Correction to: Use of a NAT-based assay to improve the surveillance system and prevent transfusion-transmitted malaria in blood banks.

An amendment to this paper has been published and can be accessed via the original article.

Use of a NAT-based assay to improve the surveillance system and prevent transfusion-transmitted malaria in blood banks.

BACKGROUND: Malaria can be transmitted by blood transfusion through donations collected from asymptomatic donors. Transfusion-transmitted malaria (TTM) poses a great risk to blood services worldwide. A good screening tool for Plasmodium spp. detection in blood banks must have a high sensitivity for prevention of TTM. However, in Brazilian blood banks, screening for malaria still relies on microscopy. METHODS: In Brazil, screening for human immunodeficiency virus type 1 (HIV), RNA/DNA for hepatitis C (HCV) and hepatitis B (HBV) viruses is mandatory for every blood donation and uses nucleic acid amplification testing (NAT). The aim of this study was to evaluate the inclusion of an assay for malaria to identify Plasmodium sp. from total nucleic acid (TNA; DNA/RNA) by targeting the 18S rRNA gene of the parasite. RESULTS: Considering the limitations of microscopy and the wide availability of the Brazilian NAT platform in the screening of blood units for HIV, HCV, and HBV, a molecular diagnostic tool was validated for detection of Plasmodium sp. in blood banks; a pilot study showed that using this novel NAT assay could reduce the risk of TTM. CONCLUSION: The prototype HIV/HCV/HBV/malaria NAT assay was effective in detecting infected candidate donors and has good prospects to be applied in routine screening for preventing TTM.

Detection of Carbapenemase Genes in Aquatic Environments in Rio de Janeiro, Brazil.

This study reveals the presence of different carbapenemase genes (blaKPC, blaNDM, blaGES, and blaOXA48-like genes) detected directly from water samples and clonal dispersion (by pulsed-field gel electrophoresis [PFGE] and multilocus sequence typing [MLST]) of KPC-2-producing Enterobacteriaceae in two important urban aquatic matrixes from Rio de Janeiro, Brazil, highlighting the role of aquatic environments as gene pools and the possibility of community spreading.

Complete substitution of the Brazilian endemic clone by other methicillin-resistant Staphylococcus aureus lineages in two public hospitals in Rio de Janeiro, Brazil.

Staphylococcus aureus is an important cause of bloodstream infections. Therefore, the main purpose of this work was to characterize a collection of 139 S. aureus isolates from bloodstream infections in two public hospitals in relation to their antimicrobial susceptibility profile, staphylococcal cassette chromosome mec types, and clonal relationship. Methicillin resistance and resistance to other 12 agents were accessed by the disk diffusion test. Minimum inhibitory concentration to mupirocin was also determined. The SCCmec types were accessed by multiplex PCR, and the clonal relationship was determined by pulsed field gel electrophoresis method and restriction modification system characterization. Besides, multilocus sequence typing was performed for representative methicillin-resistant S. aureus isolates. The military hospital showed a dissemination of the New York/Japan (USA100/ST5/CC5/SCCmecII) lineage associated to multidrug resistance, including mupirocin resistance, and the teaching hospital presented polyclonal and non-multidrug resistant MRSA isolates. Complete substitution of the Brazilian endemic clone by other lineages was found in both hospitals. These findings can highlight differences in policy control and prevention of infections used in the hospitals and a change in the epidemiological profile of MRSA in Brazilian hospitals, with the replacement of BEC, a previously well-established clone, by other lineages.

Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro hospitals.

In a collection of 50 pvl-positive Staphylococcus aureus isolates from 10 Rio de Janeiro hospitals, 18 (36%) were from bloodstream infections, and 31 (62%) carried the SCCmec IV. Among 25 (50%) isolates of the USA1100/ST30/CC30 lineage present in 8 hospitals, 1 isolate was characterized as vancomycin-intermediate S. aureus.

Complete substitution of the Brazilian endemic clone by other methicillin-resistant Staphylococcus aureus lineages in two public hospitals in Rio de Janeiro, Brazil

Abstract Staphylococcus aureus is an important cause of bloodstream infections. Therefore, the main purpose of this work was to characterize a collection of 139 S. aureus isolates from bloodstream infections in two public hospitals in relation to their antimicrobial susceptibility profile, staphylococcal cassette chromosome mec types, and clonal relationship. Methicillin resistance and resistance to other 12 agents were accessed by the disk diffusion test. Minimum inhibitory concentration to mupirocin was also determined. The SCCmec types were accessed by multiplex PCR, and the clonal relationship was determined by pulsed field gel electrophoresis method and restriction modification system characterization. Besides, multilocus sequence typing was performed for representative methicillin-resistant S. aureus isolates. The military hospital showed a dissemination of the New York/Japan (USA100/ST5/CC5/SCCmecII) lineage associated to multidrug resistance, including mupirocin resistance, and the teaching hospital presented polyclonal and non-multidrug resistant MRSA isolates. Complete substitution of the Brazilian endemic clone by other lineages was found in both hospitals. These findings can highlight differences in policy control and prevention of infections used in the hospitals and a change in the epidemiological profile of MRSA in Brazilian hospitals, with the replacement of BEC, a previously well-established clone, by other lineages.

Padronização de PCR em Tempo Real para detecção de carbapenemases dos tipos KPC e NDM produzidas por bactérias Gram negativas

Infecções relacionadas à assistência à saúde (IRAS) constituem um grave problema de saúde pública, tanto em países desenvolvidos, quanto naqueles em desenvolvimento. A vigilância sanitária possui papel fundamental para o controle e contenção da disseminação de patógenos carreadores de genes de resistência no ambiente hospitalar e na comunidade. O mecanismo mais importante associado à resistência aos ß-lactâmicos é a produção de ß-lactamases que hidrolisam sua estrutura química causando a perda de função deste antimicrobiano. Os carbapenêmicos são a principal opção para o tratamento de infecções por bactérias Gram-negativas multiresistentes. Recentemente a resistência mediada por carbapenemases tem crescido, sendo observada no Brasil e ao redor do mundo. As carbapenemases mais descritas no Brasil são as serina cabapenemases KPC e OXA-48 em enterobactérias, OXA-23 em A. baumannii e as Metalo-beta-lactamases NDM, SPM. Objetivo: Padronizar uma metodologia para detecção de carbapenemases blakpc e blandm em bacilos Gram-negativos através de PCR em Tempo Real. Metodologia: Os ensaios de padronização foram realizados no ABI PRISM Prism7500 (Applied Biosystems) com cepas controle previamente identificadas por testes fenotípicos e PCR convencional como produtoras de KPC, NDM e não-produtoras. O controle endógeno utilizado foi o gene constitutivo 16SrRNA. A padronização seguiu cinco pontos principais: 1) Metodologia de extração, através de sonicação, lise térmica e lise de colônia diretamente durante a reação de PCR. 2) Definição da metodologia de ciclagem. 3) Otimização da reação com diferentes concentrações de sondas e iniciadores. 4) Comparação entre MasterMix NAT e IBMP. 4) Bioequivalência de sondas e iniciadores de diferentes fabricantes. 5) Análise da especificidade e sensibilidade do protótipo frente a 100 cepas previamente caracterizadas. 6) determinação do Limite de detecção do protótipo Resultados: As metodologias de extração testadas se mostraram igualmente eficazes com amplificação de blakpc e blandm, entretanto a lise térmica da colônia realizada durante a ativação enzimática da enzima Taq hot start possuía maior relação custo benefício, sendo mais rápida que as demais. O protocolo de ciclagem do CDC se mostrou tão eficaz quanto o estabelecido por Bio-Manguinhos, sendo o do CDC mais rápido. Os ensaios de otimização revelaram que as melhores concentrações de sondas e iniciadores testadas foram (0,2µM e 0,4µM respectivamente) com valores médios de Ct de 17,43 para KPC e 20,53 para NDM. Mesmo após a otimização realizada com o Master Mix IBMP e adição de ROX, este não apresentou resultados tão expressivos quando comparados com o MIX NAT. Quanto a Bioequivalência os melhores resultados foram encontrados com Sondas da marca BIOSEARCH e iniciadores da marca IDT. O protótipo padronizado possuiu 100% de especificidade para a detecção de KPC e NDM e 100% de sensibilidade. Mesmo após a diluição seriada por 10 vezes, o protótipo foi capaz de detectar a presença dos genes alvos em concentrações mínimas de DNA. A partir desta primeira etapa para a validação do protótipo foi possível determinar sua eficácia frente a um grande número de cepas, sendo necessária a análise de outros parâmetros para a validação total do protótipo.