Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

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.

Impaired ankle-brachial index in antiphospholipid syndrome: Beyond the traditional risk factors.

INTRODUCTION: The patients with antiphospholipid syndrome (APS) associate an increased risk of atherosclerosis. OBJECTIVE: To determine the predictors of an abnormal ankle-brachial index (ABI), surrogate measure of atherosclerosis, in patients with APS. METHODS: The ABI was measured according to standard recommendations in 106 patients. Traditional cardiovascular risk factors were assessed in all cases. A large spectrum of APS antibodies was determined in 73 patients. RESULTS: A total of 106 patients diagnosed with APS were included. 28.3% patients included were found to have low ABI. Anti-beta 2-glycoprotein I (aß2GPI) IgG antibodies [4.00 (1.00-79.00) vs 3.00 (0.00-29.00) U/mL, P = 0.02] and antiprothrombin (aPT) IgM antibodies [4.50 (0.00-82.00) vs 3.00 (0.00-14.00) U/mL, P = 0.05] titers were found to be higher in patients with abnormal ABI. However, after multivariate regression analysis, only the aß2GPI IgG titer remained predictor of low ABI (P = 0.04). CONCLUSIONS: aß2GPI IgG associated with impaired ABI in patients with APS. This relation might reflect their involvement in the atherosclerosis occurrence.

Short communication: Could the genexpert system be a new tool for poliovirus detection in the sewage water?

Environmental enterovirus surveillance plays a key role in the detection and identification of importation of circulating vaccine-derived polioviruses into polio-free areas. In 2015, 2 cases of paralytic poliomyelitis caused by circulating vaccine-derived poliovirus type 1 (cVDPV1), from south-western Ukraine, bordering Romania, were confirmed. Romania was considered a country at risk and the environmental enterovirus surveillance was enhanced. In this context we tried to find a diagnosis algorithm for the rapid detection of poliovirus (PV) in the sewage water, using a combination between a rapid molecular method for human enterovirus (HEV) detection using the GeneXpert system and the virus isolation on cell culture lines. By using this algorithm, we would be able to give a rapid response in an emergency situation, such as the risk of polio importation.

The impact of the molecular diagnosis in the surveillance of the Clostridium difficile infection.

Clostridium difficile is the agent of many cases of antibiotic associated diarrhea. The prevalence of the toxigenic Clostridium difficile strains was assessed by real-time PCR between May 2014- January 2015, at the Emergency University Hospital, Bucharest, Romania. The incidence of the Clostridium difficile infection was 0.47%. Among the 1119 stool specimens tested, 165 (13.8%) were positive for C. difficile toxins A and B by immunochromatography test. All 165 positive samples were investigated by real time PCR and 134 (81%) samples were positive for toxin B and for the binary toxin, while the rest were positive for toxin B. A high number of samples positive for binary toxin was recorded in the General surgery department (29.85%), Neurology (13.43%), and Internal medicine (12.68%). Many of the infected patients presented as underlying pathology, cancer, diabetes mellitus and stroke. A rapid decision concerning the antibiotic therapy was made, to decrease the risk of nosocomial spread.

RAPID COMMUNICATION-- POLIO VACCINE COVERAGE IN THE ACUTE FLACCID PARALYSIS (AFP) CASES IN ROMANIA.

Poliovirus (PV), a member of the Enterovirus genus, is the etiological agent of poliomyelitis. A study carried out between 2013-2014 on 30 serum samples from acute flaccid paralysis (AFP) cases, showed a protective antibody level of 90% against poliovirus Sabin strains type 1 and type 2 and of 88% against type 3. No PV strains were isolated from 2009 to 2015 in Romania. Maintaining a high vaccine coverage level against polio is mandatory until global polio eradication, especially as the risk of polio importation remains elevated in Romania.

Status of immunity against poliomyelitis in the acute flaccid paralysis (AFP) cases in Romania between 2009-2012.

Poliovirus (PV), a member of the Enterovirus genus, is the etiological agent of poliomyelitis, an acute paralytic disease. No poliovirus strain has been isolated in Romania since 2009. A serosurvey study carried out between 2009-2012, that involved 76 serum samples from acute flaccid paralysis (AFP) cases, showed a low protection level in the examined subjects against type 1 (80%), type 2 (79%) and type 3 (71%) poliovirus Sabin strains. Samples with titers ≥ 1:8 were considered positive. Suboptimal seroprevalence in the AFP cases confirmed that a high level of immunization against polio must be assured in our country, because the risk of importation and subsequent transmission of the poliovirus remains until polio is globally eradicated.

Criteria and Non-Criteria Antiphospholipid Antibodies and Cancer in Patients with Involuntary Weight Loss.

Cancer patients have higher prevalences of antiphospholipid antibodies (aPLs), occasionally associated with thrombotic events. A cross-sectional study regarding the presence of criteria (IgG/IgM anti-cardiolipin-aCL, anti-ß2 glycoprotein I-aß2GPI) and non-criteria (IgG/IgM anti-phosphatidylserine-aPS, anti-phosphatidylethanolamine-aPE, anti-prothrombin-aPT) aPLs in 146 patients with involuntary weight loss was performed. None of the patients had thrombotic events during the study. Out of the 36 cancer patients, 33 had non-hematologic malignancies. In the cancer subgroup, 60% of the patients had at least one positive aPL, with significantly more patients being positive for aß2GPI IgG compared with the non-cancer subgroup-p = 0.03, OR = 2.23 (1.02-4.88). When evaluating the titres, aCL IgG/IgM, aß2GPI IgG, aPE IgG, and aPS IgG had significantly higher values in cancer patients, the best cancer predictor being aß2GPI IgG-AUC 0.642 (0.542-0.742). Gastrointestinal cancer patients were studied separately, and aCL IgM positivity was significantly higher-p = 0.008, OR = 6.69 (1.35-33.02). Both the titres of aCL IgM (p = 0.006) and aPS IgM (p = 0.03) were higher in the gastrointestinal cancer subgroup, with aCL IgM being the best predictor for gastrointestinal cancer development-AUC 0.808 (0.685-0.932). Despite criteria and non-criteria aPLs being frequent in cancer, their connection with thrombosis in these patients is probably dependent on other important risk factors and needs further research.

Criteria and Non-Criteria Antiphospholipid Antibodies in Antiphospholipid Syndrome: How Strong Are They Correlated?

The place of non-criteria antiphospholipid antibodies (aPLs) in the diagnosis of antiphospholipid syndrome (APS) is still debatable. The aim of this research was to evaluate the correlations between the titres of non-criteria aPLs (anti-phosphatidylethanolamine (aPE), anti-phosphatidylserine (aPS), and anti-prothrombin (aPT) antibodies), and the ones of the already studied criteria aPLs (anti-cardiolipin (aCL) and anti-ß2 glycoprotein I-aß2GPI antibodies). Altogether, 72 APS (30 primary and 42 secondary) patients were included in our study. High correlation coefficients (rs) were found between aPS IgM and aCL IgM, overall (0.77, p < 0.01), as well as in the primary (0.81, p < 0.01), and secondary (0.75, p < 0.01) APS subgroups. Low or statistically insignificant correlations were observed between IgG/IgM isotypes of aPT and aCL, or aß2GPI, in the entire study population, and when evaluating the subgroups. Therefore, moderate correlations were mainly identified between the tested non-criteria antibodies and the criteria ones, suggesting little added value for the use of the tested non-criteria aPLs, with the exception of aPT, which seems to have different kinetics and might be a promising APS diagnostic tool.

Wastewater Surveillance in Europe for Non-Polio Enteroviruses and Beyond.

Wastewater surveillance (WWS) was developed in the early 1960s for the detection of poliovirus (PV) circulation in the population. It has been used to monitor several pathogens, including non-polio enteroviruses (NPEVs), which are increasingly recognised as causes of morbidity in children. However, when applying WWS to a new pathogen, it is important to consider the purpose of such a study as well as the suitability of the chosen methodology. With this purpose, the European Non-Polio Enterovirus Network (ENPEN) organised an expert webinar to discuss its history, methods, and applications; its evolution from a culture-based method to molecular detection; and future implementation of next generation sequencing (NGS). The first simulation experiments with PV calculated that a 400 mL sewage sample is sufficient for the detection of viral particles if 1:10,000 people excrete poliovirus in a population of 700,000 people. If the method is applied correctly, several NPEV types are detected. Despite culture-based methods remaining the gold standard for WWS, direct methods followed by molecular-based and sequence-based assays have been developed, not only for enterovirus but for several pathogens. Along with case-based sentinel and/or syndromic surveillance, WWS for NPEV and other pathogens represents an inexpensive, flexible, anonymised, reliable, population-based tool for monitoring outbreaks and the (re)emergence of these virus types/strains within the general population.

Ferritin above 100 mcg/L could rule out colon cancer, but not gastric or rectal cancer in patients with involuntary weight loss.

BACKGROUND: A tenth of patients with involuntary weight loss (IWL) have gastrointestinal cancer. Ferritin is the first parameter to be modified during the process leading to iron deficiency anaemia, therefore it should be the most sensitive. The aim of this study was to assess the ability of ferritin to rule out gastrointestinal cancer in patients with involuntary weight loss. METHODS: All consecutive patients with IWL admitted in a secondary care university hospital were prospectively studied. Ferritin, haemoglobin with erythrocyte indices and serum iron were recorded for all patients. The reference standard was bidirectional endoscopy and/or 6 months follow-up. RESULTS: 290 patients were included, a quarter had cancer, of which 22 (7.6%) had gastrointestinal cancer (8 gastric cancer, 1 ileum cancer, 13 colorectal cancer). Ferritin had the best area under the curve (AUC), both for gastrointestinal cancer (0.746, CI: 0.691-0.794), and colorectal cancer (0.765, CI: 0.713-0.813), compared to the other parameters of iron deficiency. In the diagnosis of colorectal cancer, ferritin with a cut-off value of 100 mcg/L had a sensitivity of 93% (CI: 69-100%), and negative likelihood ratio of 0.13, with a negative predictive value of 99% (96-100%), while for gastrointestinal cancer, the sensitivity was lower (89%, CI: 67-95%), with a negative likelihood ratio of 0.24. There were three false negative patients, two with gastric cancer, and one with rectal cancer. CONCLUSION: In patients with involuntary weight loss, a ferritin above 100mcg/L could rule out colon cancer, but not gastric or rectal cancer.

Impact of the molecular diagnosis on the management of patients with aseptic meningitis.

Between 2007-2008, epidemic episodes of aseptic meningitis occurred in Romania. Most important strains isolated were Echoviruses. A commercial kit for rapid molecular detection of aetiological agents of aseptic meningitis was tested. The introduction of the molecular tests in clinical use is important by the efficient decision-making concerning the treatment.

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.

COVID-19 Pandemic Impact on Cord Blood Collection for Stem Cell Use and Actual Perspectives.

Cord blood collection for stem cell storage remains a popular procedure due to the advantages associated to it. In the last ten years, the therapeutic potential of umbilical cord stem cells was demonstrated in the treatment of bone pathologies, neuropsychiatry, metabolic and genetic diseases. There are several factors with direct influence on the quality and quantity of cord blood collection for stem cell storage. The collection principles should be individualized according to the maternal and fetal characteristics. Furthermore, in the context of COVID-19 pandemic, additional information can be obtained through this procedure. We present a specific case-adapted strategy for the collection of umbilical cord blood and its application in analyzing the transplacental transfer of maternal COVID-19 antibodies after vaccination. We suggest that the informed consent offered to the future parents prior to the procedure should include the history of COVID-19 during pregnancy, the vaccination status of the mother and the gestational age at the time when this event occurred.

Correlation between Osteoprotegerin Levels and Antiphospholipid Syndrome Parameters.

OBJECTIVE: To identify the osteoprotegerin (OPG) correlates with antiphospholipid syndrome (APS) parameters. METHODS: Our cohort included 40 patients with primary APS disease associated with systemic lupus erythematosus (SLE) (mean age, 43.7 years; 87% female). Data on cardiovascular risk factors and specific clinical events in APS were collected. Then we tested OPG and 10 criteria and noncriteria antiphospholipid antibodies (aPLs) on preserved specimens in all cases. RESULTS: A total of 26 patients (65%) had high serum OPG levels. Patients with high OPG were mostly overweight. In patients with SLE, the OPG levels were associated with anti-double-stranded DNA (anti-dsDNA) and anti-Sm titers. However, we did not find significant correlations of the OPG with any of the 10 aPLs tested. Also, we found no relationship regarding venous APS events. CONCLUSION: In APS, high OPG levels are not linked to serum aPL expression.

Non-polio enteroviruses associated with acute flaccid paralysis (AFP) and facial paralysis (FP) cases in Romania, 2001-2008.

Acute flaccid paralysis is a complex clinical syndrome, with a wide variety of possible etiologies and with clinical manifestations that can vary according to age or geographical region. Enteroviruses (polioviruses and non-polio enteroviruses) are among the viral agents that can cause AFP. AFP surveillance is important for public health through its use in monitoring poliomyelitis, in the context of the Global Initiative to eradicate this disease. The current paper aims to assess the non-polio enteroviruses (NPEV) association with AFP and FP cases registered in Romania in the period 2001-2008 and to identify prevalent serotypes. Within the framework of Surveillance of AFP Cases Program, were collected samples from 579 children with AFP or FP (3.069 samples). The samples were processed and inoculated onto two types of cell culture (RD and L20B), according to WHO protocol. The identification of isolated viruses has been done by the reaction of seroneutralization with pools of specific antiserum and then with monospecific antiserum for confirmation. NPEV were isolated from 58 cases (123 positive samples). During the analyzed period, 23 NPEV serotypes have circulated (15 Echo serotypes and 8 coxsackie serotypes). The most frequently identified were the Echoviruses 13 and 11 and the coxsackie A viruses. 88% of positive cases have occurred in children between 1 and 5 years. As seasonal distribution, the peak of NPEV circulation was in the months August-September (36.2%). The paper provides information about NPEV circulation in Romania in the past 8 years, about its association with the AFP and FP and it indicates the need for monitoring NPEV circulation even after the eradication of poliomyelitis.

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.

The maintaining of the active laboratory-based surveillance of the acute flaccid paralysis (AFP) cases in Romania in the framework of the strategic plan of the global polio eradication initiative.

Until 2008 poliomyelitis was controlled in Romania by predominantly using Oral Poliovirus Vaccine Sabin (OPV); the alternative vaccination schedule (IPV formalin Inactivated Poliovirus Vaccine/OPV) will be implemented starting September 2008. The vaccination coverage with 4 doses of TOPV (trivalent oral polio vaccine) in the first 14 months of life has been > 90% since 1980. In Romania, the risk of the Vaccine-Associated Paralytic Poliomyelitis cases (VAPP) decreased from less than 2 VAPP cases/year in the 1995-2006 interval to 0 VAPP cases in 2007. The serological study was performed in 2006-2007 only in cases with pair serum samples from 28 acute flaccid paralysis (AFP) cases (age = 3 months - 14 years) and from 45 facial paralysis (FP) cases (age -6 months - 4 years 9 months). A high level of vaccinal coverage was shown for all poliovirus serotypes: >95% in AFP serum samples investigated; and for FP serum samples investigated the levels of antibodies against poliovirus (PV) serotypes were 98% for PV type 1; 87% for PV type 2: and 89% for PV type 3. If the European region is polio free since 2002, the risk of wild PV importation from endemic region remains present. The laboratory capacity for the fast detection and molecular investigations of the emergence of the new epidemic strains and a high level of population immunity must be maintained. A national seroprevalence study concerning all three PV serotypes must be performed.

The integration of the molecular methods in the diagnosis algorithm for the poliovirus detection in the sewage water: comparing concentration and detection methods. A Pilot Study.

INTRODUCTION: Two cases of circulating vaccine-derived poliovirus type 1(cVDPV1), from southwestern Ukraine, bordering Romania, were confirmed in 2015 and the environmental enterovirus surveillance was enhanced in our country. The molecular detection of human enteroviruses as a screening test followed by isolation on cell culture lines or sequencing could be proposed as a new diagnosis algorithm. MATERIAL AND METHODS: The sensitivity of two molecular methods for the detection of enterovirus strains in 10 mL of sewage water (15 samples) was studied with Film Array ME panel BioFire (Biomerieux, France) and Xpert EV assay (Cepheid, USA). These are standardized methods for the detection of microorganisms in the cerebrospinal fluid (CSF). RESULTS: Of the 15 samples, six enterovirus strains were detected using Film Array ME, four enterovirus strains were detected using Xpert EV assay, while only two nonpolio enterovirus strains were isolated on RD cell line, using the standard WHO algorithm. However, only one of the strains detected by the standard WHO algorithm was detected by one of the molecular methods. CONCLUSIONS: The molecular methods for enterovirus detection are more sensitive than the virus isolation on cell culture lines, but in one case the virus isolated on RD cell line was not detected by the molecular methods. The results could be influenced by the small number of the samples investigated, by the volume and the concentration method used for samples tested, and by the limits of detection (LoD) of the enterovirus species in the samples, depending on the method used.