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.

Identification of four Treponema species in subgingival samples by nested-PCR and their correlation with clinical diagnosis.

The relationship between different species of oral Treponemas and inflammation in periodontal disease progression is complex. The purpose of this study was to analyze and compare the subgingival plaque samples collected from periodontally healthy subjects and from chronic gingivitis and periodontitis patients in order to detect the presence of T. denticola, T. pectinovorum, T. socranskii and T. vincentii using nested-PCR technology. After DNA extraction from the samples using QIAmp DNA Mini Kit (QIAGEN, the four Treponema species were determined with nested-polymerase chain reaction which requires two sets of primers to amplify a specific DNA fragment in two separate runs of PCR. Pearson chi-square was implemented to compare the three groups as to the presence of four Treponema species. Results of this investigation showed significant differences between groups regarding subject proportion of T. denticola, T. socranskii, T. pectinovorum, T. vincentii, with a higher percentage of patients from associated-disease groups of patients harboring these four species than healthy subjects. These differences were more pronounced in presence of Treponema denticola and Treponema socranskii. Our findings suggest that Treponema denticola and Treponema socranskii concurrent presence indicate more accurately the association with chronic gingivitis and periodontitis.

Identification of Treponema denticola in subgingival samples by PCR technology and its correlation with clinical diagnosis.

Treponema denticola has been associated with gingivitis and chronic periodontitis. The aim of this study was to identify Treponema denticola in subgingival samples using PCR technology and to correlate it with clinical diagnosis of subjects. The study was carried out on seventy patients (20-84 years of age; mean age, 45.06 +/- 12.58) of which 22 individuals with no detectable gingivitis or periodontitis, 4 subjects with chronic gingivitis and 44 subjects with chronic periodontitis. Subgingival plaque samples were collected from five sites in each patient. DNA was extracted from the samples using QIAamp DNA Mini Kit (QIAGEN). Treponema denticola and other four periodontopathogens were found using multiplex polymerase chain reaction followed by a reverse hybridization. The relationship between clinical diagnoses and detection of Treponema denticola was determined with Fisher exact test. The results showed significant differences between diagnostic groups regarding subject proportion. Treponema denticola was detected in 2 out of 22 subjects with no detectable gingivitis or periodontitis, 2 out of 4 subjects with chronic gingivitis, and 40 out of 44 subjects with chronic periodontitis. Our findings suggest that Treponema denticola is closely connected to the initiation and progression of periodontal disease.

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.

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.

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.

Association of Prothrombin (A20210G) and Factor V Leiden (A506G) with Recurrent Pregnancy Loss.

BACKGROUND: Inherited thrombophilias are the leading cause of maternal thromboembolism and are associated with an increased risk of recurrent spontaneous abortion (second- and third-trimester fetal loss). The purpose of this study was to investigate the effects of factor V and factor II involved in reproductive failure. Recently a possible association between unexplained infertility and genetic thrombophilia gene mutations have been reported with a significant statistically association with prothrombin A20210G. MATERIALS AND METHODS: During the period from January 2011 to December 2011, 283 patients with unexplained infertility, who had received in our hospital, were investigated for this retrospective study, and the frequency of polymorphic variations was calculated. The infertile couples with recurrent pregnancy loss (RPL), had been trying to achieve successful pregnancy for greater than 1 year without success and known causes of infertility were excluded (semen anomalies, karyotype abnormalities, uterine malformations, etc) referred to our Centre for genetic counseling. The control group consists of 100 women who had one or more children in history were investigated by DNA Strip. RESULTS: Heterozygous and normal homozygous for the factor V mutation and factor II mutation were equally distributed among patients with recurrent miscarriage and fertile patients with two or more previous births. The combination of the two polymorphisms, prothrombin (A20210G) and factor V Leiden (A506G) revealed a significant correlation between them and early fetal loss. CONCLUSIONS: The genes involved in thrombophilia could be one reason for fertility complications in some women with unexplained infertility. Our study shows that there is an association between factor II and V mutation and the risk for fetal loss.

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.