carolina Gomes; SANTOS, Pamela Andrade dos; FRANCO, Lucas Augusto Moyses; SABINO, Ester Cerdeiro; COSTA, Silvia FigueiredoBackground: Reducing the transmission of SARS-CoV-2 from asymptomatic and pre-symptomatic patients is critical in controlling the circulation of the virus.Methods: This study evaluated the prevalence of Reverse transcription polymerase chain reaction (RT-PCR) positivity in serial tests in 429 asymptomatic health care workers (HCW) and its impact on absenteeism. HCW from a COVID-19 reference hospital were tested, screened, and placed on leave. A time-series seg-mented regression of weekly absenteeism rates was used, and cases of infection among hospitalized patients were analyzed. Viral gene sequencing and phylogenetic analysis were performed on samples from HCW who had a positive result.Results: A significant decrease in absenteeism was detected 3-4 weeks after the intervention at a time of increased transmission within the city. The prevalence of RT-PCR positivity among asymptomatic professio-nals was 17.3%. Phylogenetic analyses (59 samples) detected nine clusters, two of them strongly suggestive of intrahospital transmission with strains (75% B.1.1.28) circulating in the region during this period.Conclusions: Testing and placing asymptomatic professionals on leave contributed to control strategy for COVID-19 transmission in the hospital environment, and in reducing positivity and absenteeism, which directly influences the quality of care and exposes professionals to an extra load of stress.(c) 2022 Association for Professionals in Infection Control and Epidemiology, Inc.
- SARS-CoV-2 Detection and Culture in Different Biological Specimens from Immunocompetent and Immunosuppressed COVID-19 Patients Infected with Two Different Viral Strains(2023) MENDES-CORREA, Maria Cassia; SALOMAO, Matias Chiarastelli; GHILARDI, Fabio; TOZETTO-MENDOZA, Tania Regina; VILLAS-BOAS, Lucy Santos; PAULA, Anderson Vicente de; PAIAO, Heuder Gustavo Oliveira; COSTA, Antonio Charlys da; LEAL, Fabio E.; FERRAZ, Andrea de Barros Coscelli; SALES, Flavia C. S.; CLARO, Ingra M.; FERREIRA, Noely E.; PEREIRA, Geovana M.; JR, Almir Ribeiro da Silva; FREIRE, Wilton; ESPINOZA, Evelyn Patricia Sanchez; MANULI, Erika R.; ROMANO, Camila M.; JESUS, Jaqueline G. de; SABINO, Ester C.; WITKIN, Steven S.Introduction-The dynamics of SARS-CoV-2 shedding and replication in humans remain incompletely understood. Methods-We analyzed SARS-CoV-2 shedding from multiple sites in individuals with an acute COVID-19 infection by weekly sampling for five weeks in 98 immunocompetent and 25 immunosuppressed individuals. Samples and culture supernatants were tested via RT-PCR for SARS-CoV-2 to determine viral clearance rates and in vitro replication. Results-A total of 2447 clinical specimens were evaluated, including 557 nasopharyngeal swabs, 527 saliva samples, 464 urine specimens, 437 anal swabs and 462 blood samples. The SARS-CoV-2 genome sequences at each site were classified as belonging to the B.1.128 (ancestral strain) or Gamma lineage. SARS-CoV-2 detection was highest in nasopharyngeal swabs regardless of the virus strain involved or the immune status of infected individuals. The duration of viral shedding varied between clinical specimens and individual patients. Prolonged shedding of potentially infectious virus varied from 10 days up to 191 days, and primarily occurred in immunosuppressed individuals. Virus was isolated in culture from 18 nasal swab or saliva samples collected 10 or more days after onset of disease. Conclusions-Our findings indicate that persistent SARS-CoV-2 shedding may occur in both competent or immunosuppressed individuals, at multiple clinical sites and in a minority of subjects is capable of in vitro replication.
- Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing(2023) CLARO, I. M.; RAMUNDO, M. S.; COLETTI, T. M.; SILVA, C. A. M. da; VALENCA, I. N.; CANDIDO, D. S.; SALES, F. C. S.; MANULI, E. R.; JESUS, J. G. de; PAULA, A. de; FELIX, A. C.; ANDRADE, P. D. S.; PINHO, M. C.; SOUZA, W. M.; AMORIM, M. R.; PROENCA-MODENA, J. L.; KALLAS, E. G.; LEVI, J. E.; FARIA, N. R.; SABINO, E. C.; LOMAN, N. J.; QUICK, J.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.
- Dynamics of Early Establishment of SARS-CoV-2 VOC Omicron Lineages in Minas Gerais, Brazil(2023) MENEZES, Mariane Talon de; MOREIRA, Filipe Romero Rebello; WHITTAKER, Charles; SANTOS, Franciele Martins; QUEIROZ, Daniel Costa; GEDDES, Victor; FONSECA, Paula Luize Camargos; JESUS, Jaqueline Goes de; MENDES-OLIVEIRA, Franciane; REIS-SOUZA, Valquiria; SANTOS, Bibiana; ZAULI, Danielle Alves Gomes; LIMA, Aline Brito de; MENDONCA, Cristiane de Brito; ALVIM, Luige Biciati; SILVA, Joice do Prado; MALTA, Frederico Scott Varella; FERREIRA, Alessandro Clayton de Souza; FARIA, Nuno R.; SABINO, Ester Cerdeira; AGUIAR, Renato SantanaBrazil is one of the nations most affected by Coronavirus disease 2019 (COVID-19). The introduction and establishment of new virus variants can be related to an increase in cases and fatalities. The emergence of Omicron, the most modified SARS-CoV-2 variant, caused alarm for the public health of Brazil. In this study, we examined the effects of the Omicron introduction in Minas Gerais (MG), the second-most populous state of Brazil. A total of 430 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) samples from November 2021 to June 2022 from Belo Horizonte (BH) city were sequenced. These newly sequenced genomes comprise 72% of all previously available SARS-CoV-2 genomes for the city. Evolutionary analysis of novel viral genomes reveals that a great diversity of Omicron sublineages have circulated in BH, a pattern in-keeping with observations across Brazil more generally. Bayesian phylogeographic reconstructions indicate that this diversity is a product of a large number of international and national importations. As observed previously, Sao Paulo state is shown as a significant hub for viral spread throughout the country, contributing to around 70% of all viral Omicron introductions detected in MG.
- Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil(2023) SCHIFFLER, Francine Bittencourt; PEREIRA, Asheley Henrique Barbosa; MOREIRA, Silvia Bahadian; ARRUDA, Igor Falco; MOREIRA, Filipe Romero Rebello; D'ARC, Mirela; CLARO, Ingra Morales; PISSINATTI, Thalita de Abreu; CAVALCANTE, Liliane Tavares de Faria; MIRANDA, Thamiris dos Santos; COSENTINO, Matheus Augusto Calvano; OLIVEIRA, Renata Carvalho de; FERNANDES, Jorlan; ASSIS, Matheus Ribeiro da Silva; OLIVEIRA, Jonathan Goncalves de; SILVA, Thayssa Alves Coelho da; GALLIEZ, Rafael Mello; FAFFE, Debora Souza; JESUS, Jaqueline Goes de; SILVA, Marise Sobreira Bezerra da; BEZERRA, Matheus Filgueira; FERREIRA, Orlando da Costa; TANURI, Amilcar; CASTINEIRAS, Terezinha Marta; AGUIAR, Renato Santana; FARIA, Nuno Rodrigues; ALMEIDA, Alzira Paiva de; PISSINATTI, Alcides; SABINO, Ester Cerdeira; AMENDOEIRA, Maria Regina Reis; LEMOS, Elba Regina Sampaio de; UBIALI, Daniel Guimaraes; SANTOS, Andre F. A.Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.
- Genetic differences of dengue virus 2 in patients with distinct clinical outcome(2023) MARQUES, Beatriz de Carvalho; SACCHETTO, Livia; BANHO, Cecilia Artico; ESTOFOLETE, Cassia Fernanda; DOURADO, Fernanda Simoes; CANDIDO, Darlan da Silva; DUTRA, Karina Rocha; SALLES, Flavia Cristina da Silva; JESUS, Jaqueline Goes de; SABINO, Ester Cerdeira; FARIA, Nuno Rodrigues; NOGUEIRA, Mauricio LacerdaThe genetic diversity of the dengue virus is characterized by four circulating serotypes, several genotypes, and an increasing number of existing lineages that may have differences in the potential to cause epidemics and disease severity. Accurate identification of the genetic variability of the virus is essential to identify lineages responsible for an epidemic and understanding the processes of virus spread and virulence. Here, we characterize, using portable nanopore genomic sequencing, different lineages of dengue virus 2 (DENV-2) detected in 22 serum samples from patients with and without dengue warning signs attended at Hospital de Base of Sao Jose do Rio Preto (SJRP) in 2019, during a DENV-2 outbreak. Demographic, epidemiological, and clinical data were also analyzed. The phylogenetic reconstruction and the clinical data showed that two lineages belonging to the American/Asian genotype of DENV-2-BR3 and BR4 (BR4L1 and BR4L2)-were co-circulating in SJRP. Although preliminary, these results indicate no specific association between clinical form and phylogenetic clustering at the virus consensus sequence level. Studies with larger sample sizes and which explore single nucleotide variants are needed. Therefore, we showed that portable nanopore genome sequencing could generate quick and reliable sequences for genomic surveillance to monitor viral diversity and its association with disease severity as an epidemic unfolds.
- Introduction, Dispersal, and Predominance of SARS-CoV-2 Delta Variant in Rio Grande do Sul, Brazil: A Retrospective Analysis(2023) CASTRO, Thais Regina y; PICCOLI, Bruna C.; VIEIRA, Andressa A.; CASARIN, Bruna C.; TESSELE, Luiza F.; SALVATO, Richard S.; GREGIANINI, Tatiana S.; MARTINS, Leticia G.; RESENDE, Paola Cristina; PEREIRA, Elisa C.; MOREIRA, Filipe R. R.; JESUS, Jaqueline G. de; SEERIG, Ana Paula; LOBATO, Marcos Antonio O.; CAMPOS, Marli M. A. de; GOULARTE, Juliana S.; SILVA, Mariana S. da; DEMOLINER, Meriane; FILIPPI, Micheli; PEREIRA, Vyctoria M. A. Goes; SCHWARZBOLD, Alexandre V.; SPILKI, Fernando R.; TRINDADE, Priscila A.Mutations in the SARS-CoV-2 genome can alter the virus' fitness, leading to the emergence of variants of concern (VOC). In Brazil, the Gamma variant dominated the pandemic in the first half of 2021, and from June onwards, the first cases of Delta infection were documented. Here, we investigate the introduction and dispersal of the Delta variant in the RS state by sequencing 1077 SARS-CoV-2-positive samples from June to October 2021. Of these samples, 34.7% were identified as Gamma and 65.3% as Delta. Notably, 99.2% of Delta sequences were clustered within the 21J lineage, forming a significant Brazilian clade. The estimated clock rate was 5.97 x 10-4 substitutions per site per year. The Delta variant was first reported on 17 June in the Vinhedos Basalto microregion and rapidly spread, accounting for over 70% of cases within nine weeks. Despite this, the number of cases and deaths remained stable, possibly due to vaccination, prior infections, and the continued mandatory mask use. In conclusion, our study provides insights into the Delta variant circulating in the RS state, highlighting the importance of genomic surveillance for monitoring viral evolution, even when the impact of new variants may be less severe in a given region.