Validation of an expanded, in-house library and an optimized preparation method for the identification of fungal isolates using MALDI-TOF mass spectrometry.

The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of moulds using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in a multicenter context. For that purpose, three Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5% and 94.8% and the score values were 1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (F. proliferatum, n = 1; T.interdigitale, n = 2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species.

MALDI-TOF mass spectrometry has been improved as a diagnostic method for the rapid and reliable identification of filamentous fungi by means of the creation of an expanded database containing reference protein spectra of the most clinically impacting fungal species.

Two outbreaks of Listeria monocytogenes infection, Northern Spain.

In the province of Gipuzkoa, Spain (≈700,000 inhabitants), 7-12 episodes of human listeriosis were recorded annually during 2009-2012. However, during January 2013-February 2014, 27 episodes were detected, including 11 pregnancy-associated cases. Fifteen cases in 2 epidemiologically unrelated outbreaks were caused by a rare type of Listeria monocytogenes, sequence type 87 serotype 1/2b.

Spread of blaCTX-M-9 and Other Clinically Relevant Resistance Genes, Such as mcr-9 and qnrA1, Driven by IncHI2-ST1 Plasmids in Clinical Isolates of Monophasic Salmonella enterica Serovar Typhimurium ST34.

The monophasic 4,[5],12:i:-variant of Salmonella enterica serovar Typhimurium with sequence type ST34 has become one of the most prevalent non-typhoidal salmonellae worldwide. In the present study, we thoroughly characterized seven isolates of this variant detected in a Spanish hospital and selected based on cefotaxime resistance and cefoxitin susceptibility, mediated by blaCTX-M-9. For this, conventional microbiological techniques, together with whole genome sequencing performed with the Illumina platform, were applied. All selected isolates carried the resistance region RR or variants therein, and most also contained the SGI-4 genomic island. These chromosomal elements, typically associated with monophasic S. Typhimurium ST34, confer resistance to traditional antibiotics (ampicillin, streptomycin, sulfonamides, and tetracycline) and tolerance to heavy metals (mercury, silver, and copper). In addition, each isolate carried a large IncHI2-ST1 conjugative plasmid containing additional or redundant resistance genes. All harbored the blaCTX-M-9 gene responsible for cefotaxime resistance, whereas the qnrA1 gene mediating fluoroquinolone resistance was detected in two of the plasmids. These genes were embedded in ISCR1-bearing complex class 1 integrons, specifically In60-like and In36-like. The mcr-9 gene was present in all but one of the IncHI2-ST1 plasmids found in the analyzed isolates, which were nevertheless susceptible to colistin. Most of the resistance genes of plasmid origin clustered within a highly complex and variable region. The observed diversity results in a wide range of resistance phenotypes, enabling bacterial adaptation to selective pressure posed by the use of antimicrobials.

A case of severe diarrhoea caused by Cyclospora cayetanensis in an immunocompromised patient in northern Spain.

Cyclospora cayetanensis is a parasite that causes intestinal disease that can be especially severe in immunocompromised patients. Most cases occur in tropical and subtropical areas, and in industrialized countries their diagnosis is mostly linked to international travel or the ingestion of imported food. We describe this case of severe diarrhoea in a patient with diffuse large B cell lymphoma and no epidemiological risk factors that was successfully treated with trimethoprim-sulfamethoxazole (TMP-STX). C. cayetanensis is a pathogen that should be taken into account in patients with chronic diarrhoea, especially immunocompromised patients, even when no epidemiological risk factors are present.

Clinical factors associated with a Candida albicans Germ Tube Antibody positive test in Intensive Care Unit patients.

BACKGROUND: Poor outcomes of invasive candidiasis (IC) are associated with the difficulty in establishing the microbiological diagnosis at an early stage. New scores and laboratory tests have been developed in order to make an early therapeutic intervention in an attempt to reduce the high mortality associated with invasive fungal infections. Candida albicans IFA IgG has been recently commercialized for germ tube antibody detection (CAGTA). This test provides a rapid and simple diagnosis of IC (84.4% sensitivity and 94.7% specificity). The aim of this study is to identify the patients who could be benefited by the use of CAGTA test in critical care setting. METHODS: A prospective, cohort, observational multicentre study was carried out in six medical/surgical Intensive care units (ICU) of tertiary-care Spanish hospitals. Candida albicans Germ Tube Antibody test was performed twice a week if predetermined risk factors were present, and serologically demonstrated candidiasis was considered if the testing serum dilution was ≥ 1:160 in at least one sample and no other microbiological evidence of invasive candidiasis was found. RESULTS: Fifty-three critically ill non-neutropenic patients (37.7% post surgery) were included. Twenty-two patients (41.5%) had CAGTA-positive results, none of them with positive blood culture for Candida. Neither corrected colonization index nor antifungal treatment had influence on CAGTA results. This finding could corroborate that the CAGTA may be an important biomarker to distinguish between colonization and infection in these patients. The presence of acute renal failure at the beginning of the study was more frequent in CAGTA-negative patients. Previous surgery was statistically more frequent in CAGTA-positive patients. CONCLUSIONS: This study identified previous surgery as the principal clinical factor associated with CAGTA-positive results and emphasises the utility of this promising technique, which was not influenced by high Candida colonization or antifungal treatment. Our results suggest that detection of CAGTA may be important for the diagnosis of invasive candidiasis in surgical patients admitted in ICU.

Genomic Analysis of Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 From Spanish Hospitals.

Salmonella enterica serovar Kentucky (S. Kentucky) with sequence type (ST) 198 and highly resistant to ciprofloxacin (ST198-Cip R ) has emerged as a global MDR clone, posing a threat to public health. In the present study, whole genome sequencing (WGS) was applied to characterize all Cip R S. Kentucky detected in five Spanish hospitals during 2009-2018. All Cip R isolates (n = 13) were ST198 and carried point mutations in the quinolone resistance-determining regions (QRDRs) of both gyrA (resulting in Ser83Phe and Asp87Gly, Asp87Asn, or Asp87Tyr substitutions in GyrA) and parC (with Thr57Ser and Ser80Ile substitutions in ParC). Resistances to other antibiotics (ampicillin, chloramphenicol, gentamicin, streptomycin, sulfonamides, and tetracycline), mediated by the bla TEM- 1 B , catA1, aacA5, aadA7, strA, strB, sul1, and tet(A) genes, and arranged in different combinations, were also observed. Analysis of the genetic environment of the latter resistance genes revealed the presence of multiple variants of SGI1 (Salmonella genomic island 1)-K and SGI1-P, where all these resistance genes except catA1 were placed. IS26 elements, found at multiple locations within the SGI1 variants, have probably played a crucial role in their generation. Despite the wide diversity of SGI1-K- and SGI1-P-like structures, phylogenetic analysis revealed a close relationship between isolates from different hospitals, which were separated by a minimum of two and a maximum of 160 single nucleotide polymorphisms. Considering that S. enterica isolates resistant to fluoroquinolones belong to the high priority list of antibiotic-resistant bacteria compiled by the World Health Organization, continuous surveillance of the S. Kentucky ST198-CIP R clone is required.

Molecular epidemiology of G12 rotavirus strains during eight consecutive epidemic seasons in the Basque Country (North of Spain), 2010-2018.

G12 rotaviruses were first detected in Spain (Gipuzkoa province) in December 2004. After four years with no detections, G12 strains re-emerged in the 2010-2011 epidemic season, when the first European epidemic circulation of this genotype was observed in Gipuzkoa. G12 rotaviruses were also the dominant strains in 2011-2012, 2014-2015 and 2015-2016 epidemic seasons and were sporadically detected in the remaining periods (2012-2014 and 2016-2018). The most frequently detected G-type between 2010 and 2018 was G12 (29.9%) rather than G1 rotavirus (17.8%), which historically had been the dominant genotype in our setting (1989-2009 period) and globally. Phylogenetic analysis of the VP4 and VP7 genome segments showed chronologically ordered clades, which spanned between two to four consecutive seasons. Overall, the circulating G12 rotavirus strains in Gipuzkoa between 2010 and 2018 belonged to four clades, which emerged in early 2009 potentially due to at least four importations from other regions followed by local evolution. Whole genome analysis of 16 G12 strains detected from 2010 to 2018 revealed a Wa-like genotype constellation, G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, and also showed that G12 strains from Gipuzkoa were similar to those identified in other countries. These findings suggest circulation of G12 rotavirus strains in different parts of the world leading to high genetic diversity.

Activities of fluconazole and voriconazole against bloodstream isolates of Candida glabrata and Candida krusei: a 14-year study in a Spanish tertiary medical centre.

The aim of this study was to evaluate the in vitro activities of voriconazole and fluconazole against Candida glabrata and Candida krusei isolated from blood during a 14-year period (1990-2003) at the tertiary care hospital of Cruces (Barakaldo, Spain). The in vitro activities of fluconazole and voriconazole against 28 isolates of C. glabrata and 15 isolates of C. krusei were determined by the Clinical and Laboratory Standards Institute disk diffusion method. Of the 28 C. glabrata isolates tested, 24 (85.7%) were susceptible (S) to fluconazole, 2 (7.1%) were susceptible dose-dependent (S-DD) and 2 (7.1%) were resistant (R). All C. krusei isolates were classified as R to fluconazole. Resistance to voriconazole was observed in one isolate each of C. glabrata (3.6%) and C. krusei (6.7%), and one isolate of each species was S-DD. These results were confirmed by the Sensititre YeastOne and Etest methods, with good comparative results. Voriconazole was very active in vitro against C. glabrata and C. krusei blood isolates and the resistance observed was not related to the introduction of voriconazole in the therapeutic schedule of the hospital. These facts support the usefulness of voriconazole as a therapeutic tool for candidaemia caused by these species.

Emergence and spread of G3P[8] rotaviruses possessing an equine-like VP7 and a DS-1-like genetic backbone in the Basque Country (North of Spain), 2015.

In March 2015, an atypical G3P[8] rotavirus with an equine-like VP7 gene was detected in Gipuzkoa (Basque Country, Spain) and spread contributing significantly to the seasonal epidemic. The strain was identified in fecal samples collected from 68 patients, mainly children from rural and urban settings with acute gastroenteritis, representing 14.9% of the 455 rotavirus strains genotyped between July 2014 and June 2015. Seven patients (10.3%) were hospitalized. Full genome analysis of six of these strains revealed a DS-1-like genotype constellation, G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and showed that most genome segments shared the highest nucleotide sequence identity with strains isolated in Japan, Thailand, Australia and the Philippines. The strains of Gipuzkoa were similar to novel G3P[8] reassortant rotaviruses with an equine-like VP7 gene and a DS-1-like genetic backbone that emerged in the Asia-Pacific Region in 2013. The study highlights the circulation of these atypical rotaviruses outside the Asia-Pacific Region of origin, and their emergence in a European Region. Due to their unusual genotype constellation, these strains pose a challenge for the rotavirus strain surveillance, since G-/P-typing, the most commonly used classification system, cannot identify this type of intergenogroup reassortants.

Listeriosis. Estudio de 16 años en un hospital terciario en España / Listeriosis. A 16-year survey in a tertiary hospital in Spain

Objetivo: Revisar los casos de listeriosis recogidos en nuestro hospital durante los últimos 16 años. Métodos: Se revisaron retrospectivamente los casos de listeriosis recogidos en el Hospital de Cruces (Vizcaya, España), desde enero de 1990 hasta enero de 2006. Los pacientes se dividieron en 3 grupos: adultos no gestantes (38 pacientes, 63.3%), infección perinatal (7 gestantes y 11 neonatos, 30%) e infección pediátrica no perinatal (4 niños, 6.6%). Resultados: En el grupo de adultos el 89.4% tenía algún factor predisponente, predominando la neoplasia y la hepatopatía. En 17/38 hubo afectación del SNC (13 meningitis y 4 encefalitis) y en 16/38 sepsis. Otras manifestaciones fueron: peritonitis bacteriana espontánea (4 casos) y pericarditis (1 caso). La mortalidad fue del 29%. En los pacientes oncológicos la sepsis fue más frecuente que la enfermedad neurológica (p = 0.05) y la mortalidad fue superior que en otros enfermos crónicos (p = 0.003). En las gestantes la evolución fue buena y 4/7 tuvieron recién nacidos a término sanos. En los neonatos la infección fue de comienzo precoz en 10 casos, con diagnóstico de sepsis en el 60% y de meningitis en 40%. Fallecieron 2 neonatos (18%), ambos prematuros y con sepsis de comienzo precoz. En el grupo de listeriosis pediátrica no perinatal hubo 3 casos con meningitis y uno con invaginación intestinal y aislamiento de Listeria en adenopatía mesentérica, todos con evolución favorable. La mortalidad global fue del 21.6%. El serotipo predominante fue el 4b. Conclusiones: Las infecciones por L. monocytogenes se asocian a una alta morbi-mortalidad. Los factores predisponentes han sido determinantes en la evolución de los pacientes.