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.

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.

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.

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.

Polymerase chain reaction (PCR) for detection of pathogenic microorganisms in bacteriological monitoring of dairy products.

The presence of pathogenic bacteria poses a serious problem in sustaining the safety of dairy products. Microbiological routine controls of these products make use of selective culture techniques. To detect pathogenic species, isolated colonies are characterized by specific metabolic activities and by serotyping. We present an alternative biochemical approach that does not require culture of bacteria. The total bacterial populations of food samples were isolated by centrifugation and analysed by PCRs specific for pathogenic species. A total of 90 raw milk samples and dairy products made from raw milk were screened by this method for the presence of Listeria monocytogenes, Escherichia coli, enterotoxigenic E. coli, Campylobacter jejuni and C. coli. Detection rates were 12/90 (13%) for L. monocytogenes, 41/90 (46%) for E. coli, 18/90 (20%) for enterotoxigenic E. coli producing heat-labile toxin type I or heat-stable toxin type I, and 6/90 (7%) for C. jejuni or C. coli. Except for the use of different amplification primers, this approach is identical for any bacterial species to be detected. Direct PCR analysis of food samples offers rapid screening for the presence of specific bacteria and enables selection of critical samples prior to culture.

Comparison of "Gen-Probe" DNA probe and PCR for detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese.

A comparison was made of three approaches for the detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese after enrichment. Enrichment broths were tested by plating them onto different selective agars, by "Gen Probe" DNA hybridization and by the polymerase chain reaction (PCR). Based on two-step enrichment, all three approaches showed high specificities (90% or more) in detecting L. monocytogenes. In contrast, the sensitivity of the Gen-Probe test was low (33% or less), whereas high sensitivities were obtained with selective plating and PCR (83% or more). Based on one-step enrichment, specificities again were high for selective plating and PCR assay (100%), whereas for the Gen-Probe assay the specificity was lower (88% or more). The best sensitivities were observed with selective plating (67%) and PCR (75%). In terms of sensitivity, specificity and analysis time, PCR applied to a two-step enrichment was the most powerful assay for detecting L. monocytogenes in soft and semi-soft cheese.

In vivo covalent binding of retronecine-labelled [3H]seneciphylline and [3H]senecionine to DNA of rat liver, lung and kidney.

Retronecine-labelled [3H]seneciphylline ([3H]SPH) and [3H]senecionine ([3H]SON) of high specific radioactivity (22 and 49 mCi/mmol, respectively) were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine as precursor. [2,3-3H]Putrescine was synthesized by Gabriel synthesis of 1,4-diamino-2-butene from 1,4-dibromo-2-butene and catalytic hydrogenation of the product with tritium gas. Rats of both sexes were treated with the labelled pyrrolizidine alkaloids (PAs) (75-215 microCi SPH or 40-485 microCi SON/kg body wt.) and killed after 6 h or 4-5 days. SON-treated females excreted 83.4 +/- 0.2% of applied radioactivity in faeces and urine within 4 days whereas equally treated males excreted 90.9 +/- 3.2% in the same time. Excretion of 3H-activity from SPH-treated females was completed within 5 days (104.7 +/- 6.4%). Corresponding with these results, tissue levels were highest in SON-treated females. DNA and proteins were isolated from liver, lungs and kidneys and covalent binding of the alkaloids to DNA was determined. A Covalent Binding Index (CBI, mumol alkaloid bound per mol nucleotides/mmol alkaloid administered per kg body wt.) of 210 +/- 12 was found for the liver from SON-treated females whereas binding to liver DNA of males was lower by a factor of 4. The DNA damage determined six hours after treatment persisted during the following 4 days. Administration of [3H]SPH to female and male rats resulted in a CBI of 69 +/- 7 and 73/92, respectively, for the liver DNA. Furthermore we found binding of both alkaloids to DNA of lungs and kidneys in male and female rats. The in vivo formation of [3H]SON derived DNA adducts could be proved by HPLC analysis of hydrolyzed DNA.

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat, mouse and pig.

[14C]Aflatoxin B1 (AFB1) was isolated from cultures of Aspergillus parasiticus grown on [1-14C]sodium acetate. Covalent binding of AFB1 to liver DNA of rat and mouse was determined 6-8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a 'Covalent Binding Index' (CBI), (micromol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors. The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable. Aflatoxin M1 (AFM1) is a metabolite found in the milk of cows that have been fed AFB1-contaminated diet. [14C]AFM1 was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM1 must also be regarded as a strong hepatocarcinogen. It is concluded that AFB1 contaminations should be avoided in dairy feed.

Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster.

Sixteen pyrrolizidine alkaloids (PAs) were examined for their genotoxic potency in the wing spot test of Drosophila melanogaster following oral application. This in vivo assay tests for the induction of somatic mutation and mitotic recombination in cells of the developing wing primordia. All PAs tested except the C9-monoester supinine were clearly genotoxic. Depending on their chemical structure, however, genotoxicity of the PAs varied widely in a range encompassing about three orders of magnitude. In general, macrocyclic diester-type PAs were the most and 7-hydroxy C9-monoester types the least genotoxic representatives studied, while open diesters were intermediate in this respect. Stereoisomeric PAs mostly showed similar, but sometimes also clearly unequal genotoxicity. An increasing number of hydroxy groups in the PA molecule seemed to reduce its genotoxic potency. With respect to the structure/activity relationships, there appears to be a good correlation between hepatotoxicity of PAs in experimental rodents and genotoxicity in the wing spot test of Drosophila. This suggests that PAs are bioactivated along similar pathways in the mammalian liver and in the somatic cells of Drosophila. The genotoxic potential of PAs in the Drosophila wing spot test and their carcinogenic potential in mammals also seem correlated, although the information in the literature on carcinogenicity of the non-macrocyclic PAs with moderate to low genotoxic potency is concededly limited. Comparisons with other genotoxicity tests suggest that the wing spot test is particularly suitable for genotoxins like PAs, on the one hand because of the versatile metabolic bioactivation system of Drosophila and on the other hand also because of its excellent sensitivity to the crosslinking agents among the genotoxins.

Transfer of [3H]pyrrolizidine alkaloids from Senecio vulgaris L. and metabolites into rat milk and tissues.

[3H]Retronecine and [3H]necic acid-labelled senecionine and seneciphylline were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine and [4,5-3H]isoleucine, respectively, as precursors. Lactating rats dosed with these differently labelled pyrrolizidine alkaloids (PAs) excreted within 3 h approx. 0.08% of the applied radioactivity in the milk mainly as yet not identified water-soluble retronecine-derived metabolites and with approx. 0.02% as unchanged PAs. Highest tissue levels of PAs and metabolites, 6 h after administration, were found in liver and lungs.

ELISA with enzyme amplification for sensitive detection of staphylococcal enterotoxins in food.

A highly sensitive amplified ELISA in microtitre plates for the detection of staphylococcal enterotoxins compared favourably with a commercially available kit. The amplified ELISA demonstrated a reduced effect of the food sample matrix and an ease of handling. Using the amplified ELISA, the influence of different parameters (pH, time, temperature, re-extraction, concentration procedure) on the recovery efficiency and assay reliability was tested on foods implicated in food poisoning outbreaks as well as on spiked foods containing low levels of enterotoxins.

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.

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.

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.

Detection of Escherichia coli and identification of enterotoxigenic strains by primer-directed enzymatic amplification of specific DNA sequences.

The polymerase chain reaction (PCR) was used to amplify DNA sequences from the malB operon of Escherichia coli. All E. coli strains tested yielded the specific DNA fragment. No amplification products were obtained with other Enterobacteriaceae. E. coli strains which produce enterotoxins were identified with additional primer pairs specific for the genes coding for the heat-labile toxin type I (LTI) and the heat-stable toxin type I (STI). Amplification products were identified by DNA-DNA hybridization. Alternatively, restriction endonuclease analysis was used for identification and to distinguish between different alleles of the enterotoxin genes. The detection limit was 10 bacteria. The PCR systems were validated by testing 27 E. coli of known enterotoxigenic properties. The PCR results were consistent with factual toxin production as determined by immunoassays. In addition, 58 E. coli strains isolated from soft cheese and mayonnaise were analyzed by PCR. One strain from a cheese sample was found to have the genetic information for STI production. This strain produced STI as determined by enzyme-linked immunosorbent assay.

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.

Toxicity and mutagenicity of anthraquinones from Aspergillus chevalieri.

More than 100 strains of the Aspergillus glaucus group were cultivated on synthetic media for 11 days at 28 degrees C. Organic extracts of fungal material were screened by thin-layer chromatography (TLC) for the mycotoxins aflatoxins B1,2 and G1,2, sterigmatocystin, ochratoxin A, gliotoxin, patulin, and xanthocillin X. None of these toxins were produced in detectable amounts under experimental conditions. Nevertheless, organic extracts exhibited high toxicity after intraperitoneal (i.p.) administration in mice. Aspergillus chevalieri strain ZT 8268 was selected for further investigation of its toxic metabolites. The main toxic action was attributed to the four anthraquinone derivatives, physicion, physcionanthrone B, physciondianthrone, and erythroglaucin, which were isolated and identified. No toxic effects were found after oral administration. Using the Salmonella/mammalian microsome test, mutagenic activity (frame-shift) was detected in strain TA 1537 in the presence of S-9 liver microsome preparation.

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.

Mutagenic activity of the pyrrolizidine alkaloids seneciphylline and senkirkine in Drosophila and their transfer into rat milk.

Seneciphylline and senkirkine, two pyrrolizidine alkaloids that occur in animal feeds and medicinal herbs, respectively, have been tested for their ability to produce sex-linked recessive lethals in males of Drosophila melanogaster using the Basc test (3-day feeding method). Seneciphylline was found to be mutagenic at concentrations of 10(-5), 10(-4) and 10(-3)M, which produced 3.8% sex-linked recessive lethals (983 chromosomes tested). 9.0% (708) and 15.3% (327), respectively. Senkirkine (10(-5)M) produced 4.4% sex-linked recessive lethals (2541 chromosomes tested) against 0.17% (9081) in controls. Brood pattern analysis with senkirkine showed maximum sensitivity in the late spermatid stage of spermatogenesis, which agrees with evidence that pyrrolizidine alkaloids act as indirect mutagens. Flies fed with milk from lactating rats given an oral dose of 25 mg seneciphylline/kg showed 1.2% sex-linked recessive lethals (1477 chromosomes tested), against 0.3% (1533) in controls.