Methanolobus use unspecific methyltransferases to produce methane from dimethylsulphide in Baltic Sea sediments.

BACKGROUND: In anoxic coastal and marine sediments, degradation of methylated compounds is the major route to the production of methane, a powerful greenhouse gas. Dimethylsulphide (DMS) is the most abundant biogenic organic sulphur compound in the environment and an abundant methylated compound leading to methane production in anoxic sediments. However, understanding of the microbial diversity driving DMS-dependent methanogenesis is limited, and the metabolic pathways underlying this process in the environment remain unexplored. To address this, we used anoxic incubations, amplicon sequencing, genome-centric metagenomics and metatranscriptomics of brackish sediments collected along the depth profile of the Baltic Sea with varying sulphate concentrations. RESULTS: We identified Methanolobus as the dominant methylotrophic methanogens in all our DMS-amended sediment incubations (61-99%) regardless of their sulphate concentrations. We also showed that the mtt and mta genes (trimethylamine- and methanol-methyltransferases) from Methanolobus were highly expressed when the sediment samples were incubated with DMS. Furthermore, we did not find mtsA and mtsB (methylsulphide-methyltransferases) in metatranscriptomes, metagenomes or in the Methanolobus MAGs, whilst mtsD and mtsF were found 2-3 orders of magnitude lower in selected samples. CONCLUSIONS: Our study demonstrated that the Methanolobus genus is likely the key player in anaerobic DMS degradation in brackish Baltic Sea sediments. This is also the first study analysing the metabolic pathways of anaerobic DMS degradation in the environment and showing that methylotrophic methane production from DMS may not require a substrate-specific methyltransferase as was previously accepted. This highlights the versatility of the key enzymes in methane production in anoxic sediments, which would have significant implications for the global greenhouse gas budget and the methane cycle. Video Abstract.

Evaluation of the First-Year Data from an HPV Vaccination Van Program in South Carolina, U.S.

Human papillomavirus (HPV) infections are linked to at least six different types of cancer. The Medical University of South Carolina (MUSC) Hollings Cancer Center (HCC) and Department of Pediatrics leaders identified suboptimal rates of HPV vaccinations in rural and medically underserved communities in South Carolina (SC). To address this major public health problem in SC, they received funding from the HealthyMe/HealthySC (HMHSC) program and HCC to create a statewide community engagement-focused HPV Vaccination Van Program in October 2021. The Program provides HPV vaccinations and other childhood immunizations in school districts and HMHSC health clinics throughout SC, focusing on children aged 9-18 who are eligible for the U.S. Centers for Disease Control and Prevention's Vaccines for Children Program. As of 14 December 2022, the Program administered vaccinations in 16 counties of SC to 552 participants, 243 of whom received HPV vaccinations and were predominantly female (57.2%), aged 4-18 (95.9%), and self-identified as White (44.0%), Black (33.2%), or Hispanic/Latino (15.1%). Most had Medicaid (53.1%)/no insurance coverage (25.1%). The Program is expected to expand as the Program's relationship with SC's school districts grows. The Program provides a model for delivering mobile HPV vaccinations to rural children, thus reducing their cancer risk.

Community-Onset Clostridioides Difficile Infection in Hospitalized Patients in The Netherlands.

BACKGROUND: Clostridioides difficile infection (CDI) is increasingly reported in the community. The aim of this study was to analyze characteristics of hospitalized patients with community-onset CDI (CO-CDI). METHODS: In the Netherlands, 24 hospitals (university-affiliated and general hospitals) participate in the sentinel CDI surveillance program. Clinical characteristics and 30-day outcomes of hospitalized patients >2 years old diagnosed with CDI are registered. Samples of these patients are sent to the national reference laboratory for polymerase chain reaction ribotyping. Data obtained for this surveillance from May 2012 to May 2018 were used to compare CO-CDI with hospital-onset (HO)-CDI episodes. RESULTS: Of 5405 registered cases, 2834 (52.4%) were reported as HO-CDI, 2174 (40.2%) were CO-CDI, and 339 (6.3%) had onset of symptoms in another healthcare facility (eg, nursing home). The proportion of CO-CDI increased over the years and was lower during winter months. Hospitalized patients with CO-CDI were younger (63.8 vs 68.0 years, P < .001) and more often females (53.0% vs 49.6%, P = .02) than patients with HO-CDI. Median time between onset of symptoms and CDI testing was longer in CO-CDI (4 vs 1 day, P < .001). Similar ribotypes were found in CO-CDI and HO-CDI, but ribotype 001 was more frequent among HO-CDI, whereas ribotype 023 was more frequent in CO-CDI. Six of 7 (85.7%) surgeries due to CDI, 27 of 50 (54%) ICU admissions due to CDI, and 48 of 107 (44.9%) of CDI-associated deaths were attributable to CO-CDI. CONCLUSIONS: Our study demonstrates that patients hospitalized with CO-CDI contribute substantially to the total number of CDI episodes and CDI-associated complications in hospitals, stressing the need for awareness and early testing for CDI in community and outpatient settings and also in patients admitted from community with diarrhoea. Surveillance programs that also target nonhospitalized CDI patients are needed to understand the true burden and dynamics of CDI.

Identification and validation of two peptide markers for the recognition of Clostridioides difficile MLST-1 and MLST-11 by MALDI-MS.

OBJECTIVES: Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spread of different C. difficile strains, there is a need for suitable rapid tests. The aim of this study was to identify peptide/protein markers for the rapid recognition of C. difficile strains by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). METHODS: We analysed 44 well-characterized strains, belonging to eight different multi-locus sequence types (MLST), using ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS. The amino acid sequence of two peptide markers specific for MLST-1 and MLST-11 strains was elucidated by MALDI-TOF-MS/MS. The investigation of 2689 C. difficile genomes allowed the determination of the sensitivity and specificity of these markers. C18-solid-phased extraction was used to enrich the MLST-1 marker. RESULTS: Two peptide markers (m/z 4927.81 and m/z 5001.84) were identified and characterized for MLST-1 and MLST-11 strains, respectively. The MLST-1 marker was found in 786 genomes of which three did not belong to MLST-1. The MLST-11 marker was found in 319 genomes, of which 14 did not belong to MLST-11. Importantly, all MLST-1 and MLST-11 genomes were positive for their respective marker. Furthermore, a peptide marker (m/z 5015.86) specific for MLST-15 was found in 59 genomes. We translated our findings into a fast and simple method that allowed the unambiguous identification of the MLST-1 marker on a MALDI-TOF-MS platform. CONCLUSIONS: MALDI-FTICR MS-based peptide profiling resulted in the identification of peptide markers for C. difficile MLST-1 and MLST-11.

Segmental induction heating of orthopaedic metal implants.

OBJECTIVES: Prosthetic joint infection (PJI) is a devastating complication following total joint arthroplasty. Non-contact induction heating of metal implants is a new and emerging treatment for PJI. However, there may be concerns for potential tissue necrosis. It is thought that segmental induction heating can be used to control the thermal dose and to limit collateral thermal injury to the bone and surrounding tissues. The purpose of this study was to determine the thermal dose, for commonly used metal implants in orthopaedic surgery, at various distances from the heating centre (HC). METHODS: Commonly used metal orthopaedic implants (hip stem, intramedullary nail, and locking compression plate (LCP)) were heated segmentally using an induction heater. The thermal dose was expressed in cumulative equivalent minutes at 43°C (CEM43) and measured with a thermal camera at several different distances from the HC. A value of 16 CEM43 was used as the threshold for thermal damage in bone. RESULTS: Despite high thermal doses at the HC (7161 CEM43 to 66 640 CEM43), the thermal dose at various distances from the HC was lower than 16 CEM43 for the hip stem and nail. For the fracture plate without corresponding metal screws, doses higher than 16 CEM43 were measured up to 5 mm from the HC. CONCLUSION: Segmental induction heating concentrates the thermal dose at the targeted metal implant areas and minimizes collateral thermal injury by using the non-heated metal as a heat sink. Implant type and geometry are important factors to consider, as they influence dissipation of heat and associated collateral thermal injury.Cite this article: B. G. Pijls, I. M. J. G. Sanders, E. J. Kuijper, R. G. H. H. Nelissen. Segmental induction heating of orthopaedic metal implants. Bone Joint Res 2018;7:609-619. DOI: 10.1302/2046-3758.711.BJR-2018-0080.R1.

Does compliance with healthy lifestyle behaviours cluster within individuals in Australian primary school-aged children?

BACKGROUND: The 3 "movement behaviours" of sleep, screen time, and physical activity are associated with a wide range of health outcomes in children. This study examined whether these behaviours cluster together within individuals in Australian primary school children. METHODS: Three datasets including 4,449 9- to 11-year-old children were interrogated-(a) Obesity Prevention and Lifestyle (OPAL), (b) the International Study of Children, Obesity, Lifestyle and Environment (ISCOLE), and (c) the National Children's Nutrition and Physical Activity Survey (NCNPAS). The surveys measured movement behaviours using different instruments (accelerometry, use of time recall, and questionnaires) and different operationalizations of compliance. Observed frequencies of compliance with various combinations of guidelines were compared with expected frequencies based on the assumption of independence, using chi-square tests. RESULTS: Compliance with the sleep guidelines was relatively high (72%, 75%, and 79% in the OPAL, ISCOLE, and NCNPAS datasets, respectively), and compliance with the screen (18%, 35%, and 22%) and physical activity (33%, 57%, and 87%) guidelines was generally lower. Against expectation, there was no evidence of clustering in any of the datasets (p > .99). CONCLUSIONS: Compliance with movement behaviour guidelines does not cluster within individuals in 9- to 11-year-old Australian children. It may be unlikely that fostering compliance with one guideline will have a flow-on effect to the others. Temporal trade-offs (i.e., the need to choose one movement behaviour above another) in the 24-hr day may contribute to the lack of clustering.

Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

Effectiveness of various cleaning and disinfectant products on Clostridium difficile spores of PCR ribotypes 010, 014 and 027.

BACKGROUND: In healthcare facilities, Clostridium difficile infections spread by transmission of bacterial spores. Appropriate sporicidal disinfectants are needed to prevent development of clusters and outbreaks. In this study different cleaning/disinfecting wipes and sprays were tested for their efficacy against spores of distinctive C. difficile PCR ribotypes. METHODS: Four different products were tested; 1) hydrogen peroxide 1.5%; 2) glucoprotamin 1.5%; 3) a mixture of ethanol, propane and N-alkyl amino propyl glycine; and 4) a mixture of didecyldimonium chloride, benzalkonium chloride, polyaminopropyl, biguanide and dimenthicone as active ingredients. Tiles were contaminated with a test solution containing a concentration of 5x106CFU/ml spores of C. difficile strains belonging to PCR ribotypes 010, 014 or 027. The tiles were left to dry for an hour and then wiped or sprayed with one of the sprays or wipes as intended by the manufacturers. When products neutralized after 5 min, microbiological cultures and ATP measures were performed. RESULTS: Irrespective of the disinfection method, the microbial count log10 reduction of C. difficile PCR ribotype 010 was highest, followed by the reduction of C. difficile 014 and C. difficile 027. Overall, the wipes performed better than the sprays with the same active ingredient. On average, although not significantly, a difference in relative light units (RLU) reduction between the wipes and sprays was found. The wipes had a higher RLU log10 reduction, but no significant difference for RLU reduction was observed between the different C. difficile strains (p = 0.16). CONCLUSION: C. difficile spores of PCR ribotypes 014 and 027 strains are more difficult to eradicate than non-toxigenic PCR ribotype 010. In general, impregnated cleaning/disinfection wipes performed better than ready-to-use sprays. Wipes with hydrogen peroxide (1.5%) showed the highest bactericidal activity.

Non-contact electromagnetic induction heating for eradicating bacteria and yeasts on biomaterials and possible relevance to orthopaedic implant infections: In vitro findings.

OBJECTIVES: Infection of implants is a major problem in elective and trauma surgery. Heating is an effective way to reduce the bacterial load in food preparation, and studies on hyperthermia treatment for cancer have shown that it is possible to heat metal objects with pulsed electromagnetic fields selectively (PEMF), also known as induction heating. We therefore set out to answer the following research question: is non-contact induction heating of metallic implants effective in reducing bacterial load in vitro? METHODS: Titanium alloy cylinders (Ti6Al4V) were exposed to PEMF from an induction heater with maximum 2000 watts at 27 kHz after being contaminated with five different types of micro-organisms: Staphylococcus epidermidis; Staphylococcus aureus; Pseudomonas aeruginosa; spore-forming Bacillus cereus; and yeast Candida albicans. The cylinders were exposed to incremental target temperatures (35°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C) for up to 3.5 minutes. RESULTS: There was an average linear heating rate of 0.39°C per second up to the target temperature, and thereafter the target temperature was maintained until the end of the experiment. At 60°C and higher (duration 3.5 minutes), there was a 6-log reduction or higher for every micro-organism tested. At 60°C, we found that the shortest duration of effective induction heating was 1.5 minutes. This resulted in a 5-log reduction or higher for every micro-organism tested. CONCLUSION: Non-contact induction heating of a titanium disk is effective in reducing bacterial load in vitro. These promising results can be further explored as a new treatment modality for infections of metal orthopaedic implants.Cite this article: B. G. Pijls, I. M. J. G. Sanders, E. J. Kuijper, R. G. H. H. Nelissen. Non-contact electromagnetic induction heating for eradicating bacteria and yeasts on biomaterials and possible relevance to orthopaedic implant infections: In vitro findings. Bone Joint Res 2017;6:323-330. DOI: 10.1302/2046-3758.65.BJR-2016-0308.R1.

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of Clostridium difficile isolates from three Czech hospitals in Eastern Bohemia in 2011-2012.

In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.

Whole genome sequencing reveals potential spread of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011.

Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.

Soil fungal communities of grasslands are environmentally structured at a regional scale in the Alps.

Studying patterns of species distributions along elevation gradients is frequently used to identify the primary factors that determine the distribution, diversity and assembly of species. However, despite their crucial role in ecosystem functioning, our understanding of the distribution of below-ground fungi is still limited, calling for more comprehensive studies of fungal biogeography along environmental gradients at various scales (from regional to global). Here, we investigated the richness of taxa of soil fungi and their phylogenetic diversity across a wide range of grassland types along a 2800 m elevation gradient at a large number of sites (213), stratified across a region of the Western Swiss Alps (700 km(2)). We used 454 pyrosequencing to obtain fungal sequences that were clustered into operational taxonomic units (OTUs). The OTU diversity-area relationship revealed uneven distribution of fungal taxa across the study area (i.e. not all taxa are everywhere) and fine-scale spatial clustering. Fungal richness and phylogenetic diversity were found to be higher in lower temperatures and higher moisture conditions. Climatic and soil characteristics as well as plant community composition were related to OTU alpha, beta and phylogenetic diversity, with distinct fungal lineages suggesting distinct ecological tolerances. Soil fungi, thus, show lineage-specific biogeographic patterns, even at a regional scale, and follow environmental determinism, mediated by interactions with plants.

Sequential TPF chemotherapy followed by concurrent chemoradiotherapy in locally advanced head and neck cancer--a retrospective analysis of toxicity and outcomes.

BACKGROUND AND AIMS: Phase III trials have shown that the addition of a taxane to cisplatin/5FU-based induction chemotherapy (TPF) improves response rates and overall survival in unresectable stage III/IV head and neck cancer. We sought to assess the tolerability, compliance and clinical outcomes of this treatment regime. METHODS: A retrospective study of patients treated within a single centre between September 2007 and November 2010. Toxicities were graded according to CTCAE version 3.0. Survival, distant metastasis and local control rates are expressed as percentages at two years using the Kaplan-Meier method. RESULTS: A total of 100 patients were identified (11% stage III, 86% stage IV) and 32% of patients were admitted as an emergency after TPF. The rate of neutropenic fever was 31%, this number fell to 9% when prophylactic G-CSF was used. In addition, 89% of patients underwent radical chemoradiation. Of these, 96% completed the full radiotherapy course. However, only 64% of patients received a minimum of two cycles of concurrent platinum chemotherapy. The two-year overall survival, metastasis free survival and local control rates were 62.6%, 88.5% and 73.3%, respectively. CONCLUSIONS: TPF chemotherapy can be delivered safely in a non-trial cohort of patients. There is, however, a significant reduction in concurrent chemotherapy dose intensity. The long-term impact of this remains unclear.

Epidemiology of Clostridium difficile infections in a tertiary-care hospital in Korea.

To survey healthcare-associated Clostridium difficile infection (HA-CDI) in a 900-bed tertiary-care hospital, we prospectively investigated the epidemiology of CDI and distribution of PCR-ribotypes. From February 2009 through January 2010, all patients with HA-CDI were enrolled. Epidemiological information and prescription records for antibiotics were collected. The C. difficile isolates were characterized using reference strains and were tested for antibiotic susceptibility. During the survey, incidence of HA-CDI was 71.6 per 100 000 patient-days. In total, 140 C. difficile isolates were obtained from 166 patients with HA-CDI. The PCR-ribotyping yielded 38 distinct ribotypes. The three most frequently found ribotypes made up 56.4% of all isolates; they comprised 37 isolates (26.4%) of PCR-ribotype 018, 22 (15.7%) of toxin A-negative PCR-ribotype 017, and 20 (14.3%) of PCR-ribotype 001. Clostridium difficile PCR-ribotype 018 was present in all departments throughout the hospital during the 11 months, whereas ribotype 017 and ribotype 001 appeared mostly in the pulmonary department. Hypervirulent C. difficile PCR-ribotype 027 was detected in 1 month on two wards. The incidence of CDI in each department showed a seven-fold difference, which correlated significantly with the amount of prescribed clindamycin (R = 0.783, p 0.013) or moxifloxacin (R = 0.733, p 0.025) in the departments. The rates of resistance of the three commonest ribotypes to clindamycin and moxifloxacin were significantly higher than those of other strains (92.1% versus 38.2% and 89.5% versus 27.3%, respectively). CDI is an important nosocomially acquired infection and this study emphasizes the importance of implementing country-wide surveillance to detect and control CDI in Korea.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Acquisition of Clostridium difficile by piglets.

Clostridium difficile is recognized as an important cause of nosocomial diarrhoea in humans especially in association with administration of antibiotics. In pigs, C. difficile can cause neonatal enteritis and can be isolated from faeces from both diseased and healthy animals. The presented prospective study describes how soon C. difficile can be isolated from newborn piglets after normal parturition and how C. difficile spreads within a pig farm. Six sows, their farrowing crates and their litters at one farm were sampled until C. difficile was found in all piglets. Within 48 h after birth, all 71 piglets became positive for C. difficile (two piglets were already positive within 1h post partum), all sows became positive within 113 h after parturition and the farrowing crates were found intermittently positive. C. difficile could also be detected in air samples and in samples of teats of the sows. All isolates belonged to PCR ribotype 078. Twenty-one C. difficile ribotype 078 isolates, found at the farm, were further analyzed by MLVA (multiple-locus variable-number tandem repeat analysis) and belonged to one clonal complex, except one isolate. To be sure that piglets were not born already infected with C. difficile ribotype 078, 38 caesarean derived piglets were sampled immediately after surgery. All piglets tested negative at delivery and stayed negative for C. difficile ribotype 078 during the 21 days in which they were kept in sterile incubators. This study shows that C. difficile ribotype 078 spreads easily between sows, piglets and the environment. Vertical transmission of C. difficile ribotype 078 was not found and is very unlikely to occur.

Comparison of real-time PCR techniques to cytotoxigenic culture methods for diagnosing Clostridium difficile infection.

In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027.

High occurrence of various Clostridium difficile PCR ribotypes in pigs arriving at the slaughterhouse.

BACKGROUND: Clostridium difficile is recognized as an important cause of nosocomial diarrhoea in humans, especially in association with the administration of antibiotics. Furthermore, C. difficile can not only cause neonatal enteritis in pigs but can also be found in pigs without any clinical disease symptoms. Clostridium difficile had been found on pork samples destined for human consumption. However, little is known about the risk of food-borne transmission. OBJECTIVE: To elaborate the risk of food-borne transmission of C. difficile via pigs. ANIMALS AND METHODS: The occurrence of C. difficile was assessed in pigs arriving at a slaughterhouse in the Netherlands. Rectal faecal samples from 50 pigs originating from 10 different farms were taken just after the pigs were stunned and bled. These samples were examined using a real-time PCR (BD GeneOhm™ Cdiff Assay) combined with culturing following enrichment. RESULTS: Using real-time PCR, none of the faecal samples were found positive for C. difficile while after culturing following enrichment, 14 out of 50 samples (28%) contained C. difficile. The positive samples were derived from nine different farms and encompassed seven different PCR ribotypes (015 predominant). All isolated C. difficile strains were positive for the toxin A and B genes. CONCLUSION: These results indicate that C. difficile can be found in faecal samples obtained from pigs after they were stunned and bled in a slaughterhouse. CLINICAL IMPORTANCE: The potential risk of these findings on food-borne transmission via pigs and associated impact on human health cannot be excluded and needs further study.

Considerations for pacing of the cricoarytenoid dorsalis muscle by neuroprosthesis in horses.

REASONS FOR PERFORMING STUDY: The success rate of prosthetic laryngoplasty is limited and may be associated with significant sequelae. Nerve muscle pedicle transplantation has been attempted but requires a year before function is restored. OBJECTIVE: To determine the optimal parameters for functional electrical stimulation of the recurrent laryngeal nerve in horses. METHODS: An experimental in vivo study was performed on 7 mature horses (2-21 years). A nerve cuff was placed on the distal end of the common trunk of the recurrent laryngeal nerve (RLN). In 6 horses the ipsilateral adductor branch of RLN was also transected. The electrodes were connected to programmable internal stimulator. Stimulation was performed using cathodic phase and then biphasic pulses at 24 Hz with a 0.427 ms pulse duration. Stimulation-response experiments were performed at monthly intervals, from one week following implantation. The study continued until unit failure or the end of project (12 months). Two of the horses were stimulated continuously for 60 min to assess onset of fatigue. RESULTS: Excellent arytenoid cartilage abduction (mean arytenoid angle of 52.7 degrees, range 48.5-56.2 degrees) was obtained in 6 horses (laryngeal grades I or II (n = 3) and III (n = 2). Poor abduction was obtained in grade IV horses (n = 2). Arytenoid abduction was maintained for up to a year in one horse. Technical implant failure resulted in loss of abduction in 6 horses at one week to 11 months post operatively. Mean tissue impedance was 1.06 kOhm (range 0.64-1.67 kOhm) at one week, twice this value at 2 months (mean 2.32, range 1.11-3.75 kOhm) and was stable thereafter. Maximal abduction was achieved at a stimulation range of 0.65-7.2 mA. No electrical leakage was observed. Constant stimulation of the recurrent laryngeal nerve for 60 min led to full abduction without evidence of muscle fatigue. CONCLUSIONS: Functional electrical stimulation of the recurrent laryngeal nerve leading to full arytenoid abduction can be achieved. The minimal stimulation amplitude for maximal abduction angle is slightly higher than those for man and dogs. CLINICAL RELEVANCE: This treatment modality could eventually be applicable to horses with recurrent laryngeal neuropathy.