Epidemiological characteristics and access to end-stage liver disease care for HIV-positive patients with HCV and/or HBV coinfections in Central/Eastern European and neighboring countries ­ data from the ECEE network

OBJECTIVES: There is currently an urgent need to harmonize hepatitis standards of care for HIV-positive patients across Europe. The HIV epidemic in Central and Eastern Europe has often been driven by injecting drug use, therefore a higher rate of co-infection with HCV and HBV is expected in this region. We have investigated the epidemiological prevalence and treatment availability for end-stage liver disease in HIV/HCV/HBV coinfections in countries represented in the ECEE Network Group. METHODS: The Euroguidelines in Central and Eastern Europe (ECEE) Network Group was initiated in February 2016 to compare standards of care regarding HIV infection in the region. Information about HIV/HCV/HBV co-infections and the availability for end-stage liver disease treatment for HIV-positive patients were collected through on-line surveys. The respondents were ECEE members from 16 countries of the region. The information on co-infection prevalence was sourced from WHO, national HIV programmes, articles published in international journals, single clinic reports, and personal information in ten of the participating countries (62.5%). RESULTS: The HIV/HCV co-infection rate was from 3% to 99%. The range of reported of HIV/HBV coinfection percentages was 2.3% to 40%. HIV/HCV/HBV co-infection ranged from 0% to 9%. Regarding treatment for end-stage liver disease, liver transplantation was an available option for HIV-positive patients in only three countries (19%). CONCLUSION: Our findings revealed only a limited number of treatment options for the end-stage liver disease in HIV-positive patients for the vast majority of Central and Eastern European countries. There are gaps in epidemiological surveillance in this region. It appears there are many differences in the number of co-infected patients among Central and Eastern European and neighboring countries, but there is no unification of information sources.

Comparative evaluation of aggressiveness traits in staphylococcal strains from severe infections versus nasopharyngeal carriage.

Despite their commensal status, staphylococci can become problematic pathogens expressing multiple and redundant virulence factors. This study aimed to evaluate aggressiveness markers comparatively in staphylococcal strains isolated from severe infections versus asymptomatic carriage in order to identify clinically relevant bacterial traits that could easily be detected in clinical practice and could be suggestive for particular host-pathogen interactions such as cyto-adhesion or biofilm formation, ultimately orienting the clinical decision-making process. We have used in vitro phenotypic methods to assess adhesion to and invasion of eukaryotic cells, biofilm development, and expression of soluble virulence factors in 92 Staphylococcus spp. strains. The adhesion index, invasion capacity, biofilm formation and expression of soluble factors did not differ significantly between clinical and commensal strains. The major bacterial traits we found to be significantly more prevalent in clinical staphylococci were the aggregative adhesion pattern (P = 0.012), cluster adhesion (P = 0.001) and tetrad morphology (P = 0.018). The aggregative adhesion pattern was correlated with higher cyto-adhesion (P < 0.001), higher invasion capacity (P = 0.003) and lower Carmeli scores (P = 0.002). Three major bacterial traits, namely tetrad morphology, aggregative adhesion pattern, and resistance to methicillin (acronym: TAM), can be used to compute an aggressiveness score (SAS) predictive of the staphylococcal strain's virulence and capacity to initiate and develop a biofilm-driven chronic infectious process versus a fulminant acute infection, in a susceptible host.

Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

BACKGROUND: Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. METHODS: Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. RESULTS: The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. CONCLUSIONS: Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected.

Global Dispersal Pattern of HIV Type 1 Subtype CRF01_AE: A Genetic Trace of Human Mobility Related to Heterosexual Sexual Activities Centralized in Southeast Asia.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) subtype CRF01_AE originated in Africa and then passed to Thailand, where it established a major epidemic. Despite the global presence of CRF01_AE, little is known about its subsequent dispersal pattern. METHODS: We assembled a global data set of 2736 CRF01_AE sequences by pooling sequences from public databases and patient-cohort studies. We estimated viral dispersal patterns, using statistical phylogeographic analysis run over bootstrap trees estimated by the maximum likelihood method. RESULTS: We show that Thailand has been the source of viral dispersal to most areas worldwide, including 17 of 20 sampled countries in Europe. Japan, Singapore, Vietnam, and other Asian countries have played a secondary role in the viral dissemination. In contrast, China and Taiwan have mainly imported strains from neighboring Asian countries, North America, and Africa without any significant viral exportation. DISCUSSION: The central role of Thailand in the global spread of CRF01_AE can be probably explained by the popularity of Thailand as a vacation destination characterized by sex tourism and by Thai emigration to the Western world. Our study highlights the unique case of CRF01_AE, the only globally distributed non-B clade whose global dispersal did not originate in Africa.

RegaDB: community-driven data management and analysis for infectious diseases.

SUMMARY: RegaDB is a free and open source data management and analysis environment for infectious diseases. RegaDB allows clinicians to store, manage and analyse patient data, including viral genetic sequences. Moreover, RegaDB provides researchers with a mechanism to collect data in a uniform format and offers them a canvas to make newly developed bioinformatics tools available to clinicians and virologists through a user friendly interface. AVAILABILITY AND IMPLEMENTATION: Source code, binaries and documentation are available on http://rega.kuleuven.be/cev/regadb. RegaDB is written in the Java programming language, using a web-service-oriented architecture.

Increase in transmitted resistance to non-nucleoside reverse transcriptase inhibitors among newly diagnosed HIV-1 infections in Europe.

BACKGROUND: One out of ten newly diagnosed patients in Europe was infected with a virus carrying a drug resistant mutation. We analysed the patterns over time for transmitted drug resistance mutations (TDRM) using data from the European Spread program. METHODS: Clinical, epidemiological and virological data from 4317 patients newly diagnosed with HIV-1 infection between 2002 and 2007 were analysed. Patients were enrolled using a pre-defined sampling strategy. RESULTS: The overall prevalence of TDRM in this period was 8.9% (95% CI: 8.1-9.8). Interestingly, significant changes over time in TDRM caused by the different drug classes were found. Whereas nucleoside resistance mutations remained constant at 5%, a significant decline in protease inhibitors resistance mutations was observed, from 3.9% in 2002 to 1.6% in 2007 (p = 0.001). In contrast, resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) doubled from 2.0% in 2002 to 4.1% in 2007 (p = 0.004) with 58% of viral strains carrying a K103N mutation. Phylogenetic analysis showed that these temporal changes could not be explained by large clusters of TDRM. CONCLUSION: During the years 2002 to 2007 transmitted resistance to NNRTI has doubled to 4% in Europe. The frequent use of NNRTI in first-line regimens and the clinical impact of NNRTI mutations warrants continued monitoring.

HIV-1 subtype distribution and its demographic determinants in newly diagnosed patients in Europe suggest highly compartmentalized epidemics.

BACKGROUND: Understanding HIV-1 subtype distribution and epidemiology can assist preventive measures and clinical decisions. Sequence variation may affect antiviral drug resistance development, disease progression, evolutionary rates and transmission routes. RESULTS: We investigated the subtype distribution of HIV-1 in Europe and Israel in a representative sample of patients diagnosed between 2002 and 2005 and related it to the demographic data available. 2793 PRO-RT sequences were subtyped either with the REGA Subtyping tool or by a manual procedure that included phylogenetic tree and recombination analysis. The most prevalent subtypes/CRFs in our dataset were subtype B (66.1%), followed by sub-subtype A1 (6.9%), subtype C (6.8%) and CRF02_AG (4.7%). Substantial differences in the proportion of new diagnoses with distinct subtypes were found between European countries: the lowest proportion of subtype B was found in Israel (27.9%) and Portugal (39.2%), while the highest was observed in Poland (96.2%) and Slovenia (93.6%). Other subtypes were significantly more diagnosed in immigrant populations. Subtype B was significantly more diagnosed in men than in women and in MSM > IDUs > heterosexuals. Furthermore, the subtype distribution according to continent of origin of the patients suggests they acquired their infection there or in Europe from compatriots. CONCLUSIONS: The association of subtype with demographic parameters suggests highly compartmentalized epidemics, determined by social and behavioural characteristics of the patients.

Limited cross-border infections in patients newly diagnosed with HIV in Europe.

BACKGROUND: International travel plays a role in the spread of HIV-1 across Europe. It is, however, not known whether international travel is more important for spread of the epidemic as compared to endogenous infections within single countries. In this study, phylogenetic associations among HIV of newly diagnosed patients were determined across Europe. RESULTS: Data came from the SPREAD programme which collects samples of newly diagnosed patients that are representative for national HIV epidemics. 4260 pol sequences from 25 European countries and Israel collected in 2002-2007 were included.We identified 457 clusters including 1330 persons (31.2% of all patients). The cluster size ranged between 2 and 28. A number of 987 patients (74.2%) were part of a cluster that consisted only of patients originating from the same country. In addition, 135 patients (10.2%) were in a cluster including only individuals from neighboring countries. Finally, 208 patients (15.6%) clustered with individuals from countries without a common border. Clustering with patients from the same country was less prevalent in patients being infected with B subtype (P-value <0.0001), in men who have sex with men (P-value <0.0001), and in recently infected patients (P-value =0.045). CONCLUSIONS: Our findings indicate that the transmission of HIV-1 in Europe is predominantly occurring between patients from the same country. This could have implications for HIV-1 transmission prevention programmes. Because infections through travelling between countries is not frequently observed it is important to have good surveillance of the national HIV-1 epidemics.

HIV-1 subtype B spread through cross-border clusters in the Balkans: a molecular analysis in view of incidence trends.

OBJECTIVES: To analyze phylogenetic relations and assess the role of cross-border clusters in the spread of HIV-1 subtype B across the Balkans, given the general trends of new HIV diagnoses in seven Balkan countries. DESIGN: Retrospective phylogenetic and trend analysis. METHODS: In-depth phylogenetic, phylodynamic and phylogeographic analysis performed on 2415 HIV-1 subtype B sequences from 1999 to 2019 using maximal likelihood and Bayesian methods. The joinpoint regression analysis of new HIV diagnoses by country and modes of transmission using 2004-2019 ECDC data. RESULTS: Ninety-three HIV-1 Subtype B transmission clusters (68% of studied sequences) were detected of which four cross-border clusters (11% of studied sequences). Phylodynamic analysis showed activity of cross-border clusters up until the mid-2000s, with a subsequent stationary growth phase. Phylogeography analyses revealed reciprocal spread patterns between Serbia, Slovenia and Montenegro and several introductions to Romania from these countries and Croatia. The joinpoint analysis revealed a reduction in new HIV diagnoses in Romania, Greece and Slovenia, whereas an increase in Serbia, Bulgaria, Croatia and Montenegro, predominantly among MSM. CONCLUSION: Differing trends of new HIV diagnoses in the Balkans mirror differences in preventive policies implemented in participating countries. Regional spread of HIV within the countries of former Yugoslavia has continued to play an important role even after country break-up, whereas the spread of subtype B through multiple introductions to Romania suggested the changing pattern of travel and migration linked to European integration of Balkan countries in the early 2000s.

The Predictive Value of Mutation Screening for Anticipating COVID-19 Waves.

Emerging SARS-CoV-2 strains continue to generate difficulties for authorities and health care professionals worldwide due to enhanced transmissibility and/or immune response evasion. The appearance of the Alpha and Delta strains has been associated with substantial increases in the number of COVID-19 cases and associated deaths. Whole Genome Sequencing (WGS) continues to be the gold standard for molecular surveillance of the pandemics but other assays such as mutation genotyping can be used to reduce costs and allocated time. This study investigates the efficiency of mutation screening tests compared to WGS and their predictive value to anticipate future waves. A very high degree of fidelity for this type of assay was found, regardless of the method used. The positive predictive value (PPV) of 4/5 markers was over 95% for the detection of Alpha and Delta variants. By estimating the prevalence of the Alpha and Delta strains using genotyping assays and fitting the data to a mathematical model, a five week period between the point of exponential growth of variant prevalence and a drastic increase in case numbers was found. For that reason, raising awareness about the efficacy of mutation screening could help authorities adopt better measures in the future.

Exhaustion and senescence of CD4 and CD8 T cells that express co-stimulatory molecules CD27 and CD28 in subjects that acquired HIV by drug use or by sexual route.

INTRODUCTION: The human immunodeficiency virus (HIV) infection leads to immune activation, senescence and exhaustion of T cells. Co-stimulatory molecules play important roles in controlling these processes. The CD28 signaling triggers efficient T cell activation, while CD27 provides survival signals to CD28- T cells. Loss of these molecules was associated with senescent phenotype and resistance to checkpoint inhibitors.Romania has faced an HIV outbreak among people who inject drugs (PWID), most of them chronically infected with hepatitis C virus (HCV). HIV/HCV co-infection was associated with increased immune activation and rapid disease progression. METHODS: We evaluated by flow cytometry the expression of CD27, CD28, CD38, HLA-DR, CD57 and PD-1 on CD4 and CD8 T cells from 34 subjected infected with HIV (22 PWID and 12 people who acquired HIV by sexual route - PWHS) and 18 HIV-negative individuals (controls). RESULTS: We found that as compared to controls, HIV patients, regardless of infection route, have high percentages of intermediately differentiated (CD27+CD28-) and low percentages of less differentiated (CD27+CD28+) CD8 T cells. Significantly higher levels of CD8+CD27+CD28- T cells were found in PWHS than in PWID. A lower percentage of intermediately and highly differentiated (CD27-CD28-) CD8 T cells express CD57 in people living with HIV (PLWH) than in controls. Increased levels of less and intermediately differentiated CD4 and CD8 T cells expressing PD-1 were identified in PLWH, especially in PWID; these directly correlated with HIV viral load and T cell activation and negatively correlated with CD4 counts. CONCLUSIONS: Our data show that induction of PD-1 on T cells expressing co-stimulatory molecules CD27 and/or CD28 might contribute to poor control of HIV infection and to immune activation.

Short Communication:Evidence of Novel SARS-CoV-2 Variants Circulation in Romania.

New SARS-CoV-2 variants are constantly emerging and putting a strain on public health systems by spreading faster and potentially evading immune protection through vaccination. One of these strains is the B.1.1.7 variant that has initially been described in the United Kingdom and has subsequently spread to several countries. Monitoring the amplification of the S gene-a major hotspot for molecular evolution-by reverse transcription polymerase chain reaction (RT-PCR) allows rapidly screening for such variants. This report describes the detection of sequence variants in Romania by using this strategy followed by next-generation sequencing of the entire genome for confirmation and further characterization. One B.1.1.7 and three B.1.258 sequences were confirmed. Each of these strains presented additional mutations with possible impact on the replicative capacity. Public health strategies should be devised to ensure molecular monitoring of SARS-CoV-2 evolution during the pandemic and allow adequate and rapid reaction.

Subtypes, resistance and virulence platforms in extended-drug resistant Acinetobacter baumannii Romanian isolates.

Acinetobacter baumannii has emerged worldwide as a dominant pathogen in a broad range of severe infections, raising an acute need for efficient antibacterials. This is the first report on the resistome and virulome of 33 extended drug-resistant and carbapenem-resistant A. baumannii (XDR CRAB) strains isolated from hospitalized and ambulatory patients in Bucharest, Romania. A total of 33 isolates were collected and analyzed using phenotypic antibiotic susceptibility and conjugation assays, PCR, whole-genome sequencing (WGS), pulsed-field gel electrophoresis (PFGE) and MultiLocus Sequence Typing (MLST). All isolates were extensively drug-resistant (XDR), being susceptible only to colistin. The carbapenem resistance was attributed by PCR mainly to blaOXA-24 and blaOXA-23 genes. PFGE followed by MLST analysis demonstrated the presence of nine pulsotypes and six sequence types. WGS of seven XDR CRAB isolates from healthcare-associated infections demonstrated the high diversity of resistance genes repertoire, as well as of mobile genetic elements, carrying ARGs for aminoglycosides, sulphonamides and macrolides. Our data will facilitate the understanding of resistance, virulence and transmission features of XDR AB isolates from Romanian patients and might be able to contribute to the implementation of appropriate infection control measures and to develop new molecules with innovative mechanisms of action, able to fight effectively against these bugs, for limiting the spread and decreasing the infection rate and mortality.

Circulation of influenza A viruses among patients hospitalized for severe acute respiratory infection in a tertiary care hospital in Romania in the 2018/19 season: Results from an observational descriptive epidemiological study.

ABSTRACT: The seasonal circulation of influenza viruses and the impact that this infection has on the population varies from year to year. We have prospectively captured hospital-based surveillance data describing the circulation of influenza viruses and characterizing patients with influenza admitted to a tertiary hospital in Bucharest, Romania in the 2018/19 season.We have conducted an observational descriptive epidemiological study analyzing all consecutive patients hospitalized for influenza like illness or severe acute respiratory infection at the National Institute for Infectious Diseases "Prof. Dr. Matei Bals", Bucharest, Romania, from November 2018 to April 2019. For all patients we actively collected standardized clinical information and performed real-time reverse transcription polymerase chain reaction testing of respiratory samples to identify the presence of influenza viruses and to determine the subtype/lineage.A total of 1128 hospitalized patients were tested in this study, with an influenza positivity rate of 41.2% (n = 465). We identified an exclusive circulation of influenza A viruses (A/H1 - 57.2%, A/H3 - 29.3%, A not subtyped - 13.3%), with only 1 case of influenza B detected at the end of the season (week 18/2019). Children under 5 years of age accounted for the majority of cases (40%, n = 186), and all cases had a favorable evolution. Females were more likely to test positive for influenza (53.3%) compared to males (46.7%), P = .048, and presence of asthma or chronic obstructive pulmonary disease increased the risk of influenza 4.4-fold and 2-fold, respectively (P < .001 and P = .034). Thirteen influenza patients required hospitalization in intensive care and 5 deaths were recorded (1.1%). The vaccination rate for all patients included in the study was low (4.6%). The existence of chronic conditions or age over 65 years prolonged the hospitalization period with 2 days (P < .001 each).In the 2018/19 season, we identified an important circulation of influenza A viruses among patients hospitalized for influenza like illness/severe acute respiratory infection in a tertiary care hospital in Romania, with a higher likelihood of affecting females and patients with pre-existing lung conditions. Monitoring of the clinical and epidemiological characteristics of influenza virus infection is of great interest and should be done carefully each season to better inform on the necessary measures to limit the impact that this infection may have on risk groups.

TH1/TH2 cytokine levels as an indicator for disease progression in human immunodeficiency virus type 1 infection and response to antiretroviral therapy.

A recent theory stipulates that during the course of HIV infection, there is a shift in immune response from T-helper 1 to T-helper 2 responses, characterised by elevated secretions of relevant cytokines. Cytokine profiles of 15 asymptomatic (treatment naïve) and 26 symptomatic (undergoing treatment) HIV-1 patients was determined to investigate the validity of this theory. HIV-1 RNA was quantified using the COBAS TaqMan HIV-1 test, CD4 T-cell counts with the FACSCalibur flow cytometer and IL-1, IL-4, IL-6, IL-10 and IFN-gamma cytokine levels by ELISA method. The asymptomatic group had significantly higher RNA levels (p-value; 0.000006) and lower CD4 T-cell counts than the symptomatic group indicating ongoing disease progression in the absence of antiretroviral treatment and a positive response to HIV treatment by the symptomatic group. IL-1, IL-4 and IFN-gamma were undetectable in most study subjects. IL-10 and IL-6 levels was relatively lower in the asymptomatic group (mean value; 206.352 pg/ml, 10.516 pg/ml) than the symptomatic group (mean value; 417.539, 18.387 pg/ml). Lower levels of proinflammatory cytokines (IL-1, IFN-gamma) in both study groups and elevated levels of anti-inflammatory cytokine IL-10, confirms that there is a shift in immune response as HIV infection progress to AIDS. In addition, the presence of a progressive trend of anti-inflammatory cytokine, IL-10 and proinflammatory cytokine, IL-6 in 12 symptomatic patients tested 3 months after antiretroviral therapy indicates an attempt by antiretrovirals to restore immune function.

Molecular Epidemiology Analysis of SARS-CoV-2 Strains Circulating in Romania during the First Months of the Pandemic.

BACKGROUND: The spread of SARS-CoV-2 generated an unprecedented global public health crisis. Soon after Asia, Europe was seriously affected. Many countries, including Romania, adopted lockdown measures to limit the outbreak. AIM: We performed a molecular epidemiology analysis of SARS-CoV-2 viral strains circulating in Romania during the first two months of the epidemic in order to detect mutation profiles and phylogenetic relatedness. METHODS: Respiratory samples were directly used for shotgun sequencing. RESULTS: All Romanian sequences belonged to lineage B, with a different subtype distribution between northern and southern regions (subtype B.1.5 and B.1.1). Phylogenetic analysis suggested that the Romanian epidemic started with multiple introduction events from other European countries followed by local transmission. Phylogenetic links between northern Romania and Spain, Austria, Scotland and Russia were observed, as well as between southern Romania and Switzerland, Italy, France and Turkey. One viral strain presented a previously unreported mutation in the Nsp2 gene, namely K489E. Epidemiologically-defined clusters displayed specific mutations, suggesting molecular signatures for strains coming from areas that were isolated during the lockdown. CONCLUSIONS: Romanian epidemic was initiated by multiple introductions from European countries followed by local transmissions. Different subtype distribution between northern and southern Romania was observed after two months of the pandemic.

COVID-19 Like Findings in a Fatal Case of Idiopathic Desquamative Interstitial Pneumonia Associated With IgA Glomerulonephritis in a 13-Month-Old Child.

In the COVID-19 era, patients with severe acute respiratory syndrome (SARS) are suspected to be associated with SARS-CoV-2 infection. The aim of this paper is to present a case with COVID-like pneumonia, with fatal evolution. The clinical aspects were correlated with the autopsy findings and discussed on the background of the most recent data from the medical literature. A 13-month-old girl was admitted to the emergency room with acute severe shortness of breath and pulmonary bilateral ground-glass opacities and an almost complete opacified left lung. The patient suddenly deteriorated, and death was confirmed 3 h after admission. At autopsy, severe desquamative interstitial pneumonia was diagnosed and was associated with an unusual IgA glomerulonephritis. No SARS-CoV-2 infection was detected in the lung parenchyma by RT- PCR. This is a very unusual case of rapid deterioration of an infant with idiopathic desquamative interstitial pneumonia (IDP) and multiorgan involvement. Based on immunohistochemical stains, we hypothesize that, in IDP, the hyaline membranes arise from necrotizing desquamated pneumocytes. In the COVID-19 era, such cases are extremely difficult to diagnose; they can mimic SARS-CoV-2-induced lung injuries. This pattern of hyaline membrane formation might explain the poor response to oxygen therapy. The present case highlights the importance of autopsy in such challenging cases.

Multidrug Resistant Klebsiella pneumoniae ST101 Clone Survival Chain From Inpatients to Hospital Effluent After Chlorine Treatment.

In this paper we describe the transmission of a multi-drug resistant Klebsiella pneumoniae ST101 clone from hospital to wastewater and its persistence after chlorine treatment. Water samples from influents and effluents of the sewage tank of an infectious diseases hospital and clinical strains collected from the intra-hospital infections, during a period of 10 days prior to wastewater sampling were analyzed. Antibiotic resistant K. pneumoniae strains from wastewaters were recovered on selective media. Based on antibiotic susceptibility profiles and PCR analyses of antibiotic resistance (AR) genetic background, as well as whole-genome sequencing (Illumina MiSeq) and subsequent bioinformatic analyses, 11 ST101 K. pneumoniae strains isolated from hospital wastewater influent, wastewater effluent and clinical sector were identified as clonally related. The SNP and core genome analyses pointed out that five strains were found to be closely related (with ≤18 SNPs and identical cgMLST profile). The strains belonging to this clone harbored multiple acquired AR genes [bla CTX-M- 15, bla OXA- 48, bla OXA- 1, bla SHV- 106, bla TEM- 150, aac(3)-IIa, aac(6')-Ib-cr, oqxA10, oqxB17, fosA, catB3, dfrA14, tet(D)] and chromosomal mutations involved in AR (ΔmgrB, ΔompK35, amino acid substitutions in GyrA Ser83Tyr, Asp87Asn, ParC Ser80Tyr). Twenty-nine virulence genes involved in iron acquisition, biofilm and pili formation, adherence, and the type six secretion system - T6SS-III were identified. Our study proves the transmission of MDR K. pneumoniae from hospital to the hospital effluent and its persistence after the chlorine treatment, raising the risk of surface water contamination and further dissemination to different components of the trophic chain, including humans.

Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania.

We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.

Comparative methods for genotyping hepatitis C virus isolates from Romania.

Accurate genotyping of hepatitis C virus (HCV) has clinical implications for treatment orientation and epidemiological impact in tracing the contamination sources. The aim of the study was to compare a genotyping assay by restriction fragment length polymorphism (RFLP) in the HCV 5'untranslated region (5'UTR) with sequencing in the 5'untranslated and NS5B regions. One hundred and three samples, collected between 2004 and 2006 from chronically infected patients with HCV, were tested with the 5'UTR and NS5B protocols. Of the total number of the samples tested by the 5'UTR-RFLP assay (n=103) the HCV subtype could be inferred by this method for 92 samples, by 5'UTR sequencing for 16 samples out of 23 tested (n=23) and by using the NS5B sequencing for all the samples tested (n=34). Our results showed that the HCV genotype distribution in Romania is: 1b--86.4%, 1a--10.7% and 4a--2.9%. In conclusion, RFLP screening in the 5'UTR is a convenient method for HCV genotyping and discrimination between 1b and non-1b genotypes but has a poor resolving power for subtyping and evaluation of the transmission routes. Sequencing in NS5B region is more adapted than RFLP and sequencing in 5'UTR for subtyping and epidemiological investigation.