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Clin Microbiol Infect 2012; 18: E204-E213 Abstract Underdiagnosis of Clostridium difficile infection (CDI) because of lack of clinical suspicion or the use of non-sensitive diagnostic techniques is a known problem whose real magnitude has not yet been quantified. In order to estimate the extent of this underdiagnosis, we performed C. difficile cultures on all unformed stool specimens sent-irrespective of the type of request-to a series of laboratories in Spain on a single day. The specimens were cultured, and isolates were characterized at a central reference laboratory. A total of 807 specimens from 730 patients aged ≥2 years were selected from 118 laboratories covering 75.4% of the Spanish population. The estimated rate of hospital-acquired CDI was 2.4 episodes per 1000 admissions or 3.8 episodes per 10 000 patient-days. Only half of the episodes occurred in patients hospitalized for >2 days. Two of every three episodes went undiagnosed or were misdiagnosed, owing to non-sensitive diagnostic tests (19.0%) or lack of clinical suspicion and request (47.6%; mostly young people or non-hospitalized patients). The main ribotypes were 014/020 (20.5%), 001 (18.2%), and 126/078 (18.2%). No ribotype 027 strains were detected. Strains were fully susceptible to metronidazole and vancomycin. CDI was underdiagnosed in diarrhoeic stools in a high proportion of episodes, owing to the use of non-sensitive techniques or lack of clinical suspicion, particularly in people aged <65 years or patients with community-acquired diarrhoea. C. difficile toxins should be routinely sought in unformed stools of any origin sent for microbiological diagnosis. The ribotype 027 clone has not yet disseminated in Spain. [ABSTRACT FROM AUTHOR]

Highlights the Fourth International Meeting on Bone Diagenesis which took place in Teruel, Spain in May 2000. Organizers of the meeting; Panel members.

Objective: Our previous genotyping studies suggest that some anatomical locations act as reservoirs of genotypes that may cause further candidemia, since we found identical genotypes in gastrointestinal tract or catheter tip isolates and blood cultures, in contrast, we did not find blood culture genotypes in vagina samples. We observed that some genotypes can be found in blood cultures more frequently than others, some of them being called widespread genotypes because have been found in unrelated patients admitted to different hospitals. The presence of widespread genotypes may be more frequently found because of their predisposition to cause candidemia. It is unclear whether genotypes colonizing other anatomical sites different from the gastrointestinal tract can also be detected in this way; we studied C. albicans , C. parapsilosis , and C. tropicalis colonizing genotypes to assess what proportion could be found in blood cultures and the proportion of widespread genotypes., Methods: The isolates (n= 640 Candida isolates from 323 patients) studied herein were obtained from samples processed at the Clinical Microbiology and Infectious Diseases Department of the Gregorio Marañón Hospital (Madrid, Spain) from July 1, 2016, to June 30, 2019. C. albicans (n=486), C. parapsilosis (n=94), and C. tropicalis (n=60) isolates were genotyped using species-specific microsatellite markers and sourced from blood (n=120) and colonized anatomical sites (n=520; catheter [n=50], lower respiratory tract [n=227], skin/mucosa [n=132], and urinary tract [n=111]). Isolates with identical genotypes were those presenting the same alleles for all markers or with only differences at one locus of a given marker. Identical genotypes were further classified as a match (identical genotype found in different groups of samples from a given patient) or as a cluster (identical genotype found in ≥2 patients). Finally, singletons were genotypes detected once. The genotypes found were then compared with our in-house database containing 587 blood genotypes from patients admitted to the Gregorio Marañón Hospital (2007-2023) to assess the proportion of genotypes found in colonized samples that were also found in blood cultures. Moreover, since some of our in-house database genotypes had been tagged as widespread genotypes, we compared the proportions of widespread genotypes as well as the proportions of matches, clusters, and patients involved in clusters found among exclusively colonizing genotypes, exclusively blood culture genotypes, and both colonizing and blood culture genotypes using a standard binomial method., Results: Intra-patient analysis was conducted exclusively on those patients (n=225; 69.7%) who had ≥2 isolates from a given species; the proportion of patients with matches was lower in exclusively colonized patients than in patients with candidemia and colonizing genotypes (87.3% vs. 94.1%; p = 0.126). Inter-patient analysis was conducted considering all patients (n=323) and isolates from groups 1, 2, and 3 (n=640). Overall, we detected 341 genotypes, of which 320 were singletons and 21 were clusters (6.16%). Clusters involving blood cultures and colonizing isolates sourced from catheter tips (14.6%), skin and mucosa (7.5%), urine (7.4%), and lower respiratory tract (4.6%). Cluster-involved patients had not been admitted to the same ward at the same time. Of the 290 colonizing genotypes, 91 (31.1%) were also found in blood cultures, the highest proportion being C. parapsilosis ( p < 0.05); proportions of identical genotypes found in blood cultures and catheter tips were higher than those found in blood cultures and other colonized samples (79.2% vs. 26.7%; p < 0.001). Widespread genotype ratios were significantly higher among genotypes found in both blood and colonized samples than among genotypes found exclusively in either blood culture or other colonizing genotypes (31.9% vs. 7.1% vs. 3.7%, respectively; p < 0.001)., Conclusion: We observed that 94% of patients with candidemia were colonized by a genotype causing the infection; likewise, a total of 31% of colonizing genotypes were detectable in blood cultures. Finally, identical genotypes found in both colonized samples and blood cultures had a higher probability of being widespread., Competing Interests: JG has received funds for participating in educational activities organized on behalf of Gilead, Pfizer, Mundipharma, and MSD; he has also received research funds from FIS, Gilead, F2G, Scynexis, Mundipharma, and Cidara outside the submitted work. PE has received funds for participating in educational activities organized on behalf of Gilead and has also received research funds from FIS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mesquida, Martín-Rabadán, Alcalá, Burillo, Reigadas, Muñoz, Guinea and Escribano.)

Centers for Disease Control and Prevention guidelines consider SARS-CoV-2 reinfection when sequential COVID-19 episodes occur >90 days apart. However, genomic diversity acquired over recent COVID-19 waves could mean previous infection provides insufficient cross-protection. We used genomic analysis to assess the percentage of early reinfections in a sample of 26 patients with 2 COVID-19 episodes separated by 20-45 days. Among sampled patients, 11 (42%) had reinfections involving different SARS-CoV-2 variants or subvariants. Another 4 cases were probable reinfections; 3 involved different strains from the same lineage or sublineage. Host genomic analysis confirmed the 2 sequential specimens belonged to the same patient. Among all reinfections, 36.4% involved non-Omicron, then Omicron lineages. Early reinfections showed no specific clinical patterns; 45% were among unvaccinated or incompletely vaccinated persons, 27% were among persons <18 years of age, and 64% of patients had no risk factors. Time between sequential positive SARS-CoV-2 PCRs to consider reinfection should be re-evaluated.

We have been monitoring the antifungal resistance in Candida parapsilosis isolates collected from inpatients at Madrid metropolitan area hospitals for the last 3 years. The study aimed to elucidate the presence of fluconazole-resistant C. parapsilosis genotypes in Madrid. From January 2019 to December 2021, a total of 354 C. parapsilosis isolates ( n  = 346 patients) from blood (76.6%) or intraabdominal samples were collected and genotyped using species-specific microsatellite markers. Antifungal susceptibilities to amphotericin B, the triazoles, micafungin, anidulafungin, and ibrexafungerp were performed according to EUCAST E.Def 7.3.2; the ERG11 gene was sequenced in fluconazole-resistant isolates. A total of 13.6% ( n  = 48/354) isolates (one per patient) were found to be resistant to fluconazole and non-wild-type to voriconazole but fully susceptible to ibrexafungerp. Resistant isolates were mostly sourced from blood ( n  = 45/48, 93.8%) and were detected in five hospitals. Two hospitals accounted for a high proportion of resistant isolates ( n  = 41/48). Resistant isolates harbored either the Y132F ERG11p amino acid substitution ( n  = 43) or the G458S substitution ( n  = 5). Isolates harboring the Y132F substitution clustered into a clonal complex involving three genotypes (one genotype accounted for n  = 39/43 isolates) that were found in four hospitals. Isolates harboring the G458S substitution clustered into another genotype found in a fifth hospital. C. parapsilosis genotypes demonstrating resistance to fluconazole have been spreading across hospitals in Madrid, Spain. Over the last 3 years, the frequency of isolation of such isolates and the number of hospitals affected is on the rise.

Objective: To assess the impact of COVID-19 at nine nursing homes in Madrid, Spain, during the first wave of COVID-19 infection and lockdown period when preventive measures were taken to avoid transmission among residents., Methods: Nine hundred forty-two residents and 846 staff members from nine nursing homes participated in the study (April 18 to June 20, 2020). All participants were tested for SARS-CoV-2 in the nasopharynx by PCR and for IgG antibodies detection. Microbiological status at sampling was defined as active infection (positive PCR ± presence of antibodies), past infection (negative PCR + presence of antibodies), or naïve participants (negative PCR + absence of antibodies)., Results: Laboratory results helped classify the residents as having active infection (n=224; 23.8%), past infection (n=462; 49.1%), or being naïve (n=256; 27.1%); staff members were actively infected (n=127; 15.1%), had had a past infection (n=290; 34.2%), or were naïve (n=429; 50.7%). Overall, the percentage of participants with COVID-19 was significantly higher in residents than in staff members (72.8% vs 49.2%; P=0.001). The clinical situation of residents vs staff at sampling was as follows: acute manifestations compatible with COVID-19 (7.3% vs 3.9%; P<0.01) and no manifestations of infection (92.7% vs 96.0%; P<0.01). A large proportion of both asymptomatic and symptomatic residents (69.4% vs 86.6%; P=0.015) had positive PCR results (mostly alongside positive IgG determinations)., Conclusions: COVID-19 affects 75% of the residents in nursing homes in Madrid. The high impact in these settings, despite the strict restrictions adopted during the lockdown, demonstrates the ability of SARS-CoV-2 to cause outbreaks., (©The Author 2022. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).)

Introduction: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed., Methods: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population., Results: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissions CONCLUSION: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission., (Copyright © 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved.)

The SARS-CoV-2 variant of concern (VOC) Delta (B.617.2 lineage) displaced the predominant VOC Alpha (B.1.1.7 lineage) in the United Kingdom. In Madrid, recent start of the decline of predominant VOC Alpha suggested an equivalent phenomenon. However, 11 different variants, none overrepresented in frequency, occupied progressively over a period of 7 weeks the niche previously dominated by VOC Alpha. Only after these 7 weeks, VOC Delta started to emerge. Viral competition due to the entry of VOC Delta is not the major force driving the start of VOC Alpha decline in Madrid. IMPORTANCE Our data indicate that the dynamics of SARS-CoV-2 VOCs turnover in our setting differ from those proposed for other countries. A systematic genomic analysis, updated on a weekly basis, of representative randomly selected samples of SARS-CoV-2 circulating variants allowed us to define a lapse of 7 weeks between the start of VOC Alpha decline and the final emergence of VOC Delta. During this period, VOC Alpha showed a sustained decline, while 11 VOCs, variants of interest (VOIs), and other identified variants, none overrepresented, occupied the niche left by VOC Alpha. Only after these 7 weeks, emergence of VOC Delta occurred, indicating that viral competition involving VOC Delta was not the exclusive direct driving force behind the starting of VOC Alpha decline.

Dinosaur bonebeds with amber content, yet scarce, offer a superior wealth and quality of data on ancient terrestrial ecosystems. However, the preserved palaeodiversity and/or taphonomic characteristics of these exceptional localities had hitherto limited their palaeobiological potential. Here, we describe the amber from the Lower Cretaceous dinosaur bonebed of Ariño (Teruel, Spain) using a multidisciplinary approach. Amber is found in both a root layer with amber strictly in situ and a litter layer mainly composed of aerial pieces unusually rich in bioinclusions, encompassing 11 insect orders, arachnids, and a few plant and vertebrate remains, including a feather. Additional palaeontological data-charophytes, palynomorphs, ostracods- are provided. Ariño arguably represents the most prolific and palaeobiologically diverse locality in which fossiliferous amber and a dinosaur bonebed have been found in association, and the only one known where the vast majority of the palaeontological assemblage suffered no or low-grade pre-burial transport. This has unlocked unprecedentedly complete and reliable palaeoecological data out of two complementary windows of preservation-the bonebed and the amber-from the same site., Competing Interests: SÁ, RP, EP, EB, LA, JP, CM, KT, NM, RL, RL, DP, AR, VS, CB, CM, MP, AS, CP, AA, EE, LM, XD No competing interests declared, (© 2021, Álvarez-Parra et al.)

Objectives: To quantify the workload and cost overload that the COVID-19 pandemic has meant for a Clinical Microbiology laboratory in a real-life scenario., Methods: We compared the number of samples received, their distribution, the human resources, and the budget of a Microbiology laboratory in the COVID pandemic (March-December 2020) with the same months of the previous year., Results: the total number of samples processed in the Clinical Microbiology laboratory in March to December 2020 increased 96.70% with respect to 2019 (from 246,060 to 483,993 samples), reflecting an increment of 127.50% when expressed as samples/1000 admissions (from 6057 to 13,780). The increase in workload was mainly at the expense of the virology (+2058%) and serology (+86%) areas. Despite additional personnel hiring, the samples processed per technician increased 12.5%. The extra cost attributed to Microbiology amounts to 6,616,511 euros (114.8%)., Conclusions: This is the first study to provide quantitative figures about workload and cost increase caused by the COVID-19 in a Microbiology laboratory., Competing Interests: Declaration of Competing Interests The authors report no conflicts of interest relevant to this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

SARS-CoV-2 pandemic shows the importance of having efficient virus diagnosis, especially in groups of particular relevance such as health care professionals, without involving a large economic expense. This is a prevalence study carried out in 7400 health care professionals in a 1350-bed hospital in Madrid, Spain. Pools of 10 samples were performed, using the Xpert® Xpress SARS-CoV-2 test for the diagnosis from clinical samples of nasopharyngeal exudate. A previous study was performed to evaluate the effect of the dilution in terms of sensitivity. The estimated sensitivity was over 95%. A total of 740 pools were performed, with a final result of 218 health care professionals being positive. Using the pooling system, the reagent cost reduction to the institution was 75.3%. It can be concluded that the described sample pooling system is a useful and efficient tool in the diagnosis of SARS-CoV-2 in certain groups, assuming a cost reduction without reducing the sensitivity., (Copyright © 2021. Published by Elsevier Inc.)

This document gathers the opinion of a multidisciplinary forum of experts on different aspects of the diagnosis and treatment of Clostridioides difficile infection (CDI) in Spain. It has been structured around a series of questions that the attendees considered relevant and in which a consensus opinion was reached. The main messages were as follows: CDI should be suspected in patients older than 2 years of age in the presence of diarrhea, paralytic ileus and unexplained leukocytosis, even in the absence of classical risk factors. With a few exceptions, a single stool sample is sufficient for diagnosis, which can be sent to the laboratory with or without transportation media for enteropathogenic bacteria. In the absence of diarrhoea, rectal swabs may be valid. The microbiology laboratory should include C. difficile among the pathogens routinely searched in patients with diarrhoea. Laboratory tests in different order and sequence schemes include GDH detection, presence of toxins, molecular tests and toxigenic culture. Immediate determination of sensitivity to drugs such as vancomycin, metronidazole or fidaxomycin is not required. The evolution of toxin persistence is not a suitable test for follow up. Laboratory diagnosis of CDI should be rapid and results reported and interpreted to clinicians immediately. In addition to the basic support of all diarrheic episodes, CDI treatment requires the suppression of antiperistaltic agents, proton pump inhibitors and antibiotics, where possible. Oral vancomycin and fidaxomycin are the antibacterials of choice in treatment, intravenous metronidazole being restricted for patients in whom the presence of the above drugs in the intestinal lumen cannot be assured. Fecal material transplantation is the treatment of choice for patients with multiple recurrences but uncertainties persist regarding its standardization and safety. Bezlotoxumab is a monoclonal antibody to C. difficile toxin B that should be administered to patients at high risk of recurrence. Surgery is becoming less and less necessary and prevention with vaccines is under research. Probiotics have so far not been shown to be therapeutically or preventively effective. The therapeutic strategy should be based, rather than on the number of episodes, on the severity of the episodes and on their potential to recur. Some data point to the efficacy of oral vancomycin prophylaxis in patients who reccur CDI when systemic antibiotics are required again., (©The Author 2020. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).)

Classification of patients according to their risk of poor outcomes in Clostridioides difficile infection (CDI) would enable implementation of costly new treatment options in a subset of patients at higher risk of poor outcome. In a previous study, we found that low toxin B amplification cycle thresholds (C t ) were independently associated with poor outcome CDI. Our objective was to perform a multicentre external validation of a PCR-toxin B C t as a marker of poor outcome CDI. We carried out a multicentre study (14 hospitals) in which the characteristics and outcome of patients with CDI were evaluated. A subanalysis of the results of the amplification curve of real-time PCR gene toxin B (XpertTM C. difficile) was performed. A total of 223 patients were included. The median age was 73.0 years, 50.2% were female, and the median Charlson index was 3.0. The comparison of poor outcome and non-poor outcome CDI episodes revealed, respectively, the following results: median age (years), 77.0 vs 72.0 (p = 0.009); patients from nursing homes, 24.4% vs 10.8% (p = 0.039); median leukocytes (cells/μl), 10,740.0 vs 8795.0 (p = 0.026); and median PCR-toxin B C t , 23.3 vs 25.4 (p = 0.004). Multivariate analysis showed that a PCR-toxin B C t cut-off <23.5 was significantly and independently associated with poor outcome CDI (p = 0.002; OR, 3.371; 95%CI, 1.565-7.264). This variable correctly classified 68.5% of patients. The use of this microbiological marker could facilitate early selection of patients who are at higher risk of poor outcome and are more likely to benefit from newer and more costly therapeutic options., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: To determine the Staphylococcus aureus carriage rate in wild mammals in Aragon, northern Spain, to analyse their antimicrobial resistance phenotype/genotype and to characterize the recovered isolates., Methods and Results: Nasal and rectal swabs of 103 mammals were collected in Aragón during the period 2012-2015. Antimicrobial susceptibility, the presence of antimicrobial resistance genes and virulence factors were investigated. Molecular characterization was carried out by spa, MLST, agr and SCCmec. Staphylococcus aureus were recovered from 23 animals (22%). Four of the 23 S. aureus were methicillin-resistant S. aureus (MRSA). Three MRSA were mecC-positive and were isolated from European rabbits and were typed as t843 (ascribed to CC130). The remaining MRSA was a mecA-carrying isolate from European hedgehog, typed as ST1-t386-SCCmecIVa-agrIII and it harboured the blaZ, erm(C), ant(6)-Ia and aph(3´)-IIIa resistance genes. A high diversity of spa-types was detected among the 19 methicillin-susceptible S. aureus isolates, which showed high susceptibility to the antimicrobials tested. The tst gene and different combinations of staphylococcal enterotoxins were found., Conclusions: Staphylococcus aureus were detected in nasal and rectal samples of wild mammals. Wild rabbits could be a reservoir of mecC-MRSA., Significance and Impact of the Study: This work provides information on the presence and characteristics of S. aureus from mammals in a defined geographic region in Spain., (© 2019 The Society for Applied Microbiology.)

An outbreak of Clostridium difficile infection (CDI) caused by ribotype 027 (B1/NAP1) began in our hospital in November 2014, and produced 141 episodes in the following months. The aim of this study is to describe this outbreak, assess risk factors for recurrence of CDI-027 and to analyze the implementation of a novel treatment strategy. This is a prospective study of all patients with CDI-027, from November 2014 to November 2015. The epidemiological data were collected daily for each patient. We compared clinical characteristics and treatment between patients with and without recurrence of CDI-027. Interestingly, liver cirrhosis was present in 22% of the patients, and most of them received prophylaxis for hepatic encephalopathy with rifaximin. Patients were also taking antimicrobial drugs (93.6%) and proton pump inhibitors (80.1%). Overall, 27 (23.5%) patients had a first recurrence of CDI-027. Liver cirrhosis increased the risk of recurrence (44.4% vs 14.8%). Patients treated with a prolonged oral vancomycin regimen vs the conventional regimen (oral metronidazole or 10 days of vancomycin) had fewer recurrences (8.6 versus 44.7% [p ≤ 0.01]; OR, 0.91; 95% CI, 0.028-0.294) and less attributable mortality (0% versus 7.1%; p = 0.058). We report an outbreak of CDI-027, mainly in patients with liver cirrhosis. Recurrence of CDI-027 was more common in those patients. A novel approach involving high-dose prolonged vancomycin taper as a first-line treatment, together with a bundle of outbreak measures, seemed to reduce the number of cases of CDI-027, recurrences, and attributable mortality. Nevertheless, this approach warrants further investigation.

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 (CTX R ) 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 CTX R 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.

Binary toxin (BT) has been associated with strains causing more severe Clostridium difficile infection (CDI), such as ribotype 027. Data on the outcome of patients having BT present in ribotypes other than 027 are scarce. Our objective was to investigate the association between BT isolates and outcome of CDI in a non-027 ribotype setting. We prospectively included CDI episodes (January-June 2013 and March-June 2014) from symptomatic patients aged >2 years. Epidemiological and clinical data were recorded. BT genes were detected using multiplex PCR. During the study period, we identified 326 episodes of CDI, of which 319 were available for molecular analysis. Of these, 54 (16·9%) were caused by C. difficile strains with BT. Most (90·7%) isolates with BT were ribotype 078/126. CDI patients with BT-positive strains did not differ from those with BT-negative strains in terms of recurrence (13·0% vs. 15·5%, P = 0·835), treatment failure (0·0% vs. 2·3%, P = 0·594), overall mortality (11·1% vs. 9·1%, P = 0·612), or CDI-related mortality (0·0% vs. 1·9%, P = 0·612). Multivariate regression revealed no association between BT and poor outcome. In conclusion, in a non-027 setting, we found that most BT isolates were 078/126 and were not associated with poor outcome.

A multicenter study of Clostridium difficile infection (CDI) performed during 2008 in Spain revealed that two of every three episodes went undiagnosed or were misdiagnosed owing to nonsensitive diagnostic tests or lack of clinical suspicion and request. Since then, efforts have been made to improve the diagnostic tests used by laboratories and to increase the awareness of this disease among both clinicians and microbiologists. Our objective was to evaluate the impact of these efforts by assessing the current magnitude of underdiagnosis of CDI in Spain using two point-prevalence studies performed on one day each in January and July of 2013. A total of 111 Spanish laboratories selected all unformed stool specimens received for microbiological diagnosis on these days, and toxigenic culture was performed at a central reference laboratory. Toxigenic isolates were characterized both pheno- and genotypically. The reference laboratory detected 103 episodes of CDI in patients aged 2 years or more. Half (50.5 %) of the episodes were not diagnosed in the participating laboratories, owing to insensitive diagnostic tests (15.5 %) or the lack of clinical suspicion and request (35.0 %). The main ribotypes were 014, 078/126, 001/072, and 106. Ribotype 027 caused 2.9 % of all cases. Despite all the interventions undertaken, CDI remains a highly neglected disease because of the lack of sensitive diagnostic tests in some institutions and, especially, the absence of clinical suspicion, mainly in patients with community-associated CDI. Toxigenic C. difficile should be routinely sought in unformed stools sent for microbiological diagnosis, regardless of their origin.

Objectives: Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Metronidazole and vancomycin are the mainstay of treatment, although they are associated with treatment failure and recurrence. Novel agents have emerged to address these shortcomings. We investigated the in vitro activity of a novel agent, surotomycin (formerly CB-183,315), and seven other antimicrobial agents against clinical C. difficile isolates., Methods: Antimicrobial susceptibility to surotomycin, fidaxomicin, metronidazole, vancomycin, clindamycin, rifaximin, moxifloxacin and tigecycline was determined for 100 contemporary clinical isolates of C. difficile collected in 2013. MICs were determined by agar dilution according to CLSI procedures. In addition, 10 strains with reduced susceptibility to metronidazole (n = 6) and vancomycin (n = 4) were also tested. Strains were PCR ribotyped., Results: The MICs of surotomycin for the 100 isolates ranged from ≤0.06 to 2 mg/L, with a geometric mean (GM) of 0.31 mg/L and an MIC50/90 of 0.25/0.5 mg/L. The MIC range of surotomycin was 0.25-1 mg/L (GM = 0.45 mg/L) for isolates with reduced metronidazole susceptibility and 0.125-0.5 mg/L (GM = 0.25 mg/L) for isolates with reduced vancomycin susceptibility. The three most common ribotypes were 001 (31.0%), 014/020 (17.0%) and 078/126 (17.0%). Ribotype 014/020 exhibited the lowest MICs of surotomycin (GM = 0.22 mg/L); the highest MICs were for ribotype 078/126 (GM = 0.72 mg/L)., Conclusions: Surotomycin exhibited potent in vitro activity against all the isolates tested, including those with elevated metronidazole and vancomycin MICs. The potential role of this agent in the treatment of CDI requires further clinical evaluation., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Despite the high concentration of patients with known risk factors for Clostridium difficile infection (CDI) in intensive care units (ICUs), data on ICU patients are scarce. The aim of this study was describe the incidence, clinical characteristics, and evolution of CDI in critically ill patients., Materials and Methods: From 2003 to 2012, adult patients admitted to an ICU (A-ICU) and positive for CDI were included and classified as follows: pre-ICU, if the positive sample was obtained within ±3 days of ICU admission; in-ICU, if obtained after 3 days of ICU admission and up to 3 days after ICU discharge., Results: We recorded 4095 CDI episodes, of which 328 were A-ICU (8%). Episodes of A-ICU decreased from 19.4 to 8.7 per 10000 ICU days of stay (P < .0001). Most A-ICU CDIs (66.3%) were mild to moderate. Pre-ICU episodes accounted for 16.2% and were more severe complicated than in-ICU episodes (11% vs 0%; P = .020). Overall mortality was 28.6%, and CDI-attributable mortality was only 3%., Conclusion: The incidence of A-ICU CDI has decreased steadily over the last 10 years. A significant proportion of A-ICU CDI episodes are pre-ICU and are more severe than in-ICU CDI episodes. Most episodes of A-ICU CDI were nonsevere, with low associated mortality., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We studied the molecular mechanisms of linezolid resistance in 9 isolates of toxigenic Clostridium difficile with high linezolid MICs. The activity of linezolid was determined against 891 clinical isolates of toxigenic C. difficile. The MIC50 and MIC90 of linezolid were 0.75 μg/ml and 1.5 μg/ml, respectively. Nine strains (1%) showed high linezolid MICs (6 μg/ml to 16 μg/ml) and also were resistant to clindamycin, erythromycin, and chloramphenicol. These strains were selected for molecular studies: sequencing of domain V of the 23 rRNA gene, detection of the cfr methyltransferase gene, and sequencing of the ribosomal protein genes rplC and rplD. Molecular relatedness between strains was assessed using PCR ribotyping and MLVA (multilocus variable-number tandem-repeat analysis) typing. The strains belonged to ribotypes 001 (2/9), 017 (6/9), and 078 (1/9). MLVA showed that strains of ribotype 001 and 017 belonged to the same clonal complex in each ribotype. We did not detect mutations in the 23S rRNA gene. The cfr gene was detected in 7 of 9 strains. Sequencing of cfr amplicons revealed a similarity of 100% to a fragment of transposon Tn6218 of C. difficile, which was annotated as a putative chloramphenicol/florfenicol resistance protein. We were unable to detect mechanisms of resistance to linezolid in the 2 strains belonging to ribotype 001. While the relevance of our results lies in the detection of the cfr gene as a possible mechanism of resistance to linezolid in C. difficile, our findings should be assessed by further investigations to characterize these possible cfr genes and their contribution to linezolid resistance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Introduction: Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital., Methods: From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis)., Results: Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2., Conclusion: We report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates., (Copyright © 2013 Elsevier España, S.L. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Nodosaurids are poorly known from the Lower Cretaceous of Europe. Two associated ankylosaur skeletons excavated from the lower Albian carbonaceous member of the Escucha Formation near Ariño in northeastern Teruel, Spain reveal nearly all the diagnostic recognized character that define nodosaurid ankylosaurs. These new specimens comprise a new genus and species of nodosaurid ankylosaur and represent the single most complete taxon of ankylosaur from the Cretaceous of Europe. These two specimens were examined and compared to all other known ankylosaurs. Comparisons of these specimens document that Europelta carbonensis n. gen., n. sp. is a nodosaur and is the sister taxon to the Late Cretaceous nodosaurids Anoplosaurus, Hungarosaurus, and Struthiosaurus, defining a monophyletic clade of European nodosaurids- the Struthiosaurinae.

Clostridium difficile is an emerging pathogen for humans and animals and there is concern about the possibility that livestock might serve as a reservoir of epidemic strains. In Spain, ribotype 078 is one of the most prevalent in human episodes of C. difficile infection, but the distribution of this and other ribotypes in animals is yet unknown. We present the first report on the ribotype distribution and antimicrobial susceptibility of C. difficile in swine in Spain. A total of 144 isolates were PCR ribotyped, and their MIC values for 13 antimicrobial agents were determined using the Etest. Toxins A and B production was assessed using a commercial immunoassay and, in the case of toxin B, a specific cytotoxicity test. Our results show a high prevalence of the toxigenic 078 ribotype (94.4%) and multidrug resistance (49.3%) among the studied isolates. A minority of isolates (5.6%) belonged to a mostly non-toxinogenic ribotype. All isolates were resistant to the fluoroquinolone ciprofloxacin, but susceptible to daptomycin, linezolid, meropenem, rifampicin, teicoplanin, tigecycline, metronidazole and vancomycin. Resistance to clindamycin, ertapenem, erythromycin and moxifloxacin was common (≥27.8% in all cases). Resistance rates for the different antibiotics tested were in all cases independent from the ribotype of isolates and the host's condition (diarrheic or non-diarrheic), but erythromycin and moxifloxacin resistance was associated with the geographic origin of isolates. Metronidazole heteroresistance was found among animal isolates of C. difficile. Our results highlight the role of livestock as a potential source of epidemic multidrug resistant strains in Spain., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Clostridium difficile infection (CDI) is one of the main causes of diarrhoea associated with antimicrobial therapy. Antibiotics with good 'in vitro' activity against C. difficile could protect patients from developing CDI. In this study, the potential of linezolid to protect patients with ventilator-associated pneumonia (VAP) from developing CDI was assessed. Over a 4-year period, a cohort of patients who developed VAP following major heart surgery (MHS) in Gregorio Marañón General Hospital (Madrid, Spain) was retrospectively analysed. Patients were divided into those who developed CDI in the post-operative period and those who did not. Variables associated with the development of CDI were analysed, including the role of antimicrobial therapy. Overall, 1934 patients underwent MHS; 90 patients were excluded due to intra-operative or early post-operative (first 48h) death, leaving a study population of 1844 patients, of which 105 cases had VAP. Complete clinical data were available in 91 cases. CDI occurred in 22 patients (24.2%). When comparing VAP cases with and without CDI, EuroSCORE and overall antibiotics prescribed were not significantly different. Patients with chronic renal failure (CRF) were more prone to develop CDI than those without CRF (32% vs. 13%; P=0.04), and patients without [corrected] CDI received more doses of linezolid than those with [corrected] CDI [12.4±9.7 defined daily doses (DDDs) vs. 6.7±4.0 DDDs; P=0.007]. Multivariate analysis confirmed that receiving more DDDs of linezolid protects from developing CDI (hazard ratio=0.908, 95% confidence interval 0.83-0.99; P=0.04). This work is retrospective and addresses a very particular population, but it is the first to suggest the potential impact of linezolid against CDI., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

A survey of laboratory diagnosis of Clostridium difficile infection (CDI) was conducted in 103 Spanish hospitals. A mean of 23.2 stool specimens/1000 admissions were processed to detect CDI. Overall, 35.9% of the laboratories specifically selected stool specimens for diagnostic C. difficile toxin testing. The most commonly used criteria were loose or watery stools, previous antibiotic therapy and nosocomial diarrhoea. Most laboratories (95.1%) processed the stool specimens in house, mainly five to seven days/week. All laboratories except one detected toxins directly in stool specimens, and 94.9% used enzyme immunoassays (EIAs). Only 16.3% of the laboratories used toxigenic culture as a diagnostic tool, and most of them used EIAs to detect toxins in isolates. The most common diagnostic strategy was toxin detection in stool specimens using EIA alone (79.6%), and the second most common strategy was the combination of toxin detection in stool specimens and isolates (10.2%). The estimated annual incidence of CDI was 1.71 cases/1000 admissions, and this was significantly higher in large hospitals. CDI is underdiagnosed in Spain because most laboratories use EIAs performed directly on stool specimens as the only diagnostic procedure. A national laboratory survey of diagnostic methods and testing protocols for the diagnosis of CDI is simple and inexpensive, and could act as the first step towards implementing national standardized criteria for optimal diagnosis of CDI., (Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

The very labile (decay-prone), non-biomineralized, tissues of organisms are rarely fossilized. Occurrences thereof are invaluable supplements to a body fossil record dominated by biomineralized tissues, which alone are extremely unrepresentative of diversity in modern and ancient ecosystems. Fossil examples of extremely labile tissues (e.g. muscle) that exhibit a high degree of morphological fidelity are almost invariably replicated by inorganic compounds such as calcium phosphate. There is no consensus as to whether such tissues can be preserved with similar morphological fidelity as organic remains, except when enclosed inside amber. Here, we report fossilized musculature from an approximately 18 Myr old salamander from lacustrine sediments of Ribesalbes, Spain. The muscle is preserved organically, in three dimensions, and with the highest fidelity of morphological preservation yet documented from the fossil record. Preserved ultrastructural details include myofilaments, endomysium, layering within the sarcolemma, and endomysial circulatory vessels infilled with blood. Slight differences between the fossil tissues and their counterparts in extant amphibians reflect limited degradation during fossilization. Our results provide unequivocal evidence that high-fidelity organic preservation of extremely labile tissues is not only feasible, but likely to be common. This is supported by the discovery of similarly preserved tissues in the Eocene Grube Messel biota.

Propionibacterium spp. are common flora of human skin. Nevertheless, currently recommended culture procedures do not include anaerobic processing with the result that this organism may go undetected on a colonized catheter. To determine the rate of catheter colonization by Propionibacterium spp., a sample of 1000 vascular catheters was processed by the roll-plate technique and, after conventional aerobic processing, all primary culture plates were reincubated in an anaerobic atmosphere. Propionibacterium acnes was detected in significant counts in the vascular catheters of 39 patients. This represents 14.7% (95% CI, 12.5-16.9) of all positive catheters. Propionibacterium is the second most frequent genus-colonizing catheter tips after Staphylococcus spp. Methodological shortcomings impair the detection and proper adscription of P. acnes as a potential cause of catheter-related infections.

We compared the antimicrobial co-resistance of 3,402 clinical isolates of Enterobacteriaceae resistant to third-generation cephalosporins (2,569 ESBL-producing and 833 AmpC overproducing) with that of 16,220 susceptible isolates, in order to determine the impact of resistance to third-generation cephalosporins on the likelihood of resistance to other antimicrobial classes. Enterobacteriaceae resistant to third-generation cephalosporins, independently of their mechanism of resistance, were significantly more resistant to other classes of antimicrobials than susceptible isolates (p <0.001). Percentages of co-resistance to ciprofloxacin, gentamicin, tobramycin and trimethoprim-sulfamethoxazole of resistant isolates were: 51%, 39%, 53% and 51%, respectively. However, among the susceptible isolates, percentages were 17%, 7%, 6% and 19%, respectively. Fosfomycin exhibited excellent in vitro activity against urinary isolates (92%), mainly against ESBL-producing organisms (90%), and is a good alternative treatment of infections caused by multidrug resistant Enterobacteriaceae. Amikacin and imipenem were the most active antimicrobials against all species tested.

Fossils of a giant sauropod dinosaur, Turiasaurus riodevensis, have been recovered from terrestrial deposits of the Villar del Arzobispo Formation (Jurassic-Cretaceous boundary) of Riodeva (Teruel Province, Spain). Its humerus length (1790 millimeters) and estimated mass (40 to 48 metric tons) indicate that it may have been the most massive terrestrial animal in Europe and one of the largest in the world. Phylogenetic analysis indicates that the fossil represents a member of a hitherto unrecognized group of primitive European eusauropods that evolved in the Jurassic.

The increase in the immunocompromised population and the incidence of invasive aspergillosis (IA) are leading to an overinterpretation of the potential clinical significance of many isolates of Aspergillus fumigatus. Our work prospectively assesses the workload of the isolation of A. fumigatus and its clinical significance in the microbiology laboratory of a large teaching hospital. During a 3-year period, all patients from whom A. fumigatus was isolated were prospectively monitored and classified as having IA or "nonsignificant" disease. A point score based on the prediction of five easily obtained laboratory and clinical parameters was applied. We found 404 A. fumigatus isolates in 260 patients (1/1,000 microbiology laboratory samples; 2.1 patients/10,000 admissions). A total of 90 isolates (22.3%) were from patients with IA. Of the 260 patients, 31 (12%) had invasive disease (IA), and the remaining 229 had "nonsignificant" disease. A score based on points for five parameters was applied to our population. It was constructed as follows: "sample obtained by invasive procedures" (1 point), "presence of two or more positive samples from the same patient" (1 point), "leukemia" (2 points), "neutropenia" (5 points), and "corticosteroid treatment" (2 points). Patients with a score of 0 had only a 2.5% probability of IA. Those with a score of 1 or 2 had an increased probability of 10.3%. The probabilities rose to 40% and 70%, respectively, for patients with a score of 3 or 4 or a score of > or = 5. A simple score based on five easily available parameters may be of help to microbiologists and clinicians to predict the risk of IA.

Pneumococcal bloodstream infections in pediatric patients rose from 3.6 to 14.8 episodes per 100,000 from 1986 to the year 2000 (P < 0.001) in a population of approximately 650,000 inhabitants (16% <16 years of age). Incidence in the group <24 months of age increased from 7.4 to 78.6 episodes per 100,000 per year (P < 0.001). Overall 75% of the serogroups are included in the new heptavalent vaccine.

The demolition of a maternity building at our institution provided us with the opportunity to study the load of filamentous fungi in the air. External (nearby streets) and internal (within the hospital buildings) air was sampled with an automatic volumetric machine (MAS-100 Air Samplair) at least daily during the week before the demolition, at 10, 30, 60, 90,120, 180, 240, 420, 540 and 660 min post-demolition, daily during the week after the demolition and weekly during weeks 2, 3 and 4 after demolition. Samples were duplicated to analyse reproducibility. Three hundred and forty samples were obtained: 115 external air, 69 'non-protected' internal air and 156 protected internal air [high efficiency particulate air (HEPA) filtered air under positive pressure]. A significant increase in the colony count of filamentous fungi occurred after the demolition. Median colony counts of external air on demolition day were significantly higher than from internal air (70.2 cfu/m(3) vs 35.8 cfu/m(3)) (P < 0.001). Mechanical demolition on day +4 also produced a significant difference between external and internal air (74.5 cfu/m(3) vs 41.7 cfu/m(3)). The counts returned to baseline levels on day +11. Most areas with a protected air supply yielded no colonies before demolition day and remained negative on demolition day. The reproducibility of the count method was good (intra-assay variance: 2.4 cfu/m(3)). No episodes of invasive filamentous mycosis were detected during the three months following the demolition. Demolition work was associated with a significant increase in the fungal colony counts of hospital external and non-protected internal air. Effective protective measures may be taken to avoid the emergence of clinical infections., (Copyright 2002 The Hospital Infection Society)

Background: Tuberculosis (TB) recurrences can be due to either reactivation by the same strain (standard assumption) or reinfection by a new strain. Reinfection has mainly been studied in selected populations with a high risk of reexposure to TB. Our aim was to analyze the role of reinfection in TB recurrences in unselected populations, without the clinical/epidemiological circumstances that favor the involvement of a new different strain of Mycobacterium tuberculosis in the recurrence., Methods: A molecular typing analysis was performed with 92 sequential isolates of M tuberculosis from 43 patients with recurrent TB, during a 12-year period. The subjects were both positive and negative for the human immunodeficiency virus, most did not adhere to anti-TB therapy, and they lived in an area with a moderate incidence of TB. Recurrence was considered as being caused by reinfection when the molecular fingerprints for the strains involved in the sequential episodes of TB were different., Results: In 14 (33%) of the 43 patients, different M tuberculosis strains were involved in the first and in subsequent episodes of TB. Reinfection was found for patients who were both positive and negative for the human immunodeficiency virus, and most patients did not adhere to anti-TB therapy. Differences between the reinfection and reactivation groups were not significant (P =.77) according to the time interval between episodes., Conclusions: Reinfection plays an important role in recurrent TB in a population without the clinical/epidemiological circumstances that are usually assumed to favor it. Reinfection should, thus, be considered as a cause of TB recurrences in a wider context than before.

Background: Pott's disease (PD) is an uncommon extra-pulmonary form of tuberculosis. Dissimilar data about location, diagnosis and treatment from various hospitals and different countries are reported., Patients and Method: We present our experience with Pott's disease at our institution between January 1993 to December 1999, retrospectively, based on clinical bacteriological and pathological research. We found the medical records of 14 patients diagnosed as having PD at our hospital. The following information was obtained: age, sex, symptoms and sings at presentation, PPD tuberculin skin test, imaging techniques, mycobacterium cultures and smear, histopathologic study, treatment and clinical outcome., Results: The number of microbiological diagnosed tuberculosis during the study period was of 1,400 (4 cases per 1,000 admissions). From them, 1,047 cases (74.8%) were pulmonary and 353 cases (25.2%) extra-pulmonary. We found the medical records of 14 patients diagnosed as having PD at our hospital. There was a diagnostic delay between 2 and 720 days. In 5 cases, cultures were positive (1.4% extra-pulmonary). The following information was obtained: age, sex, symptoms and signs at presentation, PPD tuberculin skin test, imaging techniques, mycobacterium cultures and smear, histopathologic study, treatment and clinical outcome. The mean age of the patients was 58 years. Pain and dorsal location were more frequent (8/14). In 6 cases, tuberculosis was diagnosed at other sites. Tuberculin skin test was positive in 7 ases (50%). The diagnosis was confirmed by positive cultures from biopsies in 5 from 8 cases performed and the histology was compatible in 8 from 11 cases performed. Diskitis was presented in 10 cases, soft tissue abscess in 9 cases (psoas abscess in 2); epidural compression in 9 cases and surgery was necessary in 9 cases (diagnostic in 3)., Conclusions: Pott's disease is a rare entity even among HIV-patients in whom extrapulmonar disease has increased. Dorsal location, diskitis and advanced forms of the disease that deserve surgical treatment are frequent.

The objective of this paper was to investigate the incidence of extra-intestinal infections caused by Clostridium difficile. During a 10-year period, the microbiology laboratory of our institution isolated 2034 isolates of C. difficile. Of the 2034 isolates, 21 (1.08%) were obtained from extra-intestinal sources. This represents an incidence of extra-intestinal isolation of four cases per 100 000 admissions. We were able to review the records of 17 patients for our study. The isolates in 12 patients were obtained from structures or fluids anatomically close to the colon and included the following infections: peritonitis in five cases (three primary and two secondary), intra-abdominal abscesses in three patients and abdominal wound infections in four cases. The infections in the other five patients were not in the anatomic vicinity of the colon. They included one case with a brain abscess, two episodes of bacteremia and two cases of foot infections (one chronic osteomyelitis). In all but one case, C. difficile isolation was obtained as part of a polymicrobial flora. The isolates were frequently non-toxigenic and the extra-intestinal infections occurred without concomitant diarrhea or prior anti-microbial therapy. Out of the 17 patients, eight died and nine survived. Death could not be directly attributed to C. difficile in any of the cases. The isolation of C. difficile outside the intestinal tract is very uncommon. Its clinical significance should be interpreted with caution.