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  • Author: Mihaela Oprea x
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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.

Abstract

The aim of this study was to determine the rate of hemolyzed specimens sent to our laboratory for coagulation testing, assess the interference of hemolysis on coagulation for patients without anticoagulant therapy and to determine the reference intervals for PT, INR and aPTT for our laboratory in order to test our own limitations.

Methods: To determine the hemolysis rate, 1,689 specimens were evaluated on a visual scale and with the hemolysis icterus lipemia (HYL) test on Architect c4000 instrument. 125 blood samples collected from subjects without anticoagulant therapy were hemolyzed in vitro and the PT, INR and aPTT results were compared before and after hemolysis.To determine reference intervals (RI) for PT, INR and aPTT in our population, 125 apparently healthy human subjects (according to CLSI C28-A2) were enrolled and tests were performed on Sysmex CS 2000i, using Siemens reagents.

Results: Out of 1,689 samples, 9.46% were assessed as hemolyzed by the visual scale, while HYL test showed a 6.63% hemolysis rate. We found a shortening of 0.1s for PT, a diminution with 0.01 units for INR and a prolongation with 0.9s for aPTT from in vitro hemolyzed compared to non-lyzed samples. As to the reference intervals, we obtained in our laboratory versus reagents producer: for PT 9.8-13.9 s vs 9.8-12.1 s, and for aPTT 19.1-31.5s vs 23-31.9 s respectively; 28.38% more PT results and 13.44% more aPTT results were within range when we used local laboratory RI, compared to the manufacturer’s RI.

Conclusions: The rate of hemolyzed coagulation samples in our laboratory is higher than the rate found in the literature. Nevertheless, for patients without anticoagulant therapy hemolyzed samples should be processed. Using our own reference interval leads to a significant reduced number of abnormal results.

Abstract

A prolonged outbreak of Healthcare-Associated Infections (HCAIs) evolved since December 2013, in a Newborns Unit from Hospital A, sited in the North-Eastern development region, Romania. A first cluster consisted of 19 cases, of which 18 infections in newborns and 1 labour infectious complication in a mother. Except for five cases declared and treated in the Neonatology Unit as hospital-acquired infections, the other cases were discharged and further required rehospitalisation and treatment.

Eight of these innitialy discharged cases were readmitted to the Pediatric Surgery Unit and two others to the Pediatrics Unit of Hospital B, while three others were readmitted to three hospitals: one to the Pediatrics Unit of Hospital C, and other two to Hospital A and Hospital D, respectively. The mother with the labour infectious complication was readmitted to the Gynecology Unit of the Hospital A.

A number of fifteen Staphylococcus aureus (S. aureus) strains isolated from the HCAI first episode and 8 strains from 7 HCWs were received by „Cantacuzino” Institute, Nosocomial Infections and Antibiotic Resistance Laboratory from the County Public Health Directorate, for confirmation and molecular typing.

After a first round of interventions for infection control, a second episode bursted in Hospital A and our laboratory received six other S. aureus isolates from newborns, hospital environment, and HCWs.

Public Health interventions based on epidemiologic data and molecular microbiology results were finally successful. The evolution of all cases was favorable.

An important factor favoring the outbreak was the moving of the Birth Unit of Hospital A to an innapropriate location for an 18-month interval, more than innitially estimated, in relation to rehabilitation of the ward.

We considered to report this episode taking into account the unusual evolution, the risk of multiresistant bacterial strains spreading, and multiple unwanted consequences caused by shortcomings in providing appropriate hygiene conditions.

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.

Abstract

The multicenter ENDOBACT study aimed at implementing molecular methods for identification of bacterial species encountered in infective endocarditis, and at attempting to reduce the number of cases with unknown etiology. For eighty seven cases was established a diagnosis of definite infective endocarditis. Thirty two of these cases had negative blood cultures. For nine cases out of 32, valve pieces were available and an attempt was made to identify the etiological agent by molecular techniques. Thirty seven available isolates were identified by phenotypical and molecular comparative methods: 16S rRNA (all available isolates), rpoB (staphylococci, streptococci and enterococci), sodA (streptococci and enterococci) genes sequencing. For eight isolates, the comparative results were discrepant. Species identification of one coagulase negative staphylococcal strain was assigned using molecular methods. Molecular identification methods applied here might represent an added value for clinical and conventional microbiological diagnosis of infective endocarditis in Romania.