Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples.

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.

Ongoing measles outbreak in Greece related to the recent European-wide epidemic.

We report an ongoing measles outbreak in Greece. The first cases were notified through the mandatory notification system on May 2017 in Northern Greece and concerned a cluster of three imported cases in unvaccinated Roma siblings. So far, 3150 cases have been reported in all 13 Regions. Initially, the outbreak mainly affected Greek Roma but as it progressed it spread to non-minority Greek nationals. The outbreak reached its ultimate peak on week 10/2018 but from week 15/2018 has been gradually declining. Most cases (60.6%) were Roma (especially children <10 year-old) followed by non-minority Greek nationals (29.3%; mostly young adults). The majority (80.4%) were unvaccinated. Interestingly, 129 (4.1%) cases were healthcare workers (HCWs). Genotype B3 was identified by molecular methods in all 87 cases tested. Overall, 61.3% of the cases were hospitalised. Complications were reported in 17.1% of the cases among which four deaths. The outbreak occurred after 3 years without local endemic measles transmission. Extensive vaccination implemented as the major public health measure managed to prevent the emergence of a large number of cases in refugee/migrant hosting sites. Mitigation efforts currently focus on raising awareness among HCWs and closing the immunisation gap in populations with suboptimal vaccination coverage.

Antiviral susceptibility profile of influenza A viruses; keep an eye on immunocompromised patients under prolonged treatment.

There was an increase in severe and fatal influenza cases in Greece during the 2011-2015 post-pandemic period. To investigate causality, we determined neuraminidase (NA) inhibitor susceptibility and resistance-conferring NA and hemagglutinin (HA) mutations in circulating influenza type A viruses during the pandemic (2009-2010) and post-pandemic periods in Greece. One hundred thirty-four influenza A(H1N1)pdm09 and 95 influenza A(H3N2) viruses submitted to the National Influenza Reference Laboratory of Southern Greece were tested for susceptibility to oseltamivir and zanamivir. Antiviral resistance was assessed by neuraminidase sequence analysis, as well as the fluorescence-based 50 % inhibitory concentration (IC50) method. Five influenza A(H1N1)pdm09 viruses (2.2 %) showed significantly reduced inhibition by oseltamivir (average IC50 300.60nM vs. 1.19nM) by Gaussian kernel density plot analysis. These viruses were isolated from immunocompromised patients and harbored the H275Y oseltamivir resistance-conferring NA substitution. All A(H1N1)pdm09 viruses were zanamivir-susceptible, and all A(H3N2) viruses were susceptible to both drugs. Oseltamivir-resistant viruses did not form a distinct cluster by phylogenetic analysis. Permissive mutations were detected in immunogenic and non immunogenic NA regions of both oseltamivir- resistant and susceptible viruses in the post-pandemic seasons. Several amino acid substitutions in the HA1 domain of the HA gene of post-pandemic viruses were identified. This study indicated low resistance to NAIs among tested influenza viruses. Antiviral resistance emerged only in immunocompromised patients under long-term oseltamivir treatment. Sequential sample testing in this vulnerable group of patients is recommended to characterise resistance or reinfection and viral evolution.

Molecular and phylogenetic analysis of Greek measles 2010 strains.

Although elimination of measles virus (MV) by 2010 was a revised target, a new epidemic has been ongoing in Greece and other European countries. The purpose of this study was the molecular and phylogenetic analysis of the Greek MV circulating strain. Twenty-four MV strains isolated from clinical samples during the 2010 outbreak were genotyped and studied in terms of nucleotide variation and phylogeny. All of the detected viruses were of the D4 genotype, which is circulating in Greece in the Roma population of Bulgarian nationality, the Greek Roma population and the Greek non-minority population, as well as in other EU countries. Phylogenetic analysis revealed that these viruses belonged to subgroup 4 of D4 MV strains. It is essential to continue epidemiological surveillance of measles in Greece to monitor the transmission pattern of the virus and the effectiveness of measles immunization, which eventually will lead to its elimination.