Multiplex planar bioassay with reduced diffusion on normal phase, identifying androgens, verified antiandrogens and synergists in botanicals via 12D hyphenation.

Hormonal active compounds affecting health by altering the hormonal system are present in food. The planar yeast antagonist androgen screen (pYAAS) bioassay is a powerful tool to detect individual hormonal active compounds in complex samples separated by high-performance thin-layer chromatography (HPTLC). Previous methods lacked either detection sensitivity or zone sharpness. To overcome diffusion caused by long bioassay incubation on the normal-phase (NP) plate, zone fixation (fix) was achieved with a new polyisobutyl methacrylate coating, leading to enhanced zone sharpness. The exclusion of false-positive antagonists was integrated in the workflow, which allowed the verification (V) of true antagonists, apart from the detection of synergists. With the new multiplex bioassay providing information on 4 activities, 68 different botanicals were screened and hormonal active zones were identified by elution from the bioautogram to orthogonal reversed-phase high performance liquid chromatography with diode array detection and high-resolution mass spectrometry including fragmentation, resulting in the 12D hyphenation NP-HPTLCfix-UV/Vis/FLD-pYAVAS-FLD-heart cut-RP-HPLC-DAD-HRMS/MS.

Multiplex planar bioassay detecting estrogens, antiestrogens, false-positives and synergists as sharp zones on normal phase.

BACKGROUND: Phytoestrogens are found in many plants used in traditional medicines. Increasingly, plant extracts (botanicals) are also being added to foods or marketed as dietary supplements. Especially such powder formulations are susceptible to adulteration and falsification, given the global processing chain. To detect estrogen-like compounds in such multicomponent mixtures, non-target screening for hormonally active or endocrine disrupting compounds in plant products is becoming more important. Unfortunately, the current planar yeast estrogen screen (pYES) is prone to zone diffusion on the normal-phase high-performance thin-layer chromatography (NP-HPTLC) plate due to long incubation times in the aqueous bioassay. PURPOSE: The present study aimed to reduce zone diffusion on NP plates, which provides the basis for extending pYES to a multiplex bioassay, offering 4 different biological activity principles, followed by targeted identification of active zones. STUDY DESIGN AND METHODS: The reduction of substance diffusion via a polyisobutyl methacrylate polymer coating was studied. After successful zone fixation (fix), a multiplex bioassay was developed, in which a 17ß-estradiol-strip was applied along each sample track to detect synergists and antagonists (A), and for verification (V), a 4-methyl umbelliferone-strip to exclude false-positives. After multiplex bioassay screening of 68 botanicals, the zones with hormonal activities were heart-cut eluted to reversed-phase high-performance liquid chromatography-diode array detection-high-resolution tandem mass spectrometry (RP-HPLC-DAD-HESI-HRMS/MS). RESULTS: The separated substances were successfully fixed by the chromatogram coating. The zone sharpness (achieved after the bioassay) made it possible to add two strips, the 17ß-estradiol-strip for antagonistic and synergistic, and the 4-methyl umbelliferone-strip for false-positive effect detection, resulting in a multiplex bioassay. Using the 12D hyphenation NP-HPTLCfix-UV/Vis/FLD-pYAVES-FLD heart-cut RP-HPLC-DAD-HESI-HRMS/MS, it was possible to obtain information on estrogens, antiestrogens, false-positives, and synergists, and (tentatively) assign 17 hormonally active compounds, of which only 7 have been known to affect the human estrogen receptor, while another 4 had structural similarity to common phytoestrogens and antiestrogens. CONCLUSIONS: The streamlined 12D hyphenation including a multiplex bioassay has been shown to differentiate hormonal effects, leading to new insights and better understanding. It can generally be used to identify unknown hormonally active compounds in complex samples.

Effect-directed profiling of 32 vanilla products, characterization of multi-potent compounds and quantification of vanillin and ethylvanillin.

Food testing is of great importance to the food industry and organizations to verify the authenticity claims, to prove the quality of raw materials and products, and to ensure food safety. The market prices of vanilla differed by a factor of about 20 in the last three decades. Therefore the risk of adulteration and counterfeiting of vanilla products is high. Instead of commonly used target analyses and sum parameter assays, a complementary non-target multi-imaging effect-directed screening was developed, which provided a new perspective on the wide range of vanilla product qualities on the market. Planar chromatography was combined with effect-directed assays, and the obtained biological and biochemical profiles of 32 vanilla products from nine different categories revealed a variety of active ingredients. Depending on the region, typical vanilla product profiles and activity patterns were obtained for pods, tinctures, paste (inner part), oleoresin and powders. However, some vanilla products showed additional active compounds and a different intensity pattern. The vanilla product profiles substantially differed from those of vanilla aroma or products containing synthetic vanillin or vanilla-flavored food products. Bioactive compounds of interest were online eluted and further characterized via HPTLC-HRMS, which allowed their tentative assignment. After purchase of the standards, these were successfully confirmed by co-chromatography. Quantification of vanillin across nine different product categories revealed levels ranging from 1 µg/g to 36 mg/g with a mean repeatability of 1.9%. The synthetic ethylvanillin was not detected in the investigated samples in significant concentrations. The assessment of differences in the activity patterns pointed to highly active compounds, which were not detected at UV/Vis/FLD but first via the biological and enzymatic assays. This effect-directed profiling bridges the gap from analytical food chemistry to food toxicology, and thus, makes an important contribution to consumer safety. In the same way, it would accelerate investigations for Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) according to Regulation (EC) No. 1907/2006.

In-process quality control of wine by planar chromatography versus micro planar chromatography.

High-performance thin-layer chromatography (HPTLC) methods were developed to determine glycerol, gluconic acid, amino acids and sugars for in-process quality control of wine. Twenty wine samples (Pfalz region, Germany) were diluted with methanol and used for quantitative analysis without any further sample preparation. The developed amino acid method provided quantitative and characteristic fingerprints of wine varieties. The amino acid assignments were verified by HPTLC-MS. The developed gluconic acid method was primarily used to control the threshold, indicating a Botrytis cinerea infection of grapes. However, this method also enabled the detection of further organic acids like malic, tartaric and citric acids. A glycerol method was developed for control of the grape must fermentation (spontaneous/regular) and for fraud detection (glycerol adulteration). The HPTLC results of the sugar contents in the wine samples were similar to those of the well-known Luff-Schoorl method. The combined use of these developed HPTLC methods allowed the fermentation control (e.g., alcoholic and malolactic fermentation) and the monitoring of the grapes' overall health status. Without modification, the HPTLC methods for sugar and amino acid analysis could be transferred to circular micro planar chromatography (µ-PLC), showing its potential and benefits in terms of an inexpensive alternative for wineries and distributors.

Effect-directed classification of biological, biochemical and chemical profiles of 50 German beers.

Biological and biochemical fingerprints were investigated for the first time for the feasibility of effect-directed classification, and thus, to allow the choice of a distinct beer with regard to beneficial health effects. A high-performance thin-layer chromatography method was newly developed and combined with in situ effect-directed analysis for profiling 50 German beers for multipotent active compounds, and thus, their health-related potential. Discovered multipotent active zones were online eluted and characterized by high resolution mass spectrometry. For example, isoxanthohumol, iso-α-ad/n-humulone or its isomers, desdimethyl-octahydro-isocohumulone and ad/n-humulone were proven as antimicrobial compounds, isoxanthohumol as an acetylcholinesterase inhibitor, and isoxanthohumol and iso-α-ad/n-humulone or its isomers as radical scavengers. Investigating multivariate data analysis of effect-directed fingerprints for the first time, the pattern recognition and classification results showed the power of clustering non-alcoholic beers from other types of beer, or it showed the differentiation of dark and non-alcoholic beers.

Effect-directed analysis of ginger (Zingiber officinale) and its food products, and quantification of bioactive compounds via high-performance thin-layer chromatography and mass spectrometry.

Decision makers responsible for quality management along the food chain need to reflect on their analytical tools that should ensure quality of food and especially superfood. The "4ables" in target analysis (stable, extractable, separable, detectable) focusing on marker compounds do not cover all relevant information about the sample. On the example of ginger, a streamlined quantitative bioprofiling was developed for effect-directed analysis of 17 commercially available ginger and ginger-containing products via high-performance thin-layer chromatography (HPTLC-UV/Vis/FLD-bioassay). The samples were investigated concerning their active profile as radical scavengers, antimicrobials, estrogen-like activators and acetylcholinesterase/tyrosinase inhibitors. The [6]-gingerol and [6]-shogaol content of the different products ranged 0.2-7.4mg/g and 0.2-3.0mg/g, respectively. Further, multipotent compounds were discovered, characterized, and for example, assigned as [8]- and [10]-gingerol via HPTLC-ESI-HRMS. The developed bioprofiling is a step forward to new analytical methods needed to inform on the true product quality influenced by cultivation, processing, and storage.

Effect-directed fingerprints of 77 botanical extracts via a generic high-performance thin-layer chromatography method combined with assays and mass spectrometry.

Quantitative effect-directed profiles of 77 industrially and freshly extracted botanicals like herbs, spices, vegetables and fruits, widely used as food ingredients, dietary supplements or traditional medicine, gave relevant information on their quality. It allows the assessment of food, dietary supplements and phytomedicines with regard to potential health-promoting activities. In contrary to sum parameter assays and targeted analysis, chromatography combined with effect-directed analysis allows fast assignment of single active compounds and evaluation of their contribution to the overall activity, originating from a food or botanical sample. High-performance thin-layer chromatography was hyphenated with UV/Vis/FLD detection and effect-directed analysis, using the 2,2-diphenyl-1-picrylhydrazyl radical, Gram-negative Aliivibrio fischeri, Gram-positive Bacillus subtilis, acetylcholinesterase and tyrosinase assays. Bioactive compounds of interest were eluted using an elution head-based interface and further characterized by electrospray ionization (high-resolution) mass spectrometry. This highly streamlined workflow resulted in a hyphenated HPTLC-UV/Vis/FLD-EDA-ESI+/ESI--(HR)MS method. The excellent quantification power of the method was shown on three compounds. For rosmarinic acid, contents ranged from 4.5mg/g (rooibos) to 32.6mg/g (rosemary), for kaempferol-3-glucoside from 0.6mg/g (caraway) to 4.4mg/g (wine leaves), and for quercetin-3-glucoside from 1.1mg/g (hawthorn leaves) to 17.7mg/g (thyme). Three mean repeatabilities (%RSD) over 18 quantifications for the three compounds were ≤2.2% and the mean intermediate precision over three different days (%RSD, n=3) was 5.2%.

Effect-directed analysis of fresh and dried elderberry (Sambucus nigra L.) via hyphenated planar chromatography.

A healthy diet is an important factor in a healthy lifestyle that is becoming increasingly important in today's society. The fruits of European elder (Sambucus nigra L.) are a rich source of bioactive compounds like anthocyanins. In this study, dried and fresh fruits of four cultivated and six wild growing plants were investigated for their anthocyanin pattern and content as well as their bioactive compounds. After separation on HPTLC plates silica gel 60 F254 with a mixture of ethyl acetate, 2-butanone, formic acid and water, the plates were quantitatively evaluated by densitometry and also subjected to various (bio)assays to investigate the samples for compounds acting as radical-scavengers, antimicrobials, estrogens, and acetylcholinesterase or tyrosinase inhibitors. The mean contents for the two most abundant anthocyanins in European elderberries, confirmed by HPTLC-ESI-MS, ranged from 159 to 647mg/100g in fresh and from 166 to 2764mg/100g in dried fruits for cyanidin-3-sambubioside, and from 112 to 521mg/100g in fresh and 95 to 226mg/100g in dried fruits for cyanidin-3-glucoside. Additionally, the anthocyanin content was higher in berries of cultivars than of wild growing plants. The anthocyanins' radical scavenging activity and antimicrobial effect against Aliivibrio fischeri were confirmed. Further, a radical scavenging compound affecting A. fischeri and acting as acetylcholinesterase inhibitor was tentatively assigned by its protonated molecule at m/z 456 as either ursolic or oleanolic acid by HPTLC-ESI-MS. HPTLC hyphenated with bioassays and mass spectrometry was selected as method of choice for fingerprinting, pattern recognition, and bioprofiling of elderberry samples as well as quantitation and confirmation of bioactive compounds therein.

Production of cyathane type secondary metabolites by submerged cultures of Hericium erinaceus and evaluation of their antibacterial activity by direct bioautography.

BACKGROUND: Fungi of the phylum Basidiomycota are well-known to form a broad spectrum of biologically active secondary metabolites, especially low molecular weight compounds such as terpenoids. Hericium erinaceus produces various cyathane type diterpenoids including erinacines. However, no quantitative data and production kinetics have been reported on the biosynthesis of the erinacines C and P in submerged cultures. In the present study, the production of erinacine C was optimized, and the product formation kinetics as well as the antimicrobial activity were studied by high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC) and direct bioautography. RESULTS: Oatmeal and Edamin® K were identified to be crucial media components for an efficient production of erinacine C. The highest concentrations of erinacine C were obtained in the optimized culture medium on the 9th culture day (approximately 260 mg L-1). The production of erinacine P was strongly time dependent. The maximum concentration of erinacine P of 184 mg L-1 was observed on the third culture day. Afterwards, the concentrations of erinacine P decreased while the concentrations of erinacine C steadily increased. Comparable results were obtained by HPTLC with UV detection and HPLC with diode-array detection (DAD) analyses. Direct bioautography allowed for an additional analysis of the antimicrobial activity of the secondary metabolites. CONCLUSIONS: The C and N sources oatmeal and Edamin® K induced the formation of erinacine C. Detailed product formation kinetics of the erinacines C and P have been reported for the first time. HPTLC combined with the Aliivibrio fischeri bioassay allowed for an instant detection of cyathane diterpenoids in crude extracts and for an evaluation of the antimicrobial activity of the secondary metabolites directly on the plate.

Engineered anisotropic microstructures for ultrathin-layer chromatography.

The strong dependence of separation behavior on ultrathin-layer chromatography (UTLC) stationary phase microstructure motivates continued UTLC plate design optimization efforts. We fabricated 4.6-5.3 mum thick normal phase silica UTLC stationary phases with several types of in-plane macropore anisotropies using the glancing angle deposition (GLAD) approach to engineering nanostructured thin films. The separation behaviors of two new media, isotropic vertical posts and anisotropic bladelike films, were compared to that of anisotropic chevron media. Channel-like structures within the anisotropic media introduced preferential mobile phase flow directions that could be exploited to give separation tracks diagonal to the development direction. Extraction of chromatograms from these angled tracks required the development of a new analytical approach that involved a commercial flatbed film scanner and custom numerical image analysis software. GLAD stationary phase performance was quantified using the Dimethyl Yellow dye separated from a lipophilic dye mixture over migration distances less than approximately 10 mm. The limits of detection were 10 +/- 4 ng for the vertical posts and 11 +/- 3 ng for the bladelike media. We obtained theoretical plate heights that varied with film microstructure between 12 and 28 mum. Unoptimized separation performance was comparable to that of other planar chromatography media. Macropore anisotropies engineered by GLAD may expand the capabilities of future UTLC stationary phases.

Molecular identification of mutations associated with anti-tuberculosis drug resistance among strains of Mycobacterium tuberculosis.

BACKGROUND: Understanding the etiologic organism, antimicrobial resistance mechanisms, and transmission of multidrug-resistant tuberculosis (MDR-TB) can be of great value in optimizing strategies to control and prevent its development and transmission. METHODS: One hundred and fifty-five Mycobacterium tuberculosis complex isolates from patients with pulmonary tuberculosis (TB) in Cairo, Egypt were studied. In vitro drug susceptibility testing against rifampin (RIF), isoniazid (INH), streptomycin (SM), ethambutol (EMB), and pyrazinamide (PZA) was performed. Resistance was studied by the standard agar proportion method. Single strand conformation polymorphism (SSCP) and DNA sequence analysis were used to detect mutations in the genes that encode resistance to rpoB, katG, rpsL, and embB. RESULTS: Among 155 consecutive M. tuberculosis isolates, 25 (16.1%) were MDR-TB; 13 of these were from newly diagnosed untreated cases, 12 were from re-treated cases, and none of the MDR-TB isolates had matching IS6110 fingerprints. Among the MDR-TB isolates, rpoB mutations were found in 76% of RIF-resistant isolates, katG mutations were found in 47.1% of INH-resistant isolates, rpsL mutations were found in 55.6% of SM-resistant isolates, and embB mutations were found in 36.4% of EMB-resistant isolates. CONCLUSIONS: No major differences were found in the frequencies of mutations or types of amino acid substitution between newly diagnosed untreated cases and re-treated cases. The high prevalence of MDR-TB at this hospital underscores the need for continuous monitoring of strains and antimicrobial resistance.

Sorbitol-fermenting enterohaemorrhagic Escherichia coli O157:H- causes another outbreak of haemolytic uraemic syndrome in children.

An outbreak of haemolytic uraemic syndrome (HUS) among children caused by infection with sorbitol-fermenting enterohaemorrhagic Escherichia coli O157:H- (SF EHEC O157:H-) occurred in Germany in 2002. This pathogen has caused several outbreaks so far, yet its reservoir and routes of transmission remain unknown. SF EHEC O157:H- is easily missed as most laboratory protocols target the more common sorbitol non-fermenting strains. We performed active case-finding, extensive exploratory interviews and a case-control study. Clinical and environmental samples were screened for SF EHEC O157:H- and the isolates were subtyped by pulsed-field gel electrophoresis. We identified 38 case-patients in 11 federal states. Four case-patients died during the acute phase (case-fatality ratio 11%). The case-control study could not identify a single vehicle or source. Further studies are necessary to identify the pathogen's reservoir(s). Stool samples of patients with HUS should be tested with an adequate microbiological set-up to quickly identify SF EHEC O157:H-.

[Risk factors for abundance of shiga toxin producing Escherichia coli in sewage water]. / Risikofaktoren der Belastung durch Shigatoxin-produzierende Escherichia coli (STEC) in Kläranlagen.

Shiga toxin producing Escherichia coli (STEC) in sewage influent into surface water are a potential source of human infections with STEC. Eight sewage treatment plants in Bavaria, Germany, were sampled at regular intervals from 2003 to 2004 in order to estimate STEC load and quantify risk factors. 95 of 378 samples (25 %) were tested positive for stx1and/or stx2 with PCR after enrichment culture. STEC elimination after treatment was 44 %. The following risk factors were analysed with logistic regression: location of sewage plant (rural vs. urban), treatment plant technology (two stage vs. three stage treatment) and sampling location (sewage input vs. sewage output). Rural plants had odds-ratios of 1,7 (95 % CI 1.03 - 2.69; p = 0.038) for a positive stx1 and/ or stx2 PCR result, sampling at sewage input of 2.1 (95 %CI 1.28 - 3.36; p = 0.003) and three stage plants of 1.51 (95 % CI 0.94 - 2.44; p = 0.087, not significant). Sampling after rain and after dry spells had no impact on STEC abundance (univariate Chi-square test = 0.01; df1; p = 0.920). Rural sewage plants had higher odds of STEC content. The influence of the sewage plant technology on the STEC load requires further clarification.

Simultaneous infection with multiple strains of Mycobacterium tuberculosis.

Drug-susceptible and drug-resistant isolates of Mycobacterium tuberculosis were recovered from 2 patients, 1 with isoniazid-resistant tuberculosis (patient 1) and another with multidrug-resistant tuberculosis (patient 2). An investigation included patient interviews, record reviews, and genotyping of isolates. Both patients worked in a medical-waste processing plant. Transmission from waste was responsible for at least the multidrug-resistant infection. We found no evidence that specimens were switched or that cross-contamination of cultures occurred. For patient 1, susceptible and isoniazid-resistant isolates, collected 15 days apart, had 21 and 19 restriction fragments containing IS6110, 18 of which were common to both. For patient 2, a single isolate contained both drug-susceptible and multidrug-resistant colonies, demonstrating 10 and 11 different restriction fragments, respectively. These observations indicate that simultaneous infections with multiple strains of M. tuberculosis occur in immunocompetent hosts and may be responsible for conflicting drug-susceptibility results, though the circumstances of infections in these cases may have been unusual.

Characterization of IS6110 restriction fragment length polymorphism patterns and mechanisms of antimicrobial resistance for multidrug-resistant isolates of Mycobacterium tuberculosis from a major reference hospital in Assiut, Egypt.

We evaluated 25 Mycobacterium tuberculosis isolates from patients at a major Egyptian reference hospital in Assiut, Egypt, who had been treated for at least 1 year for tuberculosis. Typing patterns (IS6110) were diverse, and multidrug resistance was found among 11 (44%) of the isolates. Mutations associated with antimicrobial drug resistance were found in rpoB, katG, rpsL, and embB in the resistant isolates.

Pyrazinamide-monoresistant Mycobacterium tuberculosis in the United States.

Mycobacterium bovis is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all Mycobacterium tuberculosis complex isolates demonstrating PZA monoresistance were truly M. bovis, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for pncA sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible M. tuberculosis, 1 was identified as PZA-monoresistant M. bovis, and 1 was identified as M. bovis BCG. The three remaining isolates were identified as PZA-monoresistant M. tuberculosis. Sequencing of the pncA and oxyR genes genotypically confirmed the two M. bovis and the six susceptible M. tuberculosis species. Each of the three PZA-monoresistant M. tuberculosis isolates had different, previously unreported, pncA gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser104Cys), and a base substitution at codon 90 (Ile90Ser). This study demonstrates that PZA monoresistance is not an absolute marker of M. bovis species but may also occur in M. tuberculosis, associated with a number of different mutational events in the pncA gene. It is the first report of PZA-monoresistant M. tuberculosis in the United States.

Characterization of spontaneous, In vitro-selected, rifampin-resistant mutants of Mycobacterium tuberculosis strain H37Rv.

Resistance to rifampin in Mycobacterium tuberculosis results from mutations in the gene coding for the beta subunit of RNA polymerase (rpoB). At least 95% of rifampin-resistant isolates have mutations in rpoB, and the mutations are clustered in a small region. About 40 distinct point mutations and in-frame insertions and deletions in rpoB have been identified, but point mutations in two codons, those coding for Ser(531) and His(526), are seen in about 70% of rifampin-resistant clinical isolates, with Ser(531)-to-Leu (TCG-to-TGG) mutations being by far the most common. To explore this phenomenon, we isolated independent, spontaneous, rifampin-resistant mutant versions of well-characterized M. tuberculosis laboratory strain H37Rv by plating 100 separate cultures, derived from a single low-density inoculum, onto rifampin-containing medium. Rifampin-resistant mutants were obtained from 64 of these cultures. Although we anticipated that the various point mutations would occur with approximately equal frequencies, sequencing the rpoB gene from one colony per plate revealed that 39 (60.9%) were Ser(531) to Leu. We conclude that, for unknown reasons, the associated rpoB mutation occurs at a substantially higher rate than other rpoB mutations. This higher mutation rate may contribute to the high percentage of this mutation seen in clinical isolates.

Transmission of Mycobacterium tuberculosis from medical waste.

CONTEXT: Washington State has a relatively low incidence rate of tuberculosis (TB) infection. However, from May to September 1997, 3 cases of pulmonary TB were reported among medical waste treatment workers at 1 facility in Washington. There is no previous documentation of Mycobacterium tuberculosis transmission as a result of processing medical waste. OBJECTIVE: To identify the source(s) of these 3 TB infections. DESIGN, SETTING, AND PARTICIPANTS: Interviews of the 3 infected patient-workers and their contacts, review of patient-worker medical records and the state TB registry, and collection of all multidrug-resistant TB (MDR-TB) isolates identified after January 1, 1995, from the facility's catchment area; DNA fingerprinting of all isolates; polymerase chain reaction and automated DNA sequencing to determine genetic mutations associated with drug resistance; and occupational safety and environmental evaluations of the facility. MAIN OUTCOME MEASURES: Previous exposures of patient-workers to TB; verification of patient-worker tuberculin skin test histories; identification of other cases of TB in the community and at the facility; drug susceptibility of patient-worker isolates; and potential for worker exposure to live M tuberculosis cultures. RESULTS: All 3 patient-workers were younger than 55 years, were born in the United States, and reported no known exposures to TB. We did not identify other TB cases. The 3 patient-workers' isolates had different DNA fingerprints. One of 10 MDR-TB catchment-area isolates matched an MDR-TB patient-worker isolate by DNA fingerprint pattern. DNA sequencing demonstrated the same rare mutation in these isolates. There was no evidence of personal contact between these 2 individuals. The laboratory that initially processed the matching isolate sent contaminated waste to the treatment facility. The facility accepted contaminated medical waste where it was shredded, blown, compacted, and finally deactivated. Equipment failures, insufficient employee training, and respiratory protective equipment inadequacies were identified at the facility. CONCLUSION: Processing contaminated medical waste resulted in transmission of M tuberculosis to at least 1 medical waste treatment facility worker. JAMA. 2000;284:1683-1688.

Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis.

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147-151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were > or =100 microg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 microg/ml, 3 had the same mutation (Thr47-->Ala) and 18 had the wild-type sequence. For the three Thr47-->Ala mutants PZA MICs were 12.5 microg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 microg of PZA per ml and the third was resistant to 800 microg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to > or =100 microg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47-->Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to > or =100 microg of PZA per ml.

Identification of Mycobacterium species by PCR-restriction fragment length polymorphism analyses using fluorescence capillary electrophoresis.

We developed a scheme for the rapid identification of Mycobacterium species based upon PCR amplification of polymorphic genetic regions with fluorescent primers followed by restriction and analysis by fluorescence capillary electrophoresis. Mycobacterium species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labeled [both strands] at the 5' end with 4,7,2',7'-tetrachloro-6-carboxyfluorescein) using HaeIII and BstEII and of a 475-bp hypervariable region of the 16S rRNA gene (labeled [both strands] at the 5' end with 6-carboxyfluorescein) using HaeIII and CfoI. Samples were analyzed on an automated fluorescence capillary electrophoresis instrument, and labeled fragments were sized by comparison with an internal standard. DNA templates were prepared with pure cultures of type strains. In all, we analyzed 180 strains, representing 22 Mycobacterium species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species. Three members of the Mycobacterium tuberculosis complex had a common RFLP pattern. A computerized algorithm which eliminates subjectivity from pattern interpretation and which is capable of identifying the species within a sample was developed. The convenience and short preparatory time of this assay make it comparable to conventional methodologies such as high-performance liquid chromatography and hybridization assays for identification of mycobacteria.