Reinforced poliovirus and enterovirus surveillance in Romania, 2015-2016.

Due to the risk of poliovirus importation from Ukraine in 2015, a combined surveillance program monitoring the circulation of enteroviruses (EVs) in healthy children from at-risk areas and in the environment was conducted in Romania. Virological testing of stool samples collected from 155 healthy children aged from two months to six years and of 186 sewage water samples collected from different areas was performed. A total of 58 (37.42%) stool samples and 50 (26.88%) sewage water samples were positive for non-polio EVs, but no poliovirus was detected. A high level of circulation of echovirus (E) types 6 and 7 and coxsackievirus (CV) type B5 was observed.

Application of the susceptible-infected-recovered deterministic model in a GII.P17 emergent norovirus strain outbreak in Romania in 2015.

PURPOSE: This study shows the epidemiological profile of the first gastroenteritis outbreak of GII.P17 in the Romanian territory. An outbreak with such large amplitude in a European territory was previously undocumented. PATIENTS AND METHODS: Using a cross-sectional design, with the susceptible-infected-recovered (SIR) deterministic compartmental model for a fixed population, and the cluster method for establishing the high-incidence zones, we carried out our investigation by means of questionnaires containing personal data, affected collectivities, disease onset and duration, symptoms displayed, medical assistance provided, previous antibiotic intake where applicable, food consumption and water sources, and sanitation conditions. The confirmation of cases was done based on the typical norovirus gastroenteritis symptomatology and using three laboratory confirmations (by molecular diagnosis) for GII.P17-GII.17 genotype noroviruses from three patients. RESULTS: A gastroenteritis outbreak occurred in October-November 2015, affecting 328 people in Arad, a county in Western Romania, covering 44 neighbouring localities with a total population of 35,440 people. The study detected an inter-human transmission of the infection, with an intrafamilial risk of disease of 2.26 (95% CI 1.76 to 2.90) compared with the community transmission (in school collectivity). The basic reproduction number Ro dropped from 1.26 to 0.18 during weeks 43:44, after controlling the transmission by decontamination and isolation. CONCLUSION: SIR made it possible to highlight the expansion of the emerging norovirus strain infection from community to family collectivities. This study provides practical solutions to limit disease cases, even in the absence of etiology, and shows the importance of sometimes underestimated traditional control methods.

Differential regulation of the Wnt/ß-catenin pathway by hepatitis C virus recombinants expressing core from various genotypes.

Clinical studies have suggested association of some hepatitis C virus (HCV) subtypes or isolates with progression toward hepatocellular carcinoma (HCC). HCV core protein has been reported to interfere with host Wnt/ß-catenin pathway, a cell fate-determining pathway, which plays a major role in HCC. Here, we investigated the impact of HCV core genetic variability in the dysregulation of Wnt/ß-catenin pathway. We used both transient expression of core proteins from clinical isolates of HCV subtypes 1a (Cambodia), 4a (Romania) and 4f (Cameroon) and infection systems based on a set of engineered intergenotypic recombinant viruses encoding core from these various clinical strains. We found that TCF transcription factor-dependent reporter activity was upregulated by core in a strain-specific manner. We documented core sequence-specific transcriptional upregulation of several ß-catenin downstream target genes associated with cell proliferation and malignant transformation, fibrogenesis or fat accumulation. The extent of ß-catenin nuclear translocation varied in accordance with ß-catenin downstream gene upregulation in infected cells. Pairwise comparisons of subgenotypic core recombinants and mutated core variants unveiled the critical role of core residues 64 and 71 in these dysregulations. In conclusion, this work identified natural core polymorphisms involved in HCV strain-specific activation of Wnt/ß-catenin pathway in relevant infection systems.

Impact of hepatitis C virus core mutations on the response to interferon-based treatment in chronic hepatitis C.

AIM: To determine whether hepatitis C virus (HCV) core substitutions play a role in the response to interferon-based treatment in Caucasian patients. METHODS: One hundred eight HCV chronically infected patients initiating treatment with pegylated IFN plus ribavirin for 48 wk were tested for baseline substitutions at codons 70 and 91 of the viral core protein (BigDye Terminator vers.3.1, Applied Biosystems,) and for genetic polymorphisms in host IL28B gene rs12979860 (Custom TaqMan 5' allelic discrimination assay; Applied Biosystems). RESULTS: Of the patients, all were infected with HCV genotype 1b, 44.4% had low baseline HCV viral load, and 37.9% had mild/moderate fibrosis. Only 38.9% achieved therapeutic success, defined as sustained virological response (SVR). Eighty-eight percent of the patients presented at least one substitution at core position 70 (R70Q/H) or/and position 91 (L91M). The favorable IL28B CC polymorphism was detected in only 17.6% of the patients. In the univariate analysis, young age (P < 0.001), urban residence (P = 0.004), IL28B CC genotype (P < 0.001), absence of core mutations (P = 0.005), achievement of rapid virologic response (P < 0.001) and early virological response (P < 0.001) were significantly correlated with SVR. A multivariate analysis revealed three independent predictors of therapeutic success: young age (P < 0.001), absence of core substitutions (P = 0.04) and IL28B CC genotype (P < 0.001); the model correctly classified 75.9% of SVR cases with a positive predictive value of 80.7%. CONCLUSION: HCV core mutations can help distinguish between patients who can still benefit from the affordable IFN-based therapy from those who must be treated with DAAs to prevent the evolution towards end-stage liver disease.

Retrospective study reveals the circulation of norovirus genotype GII.P21-GII.2 in Romania

Noroviruses are the leading cause of acute gastroenteritis, causing significant economic burden globally. Infection is self-limiting, occurring as sporadic cases or producing outbreaks associated with consumption of contaminated water or food. All age groups are affected and person to person transmission is frequent. Except a recent outbreak in Romania caused by the emergent genotype GII.P17-GII.17, few data regarding the circulation of noroviruses in our country are available. We retrospectively analyzed stool samples from acute gastroenteritis patients hospitalized in Romania between 2005 and 2008. Noroviruses were detected by RT-PCR and phylogenetic analysis was inferred from partial sequences spanning ORF1 and ORF2. Recombinant GII.P21-GII.2 isolates were found in two adult patients from a cluster of acute gastroenteritis in 2006. Molecular analysis based on partial genomic sequences indicated high degree of similarity between the two isolates and grouped them with cosmopolitan strains circulating in the same period of time. Along with the high rate of mutation, recombination is an important driving force in norovirus evolution. GII.P21 isolates, formerly known as GII.b recombinants, have been detected in Europe since 2000 and associated with sporadic cases and outbreaks of gastroenteritis worldwide. This is the first work describing norovirus GII.P21-GII.2 identified in Romania.

Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013.

BACKGROUND: Dengue fever is the commonest arthropod-borne infection worldwide. In recent years, rapid growth in global air travel has resulted in a considerable increase in the incidence of imported cases. In Romania it is now the second most frequent cause for hospitalization (after malaria) in patients arriving from tropical regions. METHODS: Serological and molecular diagnostics were applied to samples obtained between 2008 and 2013 from travelers with suspected dengue. Molecular typing was performed by RT-PCR followed by sequencing of the E-NS1 junction. RESULTS: Twelve of 37 suspected cases were confirmed and three remained probable. The infections were acquired in endemic regions in Asia, Africa and in Europe (Madeira Island). Dengue virus nucleic acid was detected and sequenced in nine cases. Phylogenetic analysis indicated that the viruses were of genotypes I and V of serotype 1, cosmopolitan genotype of serotype 2 and genotypes I and III of serotype 3. CONCLUSIONS: Romanian tourists traveling to dengue-endemic countries are at risk of acquiring dengue infection. Appropriate prevention measures prior to travel and upon return should be taken, particularly as the dengue secondary vector Aedes albopictus is now established in Bucharest.

SCREENING OF PROTEASE INHIBITORS RESISTANCE MUTATIONS IN HEPATITIS C VIRUS ISOLATES INFECTING ROMANIAN PATIENTS UNEXPOSED TO TRIPLE THERAPY.

Although the European recommendations include the use of new antiviral drugs for the treatment of hepatitis C, in Romania the current treatment remains interferon plus ribavirin. First generation viral protease inhibitors (i.e. boceprevir, telaprevir), which have raised the chances of obtaining viral clearance in up to 70% of infection cases produced by genotype 1 isolates, have not been introduced yet as standard treatment in our country. The success of these new antivirals is limited by the occurrence and selection of resistance mutations during therapy. We set-up a molecular study aiming to detect any resistance mutations to boceprevir and telaprevir harbored by hepatitis C isolates infecting Romanian patients naïve to viral protease inhibitors. Since these new antivirals are efficient and approved for genotype 1 infection, viral samples were genotyped following a protocol previously developed by our research group. We analyzed by both population sequencing and molecular cloning and sequencing the NS3 protease region of hepatitis C virus isolates infecting patients which were not previously exposed to boceprevir and telaprevir. All the analyzed samples were subtype 1b and resembled the samples collected in recent years from Romanian patients. Molecular cloning followed by sequencing showed great intra-host diversity, which is known to represent the source of isolates with different resistance phenotypes. Both population sequencing and molecular cloning followed by clone sequencing revealed two boceprevir resistance mutations (T54S and V55A), respectively, a telaprevir resistance mutation (T54S) in the sequences obtained from a patient with chronic hepatitis C. To our knowledge, this is the first study indicating the existence of pre-treatment resistance mutations to boceprevir and telaprevir in hepatitis C virus isolates infecting Romanian patients.

GENOMIC ANALYSIS OF HEPATITIS B VIRUS STRAINS INFECTING ROMANIAN PATIENTS.

Chronic hepatitis B is widespread and represents an important cause of morbidity and mortality due to the evolution to cirrhosis and hepatocellular carcinoma. This study was designed to improve the national laboratory surveillance of hepatitis B virus (HBV) infection, focusing on genomic analysis of isolates from Romanian patients. Sera from ten patients with HBV were collected and analyzed. Phylogenetic analysis was conducted on a DNA fragment spanning almost the entire genome. The occurrence of mutations was assessed for each open reading frame in the viral genome. Phylogenetic analysis revealed five isolates belonging to genotype A (subgenotype A2) and other five clustering with genotype D strains (subgenotype D1). Two patients treated with lamivudine were found to carry isolates harboring rtM204V lamivudine resistance mutation. An HBV isolate displaying a lamivudine complex resistance pattern, rtM204I in conjunction with rtL180M and rtA200V, was found in a lamivudine naïve patient. All samples harbored sA105P substitution, usually found in HBIg therapy escape isolates. Three of the studied strains were simultaneously displaying T1753, T1762 and A1764 mutations which in vitro induce enhanced genome replication and reduction of HBeAg expression. The sequence obtained from a patient with decompensated liver cirrhosis presents a novel type of insertion consisting of nine nucleotides between positions 260 and 261 in the X gene. Despite the small number of samples, our findings suggest the need to determine the drug resistance pattern for each patient before taking a therapeutic decision and also highlight the necessity of knowing the real level of drug resistance among HBV strains circulating in Romania.

Mutation spectrum of hepatocellular carcinoma from eastern-European patients betrays the impact of a complex exposome.

Genomic analysis of hepatocellular carcinoma (HCC) has been shown to provide clues about local risk factors. In the last decades, the mortality from malignant liver tumors increased sharply in Romania, where both hepatitis viruses and environmental pollutants are known to be highly prevalent. To date, HCC from this country has not been subject to molecular characterization. We analyzed a series of 48 consecutive HCC cases. Point mutations were searched in 9 nuclear genes and the mitochondrial D-loop. Oxidative stress response was monitored through measurement of gene expression (NRF2, KEAP1, SRXN1, and CES1) by qRT-PCR. An atypical mutation spectrum was observed, as more than 40% of DNA changes were oxidative stress-associated T>C or T>G lesions (T>S). These mutations affected primarily genes encoding for ß-catenin and NRF2 (P<0.0001). Besides, tumors from patients born in Greater Bucharest carried TP53 mutations more frequently than others (45 vs 10%, P=0.02). Finally, a R249S mutation of TP53, well-known hallmark of aflatoxin B1 exposure, was found. Our findings indicate, therefore, that distinct mutagenic processes affect Romanian patients with HCC. Further analyses are now warranted in order to identify causal lifestyle or environmental factors.

Dobrava virus carried by the yellow-necked field mouse Apodemus flavicollis, causing hemorrhagic fever with renal syndrome in Romania.

Hemorrhagic fever with renal syndrome (HFRS) has been confirmed by serological methods during recent years in Romania. In the present study, focus-reduction neutralization tests (FRNT) confirmed Dobrava hantavirus (DOBV) as the causative agent in some HFRS cases, but could not distinguish between DOBV and Saaremaa virus (SAAV) infections in other cases. DOBV was detected by a DOBV-specific TaqMan assay in sera of nine patients out of 22 tested. Partial sequences of the M genomic segment of DOBV were obtained from sera of three patients and revealed the circulation of two DOBV lineages in Romania. Investigation of rodents trapped in Romania found three DOBV-positive Apodemus flavicollis out of 83 rodents tested. Two different DOBV lineages were also detected in A. flavicollis as determined from partial sequences of the M and S genomic segments. Sequences of DOBV in A. flavicollis were either identical or closely related to the sequences obtained from the HFRS patients. The DOBV strains circulating in Romania clustered in two monophyletic groups, together with strains from Slovenia and the north of Greece. This is the first evidence for the circulation of DOBV in wild rodents and for a DOBV etiology of HFRS in Romania.

Molecular epidemiology of hepatitis C virus strains from Romania.

BACKGROUND AND AIMS: A high seroprevalence of Hepatitis C Virus (HCV) infection has been reported in Romania, with limited data on the viral subtypes' distribution. In order to detect any changes in the genetic composition of the epidemic, a survey on the recent profile of circulating HCV genotypes was conducted. METHODS: 241 hepatitis C infected patients with active viral replication diagnosed between September 2004 - October 2008 were included in a retrospective study. Genotyping using commercial Line Probe Assay (Innogenetics) was confirmed by sequencing of Core PCR products followed by phylogenetic analysis. RESULTS: HCV subtype 1b was found in 92.6% of the samples, subtype 1a in 5.4 % of the samples, subtype 4a in 1.2%, and subtype 3a in 0.8% of the samples. Chronic hepatitis C infections with subtype 1b were found in women aged 40-60 years old with a history of blood transfusions received during surgical/obstetrical interventions. No geographical clustering was evident for HCV 1b sequences. The new emerging non-1b genotypes were detected mainly in younger patients with a history of intravenous drug use. The genetic distances among the HCV 1a strains are very homogeneous and small, with a high sequence identity with other European strains, suggesting the recent entrance of this subtype in Romania from singular or limited sources of infection. CONCLUSION: The introduction of new HCV genotypes in Romania stimulates a continuous epidemiological surveillance, suggesting shifts in the transmission pathways and risk factors, with the possible emergence of recombinant strains in patients with multiple infections.

The frequency and biodiversity of poliovirus and non-polio enterovirus strains isolated from healthy children living in a limited area in Romania.

The risk of importation and transmission of poliovirus strains to small susceptible groups within populations will remain until polio is eradicated globally. We investigated the circulation and biodiversity of enteroviruses in a group of children under 6 years of age with low vaccine coverage against polio. Only vaccine Sabin strains and viruses of the human enterovirus species B were isolated from the group. Evidence of inter-human circulation of Sabin strains was found.

Laboratory diagnosis of infectious diarrhoea syndrome; a three years study in two hospitals of infectious diseases.

Infectious diarrhoea is a syndrome caused by a variety of bacterial, viral and parasitic organisms which represents a major cause of morbidity and mortality all over the world. The wide diversity of etiological agents impairs the surveillance and the diagnosis and affects the correct treatment applied to reduce the long-term complications. Besides well known enteric pathogens such as Salmonella, Shigella and Yersinia, a high number of emergent and re-emergent aetiologies are now recognised to be at the origin of diarrhoea. The lack of a correct diagnostic algorithm and adequate methods of analyses leads to under-evaluation and incertitude in an important number of clinical cases. Our study was designed as a complex analysis of the stool specimens collected from the patients, in the purpose to improve the laboratory diagnostic and to enhance the number of confirmed cases of infectious diarrhoea. A number of 756 samples from inpatients with diarrhoea were tested targeting pathogenic and opportunistic bacteria, viruses and parasites by classical and molecular methods. We documented that, in case of non-Salmonella, non-Shigella, non-Yersinia diarrhoea, the quality of diagnostic was improved by increasing the percentage of positive specimens to 22.49% compared to 11.12% when only bacteria, 5.56% when only viruses and 4.10% when only parasites were investigated. The laboratory data are of great value in evaluating the diarrhoea syndrome offering the documentation for an accurate epidemiological response and an adequate treatment.

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.

Correlation between vaccine coverage against polio and circulation and genetic evolution of the poliovirus strains isolated in Romania in the framework of the global polio eradication strategy.

Until 2008 in Romania poliomyelitis has been controlled by predominantly using trivalent oral poliovirus vaccine (TOPV). The alternative vaccination schedule (formalin inactivated poliovirus vaccine IPV/OPV) has been implemented starting September 2008 and at the begining of 2009 was decided only vaccination with IPV. Between 1995-2006 the risk of the vaccine-associated paralytic poliomyelitis (VAPP) decreased with an average of less than 2 VAPP cases/year and no VAPP case between 2007 - September 2009. Begining with 2007 the number of the poliovirus strains isolated was less. All 9 poliovirus strains (PV) isolated between 2007-2009 and investigated by RT-PCR-RFLP in VP1-2A and VP3-VP1 coding regions showed Sabin-like profiles, and only one strain poliovirus type 3 showed Sabin 2-like profile by RFLP in 3D coding ARN polymerase region. The study about the seroprevalence of antibodies against poliovirus types in serum samples from the acute flaccid paralysis (AFP), facial paralysis (FP) cases showed that the seroprevalence of antibodies against types 1 and 2 Sabin strains was higher (>90%) than for type 3 Sabin strains (average 85%). It was confirmed the necessity of maintaining a proper vaccine coverage in population, after the switch in the vaccination strategy in Romania until all threats of poliovirus are eliminated globally.

Natural genetic recombination between co-circulating heterotypic enteroviruses.

Natural recombination in poliovirus is a frequent phenomenon. In practice, whenever different genotypes have the opportunity to infect the same individual, a high proportion of viruses with recombinant genomes are excreted. To determine whether enteroviruses other than poliovirus can naturally produce viable virions with recombinant genomes, we studied the molecular features of two distant regions of the viral genomes - the VP1 coding region and the 3D polymerase coding region - of the echovirus serotypes associated with a large outbreak of aseptic meningitis. Nucleotide sequences of nine epidemic strains [belonging to echovirus serotypes 4 (E4), 7 (E7) and 30 (E30)] in the two genomic regions (300 nt of VP1 and 520 nt of 3D polymerase) were compared to prototype and field strains, and phylogenetic trees were generated from alignments. In the VP1 region, each of the three epidemic serotypes clustered with the homotypic prototype strain, whereas in the 3D polymerase region, E7 and E30 grouped as a single cluster, distant from the two corresponding prototype strains. This suggests that one of these two E7 and E30 strains has evolved through recombination with the other or that both have acquired the 3D polymerase coding region from a common ancestor. Our results suggest that such genetic recombinations between different echovirus serotypes are possible when multiple epidemic strains are circulating simultaneously.