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The photoprocesses involved in hypericin photoinactivation of three different Candida species (C. albicans, C. parapsilosis and C. krusei) have been examined. Production of singlet oxygen from the triplet state and of superoxide from both the triplet state and the semiquinone radical anion are demonstrated. Hydrogen peroxide is formed downstream of these early events. The outcome of the photodynamic treatments is dictated by the intracellular distribution of hypericin, which is different in the three species and affects the ability of hypericin to produce the different reactive oxygen species and trigger cell-death pathways. The results are in line with the previously-observed different susceptibilities of the three Candida species to hypericin photodynamic treatments.

Early and accurate diagnosis of invasive aspergillosis (IA) is one of themost critical steps needed to efficiently treat the infection and reduce the high mortality rates that can occur. We have previously found that the Aspergillus spp. secondary metabolite, bis(methylthio)gliotoxin (bmGT), can be detected in the serum from patients with possible/probable IA. Thus, it could be used as a diagnosis marker of the infection. However, there is no data available concerning the sensitivity, specificity and performance of bmGT to detect the infection. Here,we have performed a prospective study comparing bmGT detection with galactomannan (GM), the most frequently used and adopted approach for IA diagnosis, in 357 sera from 90 episodes of patients at risk of IA. Our results, involving 79 patients that finally met inclusion criteria, suggest that bmGT presents higher sensitivity and positive predictive value (PPV) than GM and similar specificity and negative predictive value (NPV). Importantly, the combination of GM and bmGT increased the PPV (100 %) and NPV (97.5 %) of the individual biomarkers, demonstrating its potential utility in empirical antifungal treatment guidance and withdrawal. These results indicate that bmGT could be a good biomarker candidate for IA diagnosis and, in combination with GM, could result in highly specific diagnosis of IA and management of patients at risk of infection., This work has been supported by Fondo Social Europeo (FSE/Gobierno de Aragón) and ARAID Foundation (J.P.)

Background Antimicrobial photodynamic therapy has been proposed as an alternative to suppress subgingival species. This results from the balance among Streptococcus sanguis, Streptococcus mutans and Candida albicans in the dental biofilm. Not all the photosensitizers have the same photodynamic effect against the different microorganims. The objective of this study is to compare in vitro the photodynamic effect of methylene blue (MB), rose Bengal (RB) and curcumin (CUR) in combination with white light on the cariogenic microorganism S. mutans, S. sanguis and C. albicans. Go to: Results Photodynamic therapy with MB, RB and CUR inhibited 6 log 10 the growth of both bacteria but at different concentrations: 0.31–0.62 μg/ml and 0.62–1.25 μg/ml RB were needed to photoinactivate S. mutans and S. sanguis, respectively; 1.25–2.5 μg/ml MB for both species; whereas higher CUR concentrations (80–160 μg/ml and 160–320 μg/ml) were required to obtain the same reduction in S. mutans and S. sanguis viability respectively. The minimal fungicidal concentration of MB for 5 log10 CFU reduction (4.5 McFarland) was 80–160 μg/ml, whereas for RB it ranged between 320 and 640 μg/ml. For CUR, even the maximum studied concentration (1280 μg/ml) did not reach that inhibition. Incubation time had no effect in all experiments. Go to: Conclusions Photodynamic therapy with RB, MB and CUR and white light is effective in killing S. mutans and S. sanguis strains, although MB and RB are more efficient than CUR. C. albicans required higher concentrations of all photosensitizers to obtain a fungicidal effect, being MB the most efficient and CUR ineffective. España, Ministerio de Ciencia e Innovación CTQ2013-48767-C3-2-R

Since the latest taxonomical changes in the genus Scedosporium by Gilgado et al. in 2010, no species-specific studies on epidemiology and antifungal susceptibility patterns (AFSP) have so far been published. This study aimed to provide qualitative epidemiological data of Scedosporium spp. isolated from cystic fibrosis (CF) patients and immunocompromised patients from Northern Spain. Isolates were identified by using amplified fragment length polymorphism (AFLP), and species-specific AFSP were generated for all currently available antifungal compounds. AFLP was a useful tool for identification to species-level and for the discrimination of inter- and intra-patient isolates. Scedosporium prolificans represents the most prevalent species in the respiratory tract of CF patients and immunocompromised patients in Northern-Spain, followed by Pseudallescheria boydii, P. apiosperma, and P. ellipsoidea. CF patients were exclusively colonised with either P. boydii or S. prolificans. Patients were colonised over years exclusively with isolates affiliated to one species, but some patients were colonised with multiple strains with different AFSP. The sum of those co-colonising strains in one patient, may appear in vitro and in vivo as a multi-resistant S. prolificans isolate, as strains are morphologically identical and might therefore be regarded as only one strain. A majority of Scedosporium strains (with exception of S. prolificans) were found susceptible for voriconazole and micafungin.

Thirteen vancomycin-resistant and teicoplanin-susceptible Enterococcus faecalis isolates were recovered from unrelated patients in three Spanish hospitals from November 2009 to December 2010. All isolates carried the vanB2 gene, showed indistinguishable or closely-related PFGE patterns and were ascribed to the sequence type ST6 (included into the high-risk clonal-complex CC2). They showed a multiresistance phenotype (erythromycin, tetracycline, ciprofloxacin and high-level-resistance to streptomycin, gentamicin and kanamycin) and harboured the aac(6')-aph(2"), ant(6)-Ia, and tet(M)+/-tet(L) genes. All isolates produced gelatinase and harboured the gelE gene, but not the esp or hyl genes. The inclusion of the vanB2 gene into the Tn5382 transposon was demonstrated in one isolate. Clonal dissemination of vanB2-containing the E. faecalis strain is demonstrated.

Resumen Antecedentes Recientemente se ha observado un incremento de las fungemias causadas por especies diferentes de Candida albicans y una disminucion de la sensibilidad de los microorganismos responsables al fluconazol. Objetivos Evaluar la epidemiologia y la sensibilidad al fluconazol de los casos de fungemia en Espana en 2009, comparando los resultados con los obtenidos entre los anos 1997-1999 (Peman J, et al. Eur J Clin Microbiol Infect Dis. 2005). Metodos Estudio prospectivo multicentrico con 44 centros participantes realizado desde enero de 2009 a febrero de 2010. Los aislamientos fungicos procedentes de hemocultivo fueron recogidos en cada centro, donde se realizo el estudio de sensibilidad antifungica mediante microdilucion colorimetrica (Sensititre Yeast One). Resultados Desde enero de 2009 a febrero de 2010 se recogieron 1.377 aislamientos en hemocultivos, correspondientes a 1.357 episodios de fungemia. Las fungemias se observaron principalmente en mayores de 64 anos (46,7%) y el 8,6% en menores de 1 ano. C. albicans (44,7%), Candida parapsilosis (29,1%), Candida glabrata (11,5%), Candida tropicalis (8,2%) y Candida krusei (1,9%) fueron las especies mas frecuentes, pero su distribucion no fue geograficamente homogenea. En los ultimos 10 anos la incidencia de C. albicans ha aumentado significativamente en Cataluna (39,1 vs. 54,7%, P = 0,03) y reducido en la Comunidad Valenciana (49,1 vs. 34,6%, P = 0,01). C. parapsilosis ha disminuido en Cataluna (29 vs. 12,4%, P = 0,002) y Extremadura (58,3 vs. 20%, P = 0,01). La sensibilidad a fluconazol fue similar en toda Espana pero en los aislamientos de C. albicans la resistencia fue diez veces superior en mayores de 64 anos. Sin embargo, la tasa de resistencia (CMI > 32 mg/L) global ha disminuido con respecto a la obtenida hace 10 anos (3,7 vs. 2,5% actual), sobre todo en C. albicans (3 vs. 1,6%). Conclusiones En los ultimos 10 anos la distribucion de las especies causantes de fungemia en Espana y la sensibilidad al fluconazol no han variado significativamente, aunque se observa una menor tasa de resistencia. La distribucion de las especies varia segun la unidad de hospitalizacion, hospital y Comunidad Autonoma.

INTRODUCTION. Since 1986 we have carried out five nationwide point-prevalence studies in Spain analyzing Staphylococcus spp. The 2006 data, corresponding to the sixth study, are presented herein. METHODS. A total of 145 hospitals from all geographic areas of the country participated in the study. We investigated 866 staphylococcal isolates (463 S. aureus). Antimicrobial susceptibility testing was performed against 16 antimicrobials by an automated microdilution method. Susceptibility to tigecycline was determined by the E-test method. RESULTS. Resistance of S. aureus to oxacillin seemed to have stabilized (31.2% in 2002 vs. 29.2% in 2006), and the same was true for resistance to erythromycin, clindamycin and ciprofloxacin. In 2006, isolates were more susceptible to gentamicin (16.9% resistance in 2002 vs. 8.6% in 2006, P < 0.001). None of the isolates presented decreased susceptibility to vancomycin, and the resistance to cotrimoxazole (0.9%) and rifampin (0.6%) was minimal. One isolate showed linezolid resistance. Resistance of coagulase negative staphylococci to oxacillin (61.3% in 2002 vs. 66.7% in 2006) and erythromycin (63.0% in 2002 vs. 66.5% in 2006) remained stable, although resistance to gentamicin (27.8% in 2002 vs. 44.2% in 2006, P < 0.001), ciprofloxacin (44.9% in 2002 vs. 54.3% in 2006, P = 0.010) and clindamycin (33.8% in 2002 vs. 46.2% in 2006, P = 0.001) has increased. Two isolates presented decreased susceptibility to teicoplanin and one was linezolid-resistant. All Staphylococcus spp. were uniformly susceptible to quinupristin-dalfopristin and tigecycline. CONCLUSIONS. Resistance of Staphylococcus spp. to oxacillin remains high in Spain, but seems to have stabilized in the last years. Linezolid resistance is emerging.

Las infecciones invasoras por hongos filamentosos han aumentado durante los ultimos 20 anos ya que la poblacion en riesgo se ha incrementado. El diagnostico y el tratamiento temprano son la clave para el exito del tratamiento. Los metodos clasicos de diagnostico microbiologico son lentos y poco sensibles. El desarrollo de nuevas tecnicas como deteccion de antigenos, galactomanano y (1,3)β-D-glucano y de ADN representan una mejora en el diagnostico de este tipo de infecciones.

Members of the fungal genus Chaetomium usually colonize cellulose-containing plant remains but on rare occasions may cause opportunistic mycoses and cutaneous infection in otherwise healthy individuals. To our knowledge, there have been only five credible descriptions of onychomycosis caused by members of this genus and only two of these contained information on therapy. We describe the first case of Chaetomium globosum onychomycosis recorded in Spain. The etiologic significance of the fungus was confirmed by its repeated isolation at different times, to the exclusion of dermatophytes. Clinically, the affected nails showed an excellent response to terbinafine and complete cure appeared to have been attained.

To get a better insight into the role of birds as reservoirs of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC β-lactamase (pAmpC) Escherichia coli producers, 100 fecal samples belonging to 15 different wild avian species from Northern Spain were analyzed. Cefotaxime-resistant (CTXR) E. coli isolates were identified in 16 of the 100 tested birds, which corresponded to 9 animal species (Gyps fulvus-griffon vulture, Larus michahellis-yellow-legged gull, Milvus migrans-black kite, Milvus milvus-red kite, Ciconia ciconia-white stork, Sturnus unicolor-spotless starling, Aquila chrysaetos-golden eagle, Cuculus canorus-common cuckoo, Tyto alba-barn owl). Fifteen isolates harbored ESBL or pAmpC-encoding genes (number of isolates): bla SHV-12 (9), bla CTX-M-1 (3), bla CTX-M-14 (2), and bla CMY-2 (1). The last CTXR isolate presented a -42-point-mutation in the chromosomal ampC promoter. Eleven out of 15 ESBL/pAmpC E. coli isolates were multiresistant (most common resistance phenotype: β-lactams-quinolones-tetracycline-sulfamethoxazole/trimethoprim). A plasmid-mediated quinolone resistance determinant (qnrS1) was identified in one E. coli from a barn owl. High genetic diversity was observed among ESBL/pAmpC E. coli isolates, with 12 different sequence types (STs), including several strains of STs frequently detected among human clinical isolates (ST38/D, ST131/B2, ST155/B1, ST10/A). The ST131 isolate belonged to the emergent ciprofloxacin-resistant H30R subclone. This study reveals a high percentage of bird as carriers of ESBL/pAmpC E. coli isolates in Spain, highlighting the elevated rate among storks, kites, and vultures. Wild birds can contribute to the global spread of ESBL/pAmpC-producing E. coli in natural ecosystems.

Onychomycosis, or fungal infection of the nails, is one of the most prevalent fungal diseases in the general population. Treatment is of limited effectiveness, tedious, and must be administered for long periods. Furthermore, systemic antifungal agents are associated with adverse effects. Photodynamic therapy (PDT) may prove to be a viable alternative in the treatment of superficial skin infections, including onychomycosis. We review articles relating to the usefulness of PDT in onychomycosis in both in vitro and in vivo settings and discuss the potential and limitations of various photosensitizing agents. In vivo, methylene blue and 5-aminolevulinic acid have led to cure rates in 80% and 43% of cases, respectively, at 12 months. Finally, based on data in the literature and our own experience, we propose a protocol of 3 PDT sessions, separated by an interval of 1 or 2 weeks, using methyl aminolevulinate 16% as a photosensitizing agent and red light (λ=630 nm, 37 J.cm(-2)). Each session is preceded by the topical application of urea 40% over several days. Clinical trials are needed to optimize PDT protocols and to identify those patients who will benefit most from this treatment.

ABSTRACT Aim: To evaluate the effectiveness of the photodynamic therapy using hypericin (HYP) against both planktonic and biofilm-forming Staphylococcus aureus. Materials & methods: HYP photoactivity was evaluated against methicillin-susceptible and resistant S. aureus. Bacterial suspension or biofilm were preincubated with HYP and subjected to LED illumination. Viable bacteria were determined by colony counting. Results: Preincubation with HYP (5 min) plus light exposure (10 min) showed bactericidal effect against planktonic methicillin-susceptible S. aureus and methicillin-resistant S. aureus. Longer preincubation times (24 h) and time light exposure (30 min) were required to reach HYP-photoactivity against S. aureus biofilms. HYP-photoactivity was correlated to the biofilm production. Conclusion: HYP could be a potential photosensitizer for the inactivation of staphylococcal biofilms forming on the surfaces accessible to visible light.

Patient 1 was a 44-year-old woman, a journalist, who was attended to at the emergency unit of Hospital Universitario Miguel Servet for two pruriginous small lesions, each with a blackish center and a hyperkeratotic halo, on the heel of the right foot and a larger lesion adjacent to the distal edge of the nail of the fifth finger, approximately 1 cm in diameter. The previous month, she had traveled to Tanzania and Zanzibar Island, performing barefoot walks on sandy beaches and rocky areas. During the trip, she had presented an episode of gener- alized pruritus. The injuries were punctured with iodine anti- septic application, and the serous material was sent to the mi- crobiology laboratory. The direct microscopic examination showed brownish structures of arthropod parts and ovoid structures (Fig. 1, left). Resolution was satisfactory.

Answer: Tunga penetrans. For both patients, considering the characteristics of the lesions, the epidemiological history of travel, and the microscopic study, a diagnosis of tungiasis was made. Tunga penetrans (sand flea) is a hematophagous flea 1 mm in length, whose habitat is warm, dry, sandy

In vitro fungistatic and fungicidal activities of voriconazole were evaluated against 114 isolates of Candida spp. MICs were determined using the NCCLS M27-A2 broth microdilution method. Minimum fungicidal concentrations (MFCs) were defined as the lowest drug concentrations that yielded ≤5 colonies (≥98% killing activity). The fungicidal activity could be only evaluated against 45.6% of the isolates (52 of 114 isolates) because trailing growth occurred, and 30 of these 52 isolates (57.69%) showed MFC values ≤1 mg/l. There was little or no fungicidal activity against C. albicans and C. tropicalis . In contrast, all C. krusei were killed by 1–2 mg/l voriconazole. In conclusion, our data show that the fungicidal effect of voriconazole against Candida spp. is species dependent.

Summary Background Tinea capitis is the most common type of dermatophytosis in children, but is uncommon in the first year of life. Objectives To review clinical, mycological and epidemiological data in a series of 10 infants aged under 1 year diagnosed as having tinea capitis in three Spanish hospitals between 1998 and 2002. Methods A retrospective case note study. Results There were six boys and four girls with a mean of age 7 months (range 1·5–12). All the children were born in Spain, but in five cases the parents were immigrants from Africa. In these cases the isolated dermatophytes were two Trichophyton tonsurans, one T. verrucosum and two Microsporum audouinii. Four autochthonous cases were caused by M. canis and one by M. audouinii (but this one was in contact with African immigrants). In two of the five cases produced by anthropophilic dermatophytes other family members were infected by the same fungus. Most cases were treated successfully with griseofulvin. Conclusions Although tinea capitis is rare in infants in their first year of life, the condition should be investigated if scaling and/or alopecia are present. A thorough epidemiological study of other family members is mandatory.

Pityriasis versicolor is a superficial infection of the stratum corneum by several yeast species formerly collectively named Malassezia furfur. The genus Malassezia has been recently enlarged with new species. With the exception of M. pachydermatis, the remaining six species have an absolute requirement in vitro for supplementation of long-chain fatty acids in media. These lipophilic yeasts comprise six species: M. furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae and M. sympodialis. The aim of this study was to establish whether there was any association between the various species of Malassezia and pityriasis versicolor lesions. Thus, we studied the isolates from 79 patients with pityriasis versicolor, both from lesions and from apparently healthy skin close to the visible lesions. In pityriasis versicolor lesions, the species most frequently isolated was M. globosa (90%), followed by M. sympodialis (41%). Almost all isolates (99%) belonged to one of these two species. The most frequent pattern was M. globosa as the sole species (58% of cases), although the association with M. sympodialis was also frequent (30%). These results confirmed M. globosa as the main agent of pityriasis versicolor and M. sympodialis as the second agent in importance. Malassezia globosa was found to be a species with high levels of esterase and lipase enzymes of probable importance in their pathogenicity.

Varon de 77 anos de edad, que presentaba alteraciones en las nas de los dedos de susmanos que habian progresado en el ultimo es. Su historia medica reciente era compleja, ya que en los ultios 10meses habia sufrido numerosos reingresos e intervenciones raiz de un primer ingreso hospitalario causado por una pancreatiis aguda litiasica. Asi, sufrio sucesivas complicaciones infecciosas osocomiales, entre las que destacan una bacteriemia asociada cateter por Staphylococcus aureus resistente a meticilina, una iarrea por Clostridium difficile y un absceso de psoas secundario una espondilodiscitis. Recibio tratamientos con diferentes antiicrobianos (piperacilina-tazobactam, ceftriaxona, vancomicina, etronidazol), y tras su alta medica, se le prescribio terapia oral de antenimiento con cotrimoxazol y rifampicina, y posteriormente on linezolid durante meses. En el examen fisico el paciente mostraba decoloracion amarilenta de la lamina ungueal, y onicodistrofia en las unas de los dedos ercero y cuarto de la mano izquierda (fig. 1A). Se realizo una toma de muestras en el laboratorio de Microbioogia, consistente en el raspado de las unas afectadas y obtencion e material del pliegue ungueal. Se sembraron en agar sangre, gar Dixon modificado, agar Sabouraud (SDA) y CHROMagar. En as muestras ungueales, la vision directa con KOH al 40% y la vision on blanco de calcofluor fueron positivas, revelando gran numero e levaduras ovales, compatibles conMalassezia (fig. 2B). En el culivo, se obtuvieron levaduras compatibles conCandida spp. en todas as muestras, pero fundamentalmente en la muestra del pliegue ngueal; mientras que unas levaduras lipofilas, compatibles con alassezia, crecieron en gran cantidad en el agar Dixon sembrado on las escamas ungueales (fig. 2A). La identificacion de espe

Resumen Antecedentes Las onicomicosis constituyen una de las infecciones micoticas mas prevalentes en la poblacion y con una mayor tasa de fracasos terapeuticos. La terapia fotodinamica (TFD) esta basada en el uso de sustancias fotosensibilizantes que, activadas con una luz de adecuada longitud de onda y en presencia de oxigeno, generan especies reactivas del oxigeno que ocasionan la muerte celular. En la actualidad se utiliza con exito en el tratamiento del cancer cutaneo no-melanocitario y se esta experimentando en otras aplicaciones, como las infecciones superficiales. Objetivos Conocer la efectividad de la TFD con metil-aminolevulinato (MAL) en un caso de onicomicosis por mohos. Metodos Una mujer de 75 anos de edad presentaba una onicomicosis por Acremonium sclerotigenum en el 5.° dedo de la mano. Fue tratada con 3 sesiones de TFD con MAL y lampara de diodo de 630 nm (Atkilite®) (37 J/cm2) separadas por 15 dias. Resultados La paciente alcanzo la cura micologica y clinica permaneciendo asintomatica tras 12 meses de seguimiento. Conclusiones Este caso ilustra la posible utilidad de la TFD-MAL en onicomicosis. Su efectividad, carencia de efectos adversos y satisfaccion por parte de medicos y pacientes hace pensar en un futuro prometedor de esta terapeutica en el manejo de las infecciones fungicas superficiales como las onicomicosis.

Summary Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole-resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole-resistant and the azole-susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light-emitting diode light fluences (18 and 37 J cm−2). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log10 CFU reduction (0.5 McFarland) was 0.62 μmol l−1 for most strains, whereas for DMMB it ranged between 1.25 and 2.5 μmol l−1. Increasing the fluence to 37 J cm−2 allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log10 reduction (4 McFarland). H2O2 was the main phototoxic species involved in the fungicidal effect of HYP-aPDT whereas 1O2 was more important for DMMB-based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.

One hundred and twenty lipid dependent Malassezia spp. isolates were obtained from the clinically normal skin of 38 healthy adult volunteers by swabbing three different body sites (back, chest and scalp). Ninety-six percent of these strains could be grouped into three biotypes on the basis of microscopic, cultural, metabolic and biochemical (catalase, esculin and lipase (C-14)) characteristics. The differential features were simple to determine and easily reproduced. Moreover, the three biotypes were referable to the species M. globosa (biotype 1), M. sympodialis (biotype 2) and M. restricta (biotype 3). Based on their microscopic features, cultural properties and body site locations, we suggest that biotype 1/ M. globosa corresponds to the description of Pityrosporum orbiculare (round yeast cells with a narrow base, very frequently found on the upper trunk), and biotype 3/ M. restricta corresponds to the concept of P. ovale (oval yeast cells with a broad budding base, located mainly on the scalp). Pleomorphic biotype 2/ M. sympodialis, most frequently found in the back, does not clearly fit into any of the Pityrosporum species.

Chronic necrotizing pulmonary aspergillosis is not common and usually involves mildly immunosuppressed patients. We present a case of a 58-year-old man with a history of mining-related pneumoconiosis and corticosteroid therapy who developed bilateral pulmonary infiltrates and subsequent cavitation. The patient was treated at first as having community-acquired pneumonia and was only belatedly diagnosed as suffering from aspergillosis after Aspergillus fumigatus precipitins appeared in blood and the same fungus grew from bronchoalveolar lavage fluid. A transthoracic needle biopsy revealed fungal filaments present in material extracted from a pulmonary lesion that was visible on scans. Treatment with amphotericin B, begun at the time that aspergillosis was diagnosed, proved to be ineffective, as was a later change to amphotericin B lipid complex. The diagnosis was confirmed at necropsy.

Nine staphylococcal strains of human and animal origin with a lincomycin-resistant/erythromycin-susceptible phenotype and carrying vga genes were characterised to determine the genetic elements involved in the dissemination of these uncommon resistance genes. These strains were typed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and/or spa typing. Antimicrobial susceptibility was studied by disk diffusion and agar dilution methods. Presence of the genes lnu(A), lnu(B), vga(A), vga(A)v, vga(B), vga(C), vga(E), lsa(B) and cfr was studied by PCR. Transformation experiments were carried out in all strains, and the plasmid or chromosomal gene location was determined by Southern blot analysis. Genetic environments of the vga genes were analysed by PCR mapping or inverse PCR and sequencing. Five meticillin-resistant Staphylococcus aureus (MRSA) ST398 strains and three Staphylococcus epidermidis strains harboured the gene vga(A), and one MRSA-ST8 strain contained the gene vga(A)v. One MRSA-ST398 strain, which also contained the gene lnu(A), showed the highest minimum inhibitory concentration (MIC) to lincomycin. The vga(A)v-positive strain presented lower MIC values than the vga(A)-positive strains. Presence of the pVGA plasmid was confirmed in two MRSA-ST398 strains. Four novel vga(A)-carrying plasmids were detected: pUR2355 (in two MRSA and one meticillin-susceptible S. epidermidis); pUR4128 (one MRSA); pUR3036 [one meticillin-resistant S. epidermidis (MRSE)]; and pUR3937 (one MRSE). The plasmid pUR4128 was very similar to pUR2355. Plasmids pUR3036 and pUR3937 were related and were very similar to plasmid pSE-12228-06. The gene vga(A)v was located in a transposon analogous to Tn5406. Therefore, four novel vga(A)-carrying plasmids and a variant of Tn5406 were identified in this study.

The virulence factor gliotoxin (GT) and its inactive derivative, bis(methylthio)gliotoxin (bmGT), are produced by pathogens of the genus Aspergillus. Here we report the detection of GT and bmGT in serum of humans at risk of invasive aspergillosis (IA) as well as in cultures of fungal isolates derived from patients with proven infection with A. fumigatus. Although both compounds are readily recoverable from spiked human serum or plasma, only bmGT is retained in whole blood, indicating that bmGT may be the better marker for in vivo detection. Accordingly, bmGT was found more frequently than GT in samples from patients at risk of IA and in cultures of clinical isolates of A. fumigatus. In some cases, bmGT was detected before mycologic evidence of infection was gained. Importantly, neither GT nor bmGT was found in serum from healthy donors or from neutropenic patients without any sign of infection. Thus, bmGT presence might provide a more reliable indicator of A. fumigatus infections than GT. Due to its simplicity and sensitivity, a diagnostic technology based on this test could be easily adopted in clinical laboratories to help in the diagnosis of this often fatal fungal infection.

Hypericin is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin PDT on various Candida spp., assessing its photocytotoxicity to keratinocytes (HaCaT) and dermal fibroblasts (hNDF) to determine possible side effects. A 3 log fungicidal effect was observed at 0.5 McFarland for two Candida albicans strains, Candida parapsilosis and Candida krusei with hypericin concentrations of 0.625, 1.25, 2.5 and 40 μm, respectively, at a fluence of 18 J cm(-2) (LED lamp emitting at 602 ± 10 nm). To obtain a 6 log reduction, significantly higher hypericin concentrations and light doses were needed (C. albicans 5 μM, C. parapsilosis 320 μM and C. krusei 320 μM; light dose 37 J cm(-2)). Keratinocytes and fibroblasts can be preserved by keeping the hypericin concentration below 1 μm and the light dose below 37 J cm(-2). C. albicans appears to be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.

Objectives: The clonal lineages, resistance mechanisms and virulence traits of tetracycline-resistant (Tet R) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in a Spanish hospital during 2009 and 2010 were investigated. Methods: Fifty-two Tet R MRSA strains from unrelated patients were included in this study. Susceptibility to 26 antimicrobial agents was determined and 24 resistance genes were tested for by PCR. The sequences of the genes grlA and gyrA were analysed in all ciprofloxacin-resistant MRSA isolates. For all strains, spa, SCCmec and agr typing was implemented. Multilocus sequence typing was performed for 16 representative strains of the different spa types. The presence of the genes tst, lukF/lukS-PV, eta, etb, etd and cna was investigated by PCR. Results: Fifteen different spa types, four of them new ones, were detected among the 52 strains, being associated with the following clonal complexes (CCs): CC398 (67.3%), CC1 (11.5%), CC5 (11.5%) and CC8 (9.6%). A novel sequence type (ST), ST2077, belonging to CC398 was identified. Most MRSA CC398 strains were typed as SCCmecV-agrI. In addition to β-lactam resistance, isolates showed resistance to (gene/number of strains): tetracycline [tet(K)/36, tet(L)/8 and tet(M)/48], macrolides and lincosamides [erm(B)/6, erm(C)/25, erm(T)/2, msr(A)/msr(B)/4 and mph(C)/4], aminoglycosides [aac(6')-Ie-aph(2')-Ia/8, ant(4')-Ia/13 and aph(3')-IIIa/15], trimethoprim [dfrS1/2 and dfrK/3] and mupirocin (mupA/3). Strains investigated for mutations mediating quinolone resistance revealed an S80F exchange in GrlA and different changes in GyrA. Three strains were Panton-Valentine leucocidin-positive (ST8 and ST94) and 41 strains were cna-positive. All MRSA isolates were negative for the genes tst, eta, etb and etd. Conclusions: Tetracycline resistance could be a good marker for MRSA CC398, although this resistance can also be found in other lineages. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Recent epidemiological surveillance studies have reported an increase in fungaemia caused by non-Candida albicans species, as well as a decrease in fluconazole susceptibility.To evaluate changes in the epidemiology of fungaemia in Spain comparing data from a new surveillance epidemiological study conducted in 2009 with a previous study carried out from 1997 to 1999 (Pemán J, et al. Eur J Clin Microbiol Infect Dis. 2005).From January 2009 to February 2010, 44 Spanish hospitals participated in a prospective multicentre fungaemia surveillance study to ascertain whether there have been changes in the epidemiology and fluconazole susceptibility. Susceptibility was determined by the colorimetric method Sensititre Yeast One. Demographic and clinical data and the first isolate of each episode were gathered.A total of 1,377 isolates from 1,357 fungaemia episodes were collected, 46.7% from patients older than 64years and 8.6% from children less than 1 year old. C. albicans (44.7%), Candida parapsilosis (29.1%), Candida glabrata (11.5%), Candida tropicalis (8.2%), and Candida krusei (1.9%) were the most frequent species isolated. Distribution varied with the geographical area. C. albicans incidence has increased significantly in the last 10years in Cataluña (39.1 vs. 54.7%, P=0.03) and decreased in the Valencian Community (49.1 vs. 34.6%, P=0.002) and Extremadura (58.3 vs. 20%, P=0.01). Susceptibility to fluconazole was similar for all geographical areas, although resistance in C. albicans was ten times greater for patients aged more than 64years. The overall rate of fluconazole resistance (MIC32 mg/L) has decreased with respect to that obtained 10years ago (3.7 vs. 2.5%) mainly in C. albicans (3 vs. 1.6%).In the last ten years, species distribution and fluconazole susceptibility have not significantly changed, although a lower rate of fluconazole resistance has been observed. Species distribution varies with hospital, hospitalization Unit and geographical area.

Onychomycosis is one of the most prevalent fungal infections in the population with a higher rate of treatment failures. Photodynamic therapy (PDT) is based on the use of photosensitizers with light of an accurate wavelength, and in the presence of oxygen, reactive oxygen species are produced which induce cell death. PDT is widely used to treat non-melanoma skin cancer, and other indications are being investigated, especially in superficial infections.To determine the effectiveness of PDT in a case of onychomycosis caused by moulds.A 75-year-old woman was diagnosed with onychomycosis caused by Acremonium sclerotigenum in the 5th finger. She was treated with 3 sessions of PDT with methyl-aminolevulinate (MAL) separated by 15 days.The patient achieved mycological and clinical cure and remains asymptomatic after 12 months of follow up.This case illustrates the potential usefulness of MAL-PDT for onychomycosis. Its effectiveness, lack of adverse effects and satisfaction on the part of physicians and patients, suggest a promising future for this therapy in the management of superficial fungal infections, such as onychomycosis.

Candidemia studies have documented geographic differences in rates and epidemiology, underscoring the need for surveillance to monitor trends. We conducted prospective candidemia surveillance in Spain to assess the incidence, species distribution, frequency of antifungal resistance, and risk factors for acquiring a Candida infection. Prospective laboratory-based surveillance was conducted from June 2008 to June 2009 in 40 medical centers located around the country. A case of candidemia was defined as the isolation of a Candida species from a blood culture. Incidence rates were calculated per 1,000 admissions. Antifungal susceptibility tests were performed by using broth microdilution assay according to the guidelines of the Clinical and Laboratory Standards Institute. We detected 984 cases, for an overall incidence of 1.09 cases per 1,000 admissions. The crude mortality was 20.20%. Candida albicans was the most common species (49.08%), followed by C . parapsilosis (20.73%), C. glabrata (13.61%), and C. tropicalis (10.77%). Overall, decreased susceptibility to fluconazole occurred in 69 (7.01%) incident isolates. Antifungal resistance was rare, and a moderate linear correlation between fluconazole and voriconazole MICs was observed. This is the largest multicenter candidemia study conducted to date and shows the substantial morbidity and mortality of candidemia in Spain.

International audience; We report a case of cavitary pneumonia caused by N. otitidiscaviarum in a man with diabetes mellitus and thrombocytopenia treated with systemic corticosteroid. Taxonomic identification involved phenotypic testing and molecular identification that was carried out by DNA sequencing of the 16SrRNA gene.

We report a case of chromoblastomycosis in a 67-year-old female farmer, which involved a large (20 x 30 cm) cicatricial erythematous plaque on the inner side of her right thigh. The lesion was initially a small nodule which gradually extended over 36 years. Direct microscopic examination revealed a granulomatous lesion with muriform cells surrounded by giant cells. The mould recovered in cultures was dark olivaceous and identified as Fonsecaea monophora by ribosomal internal transcribe spacer (ITS) sequence data. The lesion was successfully cured after 4 months treatment with itraconazole, but there was a relapse.

Here we report on two isolates of Candida glabrata recovered from urine samples collected from of an Intensive Care Unit patient. D1/D2 and ITS 1+2 rDNA sequence analysis confirmed its identification. The isolates were cholesterol dependent and resistant to Amphotericin B.

The aim of this study was to characterize the resistance mechanism in four clinical and five intestinal vancomycin-resistant Enterococcus faecium strains with VanB phenotype recovered from unrelated patients confined in two Spanish hospitals and to determine their clonal relationships. MIC values for vancomycin and teicoplanin were 16–32 and 0.5 μg ml−1, respectively. The mechanism of vancomycin resistance, as well as the genetic environment of the implicated gene, was analysed by PCR and sequencing. The vanB2 gene was detected in all nine E. faecium strains and the intergenic vanS B–Y B region showed the characteristic mutations of the vanB2 subtype. Two possibly related PFGE patterns, A (seven strains) and B (two strains), were distinguished among these enterococci. The vanX B–ORFC intergenic region was amplified in the nine strains and two amino acid changes were detected in the protein encoded by the vanX B gene in strains of pattern A with respect to those of pattern B. The vanB2 gene cluster was integrated into Tn5382 in all nine strains, being pbp5 gene-linked to this transposon. The ant(6′)-Ia, aph(3′)-IIIa and erm(B) genes were also detected in all of the strains. Both isolates with PFGE pattern B contained the esp gene. In summary, vanB2-containing E. faecium strains with indistinguishable PFGE patterns were recovered from seven patients from two Spanish hospitals.

Summary Background: Microsporum canis is a ubiquitous dermatophyte that commonly causes human infections. Since contact with infected animals is the usual way of infection, tracing its source is an essential preventive measure. Objective: To type isolates of M. canis from human patients whose skin was affected, and from some animals (dogs and cats) that were closely associated to the patients. Methods: The inter-single-sequence-repeat-PCR (ISSR-PCR) technique has been used for typing 24 isolates of M. canis. Seventeen isolates tested were from human patients, 5 from cats and 1 from a dog Results: A total of 21 genotypes were identified. The same genotype was found infecting a patient and a cat that was living closely with him, but another member of the same family proved to be infected with two genotypes different from that. Clinical specimens from two patients had been contaminated with the same genotype, probably in the laboratory where the samples were handled. Conclusion: These results demonstrate that ISSR-PCR polymorphism is a reliable method for the identification of the M. canis strains.

Liver disease is very common in patients with HIV infection. The association between chronic infection with hepatotropic viruses and being a target for other diseases, such as opportunistic infections and tumors, nonalcoholic fatty liver disease (NAFLD), and toxicity to drugs, including antiretroviral therapy (ART), results in liver disease being a leading cause of morbidity and mortality in these persons. The aim of this study was to determine the prevalence of chronic hypertransaminasemia (CHT) in a cohort of patients with HIV infection but no coinfection and to examine the factors associated with CHT. We undertook a cross-sectional, case-control study of all HIV-infected patients who regularly attended the infectious diseases office of our center from March to August 2009. A case was defined as any patient with glutamate pyruvate transaminase (GPT) values above normal (78 IU/L) at least 2 consecutive revisions (3 months or more between the 2 measurements). Patients were excluded if they had a positive serology to a hepatotropic virus or opportunistic events during the previous month. A control was defined as a patient attending the office paired for age (+5 years) and sex with a case and who had a normal GPT value, using the same exclusion criteria as for the cases. Hepatotoxicity of ART was defined as CHT due to ART, and to make this diagnosis, other causes of elevated GPT values were ruled out in all cases and NAFLD by the presence of steatosis in an abdominal ultrasound in patients consuming

Pityriasis versicolor is a superficial infection of the stratum corneum by several yeast species formerly collectively named Malasseziafurfur. The genus Malassezia has been recently enlarged with new species. With the exception of M. pachydermatis, the remaining six species have an absolute requirement in vitro for supplementation of long-chain fatty acids in media. These lipophilic yeasts comprise six species: M. furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae and M. sympodialis. The aim of this study was to establish whether there was any association between the various species of Malassezia and pityriasis versicolor lesions. Thus, we studied the isolates from 79 patients with pityriasis versicolor, both from lesions and from apparently healthy skin close to the visible lesions. In pityriasis versicolor lesions, the species most frequently isolated was M. globosa (90%), followed by M. sympodialis (41%). Almost all isolates (99%) belonged to one of these two species. The most frequent pattern was M. globosa as the sole species (58% of cases), although the association with M. sympodialis was also frequent (30%). These results confirmed M. globosa as the main agent of pityriasis versicolor and M. sympodialis as the second agent in importance. Malassezia globosa was found to be a species with high levels of esterase and lipase enzymes of probable importance in their pathogenicity.

[ES] Fundamentos: Los géneros Salmonella y Campylobacter son los principales agentes bacterianos causantes de enteritis en el hombre en los países desarrollados. El objetivo del presente trabajo es estudiar las características descriptivas de los casos de infección por Salmonella y por Campylobacter y describir su tendencia y variación estacional en la provincia de Huesca para el período 1996-1999. Método: Las fuentes de información han sido los Laboratorios Microbiológicos Hospitalarios (LMH) que realizan analíticas de coprocultivo tanto de enfermos ambulatorios como hospitalarios. Se toma el municipio como unidad de referencia para representar la distribución espacial. Para el estudio de la tendencia se utiliza un modelo determinista multiplicativo, agrupando los datos en períodos cuatrisemanales. Resultados: Se han recogido un total de 781 casos de infección por Salmonella y 654 casos de infección por Campylobacter, con unas tasas medias anuales de 95 y 79,5 105 habitantes respectivamente. La enteritis por Campylobacter se presenta mayoritariamente en menores de 5 años (73,4 %), contra el 35,5 % para la salmonelosis. La situación se invierte en el caso de porcentaje de ingreso hospitalario; así, el ingreso por salmonelosis es casi 5 veces superior. Ambos géneros presentan una clara estacionalidad, con un pico pronunciado en el mes de agosto. Conclusiones: Se observa una tendencia ascendente en el número de aislamientos positivos para ambos microorganismos y una clara estacionalidad en la época estival. [EN] Background: The Salmonella and Campylobacter genera are the main bacterial agents causing enteritis in humans in the developed countries. The purpose of this paper is to study the descriptive characteristics of the cases of infection by Salmonella and by Campylobacter and to describe their trend and seasonal variance in the province of Huesca for the 1996-1999 period. Method: The sources of information were the Hospital Microbiological Laboratories (HML's) which conduct coproculture analyses of both outpatients and inpatients. The municipality is taken as the reference unit for plotting the space distribution. For the study of the trend, a multiplicative determinist model is used for grouping the data into four-week periods. Results: A total of 781 cases of infection by Salmonella and 654 cases of infection by Campylobacter have been included, respectively showing mean annual rates of 95 and 79.5 x 10(5) inhabitants. The enteritis by Campylobacter occurs mainly in children under five years of age (73.4%), as compared to the 35.5% of the cases of salmonellosis. The situation is the reverse in the case of the hospital admission percentages, hospitalization due to salmonellosis hence being nearly five times greater. Both of these genera are of a clear-cut seasonal nature, showing a marked peak in the month of August. Conclusions: A growing trend in the number of positive isolations for both of these organisms and a clear seasonality in the summertime have been found.

Summary This report presents a rare case of tinea capitis caused by Trichophyton soudanense and Microsporum audouinii in a 31-year-old woman from Senegal. The patient showed atrophic skin lesions causing cicatricial alopecia, scarring being caused by two aetiological agents uncommon in Spain.