Reporting antimicrobial susceptibilities and resistance phenotypes in Staphylococcus spp.: a nationwide proficiency study.

OBJECTIVES: To evaluate the proficiency of microbiology laboratories in Spain in antimicrobial susceptibility testing (AST) of Staphylococcus spp. MATERIALS AND METHODS: Eight Staphylococcus spp. with different resistance mechanisms were selected: six Staphylococcus aureus (CC-01/mecA, CC-02/mecC, CC-03/BORSA, CC-04/MLSBi, CC-06/blaZ and CC-07/linezolid resistant, cfr); one Staphylococcus epidermidis (CC-05/linezolid resistant, 23S rRNA mutation); and one Staphylococcus capitis (CC-08/daptomycin non-susceptible). Fifty-one laboratories were asked to report: (i) AST system used; (ii) antimicrobial MICs; (iii) breakpoints used (CLSI or EUCAST); and (iv) clinical category. Minor, major and very major errors (mEs, MEs and VMEs, respectively) were determined. RESULTS: The greatest MIC discrepancies found were: (i) by AST method: 19.4% (gradient diffusion); (ii) by antimicrobial agent: daptomycin (21.3%) and oxacillin (20.6%); and (iii) by isolate: CC-07/cfr (48.0%). The greatest error rates were: (i) by AST method: gradient diffusion (4.3% and 5.1% VMEs, using EUCAST and CLSI, respectively); (ii) by breakpoint: 3.8% EUCAST and 2.3% CLSI; (iii) by error type: mEs (0.8% EUCAST and 1.0% CLSI), MEs (1.8% EUCAST and 0.7% CLSI) and VMEs (1.2% EUCAST and 0.6% CLSI); (iii) by antimicrobial agent: VMEs (4.7% linezolid and 4.3% oxacillin using EUCAST); MEs (14.3% fosfomycin, 9.1% tobramycin and 5.7% gentamicin using EUCAST); and mEs (22.6% amikacin using EUCAST). CONCLUSIONS: Clinical microbiology laboratories should improve their ability to determine the susceptibility of Staphylococcus spp. to some antimicrobial agents to avoid reporting false-susceptible or false-resistant results. The greatest discrepancies and errors were associated with gradient diffusion, EUCAST breakpoints and some antimicrobials (mEs for aminoglycosides; MEs for fosfomycin, aminoglycosides and oxacillin; and VMEs for linezolid and oxacillin).

Reporting identification of Acinetobacter spp genomic species: A nationwide proficiency study in Spain.

Acinetobacter baumannii is the most important genomic species of Acinetobacter from a clinical and epidemiological point of view. Nevertheless, genomic species other than A. baumannii are increasingly recognized as nosocomial pathogens. Molecular methods of identification (genotypic and proteomic assays) are more accurate and reliable and have greater discriminatory power than phenotypic methods. Eleven genomic species of Acinetobacter spp. (8 A. baumannii, 1 A. pittii, 1 A. nosocomialis and 1 A. lwoffii) with different antimicrobial resistance phenotypes and mechanisms of resistance to antimicrobial agents were sent to 48 participating Spanish centers to evaluate their ability for correct identification at the genomic species level. Identification of the genomic species was performed at the two Clinical Microbiology reference laboratories (Hospital Universitario Virgen Macarena, Seville, Spain; and Complejo Hospitalario Universitario de A Coruña, A Coruña, Spain) by partial DNA sequencing of the rpoB gene and MALDI-TOF. The mean percentage of agreement was 76.1%. Fifty percent of CC-01 (A. pittii) and 50% of CC-02 (A. nosocomialis) identification results were reported as A. baumannii. Discrepancies by type of systems used for identification were: MicroScan WA (51.1%), Vitek 2 (19.5%), MALDI-TOF (18.0%), Phoenix (4.5%), Wider (3.8%) and API 20 NE (3.0%). In conclusion, clinical microbiology laboratories must improve their ability to correctly identify the most prevalent non A. baumannii genomic species.

Challenges to accurate susceptibility testing and interpretation of quinolone resistance in Enterobacteriaceae: results of a Spanish multicentre study.

OBJECTIVES: The objective of this study was to evaluate the proficiency of Spanish laboratories with respect to accurate susceptibility testing and the detection and interpretation of quinolone resistance phenotypes in Enterobacteriaceae. METHODS: Thirteen strains of Enterobacteriaceae were sent to 62 participating centres throughout Spain; strains harboured GyrA/ParC modifications, reduced permeability and/or plasmid-mediated quinolone resistance genes. The centres were requested to evaluate nalidixic acid and five quinolones, provide raw/interpreted clinical categories and to detect/infer resistance mechanisms. Consensus results from reference centres were used to assign minor, major and very major errors (mEs, MEs and VMEs, respectively). RESULTS: Susceptibility testing in the participating centres was frequently performed using the MicroScan WalkAway, Vitek 2 and Wider systems (48%, 30% and 8%, respectively). CLSI/EUCAST breakpoints were used in 71%/29% of the determinations. The percentage of VMEs for all quinolones was well below 2%. Only ofloxacin and moxifloxacin showed higher values for raw VMEs (6.6%), which decreased to 0% and 2.9%, respectively, in the interpreted VMEs. These errors were particularly associated with the CC-03 strain [qnrS2 + aac(6')-Ib-cr]. For MEs, percentages were always <10%, except in the case of ofloxacin and nalidixic acid. There was a significantly higher percentage of all types of errors for strains whose MICs were at the border of clinical breakpoints. CONCLUSIONS: The use of different breakpoints and methods, the complexity of mutation-driven and transferable resistance mechanisms and the absence of specific tests for detecting low-level resistance lead to high variability and represent a challenge to accuracy in susceptibility testing, particularly in strains with MICs on the border of clinical breakpoints.

Challenges for accurate susceptibility testing, detection and interpretation of ß-lactam resistance phenotypes in Pseudomonas aeruginosa: results from a Spanish multicentre study.

OBJECTIVES: To evaluate the proficiency of Spanish laboratories regarding accurate susceptibility testing, detection and interpretation of Pseudomonas aeruginosa ß-lactam resistance phenotypes. METHODS: Thirteen characterized strains were sent to 54 participating centres: clinical strains producing horizontally acquired ß-lactamases [extended-spectrum ß-lactamases (ESBLs; PER-1 and OXA-161) and class A (GES-5) and B (VIM-2) carbapenemases] and mutants with combinations of chromosomal mechanisms (AmpC, OprD and/or efflux). The centres were requested to evaluate six antipseudomonal ß-lactams, provide raw/interpreted clinical categories and detect/infer the resistance mechanisms. Consensus results from reference centres were used to assign minor, major or very major errors (mEs, MEs or VMEs). RESULTS: Vitek2, MicroScan WalkAway and Wider were the most used devices (25%-30% each). CLSI/EUCAST breakpoints were used in 86%/14% of the determinations. Discrepancies exclusively due to the differential application of breakpoints were highest for aztreonam, followed by piperacillin/tazobactam. The lowest percentage of VMEs was for Vitek2, followed by Wider. The highest percentages of VMEs (6%) were for the AmpC-hyperproducing OprD(-) strain and for the GES-5 producer, while among antibiotics the highest percentage of VMEs (22%) involved piperacillin/tazobactam. Appropriate inference of resistance mechanisms was high for the VIM-2-producing strain (83%), but low (<40%) for strains producing ESBLs or non-metallo-ß-lactamase carbapenemases. CONCLUSIONS: The use of different breakpoints and devices, the complexity of mutation-driven resistance mechanisms and the lack of unequivocal tests to detect ESBLs or carbapenemases in P. aeruginosa leads to extraordinary variability and low accuracy in susceptibility testing, which may have consequences for the treatment and control of nosocomial infections.

Comparison of polymerase chain reaction-dependent methods for determining the presence of human immunodeficiency virus and hepatitis C virus in washed sperm.

OBJECTIVE(S): To evaluate the effectiveness of sperm washes by double capacitation in patients with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) and to compare between polymerase chain reaction (PCR)-dependent methods to determine viral presence after the procedure. DESIGN: Controlled prospective clinical trial. SETTING: Private and university-affiliated setting. PATIENT(S): Thirty-four HIV-positive men attending our center for assisted reproduction with their partners. INTERVENTION(S): Forty-one semen samples from seropositive males were obtained and washed through density gradients and swim-up and analyzed for the presence of HIV and HCV with either nested or one-round PCR. Qualitative and quantitative detection of DNA and RNA of HIV and RNA of HCV. RESULT(S): Of all the semen samples that were analyzed after washing, seven out of 41 samples tested positive for one or both viruses by nested PCR. The confirmation of these results with the currently used commercial methods of quantitative PCR (one-round PCR) resulted in all negatives. CONCLUSION(S): Sperm wash with nested PCR is the appropriate method to use in the assisted reproduction techniques that are offered to serodiscordant couples. The detection limits exhibited by one-round PCR do not offer a sufficient guarantee that transmission of all viral particles will be avoided since both viruses can be present in amounts lower than the detection threshold of this technique.