Continuous and Dynamic Circulation of West Nile Virus in Mosquito Populations in Bucharest Area, Romania, 2017-2023.

West Nile virus (WNV) is a mosquito-borne pathogen with a worldwide distribution. Climate change and human activities have driven the expansion of WNV into new territories in Europe during the last two decades. Romania is endemic for WNV circulation since at least 1996 when the presence of lineage 1 was documented during an unprecedented outbreak. Lineage 2 was first identified in this country during a second significant human outbreak in 2010. Its continuous circulation is marked by clade replacement, and even co-circulation of different strains of the same clade was observed until 2016. The present study aims to fill the information gap regarding the WNV strains that were circulating in Romania between 2017 and 2023, providing chiefly viral sequences obtained from mosquito samples collected in the Bucharest metropolitan area, complemented by human and bird viral sequences. WNV was detected mainly in Culex pipiens mosquitoes, the vectors of this virus in the region, but also in the invasive Aedes albopictus mosquito species. Lineage 2 WNV was identified in mosquito samples collected between 2017 and 2023, as well as in human sera from patients in southern and central Romania during the outbreaks of 2017 and 2018. Both 2a and 2b sub-lineages were identified, with evidence of multiple clusters and sub-clusters within sub-lineage 2a, highlighting the complex and dynamic circulation of WNV in Romania, as a consequence of distinct introduction events from neighboring countries followed by in situ evolution.

Water dispersible magnetite nanoparticles influence the efficacy of antibiotics against planktonic and biofilm embedded Enterococcus faecalis cells.

The objective of this study was to investigate the potential of magnetic nanoparticles to potentiate, but also to accomplish a sustained and controlled drug release and subsequently improve the efficacy of antibiotics against Enterococcus faecalis, one of the most resistant opportunistic pathogens, that poses a threat to chronically infected or immunocompromised patients and is difficult to eradicate from medical devices. To our knowledge, this is the first study trying to investigate the ability of magnetite nanoparticles to improve the anti-bacterial activity of the current antibiotics against planktonic and biofilm growing E. faecalis. Our results are suggesting that the magnetite nanoparticles may be considered an effective aminoglycoside antibiotics carrier, but a complete understanding of the way in which they selectively interact with different antibiotics and with the bacterial cell is needed, in order to obtain improved strategies for elimination of E. faecalis biofilms on biomedical devices or human tissues.

First Detection and Molecular Characterization of Usutu Virus in Culex pipiens Mosquitoes Collected in Romania.

Usutu virus (USUV) is an emergent arbovirus in Europe causing mortality in bird populations. Similar to West Nile virus (WNV), USUV is maintained in sylvatic cycles between mosquito vectors and bird reservoirs. Spillover events may result in human neurological infection cases. Apart from indirect evidence provided by a recent serological study in wild birds, the circulation of USUV in Romania was not assessed. We aimed to detect and molecular characterize USUV circulating in mosquito vectors collected in South-Eastern Romania-a well-known WNV endemic region-during four transmission seasons. Mosquitoes were collected from Bucharest metropolitan area and Danube Delta, pooled, and screened by real-time RT-PCR for USUV. Partial genomic sequences were obtained and used for phylogeny. USUV was detected in Culex pipiens s.l. female mosquitoes collected in Bucharest, in 2019. The virus belonged to Europe 2 lineage, sub-lineage EU2-A. Phylogenetic analysis revealed high similarity with isolates infecting mosquito vectors, birds, and humans in Europe starting with 2009, all sharing common origin in Northern Italy. To our knowledge, this is the first study characterizing a strain of USUV circulating in Romania.

Increasing the Awareness of Under-Diagnosed Tropical Cases of Dengue in Romania.

Global travelling increases every year and according to a report released during the COVID-19 pandemic by the UN World Tourism Organization, international travel doubled in 2022, compared to levels in 2021. his fact led also to travel-imported cases of arboviral infections and physicians are often confronted with tropical diseases, such as dengue or chikungunya. Since there is are no pathognomonic cues for these tropical illnesses, early diagnosis is still a big challenge and it depends on many factors, such as exposure risk factors, the epidemiological context, the incubation period, and the wide spectrum of differential diagnoses, including cosmopolitan or exotic infections. Since the clinical presentation of dengue is not typical and there are other febrile illnesses similar to arboviral diseases, misdiagnosis is common even among experienced doctors. Differential diagnosis needs up to date knowledge considering the short viraemic period, the antibody cross-reactivity, and the traps in recognising the nonspecific symptom picture. We present two cases of Dengue diagnosed in Romania which were initially clinically misconstrued, despite the characteristic symptom picture. The main purpose is to increase the level of awareness and to underline the difficulties that clinicians face in recognizing travel-related imported dengue virus disease.

Distribution of Insecticide Resistance Genetic Markers in the West Nile Virus Vector Culex pipiens from South-Eastern Romania.

Culex pipiens pipiens and Culex pipiens molestus mosquitoes are the vectors of West Nile virus in south-eastern Romania, an area of intense circulation and human transmission of this virus. The level of insecticide resistance for the mosquito populations in the region has not been previously assessed. Culex pipiens mosquitoes collected between 2018 and 2019 in south-eastern Romania from different habitats were subjected to biotype identification by real-time PCR. Substitutions causing resistance to organophosphates and carbamates (F290V and G119S in acetylcholinesterase 1) and to pyrethroids (L1014F in voltage gated Na+ channel) were screened by PCR or sequencing. Substitutions F290V and G119S were detected at very low frequencies and only in heterozygous state in Culex pipiens molestus biotype specimens collected in urban areas. The molestus biotype population analysed was entirely homozygous for L1014F, and high frequencies of this substitution were also found for pipiens biotype and hybrid mosquitoes collected in urban and in intensive agriculture areas. Reducing the selective pressure by limiting the use of pyrethroid insecticides only for regions where it is absolutely necessary and monitoring L1014F mutation should be taken into consideration when implementing vector control strategies.

Q fever endocarditis in Romania: the first cases confirmed by direct sequencing.

Infective endocarditis (IE) is a serious, life-threatening disease with highly variable clinical signs, making its diagnostic a real challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative. In such situations, alternative diagnostic approaches are necessary. Coxiella burnetii and Bartonella spp. are the etiological agents of blood culture-negative endocarditis (BCNE) most frequently identified by serology. The purpose of this study is to investigate the usefulness of molecular assays, as complementary methods to the conventional serologic methods for the rapid confirmatory diagnostic of Q fever endocarditis in patients with BCNE. Currently, detection of C. burnetii by culture or an antiphase I IgG antibody titers >800 represents a major Duke criterion for defining IE, while a titers of >800 for IgG antibodies to either B. henselae or B. quintana is used for the diagnosis of endocarditis due to Bartonella spp. We used indirect immunofluorescence assays for the detection of IgG titers for C. burnetii, B. henselae and B. quintana in 57 serum samples from patients with clinical suspicion of IE. Thirty three samples originated from BCNE patients, whereas 24 were tested before obtaining the blood cultures results, which finally were positive. The results of serologic testing showed that nine out of 33 BCNE cases exhibited antiphase I C. burnetii IgG antibody titer >800, whereas none has IgG for B. henselae or B. quintana. Subsequently, we used nested-PCR assay for the amplification of C. burnetii DNA in the nine positive serum samples, and we obtained positive PCR results for all analyzed cases. Afterwards we used the DNA sequencing of amplicons for the repetitive element associated to htpAB gene to confirm the results of nested-PCR. The results of sequencing allowed us to confirm that C. burnetii is the causative microorganism responsible for BCNE. In conclusion, the nested PCR amplification followed by direct sequencing is a reliable and accurate method when applied to serum samples, and it may be used as an additional test to the serological methods for the confirmatory diagnosis of BCNE cases determined by C. burnetii.

Characterization and Host-Feeding Patterns of Culex pipiens s.l. Taxa in a West Nile Virus-Endemic Area in Southeastern Romania.

Culex pipiens sensu lato has been documented as West Nile virus (WNV) vector in southeastern Romania. Bucharest, the densely populated capital city of Romania, and the surrounding Ilfov county are WNV hotspots. In this area, the morphologically indistinguishable biotypes of Cx. pipiens, namely pipiens and molestus, are usually differentiated by their behavioral and physiological traits. Their involvement in WNV transmission, as suggested by entomological investigations, was not previously documented for each biotype. We used a Real-Time PCR assay based on CQ11 microsatellite to identify the Cx. pipiens biotypes and their hybrids collected in various habitats in the Bucharest metropolitan area. A sympatric distribution of both biotypes was observed, with a preference of green areas for pipiens, and human settings and animal farmlands for molestus. In the latter habitats, pipiens and molestus were found in mixed aboveground populations. A low number of hybrids was found suggesting existence of reproductive isolation. In subway tunnels molestus was dominant with a higher number of hybrids recorded than aboveground. Blood-engorged mosquitoes were identified to biotype and the blood meal source identified by DNA barcoding. Overall, Cx. pipiens s.l. fed mainly on birds, commonly on house sparrows, collared doves, and blackbirds, which are potential WNV-amplifying hosts. The preference for avian hosts was expressed strongest by pipiens biotype, while molestus was substantially less specific, feeding on avian and mammal hosts with similar frequency, with humans representing 20% of the hosts. Hybrids had a host choice closer to that of molestus. These findings highlight the role of pipiens biotype as enzootic/epizootic vector, and specifically show molestus as the bridge vector for WNV. The pipiens and molestus biotypes show important differences in habitat preferences, including oviposition; these findings demonstrate that targeted mosquito control to limit WNV transmission may be possible.

Original Contributions to the Chemical Composition, Microbicidal, Virulence-Arresting and Antibiotic-Enhancing Activity of Essential Oils from Four Coniferous Species.

This study aimed to establish the essential oil (EO) composition from young shoots of Picea abies, Larix decidua, Pseudotsuga menziesii, and Pinus nigra harvested from Romania and evaluate their antimicrobial and anti-virulence activity, as well as potential synergies with currently used antibiotics. The samples' EO average content varied between 0.62% and 1.02% (mL/100 g plant). The mono- and sesquiterpene hydrocarbons were dominant in the composition of the studied EOs. The antimicrobial activity revealed that the minimum inhibitory concentration (MIC) values for the tested EOs and some pure compounds known for their antimicrobial activity ranged from 6.25 to 100 µL/mL. The most intensive antimicrobial effect was obtained for the Pinus nigra EO, which exhibited the best synergistic effect with some antibiotics against Staphylococcus aureus strains (i.e., oxacillin, tetracycline, erythromycin and gentamycin). The subinhibitory concentrations (sMIC) of the coniferous EOs inhibited the expression of soluble virulence factors (DN-ase, lipase, lecithinase, hemolysins, caseinase and siderophore-like), their efficiency being similar to that of the tested pure compounds, and inhibited the rhl gene expression in Pseudomonas aeruginosa, suggesting their virulence-arresting drug potential.

Screening of molecular virulence markers in Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from clinical infections.

Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts. The frequent emergence of antibiotic-resistant S. aureus and Ps. aeruginosa strains has determined the development of new strategies in order to elucidate the different mechanisms used by these bacteria at different stages of the infectious process, providing the scientists with new procedures for preventing, or at least improving, the control of S. aureus and Ps. aeruginosa infections. The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens. We used multiplex and uniplex PCR assays to detect the genes encoding different cell-wall associated and extracellular virulence factors, in order to evaluate potential associations between the presence of putative virulence genes and the outcome of infections caused by these bacteria. Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections.

Quantitative real-time PCR study of the influence of probiotic culture soluble fraction on the expression of Pseudomonas aeruginosa quorum sensing genes.

The opportunistic pathogen Pseudomonas (Ps.) aeruginosa causes severe infections, particularly in immunocompromised individuals and patients with cystic fibrosis (CF). A serious side effect of antibiotic therapy in Ps. aeruginosa infections is the development of resistance to antibiotics. During the infection process Ps. aeruginosa forms biofilms, rendering bacterial cells more resistant to disinfectants, antibiotics and the action of host immune defense effectors. Pseudomonas aeruginosa employs the intercellular communication system, known as quorum sensing (QS) to coordinate the expression of tissue-damaging factors. Since the QS systems controls the production of different virulence factors, it is possible that the inhibition of its regulatory activity to severely compromise the ability of Ps. aeruginosa to cause infections in humans. Many studies have shown that some probiotic strains exhibit inhibitory activity on different virulence properties of pathogenic bacteria (adherence to cellular or inert substrate, soluble virulence factors expression). The aim of the present study was to investigate by real-time RT-qPCR the influence of probiotic culture soluble factors on the QS genes expression in 30 Ps. aeruginosa strains isolated from patients hospitalized in the National Institute for Cardiovascular Infections, Prof. C.C. Iliescu Fundeni Hospital, Bucharest. The results of the real time RT-qPCR have shown that in all Ps. aeruginosa strains grown in the presence of probiotic culture sterile filtrates, the level of QS genes expression was reduced comparatively with those from control cultures. In conclusion, these results proved that the inhibition of virulence factors regulation mechanisms by soluble molecules secreted by probiotics could represent an interesting way pathogenicity and virulence attenuation in Ps. aeruginosa nosocomial strains.

Usefulness of complex bacteriological and serological analysis in patients with spondyloarthritis.

Spondyloarthritis (SpA) is a group of associated chronic systemic inflammatory immune-mediated rheumatic diseases affecting axial and peripheral joints and entheses. The aim of the present study was to identify what parameters are useful to determine in order to better understand the correlation between the disease activity/severity and the microbiological results/immune status against intestinal and/or urogenital pathogens. Microorganisms known to trigger SpA, including Klebsiella spp., Yersinia spp., Salmonella spp., Campylobacter spp. and Chlamydia spp., were analyzed in various specimens (stool, urine, synovial fluid and serum) collected from 27 randomly selected SpA patients and 26 healthy controls using a combined direct and indirect approach relying on conventional culture technique and nucleic acid-based assays together with serological testing by ELISA. Although Escherichia coli derived from phylogroup A prevailed in the gut microflora of the patients and controls, differences were observed regarding the representatives of the other phylogroups with a higher prevalence of E.coli members of phylogenetic group B1 in the stool specimens of patients. Antibodies against the targeted species were detected in SpA patients and controls, and the serological profiles of the former were more diverse and complex. In conclusion, the detection of anti-bacterial antibodies combined with other specific laboratory investigations should be more extensively used to monitor SpA patients in association with their symptoms and in order to determine and administer more effective therapeutics.

West Nile virus lineage 2 in Romania, 2015-2016: co-circulation and strain replacement.

BACKGROUND: West Nile virus (WNV) is endemic in southeastern Romania and, after the unprecedented urban epidemic in Bucharest in 1996 caused by lineage 1 WNV, cases of West Nile fever have been recorded every year. Furthermore, a new outbreak occurred in 2010, this time produced by a lineage 2 WNV belonging to the Eastern European clade (Volgograd 2007-like strain), which was detected in humans and mosquitoes in the following years. RESULTS: We report here, for the first time, the emergence, in 2015, of lineage 2 WNV belonging to the monophyletic Central/Southern European group of strains which replaced in 2016, the previously endemized lineage 2 WNV Volgograd 2007-like strain in mosquito populations. The emerged WNV strain harbors H249P (NS3 protein) and I159T (E glycoprotein) substitutions, which have been previously associated in other studies with neurovirulence and efficient vector transmission. CONCLUSIONS: In 2016, both early amplification of the emerged WNV and complete replacement in mosquito populations of the previously endemized WNV occurred in southeastern Romania. These events were associated with a significant outbreak of severe West Nile neuroinvasive disease in humans.

Re-emergence of severe West Nile virus neuroinvasive disease in humans in Romania, 2012 to 2017-implications for travel medicine.

BACKGROUND: In Romania, after a major outbreak in 1996, West Nile neuroinvasive disease (WNND) was reported only in a limited number of cases annually. During 2016-2017, a significant increase in the number of WNND cases was reported at the national level, associated with high mortality rates. METHODS: A retrospective analysis of all cases confirmed with WNND, hospitalized during 2012-2017 in a single tertiary facility from Bucharest was performed in order to determine the annual prevalence and mortality rate and the risk factors associated with a severe outcome. RESULTS: 47 cases were confirmed as WNND. The mortality rate was 25.5%, all death occurred during 2016-2017. Coma, confusion, obtundation, sleepiness and depressed deep tendon reflexes were symptoms predicting a severe outcome. In a univariate analysis age (p < 0.001), associated cancers (p = 0.012) and low levels of chloride in the CSF (p = 0.008) were risk factors for mortality. In a multinomial logistic analysis, age older than 75 years remained the only independent predictor of death in WNND. CONCLUSIONS: The increase in both the number and the mortality rate of WNND cases suggest a changing pattern of WNV infection in Romania. Public health authorities and clinicians should be aware of the risk of severe WNV infection in travelers returning from Romania.

First Two Imported Cases of Zika Virus Infections in Romania.

We report the first two cases of imported Zika virus (ZIKV) infection in Romanian patients returning from areas with ongoing outbreaks and challenges for laboratory diagnostic; first one with a classical pattern of acute flaviviral infection and the second one with an interesting pattern of a secondary flaviviral (ZIKV) infection in a yellow fever-vaccinated child living abroad in an endemic area.

Transmission Dynamics of the West Nile Virus in Mosquito Vector Populations under the Influence of Weather Factors in the Danube Delta, Romania.

Mosquitoes were collected in the Danube Delta during the active seasons of 2011-2013. For Culex spp. mosquitoes, the abundance was calculated. Culex pipiens (sensu lato), (s.l.) and Culex modestus pools were tested for the presence of West Nile virus (WNV) genome, and the maximum likelihood of the infection rate was established. Mean daily temperatures and precipitation were obtained for the closest meteorological station. A negative binominal model was used to evaluate linkages between the temperature/precipitation and mosquito population size. A zero-inflated negative binomial model was used to test the relationship between the temperature and the infection rate. A single complex model for infection rate prediction was also used. The linkages were calculated for lag 0 and for 10 days earlier (lag 1), 20 days earlier (lag 2), and 30 days earlier (lag 3). Significant positive linkages (P < 0.001) were detected between temperature and mosquito population size for lag 1, lag 2, and lag 3. The linkages between temperature and infection rates were positive and significant for lag 2 and lag 3. Negative significant (P < 0.001) results were detected between precipitation and infection rates for lags 1, 2, and 3. The complex model showed that the best predictors for infection rate are the temperature, 20 days earlier (positive linkage) and the precipitation, 30 days earlier (negative linkage). Positive temperature anomalies in spring and summer and rainfall decrease contributed to the increase in the Culex spp. abundance and accelerated the WNV amplification in mosquito vector populations in the following weeks.

Carbapenemase-Producing Klebsiella pneumoniae in Romania: A Six-Month Survey.

This study presents the first characterization of carbapenem-non-susceptible Klebsiella pneumoniae isolates by means of a structured six-month survey performed in Romania as part of an Europe-wide investigation. Klebsiella pneumoniae clinical isolates from different anatomical sites were tested for antibiotic susceptibility by phenotypic methods and confirmed by PCR for the presence of four carbapenemase genes. Genome macrorestriction fingerprinting with XbaI was used to analyze the relatedness of carbapenemase-producing Klebsiella pneumoniae isolates collected from eight hospitals. Among 75 non-susceptible isolates, 65 were carbapenemase producers. The most frequently identified genotype was OXA-48 (n = 51 isolates), eight isolates were positive for blaNDM-1 gene, four had the blaKPC-2 gene, whereas two were positive for blaVIM-1. The analysis of PFGE profiles of OXA-48 and NDM-1 producing K. pneumoniae suggests inter-hospitals and regional transmission of epidemic clones. This study presents the first description of K. pneumoniae strains harbouring blaKPC-2 and blaVIM-1 genes in Romania. The results of this study highlight the urgent need for the strengthening of hospital infection control measures in Romania in order to curb the further spread of the antibiotic resistance.