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  • Author: Codruța-Romanița Usein x
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xTAG Luminex multiplex assay for rapid screening of verocytotoxin-producing Escherichia coli strains

Abstract

The O26 verocytotoxin-producing Escherichia coli (VTEC)-associated outbreak of hemolytic uremic syndrome (HUS) cases in Romania during 2016 showed the need to improve the current methodology of non-O157 VTEC detection and surveillance. An in-house assay based on xTAG Luminex technology was optimized to identify seven of the most relevant diarrheagenic E.coli serogroups (O-specific wzx genes), two convenient VTEC virulence markers (eaeA and ehxA genes), and a species-specific control gene (uidA). Twenty-nine strains previously characterized in terms of serogroup and virulence genes were tested with the optimized protocol and the results were as expected. The ratio of sample signal to background varied from 66.7 (ehxA) to 7.6 (uidA) for positive samples, with a cut-off of 3. Sensitivity varied depending on the target to be amplified from approximately 102 genomic copies to approximately 104 genomic copies per reaction, respectively. The current approach seems an affordable alternative to commercially available assays that can be further exploited to improve existing autochthonous strategies to prevent future VTEC outbreaks.

Open access
Prevalence of virulence markers and pHS-2-like plasmids among Shigella sonnei and Shigella flexneri isolates originating from shigellosis cases in Romania

Abstract

The surveillance of shigellosis is carried out under the auspice of the European Centre for Disease Prevention and Control which requires a reliable laboratory-based surveillance at national level. To date, little information is published about the members of Shigella spp. responsible for Romanian cases of shigellosis which hinders the understanding of the current epidemiology of shigellosis. Consequently, this retrospective study aimed to assess the diversity of virulence profiles displayed by the strains circulating in our region, by using key chromosome- and plasmid-associated markers, and to document the prevalence of pHS-2-like plasmid previously proposed as a potential marker for reactive arthritis.

The study focused on 65 Shigella sonnei and 49 Shigella flexneri clinical isolates, originated from local patients, recovered through the national surveillance system in 2009 - 2013. PCR assays were performed for the detection of ipaH, ipaBCD, ial, sen, set1A, set1B, sat, and pic genes, and a PCR-sequencing approach on plasmid preparations was used for identifying pHS-2-specific sequences.

Overall, the virulence markers ranged in prevalence from 21% (set1A, set1B, pic) to 100% (ipaH). S. flexneri isolates displayed a higher content of virulence markers than S. sonnei, the most common genotype, detected exclusively in S. flexneri serotype 2a isolates, being defined by the association ipaH+ipaBCD+ial+sen+sat+set1A+ set1B+pic. pHS-2-like plasmids were found in S. flexneri isolates of various serotypes suggesting the potential to trigger postinfection complications in specific subjects.

This study provided baseline data regarding the molecular characteristics of the Shigella strains from Romania, useful for defining the picture of shigellosis in our region.

Open access