Breakthrough invasive fungal infection among patients with haematologic malignancies: A national, prospective, and multicentre study.

OBJECTIVES: We describe the current epidemiology, causes, and outcomes of breakthrough invasive fungal infections (BtIFI) in patients with haematologic malignancies. METHODS: BtIFI in patients with ≥ 7 days of prior antifungals were prospectively diagnosed (36 months across 13 Spanish hospitals) according to revised EORTC/MSG definitions. RESULTS: 121 episodes of BtIFI were documented, of which 41 (33.9%) were proven; 53 (43.8%), probable; and 27 (22.3%), possible. The most frequent prior antifungals included posaconazole (32.2%), echinocandins (28.9%) and fluconazole (24.8%)-mainly for primary prophylaxis (81%). The most common haematologic malignancy was acute leukaemia (64.5%), and 59 (48.8%) patients had undergone a hematopoietic stem-cell transplantation. Invasive aspergillosis, principally caused by non-fumigatus Aspergillus, was the most frequent BtIFI with 55 (45.5%) episodes recorded, followed by candidemia (23, 19%), mucormycosis (7, 5.8%), other moulds (6, 5%) and other yeasts (5, 4.1%). Azole resistance/non-susceptibility was commonly found. Prior antifungal therapy widely determined BtIFI epidemiology. The most common cause of BtIFI in proven and probable cases was the lack of activity of the prior antifungal (63, 67.0%). At diagnosis, antifungal therapy was mostly changed (90.9%), mainly to liposomal amphotericin-B (48.8%). Overall, 100-day mortality was 47.1%; BtIFI was either the cause or an essential contributing factor to death in 61.4% of cases. CONCLUSIONS: BtIFI are mainly caused by non-fumigatus Aspergillus, non-albicans Candida, Mucorales and other rare species of mould and yeast. Prior antifungals determine the epidemiology of BtIFI. The exceedingly high mortality due to BtIFI warrants an aggressive diagnostic approach and early initiation of broad-spectrum antifungals different than those previously used.

MDR Shigella sonnei in Spain: an ever-evolving emerging threat?

Background: Seven CTX-M-27-producing Shigella sonnei strains were isolated at the University Hospital Virgen del Rocío (Seville, Spain) microbiology service from October to November 2021. Objectives: To offer extensive information on the microbiological and molecular epidemiology results of the seven S. sonnei isolates and compare them with other previously documented CTX-M-27-producing S. sonnei associated with MSM transmission. Methods: S. sonnei isolated from stool samples of patients with acute diarrhoea were identified through biochemical and serological typing. Whole characterization of the seven isolates was performed by sequencing with MinION Mk1C followed by genomic and molecular analysis. Results: All the isolates were resistant to penicillins, cephalosporins, fluoroquinolones, cotrimoxazole and azithromycin. Sequencing showed the presence of several resistance determinants, outstanding bla CTX-M-27, azithromycin resistance genes [ermB and mph(A)], qnrB19 and mutations in the QRDRs. All isolates belonged to the same hierarchical clustering of cgMLST (HierCC) with five allele distance (HC5) scheme v1 from EnteroBase. However, they presented differences in plasmid composition, with all seven isolates harbouring IncFII, IncB/O/K/Z and ColE1-like while SH2, SH6 and SH7 had IncFIB only. Our isolates were closely related to others from Spain (HC5; 98748), Australia (HC5; 98748) and the UK (HC5; 98748), which were also associated with MSM transmission. Nevertheless, the structure of the non-chromosomal genetic elements and the genetic context of bla CTX-M-27 presented a certain variability compared with isolates from other countries and among them. Conclusions: This study confirms the emergence of CTX-M-27-producing S. sonnei (ST152) associated with MSM transmission in Spain, adding it to the Europe outbreak list and reinforcing the necessity of active surveillance and control of this high-risk clone.

Impact of the COVID-19 Pandemic on Antimicrobial Consumption and Hospital-Acquired Candidemia and Multidrug-Resistant Bloodstream Infections.

During the COVID-19 pandemic, the implementation of antimicrobial stewardship strategies has been recommended. This study aimed to assess the impact of the COVID-19 pandemic in a tertiary care Spanish hospital with an active ongoing antimicrobial stewardship programme (ASP). For a 20-week period, we weekly assessed antimicrobial consumption, incidence density, and crude death rate per 1000 occupied bed days of candidemia and multidrug-resistant (MDR) bacterial bloodstream infections (BSI). We conducted a segmented regression analysis of time series. Antimicrobial consumption increased +3.5% per week (p = 0.016) for six weeks after the national lockdown, followed by a sustained weekly reduction of -6.4% (p = 0.001). The global trend for the whole period was stable. The frequency of empirical treatment of patients with COVID-19 was 33.7%. No change in the global trend of incidence of hospital-acquired candidemia and MDR bacterial BSI was observed (+0.5% weekly; p = 0.816), nor differences in 14 and 30-day crude death rates (p = 0.653 and p = 0.732, respectively). Our work provides quantitative data about the pandemic effect on antimicrobial consumption and clinical outcomes in a centre with an active ongoing institutional and education-based ASP. However, assessing the long-term impact of the COVID-19 pandemic on antimicrobial resistance is required.

Efficacy and safety of a comprehensive educational antimicrobial stewardship program focused on antifungal use.

OBJECTIVE: Few data exist regarding the impact of antimicrobial stewardship programs on antifungal use. We evaluated the efficacy and safety of a comprehensive long-term antimicrobial stewardship program (ASP) focused on antifungal use. METHODS: During a 9-year period, we quarterly assessed antifungal consumption, incidence density of hospital-acquired candidemia, Candida spp. distribution, antifungal resistance, and crude death rate per 1000 occupied bed days (OBDs) of hospital-acquired candidemia. We performed segmented regression analysis of interrupted time series. RESULTS: A significant change in trend was observed for antifungal consumption, with a sustained reduction of -0.87% per quarter (95% confidence interval [CI], -1.36 -0.38, p < 0.001), accounting for a final reduction of -38.4%. The main reduction was produced in fluconazole, with a sustained reduction of -1.37% per quarter (95%CI, -1.96 -0.68, p<0.001). The incidence density of hospital-acquired candidemia decreased, with a change in slope of -5.06% cases per 1000 OBDs per year (95%CI, -8.23 -1.77, p = 0.009). The 14-day crude death rate per 1000 OBDs dropped from 0.044 to 0.017 (-6.36% deaths per 1000 OBDs per year; 95%CI, -13.45 -1.31, p = 0.09). CONCLUSIONS: This ASP has succeeded in optimizing the use of antifungal with a long-lasting reduction without increasing the incidence, neither the mortality, of hospital-acquired candidemia.

MixInYeast: A Multicenter Study on Mixed Yeast Infections.

Invasive candidiasis remains one of the most prevalent systemic mycoses, and several studies have documented the presence of mixed yeast (MY) infections. Here, we describe the epidemiology, clinical, and microbiological characteristics of MY infections causing invasive candidiasis in a multicenter prospective study. Thirty-four centers from 14 countries participated. Samples were collected in each center between April to September 2018, and they were sent to a reference center to confirm identification by sequencing methods and to perform antifungal susceptibility testing, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 6895 yeast cultures were identified and MY occurred in 150 cases (2.2%). Europe accounted for the highest number of centers, with an overall MY rate of 4.2% (118 out of 2840 yeast cultures). Of 122 MY cases, the most frequent combinations were Candida albicans/C. glabrata (42, 34.4%), C. albicans/C. parapsilosis (17, 14%), and C. glabrata/C. tropicalis (8, 6.5%). All Candida isolates were susceptible to amphotericin B, 6.4% were fluconazole-resistant, and two isolates (1.6%) were echinocandin-resistant. Accurate identification of the species involved in MY infections is essential to guide treatment decisions.

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media.

Aproximación clínica a la eosinofilia importada / Clinical approach to imported eosinophilia

La eosinofilia es frecuente en viajeros e inmigrantes, siendo las helmintosis su principal etiología. El valor predictivo positivo de la eosinofilia para una infección parasitaria es bajo en viajeros. La eosinofilia puede ser un hallazgo incidental o sintomático, y constituye un reto clínico debido a la baja sensibilidad y especificidad de las técnicas parasitológicas directas e indirectas, respectivamente. Requiere una aproximación estructurada basada en áreas geográficas, riesgos de exposición ambientales y conductuales, y síntomas asociados. La evaluación inicial debe incluir anamnesis y exploración física dirigidas, analítica básica, examen coproparasitológico completo y serología de Strongyloides stercoralis, complementada con otras pruebas según procedencia y sospecha clínica. El tratamiento empírico con albendazol y/o ivermectina (más praziquantel si hay riesgo de esquistosomiasis) es una opción en eosinofilias persistentes no filiadas tras estudio, y en personas en las que la evaluación inicial o el seguimiento no se puedan asegurar. En pacientes con riesgo de estrongiloidosis candidatos a inmunodepresión farmacológica está indicado el cribado y tratamiento previo para prevenir el síndrome de hiperinfestación

Eosinophilia is a common finding in international travelers and immigrants, being an helmintic infection its main etiology. The positive predictive value of eosinophilia for an helmintosis is low in travellers. Eosinophilia may be an incidental finding, or symptomatic, and it represents a clinical challenge due to the low sensitivity and specificity of direct and indirect parasitological diagnostic tests, respectively. It requires a structured approach based on geographical areas, environmental exposures and behavioral risks, and associated symptoms. The initial assessment should include a comprehensive and tailored anamnesis and physical examination, basic laboratory tests, a complete parasitological examination of stool samples and a Strongyloides stercoralis serology, supplemented with other explorations guided by epidemiological and clinical suspicion. Empiric treatment with albendazole and/or ivermectin (plus praziquantel if risk of schistosomiasis) is an option for unidentified persistent eosinophilia after study, and in persons in whom a proper assessment or follow-up cannot be assured. In patients at risk for estrongiloidosis who are candidates for immunosuppressive therapies, it is indicated a prior screening and treatment to prevent a future hyperinfestation syndrome

[Clinical approach to imported eosinophilia]. / Aproximación clínica a la eosinofilia importada.

Eosinophilia is a common finding in international travelers and immigrants, being an helmintic infection its main etiology. The positive predictive value of eosinophilia for an helmintosis is low in travellers. Eosinophilia may be an incidental finding, or symptomatic, and it represents a clinical challenge due to the low sensitivity and specificity of direct and indirect parasitological diagnostic tests, respectively. It requires a structured approach based on geographical areas, environmental exposures and behavioral risks, and associated symptoms. The initial assessment should include a comprehensive and tailored anamnesis and physical examination, basic laboratory tests, a complete parasitological examination of stool samples and a Strongyloides stercoralis serology, supplemented with other explorations guided by epidemiological and clinical suspicion. Empiric treatment with albendazole and/or ivermectin (plus praziquantel if risk of schistosomiasis) is an option for unidentified persistent eosinophilia after study, and in persons in whom a proper assessment or follow-up can not be assured. In patients at risk for estrongiloidosis who are candidates for immunosuppressive therapies, it is indicated a prior screening and treatment to prevent a future hyperinfestation syndrome.

Initial use of echinocandins does not negatively influence outcome in Candida parapsilosis bloodstream infection: a propensity score analysis.

BACKGROUND: Concerns have arisen regarding the optimal antifungal regimen for Candida parapsilosis bloodstream infection (BSI) in view of its reduced susceptibility to echinocandins. METHODS: The Prospective Population Study on Candidemia in Spain (CANDIPOP) is a prospective multicenter, population-based surveillance program on Candida BSI conducted through a 12-month period in 29 Spanish hospitals. Clinical isolates were identified by DNA sequencing, and antifungal susceptibility testing was performed by the European Committee on Antimicrobial Susceptibility Testing methodology. Predictors for clinical failure (all-cause mortality between days 3 to 30, or persistent candidemia for ≥72 hours after initiation of therapy) in episodes of C. parapsilosis species complex BSI were assessed by logistic regression analysis. We further analyzed the impact of echinocandin-based regimen as the initial antifungal therapy (within the first 72 hours) by using a propensity score approach. RESULTS: Among 752 episodes of Candida BSI identified, 200 (26.6%) were due to C. parapsilosis species complex. We finally analyzed 194 episodes occurring in 190 patients. Clinical failure occurred in 58 of 177 (32.8%) of evaluable episodes. Orotracheal intubation (adjusted odds ratio [AOR], 2.81; P = .018) and septic shock (AOR, 2.91; P = .081) emerged as risk factors for clinical failure, whereas early central venous catheter removal was protective (AOR, 0.43; P = .040). Neither univariate nor multivariate analysis revealed that the initial use of an echinocandin-based regimen had any impact on the risk of clinical failure. Incorporation of the propensity score into the model did not change this finding. CONCLUSIONS: The initial use of an echinocandin-based regimen does not seem to negatively influence outcome in C. parapsilosis BSI.

Impact on hospital mortality of catheter removal and adequate antifungal therapy in Candida spp. bloodstream infections.

OBJECTIVES: We set out to identify the prognostic factors in adult patients with Candida spp. bloodstream infection, assessing the impact on in-hospital mortality of catheter removal and adequacy of antifungal therapy. METHODS: Patients with positive blood culture for Candida spp. and a central venous catheter in place at the time of candidaemia were included. Data collected included demographics, underlying diseases, severity of illness, clinical presentation, catheter withdrawal and adequacy of empirical therapy. RESULTS: We included 188 patients (mortality 36.7%). The mortality rate was 34.9% (23/66) in patients with early adequate antifungal treatment and 18.9% (7/37) in patients with early adequate antifungal therapy and catheter withdrawal in the first 48 h. The APACHE (Acute Physiology and Chronic Health Evaluation) II score on the day of candidaemia [adjusted hazard ratio (aHR) 1.12; 95% CI 1.06-1.17; P < 0.001] was associated with death whereas early adequate therapy (aHR 0.4; 95% CI 0.23-0.83; P = 0.012) and catheter withdrawal (aHR 0.34; 95% CI 0.16-0.70; P = 0.03) were protective factors. In primary candidaemia, mortality was 28% (14/50) in patients with adequate therapy and decreased to 17.7% (6/34) in patients with both interventions in the first 48 h. Catheter removal was a protective factor and adequacy of antifungal therapy in the first 48 h showed a strong tendency to protection against death (aHR 0.46; 95% CI 0.19-1.08; P = 0.07). In secondary non-catheter-related candidaemia, only early adequate therapy was a protective factor for mortality. CONCLUSIONS: Delay in catheter withdrawal and in administration of adequate antifungal therapy was associated with increased mortality in candidaemic patients. Catheter management did not influence the prognosis of secondary non-catheter-related candidaemia.

Universal antifungal therapy is not needed in persistent febrile neutropenia: a tailored diagnostic and therapeutic approach.

BACKGROUND: Giving antifungal therapy exclusively to selected patients with persistent febrile neutropenia may avoid over-treatment without increasing mortality. The aim of this study was to validate an innovative diagnostic and therapeutic approach based on assessing patients' risk profile and clinical criteria in order to select those patients requiring antifungal therapy. The efficacy of this approach was compared to that of universal empirical antifungal therapy. DESIGN AND METHODS: This was a prospective study which included all consecutive adult hematology patients with neutropenia and fever refractory to 5 days of empirical antibacterial therapy admitted to a teaching hospital in Spain over a 2-year period. A diagnostic and therapeutic approach based on clinical criteria and risk profile was applied in order to select patients for antifungal therapy. The sensitivity, specificity and negative predictive value of this approach and also the overall success rate, according to the same criteria of efficacy described in classical clinical trials, were analyzed. RESULTS: Eighty-five episodes were included, 35 of them (41.2%) in patients at high risk of invasive fungal infections. Antifungal therapy was not indicated in 33 episodes (38.8%). The overall incidence of proven and probable invasive fungal infections was 14.1%, all of which occurred in patients who had received empirical antifungal therapy. The 30-day crude mortality rate was 15.3% and the invasive fungal infection-related mortality rate was 2.8% (2/72). The overall success rate following the diagnostic and therapeutic approach was 36.5% compared with 33.9% and 33.7% obtained in the trial by Walsh et al. The sensitivity, specificity and negative predictive value of the study approach were 100%, 52.4% and 100%, respectively. CONCLUSIONS: Based on the high negative predictive value of this diagnostic and therapeutic approach in persistent febrile neutropenia patients with hematologic malignancies or patients who have received a hematopoietic stem cell transplant, the approach is useful for identifying patients who are not likely to develop invasive fungal infection and do not, therefore, require antifungal therapy. The effectiveness of the strategy is similar to that of universal empirical antifungal therapy reported in controlled trials.

Candidemias: análisis multicéntrico en 16 hospitales andaluces / Candidemias: multicentre analysis in 16 hospitals in Andalusia (Spain)

Introducción La candidemia es una infección nosocomial con elevada mortalidad. Los cambios clínicos y microbiológicos descritos en otras áreas y las novedades terapéuticas de los últimos años hacen preciso conocer si la epidemiología clínica de las candidemias en nuestro medio ha cambiado. Material y métodos Estudio prospectivo, multicéntrico y observacional de todos los episodios de candidemia en pacientes adultos atendidos entre el 1 octubre 2005 y el 30 septiembre 2006 en 17 hospitales de Andalucía. Resultados El número total de episodios fue de 220, la incidencia de 0,58 episodios/por cada 1.000 altas. Candida albicans fue la etiología más frecuente (53%). El 89% de las cepas fueron sensibles a fluconazol. La sepsis fue la presentación clínica más frecuente (65,7%). El tratamiento empírico fue inapropiado en el 38,7%. La mortalidad global (..) (AU)

Introduction: Candidemia is a nosocomial infection with high associated mortality. There have been changes in microbiology, epidemiology and treatment over the last few years, which has led us to analyse our own situation. Material and methods: Prospective, multicentre and observational study. All episodes of candidemia in adult patients seen in 17 Andalusian hospitals from 1 October 2005 to 30 September 2006 were included. Results: Were detected 220 cases, the incidence was 0.58 cases/1,000 hospital discharges. Candida albicans was the most frequent species (53% of cases). The majority of isolates (89%) was susceptibility to fluconazole. Sepsis was the most frequent clinical manifestation (65.7%). The treatment was inadequate in b38.7% of cases. Overall mortality was 40%.On univarite analysis (..) (AU)

[Candidemias: multicentre analysis in 16 hospitals in Andalusia (Spain)]. / Candidemias: análisis multicéntrico en 16 hospitales andaluces.

INTRODUCTION: Candidemia is a nosocomial infection with high associated mortality. There have been changes in microbiology, epidemiology and treatment over the last few years, which has led us to analyse our own situation. MATERIAL AND METHODS: Prospective, multicentre and observational study. All episodes of candidemia in adult patients seen in 17 Andalusian hospitals from 1 October 2005 to 30 September 2006 were included. RESULTS: Were detected 220 cases, the incidence was 0.58 cases/1,000 hospital discharges. Candida albicans was the most frecuent species (53% of cases). The majority of isolates (89%) was susceptibility to fluconazole. Sepsis was the most frequent clinical manifestation (65.7%). The treatment was inadequate in 38.7% of cases. Overall mortality was 40%. On univarite analysis death was found to be significantly associated with: aged > 60 years, unknown candidemia focus, Pitt score ≥ 2, APACHE II, shock at onset, persistents positive second blood cultures, non-removal of the central venous catheter and Candida species different of C. parasilopsis, among others. In the multivariate analysis death was found to be significantly associated with: aged > 60 years, Pitt score ≥ 2, Candida species different of C.parasilopsis and inadequate treatment. CONCLUSIONS: The candidemia clinical epidemiology in our region is similar to other areas and receiving inadequate treatment is the only modifiable risk factor associated with higher odds of mortality. Therefore, this modifiable factor needs to be improved to reduce the mortality.

Risk factors for fluconazole-resistant candidemia.

Previous studies have sought to determine the risk factors associated with candidemia caused by non-albicans Candida spp. or with potentially fluconazole-resistant Candida spp. (C. glabrata and C. krusei). Non-albicans Candida strains are a heterogeneous group that includes species with different levels of virulence, and only a limited number of C. glabrata isolates are resistant to fluconazole. We set out to identify the risk factors associated with microbiologically proven fluconazole-resistant candidemia. A prospective study including adult patients with candidemia was performed. Data were collected on patient demographics; underlying diseases; exposure to corticosteroids, antibiotics, or fluconazole; and invasive procedures. Risk factors associated either with non-albicans Candida spp. or potentially fluconazole-resistant Candida spp. (C. glabrata or C. krusei) or with Candida spp. with microbiologically confirmed fluconazole resistance were assessed using logistic regressions. We included 226 candidemia episodes. Non-albicans Candida isolates accounted for 53.1% of the fungal isolates, but only 18.2% of the cases were caused by potentially fluconazole-resistant organisms. Thirty isolates exhibited microbiologically confirmed fluconazole resistance. The multivariate analysis revealed that independent predictors associated with fluconazole-resistant Candida spp. were neutropenia (odds ratio [OR]=4.94; 95% confidence interval [CI]=1.50 to 16.20; P=0.008), chronic renal disease (OR=4.82; 95% CI=1.47 to 15.88; P=0.01), and previous fluconazole exposure (OR=5.09; 95% CI=1.66 to 15.6; P=0.004). Independently significant variables associated with non-albicans Candida bloodstream infection or with potentially fluconazole-resistant Candida spp. did not include previous fluconazole exposure. We concluded that prior fluconazole treatment is an independent risk factor only for candidemia caused by microbiologically confirmed fluconazole resistant species. Our findings may be of value for selecting empirical antifungal therapy.

[Detection of methicillin resistance and identification of Staphylococcus spp. from positive blood culture bottles using the mecA and nucA genes with the LightCycler System]. / Detección de la resistencia a meticilina e identificación de Staphylococcus spp. en hemocultivos positivos amplificando los genes mecA y nucA con el sistema LightCycler.

INTRODUCTION: The aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system. METHODS: One hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene. RESULTS: All the bottles that yielded methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) or methicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNS gave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results for the mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively. CONCLUSION: This is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and -resistant strains in less than 3 hours after Gram stain.

Detección de la resistencia a meticilina e identificación de Staphylococcus spp. en hemocultivos positivos amplificando los genes mecA y nucA con el sistema Light Cycler / Detection of methicillin resistance and identification of Staphylococcus spp. from positive blood culture bottles using the mecA and nucA genes with the LightCycler System

INTRODUCCIÓN. El objetivo de este trabajo fue evaluarla eficacia del sistema LightCycler para la detección de Staphylococcus aureus y estafilococos coagulasa negativos(ECN), y la identificación de resistencia a meticilina directamente en botellas Bactec de hemocultivos positivos. MÉTODOS. Se procesaron 131 botellas de hemocultivos positivos en los que se observaron cocos grampositivos en racimos tras la tinción de Gram y 40 botellas con microorganismos diferentes a estafilococos. Para la extracción del ADN se utilizó el sistema automático MagNAPure LC (Roche Diagnostic). Los cebadores y las sondas dehibridación se diseñaron para la amplificación y detección de las secuencias específicas de un fragmento de 408 pb del gen mecA y de un fragmento de 279 pb del gen nucA. RESULTADOS. Todas las botellas con cepas de S. aureusresistente a meticilina (SARM), sensible a meticilina(SASM) o ECN resistentes a meticilina (ECNRM) fueron correctamente identificados mediante reacción en cadena de la polimerasa (PCR) de los genes nucA y mecA. Una botella con un cultivo mixto de SASM y ECNRM dio resultados positivos para ambos genes. Las 21 botellas con cepas de ECN sensible a meticilina (ECNSM) fueron correctamente identificadas con el gen nucA, pero dos de ellas fueron positivas para el gen mecA. La sensibilidad y la especificidad fueron del 100% para el gen nucA, y del 100 y 97,5%, respectivamente, para el gen mecA. CONCLUSIÓN. Este es un método sensible y específico para la identificación de estafilococos en hemocultivos, y permite también la identificación de cepas resistentes a meticilina en un tiempo máximo de 3 h a partir de la visualización de la tinción de Gram (AU)

INTRODUCTION. The aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system. METHODS. One hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene. RESULTS. All the bottles that yielded methicillin-resistant S.aureus (MRSA), methicillin-sensitive S. aureus (MSSA) ormethicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNSgave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results forthe mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively. CONCLUSION. This is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and –resistant strains in less than 3 hours after Gramstain (AU)