A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine

This work was supported by FAPERJ and PNPD-CAPES fellowships and CNPq grantFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genética Molecular de Microorganismos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genética Molecular de Microorganismos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genética Molecular de Microorganismos. Rio de Janeiro, RJ. Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microbiologia Ambiental. Ananindeua, PA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genética Molecular de Microorganismos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genética Molecular de Microorganismos. Rio de Janeiro, RJ. Brasil.Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae are difficult to differentiate phenotypically, leading to misinterpretation of their infection prevalence. We propose a multiplex PCR for blaSHV, blaLEN and blaOKP and their flanking gene (deoR). Since this scheme focuses only on chromosomal genes, it will be feasible for Klebsiella identification in the clinical routine

Virulence associated factors in bacteria from water bodies in Belem, Para, Brazil: bacteriological composition and threat to public health

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microbiologia Ambiental. Ananindeua, PA, Brasil.Federal University of Pará. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microbiologia Ambiental. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microbiologia Ambiental. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microbiologia Ambiental. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Biologia Molecular. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Microbiologia Ambiental. Ananindeua, PA, Brasil.A lack of sewage treatment contaminates water bodies threatening human health by spreading waterborne gastroenteritis. This is a particular problem for developing countries, where the risks associated with surface water contamination remain largely unknown. To understand the risk associated with sewage contamination of water bodies, we evaluated the microbiological indicators of water quality and isolated bacterial strains from water bodies from the city of Belém, Pará, Brazil. The strains were identified by biochemical and serological tests and polymerase chain reactions (PCRs). The thermotolerant coliforms and Escherichia coli presented values above 1,000 (NMP/ 100 mL) biweekly from August 2012 to November 2015, without a significant statistical difference between sampling periods (Kruskal–Wallis p > 0.05). The water of the Tucunduba river presented contamination levels similar to those in a sewage pumping station (Dunn test p > 0.05). From 240 bacterial isolates, we identified 163 Vibrio cholerae, 8 Vibrio mimicus, 24 E. coli, and 5 Salmonella spp. The isolates of V. cholerae demonstrated N-acetylglucosamine (NAG) profile (Non-O1 and NonO139) and 18 expressed the stn/sto gene. No E. coli was shown to be potentially pathogenic. The results revealed that water bodies in Belém were constantly contaminated by sewage and fecal microorganisms, including the potential circulation of pathogens in viable and cultivable form

Occurrence of norovirus Giv In environmental water samples from Belém City, Amazon Region, Brazil

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Federal University of Para. Tropical Medicine Center. Postgraduate Program in Tropical Diseases. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Noroviruses are the major cause of non-bacterial acute gastroenteritis outbreaks in humans, with few reports about the occurrence of the norovirus GIV strain. We investigated the presence of norovirus GIV in surface water (river, bay, and stream) and untreated sewage, and we determined a positivity rate of 9.4 % (9/96). The strains genotyped were GIV.1. To our knowledge, this is the first report of GIV in Brazil

Norovirus genogroups I and II in environmental water samples from Belém city, Northern Brazil

Federal University of Para State. Tropical Medicine Center. Postgraduate Program in Tropical Diseases. Belém, PA, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Oswaldo Cruz Foundation. Laboratory of Comparative and Environmental Virology. Rio de Janeiro, RJ, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Para State. Tropical Medicine Center. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.This study investigated the presence of norovirus (NoV) GI and GII in environmental samples from the northern region of Brazil. Water samples were collected monthly (November 2008/October 2010) from different sources and sewage and concentrated by the adsorption-elution method. The NoV investigation used molecular methods followed by sequencing reactions. The general positivity for NoV was 33.9% (57/168). Considering the results obtained only in the semi-nested RT-PCR (reverse transcription polymerase chain reaction) and only in the TaqMan® real-time PCR, the rates were 26.8% (45/168) and 27.4% (46/168), respectively, being for NoV GI 22.2% (10/45) and 19.6% (9/46); for GII 17.8% (8/45) and 15.2% (7/46); and for GI + GII 60% (27/45) and 65.2% (30/46), respectively. Different GI (GI.1, GI.4, GI.7 and GI.8) and GII (GII.4, GII.6, GII.9, GII.12 and GII.14) genotypes were detected. These results demonstrated the NoV was disseminated in the waters of Belém city due to a lack of sanitation that allowed the discharge of contaminated effluents into these aquatic ecosystems

Detection of viral hepatitis b and c in a metropolitan region in Northern Brazil

Executive Secretariat of Public Health. Department of Epidemiology. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal Institute of Science and Technology Education of Pará. Health manager. Belém, PA, Brazil / Faculty Porto União. Health Services Auditor. Belém, PA, Brazil.Federal Institute of Education Science and Technology of Pará . Belém, PA, Brazil.Federal Institute of Education Science and Technology of Pará . Belém, PA, Brazil.Federal Institute of Education Science and Technology of Pará . Belém, PA, Brazil.Federal Institute of Education Science and Technology of Pará . Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Geoprocessamento. Ananindeua, PA, Brasil.State University of Pará. Belém, PA, Brazil.Federal Institute of Education Science and Technology of Pará. Belém, PA, Brazil.Objective: Analyze the SINAN epidemiological database for cases of viral hepatitis B and C, using the Linkage tools between the databases of the Laboratory Environment Manager (GAL) and the data from the Foundation of Hemotherapy and Hematology Center of Pará (HEMOPA). Method: Cross-sectional and descriptive epidemiological study, based on information on HBV and HCV collected in the SINAN databases, Environment Manager of the Laboratory of the Evandro Chagas Institute (GAL-IEC), Central Laboratory of the State of Pará (GAL-Lacen) and blood donation screening sheets of the Foundation Center for Hemotherapy and Hematology of Pará (HEMOPA), with reagent results for hepatitis C anti-HCV serological markers and hepatitis B HBsAg serological markers. Results: The statistical test applied in this study proved the distance between the averages of the detection rates calculated with information from all complementary bases in relation to the information from SINAN, analyzed in all the municipalities of the Metropolitan Region I. Therefore, the rates calculated between all bases during the period 2010 to 2015 reflected an increasing trend for HBV and HCV in all municipalities. In the years 2014 and 2015, the rates were higher in all the municipalities analyzed. The data demonstrated that the detection rates of HBV and HCV were above the rate for Brazil and the state of Pará, confirming the existence of underreporting of cases. Conclusion: The cases captured in the complementary databases allowed to improve the sensitivity of the notification system of HBV and HCV in the SINAN base, of the municipalities of the Metropolitan Region I, in the state of Pará

Two-year monitoring of enterovirus and rotavirus A in recreational freshwater from an island region, Pará State, northern Brazil

Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Brazilian Ministry of Education. Federal University of Para. Institute of Biological Sciences. Virology laboratory. Belem, PA, Brazil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Enteric viruses are major causes of waterborne diseases and are present in large quantities in the stools of infected individuals. Its viability in the environment lasts for months, favoring the contamination of water used for consumption and recreation. The study aimed to monitor monthly the circulation of enterovirus (EV) and group A rotavirus (RVA) in recreational freshwater from an island region used as a bathhouse in northern Brazil, from January 2012 to December 2013. The viral RNA was obtained using guanidine isothiocyanate/silica after viral concentration by adsorption-elution method. The molecular detection was carried out by semi (EV) and nested-PCR (RVA) and the amplicons were sequenced on automated sequencer. At least one of these viruses was detected on 40.4% (42/104) of the samples. RVA was the most frequent (n = 32; 30.8%) when compared to EV (n = 20; 19.2%). Co-circulation between both was identified in 9.6% (n = 10). The highest viral positivity was found in SP02 (46.1%). The highest viral positivity was observed during high tides (57.7%; 60/104). Most EV samples were characterized as coxsackievirus (CV) A5 (85.7%, 12/14) and others as Sabin 1 poliovirus (14.2%, 2/14). The RVA positive samples were genotyped as G2, G3, G9, G12, P[8], P[4], and P[6]. These viruses were detected in 35.6% (37/104) of the samples with an acceptable concentration of fecal coliform bacteria. These results demonstrate the contamination of surface water intended for recreation by enteric viruses of Public Health concern even when bacterial indicators are within the tolerated limit, a factor that confirms the need for public policies aimed the sewage treatment before its release into water bodies

Population and Genetic Study of <i>Vibrio cholerae</i> from the Amazon Environment Confirms that the <i>WASA-1</i> Prophage Is the Main Marker of the Epidemic Strain that Circulated in the Region

<div><p><i>Vibrio cholerae</i> is a natural inhabitant of many aquatic environments in the world. Biotypes harboring similar virulence-related gene clusters are the causative agents of epidemic cholera, but the majority of strains are harmless to humans. Since 1971, environmental surveillance for potentially pathogenic <i>V. cholerae</i> has resulted in the isolation of many strains from the Brazilian Amazon aquatic ecosystem. Most of these strains are from the non-O1/non-O139 serogroups (NAGs), but toxigenic O1 strains were isolated during the Latin America cholera epidemic in the region (1991-1996). A collection of environmental <i>V. cholerae</i> strains from the Brazilian Amazon belonging to pre-epidemic (1977-1990), epidemic (1991-1996), and post-epidemic (1996-2007) periods in the region, was analyzed. The presence of genes related to virulence within the species and the genetic relationship among the strains were studied. These variables and the information available concerning the strains were used to build a Bayesian multivariate dependency model to distinguish the importance of each variable in determining the others. Some genes related to the epidemic strains were found in environmental NAGs during and after the epidemic. Significant diversity among the virulence-related gene content was observed among O1 strains isolated from the environment during the epidemic period, but not from clinical isolates, which were analyzed as controls. Despite this diversity, these strains exhibited similar PFGE profiles. PFGE profiles were significant while separating potentially epidemic clones from indigenous strains. No significant correlation with isolation source, place or period was observed. The presence of the <i>WASA-1</i> prophage significantly correlated with serogroups, PFGE profiles, and the presence of virulence-related genes. This study provides a broad characterization of the environmental <i>V. cholerae</i> population from the Amazon, and also highlights the importance of identifying precisely defined genetic markers such as the <i>WASA-1</i> prophage for the surveillance of cholera.</p> </div

Dependency model of multivariate data from strains.

<p>Bayesian network representing conditional probabilities of variables that were available for the strains. Arcs are colored according to the impact in the posterior probability of the model when the arc is removed. The network represents the end result of the evaluation of 4.5 * 10⁷ different topologies, in which the last 1.4 * 10⁷ evaluations did not yield a better model. The network was constructed using the online B-Course software [42]. </p

O1 genotypes.

<p>The presence and absence of virulence-related genes are represented, respectively, by blue and white squares. The strains are grouped in colored bars according to their PFGE cluster (Fig. 4): from top to bottom are groups 1 (purple), 2 (red), 3 (blue), 9 (yellow), 6 (orange), and 8 (green). The colors highlighting the strain keys correspond to the isolation sources. Strains highlighted pink were isolated from wastewater, blue from superficial water, green from superficial stream water, and black from clinical sources.</p

Geographical distribution of <i>V. cholerae</i> isolates.

<p>The geographical location of rivers, streams, and wastewater plants from where the strains that were used in this study were isolated are indicated in the map. The sizes of markers indicate the number of strains in each location, markers are centered in the cities where the strains were isolated (see Table S1). Belem (yellow), Barcarena (light green), Maruda (pink), Macapá (dark green), Oiapoque (light blue), Manaus (red), Tabatinga (light blue), Rio Branco (purple), and Santa Rosa (orange). Quantities of strains isolated in each period are indicated in the bar graphs. </p