Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp.

AIMS: The aim of this study was to develop a real-time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). METHODS AND RESULTS: A duplex real-time PCR specific for the genes encoding for ß-glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety-six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. CONCLUSIONS: The duplex real-time PCR assay was found to be simple, rapid, reliable and specific. SIGNIFICANCE AND IMPACT OF THE STUDY: If possible at all, delineation of so-called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno- and serotypes. This assay clearly simplifies the differentiation of both.

A multiplex one-step real-time RT-PCR assay for influenza surveillance.

For surveillance purposes real-time PCR assays for influenza viruses had to be adapted to the pandemic influenza A(H1N1)2009 strain. We combined published primers and probes for influenza A, influenza B and an internal amplification control with a detection system for influenza A(H1N1)2009 to set up a rapid, reliable, simple and cost-effective high-throughput multiplex one-step real-time RT-PCR. The workflow also includes automated sample preparation for high-throughput screening. The lower limit of detection of the multiplex assay was 3.5x10(2) RNA copies per PCR reaction. The diagnostic sensitivity of the multiplex assay was 87.7%, but increased to 99.4% for influenza-positive samples yielding C(t) values of less than 34 cycles in the respective diagnostic assay. High specificity was confirmed by sequencing and correct detection of 15 reference samples from two quality assurance studies. The multiplex PCR was introduced for surveillance of samples from a network of general practitioners and paediatricians in Bavaria, Germany during the influenza pandemic of 2009. Comparison with surveillance data from reported cases proved the reliability of the multiplex assay for influenza surveillance programmes.

Rapid detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in food, using multiplex real-time PCR.

A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.

Prevalence of emetic Bacillus cereus in different ice creams in Bavaria.

In this study, 809 samples of ice cream from different sources were investigated by using cultural methods for the presence of presumptive Bacillus cereus. Isolates from culture-positive samples were examined with a real-time PCR assay targeting a region of the cereulide synthetase gene (ces) that is highly specific for emetic B. cereus strains. The samples were collected from ice cream parlors and restaurants that produced their own ice cream and from international commercial ice cream companies in different regions of Bavaria during the summer of 2008. Presumptive B. cereus was found in 508 (62.7%) ice cream samples investigated, and 24 (4.7%) of the isolates had the genetic background for cereulide toxin production. The level of emetic B. cereus in the positive samples ranged from 0.1 to 20 CFU/g of ice cream.

Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry as a tool for rapid diagnosis of potentially toxigenic Corynebacterium species in the laboratory management of diphtheria-associated bacteria.

The rapid identification of the potentially toxigenic Corynebacterium species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis is essential for diagnosis and treatment of diphtheria and diphtheria-like diseases. We used matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDIT-OF MS) in comparison with classical microbiological and molecular methods on 116 Corynebacterium strains. All 90 potentially toxigenic Corynebacterium strains collected by the German National Consiliary Laboratory on Diphtheria in a period of more than ten years were correctly identified by MALDI-TOF MS. We propose an algorithm for fast and reliable diagnosis of diphtheria incorporating MALDI-TOF MS, real-time tox PCR and Elek testing.

Dissection of biochemical borderline phenotypes in carriers and genetic variants of medium-chain acyl-CoA dehyrogenase deficiency: implications for newborn screening [corrected].

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) represents a potentially fatal fatty acid beta-oxidation disorder. Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has been implemented worldwide, but is associated with unresolved questions regarding population heterogeneity, burden on healthy carriers, cut-off policies, false-positive and negative rates. In a retrospective case-control study, 333 NBS samples showing borderline acylcarnitine patterns but not reaching recall criteria were genotyped for the two most common mutations (c.985A>G/c.199C>T) and compared with genotypes and acylcarnitines of 333 controls, 68 false-positives, and 34 patients. c.985A>G was more frequently identified in the study group and false-positives compared to controls (1:4.3/1:2.3 vs. 1:42), whereas c.199C>T was found more frequently only within the false-positives (1:23). Biochemical criteria were devised to differentiate homozygous (c.985A>G), compound heterozygous (c.985A>G/c.199C>T), and heterozygous individuals. Four false-negatives were identified because our initial algorithm required an elevation of octanoylcarnitine (C(8)) and three secondary markers in the initial and follow-up sample. The new approach allowed a reduction of false-positives (by defining high cut-offs: 1.4 micromol/l for C(8); 7 for C(8)/C(12)) and false-negatives (by sequencing the ACADM gene of few suspicious samples). Our validation strategy is able to differentiate healthy carriers from patients doubling the positive predictive value (42-->88%) and to target NBS to MCADD-subsets with potentially higher risk of adverse outcome. It remains controversial, if NBS programs should aim at identifying all subsets of all diseases included. Because the natural course of milder variants cannot be assessed by observational studies, our strategy could serve as a general model for evaluation of MS/MS-based NBS.

Single-nucleotide polymorphism in the SCCmec-orfX junction distinguishes between livestock-associated MRSA CC398 and human epidemic MRSA strains.

A number of real-time PCR assays for direct detection of methicillinresistant (MRSA) in clinical specimens are targeting staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. When testing 184 MRSA strains of human and animal origin from geographically distinct locations, we identified several characteristic single-nucleotide polymorphisms (SNPs) within the SCCmec-orfX junction of livestock-associated (LA) MRSA CC398 which serve as suitable strain markers for screening purposes. Within an assay time of 60 minutes and an additional 10 minutes for the melting curve analysis, all MRSA CC398 isolates were correctly identified by their characteristic T(m) value in the commercial LightCycler MRSA Advanced test. Studies to confirm the diagnostic accuracy of the SNP-based strain identification assay with a larger collection of clinical and LA-MRSA strains are ongoing.

Emergency coronary stenting for dissection during percutaneous transluminal coronary angioplasty: angiographic follow-up after stenting and after repeat angioplasty of the stented segment.

OBJECTIVES: The purpose of this study was to assess the angiographic results after emergency coronary stenting and after repeat angioplasty for restenosis within the stent. BACKGROUND: There is still little angiographic information about lumen renarrowing and its correlates after emergency stenting, and data with regard to the angiographic outcome of repeat angioplasty within the stent are almost nonexistent. METHODS: This study was based on the quantitative evaluation of angiograms performed before and immediately after intervention and at 6-month follow-up. The study included 164 of the 183 eligible patients with emergency Palmaz-Schatz stent implantation and 31 of those with restenosis within the stent who had repeat angioplasty. RESULTS: Stenting produced an improvement in minimal lumen diameter from 0.82 +/- 0.41 to 2.76 +/- 0.47 mm (mean +/- SD) and in diameter stenosis from 74.9 +/- 11.5% to 18.3 +/- 8.1%. Elastic recoil was 0.51 +/- 0.34 mm, or 16%. At 6-month follow-up, 32.3% of the patients had restenosis (> or = 50% stenosis). Minimal lumen diameter decreased to 1.84 +/- 0.78 mm, and diameter stenosis increased to 41.7 +/- 21.0%. The degree of lumen loss correlated significantly with the length of the original stenosis and the initial lumen gain achieved by stenting. Thirty-one patients with in-stent restenosis underwent repeat angioplasty. The primary success rate was 100%, and no abrupt vessel closure was verified. Minimal lumen diameter increased from 0.85 +/- 0.35 to 2.18 +/- 0.39 mm, and diameter stenosis decreased from 69.7 +/- 12.9% to 28.6 +/- 9.4%. Elastic recoil was 0.82 +/- 0.38 mm, or 27%. At follow-up, 38.5% of the patients had restenosis. Minimal lumen diameter was reduced to 1.72 +/- 0.67 mm, and diameter stenosis increased to 42.4 +/- 18.1%. CONCLUSIONS: Angiographic results of emergency coronary stenting compare favorably with those of conventional angioplasty. In-stent balloon redilation in patients with restenosis is associated with excellent short-term results and a restenosis rate not different from that reported for nonstented vessels.

The Sukhumi primate monkey model for viral lymphomogenesis: high incidence of lymphomas with presence of STLV-I and EBV-like virus.

T-cell leukemia virus-like proviral sequences (STLV-I) as well as EBV-like sequences were detected in PBLs and tissues of non-human primates (Papio hamadryas baboons, Green monkeys and Macaca arctoides; Sukhumi Primate Center/Georgia) by PCR. Surprisingly, two different types of STLV-I within Papio hamadryas baboons were found. One of its represents the baboon prototype STLV-I-Su described earlier, present in lymphomatous baboons from the "high-lymphoma stock", which shows about 83% homology to HTLV-I and 85% to STLV-I in the env and tax genes. The inter-individual variability within this subtype is very low (about 1% in the tax gene). The second subtype was mainly found in asymptomatic animals from the control colony and showed in the env gene 95% homology to HTLV-I, but only 82% to the prototype baboon sequence. The presence of two subtypes within the Sukhumi baboon population might be interesting in respect to the inoculation experiments with human leukemic blood and to possible interspecies transmissions. The nature of the Herpes Papio-virus was elucidated as EBV-like and the homology to the human EBV was > 90% in the polymerase gene. The homologies between different monkey species were between 92 and 96% and also here two subtypes within the baboons were detected. This is the first direct demonstration by sequencing that the Herpes Papio virus is closely related to EBV. For further studies of this animal model, rabbits were inoculated with cells originated from lymphomatous baboons and macaques. The rabbits developed generalized lymphomas lethal within 1-2 months. EBV-like and STLV-I-like sequences could be detected by PCR and sequencing showed 99-100% identity to the inoculum, indicating in fact the transmission from monkey to rabbit. These animal models seem to be very suitable for the elucidation of the pathogenesis of human HTLV-I associated T-cell leukemia/lymphoma and might be further on used for therapeutical and preventative studies.

Late deterioration in tetralogy of Fallot: unusual findings and successful correction.

A patient with tetralogy of Fallot who underwent a successful Blalock shunt procedure as a child was evaluated 28 years later because of clinical deterioration. Results of cardiac catheterization and angiography disclosed, in addition to a functioning shunt, evidence of an infundibular, subvalvular tumor and a large anastomosis between the left circumflex coronary artery and the bronchial arteries of the right lung with the possibility of "coronary steal". At operation, the Blalock and coronary-to-bronchial artery anastomoses were ligated; and total correction was performed including resection of the pulmonary valve and infundibulum, excision of the tumor, closure of the ventricular septal defect, and application of an outflow patch to enlarge the pulmonary annulus. The patient was discharged nine days after surgical correction.

A multiplex real-time PCR method for the quantification of beef and pork fractions in minced meat.

One popular staple food in many lands is minced meat, traditionally prepared from beef and/or pork fractions. While beef is the more expensive of the two meat fractions, the possibility exists for manufacturers to fraudulently declare higher proportions of it. Additionally, the need exists to protect consumers who, out of medical or ethical reasons, reject specific meat fractions. In this work, we report on a quantitative triplex real-time PCR approach for the quantification of meat in minced meat products. With the method, beef and pork fractions are quantified employing primer and probe sequences that specifically recognise cow and pig components, against the backdrop of myostatin, a universal sequence commonly found in mammals and poultry species. The limit of detection of the qPCR method was 20 genome equivalents, while the measurement of uncertainty was determined at 1.83%. The method was validated on several commercially available minced meat products and performed well in terms of handling, reproducibility and robustness.

High-dose streptokinase therapy with three million units in one hour significantly improves early coronary patency rates in acute myocardial infarction.

In a prospective, randomized open trial, significantly higher patency rates were observed 60 minutes after beginning fibrinolytic therapy for acute myocardial infarction after administration of 3 million U streptokinase as compared to 1.5 million U (Thrombolysis in Myocardial Infarction [TIMI] grade 2 and 3 in 52% vs 26%; p = 0.04). Adverse events were observed with similar frequency in both groups.

The concept and treatment of osteoporosis.

Osteoporosis is a dynamic process, thought to be caused by an uncoupling between osteoblast and osteoclast activity. Altered pulsatile secretion of growth hormone and parathyroid hormone (PTH) have been proposed as pathogenetic factors for this unbalanced coupling. The anatomical lesions are believed to be reversible until trabecular perforations develop, if fractures already occurred the anatomical defect is permanent. It is helpful to classify osteoporosis in stages of increasing severity depending on bone density and the presence of fractures. Theoretically, if the bone density is above the fracture threshold, then the only therapeutic goal is to maintain the bone mass. If instead the mineral density is below the threshold, an active therapy is needed with drugs that can possibly increase the skeletal mass. Osteoporosis with multiple fractures cannot be reversed. The authors propose a promising pharmacologic treatment for osteoporosis, based on the combination of human PTH-(1-38) and intranasal salmon calcitonin. If started in the early stages of the osteoporotic process, this regimen may restore the initial bone mass. In more advanced stages, only a correction of the metabolic defect is possible, but the irreversible vertebral deformities are not affected. On the basis of the results, cyclic therapy with human PTH-(1-38) and salmon calcitonin represents a good treatment choice for osteoporosis.

Long-term in vitro cultivation of Borrelia burgdorferi sensu lato strains: influence on plasmid patterns, genome stability and expression of proteins.

Low (7th) and high (298th/304th) in vitro passages (cultivated over a period of 3 years) of two human Borrelia burgdorferi sensu lato strains, PKo (B. afzelii) and PBi (B. garinii) were compared by pulse-field gel electrophoresis, Southern blot, sequencing of the ospA gene, SDS-PAGE and Western blot. Digestion of genomic DNA with ApaI, BssHII, KspI, MluI, SmaI and XhoI did not reveal any differences between low and high passages. The loss of two linear plasmids with sizes of 6 and 31 kbp was detected in strain PKo between passages 34-50 and 101-304, respectively, whereas the ospA-carrying plasmid remained unchanged. In contrast, analysis of linear plasmid profiles obtained from low and high passages of B. garinii strain PBi showed no differences. Sequence analysis of the ospA gene demonstrated no difference in the strain PBi and one nucleotide exchange in the strain PKo when low and high passages were compared. The observed transition (G-A) in the third codon position did not alter the amino acid sequence. However, the rate of expression of the outer surface proteins OspA, OspB and OspC of strain PKo during low and high stages of cultivation varied significantly. In summary, our data suggest that the B. burgdorferi sensu lato genome is stable during long-term in vitro cultivation.

Malignant fibrous histiocytoma (MFH). A comparison of MFH in man and animals. A critical review.

This review gives information about localization and types of MFH in man and animals such as mouse, rat, cat, dog, opossum, cattle, horse and birds [e.g. mallard (a wild duck)]. Furthermore, this paper reports about cell culture dealing with MFH. The aim of this publication is to show that MFH originates from a primitive mesenchymal stem cell, fibroblastoid cell and fibroblasts. Histiocytes are, according to the literature in a small amount constituents of MFH and are reactive cells or without any meaning. In our own studies using rats [strain: Chbb: THOM (SPF)] the characteristic storiform or cartwheel pattern of tumour cells were evident. The cells were elongated, rich in endoplasmic reticulum and possessed no or very few lysosomes. The cells were predominantly fibroblasts and fibroblastoid cells. These cells were intermingled with giant cells. In other species mentioned above, the MFH showed very similar histological features. Our own results and findings obtained from the literature support our concept that the MFH represents a primitive phenotype or pleomorphic sarcoma which may differentiate in one or more directions. Histiocytes are not a neoplastic component.

A review of drug-induced lysosomal disorders of the liver in man and laboratory animals.

Lysosomotropic agents are selectively taken up into lysosomes following their administration to man and animals [de Duve et al. (1974) Biochem. Pharmacol. 23:2494-2531] The effects of lysosomotropic drugs studied in vivo and in vitro can be used as models of lysosomal storage diseases. These agents include many drugs still used in clinical medicine: aminoglycosides used in antibiotics [Tulkens (1988)]; phenothiazine derivatives; such antiparasitic drugs as chloroquine and suramin; antiinflammatory drugs like gold sodium thiomalate; and cardiotonic drugs like sulmazol [Schneider (1992) Arch. Toxicol. 66:23-33]. Side-effects to these drugs can be caused by their lysosomotropic properties. In addition to drugs, other compounds to which man and animals are exposed (e.g., metals, cytostatics, vitamins, hormones) are also lysosomotropic. Liver cells, especially Kuppfer cells, are known to accumulate lysosomotropic agents. Here we review studies which evaluate lysosomal changes in the liver following administration of lysosomotropic agents to experimental animals, and relate them to toxic side-effects or pharmacological action, as was suggested by de Duve et al. (1974). Common features of lysosomal changes include, the overload of liver lysosomes by non-digestible material; increased size and number of liver lysosomes; inhibition of several lysosomal enzymes; secondary increase in the activity of some lysosomal enzymes; increased autophagy, and fusion disturbances. There was no significant change in endocytosis, except for an increase in the Triton WR 1339 model.

Interaction of meloxicam with cimetidine, Maalox, or aspirin.

Meloxicam is a new enol carboxamide nonsteroidal antiinflammatory drug (NSAID). Preclinical studies have indicated that it possesses a high antiinflammatory potency and a low ulcerogenic potency. This open, randomized, crossover study was conducted to examine the effects of aspirin, the antacid Maalox (Rhone-Poulenc Rorer, Cologne, Germany), and cimetidine on the pharmacokinetics and bioavailability of a single oral dose of meloxicam 30 mg in healthy male volunteers. Plasma concentrations of meloxicam were determined and subjected to noncompartmental pharmacokinetic analysis. Meloxicam was well tolerated, and concomitant treatment with cimetidine or Maalox had little or no effect on the plasma concentration-time curves, maximum plasma concentration (Cmax), or the area under the plasma concentration-time curve (AUC0-infinity) of meloxicam. Concurrent treatment with aspirin increased plasma concentrations of meloxicam, increasing Cmax by approximately 25% and AUC0-infinity by 10%. These differences were not considered to be clinically relevant, and no adjustments of meloxicam dose should be required with coadministration of aspirin, Maalox, or cimetidine.

Pharmacokinetics and metabolite-pattern of 8-methoxypsoralen in man following oral administration as compared to the pharmacokinetics in rat and dog.

Following oral administration of 14C labelled 8-methoxypsoralen (8-MOP) in man the plasma level course, the metabolite-patterns and the elimination of the parent compound and its metabolites have been investigated. Additionally the results discovered have been compared with the data of pharmacokinetics on dog and rat. In man and rat the plasma protein binding of 8-MOP has been determined. Maximal levels of the total radioactivity in the plasma were achieved 2 h after dosing. At this time 8-MOP represents 50% of the radioactivity in the plasma. The plasma protein binding in vitro of 14C 8-MOP valued from 88% to 91% in man, and between 75% and 83% in the rat. Urinary elimination of the total radioactivity as a measure of the extent of absorption varies greatly and depends on the therapeutic formulation being employed. Following the administration of the solution 74% is recovered within 48 h. Faecal elimination of the total radioactivity reached 14% within 3 days. The metabolite-pattern does not show the unchanged 14C 8-MOP. Several polar metabolites occur in the urine among which biochemical conjugates have been recognized. Only polar metabolites are observable in the faeces from which the radioactivity is incompletely extractable. From a comparison of the metabolite profiles, the rat as well as the dog seem to be a useful animal species for experimental investigations with 8-MOP.

Distribution of [14C]meloxicam in joints of rats with adjuvant arthritis.

Using the new non-steroidal anti-inflammatory agent meloxicam (Mel), comparative investigations on pharmacokinetics and metabolism show good agreement between results in man and in the rat. To demonstrate preferential localization of the compound in inflamed sites, the distribution of the radiolabelled compound in arthritic joints of rats was studied using autoradiography. For that purpose an arthritis-inducing antigen was given to male albino rats. Twenty one days after antigen injection, all animals were given [14C]Mel orally. At three different times after receiving the labelled compound, the animals were sacrificed under anaesthesia. Tissue sections from each leg of each animal were taken for autoradiography. Sections were stained after film exposure using Masson-Goldner staining. The radiolabelled compound was preferentially localized in the areas which stained for inflamed connective tissue. The distribution pattern described persists beyond 24 h post-application. The figures indicate a highly efficient distribution of the radioactivity into chronically inflamed tissue of the hind foot-pads. Since negligible radioactivity was found in the front foot-pads, it is concluded that uptake into inflamed tissue is favoured.