Corneal Ulcer due to Monkeypox Infection.

PURPOSE: To report a rare case of keratitis due to monkeypox infection. METHODS: A 45-year-old male presented with an epithelial corneal ulcer 20 days following initial diagnosis of monkeypox from genital and perioral lesions. PCR analysis of the epithelium confirmed the presence of human monkeypox virus. RESULTS: The patient was hospitalized, and ganciclovir gel, as well as povidone iodine 0.6% and moxifloxacin eyedrops were prescribed. Oral tecovirimat 600 mg was administered during 14 days. A therapeutic contact lens was used. Twenty days after the initial diagnosis of keratitis, the corneal defect closed leaving a faint subepithelial haze, and visual acuity was 0.8. CONCLUSIONS: This is an uncommon case report of epithelial keratitis due to human monkeypox. PCR positivity for monkeypox in the corneal epithelium confirmed the presence of viral material in the cornea.

SARS-CoV-2 in Conjunctiva and Tears and Ocular Symptoms of Patients with COVID-19.

This study investigates the presence of SARS-CoV-2 in conjunctival secretions and tears and evaluates ocular symptoms in a group of patients with COVID-19. We included 56 hospitalized patients with COVID-19 in this cross-sectional cohort study. Conjunctival secretions and tears were collected using flocked swabs and Schirmer strips for SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR). Assessment of ocular surface manifestations included an OSDI (Ocular Surface Disease Index) questionnaire. Patients had been admitted to hospital for an average of 2.4 days (range 0-7) and had shown general symptoms for an average of 7.1 days (range 1-20) prior to ocular testing. Four (7.1%) of 56 conjunctival swabs and four (4%) of 112 Schirmer strips were positive for SARS-CoV-2. The mean E-gene cycle threshold values (Ct values) were 31.2 (SD 5.0) in conjunctival swabs and 32.9 (SD 2.7) in left eye Schirmer strips. Overall, 17 (30%) patients presented ocular symptoms. No association was found between positive ocular samples and ocular symptoms. This study shows that SARS-CoV-2 can be detected on the conjunctiva and tears of patients with COVID-19. Contact with the ocular surface may transmit the virus and preventive measures should be taken in this direction.

Colonisation of the male reproductive tract in asymptomatic infertile men: Effects on semen quality.

The objective was to explore presence/detection of microorganisms in the male reproductive tract (PMMRT) in asymptomatic patients undergoing infertility treatment and their effects on semen quality in our region. This study enrolled 205 men (mean age, 35.9 years) in a single-centre, tertiary university hospital from December 2015 to December 2016. We used the modified Meares-Stamey test, real-time polymerase chain reaction (rt-PCR) and the National Institutes of Health Chronic Prostatitis Sympton Index (NHI-CPSI) questionnaire to address this issue. No patient met the prostatitis criteria by the modified Meares-Stamey 4-sample test, 33 (16.1%) were positive for rt-PCR in the first-voided urine for any of the Mycoplasma (Ureaplasma urealyticum/parvum, Mycoplasma hominis/genitalium) and C. trachomatis was detected in two cases (1%), and three for rt-PCR in semen for HPV high-risk genotypes non-16/18 (1.5%). Significant statistical differences were reported among patients with and without PMMRT in terms of lower rate of progressive spermatozoa (PR) (p < .034), total motile sperm count (p < .028), normal morphologic forms, especially in the sperm head (p < .001) and highest viscosity (p < .012). It was concluded that PMMRT, specially Mycoplasmas, in asymptomatic infertility men, affects semen quality. The NIH-CPSI questionnaire was not a valid initial screening to subsequently evaluate the presence of prostatitis/PMMRT.

Prevalence of genital Mycoplasma and response to eradication treatment in patients undergoing assisted reproductive techniques / Prevalencia de micoplasmas genitales y respuesta al tratamiento de descolonización en pacientes de reproducción humana asistida

INTRODUCTION: Several studies have reported greater success of fertilisation by ART in couples who were not infected by Ureaplasma. Increased semen quality and better results have also been observed in couples who were treated with antibiotics to eradicate the infection. The aim of this study was to determine the prevalence of genital mycoplasmas in urine samples from male partners enrolled in the Assisted Reproduction Program (ARP) in our healthcare area so that, positive cases can be treated prior to the use of ART in order to increase the quality of semen, improve the embryo implantation rates and minimize the risk of adverse effects during pregnancy. MATERIAL AND METHODS: This study included couples enrolled in the ARP during 2016. Mycoplasma detection was made using real-time PCR. In positive cases, both members of the couple were treated with antibiotics until eradication of the microorganism. The antibiotics used were: azithromycin, doxycycline, levofloxacin, moxifloxacin, and clindamycin. RESULTS: Of the 205 men studied, 33 were positive: Ureaplasma urealyticum 15.1%, Mycoplasma hominis 3.9%. Eradication treatment with azithromycin failed in 50% compared to 10.2% for doxycycline. Of the 5 cases treated with levofloxacin, only 2 achieved elimination of U. urealyticum. CONCLUSIONS: We consider that genital mycoplasma routine screening could be useful in order to increase the quality of semen which could simplify the in vitro fertilisation procedures and raise the success rate of embryo implantation and pregnancy, especially when fast, sensitive and specific technics as real time PCR are used

INTRODUCCIÓN: Se han publicado estudios que demuestran mayores tasas de éxito en las técnicas reproducción asistida (TRA) en parejas no infectadas por micoplasmas. El objetivo de este estudio fue determinar la prevalencia de los micoplasmas genitales en muestras de orina del miembro masculino de las parejas incluidas en el Programa de Reproducción Asistida en nuestro Área Sanitaria realizando un tratamiento descolonizador con el fin de incrementar la calidad del semen, mejorar las tasas éxito de la embriotransferencia y minimizar los efectos adversos sobre la gestación. MATERIAL Y MÉTODOS: Participaron parejas incluidas en el Programa de Reproducción Asistida durante 2016. La detección de los micoplasmas se realizó por PCR en tiempo real. En los casos positivos, la pareja fue tratada con antibióticos hasta la erradicación del microorganismo. Los antibióticos usados fueron: azitromicina, doxiciclina, levofloxacino, moxifloxacino y clindamicina. RESULTADOS: De los 205 hombres estudiados, 33 fueron positivos: Ureaplasma urealyticum 15,1%, Mycoplasma hominis 3,9%. Azitromicina fracasó en el 50% de los casos y doxiciclina en el 10,2%. Con levofloxacino solo en 2 de 5 se consiguió la erradicación de U. urealyticum. CONCLUSIONES: El cribado de rutina de los micoplasmas genitales puede ser útil para mejorar la calidad del semen. Esto permitiría simplificar los procedimientos de fertilización in vitro e incrementar las tasas de éxito en la implantación de los embriones y en la gestación, especialmente con la aplicación de técnicas diagnósticas rápidas y específicas como la PCR en tiempo real

Occult hepatitis B and HIV infection.

INTRODUCTION: Occult hepatitis B virus (HBV) infection, so-called occult B infection (OBI), is defined by the recognition of HBV-DNA in the absence of serum hepatitis B surface antigen (HBsAg). The HBV-DNA genome in OBI is fully replication competent and produced in the liver, characteristically with low-level HBV-DNA fluctuations in the bloodstream. The OBI status remains between chronic (HBsAg +) and resolved (anti-HBs +) phases in the natural history of HBV infection. METHODS: The clinical interest in OBI has increased because of its potential for overt HBV reactivation under immunosuppression as well as for HBV transmission, well established in recipients of blood transfusions and/or organ transplants. RESULTS: Given the shared transmission routes for HIV and HBV, earlier reports claimed that OBI was more frequent in AIDS patients. By contrast, the current scenario shows that OBI is negligible in the HIV population. One explanation is that HBV immunization and recall vaccination campaigns have been very active in this group. A second and most important reason points to the wide use of antiretroviral regimens that include anti-HBV active agents, that is, tenofovir, lamivudine, and/or emtricitabine. They are recommended either as treatment for all HIV carriers or as pre-exposure prophylaxis for uninfected individuals at risk. The consequences are that HBV reactivations associated with HIV-related immunodeficiency have become very rare. Furthermore, HBV suppression with these antivirals has markedly reduced the likelihood of transmission from OBI carriers and/or acquisition by uninfected exposed individuals. CONCLUSION: Enthusiasm unabated, however, new tenofovir-sparing antiretroviral regimens are becoming popular and might account for a resurgence of OBI in the HIV setting.

Direct, indirect and total effectiveness of bivalent HPV vaccine in women in Galicia, Spain.

Bivalent human papillomavirus (HPV) vaccine was incorporated into the childhood vaccination calendar in Galicia, Spain in 2008. The objectives of this study were to estimate direct, indirect and total effectiveness of HPV vaccine and to identify sexual habits changes in the post-vaccination period in Galicia, Spain.Endocervical scrapings of 745 women attending 7 Health Areas of the Galician Public Health Service were collected in the post-vaccination period, from 2014-2017. Two groups were studied: women born between 1989 and 1993 (n = 397) and women born in 1994 or later (n = 348). Twelve high-risk human papillomavirus (HR-HPV) genotypes were detected by Cobas® 4800 HPV test (Roche Diagnostics, Mannheim, Germany). The Linear Array® HPV Genotyping Test (Roche Diagnostics) was used for HR-HPV genotype detection other than HPV 16/18. Information about sexual habits was collected by a self-filled questionnaire. Post-vaccination data were compared to previously published pre-vaccination data obtained between 2008 and 2010 in Galicia from women of the same age (18-26 years old, n = 523). The Stata 14.2 software was employed for statistical analyses.Data from 392 unvaccinated and 353 vaccinated women were compared. For unvaccinated and vaccinated women, HPV 16/18 prevalence was 9.2% and 0.8%, respectively, and HPV 31/33/45 prevalence was 8.4% and 1.1%, respectively. Direct, indirect and total effectiveness of the HPV vaccine were (%, 95% CI): 94 (72-99), 30 (-11-56) and 95 (79-99), respectively, for HPV 16/18 and 83 (46-94), -10 (-88-33) and 84 (54-94), respectively, for HPV 31/33/45. The number of women with first intercourse before 17 years old and 3 or more sexual partners along life was higher in the post-vaccination period (p < 0.05). A positive impact of bivalent HPV vaccine was observed, both on direct and cross protection. Sexual habits could have changed in the post-vaccination period.

Vigilancia de la sensibilidad a antimicrobianos de Escherichia coli productor de infecciones del tracto urinario comunitarias en Galicia / Surveillance of antimicrobial susceptibility of Escherichia coli producing urinary tract infections in Galicia (Spain)

Introducción. Escherichia coli es el microorganismo responsable de la mayoría de las infecciones del tracto urinario (ITU) comunitarias. Nuestro propósito fue conocer la sensibilidad de E. coli asociado a ITU en Galicia y considerar los antibióticos más apropiados para su tratamiento empírico. Métodos. Estudio retrospectivo durante el período 2011-2012 de los aislamientos de E. coli en muestras de orina procedentes de prácticamente toda la población gallega. Se recogieron variables demográficas, concentración mínima inhibitoria y categoría de interpretación para: amoxicilina/clavulánico, cefotaxima, gentamicina, amikacina, ciprofloxacino, cotrimoxazol, fosfomicina y nitrofurantoína. La identificación y estudios de sensibilidad se realizaron principalmente mediante sistemas automatizados. La interpretación de los resultados se realizó de acuerdo con los criterios de CLSI. Resultados: En el período estudiado se aislaron 55.046 E. coli en ITU. Los porcentajes de resistencia fueron: cotrimoxazol 30%; ciprofloxacino 33%; amoxicilina/clavulánico 23% y del 10% para cefalosporinas de 3ª generación. Fosfomicina y nitrofurantoína son los que mantienen mayor actividad, con más de un 96% de sensibilidad en nuestro estudio. La tendencia lineal de las resistencias en relación a la edad fue estadísticamente significativa (p<0,0001) y también lo fue en relación al sexo masculino (p<0,00001) para todos los antibióticos. Conclusiones. En Galicia los antibióticos más activos frente a E. coli asociado a ITU son fosfomicina y nitrofurantoína por lo que deberían ser considerados como tratamiento empírico de elección de la ITU comunitaria no complicada por E. coli (AU)

Introduction. Escherichia coli is the microorganism responsible for most of the community-acquired urinary tract infections (UTI). Our purpose was to determine the susceptibility of E. coli associated with UTI in Galicia and consider the most appropriate antibiotics for empirical treatment. Methods. Retrospective study during the period 2011-2012 of the isolation of E. coli in urine samples from almost all the Galician population. Demographic variables, minimum inhibitory concentration, and reading data were collected: amoxicillin-clavulanate, cefotaxime, gentamicin, amikacin, ciprofloxacin, cotrimoxazole, nitrofurantoin and fosfomycin. The identification and susceptibility studies were mainly conducted by automated systems. The interpretation of the results was performed according to CLSI criteria. Results. During the study period 55,046 E. coli were isolated in UTI. The percentages of resistance were: cotrimoxazole, 30%; ciprofloxacin, 33%; amoxicillin-clavulanate, 23% and 10% for 3rd generation cephalosporins. Fosfomycin and nitrofurantoin showed the highest activity with more than 96% of susceptibility in our study. The linear trend of resistance regarding age was statistically significant (p <0.0001) as it was regarding males (p <0.00001) for all antibiotics. Conclusions. In Galicia, the most active antibiotics against E. coli associated with UTI are fosfomycin and nitrofurantoin so they should be considered as empirical treatment of choice by the community-acquired UTI not complicated by E. coli (AU)

[Surveillance of antimicrobial susceptibility of Escherichia coli producing urinary tract infections in Galicia (Spain)]. / Vigilancia de la sensibilidad a antimicrobianos de Escherichia coli productor de infecciones del tracto urinario comunitarias en Galicia.

OBJECTIVE: Escherichia coli is the microorganism responsible for most of the community-acquired urinary tract infections (UTI). Our purpose was to determine the susceptibility of E. coli associated with UTI in Galicia and consider the most appropriate antibiotics for empirical treatment. METHODS: Retrospective study during the period 2011- 2012 of the isolation of E. coli in urine samples from almost all the Galician population. Demographic variables, minimum inhibitory concentration, and reading data were collected: amoxicillin-clavulanate, cefotaxime, gentamicin, amikacin, ciprofloxacin, cotrimoxazole, nitrofurantoin and fosfomycin. The identification and susceptibility studies were mainly conducted by automated systems. The interpretation of the results was performed according to CLSI criteria. RESULTS: During the study period 55,046 E. coli were isolated in UTI. The percentages of resistance were: cotrimoxazole, 30%; ciprofloxacin, 33%; amoxicillin-clavulanate, 23% and 10% for 3rd generation cephalosporins. Fosfomycin and nitrofurantoin showed the highest activity with more than 96% of susceptibility in our study. The linear trend of resistance regarding age was statistically significant (p <0.0001) as it was regarding males (p <0.00001) for all antibiotics. CONCLUSIONS: In Galicia, the most active antibiotics against E. coli associated with UTI are fosfomycin and nitrofurantoin so they should be considered as empirical treatment of choice by the community-acquired UTI not complicated by E. coli.

[Surveillance of resistance of Staphylococcus aureus to antibiotics in Galicia: 2007-2012]. / Vigilancia de la resistencia a los antibióticos en Staphylococcus aureus en Galicia: 2007-2012.

INTRODUCTION: Since 2007 the Galician Surveillance Program on Antimicrobial Resistance has been collected data of Staphylococcus aureus susceptibility patterns. The data from 2007 to 2012 have been analyzed and are reported. METHODS: A total of 4,577 different isolates of S. aureus from cerebrospinal fluid and blood cultures were included. The Institutions involved provided the information about the susceptibility patterns, the assay methods used and the interpretative guidelines followed, and demographic data of patients. RESULTS: The rate of methicillin-resistance S. aureus (MRSA) was 22% in 2007-2010 and 26% in 2011-2012, although in some areas the percentage reached 57% (2007- 2010) or 66% (2011-2012). The higher rates of resistance were found in patients older than 75 years. Gentamycin resistance was less than 9% and for quinolones were about 25%. A strong association between methicillin and quinolone-resistance were observed (91%). The resistance against linezolid and glycopeptides were exceptional. CONCLUSIONS: The percentage of MRSA has evolved slightly along the period of this study reaching no significant differences between Galicia and the global data in Spain in 2012. Nevertheless, there are significant differences among the geographic areas studied. Most MRSA isolates were recovered from hospitalized patients, but an increase in the number of MRSA among outpatients was observed, while old patients from nursing homes are included in the outpatient group, so the MRSA rate in this group could be overestimated.

Vigilancia de la resistencia a los antibióticos en Staphylococcus aureus en Galicia: 2007-2012 / Surveillance of resistance of Staphylococcus aureus to antibiotics in Galicia: 2007-2012

Introducción. Desde 2007 el Programa Gallego de Vigilancia de Resistencias Antimicrobianas recogió datos de los patrones de sensibilidad de Staphylococcus aureus. Se analizaron e informaron los datos entre 2007 y 2012. Métodos. Se incluyeron 4.577 aislamientos de S. aureus procedentes de líquido cefalorraquídeo o de sangre. Los distintos centros enviaron información sobre los patrones de sensibilidad, los métodos de ensayo, los criterios de interpretación seguidos y datos demográficos de los pacientes. Resultados. El porcentaje de aislamientos S. aureus resistentes a meticillina (SARM) fue del 22% (2007-2010) y del 26% (2011-2012), aunque en determinada área el porcentaje alcanzó el 57% (2007-2010) o 66% (2011-2012). Las tasas más altas de resistencias se encontraron en los mayores de 75 años. La resistencia a gentamicina fue menor del 9% y la de quinolonas sobre el 25%. Existe fuerte asociación entre resistencias a meticilina y quinolonas (91%). La resistencia frente a linezolid y glicopéptidos fue excepcional. Conclusiones. El porcentaje de SARM a lo largo del periodo de estudio ha presentado ciertas fluctuaciones alcanzándose en 2012 una situación similar en Galicia a la del conjunto de España. No obstante, hay importantes diferencias entre las áreas geográficas estudiadas. La mayoría de los SARM fueron aislados en pacientes hospitalizados, pero se observó un incremento entre ambulatorios. Dado que los pacientes mayores institucionalizados fueron incluidos en el grupo de los ambulatorios es posible que las tasas de SARM en este grupo hayan sido sobreestimadas (AU)

Introduction. Since 2007 the Galician Surveillance Program on Antimicrobial Resistance has been collected data of Staphylococcus aureus susceptibility patterns. The data from 2007 to 2012 have been analyzed and are reported. Methods. A total of 4,577 different isolates of S. aureus from cerebrospinal fluid and blood cultures were included. The Institutions involved provided the information about the susceptibility patterns, the assay methods used and the interpretative guidelines followed, and demographic data of patients. Results. The rate of methicillin-resistance S. aureus (MRSA) was 22% in 2007-2010 and 26% in 2011-2012, although in some areas the percentage reached 57% (2007-2010) or 66% (2011-2012). The higher rates of resistance were found in patients older than 75 years. Gentamycin resistance was less than 9% and for quinolones were about 25%. A strong association between methicillin and quinolone-resistance were observed (91%). The resistance against linezolid and glycopeptides were exceptional. Conclusions. The percentage of MRSA has evolved slightly along the period of this study reaching no significant differences between Galicia and the global data in Spain in 2012. Nevertheless, there are significant differences among the geographic areas studied. Most MRSA isolates were recovered from hospitalized patients, but an increase in the number of MRSA among outpatients was observed, while old patients from nursing homes are included in the outpatient group, so the MRSA rate in this group could be overestimated (AU)

Corneal epithelial wound healing and bactericidal effect of conditioned medium from human uterine cervical stem cells.

PURPOSE: To evaluate the effect of conditioned medium from human uterine cervical stem cells (CM-hUCESCs) on corneal epithelial healing in a rat model of dry eye after alkaline corneal epithelial ulcer. We also tested the bactericidal effect of CM-hUCESCs. METHODS: Dry eye was induced in rats by extraocular lacrimal gland excision, and corneal ulcers were produced using NaOH. Corneal histologic evaluation was made with hematoxylin-eosin (H&E) staining. Real-time PCR was used to evaluate mRNA expression levels of proinflammatory cytokines. We also studied the bactericidal effect of CM-hUCESCs in vitro and on infected corneal contact lenses (CLs) using Escherichia coli and Staphylococcus epidermidis bacteria. In addition, in order to investigate proteins from CM-hUCESCs that could mediate these effects, we carried out a human cytokine antibody array. RESULTS: After injury, dry eyes treated with CM-hUCESCs significantly improved epithelial regeneration and showed reduced corneal macrophage inflammatory protein-1 alpha (MIP-1α) and TNF-α mRNA expression as compared to untreated eyes and eyes treated with culture medium or sodium hyaluronate ophthalmic drops. In addition, we found in CM-hUCESCs high levels of proteins, such as tissue inhibitors of metalloproteinases 1 and 2, fibroblast growth factor 6 and 7, urokinase receptor, and hepatocyte growth factor, that could mediate these effects. In vitro, CM-hUCESCs showed a clear bactericidal effect on both E. coli and S. epidermidis and CLs infected with S. epidermidis. Analyses of CM-hUCESCs showed elevated levels of proteins that could be involved in the bactericidal effect, such as the chemokine (C-X-C motif) ligands 1, 6, 8, 10, and the chemokine (C-C motif) ligands 5 and 20. CONCLUSIONS: Treatment with CM-hUCESCs improved wound healing of alkali-injured corneas and showed a strong bactericidal effect on CLs. Patients using CLs and suffering from dry eye, allergies induced by commercial solutions, or small corneal injuries could benefit from this treatment.

[Plasmid-mediated AMPc producing Proteus mirabilis in the Health Care Area of Santiago de Compostela: molecular and epidemiological analysis by rep-PCR and MALDI-TOF]. / Proteus mirabilis productor de AmpC plasmídica en el Área Sanitaria de Santiago de Compostela: prevalencia y caracterización molecular por rep-PCR y MALDI-TOF MS.

INTRODUCTION: Proteus mirabilis is an important pathogen isolated from both community-acquired and health-care associated infections. Acquired AmpC-type beta-lactamases represent an important mechanism of resistance to extended-spectrum cephalosporins and are emerging in several European countries. The objective of this work was to know the prevalence of acquired AmpC beta-lactamase producing P. mirabilis over the last three years and eight months and their clonal relationships comparing MALDI-TOF and automated rep-PCR results. METHODS: P. mirabilis isolates (n= 1,396) were obtained from routine cultures at the University Hospital Complex of Santiago de Compostela from January 2006 to August 2009. Identification to the species level and antimicrobial susceptibility testing were achieved with Vitek 2. The isolates showing intermediate or total resistance to amoxicillin-clavulanic and cefoxitin, cefotaxime or ceftazidime were selected for AmpC phenotypic detection by double-disk synergy test, and molecular confirmation by multiplex PCR. Molecular typing of the isolates was performed by automated rep-PCR and MALDI-TOF. RESULTS: For the last three years and eight months, the prevalence of AmpC-producing P. mirabilis increased from 0.17% to 4.5%, mainly associated with urinary tract infection in elderly outpatients. In all cases, plasmidic AmpC belonging to LAT/CMY lineage were detected. A high genetic variability was seen with both, rep-PCR and MALDI-TOF MS. CONCLUSIONS: AmpC-producing P. mirabilis is an emergent pathogen. The high genetic variability detected suggests that the spread of the resistance mechanism is more probable than a clone dispersion. Automated rep-PCR and MALDI-TOF MS show as fast and decisive methods for bacterial strain typing in clinical microbiology laboratories.

Proteus mirabilis productor de AmpC plasmídica en el Área Sanitaria de Santiago de Compostela: prevalencia y caracterización molecular por rep-PCR y MALDI-TOF MS / Plasmid-mediated AMPc producing Proteus mirabilis in the Health Care Area of Santiago de Compostela: molecular and epidemiological analysis by rep-PCR and MALDI-TOF

Introducción: Proteus mirabilis es un patógeno de importancia creciente tanto en las infecciones nosocomiales como en las comunitarias. La producción de AmpC plasmídica es un mecanismo de resistencia frente a cefalosporinas de espectro extendido emergente en esta bacteria por lo que se consideró de gran interés estudiar su prevalencia en nuestro Área Sanitaria así como su variabilidad genética, comparando dos métodos recientemente incorporados al mercado: MALDI-TOF y rep-PCR automatizada. Métodos: Entre enero de 2006 y agosto de 2009 se recuperaron 1.396 aislamientos de P. mirabilis a partir de los cultivos de rutina realizados en Complejo Hospitalario Universitario de Santiago de Compostela. La identificación y el antibiograma se hicieron por Vitek 2. Aquellos aislamientos con sensibilidad reducida a amoxicilina-clavulánico y a cefoxitina, cefotaxima o ceftazidima fueron seleccionados para la detección fenotípica y genotípica de AmpC plasmídica mediante sinergia con doble disco y PCR múltiple, respectivamente. La tipificación molecular se llevó a cabo, comparativamente, mediante rep-PCR automatizada y MALDI-TOF. Resultados: A lo largo de tres años y ocho meses, la prevalencia de P. mirabilis productor de AmpC pasó del 0,17% al 4,5%, mayoritariamente asociado a infección urinaria en pacientes ancianos no hospitalizados. En todos los casos, AmpC plasmídica perteneció a la familia LAT/CMY. Se observó una gran variabilidad genética entre los aislamientos tanto por rep-PCR (DiversiLab) como por MALDI-TOF MS. Conclusión: P. mirabilis productor de AmpC adquirida es un patógeno emergente. La variabilidad genética de las cepas estudiadas apunta a una dispersión de este mecanismo de resistencia más que a una diseminación clonal. Rep-PCR automatizada y MALDI-TOF se muestran como métodos rápidos y resolutivos para la tipificación molecular en los laboratorios de microbiología clínica(AU)

Introduction: Proteus mirabilis is an important pathogen isolated from both community-acquired and health-care associated infections. Acquired AmpC-type beta-lactamases represent an important mechanism of resistance to extended-spectrum cephalosporins and are emerging in several European countries. The objective of this work was to know the prevalence of acquired AmpC beta-lactamase producing P. mirabilis over the last three years and eight months and their clonal relationships comparing MALDI-TOF and automated rep-PCR results. Methods: P. mirabilis isolates (n= 1,396) were obtained from routine cultures at the University Hospital Complex of Santiago de Compostela from January 2006 to August 2009. Identification to the species level and antimicrobial susceptibility testing were achieved with Vitek 2. The isolates showing intermediate or total resistance to amoxicillin-clavulanic and cefoxitin, cefotaxime or ceftazidime were selected for AmpC phenotypic detection by double-disk synergy test, and molecular confirmation by multiplex PCR. Molecular typing of the isolates was performed by automated rep-PCR and MALDI-TOF. Results: For the last three years and eight months, the prevalence of AmpC-producing P. mirabilis increased from 0.17% to 4.5%, mainly associated with urinary tract infection in elderly outpatients. In all cases, plasmidic AmpC belonging to LAT/CMY lineage were detected. A high genetic variability was seen with both, rep-PCR and MALDI-TOF MS. Conclusions: AmpC-producing P. mirabilis is an emergent pathogen. The high genetic variability detected suggests that the spread of the resistance mechanism is more probable than a clone dispersion. Automated rep-PCR and MALDI-TOF MS show as fast and decisive methods for bacterial strain typing in clinical microbiology laboratories(AU)

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF.

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.

Molecular and epidemiological analysis of nosocomial carbapenem-resistant Klebsiella spp. using repetitive extragenic palindromic-polymerase chain reaction and matrix-assisted laser desorption/ionization-time of flight.

INTRODUCTION: Infections with carbapenem-resistant enterobacteria are an emerging threat. This study reports the microbiologic, clinical, and epidemiologic features and the therapeutic outcomes of the infections caused by carbapenem- and pandrug-resistant Klebsiella emerged in our hospital. Fingerprinting analyses by automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry are also compared. MATERIALS AND METHODS: Carbapenem-resistant Klebsiella spp. affecting 13 patients were investigated using automated rep-PCR (DiversiLab System) and MALDI-TOF. Species identification was performed by Vitek 2 System and MALDI-TOF. Antimicrobial susceptibility testing was made using Vitek 2 System and Etest. Screening for extended spectrum beta-lactamase (ESBL) and carbapenemase production was made by double disk synergy and Hodge tests, respectively. Synergy studies were performed using Etest. DNA array was used for detection of KPC and ESBLs. bla(VIM-1) gene was amplified by PCR and sequencing. Use of carbapenems in the hospital was studied. RESULTS: A total of 13 patients were found to be colonized/infected with carbapenem-resistant Klebsiella. All patients were previously submitted to surgery and/or presented with severe underlying disease. After carbapenem-resistant Klebsiella isolation, the majority of the patients were treated with amikacin plus carbapenem, tigecycline, or fosfomycin. All Klebsiella isolates (n = 14), except two, had the bla(VIM-1) gene and all Klebsiella pneumoniae also had bla(SHV) gene associated with ESBL production. DiversiLab system showed higher discriminatory power than MALDI-TOF for strain typing. CONCLUSIONS: The risk of a rapid dissemination and the persistence of these multidrug-resistant strains through the time determine the need to implement routine procedures for metallo-beta-lactamase detection and measures for prevention of the spread of these microorganisms. The combined use of MALDI-TOF for species identification and DiversiLab System for clonal strain typing may be a useful tool for fast and accurate management of nosocomial outbreaks. The potential clinical utility of fosfomycin in this matter should be considered in future studies.

Emergence in Spain of a multidrug-resistant Enterobacter cloacae clinical isolate producing SFO-1 extended-spectrum beta-lactamase.

Between February 2006 and October 2009, 38 patients in different wards at the A Coruña University Hospital (northwest Spain) were either infected with or colonized by an epidemic, multidrug-resistant (MDR), and extended-spectrum-ß-lactamase (ESBL)-producing strain of Enterobacter cloacae (EbSF), which was susceptible only to carbapenems. Semiautomated repetitive extragenic palindromic sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) analysis revealed that all of the E. cloacae isolates belonged to the same clone. Cloning and sequencing enabled the detection of the SFO-1 ESBL in the epidemic strain and the description of its genetic environment. The presence of the ampR gene was detected upstream of bla(SFO-1), and two complete sequences of IS26 surrounding ampR and ampA were detected. These IS26 sequences are bordered by complete left and right inverted repeats (IRL and IRR, respectively), which suggested that they were functional. The whole segment flanked by two IS26 copies may be considered a putative large composite transposon. A gene coding for aminoglycoside acetyltransferase (gentamicin resistance gene [aac3]) was found downstream of the 3' IS26. Despite the implementation of strict infection control measures, strain EbSF spread through different areas of the hospital. A case-control study was performed to assess risk factors for EbSF acquisition. A multivariate analysis revealed that the prior administration of ß-lactam antibiotics, chronic renal failure, tracheostomy, and prior hospitalization were statistically associated with SFO-1-producing E. cloacae acquisition. This study describes for the first time an outbreak in which an SFO-1-producing E. cloacae strain was involved. Note that so far, this ß-lactamase has previously been isolated in only a single case of E. cloacae infection in Japan.

Comparación entre las pruebas para la detección de betalactamasas de espectro extendido de los sistemas Vitek 2 y Phoenix / Comparative assessment of the Vitek 2 and Phoenix systems for detection of extended-spectrum beta-lactamases

Introducción La detección de la multirresistencia a betalactámicos en Escherichia coli y Klebsiella pneumoniae es una cuestión clínicamente relevante. Por otro lado, es interesante diferenciar entre la producción de betalactamasas de espectro extendido (BLEE) y otros mecanismos de resistencia para evitar el tratamiento inadecuado de infecciones causadas por este tipo de cepas. El objetivo del presente estudio fue comparar la capacidad de las pruebas confirmatorias de los sistemas automatizados Vitek 2 y Phoenix para detectar la producción de BLEE en E. coli y K. pneumoniae. Material y métodos Se ensayaron 193 aislamientos clínicos fenotípicamente productores de BLEE (174 E. coli y 19K. pneumoniae) por Vitek 2 y BD Phoenix System y se utilizaron las tarjetas AST-N058 y los paneles UNMIC/ID-62, respectivamente. Se consideraron métodos fenotípicos de referencia la prueba de sinergia con doble disco y Etest. Como controles positivos y negativos se ensayaron 12 cepas genotípicamente caracterizadas. Resultados En el caso de los aislamientos clínicos, la sensibilidad fue del 99,5% para Vitek 2 y del 95,3% para Phoenix. En las cepas control no hubo diferencias entre ambos sistemas. La ejecución del sistema experto elevó la sensibilidad del Phoenix al 100%. Sin embargo, el sistema experto de Vitek 2 consideró incoherentes los resultados obtenidos en 7 aislamientos con la prueba para BLEE positiva. Conclusión La sensibilidad de la prueba confirmatoria para la producción de BLEE es superior en las tarjetas N-058 de Vitek. No obstante, la actuación de los sistemas expertos sitúa a ambos sistemas a la misma altura en su capacidad de detección de BLEE en E. coli y K. pneumoniae (AU)

Introduction and Purpose Detection of beta-lactam resistance in Escherichia coli and Klebsiella pneumoniae strains is clinically relevant. Moreover, it is important to differentiate between extended-spectrum beta-lactamase (ESBL) production and other mechanisms of resistance to avoid inadequate treatment of infection caused by these strains. The aim of this study was to compare the performance of the Vitek 2 and BD Phoenix automated systems for confirmatory testing of ESBL production. Material and Methods A total of 193 clinical isolates of phenotypically confirmed ESBL producers (174 E. coli and 19 K. pneumoniae) were assayed by the Vitek 2 and BD Phoenix systems using AST-N058 cards and UNMIC/ID-62 panels, respectively. The double-disk synergy test and the Etest were used as phenotype reference methods. Twelve strains characterized by genotyping were used as positive and negative controls. Results In the clinical isolates, the sensitivity of the tests was 99.5% for Vitek and 95.3% for Phoenix. There were no significant differences between the 2 systems in the control strains. Execution of the expert system raised the sensitivity of Phoenix to 100%. However, the Vitek 2 expert system considered the results obtained in 7 strains with ESBL-positive tests to be incoherent. Conclusion Confirmatory testing for ESBL production with the Vitek 2 system (AST-N058 card) showed higher sensitivity than the Phoenix (UNMIC-ID 62 panel) system. Nevertheless, the performance of the expert systems in the 2 automated tests was similar for ESBL detection in E. coli and K. pneumonia (AU)

Utilidad del medio chromID™ de betalactamasas de espectro extendido para detectar resistencia a cefalosporinas en enterobacterias inoculadas en frascos de hemocultivo / Utility of chromIDTM ESBL medium for detection of cephalosporin-resistant enterobacteria in inoculated blood culture bottles

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[Comparative assessment of the Vitek 2 and Phoenix systems for detection of extended-spectrum beta-lactamases]. / Comparación entre las pruebas para la detección de betalactamasas de espectro extendido de los sistemas Vitek 2 y Phoenix.

INTRODUCTION AND PURPOSE: Detection of beta-lactam resistance in Escherichia coli and Klebsiella pneumoniae strains is clinically relevant. Moreover, it is important to differentiate between extended-spectrum beta-lactamase (ESBL) production and other mechanisms of resistance to avoid inadequate treatment of infection caused by these strains. The aim of this study was to compare the performance of the Vitek 2 and BD Phoenix automated systems for confirmatory testing of ESBL production. MATERIAL AND METHODS: A total of 193 clinical isolates of phenotypically confirmed ESBL producers (174 E. coli and 19 K. pneumoniae) were assayed by the Vitek 2 and BD Phoenix systems using AST-N058 cards and UNMIC/ID-62 panels, respectively. The double-disk synergy test and the Etest were used as phenotype reference methods. Twelve strains characterized by genotyping were used as positive and negative controls. RESULTS: In the clinical isolates, the sensitivity of the tests was 99.5% for Vitek and 95.3% for Phoenix. There were no significant differences between the 2 systems in the control strains. Execution of the expert system raised the sensitivity of Phoenix to 100%. However, the Vitek 2 expert system considered the results obtained in 7 strains with ESBL-positive tests to be incoherent. CONCLUSION: Confirmatory testing for ESBL production with the Vitek 2 system (AST-N058 card) showed higher sensitivity than the Phoenix (UNMIC-ID 62 panel) system. Nevertheless, the performance of the expert systems in the 2 automated tests was similar for ESBL detection in E. coli and K. pneumoniae.