Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting.

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Interventions for wound healing among diabetic patients infected with Staphylococcus aureus: a systematic review.

CONTEXT AND OBJECTIVE: Staphylococcus aureus is the most frequent agent isolated in diabetic foot infections and may be associated with changes to wound healing times. The aim of this study was to perform a systematic review of the literature, including studies that assessed the efficacy of any clinical or surgical intervention, as well as oral or topical therapy for diabetic ulcers infected with S. aureus. DESIGN AND SETTING: Systematic review with a search conducted in databases. METHODS: We conducted a systematic review with a comprehensive search in the Lilacs, SciELO, PubMed/Medline, Old Medline, Embase and Cochrane Library databases, for articles published from 1966 to 2010. The articles selected were limited to studies on diabetic patients with wounds infected with S. aureus for whom their healing was followed up, with the use of either antibiotics or experimental treatments. Animal studies and those that did not report the wound healing, as well as review articles, were excluded. RESULTS: Five studies that met the inclusion and exclusion criteria were analyzed. CONCLUSIONS: There are few studies reporting the healing of wounds infected with S. aureus in diabetic patients, although this is the most commonly found pathogen in this type of wound and it frequently consists of methicillin-resistant S. aureus (MRSA). There is insufficient evidence to support early use of broad-spectrum antibiotics against MRSA to promote healing of diabetic ulcers, since antibiotic resistance may develop from such treatment. This highlights the need for further studies on the subject.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Interventions for wound healing among diabetic patients infected with Staphylococcus aureus: a systematic review / Intervenções para a cicatrização de feridas de pacientes diabéticos infectados com Staphylococcus aureus: uma revisão sistemática

CONTEXT AND OBJECTIVE: Staphylococcus aureus is the most frequent agent isolated in diabetic foot infections and may be associated with changes to wound healing times. The aim of this study was to perform a systematic review of the literature, including studies that assessed the efficacy of any clinical or surgical intervention, as well as oral or topical therapy for diabetic ulcers infected with S. aureus. DESIGN AND SETTING: Systematic review with a search conducted in databases. METHODS: We conducted a systematic review with a comprehensive search in the Lilacs, SciELO, PubMed/Medline, Old Medline, Embase and Cochrane Library databases, for articles published from 1966 to 2010. The articles selected were limited to studies on diabetic patients with wounds infected with S. aureus for whom their healing was followed up, with the use of either antibiotics or experimental treatments. Animal studies and those that did not report the wound healing, as well as review articles, were excluded. RESULTS: Five studies that met the inclusion and exclusion criteria were analyzed. CONCLUSIONS: There are few studies reporting the healing of wounds infected with S. aureus in diabetic patients, although this is the most commonly found pathogen in this type of wound and it frequently consists of methicillin-resistant S. aureus (MRSA). There is insufficient evidence to support early use of broad-spectrum antibiotics against MRSA to promote healing of diabetic ulcers, since antibiotic resistance may develop from such treatment. This highlights the need for further studies on the subject.

CONTEXTO: Staphylococcus aureus é o agente mais frequentemente isolado nas infecções de pé em pacientes diabéticos e pode estar associado a mudança no tempo de cicatrização de feridas. O objetivo deste estudo foi realizar uma revisão sistemática da literatura, incluindo estudos que avaliaram a eficácia de qualquer intervenção clínica, cirúrgica, bem como terapia oral ou tópica para o tratamento de úlceras diabéticas infectadas com o S. aureus. TIPO DE ESTUDO E LOCAL: Revisão sistemática com busca realizada em bancos de dados. MÉTODOS: Realizamos uma revisão sistemática com uma busca abrangente nos bancos de dados Lilacs, SciELO, PubMed/Medline, Old Medline, Embase e no banco de dados da biblioteca Cochrane, publicados entre 1966 e 2010. Os artigos selecionados foram limitados aos estudos com feridas infectadas por S. aureus de pacientes diabéticos, que tiveram cicatrização relatada, quer pela utilização de antibióticos ou por substâncias experimentais. Foram excluídos os estudos com animais e os que não relataram a cicatrização das feridas, bem como artigos de revisão. RESULTADOS: Foram analisados cinco estudos que obedeceram aos critérios de inclusão e exclusão. CONCLUSÕES: Raros estudos relataram cicatrização de feridas infectadas com S. aureus em pacientes diabéticos, embora este seja o patógeno mais comumente encontrado neste tipo de ferida, sendo frequentemente resistente à meticilina MRSA (methicillin-resistant S. aureus). Não há evidências suficientes que suportem a utilização precoce de antibióticos de amplo espectro contra MRSA para promoção da cicatrização de úlceras diabéticas, uma vez que o desenvolvimento de resistência a antibióticos pode decorrer desse tipo de tratamento. Isso evidencia a necessidade de novos estudos sobre o assunto.

Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose / Development of recombinant micobacteriophage for rapid detection of tubercle bacillus

O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula.

Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.