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Konell K.,Federal University of Paraná | Gelinsk M.A.,Federal University of Paraná | Benetti T.M.,State University Londrina | Abrahao W.M.,Federal University of Paraná | Abrahao W.M.,Laboratorio Central do Estado do Parana
Brazilian Journal of Microbiology | Year: 2014

The aim of this study was the detection of Campylobacter sp. in raw chicken sausages using the methods ISO 10272-1 and ISO 10272-2. The overall prevalence of Campylobacter sp. in the samples tested was 16.67%, representing a serious risk to the health of consumers, particularly if measures guaranteeing proper cooking of foods and prevention of cross-contamination are not adopted. Furthermore, the majority of campylobacteriosis cases in humans are caused by consumption or improper handling of contaminated raw or undercooked poultry meat, which constitute the main vehicle of this infection. © 2014, Sociedade Brasileira de Microbiologia.


Pontes F.L.D.,Federal University of Paraná | Gasparetto J.C.,Federal University of Paraná | de Francisco T.M.G.,Federal University of Paraná | Goetzke H.C.,Federal University of Paraná | And 3 more authors.
Food Analytical Methods | Year: 2016

A liquid chromatography tandem-mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of 22 veterinary drug residues, belonging to eight classes (coccidiostats, lincosamides, macrolides, tetracyclines, sulfonamides, benzimidazoles, diterpenes, and diaminopyrimidines), in eggs. Chromatographic separations were achieved on an XBridge BEH C18 column (150 × 2.1 mm, 3.5 μm, Waters, USA) maintained at 35 °C. The mobile phase was eluted at 400 μL min−1 in gradient mode between water and methanol/acetonitrile (20:80 v/v), both containing 0.1 % formic acid. The samples were prepared by protein precipitation with acetonitrile without additional cleanup steps. The method was successfully validated according to the Commission Decision 2002/657/EC and was demonstrated to be highly selective and free of matrix and residual effects. The method presented low limits of detection (0.37 to 7.5 μg kg−1) and quantification (1.25 to 20 μg kg−1). For banned substances, the decision limit values (CCα) and detection capability (CCβ) were 0.62–7.5 and 0.65–8.1 μg kg−1, respectively. For substances with a maximum residue limit, the CCα and CCβ values were 2.15–1061.5 and 2.3–1135.9 μg kg−1, respectively. All calibration curves showed excellent correlation (r ≥ 0.99). The recovery of the analytes and internal standards (49.0–103.7 %) was reached with high precision (RSD <8 %). At different concentration levels, the variations in precision and accuracy, in terms of repeatability and in-laboratory reproducibility, were <11 %. The new method is applicable to the routine analysis of commercial egg samples. © 2016 Springer Science+Business Media New York


Dallagassa C.B.,Federal University of Paraná | Huergo L.F.,Federal University of Paraná | Stets M.I.,Federal University of Paraná | Pedrosa F.O.,Federal University of Paraná | And 9 more authors.
Genetics and Molecular Research | Year: 2014

The mass profiles of cell-free extracts of 180 commensal and pathogenic strains of Escherichia coli were determined by MALDI-TOF mass spectrometry (MS). While some peaks were highly conserved in all E. coli, several peaks occurred only in some strains, showing heterogeneity among them. We did not detect strain-specific peaks for any of the E. coli categories tested. However, review of the fully conserved and the variable peaks suggested that MALDI-TOF MS has the potential to distinguish commensal and uropathogenic E. coli strains. Additionally, eight Shigella sonnei isolates were tested and found to be indistinguishable from E. coli by MALDI-TOF MS under the test conditions. © FUNPEC-RP.


De Almeida Torres R.S.L.,Laboratorio Of Bacteriologia | De Almeida Torres R.S.L.,Federal University of Paraná | De Almeida Torres R.S.L.,Positivo University | De Almeida Torres R.S.L.,Laboratorio Central Do Estado Do Parana | And 6 more authors.
Microbial Drug Resistance | Year: 2011

Background: Scarce data are available about the antimicrobial resistance of Group A Streptococcus in South America. Methods: This study evaluated the antimicrobial susceptibility profile of 1,112 isolates of Group A Streptococcus during the period from 1993 to 2009 in Curitiba city, Brazil. Macrolide-resistant isolates were characterized by emm typing and pulsed-field gel electrophoresis. Results: All isolates were susceptible to penicillin, vancomycin, and tigecycline. On the contrary, 18.6% of the isolates were resistant to tetracycline, presenting a minimum inhibitory concentration (MIC)50/MIC90 of 32/64 mg/L. Erythromycin resistance rose from 1.9% before 2000 to 4% after 2000 and was associated with a marked increased of MIC levels. Simultaneously, both the phenotype and genotype of macrolide resistance were modified as the M phenotypes (mef(A) genotype) were replaced by the cMLSB phenotypes (erm(B) genotype). Conclusion: This polyclonal spreading of cMLSB macrolide resistance has not been previously observed in South America and should stimulate further epidemiological surveillance in this part of the world. © 2011, Mary Ann Liebert, Inc.


Assis F.E.A.,Federal University of Paraná | Wolf S.,Federal University of Paraná | Surek M.,Federal University of Paraná | De Toni F.,Laboratorio Municipal Of Curitiba | And 5 more authors.
Journal of Infection in Developing Countries | Year: 2014

Introduction: A wide diversity of bacterial agents may cause diarrhea, presenting challenges to clinical laboratories to define a diagnosis. Considering that most stool cultures are negative, we screened stool samples from patients with diarrhea for the presence of 14 bacterial enteropathogens, aiming to establish which of them should be included in routine stool analysis.Methodology: Stool samples from 400 patients with diarrhea were analyzed for the presence of Salmonella, Shigella, Campylobacter, Aeromonas, Plesiomonas shigelloides, Vibrio, Yersinia enterocolitica, and diarrheagenic Escherichia coli using conventional microbiological methods and PCR. Two distinct samples were studied; one included predominantly patients involved in outbreaks, and the other patients of low socioeconomic status presenting sporadic cases of diarrhea.Results: In total, 86 cultures (21.5%) were positive. Mixed infections were found in five patients, leading to recovery of 91 strains of enteropathogenic bacteria: Salmonella Enteritidis (9.2%), Aeromonas (7.2%), diarrheagenic E. coli (5.2%), and C. jejuni (1%). However, Salmonella predominated, with 11.5% frequency in diarrhea outbreaks, while Aeromonas predominated among patients of lowsocioeconomic status, with 14.6% frequency.Conclusion: Aeromonas and diarrheagenic E. coli, which are not routinely screened for, deserve to be included in laboratory screening panels. © 2014 Assis et al.


Vicenzi F.J.,Laboratorio Municipal Of Curitiba | Vicenzi F.J.,Institute Pesquisa Pele Pequeno Principe | Pillonetto M.,Pontifical Catholic University of Parana | Pillonetto M.,Laboratorio Central do Estado do Parana | And 7 more authors.
Memorias do Instituto Oswaldo Cruz | Year: 2016

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recA sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recA sequencing to identify Bcc species. The recA sequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%), Burkholderia vietnamiensis (30.6%), B. cenocepacia IIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes. © 2016, Fundacao Oswaldo Cruz. All rights reserved.


Benetti T.M.,Federal University of Paraná | Monteiro C.L.B.,Federal University of Paraná | Beux M.R.,Federal University of Paraná | Abrahao W.M.,Federal University of Paraná | Abrahao W.M.,Laboratorio Central do Estado do Parana
Brazilian Journal of Microbiology | Year: 2013

This study was carried out comparing the conventional methods (ISO 11290-1 and BAM method, 2008) and system mini-Vidas® (Biomerieux), for detection of Listeria sp. and Salmonella sp. in cooled sausage. The immunoenzymatic method has shown to be effective for the detection of target pathogens, it has presented itself as an excellent screening method. © 2013, Sociedade Brasileira de Microbiologia.


Oliveira F.A.,Federal University of Paraná | Paludo K.S.,State University of Ponta Grossa | Arend L.N.V.S.,Laboratorio Central do Estado do Parana | Farah S.M.S.S.,Laboratorio Central do Estado do Parana | And 5 more authors.
Genetics and Molecular Research | Year: 2011

Eight virulence factors associated with uropathogenic Escherichia coli (UPEC) were investigated in 204 clinical isolates of E. coli recovered from urine cultures at counts ≥105. The bacteria were classified into two groups according to the number of leukocytes in urine samples from which they were isolated: group I ≤8 leukocytes/hpf, 104 strains; group II >8 leukocytes/hpf, 100 strains. Two multiplex PCR systems were used to detect genes encoding adhesin P (pap), adhesin S (sfa), afimbrial adhesin I (afa), siderophore aerobactin (aer), alpha-hemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), and traT associated with serum resistance. The PAI marker for the virulence island identified in strains CFT072 and CVD432, a marker of enteroaggregative E. coli, was also investigated using PCR. The susceptibility profile of E. coli strains was determined by disk diffusion method. Ninety percent UPEC showed at least one of the virulence genes, the prevalence being traT (76%), aer (41%), PAI (32%), sfa (26%), pap (25%), cnf1 (18%), afa (6%), and hly (5%). There was no significant difference in the distribution of virulence genes between groups I and II. A significantly higher degree of virulence was detected in UPEC group II. The CVD432 gene was not detected in any of the UPECs. Fifty-nine percent of the strains were resistant to at least one of the antimicrobials that we tested; the most common being resistance to ampicillin (51%) and trimethoprim-sulfamethoxazole (44%). © FUNPEC-RP.


Gilbet S.R.,Federal University of Paraná | Gilbet S.R.,Laboratorio Central do Estado do Parana | Alban S.M.,Federal University of Paraná | Gobor L.,Positivo University | And 3 more authors.
Revista da Sociedade Brasileira de Medicina Tropical | Year: 2013

Introduction: Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas disease, and it is estimated that approximately 5 million people in Brazil are infected with this parasite. This work aimed to compare the current diagnostic methods for Chagas disease, including conventional serological (IFAT and ELISA) and molecular techniques (PCR), to introduce PCR as an auxiliary technique. Methods: A total of 106 chagasic patients were evaluated: 88 from endemic areas of Parana, 6 from São Paulo, 3 from Minas Gerais, 3 from Rio Grande do Sul, 1 from Bahia and 5 from the Santa Catarina T. cruzi outbreak. The samples were analyzed by conventional serological methods (IFAT, ELISA), hemoculture and PCR to confirm Chagas disease. Results: When IFAT was used to determine antibody levels, the sensitivity was 81.7% for patients with the cardiac form of the disease and 100% for the other clinical forms. In contrast, ELISA showed 84% sensitivity and 100% specificity. The use of serological and molecular techniques and their implications for the diagnosis of Chagas disease in non-endemics area are discussed. Conclusions: PCR constitutes an excellent support methodology for the laboratory diagnosis of Chagas disease due to its high sensitivity and specificity.


Genotyping of hepatitis C virus (HCV) has become an essential tool for prognosis and prediction of treatment duration. The aim of this study was to compare two HCV genotyping methods: reverse hybridization line probe assay (LiPA v.1) and partial sequencing of the NS5B region.Plasma of 171 patients with chronic hepatitis C were screened using both a commercial method (LiPA HCV Versant, Siemens, Tarrytown, NY, USA) and different primers targeting the NS5B region for PCR amplification and sequencing analysis.Comparison of the HCV genotyping methods showed no difference in the classification at the genotype level. However, a total of 82/171 samples (47.9%) including misclassification, non-subtypable, discrepant and inconclusive results were not classified by LiPA at the subtype level but could be discriminated by NS5B sequencing. Of these samples, 34 samples of genotype 1a and 6 samples of genotype 1b were classified at the subtype level using sequencing of NS5B.Sequence analysis of NS5B for genotyping HCV provides precise genotype and subtype identification and an accurate epidemiological representation of circulating viral strains.

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