Hector Rodriguez-Villalobos

Rapid Detection of Candida albicans in Clinical Blood Samples by Using a TaqMan-Based PCR Assay

ABSTRACT We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.

Detection and characterization of class A extended-spectrum-β-lactamase-producing Pseudomonas aeruginosa isolates in Belgian hospitals

OBJECTIVES To investigate the presence of extended-spectrum beta-lactamases (ESBLs) among Pseudomonas aeruginosa clinical isolates referred to two Belgian reference laboratories. METHODS Antibiograms were analysed for P. aeruginosa isolates referred between 2004 and 2008. Isolates resistant to ceftazidime (MIC > 8 mg/L) and with a positive double-disc synergy test between ceftazidime and clavulanate were serotyped and screened for the presence of ESBL-encoding genes. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought by PCR in ESBL-producing isolates with positive imipenem/EDTA synergy tests. PFGE of SpeI-digested genomic DNA was used to compare isolates and selected strains were characterized by multilocus sequence typing. RESULTS Forty-eight (2.2%) of 2150 P. aeruginosa isolates were confirmed as class A ESBL-producing isolates by molecular testing. bla(BEL) and bla(PER) alleles were detected, respectively, in 39 and 10 P. aeruginosa isolates originating from 16 hospitals (two isolates were simultaneously positive for BEL and PER). Fifteen of the isolates were found to co-produce ESBLs and VIM carbapenemases. These strains were pan-resistant and remained susceptible only to colistin (MICs <or= 2 mg/L). The majority of the ESBL-producing isolates belonged to the same PFGE clone and were identified as ST235; serotype O11. CONCLUSIONS BEL enzymes were produced by 80% of P. aeruginosa isolates with phenotypic evidence of ESBL production. BEL or PER ESBLs co-existed with VIM carbapenemases in 15 isolates and caused outbreaks in four hospitals. Our data further highlight the epidemic potential of the international clone ST235, which may have acquired bla(BEL-1) gene cassettes from a yet unidentified local gene reservoir.

Intensive care unit outbreak of extended-spectrum β-lactamase- producing Klebsiella pneumoniae controlled by cohorting patients and reinforcing infection control measures

OBJECTIVE To describe an outbreak of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the intensive care units (ICUs) of a hospital and the impact of routine and reinforced infection control measures on interrupting nosocomial transmission. DESIGN Outbreak report. SETTING A 31-bed intensive care department (composed of 4 ICUs) in a university hospital in Belgium. INTERVENTION After routine infection control measures (based on biweekly surveillance cultures and contact precautions) failed to interrupt a 2-month outbreak of ESBL-producing K. pneumoniae, reinforced infection control measures were implemented. The frequency of surveillance cultures was increased to daily sampling. Colonized patients were moved to a dedicated 6-bed ICU, where they received cohorted care with the support of additional nurses. Two beds were closed to new admissions in the intensive care department. Meetings between the ICU and infection control teams were held every day. Postdischarge disinfection of rooms was enforced. Broad-spectrum antibiotic use was discouraged. RESULTS Compared with a baseline rate of 0.44 cases per 1,000 patient-days for nosocomial transmission, the incidence peaked at 11.57 cases per 1,000 patient-days (October and November 2005; rate ratio for peak vs baseline, 25.46). The outbreak involved 30 patients, of whom 9 developed an infection. Bacterial genotyping disclosed that the outbreak was polyclonal, with 1 predominant genotype. Reinforced infection control measures lasted for 50 days. After the implementation of these measures, the incidence fell to 0.08 cases per 1,000 patient-days (rate ratio for after the outbreak vs during the outbreak, 0.11). CONCLUSION These data indicate that, in an intensive care department in which routine screening and contact precautions failed to prevent and interrupt an outbreak of ESBL-producing K. pneumoniae, reinforced infection control measures controlled the outbreak without major disruption of medical care.

Trends in production of extended-spectrum beta-lactamases among Enterobacteriaceae of clinical interest: results of a nationwide survey in Belgian hospitals.

OBJECTIVES to assess the frequency and diversity of extended-spectrum β-lactamases (ESBLs) in Enterobacteriaceae isolates in Belgium. METHODS during 2006 and 2008, non-duplicate clinical isolates of Enterobacteriaceae resistant to ceftazidime and/or cefotaxime were collected in 100 Belgian hospitals. ESBL production was confirmed by phenotypic and genotypic tests. MICs of 13 antimicrobial agents were determined by Etest. ESBL-encoding genes were identified by PCR sequencing and the bla(CTX-M) environment was characterized by PCR mapping. Selected isolates were genotyped by PFGE, multilocus sequence typing analysis and phylogenetic grouping by PCR. RESULTS overall, 733 isolates were confirmed as ESBL producers. Carbapenems and temocillin were active against ≥ 95% of all tested isolates. Co-resistance to co-trimoxazole and to ciprofloxacin was found in almost 70% and 80% of the strains, respectively. Overall, Escherichia coli (49%), Enterobacter aerogenes (32%) and Klebsiella pneumoniae (9%) represented the most prevalent species. Isolates harboured predominantly TEM-24 (30.7%), CTX-M-15 (24.2%) and TEM-52 (12.1%). Compared with 2006, the proportion of CTX-M-type enzymes increased significantly in 2008 (54% versus 23%; P < 10(-6)), mostly linked to a rising proportion of CTX-M-15-producing E. coli. TEM-24 decreased (19% in 2008 versus 43% in 2006; P < 10(-6)) during the same period, while the prevalence of TEM-52 remained unchanged (10% in 2008 versus 14% in 2006; not significant). Over 80% of the CTX-M-15-producing E. coli isolates clustered into a single PFGE type and phylogroup B2, corresponding to the sequence type (ST) 131 clone. Intra- and inter-species gene dissemination (CTX-M-15, CTX-M-2 and CTX-M-9) and wide epidemic spread of the CTX-M-15-producing E. coli ST131 clone in several Belgian hospitals were observed. CONCLUSIONS the rapid emergence of multiresistant CTX-M-15-producing E. coli isolates is of major concern and highlights the need for further surveillance in Belgium.

Controlled performance evaluation of the DiversiLab repetitive-sequence-based genotyping system for typing multidrug-resistant health care-associated bacterial pathogens.

ABSTRACT Fast, reliable, and versatile typing tools are essential to differentiate among related bacterial strains for epidemiological investigation and surveillance of health care-associated infection with multidrug-resistant (MDR) pathogens. The DiversiLab (DL) system is a semiautomated repetitive-sequence-based PCR system designed for rapid genotyping. The DL system performance was assessed by comparing its reproducibility, typeability, discriminatory power, and concordance with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and by assessing its epidemiological concordance on well-characterized MDR bacterial strains (n = 165). These included vanA Enterococcus faecium, extended-spectrum β-lactamase (ESBL)-producing strains of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii, and ESBL- or metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa strains. The DL system showed very good performance for E. faecium and K. pneumoniae and good performance for other species, except for a discrimination index of <95% for A. baumannii and E. coli (93.9% and 93.5%, respectively) and incomplete concordance with MLST for P. aeruginosa (78.6%) and E. coli (97.0%). Occasional violations of MLST assignment by DL types were noted for E. coli. Complete epidemiological concordance was observed for all pathogens, as all outbreak-associated strains clustered in identical DL types that were distinct from those of unrelated strains. In conclusion, the DL system showed good to excellent performance, making it a reliable typing tool for investigation of outbreaks caused by study pathogens, even though it was generally less discriminating than PFGE analysis. For E. coli and P. aeruginosa, MLST cannot be reliably inferred from DL type due to phylogenetic group violation or discordance.

Nosocomial infections caused by multidrug-resistant Pseudomonas putida isolates producing VIM-2 and VIM-4 metallo-β-lactamases

1. DANMAP. DANMAP 2005-Use of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Bacteria from Food Animals, Foods, and Humans in Denmark. Danish Institute for Food and Veterinary Research, Copenhagen, Denmark, 2006. 2. Jensen LB, Hasman H, Agerso Y et al. First description of an oxyimino-cephalosporin-resistant, ESBL-carrying Escherichia coli isolated from meat sold in Denmark. J Antimicrob Chemother 2006; 57: 793–4. 3. Aarestrup FM, Hasman H, Agerso Y et al. First description of blaCTX-M-1-carrying Escherichia coli isolates in Danish primary food production. J Antimicrob Chemother 2006; 57: 1258–9. 4. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15. CLSI, Wayne, PA, USA, 2005. 5. Hasman H, Mevius D, Veldman K et al. b-Lactamases among extended-spectrum b-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands. J Antimicrob Chemother 2005; 56: 115–21. 6. Olesen I, Hasman H, Aarestrup FM. Prevalence of b-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark. Microb Drug Resist 2004; 10: 334–40. 7. Jorgensen CJ, Cavaco LM, Hasman H et al. The occurrence of CTX-M-1-producing Escherichia coli in pigs treated with ceftiofur. J Antimicrob Chemother 2007; 59: 1040–2.

Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing KPC-2 carbapenemase in Belgium.

Sir, The Klebsiella pneumoniae carbapenemase (KPC) is a class A b-lactamase that confers resistance to virtually all b-lactams including carbapenems. Initially reported in the USA in a Klebsiella isolate in 1996, KPC-producing K. pneumoniae have been responsible for large nosocomial outbreaks in the USA, Israel and Greece where these isolates are now endemic in hospitals. The rapid dissemination of KPC enzymes among and across different enterobacterial species is likely to be due to the localization of blaKPC genes on transferable broad host range plasmids and their association with a transposon, but is also linked with a disseminated international clone of KPC-producing K. pneumoniae isolates of sequence type (ST) 258. Here we report on the first three cases of KPC-2-producing ST258 K. pneumoniae that were detected in Belgium, subsequent to the transfer of patients from three Greek hospitals. In 2009, a patient with high-grade bladder neoplasm with pelvic metastases was transferred from a hospital in Greece to St-Pierre University Hospital in Brussels. Six weeks later, a patient with severe trauma was transferred from another hospital in the same region of Greece to the intensive care unit (ICU) of the same Brussels hospital. Finally, a few days later, the third polytrauma patient was transferred from the ICU of a hospital in Rhodes to the ICU of Erasme University Hospital in Brussels. Upon admission, a urine culture for the first patient and rectal swabs from the two other patients revealed the presence of multidrug-resistant K. pneumoniae (isolates Hk1, Hk2 and Rh1, respectively) with reduced susceptibility to carbapenems. No secondary local transmission occurred in the two hospitals following the rapid implementation of strict infection control measures. MICs produced using the Vitek2 system (N046 AST cards) and Etest (AB BIODISK, Solna, Sweden) were interpreted according to clinical breakpoints from the European Committee for Antimicrobial Susceptibility Testing (EUCAST). The three multidrug-resistant isolates showed high-level resistance to all penicillins (including temocillin) and to all expanded-spectrum cephalosporins except cefepime to which they all remained borderline susceptible (MICs of 8–16 mg/L) (Table 1). Interestingly, the three isolates appeared to be of intermediate susceptibility to meropenem using the Vitek2 system (MIC of 4–8 mg/L) while they were resistant to meropenem by Etest (MIC of 32 mg/L). All the isolates remained susceptible to gentamicin but were of intermediate

Prevalence and mechanisms of resistance to carbapenems in Enterobacteriaceae isolates from 24 hospitals in Belgium.

OBJECTIVES To determine the point prevalence of carbapenem-non-susceptible Enterobacteriaceae (CNSE) and carbapenemase-producing Enterobacteriaceae (CPE) isolates among hospitalized patients in Belgium. METHODS Twenty-four hospital-based laboratories prospectively collected 200 non-duplicated Enterobacteriaceae isolates from clinical specimens of hospitalized patients over a 2 month period. All isolates were screened locally for decreased susceptibility to carbapenem drugs using a disc diffusion method according to CLSI interpretative criteria. CNSE strains were referred centrally for confirmation of carbapenemase by phenotypic and molecular testing. RESULTS From February to April 2012, 158 of the 4564 screened Enterobacteriaceae isolates were categorized as non-susceptible to carbapenems, resulting in a point prevalence of CNSE of 3.5% (95% CI: 2.9%-4.2%; range per centre: 0.5%-8.5%). Of the 125 referred CNSE isolates, 11 Klebsiella pneumoniae isolates [OXA-48 (n = 7), KPC type (n = 3) and NDM type (n = 1)], 1 OXA-48-positive Escherichia coli isolate and 1 KPC-positive Klebsiella oxytoca isolate were detected in eight hospitals. None of the 72 carbapenem-non-susceptible Enterobacter spp. isolates were confirmed as CPE. The minimal estimated point prevalence of CPE isolates was 0.28% (13/4564; 95% CI: 0.13%-0.44%) overall (range per centre: 0%-1.5%). CONCLUSIONS Despite the overall low prevalence of CNSE found in this study, the detection of CPE isolates in one-third of the participating centres raises concerns and highly suggests the spread and establishment of CPE in Belgian hospitals.

Streptococcus gordonii septic arthritis: two cases and review of literature.

BackgroundDespite advances in antimicrobial and surgical therapy, septic arthritis remains a rheumatologic emergency that can lead to rapid joint destruction and irreversible loss of function. In adults, Staphylococcus aureus is the most common microorganism isolated from native joints. Streptococcus gordonii is a prominent member of the viridans group of oral bacteria and is among the bacteria most frequently identified as being primary agent of subacute bacterial endocarditis. To the best of our knowledge, Streptococcus gordonii has not yet been described as agent of septic arthritis.Case PresentationWe describe here two cases of septic arthritis due to Streptococcus gordonii. It gives us an opportunity to review epidemiology, diagnosis criteria and management of septic arthritis.ConclusionAlthough implication of S. gordonii as aetiologic agent of subacute endocarditis is well known, this organism is a rare cause of septic arthritis. In this case, the exclusion of associated endocarditis is warranted.

Haemophilus parainfluenzae: an Underdiagnosed Pathogen of Biliary Tract Infections?

Abstract Presented here is a case of monobacterial peritonitis complicating cholecystitis and caused by an uncommon agent of gastrointestinal infections, Haemophilus parainfluenzae. The pathogenic role of this organism in digestive infections, particularly in those of the biliary tract, has been reported increasingly though sporadically. Indeed, it has been shown to be a coloniser of the gastrointestinal tract, and a recent hypothesis of an ascending route of infection to the biliary tract has been postulated to partly explain its pathogenicity. More frequent identification of Haemophilus parainfluenzae as a causal agent of biliary tract infection would probably be obtained through the use of specific culture media, since its potential implication has been demonstrated.