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.

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.

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, 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.

INTRODUÇÃO: Vigilância com base em detecção laboratorial é um componente importante no controle de enterococos resistentes a vancomicina (ERV). MÉTODOS: Avaliamos procedimento da reação em cadeia da polimerase real time (PCR-RT) (genes vanA-vanB) para detecção de ERV em 115 swabs de pacientes incluídos em um programa de vigilância. RESULTADOS: A sensibilidade do RT-PCR foi semelhante a da cultura primária (75% e 79,5%, respectivamente) quando comparada com a cultura em caldo enriquecido, enquanto a especificidade foi de 83,1%. CONCLUSÕES: O RT-PCR fornece resultados no mesmo dia, contudo mostra baixa sensibilidade para a detecção de VRE.

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

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.

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.

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% 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% 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% 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.

INTRODUÇÃO: Staphylococcus pettenkoferi foi originalmente isolado e descrito por Trülzsch et al (2002). Neste estudo, caracterizamos dois isolados dessa nova espécie. MÉTODOS: As hemoculturas foram inicialmente processadas usando o instrumento BacT/ALERT® e os isolados foram inicialmente caracterizados pelo sistema de identificação Vitek2. RESULTADOS: A caracterização inicial revelou um coco Gram-positivo de crescimento lento com formação de colônias opacas em agar sangue de carneiro. Outros testes fenotípicos/genotípicos foram realizados. CONCLUSÕES: Gostaríamos de enfatizar que esta nova espécie de Staphylococcus é fenotipicamente similar a outros CoNS, especialmente, S. auricularis, que poderia levar a um erro na identificação dessas espécies incomuns.

INTRODUCTION: Staphylococcus pettenkoferi was originally isolated and described by Trülzsch et al (2002). In this study, we characterized two isolates of this newly described species. METHODS: Blood cultures were initially processed using the BacT/ALERT® device, and the isolates were initially characterized using the Vitek2 identification system. RESULTS: The initial characterization revealed slow-growing Gram-positive cocci that formed opaque colonies on sheep blood agar. Other phenotypic/genotypic tests were performed. CONCLUSIONS: We would like to emphasize that this new staphylococcus species is phenotypically similar to other CoNS, especially S. auricularis. This could potentially lead to misidentification of these uncommon species.

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%), enquanto a comparação entre as PCRs e a CH2 mostrou concordância razoável entre os resultados (PCRc = 90,2% e PCR-TR = 87,8%). 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%), whereas the comparison between PCR and HC2 showed a fair correlation (90.2% and 87.8% 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.

This study determined the prevalence of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in two hospitals located in the Southern part of Brazil and compare the performance of two different phenotypic tests. Thirty-one non-repetitive Pseudomonas aeruginosa isolates from various clinical samples from patients admitted to two hospitals located in Rio Grande do Sul, Brazil (twenty-three from a hospital in Porto Alegre City and eight isolates from a hospital in Vale dos Sinos Region). All strains suggestive of possessing MBLs by phenotypic methods were included in this study. Phenotypic detection of MBLs was carried out simultaneously by using both the MBL Etest® and disk approximation test using 2-mercaptopropionic acid close to a ceftazidime disk. Strains positive were further confirmed using molecular techniques for blaVIM, blaIMP and blaSPM-1. The prevalence of MBLs from samplesof inpatients from the hospital located in Porto Alegre was 30.4% and that of inpatients from Vale dos Sinos hospital was only 3.1%. Only MBL type SPM-1 was detected in these samples by molecular analysis and all were detected by the Etest® MBL strips. The prevalence of P. aeruginosa that produce MBLs can be markedly different in distinct geographical areas, even among different hospitals in the same area. In our study, the EDTA-based method was the only method able to detect all strains harboring the SPM-1 enzyme.

Descrevemos infecção de lesão neoplásica em paciente masculino de 27 anos, envolvendo pele e partes moles, causada por Corynebacterium striatum, um microrganismo raramente descrito como patógeno humano. A identificação foi confirmada por seqüenciamento de DNA. O paciente foi tratado com penicilina, com sucesso. O papel do C. striatum como patógeno oportunista é discutido.

We described a case of a 27-year old male patient with skin and soft tissue infection of a neoplastic lesion caused by Corynebacterium striatum, an organism which has been rarely described as a human pathogen. Identification was confirmed by DNA sequencing. Successful treatment with penicillin was achieved. The role of the C. striatum as an emerging opportunistic pathogen is discussed.

Relatamos um caso de conjuntivite ocasionada por Achromobacter xylosoxidans em paciente imunocompetente usuária de lentes de contato rígidas. A bactéria foi isolada da solução utilizada para a desinfecção das lentes bem como do raspado conjuntival. A. xylosoxidans tem sido descrita em infecções oportunistas em pacientes imunodeprimidos, contudo pode ser confundida com outros bacilos gram-negativos, principalmente Pseudomonas aeruginosa, isoladas de infecções oculares em pacientes imunocompetentes. Devido ao reduzido perfil de sensibilidade aos antimicrobianos demonstrado pelo A. xylosoxidans, torna-se importante a identificação deste agente etiológico em quadros de conjuntivite.

We report here a case of conjunctivitis in an immunocompetent patient due to Achromobacter xylosoxidans, which was associated with the use of rigid contact lenses. The bacteria were isolated from the scraped conjunctival swab as well as from the lens cleaning fluid. A. xylosoxidans is an opportunistic pathogen, especially in immunocompromised patients; however, in isolates of ocular infections, from immunocompetent patients, it may be confused with other gram-negative organisms, particularly Pseudomonas aeruginosa. Due to an increased resistance against different antimicrobial agents, A. xylosoxidans must be fully identified and differentiated from other gram-negative isolates from ocular infections.

The NCCLS (2004) presented a new methodology to detect, by disk-diffusion agar, oxacillin-resistance using a cefoxitin disk. We identified coagulase-negative staphylococci (SCoN) to the species level and compared the use of cefoxitin disks (30 µg) with oxacillin disks (1 µg), agar dilution (minimum inhibitory concentration of oxacillin) and mecA gene detection in isolates of coagulase-negative bacteria other than Staphylococcus epidermidis (SCoNne). A total of 238 SCoNne was evaluated; oxacillin-resistance (the mecA gene) was detected in 71% of the isolates. All methods gave 100% sensitivity, based on presence of the mecA gene. The specificity of the cefoxitin disk was 100%, while the oxacillin disk gave a specificity of 91% and agar dilution oxacillin gave a specificity of 88%. We conclude that the cefoxitin disk is an efficient test, and it is an easy method for use in clinical laboratories to detect oxacillin-resistance in staphylococci.

Reportamos o isolamento de Corynebacterium pseudodiphtheriticum de um caso de infecção cutânea. O paciente apresentou-se ao laboratório clínico com uma úlcera na perna esquerda. A coloração de Gram do material revelou a presença de bacilos Gram-positivos e ausência de outras espécies bacterianas. O organismo foi isolado em cultura pura no ágar sangue de carneiro e foi identificado como C. pseudodiphtheriticum através de um sistema de identificação comercial (API-Coryne, BioMérieux, França). A infecção foi tratada com sucesso através do uso de ciprofloxacina. Este caso reforça a importância do laboratório de microbiologia clínica na identificação de organismos Gram-positivos isolados de cultura pura de amostras de úlceras cutâneas.

We report here a rare case of cutaneous infection due to Corynebacterium pseudodiphtheriticum. The patient presented to the clinical laboratory with a skin ulcer on his left leg. Gram-stained preparation of the purulent secretion revealed the presence of numerous rod-shaped Gram-positive organisms in the absence of any other species. The organism was grown in pure culture on sheep blood agar and was further identified as C. pseudodiphtheriticum using a commercial identification system (API-Coryne, BioMérieux, France). The infection was successfully treated with ciprofloxacin. This case emphasizes the importance of the clinical microbiology laboratory in correctly identifying Gram-positive organisms obtained in pure culture from skin ulcers.