Identifying Borrelia burgdorferi as the causative agent of Lyme disease in 1981 was a watershed moment in understanding the major impact that tick-borne zoonoses can have on public health worldwide, particularly in Europe and the USA. The medical importance of tick-borne diseases has long since been acknowledged, yet little is known regarding the occurrence of emerging tick-borne pathogens such as Borrelia spp., Anaplasma phagocytophilum, Rickettsia spp., Bartonella spp., “Candidatus Neoehrlichia mikurensis”, and tick-borne encephalitis virus in questing ticks in Romania, a gateway into Europe. The objective of our study was to identify the infection and co-infection rates of different Borrelia genospecies along with other tick-borne pathogens in questing ticks collected from three geographically distinct areas in eastern Romania. We collected 557 questing adult and nymph ticks of three different species (534 Ixodes ricinus, 19 Haemaphysalis punctata, and 4 Dermacentor reticulatus) from three areas in Romania. We analyzed ticks individually for the presence of eight different Borrelia genospecies with high-throughput real-time PCR. Ticks with Borrelia were then tested for possible co-infections with A. phagocytophilum, Rickettsia spp., Bartonella spp., “Candidatus Neoehrlichia mikurensis”, and tick-borne encephalitis virus. Borrelia spp. was detected in I. ricinus ticks from all sampling areas, with global prevalence rates of 25.8%. All eight Borrelia genospecies were detected in I. ricinus ticks: Borrelia garinii (14.8%), B. afzelii (8.8%), B. valaisiana (5.1%), B. lusitaniae (4.9%), B. miyamotoi (0.9%), B. burgdorferi s.s (0.4%), and B. bissettii (0.2%). Regarding pathogen co-infection 64.5% of infected I. ricinus were positive for more than one pathogen. Associations between different Borrelia genospecies were detected in 9.7% of ticks, and 6.9% of I. ricinus ticks tested positive for co-infection of Borrelia spp. with other tick-borne pathogens. The most common association was between B. garinii and B. afzelii (4.3%), followed by B. garinii and B. lusitaniae (3.0%). The most frequent dual co-infections were between Borrelia spp. and Rickettsia spp., (1.3%), and between Borrelia spp. and “Candidatus Neoehrlichia mikurensis” (1.3%). The diversity of tick-borne pathogens detected in this study and the frequency of co-infections should influence all infection risk evaluations following a tick bite.

BackgroundThe epidermal growth factor receptor (EGFR) mutation status assessment has become increasingly important given the significant impact of tyrosine kinase inhibitors in lung cancer management.Our aim was to compare real life operational characteristics for three EGFR mutation assays - two targeted approaches and a next generation sequencing (NGS) technique.MethodsEGFR mutation status was assessed for lung adenocarcinoma samples (formalin fixed- paraffin embedded samples) using qPCR, SNaPshot and NGS (Ion Torrent™) techniques.ResultsA total of 15 high clinical significance mutations were identified by at least one technique from the total of 64 samples. All mutations were identified by the TaqMan qPCR technique while SNaPshot in conjunction with fragment analysis identified 11 EGFR mutations. The NGS approach followed by an automatic analysis using the default calling parameters identified 10 mutations from the SNaPshot/qPCR panel and other three insertions, five point mutations and 58 silent variants; manual data review identified all 15 high significance mutations.ConclusionsPerformance was similar for high tumor content samples but careful data analysis and post hoc variant calling filter alterations were necessary in order to obtain robust results from low tumor content samples by NGS. NGS is able to generate a comprehensive mutational profile albeit at a higher cost and workload. Result interpretation should take into account not only general run parameters such as mean read depth but also relative coverage and read distribution; currently there is an acute need to define firm recommendations/standards concerning NGS data interpretation and quality control.

The members of the Anopheles maculipennis complex have been incriminated for the transmission of the malaria in Europe, which was endemic until the middle of the century. The global warming and the intensification of the intercontinental travel constitute a risk of the re-emergence of the malaria in Europe, given the presence of the Anopheles vectors. The study has attempted the identification by using the PCR (Polymerase Chain Reaction) of the members of the Anopheles maculipennis complex from the North-eastern area of Romania from the city of Iaşi. In total there have been identified by using the PCR amplifying the ITS2 sequence of the ribosomal DNA, 217 specimens belonging to the complex of A. maculipennis among which: 58 A. atroparvus, 18 A. melanoon, 2 A. labranchiae, 52 A. maculipennis and 87 A. messeae. The ITS2 sequences of the ribosomal DNA have been compared to those of the species belonging to the A. maculipennis available in GenBank. The Species A. labranchiae is reported for the first time in Romania, being identified in the larval stage IV. The adaptation of a new species to the climatic conditions present in the North-eastern Romania, confirms the phenomenon of global warming and also the intensification of the travelling. As a result of the analysis of the A. labranchiae sequence, this one corresponds to the extent of 96% to the species from Italy, registered in GenBank, given the fact that a high number of the inhabitants of the municipality of Iaşi are working in this country.

Mycobacteria culture remains the cornerstone of tuberculosis diagnosis. Naturally contaminated samples need pre-inoculation processing but some economically challenged medical facilities may benefit from a simpler and cheaper sputum decontamination procedure. The aim of this study was to test a simple decontamination method lacking a centrifugation step to be used in conjunction with the culture on Löwenstein-Jensen medium. A total of 7446 sputum samples collected from 3229 patients were microscopically examined and then cultured on Löwestein-Jensen medium using a simplified Petroff method. All positive cultures were confirmed by direct microscopic examination and biochemical identification. Culture and microscopic status and time to positivity were recorded. Mean and median times to culture and contamination rate were similar as compared to classical Löwenstein-Jensen culture method. Overall results suggest that the described modified of Petroff method may be used with adequate results in resource poor settings as the method does not require an aerosol safe centrifuge and relies on cheap, stable and readily available reagents.

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