Multilocus sequence typing and CTX-M characterization of ESBL-producing E. coli: a prospective single-centre study in Lower Saxony, Germany.

The increasing prevalence of extended-spectrum ß-lactamase (ESBL)-producing Gram-negative bacteria is a serious threat for current healthcare settings. In this study we investigated the molecular epidemiology of ESBL-producing E. coli at the University Medical Center Göttingen in Lower Saxony, Germany. All E. coli isolates with an ESBL phenotype were collected during a 6-month period in 2014. Multilocus sequence typing and CTX-M characterization were performed on 160 isolates. Of the ESBL-producing isolates 95·6% were CTX-M positive. Compared to recent Germany-wide studies, we found CTX-M-1 to occur in higher frequency than CTX-M-15 (44·4% vs. 34·4%). CTX-M-14 and CTX-M-27 were detected at 9·4% and 5·0%, respectively. The globally dominant sequence type (ST) 131, which is often associated with CTX-M-15, occurred at a relatively low rate of 24%. Major non-ST131 sequence types were ST101 (5%), ST58 (5%), ST10 (4·4%), ST38 (4·4%), ST410 (3·8%) and ST453 (3·1%). Several of these major sequence types were previously shown to be associated with livestock farming. Together, our study indicates that E. coli lineage distribution in individual healthcare settings can significantly differ from average numbers obtained in nationwide studies.

Diphosphonate therapy of paget's disease of bone.

The use of disodium ethane-1 hydroxy-1, 1-diphosphonate (EHDP) therapy for Paget's disease of bone was examined in 75 affected patients. Forty-eight patients received randomly assigned oral doses of either 0, 2.5, 5, 10, or 20 mg/kg/day in a controlled, double-blind protocol, and the remainder received either 10 or 20 mg/kg/day in a non-random protocol. The clinical status of the patients and appropriate laboratory tests were evaluated before treatment and at frequent intervals during a six-month period of initial therapy. There were no significant changes in either urinary hydroxyproline or serum alkaline phosphatase in those patients receiving placebos, while both these parameters decreased significantly at all dose levels of EHDP, with the greatest decline noted in the highest dose group. However, statistical analysis of the data related to changes in symptoms in the double-blind study revealed that patients receiving the higher dose of EHDP (10 or 20 mg/kg/day) had less favorable outcomes than those receiving the lower doses (2.5 or 5 mg/kg/day). The high does group had a relatively lower rate of symptom improvement and a relatively greater rate of deterioration than did the low dose group. Twenty-one of forty-nine patients followed for at least 18 months have shown a sustained suppression of their serum alkaline phosphatase and urinary hydroxyproline values for 12 months following cessation of EHDP, while therapy has been reinstituted for the other 28 patients because of increases in these measurements, with or without accompanying symptomatic deterioration. Eight patients sustained fractures through Pagetic bone during the period of study and all of these were treated with higher doses of EHDP. On the basis of the biochemical and clinical data in this study it appears that initial therapy of Paget's disease of bone with 5 mg EHDP/kg/day maximizes benefits while minimizing possible adverse effects.

Toxoplasmosis: a persisting challenge.

The sixth biennial International Congress on Toxoplasmosis, organized by Uwe Gross (University of Göttingen, Germany), was held on 21-25 May 2001 in Freising, Germany. The first meeting of this kind in 1990 was attended by only 26 investigators and this year there were 115 participants covering various research topics including the immunology, epidemiology, cellular and molecular biology of Toxoplasma gondii.

Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements.

BAG1 is a small heat-shock protein of Toxoplasma gondii that is specifically expressed in the cyst-forming bradyzoite stage of the parasite. Upregulation of BAG1 mRNA occurs early during the differentiation pathway from tachyzoites to bradyzoites. In order define genomic elements involved in bradyzoite-specific gene regulation, chloramphenicol acetyltransferase (CAT)-reporter gene studies were performed with 5' flanking sequences of the BAG1 gene. Tachyzoites, transiently transfected with the BAG1/cat construct, exhibited very low CAT activity (200 fold less than in parasites transfected with a tubulin promoter/cat construct). After induction of bradyzoite differentiation by alkaline pH shift, however, CAT activity increased 50 fold, demonstrating bradyzoite-specific expression of the CAT reporter gene under control of 5' flanking sequences of BAG1. Stage-specific regulation of BAG1/CAT was independent of the 3'-flanking region, since constructs containing 3'-flanking sequences of the tachyzoite-specific SAG1 gene showed identical regulation to those containing the BAG1 3'-flanking region. The kinetics of BAG1/CAT induction in stably transfected parasites is similar to the kinetics of endogenous BAG1 expression: increased CAT activity was first detected on day 3 after alkaline pH shift (20 fold) and was dramatically upregulated 250 fold on day 4. A series of deletions in the BAG1 5'-flanking sequences demonstrated that a 324 nucleotide (nt) fragment, starting 60 nt upstream of the BAG1 transcription start, is sufficient to confer stage-specific regulation on the CAT reporter. These deletion analyses demonstrate that bradyzoite-specific expression of a heterologeous reporter gene requires only minimal genomic sequences.

Stage-specific expression of a selectable marker in Toxoplasma gondii permits selective inhibition of either tachyzoites or bradyzoites.

The establishment of culture conditions suitable for inducing differentiation of Toxoplasma gondii tachyzoites into parasites resembling the latent bradyzoite form has opened this important developmental transition to experimental analysis. In order to develop a genetic marker suitable for positive and negative selection during parasite differentiation. the T. gondii HXGPRT gene was placed under control of 5' flanking sequences derived from two bradyzoite-specific genes: BAG1 and LDH2. Random transgene integration at undefined genomic loci resulted in modest regulation (approximately 5-6-fold induction) above relatively high background levels (approximately 4% of wild-type controls). Integration of transgenes at a defined genomic position was achieved by targeting the uracil phosphoribosyl transferase (UPRT) locus using flanking homologous sequences and fluorouracil selection. This strategy was found to provide the added advantage of enhancing bradyzoite induction frequencies under conditions of pyrimidine starvation (low CO2). Constructs integrated in the direction of normal UPRT transcription exhibited moderate levels of inducibility, but transgenes integrated in the opposite direction were dramatically induced under differentiation conditions: 50-100-fold above the very low levels observed in tachyzoites (< 1% control). Positive selection (using mycophenolic acid) was shown to inhibit tachyzoites but not bradyzoites, while negative selection (using 8-azahypoxanthine) inhibited bradyzoites only. Stage-specific regulation of the HXGPRT selectable marker should permit genetic selections for the identification of mutants in the bradyzoite differentiation process.

Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii.

Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite 'tissue cyst' form of Toxoplasma gondii, first appearing approximately 2-3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture.

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.

Stage differentiation of the protozoan parasite Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii is able to persist lifelong in its hosts by differentiating from the replicative tachyzoite stage into cyst forming latent bradyzoites. Beside the clinical relevance of stage conversion and its importance for pathogenesis and prevention of toxoplasmic encephalitis, reversible stage differentiation in T. gondii is an interesting model system of protozoan differentiation in general. In recent years a variety of molecular techniques have been developed for T. gondii, including transfection systems and the development of many selectable markers. Together with tissue culture models in which stage differentiation from tachyzoites to bradyzoites can be induced these techniques provide the tools for a molecular dissection of the differentiation pathways. Three aspects of stage conversion are highlighted in this review, including the alteration of the parasite surface, alterations in parasite metabolism and the induction of genes associated with stress response.

The Syme amputation in children.

Twenty-three children had proportionate growth of bone and soft tissue between two and eleven years after a Syme ankle disarticulation. All walked with permanent prostheses within three months after operation. Ischemic necrosis occurred twice after inadvertent ligation of the posterior tibial artery. Suture of the extensor tendons into the heel pad eliminated its posterior migration. Prosthetic realignment compensated for progressive genu valgum in ten of fourteen amputees with fibular hemimelia. On the basis of these gratifying results, the Syme ankle disarticulation is suggested as early treatment for certain congenital anomalies of the lower extremity.

Spontaneous osteonecrosis of the knee.

Spontaneous osteonecrosis was diagnosed in ninety knees in which a roentgenographically visible lesion had developed within six months after onset of symptoms. Scintimetry can be used to differentiate between osteonecrosis and other painful conditions of the knee. The prognosis of osteonecrosis is dependent on the size of the radiolucent lesion. In knees with a lesion that is larger than 2.3 square centimeters, osteoarthritis is likely to develop. For forty-two knees the patients were treated with analgesics, partial weight-bearing, and quadriceps exercises. Forty-eight knees were treated surgically. Indications for surgical treatment were persistent severe complaints in spite of conservative treatment and a large osteonecrotic lesion with collapse of the condyle resulting in a varus deformity. The best results were achieved with osteotomy together with arthrotomy, and by knee replacement arthroplasty. Arthrotomy and drilling alone were ineffective.

Ulnar dysmelia.

Eleven patients with hypoplasia and partial or complete aplasia of the ulna (examples of a complex spectrum of postaxial forearm and hand abnormalities) were reviewed. Three types of ulnar deformity were observed: (1) hypoplasia, (2) partial aplasia (ossification of the proximal part of the ulna present at birth); and (3) total aplasia (ossification not development). The roentgenographically "absent" segment of the ulna may be a large fibrocartilaginous anlage attached distally to the distal radial epiphysis or the ulnar side of the carpus, or both. The tethering effect of this band may cause ulnar deviation of the wrist (and hand) and dislocation of th badial head in utero as well as progression of these deformities after birth. Resection of the distal end of the fibrocartilaginous anlage during the first to second year of life is recommended, since the results of this procedure suggest that it reduces the angular growth deformities. It is also suggested that if the one-bone-forearm operation is indicated, it should be deferred until a later age, since complications may be less likely to occur then than at the time that the anlage is resected.

Macrodystrophia lipomatosa of the foot: a report of three cases and literature review.

Macrodystrophia lipomatosa is a distinct clinical entity often misdiagnosed as other forms of macrodactyly. The most specific finding is an overabundance of fibrofatty tissue on the plantar aspect of the foot. Three cases, with the diagnoses made from tissue specimens, are presented in this article. The clinical, pathologic, and roentgenographic findings are discussed and a review of the literature is provided.

Action of the peroneus longus tendon on the first metatarsal against metatarsus primus varus force.

A medially directed force was applied to the first metatarsal in 10 cadaver feet. The peroneus longus tendon was subjected to a pull of 5 pounds. The soft tissues between the first and second metatarsals were cut sequentially, starting with the skin on the dorsal and plantar aspect, followed by the intermetatarsal ligament and adductor hallucis tendon, and, finally, the peroneus longus tendon at its distal insertion. Dorsoplantar radiographs while weightbearing were taken after each sectioning. A statistically significant varus displacement of the first metatarsal was observed only after transection of the peroneus longus tendon. It was concluded that the peroneus longus tendon is a strong retaining mechanism of the first metatarsal.

Anatomy of the Achilles tendon and plantar fascia in relation to the calcaneus in various age groups.

Ten adult cadaver feet, three neonatal feet, and the feet of two fetuses were dissected to investigate whether an anatomical continuity exists between the fibers of the Achilles tendon and the plantar fascia. Histologic sections of the feet were done in three age groups: neonate, persons in their mid-20s, and the elderly. As the foot ages, there appears to be continued diminution of the number of fibers connecting the Achilles tendon and plantar fascia. The neonate has a thick continuation of fibers, while the middle-aged foot has only superficial periosteal fibers that continue from tendon to fascia. The elderly feet show simply an insertion of fibers of both structures into the calcaneus with periosteum in between.

Spindle cell lipoma of the foot: a case report and literature review.

Lipomas are among the most commonly encountered soft tissue tumors in clinical practice, though they are rare in the foot. Although a presumptive diagnosis is typically made clinically, those tumors with atypical clinical features may require radiological consultation. Difficulty arises when radiographic features are not typical of lipoma. We present a fatty soft tissue tumor of the foot with nonadipose elements on magnetic resonance imaging evaluation. Differentiation of lipoma variants (e.g., spindle cell lipoma, atypical lipoma, pleomorphic lipoma, lipoblastoma, angiolipoma) from liposarcoma based on imaging features is not possible, necessitating surgical resection for definitive histological diagnosis.

The superior peroneal retinaculum: an anatomic study.

The superior peroneal retinaculum is thought to be the primary restraint to subluxation and/or dislocation of the peroneal tendons as they pass posterior to the distal fibula. The descriptions in anatomy texts vary widely. This anatomical study describes the different patterns of insertions for the superior peroneal retinaculum and its relationship to the peroneal tendons and the ligaments of the lateral ankle.

Ganglion cysts of the lower extremity: an analysis of 54 cases and review of the literature.

This article reviews 54 consecutive patients with lower extremity ganglion cysts that were surgically removed and histologically confirmed at the Hospital for Special Surgery from 1981 to 1993. Lower extremity ganglia were more common among women. Patients' ages ranged from 13 to 80 years, with the fifth and sixth decades being the most common. Size of the cysts ranged from 3 cm to 10 cm (average: 2.9 cm). Thirty-six (67%) patients had ganglion cysts of the foot and ankle, and 18 (33%) patients had ganglion cysts of the knee area. Four (7%) patients had intraosseous ganglia located in the proximal tibia, patella, and the first metatarsal head. Follow-up data of 40 (74%) patients at an average of 5.9 years (range: 1 to 12.5 years) were obtained. Satisfaction was reported by 83% of patients. Recurrence was seen in 10% of patients, and a report of no or mild pain was given by 86% of the group. Patients who underwent revision ganglion excision had inferior results. Only 25% reported satisfaction and 50% reported no or mild pain. Patients who underwent curettage of an intraosseous ganglion appeared to have superior results. All patients reported satisfaction and no or mild pain. The performance of a concomitant surgical procedure, the anatomic region of the ganglion, or type of postoperative immobilization did not appear to affect the outcome.

Toxoplasma gondii inhibits the in vitro induced apoptosis of HL-60 cells.

The integrity of the host cell may represent an important prerequisite for the intracellular survival and development of obligate intracellular pathogens. In the present study, we investigated the influence of infections with the protozoan parasite Toxoplasma gondii on the rate of apoptosis in the human leukemia cell line HL-60. After infection with T. gondii tachyzoites of the strain NTE and in uninfected controls, less than 2% of the host cells showed typical signs of apoptosis, i.e. condensation of chromatin after staining with Hoechst 33258 or internucleosomal DNA fragmentation after agarose gel electrophoresis of genomic DNA. After treatment with 0.1 to 0.5 microg/ml actinomycin D for up to 16 hours, HL-60 cells considerably underwent apoptosis. However, this actinomycin D-induced apoptosis was clearly reduced after concomitant infection with T. gondii as shown by staining with Hoechst 33258 and by DNA fragmentation assay. Inhibition of apoptosis by the intracellular pathogen T. gondii might be recognized as an evasion mechanism that enables intracellular survival and establishes long-lasting persistence.

[Epidemiologic investigations on caries prevalence among schoolchildren in Brittany (France)]. / Epidemiologische Erhebungen zur Kariesprävalenz schulpflichtiger Kinder in der Bretagne (Frankreich).

1421 randomly selected 6- to 15-year-old Breton children were examined in 1987 in order to assess the prevalence of caries in permanent teeth. Methods proposed by Turlot & Cahen (1989) were used. At 6, 85.9% of all children were caries-free and at 15, only 5.3%. At 14, DMFT and DMFS rates reached 6.46 and 12.36, respectively. The differences of scores stated between boys and girls and between children from urban and rural regions were not statistically significant. Pits and fissures were more frequently affected than approximal and smooth surfaces. Since 1986, fluoridated domestic salt is available in France. Thus, the present investigation will serve to study the effects of this recently adopted preventive measure.