Dragoş Florea

Nasopharyngeal carriage of Streptococcus pneumoniae in Romanian children before the introduction of the pneumococcal conjugated vaccination into the national immunization programme: a national, multi-centre, cross-sectional observational study.

OBJECTIVES We analysed the distribution of vaccine and non-vaccine Streptococcus pneumoniae serotypes and the antimicrobial susceptibility of pneumococcal strains isolated from healthy Romanian children. METHODS A multi-centre cross-sectional study was performed in four counties to evaluate carried strains of S. pneumoniae isolated from 2000 children aged 0-5 years. RESULTS S. pneumoniae carriage was detected in 25.25% of the tested children. Carriage increased from 16.7% among infants to 29.4% in 3-5-year-old children (p<0.0001). The proportions of the serotypes included in pneumococcal conjugate vaccines PCV7, PCV10, and PCV13 among our isolates were 39.9%, 40.1%, and 58.7%, respectively. Erythromycin resistance was 72.5%, and it was significantly lower in non-vaccine serotypes compared with PCV13 serotypes: 57.3% versus 83.6% (p<10(-7)). Penicillin minimum inhibitory concentrations (MICs) >0.064mg/l were recorded in 71.6%, but the penicillin MIC was >2mg/l for only 8.4% of tested isolates. CONCLUSIONS In Romanian children, the majority of carried S. pneumoniae isolates are vaccine serotypes. The isolates with MICs defining macrolide resistance were very frequent, as well as the isolates with MICs defining penicillin resistance in the case of meningitis or penicillin dose-dependent susceptibility for other infections, mainly for the strains belonging to PCV13 serotypes. The implementation of PCV13 within the Romanian national immunization programme could reduce the circulation of these strains with higher macrolide and/or penicillin MICs.

Liver abscess associated with severe myopathy caused by Klebsiella pneumoniae serotype K1 in Romania

Liver abscess was diagnosed in a man presenting with fever, chills and severe myopathy. The K. pneumoniae isolated from blood cultures belonged to the K1 serotype. The patient responded favourably to percutaneous drainage of the abscess and antibiotics. This is the first documented report of Klebsiella pneumoniae liver abscess syndrome (KLAS) described in Romania and may indicate the emergence of this syndrome in Eastern Europe.

Clinical and microbiological characterization of Clostridium difficile infection in Romania (2013-2014); a hospital-based study

Methods We collected data for all 398 confirmed or probable cases of CDI admitted during 15 November 2013-28 February 2014 in 11 hospitals: 5 from Bucharest, and 7 from Cluj, Iasi, Timisoara, Târgu Mures and Brasov. PCR ribotyping was performed at Cantacuzino Institute and E-test (for moxifloxacin and metronidazole) and binary toxin gene identification (PCR) were performed at Matei Bals Institute. The hospitals sent a maximum 20 feces samples for each test.

Clinical utility of the GeneXpert assay for the diagnosis of Clostridium difficile infections

Background Since 2011 a rapid increase of cases with Clostridium difficile infection (CDI) was observed in the National Institute for Infectious Diseases (NIID), the largest tertiary level infectious diseases hospital in Romania. The need of a fast and accurate diagnosis of CDI and the low sensitivity of the available rapid immunoassays supported the introduction of a molecular assay as a part of the CDI diagnostic strategy. The present study aimed to evaluate the clinical utility of the GeneXpert Clostridium difficile (Cepheid, Sunnyvale, CA) assay in the diagnosis of CDI cases in NIID.

Broad-range PCR coupled with mass-spectrometry for the detection of Mycobacterium tuberculosis drug resistance

BACKGROUND The need to limit the spread of drug-resistant Mycobacterium tuberculosis requires rapid detection of resistant strains. The present study aimed to evaluate a commercial assay using broad-range PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) for the rapid detection of isoniazid (INH) and rifampin (RIF) resistance in M. tuberculosis strains isolated from Romanian patients with pulmonary tuberculosis. METHODS PCR/ESI-MS was used to detect genotypic resistance to RIF and INH in a panel of 63 M. tuberculosis isolates phenotypically characterized using the absolute concentration method on Löwenstein-Jensen medium. RESULTS Thirty-eight (60%) strains were susceptible to both drugs, 22 (35%) were RIF and INH resistant, one was INH mono-resistant and two were RIF mono-resistant. The sensitivity for INH and RIF resistance mutations detection were 100% and 92% respectively, with a specificity of more than 95% for each drug. CONCLUSION PCR/ESI-MS is a good method for the detection of RIF and INH resistance and might represent an alternative to other rapid diagnostic tests for the detection of genetic markers of resistance in M. tuberculosis isolates.

Emergence of Carbapenemase-producing Enterobacteriaceae, a Public Health Threat: a Romanian Infectious Disease Hospital Based Study / Emergenţa Enterobacteriaceaelor producătoare de carbapenemaze, o ameninţare pentru sănătatea publică: un studiu realizat într-un spital romanesc de boli infectioase

Abstract Introduction: Hospital-acquired infections caused by Enterobacteriaceae producing different types of carbapenem- hydrolizing enzymes are now commonly observed and represent a great limitation for antimicrobial therapy. The purpose of the study was to evaluate the emergence of carbapenem-resistant Enterobaceriaceae among the strains isolated from hospitalized patients to the National Institute of Infectious Diseases, Bucharest (NIID) and the identification of different types of carbapenemases, using phenotypic methods. Materials and methods: Between January - June 2014, 587 strains of Klebsiella pneumoniae, Enterobacter species and E.coli were isolated from various clinical specimens. We were included all non-susceptible strains to carbapenems, according to EUCAST 2014 clinical breakpoints, as determined by using microdilution MicroScan Panels (Siemens Healthcare Diagnostics). The modified Hodge test (MHT) was performed as phenotypic confirmatory test for carbapenemase production according to CLSI guidelines and the combination disk test (KPC, MBL , OXA-48 Confirm kit, Rosco Diagnostica) according to EUCAST guidelines. Results: A total of 45 non-repeat Enterobaceriaceae (32 strains Klebsiella pneumoniae, 5 strains E.coli, 8 strains Enterobacter spp) were identified as non-susceptibile to one or more carbapenems (93,33% ertapenem, 53,33% meropenem, 48,88% imipenem). Most strains were isolated from urine (75,55%). MHT was positive in 55,6% (25/45) of carbapenem-resistant strains; in 24 cases the carbapenem-hydrolizing enzyme was identified as: OXA-48-like (n=16), KPC (n=4), MBL (n=1), KPC + MBL (n=2) and MBL + OXA-48-like (n=1). All carbapenemase- positive strains were 100% resistant to 3rd and 4th generation cephalosporins, showing less resistance to tigecycline (12,5% resistant and 25% intermediate), colistin (37,5%) and fosfomycin (41,6%). Conclusion: During 6 months period, there were isolated 7,66% (45/587) carbapenem-resistant Enterobacteriaceae (K. pneumoniae 21,47%, E. coli 1,23%). Twenty four strains were carbapenemase-producers. The most frequent carbapenemase isolated in our study was OXA-48-like. Rezumat Introducere: Infecţiile produse de Enterobacteriaceae producătoare de carbapenemaze sunt tot mai frecvent întâlnite în practica medicală şi limitează serios antibioticoterapia. Am evaluat emergenţa enterobacteriilor rezistente la carbapeneme din tulpinile izolate de la pacienţii internaţi în Institutul Naţional de Boli Infecţioase (INBI) din Bucureşti şi identificarea diferitelor tipuri de carbapenemaze, utilizând metode fenotipice. Materiale si metode: În perioada ianuarie-iunie 2014 au fost izolate 587 tulpini de Klebsiella pneumoniae, Enterobacter species şi E.coli din diferite prelevate clinice. În studiu au fost incluse toate tulpinile rezistente la unul sau mai multe carbapeneme conform indicaţiilor din EUCAST 2014. Testul Hodge modificat (MHT) a fost efectuat pentru confirmarea fenotipică a producerii de carbapenemază conform ghidului CLSI, iar combinaţia de discuri Rosco (KPC, MBL, OXA-48 Confirm kit, Rosco Diagnostica) s-a utilizat conform ghidului EUCAST în acelaşi scop. Rezultate: Un total de 45 tulpini non-duplicate de Enterobacteriaceae (32 tulpini Klebsiella pneumoniae, 5 tulpini E.coli, 8 tulpini Enterobacter spp.) au fost identificate ca rezistente sau intermediare la unul sau mai multe carbapeneme (93,33% ertapenem, 53,33% meropenem, 48,88% imipenem). Majoritatea tulpinilor au fost izolate din urină (75,55%). MHT a fost pozitiv în 55,6% (25/45) dintre tulpinile rezistente la carbapeneme ; la 24 dintre ele a fost identificată enzima care hidrolizează carbapenemele : tip OXA-48 (n=16), KPC (n=4), MBL (n=1), KPC + MBL (n=2) şi MBL + OXA-48-like (n=1). Toate tulpinile producătoare de carbapenemază au fost 100% rezistente la cefalosporinele de generaţia a 3-a şi a 4-a, cea mai mică rezistenţă fiind la tigeciclină (12,5% rezistent, 25% intermediar), colistin (37,5%), fosfomicină (41,6%). Concluzii: Într-o perioadă de 6 luni s-au izolat 7,66% (45/587) Enterobacteriaceae rezistente la carbapeneme (K. pneumoniae 21,47%, E. coli 1,23%). Dintre acestea aproximativ jumătate au fost producătoare de carbapenemază. Cel mai frecvent tip de carbapenemază detectat a fost OXA-48.

PCR coupled with mass-spectrometry for detection of Clostridium difficile virulence markers during the emergence of ribotype 027 in Bucharest area

Abstract In recent years Clostridium difficile infection (CDI) has represented a serious public health issue, mainly due to the global spread of the hypervirulent strain NAP1/027/BI. The purpose of the present study was to evaluate the utility of a PCR coupled with electrospray ionization mass spectrometry (ESI-MS) commercial assay for the detection of C. difficile virulence markers. Non-duplicative C. difficile isolates from patients with CDI diagnosed in a tertiary level hospital from Bucharest were tested for toxin A, toxin B, binary toxin genes and deletion in tcdC gene using PCR/capillary gel electrophoresis and PCR/ESI-MS. The study analysed 45 non-duplicative isolates, 33 strains (73.3%) belonging to ribotype 027. The concordance between PCR/capillary gel electrophoresis and PCR/ESI-MS was 100% for toxin A gene, 97.8% for toxin B gene, 91.1% for binary toxin subunit A gene and 95.6% for binary toxin subunit B gene. The general concordance for the complete panel of markers was 88.9% but was 100% for ribotype 027 isolates. PCR/ESI-MS might be a valid method for the detection of C. difficile virulence markers, including binary toxin.

The Recent Emergence of Clostridium Difficile Infection in Romanian Hospitals is Associated With a High Prevalence of Polymerase Chain Reaction Ribotype 027

Aims: To investigate the epidemiology of Clostridium difficile infection in Romanian hospitals. Methods: A survey was conducted at nine hospitals throughout Romania between November 2013 and February 2014. Results: The survey identified 393 patients with Clostridium difficile infection. The median age was 67 years (range: 2-94 years); 56% of patients were aged >65 years. The mean prevalence of Clostridium difficile infection was 5.2 cases per 10.000 patient-days. The highest prevalences were 24.9 and 20 per 10.000 patient-days in hospitals specializing in gastroenterology and infectious diseases, respectively. Clostridium difficile infections were health care-associated in 70.5% patients and community-acquired in 10.2%. The origin was not determined in 19.3%. Clostridium difficile infection was severe in 12.3% of patients, and the in-hospital all-cause mortality was 8.8%. Polymerase chain reaction ribotype 027 had the highest prevalence in all participating hospitals and represented 82.6% of the total ribotyped isolates. The minimum inhibitory concentration of moxifloxacin was >4 μg/mL for 59 of 80 tested isolates (73.8%). Of 59 isolates, 54 were highly resistant to moxifloxacin (minimum inhibitory concentration ≥32 μg/mL), and the majority were polymerase chain reaction ribotype 027 (p<0.0001). Conclusion: The ribotype 027 was the predominant cause of Clostridium difficile infections in Romania. In some specialized hospitals, the prevalence of Clostridium difficile infection was higher than the European mean prevalence, and this demonstrates the need for strict adherence to infection control programs.

Antiviral resistance in HCV strains isolated from Romanian patients with limited treatment options for chronic HCV infection

Results We assessed data from 12 patients (gender ratio 1:1), of which 10 had HCV monoinfection and 2 had HCV+HIV coinfection. The mean age was 43.8 ± 16.5 years (range 19-71 years). Eleven patients were infected with HCV genotype 1b, and one patient had been coinfected with genotype 2a/2c but had spontaneously cleared 2a infection and was now monoinfected with HCV 2c. Only one patient had IL28-B genotype CC, 4 patients CT and 5 patients TT (in two cases data on IL28-B were not available). Seven of the patients had received prior anti-HCV therapy: 2 with peg-interferon+ribavirin, 2 with faldaprevir-based regimens and 3 with telaprevir-based regimens. Of them, 3 had been non-responders and 4 had been relapsers. The mean plasma HCV-RNA was 6.1±0.7 log10 IU/ mL. Patients were distributed over the whole range of fibrosis values on FibroMax, with a slight predominance of advanced fibrosis: F3 (2 patients) and F4 (3 patients). Resistance to boceprevir or simeprevir was identified in 3/12 cases (fold-change range: 4-24) and 2/12 cases (fold-change range: 32-38), respectively, although none of the patients had received prior therapy with these antivirals. Resistance to telaprevir was identified in 3/12 cases (surprisingly, none of the cases with telaprevir therapy). Possible resistance was identified in another 6 cases (including cases treated with telaprevir and faldaprevir). The overall fold-change range was 1.8-22.4. Resistance to faldaprevir was identified in 3/12 cases (surprisingly, none of the cases with faldaprevir therapy), with a fold-change range of 1.2-360.0.

Low level viraemia and the risk of virological failure in HIV infected patients

Objective: to estimate the rate and the significance of low level viraemia in HIV positive patients registered in the National Institute for Infectious Diseases “Prof. Dr. Matei Bals”, Bucharest, Romania. We retrospectively analysed the rate of HIV viral loads (VL) 200 and >1000 copies/mL) over a 12 months period. A low level VL was detected in 16.2% of the 3,916 evaluated patients, the rate of HIV RNA of 51-200 copies/mL being two times higher than the rate of a VL <50 copies/mL. A number of 84 patients had a VL<200 copies/mL after being HIV RNA undetectable. In these patients the risk of virological failure over a 12 months period was not correlated with the HIV VL group or the used antiretroviral treatment (protease inhibitors PI vs. non-PI containing regimen). A low level viraemia is a rather common event in the studied HIV patients. In previously undetectable patients a HIV RNA <200 copies/mL is not associated with an increased risk of virological failure in the next year.