Testing for viral penetration of non-latex surgical and examination gloves: a comparison of three methods.

Currently, there are no international standards based on microbiological methodology for testing the ability of medical examination or surgical gloves to prevent the passage of viruses. Three protocols for the direct examination of the viral barrier properties of non-latex gloves were compared with 1080 gloves (270 gloves from each of two surgical brands and two medical examination brands). In two of the methods, gloves were filled with and suspended in a nutrient broth solution, and bacteriophage phiX174 was placed either inside or outside the glove, while the entire test vessel was agitated. Gloves tested using the third method were filled with a suspension of bacteriophage and allowed to rest in a vessel containing nutrient broth. Gloves were tested directly from the manufacturer's packaging, or after being punctured intentionally or subjected to a stress protocol. The passage of bacteriophage was detected with plaque assays. Significant differences in failure rates between glove brands were apparent only among gloves that had been subjected to the stress protocol. Overall, the two methods in which bacteriophage were placed inside the gloves provided more sensitivity than the method in which bacteriophage was spiked into broth outside the gloves. Thus the placement of bacteriophage inside test gloves (or the use of pressure across the glove barrier during testing), and the use of a standardised stress protocol, will improve significantly the ability of a glove test protocol to determine the relative quality of the barrier offered by medical examination and surgical gloves. Further research is needed to provide test methods that can incorporate reproducibly both the use of bacteriophage and simulated glove use in an industrial quality control setting.

A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine.

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K(D)) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC50 of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 microg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).

Transcriptional organization and regulation of a polycistronic cold shock operon in Sinorhizobium meliloti RM1021 encoding homologs of the Escherichia coli major cold shock gene cspA and ribosomal protein gene rpsU.

A homolog of the major eubacterial cold shock gene cspA was identified in Sinorhizobium meliloti RM1021 by luxAB reporter transposon mutagenesis. Here we further characterize the organization and regulation of this locus. DNA sequence analysis indicated that the locus includes three open reading frames (ORFs) encoding homologs corresponding to CspA, a novel 10.6-kDa polypeptide designated ORF2, and a homolog of the Escherichia coli ribosomal protein S21. Transcription analysis indicated that this locus produced two different-sized cspA-hybridizing transcripts upon cold shock, a 400-nucleotide (nt) RNA encoding cspA alone and a 1, 000-nt transcript encoding cspA-ORF2-rpsU. The sizes of the transcripts agreed with the location of the transcription start site determined by primer extension and the locations of two putative transcriptional terminators. The promoter of the cspA-ORF2-rpsU locus had -10 and -35 elements similar to the E. coli sigma(70) consensus promoter and, like the cspA locus of E. coli, included an AT-rich region upstream of the -35 hexamer. The promoter of the S. meliloti cspA locus was found to impart cold shock-induced mRNA accumulation. In addition, the 5'-untranslated region (5' UTR) was found to increase the fold induction of cspA transcripts after cold shock and depressed the level of luxAB mRNA prior to cold shock, another feature similar to cspA regulation in E. coli. No "cold box" was identified upstream of the S. meliloti cspA gene, however, and there was no other obvious sequence identity between the S. meliloti 5' UTR and that of E. coli. DNA hybridization analysis indicated that outside the cspA-ORF2-rpsU cold shock locus there are several additional cspA-like genes and a second rpsU homolog.

Identification of cold shock gene loci in Sinorhizobium meliloti by using a luxAB reporter transposon.

Using a luxAB reporter transposon, seven mutants of Sinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15 degrees C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the three rrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrn operons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene, cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15 degrees C.

Assessment of an automated solid phase competitive fluoroimmunoassay for benzoylecgonine in untreated urine.

A new solid phase fluoroimmunoassay using a fully automated flow fluorometer adapted for urinalysis of drug metabolites is described. Fluorescein-conjugated benzoylecgonine (FL-BE) and monoclonal antibodies (mAb) against benzoylecgonine (BE) were the reagents used for demonstration. The solid phase consisted of anti-BE mAbs immobilized on the surface of polymethyl methacrylate (PMMA) beads. Free BE in solution competed with FL-BE and reduced bead-bound fluorescence in a concentration-dependent manner. The binding of FL-BE to the anti-BE mAb reached steady-state within minutes. FL-BE was not bound by uncoated beads nor beads coated with non-specific proteins or IgG. The signal-to-noise ratio was 33, and the sensitivity of the assay was 2 ng ml(-1) for BE. The effective concentration of BE was 1 to 100 ng ml(-1), with an IC50 value of 12 ng ml(-1). The mAb showed equal affinities for BE, cocaine, and cocaethylene, but a five order-of-magnitude lower affinity for ecgonine and ecgonine methylester. In a double-blind comparison using clinical urine samples, the data from this single-step competitive assay had excellent agreement with results obtained using a fiber-optic biosensor (FOB), and the EMIT assay performed commercially. The assay provided kinetic data rapidly and can be used to detect small analytes for which antibodies and fluorescein conjugates are available. The affinity of the mAb for FL-BE, calculated from kinetic analysis of the time course of the on and off reaction, was 2.25 x 10(-9) M.

Foliar Chlorosis in Symbiotic Host and Nonhost Plants Induced by Rhizobium tropici Type B Strains.

Rhizobium tropici CIAT899 induced chlorosis in the leaves of its symbiotic hosts, common bean (Phaseolus vulgaris L.), siratro (Macroptilium atropurpureum Urb.), and Leucaena leucocephala (Lam.) de Wit. Chlorosis induction by strains CIAT899 and CT9005, an exopolysaccharide-deficient mutant of CIAT899, required carbon substrate. When the bacteria were added at planting in a solution of mannitol (50 g/liter), as few as 10 cells of CIAT899 were sufficient to induce chlorosis in bean plants. All carbon sources tested, including organic acids and mono- and disaccharides, supported chlorosis induction. The addition of a carbon source did not affect the growth rate or the population density of CT9005 in the bean plant rhizosphere. Cell-free filtrates of cultures of CT9005 did not induce detectable chlorosis. All type B strains of R. tropici tested also induced chlorosis in common bean. Type A strains of R. tropici and all other species of bacteria tested did not induce chlorosis. Several lines of evidence indicated that nodulation was not required for chlorosis induction. Strain RSP900, a pSym-cured derivative of CIAT899, induced chlorosis in wild-type P. vulgaris. In addition, NOD125, a nodulation-defective line of common bean, developed chlorosis when inoculated with CIAT899, but did not develop nodules. CIAT899 consistently induced severe chlorosis in the leaves of the nonhost legumes alfalfa (Medicago sativa L.) and Berseem clover (Trifolium alexandrinum L.), and induced chlorosis in 29 to 58% of the plants tested of sunflower, cucumber, and tomato seedlings, but it did not induce chlorosis in the leaves of corn or wheat. Chlorosis induction in nonhost plants also required carbon substrate. The data are consistent with the hypothesis that R. tropici type B strains produce a chlorosis-inducing factor that affects a wide range of plant species.

chvA locus may be involved in export of neutral cyclic beta-1,2-linked D-glucan from Agrobacterium tumefaciens.

Extracellular and intracellular neutral beta-1,2-linked D-glucan content was determined in a virulent, attachment-deficient mutants of Agrobacterium tumefaciens that map in the chvA locus. chvA mutants contained approximately the same amount of intracellular glucan as cells of the virulent control strain A759, but released into the culture medium only 2% of the glucan released by strain A759. Introduction of a cosmid carrying the wild-type chv region restored attachment and virulence and restored extracellular glucan production to chvA mutant A2505. Exogenous glucan did not enhance or inhibit attachment or tumorigenesis of the virulent control strain or the chvA or chvB mutants. Our results suggest that the chvA locus is involved in the export of glucan from the cell and that export may be required for tumorigenesis.