RESUMEN
Stool samples are alternatives to respiratory samples for bacteriological confirmation of childhood tuberculosis but require intensive laboratory processing before molecular testing to remove PCR inhibitors and debris. We aimed to develop a centrifuge-free processing method for use in resource-limited settings based on a sucrose-flotation method that showed good sensitivity for childhood tuberculosis diagnosis. In an in vitro study using Xpert MTB/RIF Ultra on stool samples spiked with defined bacterial concentrations of Mycobacterium tuberculosis (MTB), we compared different simplification parameters to the reference sucrose-flotation method. Best methods were selected based on the rate of invalid/error results and on sensitivity, compared to the reference method on stools spiked at 103 colony forming units (CFU)/g MTB. For final selection, we tested the best parameter combinations at 102 CFU/g. Out of 13 different parameter combinations, three were tested at 102 CFU/g. The best combination used 0.5 g stool, manual shaking, no filtration, 30-min sedimentation, and a 1:3.6 dilution ratio. This method gave 10% invalid/error results and a sensitivity of 70% vs 63% at 103 CFU/g and 53% vs 58% at 102 CFU/g compared to the reference method. This pre-clinical study was able to develop a centrifuge-free processing method to facilitate stool Xpert Ultra testing.
Asunto(s)
Heces/microbiología , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Niño , Humanos , Tuberculosis/microbiologíaRESUMEN
We detected for the first time blaNDM-5 and blaOXA-181 in Escherichia coli isolates from hospitalized patients and healthy volunteers in Chad. These resistance genes were located on IncX3 and IncF plasmids. Despite the large diversity of E. coli clones, the identified resistant intestinal isolates belonged mainly to the same sequence type.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , beta-Lactamasas/genética , Antibacterianos/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Chad , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli/efectos de los fármacos , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Plásmidos/genéticaRESUMEN
Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.
