Performance of rapid tests for carbapenemase detection among Brazilian Enterobacteriaceae isolates

ABSTRACT The global emergence of carbapenemases led to the need of developing new methods for their rapid detection. The aim of this study was to evaluate the performance of the rapid tests for carbapenemase-producing and non-producing Enterobacteriaceae. Carbapenem non-susceptible Enterobacteriaceae from a surveillance study submitted to a multiplex real time PCR for carbapenemase detection were included in this study. The isolates were subjected to the rapid phenotypic tests Carba NP, Blue-Carba and Carbapenem Inactivation Method (CIM). A total of 83 carbapenemase-producing (43) and non-producing (40) isolates were included in the study. The sensitivity/specificity were 62.7%/97.5%, 95.3%/100%, and 74.4%/97.5% for Carba NP, Blue-Carba and CIM, respectively. Both Carba NP and Blue-Carba presented their final results after 75 min of incubation; the final results for CIM were obtained only after 8 h. Failure to detect OXA-370 carbapenemase was the main problem for Carba NP and CIM assays. As the Blue-Carba presented the highest sensitivity, it can be considered the best screening test. Conversely, CIM might be the easiest to perform, as it does not require special reagents. The early detection of carbapenemases aids to establish infection control measures and prevent carbapenemases to spread reducing the risk of healthcare associated infections and therapeutic failure.

Influência de TLR3 em modelo de Paracoccidioidomicose experimental / TLR3 influence in experimental PCM model

Os receptores do tipo Toll compreendem a família de receptores de reconhecimento de padrões melhor caracterizados, que podem ativar diferentes respostas imunes, dependendo de quais receptores e conjuntos de adaptadores são utilizados. Os TLRs, como TLR2, TLR4 e TLR9, e sua sinalização foram implicados no reconhecimento de P. brasiliensis e na regulação da resposta imune, no entanto, o papel do TLR3 ainda não está claro. Assim, a compreensão da função endossomal do TLR3 na PCM experimental é crucial. Utilizamos modelos in vitro e in vivo de infecção por P. brasiliensis, camundongos C57Bl/6 e TLR3-/-, para avaliar a contribuição da TLR3 no desenvolvimento da infecção. Mostramos que ausência de TLR3 leva o aumento de óxido nítrico e a capacidade fagocítica por macrófagos nas primeiras 4 horas de interação com leveduras P. brasiliensis. Mostramos ainda que os camundongos TLR3-/- desempenham papel protetor após 30 dias de infecção intratraqueal com P. brasiliensis, mostrando diminuição do aumento de CFU, perfil de resposta Th1 e Th17, bem como aumento de células citotóxicas T CD8+ produtoras de IFN-γ e IL-17. As células citotóxicas T CD8+ mostraram ser essenciais para o controle da infecção nos camundongos TLR3-/-, uma vez que a depleção dessas células levou a progressão da doença. Em estágios iniciais, 3 e 5 dias de infecção, observamos aumento do recrutamento de neutrófilos para o pulmão. Estudos recentes indicam que o TLR3 é um receptor importante para a resposta imune na micose e sua ausência favorece a infecção por fungos. Em contraste, nossos resultados mostram que, no caso do PCM, o TLR3 é prejudicial ao hospedeiro, sugerindo que a ativação do TLR3 pode ser um possível mecanismo de escape de P. brasiliensis

Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, C57Bl/6 and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show that absence of TLR3 leads to increased nitric oxide and phagocytic capacity by macrophages in the first 4 hours of interaction with yeasts P. brasiliensis. We also showed that TLR3-/- mice play a protective role after 30 days of intratracheal infection with P. brasiliensis, showing a decrease in the CFU increase, Th1 and Th17 response profile, as well as an increase in cytotoxic CD8+ cells producing IFN-γ and IL-17. The cytotoxic T CD8+ cells were shown to be essential for the control of infection in TLR3-/- mice, since the depletion of these cells led to the progression of the disease. In the initial stages, 3 and 5 days of infection, we observed increased recruitment of neutrophils to the lung. Recent studies indicate that TLR3 is an important receptor for the immune response in mycosis and its absence favors fungal infection. In contrast, our results show that in the case of PCM, TLR3 is detrimental to the host, suggesting that TLR3 activation may be a possible escape mechanism of P. brasiliensis

SIRT1 negatively regulates amyloid-beta-induced inflammation via the NF-B pathway

Chronic inflammation induced by amyloid-beta (Aβ) plays a key role in the development of age-related macular degeneration (AMD), and matrix metalloproteinase-9 (MMP-9), interleukin (IL)-6, and IL-8 may be associated with chronic inflammation in AMD. Sirtuin 1 (SIRT1) regulates inflammation via inhibition of nuclear factor-kappa B (NF-κB) signaling, and resveratrol has been reported to prevent Aβ-induced retinal degeneration; therefore, we investigated whether this action was mediated via activation of SIRT1 signaling. Human adult retinal pigment epithelial (RPE) cells were exposed to Aβ, and overactivation and knockdown of SIRT1 were performed to investigate whether SIRT1 is required for abrogating Aβ-induced inflammation. We found that Aβ-induced RPE barrier disruption and expression of IL-6, IL-8, and MMP-9 were abrogated by the SIRT1 activator SRT1720, whereas alterations induced by Aβ in SIRT1-silenced RPE cells were not attenuated by SRT1720. In addition, SRT1720 inhibited Aβ-mediated NF-κB activation and decrease of the NF-κB inhibitor, IκBα. Our findings suggest a protective role for SIRT1 signaling in Aβ-dependent retinal degeneration and inflammation in AMD.

Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.

Reverse transcriptase -polymerase chain reaction and DNA enzyme immunoassay for diagnosis of dengue fever and for dengue virus typing

In this study the use of commercially prepared reagents for detection and typing of dengue viruses from clinical samples using reverse transcriptase- polymerase chain reaction [RT- PCR] was evaluated on local dengue strains isolated in Jeddah Saudi Arabia over the period from February 1994 to July 1999. Total of 80 dengue culture confirmed cases, and 30 culture negative and IgM negative non dengue cases, and additionaly dengue virus infected, 20, and non infected, 20, tissue culture supernatants from C6/36 cell line, were used. All dengue culture positive cases were detected by RT- PCR followed by gel electrophoresis and or DNA enzyme immunoassay [DEIA], except 1 sample, which was negative by RT-PCR but was positive in culture in 1994 epidemic, with a sensitivity of 98.7% and specificity of 100%. All positive cases were correctly typed using gel electrophoresis following nested PCR and also by DEIA from first amplification product, without the need of nested PCR. This study showed that the commercially availabe reagents for extraction, RT- PCR, and detection are effective methods for the rapid and sensitive as well as specific diagnosis of dengue virus infection and are also effective in rapid typing of the infecting serotype, and that the use of DEIA in detection and typing from first amplification products is as effective as nested PCR using specific primers followed by gel electrophoresis or culture followed by antigen detection using polyclonal and monoclonal antibodies in an indirect immunofluorescent assay