Phylogenetic Analysis of Mycobacterium tuberculosis Strains in Wales by Use of Core Genome Multilocus Sequence Typing To Analyze Whole-Genome Sequencing Data.

An inability to standardize the bioinformatic data produced by whole-genome sequencing (WGS) has been a barrier to its widespread use in tuberculosis phylogenetics. The aim of this study was to carry out a phylogenetic analysis of tuberculosis in Wales, United Kingdom, using Ridom SeqSphere software for core genome multilocus sequence typing (cgMLST) analysis of whole-genome sequencing data. The phylogenetics of tuberculosis in Wales have not previously been studied. Sixty-six Mycobacterium tuberculosis isolates (including 42 outbreak-associated isolates) from south Wales were sequenced using an Illumina platform. Isolates were assigned to principal genetic groups, single nucleotide polymorphism (SNP) cluster groups, lineages, and sublineages using SNP-calling protocols. WGS data were submitted to the Ridom SeqSphere software for cgMLST analysis and analyzed alongside 179 previously lineage-defined isolates. The data set was dominated by the Euro-American lineage, with the sublineage composition being dominated by T, X, and Haarlem family strains. The cgMLST analysis successfully assigned 58 isolates to major lineages, and the results were consistent with those obtained by traditional SNP mapping methods. In addition, the cgMLST scheme was used to resolve an outbreak of tuberculosis occurring in the region. This study supports the use of a cgMLST method for standardized phylogenetic assignment of tuberculosis isolates and for outbreak resolution and provides the first insight into Welsh tuberculosis phylogenetics, identifying the presence of the Haarlem sublineage commonly associated with virulent traits.

Health sequelae of human cryptosporidiosis-a 12-month prospective follow-up study.

To investigate long-term health sequelae of cryptosporidiosis, with especial reference to post-infectious irritable bowel syndrome (PI-IBS). A prospective cohort study was carried out. All patients with laboratory-confirmed, genotyped cryptosporidiosis in Wales, UK, aged between 6 months and 45 years of age, over a 2-year period were contacted. Five hundred and five patients agreed to participate and were asked to complete questionnaires (paper or online) at baseline, 3 and 12 months after diagnosis. The presence/absence of IBS was established using the Rome III criteria for different age groups. Two hundred and five of 505 cases completed questionnaires (40% response rate). At 12 months, over a third of cases reported persistent abdominal pain and diarrhoea, 28% reported joint pain and 26% reported fatigue. At both 3 and 12 months, the proportion reporting fatigue and abdominal pain after Cryptosporidium hominis infection was statistically significantly greater than after C. parvum. Overall, 10% of cases had sufficient symptoms to meet IBS diagnostic criteria. A further 27% met all criteria except 6 months' duration and another 23% had several features of IBS but did not fulfil strict Rome III criteria. There was no significant difference between C. parvum and C. hominis infection with regard to PI-IBS. Post-infectious gastrointestinal dysfunction and fatigue were commonly reported after cryptosporidiosis. Fatigue and abdominal pain were significantly more common after C. hominis compared to C. parvum infection. Around 10% of people had symptoms meriting a formal diagnosis of IBS following cryptosporidiosis. Using age-specific Rome III criteria, children as well as adults were shown to be affected.

Complete excision of the infrapatellar fat pad is associated with patellar tendon shortening after primary total knee arthroplasty.

BACKGROUND: Patella baja is a potentially disabling and difficult-to-treat complication following total knee arthroplasty (TKA). We investigated whether complete or partial excision of the fat pad has an effect on the length of the patellar tendon following TKA. METHODS: A retrospective review of patient radiographs was performed. One hundred and eleven consecutive patients who underwent primary TKA for osteoarthritis using the same components by two consultants were selected. Seventy-two patients underwent complete excision of the fat pad, whilst 39 had partial excision as per consultant practice. Patellar height was measured using the Caton-Deschamps Index (CDI) on immediate postoperative radiographs and at a minimum follow-up of 1 year. RESULTS: In the complete excision group, the mean CDI changed from 0.54 immediately postoperatively to 0.45 at a minimum follow-up of 1 year (p < 0.0001) indicating shortening of the patellar tendon, and eight patients reported anterior knee pain. The partial excision group's mean CDI changed from 0.76 to 0.75 (p = 0.231). An analysis of variance showed that the effect of complete fat pad excision on patella tendon length was significant, F(1, 109) = 15.273, p < 0.001. CONCLUSION: At a minimum follow-up of 1 year, the patellar tendon length shortened significantly in the group of patients with complete fat pad excision. With partial excision, there was no significant change in tendon length. Complete excision of the fat pad should therefore be avoided wherever possible in an attempt to avoid patella baja. LEVEL OF EVIDENCE: II.

Using semifield studies to examine the effects of pesticides on mobile terrestrial invertebrates.

Many farmers rely on regular pesticide applications to avoid losses from arthropod pests and the diseases they vector. However, widespread and injudicious use of pesticides is detrimental to the environment, poses a health risk, and undermines biocontrol services. Researchers are increasingly required to develop techniques to quantify the trade-offs and risks associated with pesticides. Laboratory studies, though useful for assessing short-term impacts (e.g., mortality), cannot detect longer-term or indirect effects that can potentially be assessed using semifield studies. Here we review the range and scope of studies that have used semifield methods for regulatory testing and risk assessment of pesticides and for understanding the community-level effects of pesticide use in agricultural landscapes. We include studies on target and nontarget species, with an emphasis on quantifying effects when the target species is highly mobile. We suggest improvements in the design and analysis of semifield studies to more effectively assess effects on highly mobile species.

Clinical laboratory practices for detection and reporting of Cryptosporidium in community cases of diarrhoea in the United Kingdom, 2008.

To identify procedures employed by publicly funded clinical diagnostic laboratories in the United Kingdom (UK) for the detection of Cryptosporidium in community cases of diarrhoea, a telephone survey was conducted between August 2008 and January 2009 of all such laboratories that test stools from community-based patients. All 200 laboratories responded: 145 (72.5%) tested all stool samples for Cryptosporidium, while 55 (27.5%) applied selection criteria. There were country and regional differences in the proportion of laboratories selectively testing stools, which were significantly correlated with Cryptosporidium report rates to national surveillance (Spearman's rank correlation coefficient (rs)=0.61, degrees of freedom (df)=11, p=0.03). Understanding of laboratory practice is fundamental to interpreting trends in surveillance data, estimating disease burden and identifying outbreaks, as well as providing important background information against which changes and effects of new public health regulations can be measured.

Trichogramma (Hymenoptera: Trichogrammatidae) ecology in a tropical bt transgenic cotton cropping system: sampling to improve seasonal pest impact estimates in the Ord River Irrigation Area, Australia.

Trichogramma Westwood (Hymenoptera: Trichogrammatidae) cause high mortality rates in the potentially resistant pest species, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), and are considered integral to the resistance management plan for Bacillus thuringiensis transgenic cotton, Gossypium hirsutum L., production in the Ord River Irrigation Area (ORIA), Western Australia. Measured as percentage of parasitism, Trichogramma activity seems highly variable over time; yet, it contributes significantly to pest suppression at peak insect pest density. Environmental constraints on Trichogramma survival, especially insecticide applications, may limit their effectiveness. The decision to initiate insecticide applications in ORIA cotton crops is best delayed unless absolutely necessary to avoid disruption of Trichogramma impact on pests. Trichogramma disperse into young crops and display high intrinsic rates of increase effectively stifling Helicoverpa (Hardwick) population increase after initial egg lay during high-density years in the ORIA, and evidence suggests a possible preference for H. armigera host eggs.

Causes of failure of the Kinemax Plus total knee replacement in the first five years.

In order to investigate whether any single batch of polyethylene inserts was responsible for premature failure of total knee replacements (TKR), we performed a retrospective database review of 1133 consecutive primary Kinemax Plus TKR performed by 2 surgical teams between January 1992 and January 2001 at our institution. Cases re-operated in any way within 5 years of the index arthroplasty were identified, case notes studied and radiographs reviewed. The causes contributing to the premature failure of the implants were identified where possible and the polyethylene lot numbers were recorded. Nineteen cases were known to have been revised, 7 cases for deep infection of the prosthesis and 12 for aseptic failure. In 10 cases, an identifiable technical error had contributed to the early failure of the implant. In 4 of these cases, there was inadequate tissue balancing in patients with a significant pre-operative valgus deformity. In 2 cases, the tibial base plate was inserted in a varus mal-alignment. There was one extended tibial component, one internally rotated femoral component, one oversized femoral component and one femoral component with inadequate fixation. Two further cases failed aseptically, one with a fracture of the medial tibial plateau and one idiopathic. The lot numbers of the polyethylene inserts were all different. This study confirms that the premature failure rate of the Kinemax Plus TKR is low. Although a "bad batch" of polyethylene has been suggested as a cause for early failure in TKR, in this series, surgical error was the predominant cause.

Biofilm formation in medical device-related infection.

Medical device-associated infections, most frequently caused by coagulase-negative staphylococci, especially Staphylococcus epidermidis, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of medical device-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. There is urgent need for an increased understanding of the functional factors involved in biofilm formation, the regulation of their expression, and the interaction of those potential virulence factors in device related infection with the host. Significant progress has been made in recent years which may ultimately lead to new rational approaches for better preventive, therapeutic, and diagnostic measures.

A rapid single-step multiplex method for discriminating between Trichogramma (Hymenoptera: Trichogrammatidae) species in Australia.

Inaccurate species identification confounds insect ecological studies. Examining aspects of Trichogramma ecology pertinent to the novel insect resistance management strategy for future transgenic cotton, Gossypium hirsutum L., production in the Ord River Irrigation Area (ORIA) of Western Australia required accurate differentiation between morphologically similar Trichogramma species. Established molecular diagnostic methods for Trichogramma identification use species-specific sequence difference in the internal transcribed spacer (ITS)-2 chromosomal region; yet, difficulties arise discerning polymerase chain reaction (PCR) fragments of similar base pair length by gel electrophoresis. This necessitates the restriction enzyme digestion of PCR-amplified ITS-2 fragments to readily differentiate Trichogramma australicum Girault and Trichogramma pretiosum Riley. To overcome the time and expense associated with a two-step diagnostic procedure, we developed a "one-step" multiplex PCR technique using species-specific primers designed to the ITS-2 region. This approach allowed for a high-throughput analysis of samples as part of ongoing ecological studies examining Trichogramma biological control potential in the ORIA where these two species occur in sympatry.

An unusual lymphocytic perivascular infiltration in tissues around contemporary metal-on-metal joint replacements.

BACKGROUND: Metal-on-metal bearing surfaces have been reintroduced for use during total hip replacement. To assess tissue reactions to various types of articulations, we studied the histological appearance of periprosthetic tissues retrieved from around metal-on-metal and metal-on-polyethylene total hip replacements and compared these findings with the appearance of control tissues retrieved at the time of primary arthroplasty. METHODS: Periprosthetic tissues were obtained at the time of revision of twenty-five cobalt chromium-on-cobalt chromium, nine cobalt chromium-on-polyethylene, and ten titanium-on-polyethylene total hip arthroplasties. Control tissues were obtained from nine osteoarthritic hips at the time of primary total hip arthroplasty. Each tissue sample was processed for routine histological analysis, and sections were stained with hematoxylin and eosin. Quantitative stereological analysis was performed with use of light microscopy. RESULTS: Tissue samples obtained from hips with metal-on-metal implants displayed a pattern of well-demarcated tissue layers. A prominent feature, seen in seventeen of twenty-five tissue samples, was a pattern of perivascular infiltration of lymphocytes. In ten of the tissue samples obtained from hips with metal-on-metal prostheses, there was also an accumulation of plasma cells in association with macrophages that contained metallic wear-debris particles. The surfaces of tissues obtained from hips with metal-on-metal prostheses were more ulcerated than those obtained from hips with other types of implants, particularly in the region immediately superficial to areas of perivascular lymphocytic infiltration. The lymphocytic infiltration was more pronounced in samples obtained at the time of revision because of aseptic failure than in samples retrieved at the time of autopsy or during arthrotomy for reasons other than aseptic failure. Total-joint-replacement and surface-replacement designs of metal-on-metal prostheses were associated with similar results. Tissue samples obtained from hips with metal-on-polyethylene implants showed far less surface ulceration, much less distinction between tissue layers, no pattern of lymphocytic infiltration, and no plasma cells. The inflammation was predominantly histiocytic. Tissues retrieved from hips undergoing primary joint replacement showed dense scar tissue and minimal inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: The pattern and type of inflammation seen in periprosthetic tissues obtained from hips with metal-on-metal and metal-on-polyethylene implants are very different. At the present time, we do not know the prevalence or clinical implications of these histologic findings, but we suggest that they may represent a novel mode of failure for some metal-on-metal joint replacements.

Metal-specific differences in levels of DNA damage caused by synovial fluid recovered at revision arthroplasty.

Previous research has shown an increase in chromosomal aberrations in patients with worn implants. The type of aberration depended on the type of metal alloy in the prosthesis. We have investigated the metal-specific difference in the level of DNA damage (DNA stand breaks and alkali labile sites) induced by culturing human fibroblasts in synovial fluid retrieved at revision arthroplasty. All six samples from revision cobalt-chromium metal-on-metal and four of six samples from cobalt-chromium metal-on-polyethylene prostheses caused DNA damage. By contrast, none of six samples from revision stainless-steel metal-on-polyethylene prostheses caused significant damage. Samples of cobalt-chromium alloy left to corrode in phosphate-buffered saline also caused DNA damage and this depended on a synergistic effect between the cobalt and chromium ions. Our results further emphasise that epidemiological studies of orthopaedic implants should take account of the type of metal alloy used.

Lipoma arborescens of the knee.

Lipoma arborescens (diffuse articular lipomatosis) is a rare, benign intra-articular lesion of unknown aetiology. It is characterised by villous proliferation of the synovium and diffuse replacement of the subsynovial tissue by mature fat cells. It forms part of the differential diagnosis for a slowly progressive chronically swollen knee. We present a very rare case of bilateral involvement of the knee and discuss the symptoms, diagnosis and treatment of this condition.

Expanding mesh stent in the emergency treatment of Boerhaave's syndrome.

Boerhaave's syndrome, or spontaneous esophageal perforation, is a life threatening condition demanding early diagnosis and rapid aggressive management to prevent fulminant mediastinitis and death. We describe a patient treated in an emergency situation with an expanding esophageal mesh stent to bridge the esophageal rent together with chest and mediastinal drainage, resulting in complete recovery and return to the community. We also describe the partial recovery of the stent in the convalescent period.

Haemophilus paraphrophilus; a rare cause of intracranial abscess.

We report a case of a 42-year-old man man who presented with neurological symptoms and was found to have an intracranial abscess. A stereotactic aspiration of the abscess yielded a pure growth of Haemophilus paraphrophilus. The patient responded to treatment with cefotaxime. We postulate the mechanism of infection in this patient.

Length of time to laboratory diagnosis of Mycobacterium tuberculosis infection: comparison of in-house methods with reference laboratory results.

OBJECTIVES: To audit the time taken to obtain laboratory confirmation of infection with Mycobacterium tuberculosis using in-house methods of polymerase chain reaction (PCR) and culture and referral to a reference laboratory. METHODS: Retrospective collection of data from laboratory records covering a period of 1 year. RESULTS: Median time to microbiological diagnosis of a new infection using the in-house services in addition to the reference laboratory was 22.0 days. Using reference laboratory results alone, median time to diagnosis would have been 61.5 days. CONCLUSIONS: Development of on-site laboratory facilities to identify Mycobacterium tuberculosis can reduce the time to its identification by almost two-thirds.

Comparison of fitness of two isolates of Mycobacterium tuberculosis, one of which had developed multi-drug resistance during the course of treatment.

OBJECTIVES: We report the cases of two patients, brother and sister, both with pulmonary tuberculosis. Both patients complied poorly with treatment. One developed multi-drug resistant disease, whilst the other did not. We aimed to show that the two infecting strains were the same, and then to compare the fitness of the resistant strain to that of the sensitive strain. METHODS: The isolates were typed by RFLP. The fitness of the multi-drug resistant tuberculosis strain was determined by calculating the ratio of generation produced by the drug-resistant and a drug-susceptible strain in a mixed culture. The number of bacteria present in this broth culture was estimated using the Miles and Misra technique. The number of drug-resistant bacteria present was determined by inoculating aliquots of broth onto Middlebrook 7H10 agar with 5mg/l rifampicin. RESULTS: The infecting strain of Mycobacterium tuberculosis was shown to be the same on RFLP typing in both cases. It was found that the multi-drug resistant organism had decreased fitness compared to the sensitive organism. CONCLUSION: The decreased relative fitness of the resistant strain implies a physiologic cal cost for the development of drug resistance.

Plain radiography in the degenerate knee. A case for change.

We took posteroanterior weight-bearing radiographs, both with the joint fully extended and in 30 degrees of flexion, in a consecutive series of 50 knees in 37 patients referred for the primary assessment of pain and/or stiffness. These radiographs were reported 'blind' both by an orthopaedic surgeon and a radiologist. Direct measurement of the joint space, together with grading of the severity of erosion according to the Ahlback criteria, was undertaken. Any other abnormality present was also documented. The radiographs of the knees in 30 degrees of flexion consistently showed more advanced erosion in both the medial (p = 0.001) and the lateral (p = 0.0001) tibiofemoral compartments, when compared with those of knees in full extension. The Ahlback classification of 25 joints was altered, in some cases by several grades, by the flexed position of the joint. In every case in which another abnormality was identified on the radiograph in full extension, it was also noted on that of the knee in 30 degrees of flexion. In a further four cases, additional pathology could only be seen in the flexed knee. Every patient was able to complete the radiological examination without difficulty. Our study supports the adoption of a weight-bearing view in 30 degrees of flexion as the standard posteroanterior radiograph for the assessment of tibiofemoral osteoarthritis in patients over 50 years of age.

The sulcus angle and malalignment of the extensor mechanism of the knee.

Anterior knee pain due to dysplasia of the extensor mechanism is common. We have studied 137 knees (103 patients) in order to identify a rapid and reproducible radiological feature which would indicate the need for further analysis. Overall, 67 knees (49%) had at least one radiological abnormality; 70 (51%) were considered normal. There were five cases of Dejour type-3 dysplasia of the femoral trochlea, nine of type-2 and 12 of type-1. There were 49 cases of patella alta and five of patella infera. Four knees had an abnormal lateral patellofemoral angle (patellar tilt), and in 15 knees there was more than one abnormality. Classification of trochlear dysplasia was difficult and showed poor reproducibility. This was also true for the measurement of the lateral patellofemoral angle. Patellar height was more easily measured but took time. The sulcus angle is an easily and rapidly measurable feature which was reproducible and was closely related to other features of dysplasia of the extensor mechanism. The finding of a normal sulcus angle suggested that seeking other radiological evidence of malalignment of the extensor mechanism was unlikely to reveal additional useful information. The severity of other features of dysplasia of the extensor mechanism correlated with increasing sulcus angle.

Inter- and intraexaminer variability in assessment of orthodontic treatment need.

Two examiners assessed subjectively the orthodontic treatment need of a group of 183 10-year-old children. The examiners did not standardize the criteria establishing treatment need. Intraexaminer agreement was reached in 79.8% of the cases. Intraexaminer agreement was 91.7% for Examiner A and 93.4% of Examiner B. The paper highlights the subjective element in deciding orthodontic treatment need.

Glucagon-producing pancreatic endocrine tumors in two dogs with superficial necrolytic dermatitis.

A newly recognized disease in dogs, ulcerative dermatosis associated with diabetes mellitus (diabetic dermatopathy), was diagnosed in 2 dogs with pancreatic endocrine tumors that had immunohistologic evidence of glucagon production. Dogs developed diabetes mellitus in the later stages of the illness, months after the skin disease was first observed. Liver disease was identified and characterized by high serum alkaline phosphatase and alanine transaminase activities. Clinically, erythema and crusting involved the footpads, the face, perioral and genital skin, and ventrum. Histologically, skin lesions were intercellular and intracellular edema and necrosis of the upper half of the epidermis and diffuse parakeratosis. Clinically and histologically, skin lesions closely resembled necrolytic migratory erythema of people, a skin disease that usually is associated with a glucagon-secreting pancreatic endocrine tumor and diabetes mellitus (glucagonoma syndrome): The morphologically descriptive term, superficial necrolytic dermatitis, was preferred over the previously proposed names hepatocutaneous syndrome and diabetic dermatopathy, which each connote only a single feature of the disease.