Specific pathway abundances in the neonatal calf faecal microbiome are associated with susceptibility to Cryptosporidium parvum infection: a metagenomic analysis.

BACKGROUND: Cryptosporidium parvum is the main cause of calf scour worldwide. With limited therapeutic options and research compared to other Apicomplexa, it is important to understand the parasites' biology and interactions with the host and microbiome in order to develop novel strategies against this infection. The age-dependent nature of symptomatic cryptosporidiosis suggests a link to the undeveloped immune response, the immature intestinal epithelium, and its associated microbiota. This led us to hypothesise that specific features of the early life microbiome could predict calf susceptibility to C. parvum infection. RESULTS: In this study, a single faecal swab sample was collected from each calf within the first week of life in a cohort of 346 animals. All 346 calves were subsequently monitored for clinical signs of cryptosporidiosis, and calves that developed diarrhoea were tested for Rotavirus, Coronavirus, E. coli F5 (K99) and C. parvum by lateral flow test (LFT). A retrospective case-control approach was taken whereby a subset of healthy calves (Control group; n = 33) and calves that went on to develop clinical signs of infectious diarrhoea and test positive for C. parvum infection via LFT (Cryptosporidium-positive group; n = 32) were selected from this cohort, five of which were excluded due to low DNA quality. A metagenomic analysis was conducted on the faecal microbiomes of the control group (n = 30) and the Cryptosporidium-positive group (n = 30) prior to infection, to determine features predictive of cryptosporidiosis. Taxonomic analysis showed no significant differences in alpha diversity, beta diversity, and taxa relative abundance between controls and Cryptosporidium-positive groups. Analysis of functional potential showed pathways related to isoprenoid precursor, haem and purine biosynthesis were significantly higher in abundance in calves that later tested positive for C. parvum (q ≤ 0.25). These pathways are either absent or streamlined in the C. parvum parasites. Though the de novo production of isoprenoid precursors, haem and purines are absent, C. parvum has been shown to encode enzymes that catalyse the downstream reactions of these pathway metabolites, indicating that C. parvum may scavenge those products from an external source. CONCLUSIONS: The host has previously been put forward as the source of essential metabolites, but our study suggests that C. parvum may also be able to harness specific metabolic pathways of the microbiota in order to survive and replicate. This finding is important as components of these microbial pathways could be exploited as potential therapeutic targets for the prevention or mitigation of cryptosporidiosis in bovine neonates.

Medicine and the Law.

Congenital high airway obstruction syndrome (CHAOS) is a rare condition that can be diagnosed antenatally by ultrasound. It is usually lethal without immediate intervention at delivery. A 24-year-old woman was diagnosed with fetal CHAOS at 27 weeks' gestation. The couple declined termination of pregnancy. A multidisciplinary team including obstetricians, geneticists, paediatric surgeons, neonatologists and anaesthetists was constituted to plan an ex utero intrapartum treatment (EXIT) procedure. After several simulations, a caesarean section was performed at 38 weeks' gestation under deep inhalational anaesthesia. The fetus was fully delivered with placenta remaining in utero to maintain perfusion. A surgical airway was established via tracheostomy in approximately 5 minutes. The operation was then completed with no maternal complications. The child remains well at 3 years of age. To our knowledge, this is the first EXIT procedure performed for CHAOS in the public sector. This procedure can be lifesaving and is possible with proper planning.

Shifting sows: longitudinal changes in the periparturient faecal microbiota of primiparous and multiparous sows.

Knowledge of periparturient longitudinal changes in sow microbiota composition is necessary to fully understand her role in the development of the piglet microbiota, but also to improve gut health and performance of the sow in lactation. Primiparous sows face the challenge of partitioning nutrients to support maternal growth in addition to supporting foetal growth and the demands of lactation. Additional metabolic stress present during the periparturient period may induce changes in the microbiota profile between primiparous and multiparous sows. Using 16S rRNA gene sequencing, the study aimed to characterise the longitudinal changes in the periparturient microbiota and identify differences within the sow microbiota profile associated with parity. Faecal samples from primiparous (n = 13) and multiparous (n = 16) sows were collected at four different time points (day -6, -1, 3 and 8) in relation to farrowing (day 0). Microbiota richness was lowest on day 3 and -1 of the periparturient period (P < 0.05). Microbiota community composition, assessed by weighted and unweighted UniFrac distances, demonstrated longitudinal changes, with day 3 samples clustering away from all other sampling time points (P < 0.05). The relative abundance of several genera segregated gestation from lactation samples including Roseburia, Prevotella 1, Prevotella 2, Christensenellaceae R-7 group, Ruminococcaceae UCG-002 and Ruminococcaceae UCG-010 (P < 0.01). Furthermore, day 3 was characterised by a significant increase in the relative abundance of Escherichia/Shigella, Fusobacterium and Bacteroides, and a decrease in Alloprevotella, Prevotellaceae UCG-003 and Ruminococcus 1 (P < 0.001). Primiparous sows had overall lower periparturient microbiota diversity (P < 0.01) and there was a significant interaction between parity and sampling time point, with primiparous sows having lower microbiota richness on day -6 (P < 0.001). There was a significant interaction between sow parity and sampling time point on microbiota composition on day -6 and -1 (unweighted UniFrac distances;  ≤ 0.01) and day 8 (weighted and unweighted UniFrac distances; P < 0.05). Whilst no significant interactions between sow parity and sampling day were observed for genera relative abundances, multiparous sows had a significantly higher relative abundance of Bacteroidetes dgA-11 gut group and Prevotellaceae UCG-004 (P < 0.01). This study demonstrates that the sow microbiota undergoes longitudinal changes, which are collectively related to periparturient changes in the sow environment, diet and physiological changes to support foetal growth, delivery and the onset of lactation, but also sow parity.

Association between pelvic inflammatory disease, infertility, ectopic pregnancy and the development of ovarian serous borderline tumor, mucinous borderline tumor and low-grade serous carcinoma.

OBJECTIVE: Risk factors for ovarian borderline tumors and low-grade serous carcinoma (LGSC) are poorly understood. The aim of this study was to examine the association between infertility, pelvic inflammatory disease (PID), endometriosis, ectopic pregnancy, hysterectomy, tubal ligation and parity and the risk of serous borderline tumor (SBT), mucinous borderline tumor (MBT) and LGSC. METHODS: This was a population-based cohort study using linked administrative and hospital data. Participants were 441,382 women born between 1945 and 1975 who had been admitted to hospital in Western Australia between 1 January 1980 and 30 June 2014. We used Cox regression to estimate hazard ratios (HRs). RESULTS: We observed an increased rate of SBT associated with infertility, PID and ectopic pregnancy (HRs and 95% CIs were, respectively, 1.98 (1.20-3.26); 1.95 (1.22-3.10) and 2.44 (1.20-4.96)). We did not detect an association between any of the factors under study and the rate of MBT. A diagnosis of PID was associated with an increased rate of LGSC (HR 2.90, 95% CI 1.21-6.94). CONCLUSIONS: The association with PID supports the hypothesis that inflammatory processes within the upper gynaecological tract and/or peritoneum may predispose to the development of SBT and LGSC.

'Bastard bush': acacia thorn injuries and management.

This case report outlines the presentation and management of a young soldier who sustained a lower limb acacia thorn injury while on exercise in Kenya. The injury failed to heal with a subsequent ultrasound scan revealing a large retained thorn requiring surgical removal and wash out. From this case, lessons can be learnt regarding the management of thorn injuries, which are common in exercising troops in Kenya and indeed around the world. The key take-home messages are always consider a retained thorn if wounds fail to settle, use ultrasound as the imaging modality of choice, always remove identified retained thorns and if antibiotics are required use broad-spectrum antibiotics pending culture results.

Circulating 25-hydroxyvitamin D, nasopharyngeal airway metabolome, and bronchiolitis severity.

Low circulating 25-hydroxyvitamin D (25OHD) levels are a risk factor for acute respiratory infection (eg, bronchiolitis) in children. However, little is known about the relation of circulating 25OHD with the many downstream functional molecules in target organs-such as the airway-and with clinical outcomes. In this prospective multicenter study of infants (age <1 year) hospitalized with bronchiolitis, we measured serum 25OHD levels and profiled the metabolome of 144 nasopharyngeal airway samples. Among 254 metabolites identified, we defined a set of 20 metabolites that are related to lower serum 25OHD and higher vitamin D-binding protein levels. Of these metabolites, 9 metabolites were associated with a significantly higher risk of positive pressure ventilation use. These metabolites were glycerophosphocholines esterified with proinflammatory fatty acids (palmitate, arachidonate, linoleate, and stearate), sphingomyelins, alpha-hydroxyisovalerate, 2-hydroxybutyrate, and 3-(4-hydroxyphenyl)lactate (all FDR<0.05). Based on the multicenter data, vitamin D-related airway metabolites were associated with risks of bronchiolitis severity.

Liposuction as an effective treatment for lower extremity lymphoedema: A single surgeon's experience over nine years.

BACKGROUND: Lymphoedema is a chronic, debilitating condition caused by a compromised lymphatic system. In recent years, the success of treating upper extremity lymphoedema with liposuction has been translated to patients with lower extremity lymphoedema (LEL), yet there remains a paucity of clinical evidence firmly supporting its use within this patient group. METHODS: 69 patients with LEL (72 legs) were consecutively treated with liposuction by a single surgeon. Compression garments were applied in theatre and continued postoperatively. RESULTS: Mean preoperative volume of oedema was 4372 mL (range 229-15,166 mL), and mean volume of aspirate was 4550 mL (range 575-12,150 mL). There were no major surgical complications. An average reduction in volume of leg oedema of 85% was found at 3 months (n = 72), 88% at 1 year (n = 60), 94% at 2 years (n = 41) and 90% at 5 years (n = 15). CONCLUSIONS: We have demonstrated that liposuction combined with continuous compression therapy (CCT) is a safe and effective technique for treatment of primary and secondary LEL, with a significant reduction of the original excess limb volume. Male patients with primary lymphoedema have the poorest outcomes. Limbs with secondary lymphoedema respond best to this treatment.

Abnormal loading and functional deficits are present in both limbs before and after unilateral knee arthroplasty.

Unilateral knee replacement is often followed by a contralateral replacement in time and the biomechanics of the other knee before and after knee replacement remains poorly understood. The aim of this paper is to distinguish the features of arthritic gait in the affected and unaffected legs relative to a normal population and to assess the objective recovery of gait function post-operatively, with the aim of defining patients at risk of poor post-operative function. Twenty patients with severe knee OA but no pain or deformity in any other lower limb joint were compared to twenty healthy subjects of the same age. Gait analysis was performed and quadriceps and hamstrings co-contraction was measured. Fifteen subjects returned 1year following knee arthroplasty. Moments and impulses were calculated, principal component analysis was used to analyse the waveforms and a classification technique (the Cardiff Classifier) was used to select the most discriminant data and define functional performance. Comparing pre-operative function to healthy function, classification accuracies for the affected and unaffected knees were 95% and 92.5% respectively. Post-operatively, the affected limb returned to the normal half of the classifier in 8 patients, and 7 of those patients returned to normal function in the unaffected limb. Recovery of normal gait could be correctly predicted 13 out of 15 times at the affected knee, and 12 out of 15 times at the unaffected knee based on pre-operative gait function. Focused rehabilitation prior to surgery may be beneficial to optimise outcomes and protect the other joints following knee arthroplasty.

Gut microbiota of Type 1 diabetes patients with good glycaemic control and high physical fitness is similar to people without diabetes: an observational study.

AIM: Type 1 diabetes is the product of a complex interplay between genetic susceptibility and exposure to environmental factors. Existing bacterial profiling studies focus on people who are most at risk at the time of diagnosis; there are limited data on the gut microbiota of people with long-standing Type 1 diabetes. This study compared the gut microbiota of patients with Type 1 diabetes and good glycaemic control and high levels of physical-fitness with that of matched controls without diabetes. METHODS: Ten males with Type 1 diabetes and ten matched controls without diabetes were recruited; groups were matched for gender, age, BMI, peak oxygen uptake (VO2max ), and exercise habits. Stool samples were analysed using next-generation sequencing of the 16S rRNA gene to obtain bacterial profiles from each individual. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was implemented to predict the functional content of the bacterial operational taxonomic units. RESULTS: Faecalibacterium sp., Roseburia sp. and Bacteroides sp. were typically the most abundant members of the community in both patients with Type 1 diabetes and controls, and were present in every sample in the cohort. Each bacterial profile was relatively individual and no significant difference was reported between the bacterial profiles or the Shannon diversity indices of Type 1 diabetes compared with controls. The functional profiles were more conserved and the Type 1 diabetes group were comparable with the control group. CONCLUSIONS: We show that both gut microbiota and resulting functional bacterial profiles from patients with long-standing Type 1 diabetes in good glycaemic control and high physical fitness levels are comparable with those of matched people without diabetes.

Microbiological profiles of sputum and gastric juice aspirates in Cystic Fibrosis patients.

Gastro-Oesophageal Reflux (GOR) is a key problem in Cystic Fibrosis (CF), but the relationship between lung and gastric microbiomes is not well understood. We hypothesised that CF gastric and lung microbiomes are related. Gastric and sputum cultures were obtained from fifteen CF patients receiving percutaneous endoscopic gastrostomy feeding. Non-CF gastric juice data was obtained through endoscopy from 14 patients without lung disease. Bacterial and fungal isolates were identified by culture. Molecular bacterial profiling used next generation sequencing (NGS) of the 16S rRNA gene. Cultures grew bacteria and/or fungi in all CF gastric juice and sputa and in 9/14 non-CF gastric juices. Pseudomonas aeruginosa(Pa) was present in CF sputum in 11 patients, 4 had identical Pa strains in the stomach. NGS data from non-CF gastric juice samples were significantly more diverse compared to CF samples. NGS showed CF gastric juice had markedly lower abundance of normal gut bacteria; Bacteroides and Faecalibacterium, but increased Pseudomonas compared with non-CF. Multivariate partial least squares discriminant analysis demonstrated similar bacterial profiles of CF sputum and gastric juice samples, which were distinct from non-CF gastric juice. We provide novel evidence suggesting the existence of an aerodigestive microbiome in CF, which may have clinical relevance.

Microsurgical skill acquisition in a one-day introductory course with performance evaluation using software-assisted scoring system.

BACKGROUND: Recent emphasis on microsurgical skill acquisition at an earlier stage of plastic surgery training has seen a shift toward objective competence-based assessment. Yet no objective measures of spacing or alignment exist, with few validated models that assess ability. The authors propose a novel software analysis scoring system to objectively measure spacing, alignment and the overall improvement in a 1-day, introductory course setting. METHODS: Images of standard 4-mm latex strips that had been sutured by participants using the Microtrainer system were uploaded onto calibrated, online software. Sutures were analysed with regard to spacing, alignment and density. From these measurements, a total score was calculated, one on initial assessment at the course beginning (Score 1) and another on final assessment at the course end (Score 2), thereby facilitating measurement of the overall improvement. RESULTS: A total of 38 microsurgical anastomoses from 19 participants ranging from postgraduate years 1-7 were analysed. Seventeen participants had no previous experience of microsurgery. The mean average Score 1 of participants was -2 (range -12 to +22) and Score 2 was 22 (range +12 to +32), thus showing a significant improvement in candidate ability throughout the course of the day (p < 0.0001). CONCLUSIONS: Microtrainer system software analysis provides a novel, reliable, and consistent objective assessment for surgical trainees at all stages of training, without risk to patients. It has an associated cost for the initial setup, yet is timely, repeatable and can efficiently demonstrate progress in a 1-day course setting.

Novel BRAF and KRAS Mutations in Papillary Thyroid Carcinoma Arising in Struma Ovarii.

Papillary carcinomas of thyroid type rarely arise within struma ovarii. There are limited data on the immunohistochemical and molecular features of these tumors. Three cases of papillary carcinoma arising in struma ovarii (PCSO) were identified. The clinicopathological features were reviewed and immunohistochemical staining for HBME-1, cytokeratin (CK) 19, and CD56 was performed. Tumor DNA was sequenced for somatic mutations using a panel of 26 oncogenes, with a particular focus on BRAF and KRAS mutations. The patients were aged 22, 48, and 55 years. All cases were FIGO stage IA. Two tumors were of classical histological type, and one was a follicular variant papillary carcinoma. All tumors expressed HBME-1 and two were positive for CK19. CD56 was negative in all three cases. One tumor demonstrated a BRAF G469A mutation in exon 11, and in a second case, a KRAS Q61K double base mutation in exon 3 was detected. These mutations have not been described previously in PCSO. No mutations were detected in the benign follicular components of the tumors adjacent to the malignant papillary tissue. None of the patients had tumor recurrence on clinical follow-up (range 11 months to 8½ years). HBME-1, CK19, and CD56 are useful immunohistochemical markers of PCSO. Novel BRAF and KRAS mutations were identified in two of three tumors suggesting that mutations in PCSO may differ from those commonly identified in papillary carcinoma of the eutopic thyroid. The clinical significance of these mutations is uncertain but follow-up data in this small series support the generally good prognosis of PCSO.

Effect of crop management and sample year on abundance of soil bacterial communities in organic and conventional cropping systems.

AIMS: To identify changes in the bacterial community, at the phylum level brought about by varied crop management. METHODS AND RESULTS: Next-generation sequencing methods were used to compare the taxonomic structure of the bacterial community within 24 agricultural soils managed with either organic or conventional methods, over a 3-year period. Relative abundance of the proportionately larger phyla (e.g. Acidobacteria and Actinobacteria) was primarily affected by sample year rather than crop management. Changes of abundance in these phyla were correlated with changes in pH, organic nitrogen and soil basal respiration. Crop management affected some of the less dominant phyla (Chloroflexi, Nitrospirae, Gemmatimonadetes) which also correlated with pH and organic N. CONCLUSION: Soil diversity can vary with changing environmental variables and soil chemistry. If these factors remain constant, soil diversity can also remain constant even under changing land use. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of crop management on environmental variables must be considered when interpreting bacterial diversity studies in agricultural soils. Impact of land use change should always be monitored across different sampling time points. Further studies at the functional group level are necessary to assess whether management-induced changes in bacterial community structure are of biological and agronomic relevance.

Comparison of cold knife cone biopsy and loop electrosurgical excision procedure in the management of cervical adenocarcinoma in situ: What is the gold standard?

OBJECTIVE: To compare the outcomes of patients with cervical adenocarcinoma in situ (ACIS) treated with cold knife cone (CKC) biopsy or loop electrosurgical excision procedure (LEEP) for the treatment of cervical adenocarcinoma in situ (ACIS). STUDY DESIGN: This is a retrospective, population-based cohort study of Western Australian patients with ACIS diagnosed between 2001 and 2012. Outcomes included pathological margin status and the incidence of persistent or recurrent endocervical neoplasia (ACIS and adenocarcinoma) during follow-up (<12 months) and surveillance (≥12 months) periods. RESULTS: The study group comprised 338 patients including 107 (32%) treated initially by LEEP and 231 (68%) treated by CKC biopsy. The mean age was 33.2 years (range 18 to 76 years) and median follow-up interval was 3.6 years (range <1 year to 11.8 years). Overall, 27 (8.0%) patients had ACIS persistence/recurrence while 9 (2.7%) were diagnosed with adenocarcinoma during the follow-up and surveillance periods. No patient died of cervical cancer within the study period. There were no significant differences in the incidence of persistent and/or recurrent endocervical neoplasia according to the type of excisional procedure. Patients with positive biopsy margins were 3.4 times more likely to have disease persistence or recurrence. CONCLUSION(S): LEEP and CKC biopsy appear equally effective in the treatment of ACIS for women wishing to preserve fertility. Patients undergoing conservative management for ACIS should be closely monitored, particularly if biopsy margins are positive in initial excision specimens. Patients and their clinicians should be aware of the potential risks of residual and recurrent disease.

NUT protein immunoreactivity in ovarian germ cell tumours.

The aim of this study was to investigate NUT (nuclear protein in the testis) expression in ovarian germ cell tumours (GCTs). Immunostaining for NUT protein was performed in 10 mature cystic teratomas and in 49 malignant ovarian GCTs including 15 pure dysgerminomas, six dysgerminomas associated with gonadoblastoma, nine yolk sac tumours, 12 immature teratomas, and seven mixed malignant tumours. Only nuclear staining was considered a positive finding although cytoplasmic staining was noted when present. Thirty-seven (76%) malignant GCTs were NUT positive but staining was usually of weak to moderate intensity and observed in a relatively small proportion of neoplastic cells. Staining in immature teratomas and yolk sac tumours was restricted to foci of hepatoid and intestinal/glandular differentiation, where both nuclear and cytoplasmic reactivity were observed. In dysgerminoma associated with gonadoblastoma only the in situ and invasive germ cell elements were NUT positive. Nuclear staining was not seen in benign teratomas. Most malignant ovarian GCTs express NUT protein, albeit focally, and this should be considered when evaluating immunostaining in the differential diagnosis of poorly differentiated malignancies, particularly NUT midline carcinoma. Since NUT protein appears to play a role in normal germ cell maturation it may influence intestinal or hepatoid differentiation within malignant GCTs.

Fascin expression in cervical normal squamous epithelium, cervical intraepithelial neoplasia, and superficially invasive (stage IA1) squamous carcinoma of the cervix.

The aims of this study were to: investigate fascin expression in normal cervix, cervical intraepithelial neoplasia (CIN), and stage IA1 squamous cell carcinoma (SCC).Fascin immunostaining was performed in cervical biopsies showing normal squamous epithelium (n=10), CIN 1 (n=10), CIN 2-3 without invasion (n=11), and CIN 2-3 adjacent to SCC (n=40); SCC was also present in 27 of the latter cases.Fascin expression in normal squamous epithelium was restricted to basal and parabasal cells, whereas there was increased staining in immature squamous metaplasia and in most CIN lesions. Full thickness staining was more frequent in high grade CIN adjacent to invasion than in CIN 2-3 alone. Eighteen SCCs (67%) were fascin positive and seven cases showed accentuated staining at the tumour-stromal interface (invasive front). There was no consistent relationship between fascin expression in CIN lesions and in corresponding carcinomas. Fascin staining in reactive stromal cells sometimes made identification of the invasive neoplastic cells difficult.Fascin is overexpressed in most CIN lesions and this may be a marker of increased invasive potential in high grade CIN. However, fascin staining does not distinguish low and high grade CIN or in situ and invasive squamous neoplasia. Therefore fascin has limited diagnostic utility in demonstrating superficial stromal invasion.

Exercise-induced dehydration does not alter time trial or neuromuscular performance.

This study examined the effect of exercise-induced dehydration by ~4% body mass loss on 5-km cycling time trial (TT) performance and neuromuscular drive, independent of hyperthermia. 7 active males were dehydrated on 2 occasions, separated by 7 d. Participants remained dehydrated (DEH, -3.8±0.5%) or were rehydrated (REH, 0.2±0.6%) over 2 h before completing the TT at 18-25 °C, 20-30% relative humidity. Neuromuscular function was determined before dehydration and immediately prior the TT. The TT started at the same core temperature (DEH, 37.3±0.3°C; REH, 37.0±0.2 °C (P>0.05). Neither TT performance (DEH, 7.31±1.5 min; REH, 7.10±1.3 min (P>0.05)) or % voluntary activation were affected by dehydration (DEH, 88.7±6.4%; REH, 90.6±6.1% (P>0.05)). Quadriceps peak torque was significantly elevated in both trials prior to the TT (P<0.05), while a 19% increase in the rate of potentiated peak twitch torque development (P<0.05) was observed in the DEH trial only. All other neuromuscular measures were similar between trials. Short duration TT performance and neuromuscular function are not reduced by dehydration, independent of hyperthermia.