A point-prevalence study on community and inpatient Clostridioides difficile infections (CDI): results from Combatting Bacterial Resistance in Europe CDI (COMBACTE-CDI), July to November 2018.

BackgroundThere is a paucity of data on community-based Clostridioides difficile infection (CDI) and how these compare with inpatient CDI.AimTo compare data on the populations with CDI in hospitals vs the community across 12 European countries.MethodsFor this point-prevalence study (July-November 2018), testing sites sent residual diagnostic material on sampling days to a coordinating laboratory for CDI testing and PCR ribotyping (n = 3,163). Information on whether CDI testing was requested at the original site was used to identify undiagnosed CDI. We used medical records to identify differences between healthcare settings in patient demographics and risk factors for detection of C. difficile with or without free toxin.ResultsThe CDI positivity rate was 4.4% (country range: 0-16.2) in hospital samples, and 1.3% (country range: 0-2.2%) in community samples. The highest prevalence of toxinotype IIIb (027, 181 and 176) was seen in eastern European countries (56%; 43/77), the region with the lowest testing rate (58%; 164/281). Different predisposing risk factors were observed (use of broad-spectrum penicillins in the community (OR: 8.09 (1.9-35.6), p = 0.01); fluoroquinolones/cephalosporins in hospitals (OR: 2.2 (1.2-4.3), p = 0.01; OR: 2.0 (1.1-3.7), p = 0.02)). Half of community CDI cases were undetected because of absence of clinical suspicion, accounting for three times more undiagnosed adults in the community compared with hospitals (ca 111,000 vs 37,000 cases/year in Europe).ConclusionThese findings support recommendations for improving diagnosis in patients presenting with diarrhoea in the community, to guide good practice to limit the spread of CDI.

Two Distinct Patterns of Clostridium difficile Diversity Across Europe Indicating Contrasting Routes of Spread.

Background: Rates of Clostridium difficile infection vary widely across Europe, as do prevalent ribotypes. The extent of Europe-wide diversity within each ribotype, however, is unknown. Methods: Inpatient diarrheal fecal samples submitted on a single day in summer and winter (2012-2013) to laboratories in 482 European hospitals were cultured for C. difficile, and isolates the 10 most prevalent ribotypes were whole-genome sequenced. Within each ribotype, country-based sequence clustering was assessed using the ratio of the median number of single-nucleotide polymorphisms between isolates within versus across different countries, using permutation tests. Time-scaled Bayesian phylogenies were used to reconstruct the historical location of each lineage. Results: Sequenced isolates (n = 624) were from 19 countries. Five ribotypes had within-country clustering: ribotype 356, only in Italy; ribotype 018, predominantly in Italy; ribotype 176, with distinct Czech and German clades; ribotype 001/072, including distinct German, Slovakian, and Spanish clades; and ribotype 027, with multiple predominantly country-specific clades including in Hungary, Italy, Germany, Romania, and Poland. By contrast, we found no within-country clustering for ribotypes 078, 015, 002, 014, and 020, consistent with a Europe-wide distribution. Fluoroquinolone resistance was significantly more common in within-country clustered ribotypes (P = .009). Fluoroquinolone-resistant isolates were also more tightly clustered geographically with a median (interquartile range) of 43 (0-213) miles between each isolate and the most closely genetically related isolate, versus 421 (204-680) miles in nonresistant pairs (P < .001). Conclusions: Two distinct patterns of C. difficile ribotype spread were observed, consistent with either predominantly healthcare-associated acquisition or Europe-wide dissemination via other routes/sources, for example, the food chain.

Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013.

There are limited national epidemiological data for community-associated (CA)-Clostridium difficile infections (CDIs). Between March 2011 and March 2013, laboratories in England submitted to the Clostridium difficile Ribotyping Network (CDRN) up to 10 diarrhoeal faecal samples from successive patients with CA-CDI, defined here as C. difficile toxin-positive diarrhoea commencing outside hospital (or less than 48 hours after hospital admission), including those cases associated with community-based residential care, with no discharge from hospital within the previous 12 weeks. Patient demographics and C. difficile PCR ribotypes were compared for CA-CDIs in our study and presumed healthcare-associated (HA) CDIs via CDRN. Ribotype diversity indices, ranking and relative prevalences were very similar in CA- vs HA-CDIs, although ribotypes 002 (p ≤ 0.0001),020 (p = 0.009) and 056 (p < 0.0001) predominated in CA-CDIs; ribotype 027 (p = 0.01) predominated in HA-CDIs. Epidemic ribotypes 027 and 078 predominated in institutional residents with CDI (including care/nursing homes) compared with people with CDI living at home. Ribotype diversity decreased with increasing age in HA-CDIs, but not in CA-CDIs. Ribotype 078 CA-CDIs were significantly more common in elderly people (3.4% (6/174) vs 8.7% (45/519) in those aged < 65 and ≥ 65 years, respectively; p = 0.019). No antibiotics were prescribed in the previous four weeks in about twofold more CA-CDI vs HAs (38.6% (129/334) vs 20.3% (1,226/6,028); p < 0.0001). We found very similar ribotype distributions in CA- and HA-CDIs, although a few ribotypes significantly predominated in one setting. These national data emphasise the close interplay between, and likely common reservoirs for, CDIs, particularly when epidemic strains are not dominant.

Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013.

Clostridium difficile infection (CDI) is the major cause of infective diarrhoea in healthcare environments. As part of the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), the largest C. difficile epidemiological study of its type, PCR ribotype distribution of C. difficile isolates in Europe was investigated. PCR ribotyping was performed on 1,196 C. difficile isolates from diarrhoeal samples sent to the European coordinating laboratory in 2012-13 and 2013 (from two sampling days) by 482 participating hospitals from 19 European countries. A total of 125 ribotypes were identified, of which ribotypes 027 (19%, n =222), 001/072 (11%, n = 134) and 014/020 (10%, n = 119) were the most prevalent. Distinct regional patterns of ribotype distribution were noted. Of 596 isolates from patients with toxin-positive stools (CDI cases), ribotype 027 accounted for 22% (32/144) of infections in cases aged from 18 to less than 65 years, but the prevalence decreased in those aged ≥ 65 years (14% (59/412)) and further decreased in those aged ≥ 81 years (9% (18/195)). The prevalence of ribotype 027 and 176, but not other epidemic strains, was inversely proportional to overall ribotype diversity (R(2) = 0.717). This study highlights an increased diversity of C. difficile ribotypes across Europe compared with previous studies, with considerable intercountry variation in ribotype distribution. Continuous surveillance programmes are necessary to monitor the changing epidemiology of C. difficile.

Nematodes from galls on Myrtaceae. IV. Fergusobia from flat leaf galls on Eucalyptus and Corymbia, with descriptions of two new species.

Two new species of Fergusobia are described. Both were collected from flat leaf galls from South Australia, one on Eucalyptus microcarpa and the other on E. porosa. Fergusobia microcarpae n. sp. Davies is characterised by the combination of a C-shaped parthenogenetic female with a short, broadly rounded conoid tail, an arcuate to open C-shaped infective female with an hemispherical tail tip, and arcuate to J-shaped males with angular spicules and short peloderan bursa. Fergusobia porosae n. sp. Davies is similar in having an arcuate to C-shaped parthenogenetic female with a small conoid tail, an almost straight to arcuate infective female with an hemispherical tail tip, and males that are almost straight to barely J-shaped with angular spicules and short peloderan bursa. They differ in that the bodies of parthenogenetic and infective females of F. microcarpae n. sp. are more curved than in F. porosae n. sp. Other known similar forms of Fergusobia/Fergusonina galls are outlined and the larval shield morphologies of their associated mutualistic Fergusonina fly species are discussed where known. An inventory of all known Fergusobia/Fergusonina associations from flat leaf galls from Corymbia spp. and Eucalyptus spp. is presented. Relationships of Fergusobia nematodes were inferred from analysis of sequences of 28S rDNA D2/D3 domains and a portion of mitochondrial DNA cytochrome oxidase subunit I (mtCOI). Nematodes from flat leaf galls appeared in two clades. 

Nematodes from galls on Myrtaceae. V. Fergusobia from large multilocular shoot bud galls from Angophora and Eucalyptus in Australia, with descriptions of six new species.

Six new species of Fergusobia, from large multilocular shoot bud galls on two species of Angophora and four species of Eucalyptus from both subgenera Eucalyptus and Symphyomyrtus, are described. Fergusobia cosmophyllae Davies n. sp. is characterized by the combination of a C-shaped parthenogenetic female with a short arcuate conoid tail, a broad (small a ratio) arcuate infective female with an hemispherical tail tip, and an arcuate to J-shaped male with broad, angular spicules and short bursa.  Fergusobia delegatensae Davies n. sp. has an open C-shaped parthenogenetic female with a broadly conoid tail, an infective female of variable shape with an hemispherical tail tip, and a male of open C-shape with a crenate bursa that arises 40-70% along the length of the body from the tail tip and terminates just anterior to the cloaca. Fergusobia diversifoliae Davies n. sp. has a C-shaped parthenogenetic female with a conoid tail, an arcuate infective female with a hemispherical tail tip, and an arcuate, C- or J-shaped male with angular spicule and a long peloderan bursa. Fergusobia floribundae Davies n. sp. has a C-shaped parthenogenetic female with a narrow, arcuate, conoid tail, an arcuate infective female with a hemispherical tail tip, and an arcuate or J-shaped male with an angular spicule and a short to mid-body length peloderan bursa. Fergusobia minimus Lisnawita n. sp. has a C-shaped parthenogenetic female with a conoid tail, an arcuate to open C-shaped infective female with a hemispherical tail tip, and an arcuate to open C-shaped male with an angular spicule and a peloderan bursa arising at about 10-30% of body length. Fergusobia pimpamensis Davies n. sp. has an open C to C-shaped parthenogenetic female with a narrow conoid tail, an arcuate to open C-shaped infective female with a hemispherical tail tip, and an arcuate to C-shaped male with an arcuate spicule and a long, crenate, peloderan bursa. An inventory of all known Fergusobia/Fergusonina associations from terminal shoot bud galls is presented. The larval shield morphology of the associated mutualistic Fergusonina species is discussed where known. Analyses of DNA sequences of D2/D3 and COI suggested that the six new species are distributed between three clades of Fergusobia.

Impact of testing on Clostridioides difficile infection in hospitals across Europe: a mathematical model.

OBJECTIVES: The prevalence of Clostridioides difficile infection (CDI) has been shown to vary markedly between European countries, both in hospitals and in the community. Determining the true prevalence has proven challenging. Without systematic testing in hospitals, the unchecked transmission of CDI can lead to large outbreaks in more susceptible cohorts. We investigate the success of CDI surveillance and control measures across Europe, by examining the dynamics of disease spread from the community into a hospital setting. We focus on national differences, such as variability in testing and sampling, disease prevalence in communities and hospitals, and antimicrobial usage. METHODS: We developed a stochastic, compartmental, dynamic mathematical model parameterized using sampling and testing rate data from COMBACTE-CDI, a multicountry study in which all diarrhoeal stool samples (N = 3163) from European laboratories were tested for CDI, and data for antimicrobial usage and incidence of hospital cases sourced from the European Centre for Disease Prevention and Control. RESULTS: The framework estimates the prevalence of CDI among hospital patients across European countries and explores how national differences impact the dynamics, transmission, and relative incidence of CDI within the hospital setting. The model illustrates the mechanisms influencing these national differences, namely, antimicrobial usage rates, national sampling and testing rates, and community prevalence of CDI. DISCUSSION: Differential costs for testing and practicalities of scaling up testing mean every country needs to consider balancing CDI testing costs against the costs of treatment and care of patients with CDI.

New species of gall fly (Diptera: Fergusoninidae) from Eucalyptus camaldulensis (Myrtaceae) in southern Australia and its associated parasitoids and inquilines.

A new species of gall fly, Fergusonina nodulosa sp. nov. (Diptera: Fergusoninidae) is described from Eucalyptus camaldulensis Dehnh. (Myrtaceae) in southern Australia. The adult fly is yellow with dark brown to black markings, the male postgonites have one large distal tooth and an ovoid subapical lobe with two lateral setae, the female postabdomen is short and sparsely setose, and the wing has a posterior cross vein. The larva possesses a dorsal shield comprising rows of raised spicules on thoracic segments 1-3 and abdominal segments 1-7, and the puparium is heavily sclerotised dark brown, barrel-shaped, bearing the dorsal shield. Fergusonina nodulosa sp. nov. forms nodular 'stem' galls initiating from the axial shoot buds. The galls are 2-8 mm in diameter with an average volume of 123 (range 25-480) mm3. Thirteen species of Hymenoptera, comprising primary parasitoids and hyperparasitoids of the gall fly and associated lepidopteran inquilines, and three species of Lepidoptera inquilines were reared from galls of this new species. Fergusonina nodulosa sp. nov. is associated with the obligate mutualist nematode, Fergusobia camaldulensae Davies.

The predictive value of quantitative nucleic acid amplification detection of Clostridium difficile toxin gene for faecal sample toxin status and patient outcome.

BACKGROUND: Laboratory diagnosis of Clostridium difficile infection (CDI) remains unsettled, despite updated guidelines. We investigated the potential utility of quantitative data from a nucleic acid amplification test (NAAT) for C. difficile toxin gene (tg) for patient management. METHODS: Using data from the largest ever C. difficile diagnostic study (8853 diarrhoeal samples from 7335 patients), we determined the predicative value of C. difficile tgNAAT (Cepheid Xpert C.diff) low cycle threshold (CT) value for patient toxin positive status, CDI severity, mortality and CDI recurrence. Reference methods for CDI diagnosis were cytotoxicity assay (CTA) and cytotoxigenic culture (CTC). RESULTS: Of 1281 tgNAAT positive faecal samples, 713 and 917 were CTA and CTC positive, respectively. The median tgNAAT CT for patients who died was 25.5 vs 27.5 for survivors (p = 0.021); for toxin-positivity was 24.9 vs 31.6 for toxin-negative samples (p<0.001) and for patients with a recurrence episode was 25.6 vs 27.3 for those who did not have a recurrent episode (p = 0.111). Following optimal cut-off determination, low CT was defined as ≤25 and was significantly associated with a toxin-positive result (P<0.001, positive predictive value 83.9%), presence of PCR-ribotype 027 (P = 0.025), and mortality (P = 0.032). Recurrence was not associated with low CT (p 0.111). CONCLUSIONS: Low tgNAAT CT could indicate CTA positive patients, have more severe infection, increased risk of mortality and possibly recurrence. Although, the limited specificity of tgNAAT means it cannot be used as a standalone test, it could augment a more timely diagnosis, and optimise management of these at-risk patients.

Nematodes from galls on Myrtaceae. XI. Descriptions of five new species of Fergusobia from Australia.

Five new species of Fergusobia are described. Fergusobia janetae Davies n. sp. is characterized by the combination of an arcuate to straight, spindle-shaped parthenogenetic female with a relatively small oesophageal gland, an extensile uterus and a conoid tail, an arcuate infective female with a bluntly rounded tail tip, and a C-shaped male with an arcuate to angular spicule and bursa arising at more than 90% of body length. Fergusobia robustae Davies n. sp. is characterized by the combination of a small, C-shaped parthenogenetic female with a short cylindroid tail with a broadly rounded tip; an arcuate to C-shaped, relatively broad, infective female with a tail tip that is almost hemispherical; and an arcuate male with a strong angular spicule and a bursa arising at 30-40% of body length. Fergusobia pruinosae Davies n. sp. is characterized by the combination of a medium sized, open C-shaped, parthenogenetic female in which the cuticle does not swell upon fixation, with a strongly sclerotised stylet, with a more or less narrowly conoid tail with a bluntly or broadly rounded tip; infective female that is arcuate to J-shaped with a notched tail tip; and open C-shaped males with stout, angular spicules and bursa arising near the secretory/excretory pore. Fergusobia pauciflorae Davies n. sp. is characterized by the combination of a medium sized, arcuate, parthenogenetic female with a small stylet, and a conoid tail with a bluntly rounded tip; a small, open C to J-shaped infective female with a broadly rounded to almost hemispherical tail tip; and straight to arcuate males with stout, angular spicules and bursa at ~70% of body length anterior to the cloaca. Fergusobia obliquae Davies n. sp. has a relatively broad, arcuate, parthenogenetic female having a small but strongly sclerotised stylet, and a short conoid tail with a bluntly rounded tip; an arcuate, infective female with an almost hemispherical tail tip; and C-shaped males with arcuate to angular (not heavily sclerotised) spicules and bursa arising 50-80% of body length anterior to tail. Sequencing of the 18S and 28S rDNA domains for F. janetae n. sp. and of mitochondrial DNA cytochrome oxidase subunit I (mtCOI) for F. pauciflorae n. sp. confirm that they are distinct species.

Tripylella australis sp. nov. and Tripyla setifera Bütschli 1873 (Nematoda: Triplonchida: Tripylidae) from Australia.

This paper describes a new species of Tripylella and gives a new record of Tripyla (family Tripylidae) in Australia, and provides a key to species in the genus Tripylella. Tripylella australis sp. nov. is characterized by having females with a long body (1804-1832 µm), a=41-42, b= 4.4-4.5, c=10.3-11.3, c'=5.4-6.4 and V=51-54%, outer labial setae 13-14 µm long and cephalic setae 3.7-4.5 µm long in one whorl, dorsal tooth posterior to two subventral teeth, tail tapering uniformly, and four single caudal setae on the dorsal part of the tail. Males are similar to females, and have a long body (1870-2014 µm), a=50-55, b= 4.7-4.9, c=11.3-11.4, c'=5.8-6.0 and T=53-56%, horn-shaped spicules, 36-38 µm long, a straight gubernaculum 14-16 µm in length, tail tapering almost uniformly, and four caudal setae on the dorsal part of the tail. A key for identification of Tripylella species is presented. Tripyla setifera Bütschli 1873 is recorded for the first time in Australia, and is re-described.

New plant-parasitic nematode from the mostly mycophagous genus Bursaphelenchus discovered inside figs in Japan.

A new nematode species, Bursaphelenchus sycophilus n. sp. is described. The species was found in syconia of a fig species, Ficus variegata during a field survey of fig-associated nematodes in Japan. Because it has a well-developed stylet and pharyngeal glands, the species is considered an obligate plant parasite, and is easily distinguished from all other fungal-feeding species in the genus based upon these characters. Although B. sycophilus n. sp. shares an important typological character, male spicule possessing a strongly recurved condylus, with the "B. eremus group" and the "B. leoni group" of the genus, it was inferred to be monophyletic with the "B. fungivorus group". The uniquely shaped stylet and well-developed pharyngeal glands is reminiscent of the fig-floret parasitic but paraphyletic assemblage of "Schistonchus". Thus, these morphological characters appear to be an extreme example of convergent evolution in the nematode family, Aphelenchoididae, inside figs. Other characters shared by the new species and its close relatives, i.e., lack of ventral P1 male genital papilla, female vulval flap, and papilla-shaped P4 genital papillae in males, corroborate the molecular phylogenetic inference. The unique biological character of obligate plant parasitism and highly derived appearance of the ingestive organs of Bursaphelenchus sycophilus n. sp. expands our knowledge of the potential morphological, physiological and developmental plasticity of the genus Bursaphelenchus.

Nematodes from galls on Myrtaceae. IX. Fergusobia rosettae n. sp. on Melaleuca quinquenervia and F. tolgaensis n. sp. on Syzygium luehmannii, from Queensland.

Two new species of Fergusobia, collected from 'rosette' shoot bud galls on Melaleuca quinquenervia, and from leaf, stem, leaf and flower bud galls on Syzygium luehmannii, both from the Cairns region of Queensland, Australia, are described. Fergusobia rosettae Davies n. sp. is characterised by the combination of a small, arcuate parthenogenetic female having a short conoid tail with a bluntly rounded tip, an arcuate, relatively slender, infective female with an almost hemispherical tail tip, and arcuate males with arcuate to angular (not heavily sclerotised) spicules and leptoderan bursa arising at 40-50% of body length from tail tip. Fergusobia tolgaensis Davies n. sp. is characterised by the combination of a small open C-shaped parthenogenetic female with a broadly conoid tail, an arcuate infective female with a broadly rounded tail tip, and arcuate males with angular (not heavily sclerotised) spicules and short to mid-length leptoderan bursa. These two species of nematodes are associated with fly larvae that have dorsal shields comprising bars of raised cuticular ridges and spicules, similar to that of fly larvae from the M. leucadendra species group. The shield morphologies of these fly larvae and their possible genetic relationships are discussed. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants.

Nematodes from galls on Myrtaceae. X. Fergusobia from galls on narrow-leaved Melaleuca spp. in Australia, with descriptions of three new species .

Three new species of Fergusobia, respectively collected from shoot bud galls on narrow-leaved Melaleuca spp. in Australia, are described. Fergusobia armillarisae n. sp. Davies is characterised by the combination of an arcuate to open C-shaped parthenogenetic female with an extensile uterus and a short, conoid tail, an arcuate infective female with a broadly rounded tail tip, and an arcuate male with an angular spicule and bursa arising at 50-80% of body length. Fergusobia decorae n. sp. Davies has an arcuate parthenogenetic female with a non-extensile uterus and a broadly conoid tail, an arcuate infective female with most curvature behind the vulva and a short tail with a broadly rounded tip, and an arcuate male with an arcuate spicule and bursa arising at 40-50% of body length. Fergusobia linariifoliae n. sp. Davies is characterised by the combination of an arcuate parthenogenetic female with an extensile uterus and a short, conoid tail with a bluntly rounded tip, a barely arcuate infective female with a broadly rounded tail tip, and an arcuate male with an angular spicule and bursa arising at 40-50% of body length. Earlier molecular analyses inferred from DNA sequencing of 28S rDNA D2/D3 domains and a portion of mitochondrial DNA cytochrome oxidase subunit I (mtCOI) are further discussed. 

Nematodes from galls on Myrtaceae. VI. Fergusobia from galls on Angophora in Australia, with description of F. colbrani n. sp. and key.

Collection data and biological information is presented on the Fergusobia (Nematoda: Neotylenchidae)/ Fergusonina (Diptera: Fergusoninidae) mutualism inducing galls on Angophora in Australia. Three species and two morphospecies have been recognised. Fergusobia colbrani Davies n. sp. is described from soft spheroid leaf galls on Angophora floribunda. It is characterised by a combination of morphological characters including a small C-shaped parthenogenetic female with a short broadly conoid tail, an arcuate infective female with an almost hemispherical tail tip, and an arcuate to barely J-shaped male with an angular spicule having a notched tip and mid-length leptoderan bursa. A key to the species and morphospecies of nematodes collected from Angophora is presented. Possible relationships of these organisms are discussed based on evidence from the nematode morphology, gall forms, and the morphology of the dorsal shield of the associated Fergusonina fly larvae. 

Distribution and diversity of soil microfauna from East Antarctica: assessing the link between biotic and abiotic factors.

Terrestrial life in Antarctica has been described as some of the simplest on the planet, and mainly confined to soil microfaunal communities. Studies have suggested that the lack of diversity is due to extreme environmental conditions and thought to be driven by abiotic factors. In this study we investigated soil microfauna composition, abundance, and distribution in East Antarctica, and assessed correlations with soil geochemistry and environmental variables. We examined 109 soil samples from a wide range of ice-free habitats, spanning 2000 km from Framnes Mountains to Bailey Peninsula. Microfauna across all samples were patchily distributed, from complete absence of invertebrates to over 1600 specimens/gram of dry weight of soil (gdw), with highest microfauna abundance observed in samples with visible vegetation. Bdelloid rotifers were on average the most widespread found in 87% of sampled sites and the most abundant (44 specimens/gdw). Tardigrades occurred in 57% of the sampled sites with an abundance of 12 specimens/gdw. Nematodes occurred in 71% of samples with a total abundance of 3 specimens/gdw. Ciliates and mites were rarely found in soil samples, with an average abundance of 1.3 and 0.04 specimens/gdw, respectively. We found that microfaunal composition and abundance were mostly correlated with the soil geochemical parameters; phosphorus, NO3 (-) and salinity, and likely to be the result of soil properties and historic landscape formation and alteration, rather than the geographic region they were sampled from. Studies focusing on Antarctic biodiversity must take into account soil geochemical and environmental factors that influence population and species heterogeneity.

Nematodes from galls on Myrtaceae. VIII. Fergusobia from small galls on shoot buds, with descriptions of four new species.

Small shoot bud galls induced by the Fergusobia (Nematoda: Neotylenchidae)/Fergusonina (Diptera: Fergusoninidae) mutualism occur on various Eucalyptus spp. Four new species of Fergusobia, collected from small shoot bud galls on Eucalyptus camaldulensis, E. gomphocephala and E. leucoxylon, are described. Fergusobia gomphocephalae Davies n. sp. is morphologically characterized by a combination of a small C-shaped parthenogenetic female with a variable, conoid tail, a small C-shaped infective female with a hemispherical tail tip, and an arcuate or J-shaped male with a broad tail, angular spicule and short peloderan bursa. Fergusobia leucoxylonae Davies n. sp. has a C-shaped parthenogenetic female with a conoid tail with a narrowly rounded tip, an arcuate infective female with a broadly rounded tail tip, and an almost straight to barely J-shaped male with angular (not heavily sclerotised) spicule and short bursa. Fergusobia schmidti Davies & Bartholomaeus n. sp. has an arcuate to open C-shaped parthenogenetic female with a relatively large body diameter, relatively long stylet and small tail with a broadly rounded tail tip, an open C-shaped infective female with a broadly rounded to hemispherical tail tip, and an arcuate to barely J-shaped male with spicules angular at about 33% of their length and peloderan bursa arising at about half body length. Fergusobia sporangae Davies n. sp. has an arcuate to open C-shaped parthenogenetic female with a relatively long stylet and a broadly rounded tail tip, an arcuate infective female with a short tail with a broadly rounded to hemispherical tip, and an arcuate to barely J-shaped male with angular (not heavily sclerotised) spicule and short peloderan bursa. Various forms of small shoot bud galls are described. From phylogenetic analyses based on sequences of the D2/D3 expansion segment of the large subunit rRNA gene, the four new species belong to two sister clades of Fergusobia. The larval shield morphology of their associated fly species and possible genetic relationships are discussed. 

Nematodes from galls on Myrtaceae. VII. Fergusobia from 'leafy' leaf bud galls in Australia, with re-description of Fergusobia tumifaciens (Currie 1937) Wachek 1955 and descriptions of Fergusobia planchonianae n. sp. and Fergusobia viminalisae n. sp.

Fergusobia tumifaciens (Currie 1937) Wachek 1955, the type species for the genus Fergusobia, is re-described from specimens collected from 'leafy' leaf bud galls on Eucalyptus bridgesiana near Albury in New South Wales, Australia. It is morphologically characterized by the combination of an open C-shaped parthenogenetic female with a small broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with angular spicules, not heavily sclerotised, and short to mid-length peloderan bursa. Two new species of Fergusobia, collected from 'leafy' leaf bud galls on, respectively, Eucalyptus planchoniana in Queensland, and E. viminalis in South Australia, Australia, are described. Fergusobia planchonianae Davies n. sp. is characterised by the combination of a C-shaped parthenogenetic female with a conoid tail, an arcuate infective female with an hemispherical tail tip, and an almost straight to arcuate to C-shaped male with an angular spicule, a long peloderan bursa and a narrow tail. Fergusobia viminalisae Davies n. sp. is characterised by the combination of an open C-shaped parthenogenetic female with a broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with an angular (not heavily sclerotised) spicule and short to mid-length peloderan bursa. The shield morphologies of the fly larvae associated with the 'leafy' leaf bud galls and their possible relationships are outlined. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants. 

Underdiagnosis of Clostridium difficile across Europe: the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID).

BACKGROUND: Variations in testing for Clostridium difficile infection can hinder patients' care, increase the risk of transmission, and skew epidemiological data. We aimed to measure the underdiagnosis of C difficile infection across Europe. METHODS: We did a questionnaire-based study at 482 participating hospitals across 20 European countries. Hospitals were questioned about their methods and testing policy for C difficile infection during the periods September, 2011, to August, 2012, and September, 2012, to August, 2013. On one day in winter, 2012-13 (December, 2012, or January, 2013), and summer, 2013 (July or August), every hospital sent all diarrhoeal samples submitted to their microbiology laboratory to a national coordinating laboratory for standardised testing of C difficile infection. Our primary outcome measures were the rates of testing for and cases of C difficile infection per 10 000 patient bed-days. Results of local and national C difficile infection testing were compared with each other. If the result was positive at the national laboratory but negative at the local hospital, the result was classified as undiagnosed C difficile infection. We compared differences in proportions with the Mann-Whitney test, or McNemar's test if data were matched. FINDINGS: During the study period, participating hospitals reported a mean of 65·8 tests (country range 4·6-223·3) for C difficile infection per 10 000 patient-bed days and a mean of 7·0 cases (country range 0·7-28·7) of C difficile infection per 10 000 patient-bed days. Only two-fifths of hospitals reported using optimum methods for testing of C difficile infection (defined by European guidelines), although the number of participating hospitals using optimum methods increased during the study period, from 152 (32%) of 468 in 2011-12 to 205 (48%) of 428 in 2012-13. Across all 482 European hospitals on the two sampling days, 148 (23%) of 641 samples positive for C difficile infection (as determined by the national laboratory) were not diagnosed by participating hospitals because of an absence of clinical suspicion, equating to about 74 missed diagnoses per day. INTERPRETATION: A wide variety of testing strategies for C difficile infection are used across Europe. Absence of clinical suspicion and suboptimum laboratory diagnostic methods mean that an estimated 40 000 inpatients with C difficile infection are potentially undiagnosed every year in 482 European hospitals. FUNDING: Astellas Pharmaceuticals Europe.

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection.

BACKGROUND: Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. METHODS: In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12,420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. FINDINGS: Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12-2·31). Multistage algorithms performed better than did standalone assays. INTERPRETATION: We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection.