Aplicabilidade da PCR em tempo real ao diagnóstico da leptospirose humana e ao monitoramento da contaminação ambiental por espécies patogênicas de Leptospira

Orientador : Prof. Dr. Alexander W. BiondoOrientador : Prof. Albert Icksang KoCoorientador : Prof. Dr. Marco Aurélio KriegerCoorientador : Dr. Alex HoffmasterTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 27/02/2012Inclui referências : f. 150-169Resumo: cuja sintomatologia é inespecífica. Humanos desenvolvem formas graves de leptospirose e os métodos diagnósticos atuais não são eficazes para a detecção precoce da doença. O uso de um método de PCR em tempo real pode permitir o diagnóstico precoce e a quantificação das bactérias em amostras clínicas; além da quantificação do risco ambiental em amostras de água. Foi demonstrado que o método permite a detecção das leptospiras em amostras de sangue total coletadas nos primeiros seis dias de doença, após os quais a carga bacteriana decai de modo inversamente proporcional ao título de anticorpos. O limite mínimo de detecção foi estimado em 280 GEq/mL. O ponto de corte prognóstico foi estimado em 159 GEq/mL para o desenvolvimento de LPHS, com sensibilidade de 64,3% (IC 95% 35,1-87,2%), especificidade de 68,8% (IC 95% 62,8-74,3%) e acurácia 68,0%. Para óbito, o ponto de corte prognóstico foi definido em 1.181 GEq/mL, com sensibilidade de 76,5% (IC 95% 50,1-93,2%), especificidade de 89,3% (IC 95% 85,0-92,8%) e acurácia de 88,3%. A seleção do kit para extração do DNA e estabelecimento de parâmetros de concentração, volume inicial de amostra e limite mínimo de detecção permitiu a adaptação do método de qPCR para a quantificação das leptospiras em amostras ambientais. O método adaptado foi validado em estudo conduzido com amostras ambientais coletadas na comunidade de Pau da Lima, em Salvador (Bahia, Brasil). Os resultados apontaram maior positividade entre as amostras coletadas em baixa altitude (50% das amostras positivas), bem como entre as amostras coletadas pela manhã em comparação com a tarde (17,7% vs. 5,8%). A positividade foi maior entre amostras de esgoto do que entre amostras de água empoçada (17,7% vs. 5,6%). Também houve diferença de positividade entre as amostras coletadas nos períodos úmido e com umidade intermediária (16,0% vs. 9,7%). Assim, concluímos que o método de qPCR é uma ferramenta útil para o diagnóstico e avaliação prognóstica em pacientes com suspeita clínica de leptospirose. O método também foi eficaz na identificação de possíveis focos de infecção a partir da avaliação de amostras de água coletadas em área endêmica. Palavras-chave: Leptospira, leptospirose, PCR em tempo real, sangue total, carga bacteriana, prognóstico, amostra ambiental de água.Abstract: are non-specific. Humans develop severe forms of leptospirosis and the current diagnostic methods are not suited for the early diagnosis of the disease. The use of a real-time PCR method allowed for an early diagnosis and bacterial quantification in clinical samples, as well as quantification of environmental burden in water samples. It has been shown that this method allows for the detection of leptospires in whole blood samples obtained during the first six days of illness, after which the bacterial load decrease is inversely proportional to the raise in antibody titer. The lower limit of detection was estimated to be 280 GEq/mL. The prognostic threshold was estimated at 159 GEq/mL for the development of LPHS, with sensitivity of 64.3% (95% CI 35.1- 87.2%), specificity of 68.8% (95% CI 62.8-74.3%) and accuracy of 68.0%. For death, the prognostic threshold was set at 1.181 GEq/mL, with sensitivity of 76.5% (95% CI 50.1-93.2%), specificity of 89.3% (95% CI 85.0-92.8%) and accuracy of 88.3%. Selection of the commercial kit for DNA extraction, and establishment of concentration parameters, initial sample volume and lower limit of detection led to the adaption of the qPCR method to allow for the quantification of leptospires in environmental samples. The adapted method was validated in a study conducted with environmental samples collected in the slum community of Pau da Lima, in Salvador (Bahia, Brazil). Results showed a higher positivity for samples collected at low altitudes (positivity of 50%), as well as for samples collected in the morning when compared to those collected in the afternoon (17.7% vs. 5.8%). Positivity was also higher for sewage than for puddle water (17.7% vs. 5.6%). There was also difference in positivity for samples collected during the wet season when compared to those collected during the period with intermediate humidity (16.0% vs. 9.7%). Therefore, we conclude that the qPCR method is a useful tool for the diagnosis and prognostic evaluation of patients with a clinical suspicion of leptospirosis. The method was also reliable in identifying possible infection sites through the evaluation of water samples collected from an endemic area. Keywords: Leptospira, leptospirosis, real time PCR, whole blood, bacterial load, prognosis, environmental water sample

Comparação dos diagnósticos sorológico e molecular da leptospirose humana na região metropolitana de Curitiba, Paraná

Orientadora : Prof. Dr. Alexander Welker BiondoDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 2007Inclui bibliografiaA leptospirose é uma zoonose de distribuição mundial que apresenta sintomas clínicos não-específicos, cujo diagnóstico definitivo depende de testes laboratoriais. Amostras de sangue total, plasma e soro contaminadas in vitro foram submetidas à PCR em sistemas monoplex e duplex, utilizando dois conjuntos de iniciadores previamente descritos (G1/G2 e A/B). Posteriormente, amostras clínicas de sangue e urina foram amplificadas com os mesmos iniciadores para o diagnóstico da leptospirose. Amplificações com os iniciadores G1/G2 apresentaram sensibilidade de 60,7% e especificidade de 65,8%, contra 7,1% e 94,7% obtidas com os iniciadores A/B. Sensibilidade e especificidade gerais da PCR foram 53,6% e 63,2%, respectivamente. Sessenta e seis amostras clínicas foram testadas por ELISA IgM, MAT e PCR de sangue e urina, acompanhadas de 15 amostras pareadas. A detecção do DNA das leptospiras por PCR ocorreu a partir do primeiro dia de doença, enquanto a detecção dos anticorpos pelo MAT só foi possível a partir do quinto dia. Nossos resultados sugerem que amostras de sangue são mais apropriadas para a investigação clínica da leptospiremia, frente a amostras de plasma ou soro. Essas amostras apresentaram limite mínimo de detecção de 5x10³, 5x10 elevado a 4 e 5x10 elevado a 6 células/ml, respectivamente. Apesar da amplificação parcial do gene secY ser utilizada para a detecção de leptospiras patogênicas, o seqüenciamento desses produtos de PCR não permitiu a genotipagem da cepa infectante. A PCR foi menos sensível (53,6%) do que o ELISA IgM (85,7%) ao longo do curso da doença. Entretanto, a PCR realizada em amostras de sangue ou urina permitiu o diagnóstico precoce em 71,4% dos pacientes cuja soroconversão foi confirmada pelo MAT. Assim, a PCR constitui uma ferramenta complementar na primeira fase da doença, especialmente quando não é possível detectar anticorpos específicos pelas técnicas sorológicas, permitindo a confirmação precoce da infecção e o diagnóstico diferencial de outras doenças febris.Leptospirosis is a worldwide zoonosis of nonspecific clinical symptoms, which definitive diagnosis relies on laboratorial tests. Whole blood, plasma and serum samples in vitro contaminated were submitted to both monoplex and duplex PCR assays using previously described protocols (G1/G2 and A/B). Next, whole blood and urine clinical specimens were amplified with the same primers for diagnosis of leptospirosis. G1/G2-primed amplifications showed sensitivity of 60.7% and specificity of 65.8%, compared to 7.1% and 94.7% of the A/B-primed reactions. Overall PCR sensitivity and specificity was 53.6% and 63.2%, respectively. Sixty-six clinical samples tested by IgM ELISA, MAT and blood and urine PCR, along with 15 paired samples. PCR detection of leptospiral DNA occurred from the first day of illness, while antibodies detection by MAT was possible from the fifth day. Our results suggest that whole blood specimens are more appropriate for clinical investigation of leptospiremia, when compared to plasma or serum samples. Those samples had minimal detection limit of 5x10³, 5x10 to the power of 4 e 5x10 to the power of 6 cells/ml, respectively. Although PCR of secY gene fragment has been used for diagnosis of pathogenic leptospires, sequencing of respective amplicons does not allow genotyping of the infecting strain. PCR was less sensitive (53.6%) than IgM ELISA (85.7%) throughout the course of the disease. However, PCR performed on blood or urine samples permitted early diagnosis in 71.4% of patients whose seroconversion was confirmed by MAT. Thus, PCR constitutes a complementary tool in the first phase of the illness, especially when no specific antibodies can be detected by serological techniques, allowing early confirmation of the infection and differential diagnosis from other infectious febrile diseases

Molecular Investigation Confirms Myotis Genus Bats as Common Hosts of Polychromophilus in Brazil

Plasmodium spp. and some other blood parasites belonging to the order Haemosporida are the focus of many epidemiological studies worldwide. However, haemosporidian parasites from wild animals are largely neglected in scientific research. For example, Polychromophilus parasites, which are exclusive to bats, are described in Europe, Asia, Africa, and Oceania, but little is known about their presence and genetic diversity in the New World. In this study, 224 samples of bats from remaining fragments of the Atlantic Forest and Pantanal biomes, as well as urbanized areas in southern and southeastern Brazil, were analyzed for the presence of haemosporidian parasites by PCR of the mitochondrial gene that encodes cytochrome b (cytb). The PCR fragments of the positive samples were sequenced and analyzed by the Bayesian inference method to reconstruct the phylogenetic relationships between Polychromophilus parasites from bats in Brazil and other countries. Sequences from Brazilian lineages of Polychromophilus were recovered in a clade with sequences from Polychromophilus murinus and close to the one Polychromophilus sequence obtained in Panama, the only available sequence for the American continent. This clade was restricted to bats of the family Vespertilionidae and distinct from Polychromophilus melanipherus, a parasite species mainly found in bats of the family Miniopteridae. The detection of Polychromophilus and the genetic proximity to P. murinus were further confirmed with the amplification of two other genes (clpc and asl). We also found a Haemosporida parasite sequence in a sample of Noctilio albiventris collected in the Pantanal biome, which presents phylogenetic proximity with avian Haemoproteus sequences. Morphological and molecular studies are still needed to conclude and describe the Polychromophilus species in Brazilian Myotis bats in more detail and to confirm Haemoproteus parasites in bats. Nevertheless, these molecular results in Brazilian bats confirm the importance of studying these neglected genera.Fil: da Silva Mathias, Bruno. Universidade de Sao Paulo; BrasilFil: Minozzo, Guilherme Augusto. No especifíca;Fil: Biondo, Alexander Welker. Universidade Federal do Paraná; BrasilFil: de Oliveira Jorge Costa, Jaciara. Universidade de Sao Paulo; BrasilFil: Sousa Soares, Herbert. Universidade de Santo Amaro; BrasilFil: Marcili, Arlei. Universidade de Sao Paulo; BrasilFil: de Oliveira Guimarães, Lilian. Instituto Pasteur; BrasilFil: a Clares dos Anjos, Carolina. Universidade de Sao Paulo; BrasilFil: Pires Dos Santos, Andrea. Purdue University; Estados UnidosFil: Riediger, Irina Nastassja. No especifíca;Fil: Fecchio, Alan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Centro de Investigación Esquel de Montaña y Estepa Patagónica. Universidad Nacional de la Patagonia "San Juan Bosco". Centro de Investigación Esquel de Montaña y Estepa Patagónica; ArgentinaFil: Bueno, Marina Galvão. Fundación Oswaldo Cruz; BrasilFil: Pinho, João Batista. Universidade Federal do Mato Grosso do Sul; BrasilFil: Kirchgatter, Karin. Universidade de Sao Paulo; Brasi

Genomic epidemiology unveils the dynamics and spatial corridor behind the Yellow Fever virus outbreak in Southern Brazil

Despite the considerable morbidity and mortality of yellow fever virus (YFV) infections in Brazil, our understanding of disease outbreaks is hampered by limited viral genomic data. Here, through a combination of phylogenetic and epidemiological models, we reconstructed the recent transmission history of YFV within different epidemic seasons in Brazil. A suitability index based on the highly domesticated Aedes aegypti was able to capture the seasonality of reported human infections. Spatial modeling revealed spatial hotspots with both past reporting and low vaccination coverage, which coincided with many of the largest urban centers in the Southeast. Phylodynamic analysis unraveled the circulation of three distinct lineages and provided proof of the directionality of a known spatial corridor that connects the endemic North with the extra-Amazonian basin. This study illustrates that genomics linked with eco-epidemiology can provide new insights into the landscape of YFV transmission, augmenting traditional approaches to infectious disease surveillance and control

Comparação dos diagnósticos sorológico e molecular da leptospirose humana na região metropolitana de Curitiba, Paraná

Orientadora : Prof. Dr. Alexander Welker BiondoDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 2007Inclui bibliografiaA leptospirose é uma zoonose de distribuição mundial que apresenta sintomas clínicos não-específicos, cujo diagnóstico definitivo depende de testes laboratoriais. Amostras de sangue total, plasma e soro contaminadas in vitro foram submetidas à PCR em sistemas monoplex e duplex, utilizando dois conjuntos de iniciadores previamente descritos (G1/G2 e A/B). Posteriormente, amostras clínicas de sangue e urina foram amplificadas com os mesmos iniciadores para o diagnóstico da leptospirose. Amplificações com os iniciadores G1/G2 apresentaram sensibilidade de 60,7% e especificidade de 65,8%, contra 7,1% e 94,7% obtidas com os iniciadores A/B. Sensibilidade e especificidade gerais da PCR foram 53,6% e 63,2%, respectivamente. Sessenta e seis amostras clínicas foram testadas por ELISA IgM, MAT e PCR de sangue e urina, acompanhadas de 15 amostras pareadas. A detecção do DNA das leptospiras por PCR ocorreu a partir do primeiro dia de doença, enquanto a detecção dos anticorpos pelo MAT só foi possível a partir do quinto dia. Nossos resultados sugerem que amostras de sangue são mais apropriadas para a investigação clínica da leptospiremia, frente a amostras de plasma ou soro. Essas amostras apresentaram limite mínimo de detecção de 5x10³, 5x10 elevado a 4 e 5x10 elevado a 6 células/ml, respectivamente. Apesar da amplificação parcial do gene secY ser utilizada para a detecção de leptospiras patogênicas, o seqüenciamento desses produtos de PCR não permitiu a genotipagem da cepa infectante. A PCR foi menos sensível (53,6%) do que o ELISA IgM (85,7%) ao longo do curso da doença. Entretanto, a PCR realizada em amostras de sangue ou urina permitiu o diagnóstico precoce em 71,4% dos pacientes cuja soroconversão foi confirmada pelo MAT. Assim, a PCR constitui uma ferramenta complementar na primeira fase da doença, especialmente quando não é possível detectar anticorpos específicos pelas técnicas sorológicas, permitindo a confirmação precoce da infecção e o diagnóstico diferencial de outras doenças febris.Leptospirosis is a worldwide zoonosis of nonspecific clinical symptoms, which definitive diagnosis relies on laboratorial tests. Whole blood, plasma and serum samples in vitro contaminated were submitted to both monoplex and duplex PCR assays using previously described protocols (G1/G2 and A/B). Next, whole blood and urine clinical specimens were amplified with the same primers for diagnosis of leptospirosis. G1/G2-primed amplifications showed sensitivity of 60.7% and specificity of 65.8%, compared to 7.1% and 94.7% of the A/B-primed reactions. Overall PCR sensitivity and specificity was 53.6% and 63.2%, respectively. Sixty-six clinical samples tested by IgM ELISA, MAT and blood and urine PCR, along with 15 paired samples. PCR detection of leptospiral DNA occurred from the first day of illness, while antibodies detection by MAT was possible from the fifth day. Our results suggest that whole blood specimens are more appropriate for clinical investigation of leptospiremia, when compared to plasma or serum samples. Those samples had minimal detection limit of 5x10³, 5x10 to the power of 4 e 5x10 to the power of 6 cells/ml, respectively. Although PCR of secY gene fragment has been used for diagnosis of pathogenic leptospires, sequencing of respective amplicons does not allow genotyping of the infecting strain. PCR was less sensitive (53.6%) than IgM ELISA (85.7%) throughout the course of the disease. However, PCR performed on blood or urine samples permitted early diagnosis in 71.4% of patients whose seroconversion was confirmed by MAT. Thus, PCR constitutes a complementary tool in the first phase of the illness, especially when no specific antibodies can be detected by serological techniques, allowing early confirmation of the infection and differential diagnosis from other infectious febrile diseases

Whole-Genome Characterization of a Novel Human Influenza A(H1N2) Virus Variant, Brazil

We report the characterization of a novel reassortant influenza A(H1N2) virus not previously reported in humans. Recovered from a a pig farm worker in southeast Brazil who had influenza-like illness, this virus is a triple reassortant containing gene segments from subtypes H1N2 (hemagglutinin), H3N2 (neuraminidase), and pandemic H1N1 (remaining genes)

Molecular identification and typing of Mycobacterium massiliense isolated from postsurgical infections in Brazil

AbstractObjectiveOne hundred thirty-one cases of postsurgical infections were reported in Southern Region of Brazil between August 2007 and January 2008. Thirty-nine (29.8%) cases were studied; this report describes epidemiological findings, species identification, antimicrobial susceptibility and clonal diversity of rapidly growing mycobacteria isolated in this outbreak.MethodsAll 39 isolates were analyzed by Ziehl-Nielsen stained smear, bacterial culture and submitted to rpoB partial gene sequencing for identification. The isolates were also evaluated for their susceptibility to amikacin, cefoxitin, clarithromycin, ciprofloxacin, doxycycline, tobramycin and sulfamethoxazole.ResultsThirty-six isolates out of the confirmed cases were identified as Mycobacterium massiliense and the remaining three were identified as Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum. All M. massiliense isolates were susceptible to amikacin (MIC90=8μg/mL) and clarithromycin (MIC90=0.25μg/mL) but resistant to cefoxitin, ciprofloxacin, doxycycline, tobramycin and sulfamethoxazole. Molecular analysis by pulsed-field gel electrophoresis clustered all 36 M. massiliense isolates and showed the same pattern (BRA 100) observed in three other outbreaks previously reported in Brazil.ConclusionsThese findings suggest a common source of infection for all patients and reinforce the hypotheses of spread of M. massiliense BRA100 in Brazilian hospital surgical environment in recent years

Identification of a novel alphavirus related to the encephalitis complexes circulating in southern Brazil

Submitted by Manoel Barata ([email protected]) on 2019-07-22T17:44:10Z No. of bitstreams: 1 222217512019.pdf: 4079083 bytes, checksum: 65970f6566a9f9aee02974252f0933b3 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-07-22T18:15:37Z (GMT) No. of bitstreams: 1 222217512019.pdf: 4079083 bytes, checksum: 65970f6566a9f9aee02974252f0933b3 (MD5)Made available in DSpace on 2019-07-22T18:15:37Z (GMT). No. of bitstreams: 1 222217512019.pdf: 4079083 bytes, checksum: 65970f6566a9f9aee02974252f0933b3 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Universidade Federal do Rio de Janeiro. Departamento de Genética. Instituto de Biologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Secretaria da Saúde do Estado do Paraná. Laboratório Central. São José dos Pinhais, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Regulação da Expressão Gênica. Curitiba, PR, Brasil.Secretaria da Saúde do Estado do Paraná. Laboratório Central. São José dos Pinhais, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.In early 2017, an outbreak caused by an unknown and supposedly viral agent in the Marilena region of southern Brazil was investigated. Since the etiological agent causing the outbreak was not identified from human samples, mosquitoes from this region were collected. Three out of 121 mosquito pools collected from the region tested positive for alphavirus in molecular tests. Next generation sequencing results revealed the presence of a novel alphavirus, tentatively named here as Caainguá virus (CAAV). DNA barcoding analyses indicated that different species of Culex are hosts for CAAV. This new virus was basal to the New World encephalitic alphaviruses in a comprehensive and robust phylogenetic approach using complete genomes. Viral particles were observed in the cytosol and inside of intracellular compartments of cells in mosquito-derived cell cultures. Despite being noninfectious in vertebrate derived cell cultures, primary culturing of CAAV in human mononuclear cells suggests monocytes and lymphocytes as CAAV targets. However, the epidemiological link of CAAV on the human outbreak should be further explored