RESUMO
In 2020, a global pandemic caused by SARS-CoV-2 was declared. Different institutes proposed diagnostic molecular methods to detect the virus in clinical samples. This study aims to validate and standardize the use of a loop-mediated isothermal amplification (LAMP)-based methodology targeting the viral RP gene, as a faster and low-cost diagnostic method for SARS-CoV-2 infections. The results obtained with RT-LAMP (Reverse Transcriptase) were compared to the results of real-time polymerase chain reaction (RT-PCR) to assess its sensitivity and specificity. In total, 115 samples (nasopharyngeal samples) were used for detecting SARS-CoV-2 by RT-LAMP, with 43 positives and 72 negatives. The study showed a positive predictive value (PPV) of 90.7% and a negative predictive value (VPN) of 100%. The LAMP assay also demonstrated a high sensitivity of 90.7% and a specificity of 100% (confidence interval 77.9-97.4%) when using the lower detection limit of 40 copies/µL. The RT-LAMP described here has the potential to detect even the new variants of SARS-CoV-2, suggesting that it may not be significantly affected by gene mutations. The RT-LAMP targeting the RP viral region is faster and less expensive than other molecular approaches, making it an alternative for developing countries.
Assuntos
COVID-19 , Transcrição Reversa , Humanos , RNA Viral/genética , COVID-19/diagnóstico , SARS-CoV-2/genética , RNA Polimerases Dirigidas por DNARESUMO
In 2015, two new species related to the Staphylococcus aureus were proposed. We describe five isolates of the new species Staphylococcus argenteus cultured from human cases of bacteremia and skin and soft tissue infections. This is the first report of S. argenteus, from South America, causing community-acquired and nosocomial infections.
Assuntos
Infecções Comunitárias Adquiridas , Infecções Estafilocócicas , Humanos , Brasil/epidemiologia , Staphylococcus , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus , Infecções Comunitárias Adquiridas/epidemiologiaRESUMO
The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.
Assuntos
COVID-19 , SARS-CoV-2 , Brasil , Genômica , HumanosRESUMO
Brazil has experienced some of the highest numbers of COVID-19 cases and deaths globally and from May 2021 made Latin America a pandemic epicenter. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of virus transmission dynamics at the national scale. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and a bordering country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed under an absence of effective restriction measures, there was a local emergence and onward international spread of Variants of Concern (VOC) and Variants Under Monitoring (VUM), including Gamma (P.1) and Zeta (P.2). In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the usefulness and need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring that provides a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies.
RESUMO
The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.
RESUMO
Background: The efficiency of isolation and purification of the viral genome is a critical step to the accuracy and reliability of RT-qPCR to detect SARS-CoV-2. However, COVID-19 testing laboratories were overwhelmed by a surge in diagnostic demand that affected supply chains especially in low and middle-income facilities. Objectives: Thus, this study compares the performance of alternative methods to extraction and purification of viral RNA in samples of patients diagnosed with COVID-19. Study design: Nasopharyngeal swabs were submitted to three in-house protocols and three commercial methods; viral genome was detected using the primer-probe (N1 and N2) described by CDC and viral load of samples were determined. Results: The in-house protocols resulted in detection of virus in 82.4 to 86.3% of samples and commercial methods in 94.1 to 98%. The disagreement results were observed in samples with low viral load or below the estimated limit of detection of RT-qPCR. Conclusion: The simplified methods proposed might be less reliable for patients with low viral load and alternative commercial methods showed comparable performance.
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Background: In Sub-Saharan Africa, including Mozambique, acute bacterial meningitis (ABM) represents a main cause of childhood mortality. The burden of ABM is seriously underestimated because of the poor performance of culture sampling, the primary method of ABM surveillance in the region. Low quality cerebrospinal fluid (CSF) samples and frequent consumption of antibiotics prior to sample collection lead to a high rate of false-negative results. To our knowledge, this study is the first to determine the frequency of ABM in Mozambique using real-time polymerase chain reaction (qPCR) and to compare results to those of culture sampling. Method: Between March 2013 and March 2014, CSF samples were collected at 3 regional hospitals from patients under 5 years of age, who met World Health Organization case definition criteria for ABM. Macroscopic examination, cytochemical study, culture, and qPCR were performed on all samples. Results: A total of 369 CSF samples were collected from children clinically suspected of ABM. qPCR showed a significantly higher detection rate of ABM-causing pathogens when compared to culture (52.3% [193/369] versus 7.3% [27/369], p = 0.000). The frequency of Streptococcus pneumoniae, Haemophilus influenzae, group B Streptococci, and Neisseria meningitidis were 32.8% (121/369), 12.2%, (45/369), 3.0% (16/369) and 4.3% (11/369), respectively, significantly higher compared to that obtained on culture (p < 0.001 for each). Conclusion: Our findings demonstrate that culture is less effective for the diagnosis of ABM than qPCR. The common use of culture rather than qPCR to identify ABM results in serious underestimation of the burden of the disease, and our findings strongly suggest that qPCR should be incorporated into surveillance activities for ABM. In addition, our data showed that S. pneumoniae represents the most common cause of ABM in children under 5 years of age.
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Pré-Escolar , DNA Bacteriano/líquido cefalorraquidiano , Meningites Bacterianas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estações do Ano , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/genética , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Índice de Gravidade de Doença , DNA Bacteriano/genética , Testes de Sensibilidade Microbiana , Haemophilus influenzae/isolamento & purificação , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Análise Multivariada , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/epidemiologia , Farmacorresistência Bacteriana , Moçambique/epidemiologia , Neisseria meningitidis/isolamento & purificação , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/genéticaRESUMO
INTRODUCTION: The genera Enterococcus, Staphylococcus and Streptococcus are recognized as important Gram-positive human pathogens. The aim of this study was to evaluate the performance of Vitek 2 in identifying Gram-positive cocci and their antimicrobial susceptibilities. METHODS: One hundred four isolates were analyzed to determine the accuracy of the automated system for identifying the bacteria and their susceptibility to oxacillin and vancomycin. RESULTS: The system correctly identified 77.9% and 97.1% of the isolates at the species and genus levels, respectively. Additionally, 81.8% of the Vitek 2 results agreed with the known antimicrobial susceptibility profiles. CONCLUSION: Vitek 2 correctly identified the commonly isolated strains; however, the limitations of the method may lead to ambiguous findings.
Assuntos
Antibacterianos/farmacologia , Cocos Gram-Positivos/efeitos dos fármacos , Testes de Sensibilidade Microbiana/instrumentação , Oxacilina/farmacologia , Software , Vancomicina/farmacologia , Automação Laboratorial , Cocos Gram-Positivos/classificação , Humanos , Testes de Sensibilidade Microbiana/métodos , Reprodutibilidade dos TestesRESUMO
A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.
Um novo teste baseado na reação em cadeia da polimerase em tempo real (qPCR) com SYBR ® Green foi desenvolvido para detectar duas espécies de Bartonella, B. henselae e B. clarridgeiae, diretamente em amostras de sangue. Este teste foi utilizado em amostras de sangue obtidas de gatos que vivem em abrigos de animais do sul do Brasil. Os resultados foram comparados aos obtidos pelo PCR convencional utilizado para a detecção de Bartonella spp. Das 47 amostras analisadas, oito foram positivas no PCR convencional e 12 foram positivas para qPCR. A reação de qPCR, permitiu a detecção da presença simultânea de B. henselae e B. clarridgeiae em duas destas amostras. Os resultados mostram que a qPCR aqui descrita pode ser uma ferramenta confiável para a detecção e diferenciação de duas espécies importantes de Bartonella spp.
Assuntos
Animais , Gatos , Infecções por Bartonella/veterinária , Bartonella/genética , Bartonella/isolamento & purificação , Doenças do Gato/microbiologia , DNA Bacteriano/sangue , Reação em Cadeia da Polimerase Multiplex/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Infecções por Bartonella/microbiologia , Bartonella henselae/genética , Bartonella henselae/isolamento & purificação , Especificidade da EspécieRESUMO
A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.
Assuntos
Infecções por Bartonella/veterinária , Bartonella/genética , Bartonella/isolamento & purificação , Doenças do Gato/microbiologia , DNA Bacteriano/sangue , Reação em Cadeia da Polimerase Multiplex/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Infecções por Bartonella/microbiologia , Bartonella henselae/genética , Bartonella henselae/isolamento & purificação , Gatos , Especificidade da EspécieRESUMO
Real-time PCR based on the recN and gyrB genes was developed to detect four Streptococcus bovis/Streptococcus equinus complex (SBEC) subspecies from rectal swab specimens. The overall prevalence was 35.2%: Streptococcus gallolyticus subsp. gallolyticus (11.1%), S. gallolyticus subsp. pasteurianus (13%), Streptococcus infantarius subsp. coli (20.4%), and S. infantarius subsp. infantarius (11.1%). To conclude, these real-time PCR assays provide a reliable molecular method to detect SBEC pathogenic subspecies from rectal swab specimens.
Assuntos
Técnicas Bacteriológicas/métodos , Portador Sadio/diagnóstico , Fezes/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Estreptocócicas/diagnóstico , Streptococcus/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , DNA Girase/genética , Enzimas de Restrição do DNA/genética , DNA Bacteriano/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sondas de Oligonucleotídeos/genética , Prevalência , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Streptococcus/genéticaRESUMO
The aims of this research were to screen and characterize a new microbial source of γ-PGA, to optimize aspects of culture conditions and medium composition using central composite design and response surface methodologies. The influence of bioreactor stirring rates on the production of γ-PGA was also investigated and the oxygen volumetric mass transfer coefficients (k La) were established. The most productive strain was identified by 16S rDNA analysis as Bacillus subtilis, and its γ-PGA production in rotatory shaker was threefold increased under optimized conditions (37 °C, pH 6.9, and 1.22 mM Zn(2+)), compared to conventional medium. In bioreactor, the γ-PGA production was further increased, reaching 17 g l(-1), 70 % higher than shaker cultures. γ-PGA production showed high dependency on oxygen transfer. At k La of 210 h(-1), the cultivation time could be reduced to 48 h, about 50 % of the time required for operations at k La 55 h(-1).
Assuntos
Bacillus subtilis/metabolismo , Ácido Poliglutâmico/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Reatores Biológicos , Brasil , Meios de Cultura , DNA Ribossômico/genética , Cinética , RNA Ribossômico 16S/genéticaRESUMO
The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5% of samples and hBoV in 13.2%. A unique causative viral agent was identified in 46.2% samples and the coinfection rate was 43.7%. For hBoV, 98.3% of all positive samples were from patients with mixed infections. Similarly, 84.8% of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.
Assuntos
Bocavirus Humano/isolamento & purificação , Metapneumovirus/isolamento & purificação , Nasofaringe/virologia , Infecções Respiratórias/virologia , Doença Aguda , Feminino , Bocavirus Humano/genética , Humanos , Lactente , Masculino , Metapneumovirus/genética , Reação em Cadeia da Polimerase , Infecções Respiratórias/diagnóstico , Estações do Ano , População UrbanaRESUMO
The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5 percent of samples and hBoV in 13.2 percent. A unique causative viral agent was identified in 46.2 percent samples and the coinfection rate was 43.7 percent. For hBoV, 98.3 percent of all positive samples were from patients with mixed infections. Similarly, 84.8 percent of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.
Assuntos
Feminino , Humanos , Lactente , Masculino , Bocavirus Humano , Metapneumovirus , Nasofaringe , Infecções Respiratórias , Doença Aguda , Bocavirus Humano , Metapneumovirus , Reação em Cadeia da Polimerase , Infecções Respiratórias , Estações do Ano , População UrbanaRESUMO
A avaliação interlaboratorial ou Teste de Proficiência (TP) é uma ferramenta utilizada como avaliação externa da qualidade, visando comparar o desempenho dos resultados obtidos pelos laboratórios de análises clínicas (LACs). Este trabalho objetivou realizar um programa piloto para comparação interlaboratorial em ensaios de Bioquímica, de acordo com os documentos da National Association os Testing Authorities NATA (2004) e Associação Brasileira de Normas Técnicas ABNT (1999). Participaram desse estudo seis LACs, sediados na região metropolitana de Porto Alegre. Como amostras para essa avaliação foram utilizados três lotes de pool de soros onde se avaliaram glicose, creatinina, ureia e colesterol total. Os dados enviados por cada participante foram avaliados utilizando-se estatística robusta, e seu desempenho classificado como Satisfatório, Questionável ou Insatisfatório, segundo o Escore Z obtido. Verificou-se que todos os laboratórios participantes obtiveram resultado Satisfatório para os parâmetros colesterol total e ureia. Para a glicose, um dos participantes obteve resultado Questionável (Z= 2). Resultado idêntico foi encontrado para acreatinina, onde um laboratório obteve escore Z= 2,6, classificado como Questionável. Com base nestes dados, concluímos que é possível utilizar, com sucesso, o pool de soros para ensaios de proficiência, permitindo ao laboratório comparar o seu desempenho com o de outros laboratórios semelhantes, implementando ações preventivas visando à melhoria dos seus procedimentos.
The interlaboratorial assessment or Proficiency Test (PT) is a tool used as external quality assessment, comparing the results obtained by the performance of clinical laboratories (LACs). The aim of this study was propose a pilot program for intercomparison trials of Biochemistry, according to the documents of the National Association os Testing Authorities NATA (2004) and Associação Brasileira de Normas Técnicas ABNT (1999). Participated in this study 6 LACs, based in the metropolitan region of Porto Alegre. As samples for this evaluation was used 3 batches of pool of blood serum which evaluated glucose, creatinine, urea and total cholesterol. The data sent by each participant were assessed using a robust statistical, and its performancerated as Satisfactory, Unsatisfactory or Questionable, according to the Z-score obtained. It was found that all participating laboratories obtained satisfactory results for the parameters total cholesterol and urea. For glucose, a result of the participants got questionable (Z = 2). Identical results was found for creatinine, where a laboratory had Z score=2.6, classified as Questionable. Based on these data, we conclude that it is possible to use successfully, the pool of blood serum for proficiency testing, allowing the laboratory to compare its performance with other similar laboratories, implementing preventive actions aimed at improving their procedures.
Assuntos
Bioquímica , Coleta de Amostras Sanguíneas , Ensaio de Proficiência Laboratorial , Controle de QualidadeRESUMO
Bartonella spp are the causative agent of cat scratch disease in humans. Cats are the natural reservoir of these bacteria and may infect humans through scratches, bites or fleas. Blood samples from 47 cats aged up to 12 months were collected for this study. All animals were lodged in municipal animal shelters in the Vale do Sinos region, Rio Grande do Sul, Brazil. Bartonella spp were detected by genus-specific polymerase chain reaction (PCR) and when the PCR was positive, the species were determined by DNA sequencing. A Giemsa-stained blood smear was also examined for the presence of intraerythrocytic elements suggestive of Bartonella spp infection. Phylogenetic analysis was also performed for all positive samples. Using molecular detection methods, Bartonella spp were detected in 17.02% (8/47) of the samples. In seven out of eight samples confirmed to be positive for Bartonella spp, blood smear examination revealed the presence of intraerythrocytic elements suggestive of Bartonella spp. Phylogenetic analysis characterized positive samples as Bartonella henselae (5) or Bartonella clarridgeiae (3). To the best of our knowledge, this is the first molecular study demonstrating the presence of Bartonella spp in cats from the Southern Region of Brazil.
Assuntos
Infecções por Bartonella/veterinária , Bartonella/genética , Doenças do Gato/microbiologia , Animais , Bartonella/classificação , Bartonella/isolamento & purificação , Infecções por Bartonella/epidemiologia , Infecções por Bartonella/microbiologia , Bartonella henselae/genética , Bartonella henselae/isolamento & purificação , Brasil/epidemiologia , Doenças do Gato/epidemiologia , Gatos , DNA Bacteriano/sangue , DNA Bacteriano/genética , Filogenia , Reação em Cadeia da Polimerase , PrevalênciaRESUMO
Bartonella spp are the causative agent of cat scratch disease in humans. Cats are the natural reservoir of these bacteria and may infect humans through scratches, bites or fleas. Blood samples from 47 cats aged up to 12 months were collected for this study. All animals were lodged in municipal animal shelters in the Vale do Sinos region, Rio Grande do Sul, Brazil. Bartonella spp were detected by genus-specific polymerase chain reaction (PCR) and when the PCR was positive, the species were determined by DNA sequencing. A Giemsa-stained blood smear was also examined for the presence of intraerythrocytic elements suggestive of Bartonella spp infection. Phylogenetic analysis was also performed for all positive samples. Using molecular detection methods, Bartonella spp were detected in 17.02 percent (8/47) of the samples. In seven out of eight samples confirmed to be positive for Bartonella spp, blood smear examination revealed the presence of intraerythrocytic elements suggestive of Bartonella spp. Phylogenetic analysis characterized positive samples as Bartonella henselae (5) or Bartonella clarridgeiae (3). To the best of our knowledge, this is the first molecular study demonstrating the presence of Bartonella spp in cats from the Southern Region of Brazil.
Assuntos
Animais , Gatos , Infecções por Bartonella/veterinária , Bartonella , Doenças do Gato , Infecções por Bartonella , Infecções por Bartonella , Bartonella henselae , Bartonella henselae , Bartonella , Bartonella , Brasil , Doenças do Gato , DNA Bacteriano/sangue , DNA Bacteriano , Filogenia , Reação em Cadeia da Polimerase , PrevalênciaRESUMO
INTRODUCTION: Norovírus was recently identified as the main cause of outbreaks of acute gastroenteritis of non-bacterial origin worldwide and it is involved in episodes of foodborne origin. In this study, patients with symptoms of acute gastroenteritis were evaluated over a one-year period, in order to evaluate two methods for identifying norovírus (real-time and conventional polymerase chain reaction), along with its incidence, seasonality and predominant genotype. METHODS: After RNA extraction, 50 samples were analyzed using conventional PCR and 365 were analyzed using real-time PCR. All the samples that presented positive results using both methods or discordant results were sequenced. In all, 13 samples were sequenced. RESULTS: Out of the 50 samples tested using both methods, seven presented a positive result from the conventional method and 15 from real-time PCR. Out of the total of 365 samples tested using real-time PCR, 48 were positive. All of the sequenced samples were shown to present norovírus of genogroup II. Regarding the distribution of norovírus-positive sample incidence over the course of the year, higher frequency of positive cases was observed during the southern hemisphere spring, reaching 29.7% in November. CONCLUSIONS: We observed that real-time PCR was more sensitive for identifying norovírus. The incidence of norovírus was 13.2% and genogroup II predominated among the population evaluated, with the greatest infection rate in the southern hemisphere spring.
Assuntos
Fezes/virologia , Gastroenterite/virologia , Norovirus/genética , RNA Viral/análise , Doença Aguda , Brasil/epidemiologia , Genótipo , Humanos , Incidência , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Estações do AnoRESUMO
INTRODUÇÃO: O norovírus foi recentemente identificado como o principal causador de surtos de gastroenterite aguda de origem não bacteriana em todo o mundo e está envolvido em episódios de origem alimentar. Neste estudo, foram avaliados pacientes com sintomas de gastroenterite aguda pelo período de um ano, a fim de se avaliar duas metodologias na identificação do NoV - a reação em cadeia por polimerase convencional e em tempo real -, incidência, sazonalidade e genótipo predominante. MÉTODOS: Após a extração do RNA, 50 amostras foram analisadas pela metodologia de PCR convencional e 365 amostras foram analisadas pela metodologia de PCR em tempo real. Todas as amostras que apresentaram resultado positivo pelas duas metodologias ou discordante foram sequenciadas, ao todo, 13 amostras foram sequenciadas. RESULTADOS: Das 50 amostras testadas pelas duas metodologias, 7 apresentaram resultado positivo pelo método convencional e 15 pelo método da PCR em tempo real. Do total de 365 amostras testadas pela metodologia de PCR, em tempo real, 48 foram positivas. Em relação às amostras sequenciadas, todas mostraram ser NoV do genogrupo II. Em relação à distribuição da incidência de amostras, positivas para NoV, ao longo do ano, pôde ser observada uma frequência de casos positivos maior na primavera, chegando a 29,7 por cento em novembro. CONCLUSÕES: Observamos que o PCR em tempo real é o método mais sensível para a identificação do Nov, que a incidência do NoV é de 13,2 por cento e o genogrupo II prevalece na população avaliada, sendo a primavera o período de maior taxa de infecção.
INTRODUCTION: Norovírus was recently identified as the main cause of outbreaks of acute gastroenteritis of non-bacterial origin worldwide and it is involved in episodes of foodborne origin. In this study, patients with symptoms of acute gastroenteritis were evaluated over a one-year period, in order to evaluate two methods for identifying norovírus (real-time and conventional polymerase chain reaction), along with its incidence, seasonality and predominant genotype. METHODS: After RNA extraction, 50 samples were analyzed using conventional PCR and 365 were analyzed using real-time PCR. All the samples that presented positive results using both methods or discordant results were sequenced. In all, 13 samples were sequenced. RESULTS: Out of the 50 samples tested using both methods, seven presented a positive result from the conventional method and 15 from real-time PCR. Out of the total of 365 samples tested using real-time PCR, 48 were positive. All of the sequenced samples were shown to present norovírus of genogroup II. Regarding the distribution of norovírus-positive sample incidence over the course of the year, higher frequency of positive cases was observed during the southern hemisphere spring, reaching 29.7 percent in November. CONCLUSIONS: We observed that real-time PCR was more sensitive for identifying norovírus. The incidence of norovírus was 13.2 percent and genogroup II predominated among the population evaluated, with the greatest infection rate in the southern hemisphere spring.
Assuntos
Humanos , Fezes/virologia , Gastroenterite/virologia , Norovirus/genética , RNA Viral/análise , Doença Aguda , Brasil/epidemiologia , Genótipo , Incidência , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Estações do AnoRESUMO
INTRODUÇÃO E OBJETIVOS: São conhecidos mais de 100 tipos de papilomavírus humano (HPV), dos quais 30 têm sido reportados em infecções anogenitais. A infecção tem importância clínica, pois alguns tipos virais estão associados a lesões que podem progredir para o câncer cervical. Sabe-se que os métodos moleculares são muito importantes para o diagnóstico dessa infecção. O objetivo do estudo é comparar a detecção de HPV de alto risco pelo método de captura híbrida 2 (CH2) com a detecção do vírus pela reação em cadeia da polimerase convencional (PCRc) e em tempo real (PCR-TR). METODOLOGIA: Foram analisadas 56 amostras ectocervicais por CH2 e, após, por PCRc e PCR-TR. RESULTADOS: Ambas, PCRc e PCR-TR, apresentaram alta concordância entre si (95,1 por cento), enquanto a comparação entre as PCRs e a CH2 mostrou concordância razoável entre os resultados (PCRc = 90,2 por cento e PCR-TR = 87,8 por cento). DISCUSSÃO E CONCLUSÃO: A CH é aceita para a detecção do HPV, entretanto pode ser menos sensível em comparação com as técnicas de PCR. A PCR-TR tem a vantagem sobre a PCRc em termos de velocidade, sendo também um pouco mais sensível. Devido à alta sensibilidade e à rapidez, os métodos de PCR poderiam ser usados para a triagem de HPV em amostras ectocervicais.
INTRODUCTION AND OBJECTIVE: More than 100 types of human papillomaviruses (HPV) are known, of which 30 have been reported in anogenital infections. The infection has clinical importance, inasmuch as some viral types are associated with lesions that can progress to cervical cancer. Molecular methods are considered an important tool for the diagnosis of this infection. The objective of this study was to compare the detection of high risk HPV using hybrid capture 2 with HPV detection by conventional and real time PCR. METHODOLOGY: 56 ectocervical samples were analyzed by hybrid capture and after that by conventional and real time PCR. RESULTS: Both PCR and RT-PCR showed a high degree of correlation (95.1 percent), whereas the comparison between PCR and HC2 showed a fair correlation (90.2 percent and 87.8 percent for PCR and RT-PCR, respectively). DISCUSSION AND CONCLUSIONS: HC is widely accepted for the detection of HPV, however, it may lack sensitivity in comparison with PCR techniques. RT-PCR has further advantages over the conventional PCR in terms of speed as well as it is slightly more sensitive. Due to their high sensitivity and fast response, PCR methods could be used as a screening method for HPV detection in ectocervical samples.