Evaluation of real time polymerase chain reaction assays for confirmation of Neisseria gonorrhoeae in clinical samples tested positive in the Roche Cobas Amplicor assay.

OBJECTIVE: Development of a rapid, sensitive, and accurate assay for confirmation of Neisseria gonorrhoeae in clinical samples. METHOD: Two real time polymerase chain reaction (PCR) assays, developed on the LightCycler for amplification of the N gonorrhoeae cppB gene, were utilised for confirmation of this bacterial pathogen in samples positive by the Roche Cobas Amplicor assay. Performance characteristics of the two assays were compared with other commercial nucleic acid amplification assays, including the Abbott LCx and Roche 16S rRNA tests. RESULTS: All related Neisseria as well as other bacterial species tested negative by both cppB gene based assays, whereas 120 N gonorrhoeae clinical isolates from various geographical regions gave in positive results. Both assays had a sensitivity of one copy per reaction. 122 clinical samples positive and another 50 samples negative for N gonorrhoeae by Roche Cobas Amplicor were selected from a specimen pool of more than 3000 women tested previously. Overall, 73 of 122 (59.8%) samples were confirmed as positive. The two real time assays had sensitivities of 99% and 100% and specificities of 98% and 100%, respectively. The 16S and LCx assays produced similar results to the real time assays, indicating a similar sensitivity to and specificity of both real time assays. CONCLUSION: The data from this study highlight the need to confirm N gonorrhoeae positive Cobas Amplicor PCR results as an important part of the testing algorithm of all diagnostic laboratories utilising this assay.

Infrared spectroscopic study of cervical smears in patients with HIV: implications for cervical carcinogenesis.

Patients with HIV have an increased incidence of cervical cancer, necessitating increased surveillance. Infrared spectroscopy (IRS) has the potential of aiding the diagnosis of cervical neoplasia and also of providing clues into its pathogenesis. We studied by IRS cervical scrapings from 22 HIV-infected and 23 control women; 8 of the former and none of the latter had dysplasia. The infrared spectra followed three patterns, designated pattern I (similar to that previously associated with normal cervical samples), pattern II (intermediate between patterns I and III), and pattern III (associated with cervical neoplasia). Compared with HIV-negative controls, HIV-infected women had a higher prevalence of pattern III and a lower prevalence of pattern II; these differences were statistically significant (P = .015 by chi2 analysis). Similar spectroscopic changes were present even when only the cytologically normal samples from HIV-positive and HIV-negative women were analyzed. We speculate that these changes may reflect early structural changes associated with cervical neoplasia that are not detectable cytologically. The infrared spectra in the region 950 to 1,300 cm(-1) could not differentiate cervical samples from HIV-infected and uninfected patients. The potential practical applications of IRS in HIV cervical disease are discussed.

Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: implications for tumor biology.

Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2, 000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (i) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (ii) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.

Infrared spectroscopy of normal and abnormal cervical smears: evaluation by principal component analysis.

Fourier-transform infrared (FT-IR) spectra of malignant and dysplastic cervical scrapings were abnormal, as first described in our study of a limited number of samples, where the spectra were evaluated by visual inspection and peak intensity ratios. We have expanded our study to evaluate more cervical conditions, and to analyze the spectra by a chemometric approach (principal component analysis [PCA]). Cervical samples from 436 females were evaluated by FT-IR and Papanicolaou testing; 40/436 spectra were nonanalyzable. The remaining were as follows: normal, 174; malignant, 19; dysplasia, 8; atypia, 113; atrophy, 19; inflammatory, 47; bloody smear, 12; hypocellular, 4. PCA analysis followed by chi2 test revealed that statistically significant frequencies of being predicted malignant by FT-IR were associated with samples diagnosed as malignant (P < 0.0001), and also those diagnosed as "atrophy" (P < 0.001), "atypical with bloody smear" (P < 0.05), "atypical with atrophic pattern" (P < 0.05), and "dysplasia" (P < 0.05). Based on these findings, for the diagnosis of cervical cancer by FT-IR, as defined here, the sensitivity is 79%, the specificity is 77%, the positive predictive value is 15%, and the negative predictive value is 98.6%. Our findings (a) demonstrate the application of a chemometric approach to the study of cervical FT-IR spectra; (b) assess its potential diagnostic role; (c) suggest that atrophic and neoplastic samples share structural features; and (d) suggest that blood may interfere with such spectroscopic evaluation. These findings warrant further evaluation of FT- IR spectroscopy in cervical and other malignancies.

Colorimetric assay for free and bound L-fucose.

A novel, rapid, and reliable colorimetric method for measuring L-fucose has been developed. This method utilizes NADH formed from the interaction of L-fucose with fucose dehydrogenase and NAD to generate color in a reaction involving CuSO4 and neocuproine. NADH reduces Cu2+ to Cu1+ and the latter interacts with neocuproine to yield a complex with a maximal absorption at 455 nm. The reaction of NADH with copper-neocuproine is immediate and under the conditions of the assay the color formed remains stable for at least 2 h. When the assay is used to determine levels of L-fucose, the absorbance is found to be linearly proportional to exogenously added fucose concentrations from 16 to 179 nmol with resulting molar extinction coefficient of 13,660. Using this procedure, L-fucose released by acid hydrolysis from porcine submaxillary mucin, and by alpha-L-fucosidase from p-nitrophenyl-alpha-L-fucopyranoside, was quantitated.

A colorimetric procedure for measuring beta-lactamase activity.

The enzymatic hydrolysis of benzylpenicillin was measured by a novel colorimetric procedure. The penicilloic acid generated from the hydrolysis of penicillin was reacted with CuSO4 and neocuproine to form a colored complex having a maximal absorption at 454.5 nm. A plot of absorbance versus beta-lactamase activity yielded a straight line from 1 to 5 mU of enzyme. Using TEM-1 as the model beta-lactamase, a Km of 46 microM was observed with benzylpenicillin serving as the substrate. When the assay was used to determine levels of benzylpenicillin, the absorbance was found to be linearly proportional to exogenously added penicillin from 2.8 to 88 microM. This procedure is simple to use and can be employed to measure the hydrolysis of other beta-lactam antibiotics.

Preliminary evaluation of a rapid colorimetric method for identification of pathogenic Neisseria.

A rapid colorimetric method for the identification of pathogenic Neisseria (Identicult-Neisseria; Scott Laboratories, Inc.) based on beta-galactosidase, gamma-glutamylaminopeptidase, and gamma-prolylaminopeptidase is described. All 82 clinical isolates of Neisseria gonorrhoeae, 9 clinical isolates of N. meningitidis, and 5 clinical isolates of N. lactamica were correctly determined to the species level, as were 4 isolates of Branhamella catarrhalis. Reactions were prompt and easily interpreted. The system should be extremely useful in clinical laboratories.

Increased detection of prolylaminopeptidase in Neisseria meningitidis by Identicult-Neisseria.

Identicult-Neisseria (Scott Laboratories, Inc., Fiskeville, R.I.), a rapid enzymatic method with chromogenic substrates, was tested in our laboratories for the identification of Neisseria gonorrhoea, Neisseria meningitidis, and Neisseria lactamica. The test correlated very highly in its identification of pathogenic Neisseria spp. with modified New York City fermentation medium. Identicult-Neisseria appeared to be more sensitive in its detection of prolylaminopeptidase activity in N. meningitidis than most of the currently available systems.

Effect of immobilization on the physical and kinetic properties of soluble and insoluble trypsin-albumin polymers.

Bovine trypsin was crosslinked to human serum albumin (HSA) with glutaraldehyde to form soluble and insoluble copolymers. The physical and kinetic properties of trypsin and trypsin-HSA polymers were compared. Trypsin was heat labile, retaining only 24% of its enzymic activity after heating for 5 min at 60 degrees C. In contrast, under the same condition both the soluble and insoluble trypsin-HSA polymers showed enhanced resistance to heat in-activation, retaining 81 and 100% of their original activities, respectively. The trypsin-HSA polymers also showed shifts in pH optima, an increase in activation energy, and a broadening of their pH stability profiles.

Preliminary evaluation of a rapid colorimetric method for the presumptive identification of group A streptococci and enterococci.

A rapid colorimetric method for the presumptive identification of group A streptococci and enterococci based upon pyroglutamyl aminopeptidase activity is described. Of 76 group A streptococcal isolates from primary plates, 83 gave positive reactions, and the remaining 7 were positive on retesting in pure culture. Of the 31 enterococcal isolates tested, all gave positive reactions. Despite occasional positive reactions with staphylococci and Klebsiella pneumoniae, the test could be useful and cost-effective in the clinical laboratory.

Five novel plasmid-determined beta-lactamases.

Five novel plasmid-determined beta-lactamases named TLE-1, OXA-4, OXA-5, OXA-6, and OXA-7 were detected in ampicillin-resistant isolates of Escherichia coli and carbenicillin-resistant strains of Pseudomonas aeruginosa. TLE-1 resembled TEM-1 in substrate profile and reactions with inhibitors but differed in isoelectric point (5.55) and enzyme banding pattern on flat-bed electrofocusing.OXA-4, OXA-5, OXA-6, and OXA-7 hydrolyzed oxacillin, methicillin, and cloxacillin readily but differed from OXA-1, OXA-2, and OXA-3 in substrate profiles, inhibitor reactions, and isoelectric points (7.5 to 7.8).OXA-4 and OXA-6 were unusual for members of the OXA group in their sensitivity to inhibition by cloxacillin. OXA-5 and OXA-7 had isoelectric points close to that of SHV-1, emphasizing the need in beta-lactamase classification for studies in addition to isoelectric focusing. These five new enzymes bring the number of plasmid-determined beta-lactamases known in gram-negative organisms to more than 20. The evolution of such enzymatic diversity remains to be explored.

Genetic and biochemical properties of AER-1, a novel carbenicillin-hydrolyzing beta-lactamase from Aeromonas hydrophila.

A novel carbenicillin-hydrolyzing beta-lactamase has been discovered in a blood isolate of Aeromonas hydrophila. The enzyme resembles plasmid-determined carbenicillinases in substrate profile but differs in isoelectric point (pI 5.9) and molecular weight (22,000) and has been termed AER-1. No evidence for a plasmid location could be obtained in A. hydrophila, but the AER-1 gene and resistance to chloramphenicol, streptomycin, and sulfonamide could be transferred by mobilization with IncP plasmids to Escherichia coli, where the gene cluster inserted at a unique chromosomal site. The linked resistances are similar to those found on multiresistance beta-lactamase transposons, but since insertion of the A. hydrophila gene cluster was site specific and recA+ dependent, the cluster is not a functional transposon.

Effect of immobilization on stability and kinetic properties of alpha-L-fucosidase from Turbo cornutus.

An amount of alpha-L-fucosidase from T. cornutus liver was copolymerized with glutaraldehyde using bovine serum albumin as the carrier protein. The properties of the native, the soluble enzyme polymer complex, and the insoluble enzyme polymer complex were studied and compared under various conditions of pH, temperature, substrate, and inhibitor concentration. Native alpha-L-fucosidase was heat labile and lost more than 85% of its activity when incubated at 55 degrees C for 5 min. In contrast, under equivalent incubation conditions, both the soluble and the insoluble enzyme polymer complexes exhibited enhanced resistance to thermal inactivation and after 5 min lost only 65 and 40% of their original activity, respectively. Polymerzation also resulted in the shift of pH optima towards the acidic range, a decrease in activation energy and a change in the apparent K(m) values towards the p-nitrophenyl-alpha-L-fucopyranoside substrate.

Nonenzymatic glycosylation of human IgG: in vitro preparation.

Incubation of human serum with either D- (1-14C) galactose (5 mM), D- (1-14C) glucose (5 mM) or L- (1-14C) fucose (5 mM) in vitro for 7 days under physiological conditions resulted in the accumulation of radioactivity into trichloroacetic acid precipitable material. Separation of the serum proteins by Sephadex G-200 chromatography, revealed the association of radioactivity with the albumin fraction (95%) and to a lesser extent with IgG (4%) and IgM (1%). D-galactose glycosylated purified human IgG at 2 to 3 fold the rate of D-glucose of L-fucose. The rate of glycose incorporation into IgG increased parabolically with increasing pH and temperature of incubation, and followed a first order dependence with either the glycose or the IgG concentration. The post-translational modification of IgG through nonenzymatic glycosylation may affect its immunological properties in clinical conditions associated with increased blood sugar concentrations.

Nonenzymatic galactosylation of human serum albumin. In vitro preparation.

Incubation of purified human serum albumin with D-[1-14C]galactose (5 mM) or D-[1-14C]glucose (5 mM) in vitro for 7 days under physiological conditions resulted in the time-dependent accumulation of radioactivity into trichloroacetic acid-precipitable material. Comparative studies indicated that the rate of sugar incorporation into albumin increased with increasing pH and temperature of incubation and followed a first order dependence with regard to monosaccharide and albumin concentrations. The extent of nonenzymatic galactosylation of human albumin was approximately 300% greater than the extent of nonenzymatic glucosylation under equivalent experimental conditions. Prolonged dialysis of the modified albumins against a large excess of the unlabeled monosaccharides failed to alter the amount of protein-bound radiolabeled carbohydrate, suggesting that the linkage between sugar and albumin is covalent in nature. The post-translational modification of proteins by nonenzymatic galactosylation may be of physiological significance in individuals with reduced galactokinase or galactose-1-phosphate uridyl transferase activities.