Comparative study between virus neutralisation testing and other serological methods detecting anti-SARS-CoV-2 antibodies in Europe, 2021.

One consequence of the ongoing coronavirus disease pandemic was the rapid development of both in-house and commercial serological assays detecting anti-SARS-CoV-2 antibodies, in an effort to reliably detect acute and past SARS-CoV-2 infections. It is crucial to evaluate the quality of these serological tests and consequently the sero-epidemiological studies that are performed with the respective tests. Here, we describe the set-up and results of a comparative study, in which a laboratory contracted by the European Centre for Disease Prevention and Control offered a centralised service to EU/EEA Member and pre-accession Member States to test representative serum specimens with known serological results, with the gold standard technique (virus neutralisation tests) to determine the presence of neutralising antibodies. Laboratories from 12 European countries shared 719 serum specimens with the contractor laboratory. We found that in-house serological tests detecting neutralising antibodies showed the highest percent agreement, both positive and negative, with the virus neutralisation test results. Despite extensive differences in virus neutralisation protocols neutralisation titres showed a strong correlation. From the commercial assays, the best positive percent agreement was found for SARS-CoV-2 IgG (sCOVG) (Siemens - Atellica IM Analyzer). Despite lower positive percent agreement of LIAISON SARS-CoV-2 TrimericS IgG kit (Diasorin Inc.), the obtained results showed relatively good correlation with neutralisation titres. The set-up of this study allowed for high comparability between laboratories and enabled laboratories that do not have the capacity or capability to perform VNTs themselves. Given the variety of in-house protocols detecting SARS-CoV-2 specific neutralising antibodies, including the virus strain, it could be of interest to select reference isolates for SARS-CoV-2 diagnostic to be made available for interested EU Member States and pre-accession countries.

Assessment of the Humoral Immune Response Following COVID-19 Vaccination in Healthcare Workers: A One Year Longitudinal Study.

The continuous variability of SARS-CoV-2 and the rapid waning of specific antibodies threatens the efficacy of COVID-19 vaccines. We aimed to evaluate antibody kinetics one year after SARS-CoV-2 vaccination with an mRNA vaccine in healthcare workers (HCW), with or without a booster. A marked decline in anti-Spike(S)/Receptor Binding Domain (RBD) antibody levels was registered during the first eight months post-vaccination, followed by a transitory increase after the booster. At three months post-booster an increased antibody level was maintained only in HCW vaccinated after a prior infection, who also developed a higher and long-lasting level of anti-S IgA antibodies. Still, IgG anti-nucleocapsid (NCP) fades five months post-SARS-CoV-2 infection. Despite the decline in antibodies one-year post-vaccination, 68.2% of HCW preserved the neutralization capacity against the ancestral variant, with a decrease of only 17.08% in the neutralizing capacity against the Omicron variant. Nevertheless, breakthrough infections were present in 6.65% of all participants, without any correlation with the previous level of anti-S/RBD IgG. Protection against the ancestral and Omicron variants is maintained at least three months after a booster in HCW, possibly reflecting a continuous antigenic stimulation in the professional setting.

Detection of anti-SARS-CoV-2-Spike/RBD antibodies in vaccinated elderly from residential care facilities in Romania, April 2021.

Introduction: SARS-CoV-2 infection rates and related mortality in elderly from residential care facilities are high. The aim of this study was to explore the immune status after COVID-19 vaccination in people 65 years and older. Methods: The study involved volunteer participants living in residential care facilities. The level of anti-Spike/RBD antibodies was measured at 2-12 weeks after complete vaccination, using chemiluminescent microparticle immunoassay (SARS-CoV-2 IgG II Quant Abbott). Results: We have analyzed 635 serum samples collected from volunteers living in 21 Residential Care Facilities. With one exception, in which the vaccination was done with the Moderna vaccine, all volunteers received the Pfizer-Comirnaty vaccine. Individuals enrolled in the study had ages between 65-110 years (median 79 years). Of the people tested, 54.8% reported at least one comorbidity and 59.2% reported having had COVID-19 before vaccination. The presence of anti-S/RBD antibodies at a protective level was detected in 98.7% of those tested (n = 627 persons) with a wide variation of antibody levels, from 7.1 to 5,680 BAU/ml (median 1287 BAU/ml). Antibody levels appeared to be significantly correlated to previous infection (r = 0.302, p = 0.000). Conclusions: The study revealed the presence of anti-SARS CoV-2 antibodies in a significant percentage of those tested (98.7%). Of these, more than half had high antibody levels. Pre-vaccination COVID-19 was the only factor found to be associated with higher anti-S/RBD levels. The significant response in elderly people, even in those with comorbidities, supports the vaccination measure for this category, irrespective of associated disabilities or previous infection.

Indwelling Device-Associated Biofilms in Critically Ill Cancer Patients-Study Protocol.

Health care-associated infections are a leading cause of inpatient complications. Rapid pathogen detection/identification is a major challenge in sepsis management that highly influences the successful outcome. The current standard of microorganism identification relies on bacterial growth in culture, which has several limitations. Gene sequencing research has developed culture-independent techniques for microorganism identification, with the aim to improve etiological diagnosis and, therefore, to change sepsis outcome. A prospective, observational, non-interventional, single-center study was designed that assesses biofilm-associated pathogens in a specific subpopulation of septic critically ill cancer patients. Indwelling device samples will be collected in septic patients at the moment of the removal of the arterial catheter, central venous catheter, endotracheal tube and urinary catheter. Concomitantly, clinical data regarding 4 sites (nasal, pharyngeal, rectal and skin) of pathogen colonization at the time of hospital/intensive care admission will be collected. The present study aims to offer new insights into biofilm-associated infections and to evaluate the infection caused by catheter-specific and patient-specific biofilm-associated pathogens in association with the extent of colonization. The analysis relies on the two following detection/identification techniques: standard microbiological method and next generation sequencing (NGS). Retrospectively, the study will estimate the clinical value of the NGS-based detection and its virtual potential in changing patient management and outcome, notably in the subjects with missing sepsis source or lack of response to anti-infective treatment.

Carbapenemase-producing Enterobacteriaceae: a 2-year surveillance in a hospital in Iasi, Romania.

AIM: Limited information is currently available about the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) in Romania. MATERIALS & METHODS: Routine tests of 1,993 clinical isolates at a hospital in Iasi yielded 46 isolates that were resistant to carbapenems. All 46 isolates were phenotypically and genotypically analyzed using VITEK-2 and DNA microarray-based assays. RESULTS: Isolates were assigned to Klebsiella pneumoniae and Enterobacter cloacae. For 39 isolates, carbapenem resistance was confirmed and 37 harbored at least one carbapenem resistance gene. Two isolates were probably resistant due to AmpC ß-lactamases in combination with a porin loss. The overall concordance between detected phenotype and genotype was 95%. CONCLUSION: Our data show that carbapenemase-producing isolates with different underlying resistance mechanisms are still rare in Iasi, but the global rise of CPE warrants intensified surveillance.

Detection of Aminoglycoside and Macrolide Resistance Mechanisms in Methicillin-Resistant Staphylococcus Aureus.

Methicillin-resistant Staphylococcus aureus can become resistant to many different classes of antibiotics. Objective: To characterize aminoglycoside and macrolide resistance mechanisms in MRSA strains in relation to antibiotic susceptibility patterns. Materials and methods: We tested 86 MRSA strains using multiplex PCR for detection of genes mecA, aac(6')-Ie/aph(2″), ant(4')-Ia, aph(3')-IIIa, ermA, ermC and msrA. Results: There was a prevalence of msrA (32.5%), ermC (30.2%) and aph(3')-IIIa (61.6%) genes, which are less frequently reported in MRSA. Most msrA genes was detected in PVL positive strains (92.8%) and was associated only with non-MDR strains, while ermC genes were associated with MDR strains. PVL producing strains were characterized by the presence of aph(3')-IIIa (93.1%) and msrA genes (93.1%), being phenotypically susceptible to clindamycin. Conclusions: Detection of aminoglycoside and macrolide resistance genes allowed us to establish the concordance between genotypic and phenotypic methods and to correlate the presence of certain resistance genes with the type of circulating strain and the production of virulence factors.

Characterization of MRSA strains by phenotypic and OCR-based methods.

With the emergence and spread of new methicilin resistant Staphylococcus aureus (MRSA) strains, control of dissemination, both in hospitals and in the community, requires the molecular characterization of the circulating strains in order to establish their dynamics and identify the sources of infection. During this study we analyzed the MRSA isolates by means of PCR-based methods in order to improve epidemiological surveillance and early application of prevention measures. The presence of mecA, nuc, lukF-PV and lukS-PV genes, as well as SCCmec types was assessed in relation to clinical characteristics and multidrug resistance (MDR) for 86 MRSA isolates and showed that 51% of MDR strains were carriers of mobile genetic elements SCCmec IV and the majority of non-MDR SCCmec type IV strains were PVL-positive (81.8%). Comparison of diagnostic methods showed that PBP2 detection represents an extremely useful alternative to PCR for the rapid screening of MRSA isolates, in laboratories that lack facilities necessary for molecular diagnosis, such as PFGE (Pulse Field Gel Electrophoresis), spa-typing and/or MLST (Multilocus Sequence Typing).

Molecular typing of MRSA and of clinical Staphylococcus aureus isolates from Iasi, Romania.

Romania is one of the countries with the highest prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the world. To obtain data on affiliation of MRSA to strains and clonal complexes and on the population of methicillin susceptible S. aureus (MSSA), clinical isolates from bloodstream infections, skin and soft tissue infections as well as from screening swabs were collected at hospitals in Ia?i, a city in the North-Eastern part of Romania. Isolates were characterised by microarray hybridisation. Nearly half of all isolates (47%), and about one third (34%) of bloodstream isolates were MRSA. The prevalence of the Panton-Valentine leukocidin (PVL) was also high (31% among MRSA, 14% among MSSA). The most common MRSA strain was a PVL-negative CC1-MRSA-IV that might have emerged locally, as a related MSSA was also common. PVL-positive CC8-MRSA-IV ("USA300") and PVL-negative ST239-like MRSA-III were also frequently found while other MRSA strains were only sporadically detected. Among MSSA, PVL-positive CC121 as well as PVL-negative CC1, CC22 and CC45 predominated. Although this study provides only a snapshot of S. aureus/MRSA epidemiology in Romania, it confirms the high burden of MRSA and PVL on Romanian healthcare settings.

Methicillin-resistant Staphylococcus aureus carriage in infants with protein-energy malnutrition.

AIM: The aim of the present study was to investigate the frequency of methicillin-resistant S. aureus (MRSA) colonization in infants with protein-energy malnutrition (PEM) and to characterize the antibiotic susceptibility patterns in isolated strains. MATERIAL AND METHODS: The study included 123 infants with PEM, admitted to the Paediatric Rehabilitation Department of,,St. Mary" Emergency Clinical Children's Hospital Iasi, during October 1st 2010 and August 30th 2011. Nasal, pharyngeal and conjunctival swabs were collected for detection of colonization upon admission and at discharge. RESULTS: The study revealed a high rate of MRSA carriage among infants with PEM (62.60%), most of the isolated strains being multidrug-resistant (86.13%), with additional resistance to aminoglycosides, macrolides and inducible resistance to clindamycin. 88% of colonized infants were nasal carriers. CONCLUSIONS: Identification of MRSA carriers permits the implementation of measures that decrease the risk for subsequent infection.

Emergence of a new group CTX-M enzyme in Romania and risk factors for extended spectrum beta-lactamase producing E. coli infections.

Antibiotic resistance rates in E. coli are rapidly rising, with worrisome aspects especially regarding community--acquired resistance to third- and fourth-generation cephalosporins and fluoroquinolones. The objectives of this prospective cohort study was to determine the resistance profile of E. coli for two categories of patients (< 49 years and > or = 50 years), risk factors for ESBL positivity and to investigate the molecular epidemiology of ESBL type CTX-M enzymes. A total of 885 strains of E. coli were isolated in the Infectious Diseases Hospital laboratory between June 2008 and June 2011 and E. coli resistance due to ESBL production was noted in 17% of cases. We found that previous therapy with cephalosporins, hospitalization and urinary catheter were risk factors for ESBL positivity. We noted significant differences concerning resistance rate between patients under 49 years and aged more than 50 years for ciprofloxacin (19% and 38%, respectively, p = 0,0001), for gentamicin (15% and 23%, p = 0,008), ceftazidime (15% and 24%, p = 0,001) and ESBL positivity (14% and 20%, p = 0.009). This study highlights the predominance of CTX-M producing strains (92.5% of ESBLs-positive E. coli harboured bla CTX-M genes); CTX-M-15 producing isolates were the most common, accounting for 96% of isolates. Only 4% were belonging to CTX-M group-9, an emerging ESBL group which is newly described in Romania.

Triplex real-time pcr for detection of methicillin-resistant staphylococcus aureus and Panton-Valentine leukocidin in North-East Romania.

UNLABELLED: The aim of the present study was to investigate S. aureus isolates for the presence of methicillin-resistance and Panton-Valentine leukocidin (PVL) genes and to further characterize positive strains by means of antibiotic resistance patterns. MATERIAL AND METHODS: We used a triplex Real-Time PCR method for simultaneous detection of nuc, mecA and pvl genes in clinical isolates from 188 patients admitted to "Sf. Parascheva" Infectious Diseases Hospital lasi, during a 3 year period (2008-2010). RESULTS: The study revealed a relatively high rate of PVL-producing strains (23.93%), mainly community-associated (CA-MRSA) (51.11%). Most pvl-positive CA-MRSA isolates were resistant to erythromycin (91.3%), but none was resistant to clindamycin, fluoroquinolones, rifampicin, chloramphenicol or fusidic acid. CONCLUSIONS: Antibiotic susceptibility testing showed a high rate of multidug-resistance among strains classified as CA-MRSA (54.83%), but not among PVL-producers (4.44%). Although resistance to fusidic acid was previously proposed as a marker for PVL-producing CA-MRSA, our data suggest that we cannot rely on resistance to fusidic'acid to screen for PVL-producing CA-MRSA in our setting.

[Use of real time PCR for testing Staphylococcus aureus isolates from the Iasi Infectious Diseases Hospital]. / Utilizarea real time PCR pentru testarea izolatelor clinice de Staphylococcus aureus din Spitalul Clinic de Boli Infectioase Iasi.

UNLABELLED: S. aureus is capable of producing a wide spectrum of diseases and can quickly develop resistance to antibiotics. These features require a careful monitoring of these organisms, by detection of resistance genes and virulence factors, such as Panton-Valentine leukocidin (PVL). AIM: To determine the presence of mecA and pvl genes in S. aureus isolates by a Real Time-PCR technique (RT-PCR) in order to shorten the detection time. MATERIALS AND METHODS: We tested 119 strains isolated from pus, using phenotypic methods for methicillin resistance characterization, according to CLSI 2008-2010 guidelines. Detection of mecA and pvl genes was done with hydrolysis probes. RESULTS: The prevalence rate of methicilin resistant S. aureus (MRSA) was 40,33%, and pvl was detected in 52,08% of those strains. The results of the conventional methods for methicillin resistance detection were validated by those obtained by RT-PCR CONCLUSIONS: RT-PCR is useful in epidemiological surveillance of MRSA and PVL-producing strains and validation of test results for phenotypic resistance to oxacillin.

[Cross-sectional study to evaluate risk factors in infant malnutrition]. / Studiu transversal de evaluare a unor factori de risc implicati în aparitia malnutritiei la sugar.

UNLABELLED: Malnutrition is a major health problem in our country by maintaining a high number of infants with poor nutritional status. Various studies have highlighted the role of infant's malnutrition in the development of adult diseases. METHODS: We made a cross-sectional study during six months (October 2010 - March 2011) on a group of 63 infants admitted in Pediatric Recovery Department-Children's Hospital, Iasi; we evaluated the presence of risk factors for malnutrition. The data were processed using SPSS 16 and Epilnfo 3.5.2. (December 2010). RESULTS: The infants were predominantly female (52,4%); the most affected age group was 5-24 weeks (84,11%). Most of them came from rural areas (79,4%), from families with low socioeconomic income (95,2%), mothers with a low educational status (63,4%), housewives (88,9%). We noted the presence of previous diseases in 71,4% infants. Only 12 infants received breast milk for a short time (three weeks), the other 51 infants have been bottle-fed since birth. Complementary food was incorrect in 68,42% cases. With a proper diet the mean weight gain was 895,68 g and the Z score values (weight for age, height for age, weight for height) have improved during an average of 34,15 days; positive correlation between these factors is strong. CONCLUSIONS: Low socio-economic income, rural areas, low maternal educational level, diet errors, small infants with multiple previous diseases are the main risk factors in malnutrition's occurrence. We consider particularly important to solve social problems too, not only the medical, because when the infant returns in the same disadvantaged family there is an increased risk for malnutrition to recur.

Optimization of triplex real time PCR for detecting Staphylococcus aureus mecA, pvl and nuc genes.

Multiplex polymerase chain reaction (PCR) allows simultaneous detection of two or more genes, using the same reaction conditions, and so it is possible the rapid detection of methicillin resistant Staphylococcus aureus strains (MRSA) in clinical specimens. This study aimed to implement, for the first time in our laboratory, a triplex real time PCR (RT-PCR) technique for detection of genes encoding resistance to oxacillin and synthesis of Panton Valentine leukocidin (pvl), a pathogenicity factor characteristic for community acquired strains (CA-MRSA). The application of this method will permit the epidemiological surveillance of circulating strains and early application of prevention measures.

[Carbapenem resistance in Klebsiella pneumoniae clinical isolates from urinary tract infections]. / Emergenta rezistentei la carbapeneme la tulpini de Klebsiella pneumoniae izolate din infectii ale tractusului urinar.

UNLABELLED: Resistance to carbapenems by KPC (Klebsiella pneumoniae carbapenemase) production in Klebsiella pneumoniae clinical isolates was first described ten years ago in the U.S.A. and recently reported in other countries. This enzyme inactivates all beta-lactam antibiotics and is associated with fluoroquinolone and aminoglycoside resistance. MATERIAL AND METHODS: We investigated the carbapenem resistance in 498 Escherichia coli and Klebsiella pneumoniae strains isolated from urinary tract infections during 2009 in the lasi "Dr. C. I. Parhon" Clinical Hospital. Antimicrobial susceptibility testing was performed according to CLSI guidelines. To detect ESBL (extended spectrum beta-lactamase) and KPC production we used phenotypic tests and molecular biology methods (PCR). RESULTS: From all tested strains, only two K. pneumoniae strains showed modified susceptibility to carbapenems. The modified Hodge test was positive for the strain resistant to ertapenem, meropenem, imipenem (KA) and negative for the strain resistant only to ertapenem (K(B)). Both KA and K(B) isolates were negative for blaKPC and blaTEM genes, but harbored blaSHV and bla(CTX-M) genes, respectively. CONCLUSIONS: We report the first carbapenem resistant K. pneumoniae strains from Northeast Romania. The resistance is not mediated by KPC-carbapenemase; the possibility of dual mechanisms through cefalosporinases production and porins loss is suggested.

[MecA and pvl genes detection in Staphylococcus aureus strains isolated from lower respiratory tract infections]. / Detectarea genelor mecA si pvl la tulpini de Staphylococcus aureus izolate din infectii ale tractului respirator inferior.

UNLABELLED: Although community-acquired Staphylococcus aureus pneumonia with highly virulent Panton-Valentine leukocidin (PVL)-positive strains, a severe disease with significant lethality, is rare, especially in adult and adolescent patients, recent reports highlight that these infections are on the rise. On the other hand, methicillin-resistant S. aureus (MRSA) is one of the high-risk and potential multi-drug resistant microorganisms. OBJECTIVES: The aim of this study was to investigate the prevalence of mecA and pvl genes in S. aureus strains isolated from lower respiratory tract infections and to assess the antibiotic resistance profile of these strains. MATERIAL AND METHOD: The antibiotic susceptibility testing was performed by the disk diffusion method according to CLSI recommendations in 32 consecutive non-repeated S. aureus strains isolated from sputum specimens and endotracheal aspirates of hospitalized patients over the period from January 2005 to December 2009. Only 20 strains (2008-2009) were tested for the presence of mecA and pvl genes by real-time PCR and detection with specific fluorescence probes. RESULTS: Of the 32 S. aureus isolates, 68.7% were MRSA. MRSA strains showed higher resistance rates to gentamicin, tetracycline, rifampicin, fluoroquinolones comparing to the methicillin susceptible ones. Only one strain produced pvl; it was isolated from a 7 year old child with lethal sepsis with pulmonary and meningeal secondary localisations. CONCLUSIONS: Glicopeptides and linezolid are therapeutic options indicated in the treatment of staphylococcal pneumonia produced either by MRSA strains or pvl positive S. aureus strains.