Detection and identification of Mycobacterium spp. in clinical specimens by combining the Roche Cobas Amplicor Mycobacterium tuberculosis assay with Mycobacterium genus detection and nucleic acid sequencing.

We have recently developed a PCR assay for detection of Mycobacterium spp. at the genus level based on the Cobas Amplicor platform. The sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. The specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. In a follow-up study, we have systematically evaluated the Mycobacterium genus assay in parallel with the Cobas Amplicor Mycobacterium tuberculosis assay on 2,169 clinical specimens, including respiratory and nonrespiratory specimens. Based on the genus assay, nontuberculous mycobacteria were readily detected and identified to the species level by PCR-mediated sequencing. In addition, our data point to a limited specificity of the Cobas Amplicor M. tuberculosis assay.

Development and evaluation of a molecular assay for detection of nontuberculous mycobacteria by use of the cobas amplicor platform.

We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the Cobas Amplicor Mycobacterium tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the Cobas Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of Cobas Amplicor M. tuberculosis detection. Four hundred sixteen clinical specimens were evaluated for the presence of NTM DNA. Sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. Specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. The genus assay is easy to perform, produces reliable results, and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTM not routinely recovered by culture and to discover new mycobacterial species.

[Spondylodiscitis without fever: a diagnostic challenge]. / Spondylodiszitis ohne Fieber: Eine diagnostische Herausforderung.

HISTORY AND CLINICAL FINDINGS: Three patients with different forms of vertebral osteomyelitis are presented, two with hematogenous infections caused by Streptococcus pneumoniae and Staphylococcus aureus and one with postsurgical infection after excision of a vertebral disc caused by coagulase-negative staphylococci. None of the patients was initially febrile, but all had localized back pain and a restricted range of movement of the vertebral column. EXAMINATIONS, DIAGNOSIS: In all three patients the MRI of the affected vertebral column was consistent with vertebral osteomyelitis. Microbiological diagnosis was made by bone biopsy in all patients and by blood cultures in two of them. TREATMENT AND COURSE: Antibiotics were administered for 4-6 weeks. At follow-up two patients were without symptoms, but the third patient had persistent back pain without radiological signs of vertebral osteomyelitis. CONCLUSION: In patients with localized back pain vertebral osteomyelitis should be included in the differential diagnosis, even if there is no fever and no increase in white cell count, the erythrocyte sedimentation rate or C-reactive protein level and radiography is normal. Specific bacterial diagnosis should be made by multiple bone biopsy or blood cultures, before starting appropriate antibiotics.

Rapid detection of diarrheagenic E. coli by real-time PCR.

Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.

[Safety aspects of folic acid for the general population]. / Sicherheitsaspekte der Folsäure für die Gesamtbevölkerung.

Periconceptional use of folic acid reduces the risk of neural tube defects considerably. In Switzerland, implementation of these findings could be improved through fortification of a staple food with folic acid. The present paper reviews possible hazards associated with high intake of folic acid in the general population. Among the potential safety issues are interaction between folic acid and zinc, interaction between folic acid and drugs (phenytoin, methotrexate etc.) and hypersensitivity to folic acid. Of main concern are adverse effects of folic acid in cobalamin deficiency. Solutions are discussed.

The role of folate, antioxidant vitamins and other constituents in fruit and vegetables in the prevention of cardiovascular disease: the epidemiological evidence.

Evidence that fruit and vegetables may protect against coronary heart disease is accumulating. It is unclear which constituents of fruit and vegetables are responsible for this protective effect. Folate as a co-substrate in homocysteine metabolism may be important. An intake of about 400 micrograms folate equivalents/day seems to be required to achieve stable low homocysteine blood levels. Five of eight epidemiologic studies show significant inverse associations between folate and cardiovascular disease. These associations could be confounded by antioxidant vitamins and/or other substances. In trials examining an association between folate and cardiovascular disease such confounding must be excluded, before specific recommendations can be given. Observational studies suggest that vitamin C plays a role in the aetiology of cardiovascular disease, but there are no completed intervention trials of this vitamin alone. With regard to vitamin E two cohort studies point to cardiovascular benefits with the long-term use of supplements of at least 100 IU/day, but the results of controlled trials are inconclusive. There is some evidence from observational studies of an inverse association between beta-carotene and cardiovascular disease, particularly in smokers. Intervention trials do not support this hypothesis, rather, they suggest a possible harmful effect of beta-carotene supplements in smokers. Nevertheless, protective effects of beta-carotene and vitamin E in different dosages, durations of administration, or different combinations are still possible. The last paragraph of this review discusses limitations of the present and priorities of future research.

Quantitative competitive (QC) PCR for quantification of porcine DNA.

Many meat products nowadays may contain several species in different proportions. To protect consumers from fraud and misdeclarations, not only a qualitative but also a quantitative monitoring of ingredients of complex food products is necessary. DNA based techniques like the polymerase chain reaction (PCR) are widely used for identification of species but no answer to the proportional amount of a certain species could be given using current techniques. In this study we report the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of porcine DNA using a new porcine specific PCR system based on the growth hormone gene of sus scrofa. A DNA competitor differing by 30 bp in length from the porcine target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle, sheep, chicken and turkey. The competitor concentration was adjusted to porcine DNA contents of 2 or 20% by coamplification of mixtures containing porcine and corresponding amounts of bovine DNA in defined ratios.

Folate and the risk of colorectal, breast and cervix cancer: the epidemiological evidence.

It is only recently that folate deficiency has been implicated in the development of cancer. The mechanisms by which folate might protect against cancer are not clear but may relate to its role in DNA methylation and DNA synthesis. All case-control, cohort and intervention trials reported in English, French, or German, on folate intake or blood levels in relation to the risk of colorectal, breast, and cervix cancer were reviewed. Twenty case-control, and 12 nested case-control or cohort studies were identified. The epidemiological studies consistently show an inverse association between intake and/or levels of folate and the frequency of colorectal carcinomas, and less clearly of adenomas. Long-term use of supplements of folate seems to be of greater benefit than dietary intake. The effect of folate seems to be modulated by alcohol, methionine, and MTHFR polymorphisms. Results from animal studies suggest that folate supplementation might decrease or increase cancer risk depending on dosage and timing. Recent studies also suggest an inverse association between folate intake and breast cancer among women who regularly consume alcohol. Conversely, epidemiological evidence remains uncertain for the role of folate in cervical cancer prevention; the results of two intervention trials on rates of cervical intraepithelial neoplasia regression or progression were negative. An effect of folate later in carcinogenesis is not supported by the few (nested) case-control studies on invasive cervical cancer. Some of the conflicting results may be due to the fact that dietary intake or blood levels of folate do not accurately reflect folate concentrations in the cells of cancer origin. Furthermore, only a few studies have taken into account the modulating effect of alcohol, methionine, and MTHFR polymorphisms in their analyses. The observed inverse associations between folate and risk of cancer, on the other hand, may be confounded by various factors, especially by other potentially protective constituents in fruits and vegetables. Ongoing intervention studies can strengthen evidence for causality by excluding such confounding, but the optimal dose, duration, and stage of carcinogenesis and the appropriate (genetically predisposed) study group for folate chemoprevention are not yet defined.

Outbreak of viral gastroenteritis due to sewage-contaminated drinking water.

In August 1998, a large outbreak of gastroenteritis occurred in a Swiss village of 3500 inhabitants whereof more than 50% were affected. A high contamination of drinking water with faecal coliforms revealed a defect in the waste water system. The objective of the present study was to investigate the outbreak in respect of the presence of human pathogenic viruses. Drinking water and clinical samples from patients were examined for the presence of 'Norwalk-like viruses' (NLVs) and enteroviruses. NLVs and enteroviruses were detected by reverse transcription-polymerase chain reaction (RT-PCR) in one of two drinking water samples. Five of seven stool samples from ill persons were positive for NLVs. Typing of NLV-specific RT-PCR products by DNA sequencing revealed the presence of an identical genogroup-1 strain closely related to Southampton virus in both the water and one of the stool samples. A genogroup-2 NLV strain was identified in all positive stool samples. The enteroviral amplicon showed high sequence similarity with swine vesicular disease virus. These results demonstrate that the drinking water was highly contaminated with enteric viruses and that at least two NLV strains were involved in this outbreak of gastroenteritis.

Study of the presence of Campylobacter jejuni and C. coli in sand samples from four Swiss chicken farms.

Chicken farms are frequently infected with Campylobacter jejuni and Campylobacter coli. The objective of the present study was to investigate environmental samples from chicken farms for the presence of C. jejuni and C. coli. Every week between July and November 1997, three sand samples from the runs of four chicken farms were analyzed by culture and directly by polymerase chain reaction (PCR). These two detection methods were compared to each other. A total of 231 samples were tested. Eleven samples (4%) were found to contain Campylobacter cells by culture, whereas 157 samples (68%) were positive by PCR. All samples which were positive by culture were also positive by PCR. All direct PCR products were further typed by restriction fragment length polymorphism (RFLP). Three different RFLP types and mixtures of these types were observed. Direct PCR products of one chicken farm were further typed by direct sequencing and two temporally separated sequence types could be distinguished. Campylobacter strains isolated by culture were also typed by RFLP and direct sequencing revealing close accordance with the corresponding direct PCR products.

[Epidemiology of overweight in Switzerland: results of the Swiss National Health Survey 1992-93]. / Epidemiologie des Ubergewichts in der Schweiz: Resultate der Schweizerischen Gesundheitsbefragung 1992/93.

UNLABELLED: In many western countries more than half of the population is considered overweight (BMI > or = 25). METHODS: Data from the first national representative health survey for Switzerland (conducted 1992/93), including 7930 men and 7358 women (response rate 71%) aged 15 and over, were used. Means and percentiles of body weight, body height and BMI were calculated for men and women of different age groups. Overweight was defined as a BMI > or = 25, graded as overweight grade I (25.00-29.99), grade II (30.00-39.99), and grade III (BMI > or = 40.00) and analyzed according to gender, age, etc. RESULTS: Mean values for height decreases with increasing age, and body weight increases up to the age of 55-64 years in men and women. BMI values of all percentiles and age groups are lower for women than for men with the exception of age groups of 55 years or older and the 90 percentile. The prevalence of grade I overweight is 33.1% for men and 17.1% for women, of grade II overweight 5.8% and 4.5%, and of grade III overweight 0.3% and 0.2% respectively. Overall prevalence of overweight is 21.8% for women and 39.2% for men. Increasing age is associated with a higher prevalence of overweight. The prevalence of overweight in men increases with age up to 55-64 years, then levels off. In women, prevalence continues to rise. Overweight is more common in men of all age groups than in women. CONCLUSIONS: Periodic studies of the national prevalence of overweight are essential for monitoring the magnitude, and changes in the magnitude, of this serious public health problem.

PCR-based detection of verotoxin-producing Escherichia coli (VTEC) in ground beef.

Pathogenic strains of Escherichia coli producing verotoxins (VTs) have been recognized as a cause of human disease, and rapid and sensitive detection tests are urgently needed to ensure the safety of food, especially ground beef. We applied two nested polymerase chain reaction (PCR) assays to detect the genes encoding VT1 and VT2 irrespective of the bacterial serotype. In combination with a direct sample preparation protocol, we were able to uncover the presence of about 110 CFU of verotoxinogenic E. coli (VTEC) in 10 g of ground beef. When a six-hour enrichment was included, we found the detection limit to be in the range of 1 to 10 bacterial cells per 10 g of ground beef. To evaluate our detection system, we tested 30 ground beef samples originating from butcher shops in Berne, Switzerland. One sample yielded positive PCR results for both the VT1 and VT2 genes, indicating the presence of verotoxinogenic E. coli. Finally, 20 food homogenates, shown to contain E. coli strains by standard culture, were analysed with our method, and the gene encoding VT2 was detected in one cheese sample. The results suggest that the described PCR method can serve as a valuable tool for the surveillance of VTEC contamination of foods.

Three-step isolation method for sensitive detection of enterovirus, rotavirus, hepatitis A virus, and small round structured viruses in water samples.

Control of drinking or bathing water quality in respect to viral contamination remains an unsolved problem. A highly sensitive isolation protocol was developed for concentration and detection of different enteric viruses from water samples. The three-step isolation procedure combines filtration with a positively charged nylon membrane, ultrafiltration and clean-up of the viral RNA with a silica based membrane. Detection of the viral RNA is accomplished by reverse-transcription polymerase chain reaction (RT-PCR). Detection limits were determined to be one 50% tissue culture infective dose (TCID50) of seeded coxsackievirus B2 or hepatitis A Virus per litre of tap water by RT-PCR compared to two orders of magnitude lower sensitivity for culture in the case of coxsackievirus B2. The isolation procedure is highly sensitive, easy to perform and allows the detection of different human pathogenic virus groups in one water sample. The application of the isolation procedure to six river water samples and subsequent detection with nested or semi-nested PCR revealed enterovirus in 6/6 (100%), rotavirus in 6/6 (100%), hepatitis A virus in 0/6 (0%), small round structured virus genotype I in 6/6 (100%) and small round structured virus genotype II in 2/6 (33%) of the samples. These findings suggest that first, we have developed a very sensitive detection procedure and second, that river water in Switzerland-where most of the wastewater is handled by sewage treatment plants-shows a high contamination rate with enteric viruses.

Seminested RT-PCR systems for small round structured viruses and detection of enteric viruses in seafood.

Highly sensitive seminested RT-PCR systems for the specific detection of genotype I and II small round structured viruses (SRSVs) were developed based on the nucleic acid information deposited in the databanks. SRSVs could be detected in 10(7)-fold dilutions of three different stool samples. In addition, a rapid and simple purification protocol for enteric viruses from seafood tissues was elaborated using poliovirus (PV) as model. The virus isolation and viral RNA purification include the following steps: elution of the viruses from the seafood tissue with glycine buffer, their concentration by PEG-precipitation, lysis of viral particles with guanidine hydrochloride and viral RNA isolation using a silica based membrane. The detection limit was 3 to 30 TCID50 of poliovirus in 1.25 g of seeded seafood tissues without marked food matrix differences, whereas SRSV viruses were 10- and 100-fold better detected in mussels than in shrimps and oysters, respectively. The newly developed purification method, which was shown to remove potential RT-PCR inhibitors present in mussel tissue samples, was applied in a small market survey. 15 mussels, 15 oysters and 12 shrimps were examined for the presence of Hepatitis A virus (HAV), Enterovirus (EV), Rotavirus (RV) and SRSV using specific RT-PCR detection systems. The finding of three oyster samples positive for Rotavirus demonstrated the successful application of our method for the detection of enteric viruses in naturally contaminated seafood samples. The rapid isolation method might be suitable for application in routine testing laboratories and will help to improve public health controls for seafood.

Genotoxicity of ethyl carbamate (urethane) in Salmonella, yeast and human lymphoblastoid cells.

Ethyl carbamate is a known carcinogen occurring in fermented food and beverages and is therefore of interest for food safety assurance. We studied the genotoxicity of ethyl carbamate in Salmonella typhimurium, in Saccharomyces cerevisiae and in human lymphoblastoid TK6 cells. In absence of cytochrome P450 enzymes, no ethyl carbamate-mediated genotoxicity was observed in any of the three test systems in the non-cytotoxic range. In the presence of an activating system, ethyl carbamate was found to be mutagenic in Salmonella typhimurium strain TA100 but not in strains TA98 and TA102, indicating base-pair substitutions at G-C base pairs. In contrast, no significant mutagenicity of ethyl carbamate could be detected in human lymphoblastoid TK6 cells. However, applied in cytotoxic concentrations, ethyl carbamate was genotoxic for Saccharomyces cerevisiae in the absence of P450-mediated metabolic activation. Inhibitors of P450IIE1 (DMSO, ethanol and dithiodiethylcarbamate) diminished ethyl carbamate-mediated mutagenicity in Salmonella typhimurium strain TA100 in a dose dependent manner, suggesting that P450IIE1 is the activating enzyme.

Polymerase chain reaction (PCR) in the quality and safety assurance of food: detection of soya in processed meat products.

A new method for the specific and sensitive detection of soya (Glycine max) in processed meat products has been developed using polymerase chain reaction (PCR) technology. The presence of soya deoxyribonucleic acid (DNA) from several soya protein concentrates was determined with two pairs of specific oligonucleotides yielding a 414-bp (bp = base pair) fragment and an internal 118-bp fragment amplified from the soya lectin Le1 gene. The test detected DNA from textured soya protein concentrates in meat products at a level of 1% and was confirmed by a commercially available enzyme-linked immunosorbent assay (ELISA).

[Value of Doppler ultrasonic studies in the diagnosis of deep venous thrombosis of the lower limbs]. / Valeur de l'écho-Doppler pour le diagnostic de thromboses veineuses profondes des membres inférieurs.

To compare the operating characteristics of the venous echo-doppler and that of venous phlebography in our setting, 90 consecutive patients admitted on suspicion of deep vein thrombosis of one leg underwent both examinations. Phlebography confirmed the diagnosis in 46 patients (51%). The sensitivity, specificity, and positive and negative predictive values of the echo-doppler were 91, 92, 94 and 90% respectively. For the subgroup of patients with proximal deep vein thrombosis, these values were 97, 97, 97 and 97%, respectively; for the subgroup with distal deep vein thrombosis, these values were 60, 95, 75 and 90%, respectively. This study confirms the excellent performance of the venous echo-doppler for the diagnosis of deep vein thrombosis of the leg in our hospital. However, in the subgroup of patients with distal deep vein thrombosis, only the sensitivity of the venous echo-doppler is relatively low and the diagnosis may still require color echo-doppler or phlebography.