Molecular typing of Leptospira spp. in farmed and wild mammals reveals new host-serovar associations in New Zealand.

AIMS: To apply molecular typing to DNA isolated from historical samples to determine Leptospira spp. infecting farmed and wild mammals in New Zealand. MATERIALS AND METHODS: DNA samples used in this study were extracted from urine, serum or kidney samples (or Leptospira spp. cultures isolated from them) collected between 2007 and 2017 from a range of domestic and wildlife mammalian species as part of different research projects at Massey University. Samples were included in the study if they met one of three criteria: samples that tested positive with a lipL32 PCR for pathogenic Leptospira; samples that tested negative by lipL32 PCR but were recorded as positive to PCR for pathogenic Leptospira in the previous studies; or samples that were PCR-negative in all studies but were from animals with positive agglutination titres against serogroup Tarassovi. DNA samples were typed using PCR that targeted either the glmU or gyrB genetic loci. The resulting amplicons were sequenced and typed relative to reference sequences. RESULTS: We identified several associations between mammalian hosts and Leptospira strains/serovars that had not been previously reported in New Zealand. Leptospira borgpetersenii strain Pacifica was found in farmed red deer (Cervus elaphus) samples, L. borgpetersenii serovars Balcanica and Ballum were found in wild red deer samples, Leptospira interrogans serovar Copenhageni was found in stoats (Mustela erminea) and brushtail possums (Trichosurus vulpecula), and L. borgpetersenii was found in a ferret (Mustela putorius furo). Furthermore, we reconfirmed previously described associations including dairy cattle with L. interrogans serovars Copenhageni and Pomona and L. borgpetersenii serovars Ballum, Hardjo type bovis and strain Pacifica, sheep with L. interrogans serovar Pomona and L. borgpetersenii serovar Hardjo type bovis, brushtail possum with L. borgpetersenii serovar Balcanica, farmed deer with L. borgpetersenii serovar Hardjo type bovis and hedgehogs (Erinaceus europaeus) with L. borgpetersenii serovar Ballum. CONCLUSIONS: This study provides an updated summary of host-Leptospira associations in New Zealand and highlights the importance of molecular typing. Furthermore, strain Pacifica, which was first identified as Tarassovi using serological methods in dairy cattle in 2016, has circulated in animal communities since at least 2007 but remained undetected as serology is unable to distinguish the different genotypes. CLINICAL RELEVANCE: To date, leptospirosis in New Zealand has been diagnosed with serological typing, which is deficient in typing all strains in circulation. Molecular methods are necessary to accurately type strains of Leptospira spp. infecting mammals in New Zealand.

Carriage of Staphylococcus pseudintermedius by clinically normal dogs in Canterbury, New Zealand.

AIMS: To investigate the frequency of carriage of methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius (MRSP) in a population of clinically normal dogs within the Christchurch and wider Canterbury region, an area in which MRSP has been detected. METHODS: Buccal and perianal swabs were collected from 126 clinically normal dogs presenting at veterinary clinics in the Christchurch/Canterbury region for de-sexing or routine vaccination. S. pseudintermedius was isolated by selective culture. Isolates were tested for susceptibility to 12 antimicrobials by disc diffusion. RESULTS: S. pseudintermedius was isolated from 92/126 (73.0 (95% CI = 64.4-80.5)%) dogs, with 38/126 (30.2 (95% CI = 22.3-39.0)%) positive dogs carrying S. pseudintermedius at both sampled sites. More animals (78/126; 61.9 (95% CI = 52.8-70.4)%) had positive mouth cultures than positive perianal region cultures (52/126; 41.3 (95% CI = 32.6-50.4)%). No MRSP was isolated from clinically normal dogs. However, resistance to penicillin (106/130 (85.1%) swabs) and tetracycline (33/130 (25.4%) swabs) was seen. CONCLUSIONS: The majority of the dogs in this sample were carriers of S. pseudintermedius. However, none of these isolates were MRSP. CLINICAL RELEVANCE: While most clinically normal dogs in the studied region are likely to be carriers of S. pseudintermedius, only a small proportion, if any, are likely to be carriers of MRSP. Antibiotic stewardship practices may be important to maintain low-level circulation of drug-resistant bacterial lineages.

Isolation of Campylobacter hepaticus from free-range poultry with spotty liver disease in New Zealand.

Case history: In October 2019, a free-range egg laying flock suffering an outbreak of spotty liver disease was investigated. Eight 32-week-old hens were examined post-mortem. Clinical and pathological findings: Five of the eight hens had sparse, focal, gross hepatic lesions typical of spotty liver disease. Histopathology of the liver showed random, focal hepatic necrosis, lymphoplasmacytic cholangitis/pericholangitis and, in one hen, severe lymphoplasmacytic cholecystitis. Campylobacter-like organisms were grown from all eight bile samples which were confirmed by PCR as Campylobacter hepaticus. The genome of C. hepaticus isolates from the outbreak were sequenced and compared to those of isolates from Australia and the United Kingdom. Phylogenetic analysis based on single nucleotide polymorphisms showed that the C. hepaticus isolates from this outbreak were most closely related to isolates from Australia. Diagnosis: Campylobacter hepaticus focal hepatic necrosis. Clinical relevance: This is the first report of an outbreak of spotty liver disease confirmed to be caused by C. hepaticus in poultry in New Zealand. Therefore infection with C. hepaticus should be considered as a differential diagnosis for mortality in laying hens around peak lay in New Zealand.

Source attributed case-control study of campylobacteriosis in New Zealand.

BACKGROUND: Following an initial reduction in human campylobacteriosis in New Zealand after the implementation of poultry food chain-focused interventions during 2006-2008, further decline has been relatively small. We report a year-long study of notified campylobacteriosis cases, incorporating a case control study combined with a source attribution study. The purpose was to generate up-to-date evidence on the relative contributions of different sources of campylobacteriosis in New Zealand. METHODS: The study approach included: • A case-control study of notified cases (aged six months or more) sampled in a major urban centre (Auckland, every second case) and a mixed urban/rural area (Manawatu/Whanganui, every case), between 12 March 2018 and 11 March 2019. • Source attribution of human campylobacteriosis cases sampled from these two regions over the study period by modelling of multilocus sequence typing data of Campylobacter jejuni and C. coli isolates from faecal samples of notified human cases and relevant sources (poultry, cattle, sheep). RESULTS: Most cases (84%) were infected with strains attributed to a poultry source, while 14% were attributed to a cattle source. Approximately 90% of urban campylobacteriosis cases were attributed to poultry sources, compared to almost 75% of rural cases. Poultry consumption per se was not identified as a significant risk factor. However specific risk factors related to poultry meat preparation and consumption did result in statistically significantly elevated odds ratios. CONCLUSIONS: The overall findings combining source attribution and analysis of specific risk factors indicate that poultry meat remains a dominant pathway for exposure and infection.

Radiation-Induced Imaging Changes and Cerebral Edema following Stereotactic Radiosurgery for Brain AVMs.

BACKGROUND AND PURPOSE: T2 signal and FLAIR changes in patients undergoing stereotactic radiosurgery for brain AVMs may occur posttreatment and could result in adverse radiation effects. We aimed to evaluate outcomes in patients with these imaging changes, the frequency and degree of this response, and factors associated with it. MATERIALS AND METHODS: Through this retrospective cohort study, consecutive patients treated with stereotactic radiosurgery for brain AVMs who had at least 1 year of follow-up MR imaging were identified. Logistic regression analysis was used to evaluate predictors of outcomes. RESULTS: One-hundred-sixty AVMs were treated in 148 patients (mean, 35.6 years of age), including 42 (26.2%) pediatric AVMs. The mean MR imaging follow-up was 56.5 months. The median Spetzler-Martin grade was III. The mean maximal AVM diameter was 2.8 cm, and the mean AVM target volume was 7.4 mL. The median radiation dose was 16.5 Gy. New T2 signal and FLAIR hyperintensity were noted in 40% of AVMs. T2 FLAIR volumes at 3, 6, 12, 18, and 24 months were, respectively, 4.04, 55.47, 56.42, 48.06, and 29.38 mL Radiation-induced neurologic symptoms were encountered in 34.4%. In patients with radiation-induced imaging changes, 69.2% had new neurologic symptoms versus 9.5% of patients with no imaging changes (P = .0001). Imaging changes were significantly associated with new neurologic findings (P < .001). Larger AVM maximal diameter (P = .04) and the presence of multiple feeding arteries (P = .01) were associated with radiation-induced imaging changes. CONCLUSIONS: Radiation-induced imaging changes are common following linear particle accelerator-based stereotactic radiosurgery for brain AVMs, appear to peak at 12 months, and are significantly associated with new neurologic findings.

Primary trauma care: a 20-year review.

This year marks the 20th anniversary of the Primary Trauma Care Foundation (PTC), which provides a course aimed at providing a systematic approach to trauma and the unwell patient in low- and middle-income countries. The course is based on the original 36-page PTC manual and a key ethos of being appropriate to the target audience, affordable to those who need it, adaptable to the needs of each local area and sustainable to perpetuate itself. The concept is simple: a two-day course for candidates, followed by a one-day instructor course, and finally a first course delivered to local healthcare staff by the first cohort. Now in its 20th year, its reach spans 76 countries, is ingrained in the syllabus of many healthcare systems and continues to reach new territories with a programme shown to produce a measurable improvement in the care delivered.

Dosimetric comparison of 106Ru and 125I plaques for treatment of shallow (

The aim of this study was to compare dosimetric parameters between iodine-125 ((125)I) and ruthenium-106 ((106)Ru) plaques of similar sizes in the treatment of choroidal melanomas. The study design included retrospective double planning of each case. 26 consecutive patients with choroidal melanomas measuring 5 mm or less in thickness were included. Dose distributions were calculated using Plaque Simulator treatment-planning software for a prescription of 85 Gy to the tumour apex. Doses to the outer sclera, lens, fovea and optic disc were obtained for each case using appropriately sized plaques of each isotope. Statistical inferences were made using the non-parametric Wilcoxon signed rank test. The mean dose to the macula, disc and lens was 18%, 53% and 89% less, respectively, with (106)Ru than with (125)I. Scleral doses were greater for (106)Ru. The use of collaborative ocular melanoma study dosimetry results in even higher mean doses to the macula, disc, lens and sclera. Two-dimensional dose distributions and dose volume histograms demonstrated the increase in dose outside the tumour volume using (125)I. This comparison shows that, for tumours not exceeding 5 mm in thickness, the use of (106)Ru plaques has the potential to reduce the radiation dose to nearby normal structures and possibly lower the risk of radiation-induced visual loss.

High dose rate (HDR) brachytherapy quality assurance: a practical guide.

The widespread adoption of high dose rate brachytherapy with its inherent dangers necessitates adoption of appropriate quality assurance measures to minimize risks to both patients and medical staff. This paper is aimed at assisting someone who is establishing a new program or revising one already in place into adhere to the recently issued Nuclear Regulatory Commission (USA) regulations and the guidelines from the American Association of Physicists in Medicine.

Seizures and the pituitary gland during pregnancy.

We present the anaesthetic management of a woman who, at 10 days post partum, suffered a series of convulsions in the context of two episodes of post partum haemorrhage. The probable aetiology of the convulsions is discussed.

High dose rate (192)Ir afterloading brachytherapy for cancer of the vagina.

We report results of brachytherapy for carcinoma of the vagina, utilizing a Nucletron high dose rate system for Delclos Vaginal Applicators (cylinder) and Syed Template Applicators (interstitial). The linear quadratic (LQ) model was used to determine the optimum time-dose-fractionation schedules. Interstitial doses were determined at the isodose line that included gross tumour. Cylinder doses were determined either at the vaginal surface (5 cases), at 0.5 cm depth (5 cases), or at 1.0 cm depth (1 case). For the first treatment (n=19), interstitial templates were utilized in 8 patients and vaginal cylinders in 11. 11 patients received second treatments: 6 templates and 5 cylinders. The median dose of external beam radiation (n=15) was 40.0 Gy followed, after a median 23 day interval, by high dose rate brachytherapy (HDRB) of 4 fractions in 30-42 h; then a median interval gap of 25 days, followed by repeat HDRB. The median total fractionated HDRB dose per patient was 23.0 Gy (range: 6.9 Gy to 40.4 Gy; calculated low dose rate equivalent of 29.8 Gy). Tumour histologies included 14 squamous cell carcinomas, 2 adenocarcinomas, 2 melanomas, and 1 small cell tumour. Three patients experienced early brachytherapy-related complications (diarrhoea, dysuria and labial dermatitis). Three patients (15.8%) developed serious/late complications including ureteral stenosis, painful vaginal necrosis and small bowel obstruction. The first of these patients received 2 templates, the second a cylinder followed by a template and a cylinder, and the third a single cylinder. The 2 year progression-free survival was 39.3% (median 15.7 months), while the 2 year overall survival was 66.1% (median 29.9 months). (192)Ir afterloading HDRB is a feasible approach to women with vaginal cancer with acceptable toxicity and tumour response. Potential advantages include patient preference, outpatient cost-effectiveness in the case of cylinder technique, and no radiation exposure to hospital personnel. Long-term follow-up is needed to further assess late complications, and larger studies are needed to confirm our results.

Dosimetric comparison of pre-planned and or-planned prostate seed brachytherapy.

PURPOSE: To compare the dosimetry of the traditional two step procedure (volume study + treatment planning several weeks later) with that of an OR-based single procedure in which these two steps follow one another immediately. Computer generated treatment plans were used in both procedures. METHODS AND MATERIALS: Several dosimetric parameters relating to target coverage were obtained from dose volume histograms of CT-based evaluation plans developed either 1 or 3 days following seed implantation. A total of 113 patients with early stage (T1C, T2A) prostate cancer were used for this retrospective study. RESULTS: The fraction of target (prostate) covered by the prescription dose (144 Gy), 90% of the prescription dose (115 Gy), and the dose encompassing 90% of the target in the evaluation plan were all statistically significantly improved for OR-based plans compared to pre-planned cases. CONCLUSION: In our hands, there is a small but significant improvement in dose coverage of the prostate when the ultrasound volume study and treatment planning are combined into a single procedure.

Conformal prostate treatment planning using a low-energy (6-MV) beam.

A 4-field noncoplanar technique for treatment of prostate cancer developed at the University of Michigan was modified for use with low-energy (6 MV) beams. These modifications include the use of wedges on the 2 anterior inferior-superior oblique fields and adjusting the weights of the oblique and lateral fields appropriately. A margin of 1.5 cm around the physician-defined target region was used to define the blocks on each beam's-eye view. Dose distributions produced using this technique with 6-MV and 24-MV beams were compared visually on several dose planes (transverse and sagittal) and quantitatively by dose volume histograms (target, rectum, and bladder). These comparisons showed insignificant differences between the high-energy and low-energy treatment plans. Much larger differences were observed in comparisons of 2 types of coplanar plans with the noncoplanar setup for the 6-MV photon machine. Rectal doses measured in situ were used to help validate the dose distribution predicted by the treatment planning system for the 6-MV noncoplanar technique.

Effects of using recommended coping strategies on drinking outcome following a brief intervention.

AIMS: To explore the unique contribution to outcome drinking of clients' use of six strategies for moderating drinking, after statistically accounting for variance explained by some client and intervention variables. DESIGN: An exploratory hierarchical regression analysis assessed the contributions to variance in drinking outcome of pre-intervention (client characteristics, baseline drinking), assignment (level of assessment, therapist experience) and early follow-up variables. Data came from an experimental trial which evaluated the effect of adding assessment to provision of a self-help book to heavy drinkers. SETTING: Diverse Ontario communities. PARTICIPANTS: Heavy drinkers (99 males, 56 females) were selected from 185 media-recruited applicants who were screened by telephone to exclude cases with severe alcohol-related problems. Their mean (+/- SD) pre-admission weekly quantity of alcohol consumed was 22 +/- 15 drinks. Follow-up rates at 3 and 12 months were 92% and 88%. MEASUREMENTS: Regressed onto weekly quantity at follow-up were: client characteristics, previous measures of weekly quantity, experimental condition and use of the menu of strategies (setting goals for drinking, keeping track of drinking, pacing drinking, planning ahead to avoid heavy drinking, developing free-time activities and coping with problems without drinking). FINDINGS: At 3 months the variables predicting lower weekly quantity were: pre-intervention weekly quantity, developing free-time activities, setting goals for drinking and condition. Lower weekly quantity at 12 months was predicted by lower 3-month and pre-intervention weekly quantity, and regular use of: coping with problems without drinking, setting goals for drinking and keeping track of drinking. CONCLUSIONS: This descriptive study revealed a positive association between level of use of recommended coping strategies at follow-up and drinking outcome. Controlled studies of the effects of strategy use on drinking outcome are therefore warranted.

Cost comparison of radical prostatectomy and transperineal brachytherapy for localized prostate cancer.

OBJECTIVES: To compare perioperative costs associated with radical retropubic prostatectomy (RRP) to transperineal brachytherapy (BXRT) with iodine-125 (125I) seeds in the treatment of localized prostate cancer. METHODS: Actual costs per case for the perioperative period were compiled prospectively for 583 consecutive patients undergoing RRP or BXRT between January 1, 1997 and October 30, 1998 using a hospital-wide cost accounting system. The total cost per case included both technical and professional components. The technical costs included those incurred for anesthesiology, laboratory medicine, medicine, pharmacy, nursing, radiology, 125I seeds, and BXRT technicians. Professional costs included fees from anesthesiology, laboratory, medicine, urology, radiation oncology, and physics. Cases were divided into three groups for analysis: group 1, RRP (n = 404); group 2, BXRT with planning ultrasound performed in the office setting before implantation (n = 107); and group 3, BXRT with planning ultrasound performed in the operating room at the time of implantation (n = 72). Results are reported as relative cost ratios, with RRP assigned a relative cost of 1.0. RESULTS: The total relative perioperative cost for BXRT exceeded that for RRP by 85% to 105%. Technical cost, exclusive of 125I seeds, was substantially lower for BXRT (relative cost 0.36 to 0.42) but was more than offset by the cost of the seeds when comparing total cost with RRP. Performance of the planning ultrasound in the operating room (group 3) increased the total cost by 20%. The categorical technical costs for both BXRT groups were significantly lower for anesthesiology, laboratory medicine, medicine, pharmacy, and nursing but were significantly higher for radiology. The total professional costs were similar for all groups. CONCLUSIONS: Perioperative costs of BXRT with 125I seeds are substantially higher than RRP in the treatment of localized prostate cancer, primarily because of the cost of the seeds.

Virulent Treponema pallidum, lipoprotein, and synthetic lipopeptides induce CCR5 on human monocytes and enhance their susceptibility to infection by human immunodeficiency virus type 1.

Treponema pallidum, its membrane lipoproteins, and synthetic lipoprotein analogues (lipopeptides) were each examined to determine whether they induced CCR5 expression on human peripheral blood mononuclear cells (PBMC). Reverse transcription-polymerase chain reaction for CCR5 gene transcripts, macrophage inflammatory protein (MIP)-1beta binding assays, and flow cytometry revealed that either T. pallidum, a representative treponemal lipoprotein, or a corresponding synthetic lipopeptide induced CCR5 on CD14 monocytes but not on CD3 lymphocytes. CXCR4, the coreceptor for T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), was not induced on PBMC by treponemes or by lipoproteins or lipopeptides. Consistent with these findings, T. pallidum, lipoprotein, and synthetic lipopeptide all promoted the entry of a macrophage-tropic, but not a T cell-tropic, strain of HIV-1 into monocytes. These combined results imply that T. pallidum and its constituent lipoproteins likely induce the expression of CCR5 on macrophages in syphilitic lesions, thereby enhancing transmission of macrophage-tropic HIV-1.

Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening.

The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237. The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.

The tetraspanin CD9 influences the adhesion, spreading, and pericellular fibronectin matrix assembly of Chinese hamster ovary cells on human plasma fibronectin.

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and naïve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.

A CD9, alphaIIbbeta3, integrin-associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by alphaIIbbeta3 conformational states.

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor alphaIIbbeta3, integrin-associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta3 (AP3) and alphaIIb (C3). Additionally, the association between CD9 and alphaIIbbeta3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (alphavbeta3) suggesting that CD9/alphaIIbbeta3 association was alphaIIb-subunit or alphaIIbbeta3-complex dependent. D3, an alphaIIbbeta3-activating mAb that is also an anti-LIBS (ligand-induced binding site), immunoprecipitated primarily alphaIIbbeta3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct alphaIIbbeta3 activation and was upregulated by the alphaIIbbeta3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on alphaIIbbeta3 by D3 binding not only influenced the CD9 complex but also affected alphaIIbbeta3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alphaIIbbeta3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alphaIIbbeta3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of alphaIIbbeta3.