Evaluation of Alinity m CMV assay performance for detecting CMV in plasma, cerebrospinal fluid, and bronchoalveolar lavage specimens.

Accurate detection and quantification of cytomegalovirus (CMV) is crucial to preventing adverse outcomes in immunocompromised individuals. Current assays were developed for use with plasma specimens, but CMV may be present in bronchoalveolar lavage (BAL) fluid and cerebrospinal fluid (CSF). We evaluated the performance of the Abbott Alinity m CMV assay compared to the Abbott RealTime CMV assay for quantification of CMV in plasma, BAL, and CSF specimens. To evaluate clinical performance, 190 plasma, 78 BAL, and 20 CSF specimens were tested with the Alinity m assay and compared to the RealTime assay. The Alinity m CMV assay showed high precision (SD <0.01 to 0.13) for all 3 specimen types. Clincal plasma and BAL specimens with quantifiable CMV DNA demonstrated strong correlation to RealTime CMV assay results (r2 = 0.9779 for plasma, r2 = 0.9373 for BAL). The Alinity m CMV assay may be useful for quantification of CMV in plasma, BAL, and CSF specimens.

Clinical Evaluation of the Alinity m STI Multiplex PCR Assay.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are routinely tested and reported; however, Trichomonas vaginalis (TV) is the most common sexually transmitted infection (STI) in the United States and the prevalence of Mycoplasma genitalium (MG) infections is likely higher than estimated. We examined the clinical performance of the Alinity m STI assay for detection and surveillance of CT/NG/TV/MG in urine specimens from patients at a large academic medical center. METHODS: Urine specimen from 198 patients was tested in this evaluation. Alinity m STI and Aptima Combo 2 CT/NG and TV assay (Panther System) results were compared, with discrepant results run on the cobas 6800 CT/NG, TV/MG assays. Analyzer turnaround times, time from loading the specimen on the analyzer to results reporting, were determined for Alinity m and Panther systems. RESULTS: Overall percent agreements of the Alinity m in comparison with the Aptima and cobas assays for CT, NG, TV, and MG were 99.5% (97.2%, 99.9%), 99.5% (97.2%, 99.9%), 98.4% (95.5%, 99.5%), and 86.4% (66.7%, 95.3), respectively. There were 5 discrepant samples (CT, 1; NG, 1; TV, 3) between the Alinity m and the Aptima assays, and 3 MG discrepant samples between the Alinity m STI and cobas 6800. Two of the 5 Aptima and Alinity m discrepant samples were resolved as they yielded similar results on both Alinity m and cobas 6800. TV and MG infections comprised 54% of the positive samples and were more often asymptomatic than CT and NG infections. Analyzer turnaround time was 3 hours 25 minutes for the Aptima CT/NG, 3 hours 25 minutes for Aptima TV, and 1 hour 55 minutes for Alinity m STI assay. CONCLUSIONS: The Alinity m STI assay allows for fast and simultaneous detection of the 4 major STI pathogens, which can facilitate surveillance and provide accurate results to help clinicians diagnose for initiation of appropriate treatment.

Performance of the Abbott RealTime MTB RIF/INH resistance assay when used to test Mycobacterium tuberculosis specimens from Bangladesh.

INTRODUCTION: The Abbott RealTime MTB RIF/INH Resistance Assay (RT MTB RIF/INH) is an assay for the detection of rifampicin (RIF)- and/or isoniazid (INH)-resistant Mycobacterium tuberculosis (MTB). The assay can be used to test sputum, bronchial alveolar lavage, and N-Acetyl-L-Cysteine (NALC)/NaOH pellets prepared from these samples. The assay can be used in direct testing mode, or in reflex mode following a MTB positive result produced by its companion assay, Abbott RT MTB. METHODS: In this study, the direct testing mode was used to test paired sputum and NALC/NaOH pellets prepared from sputum collected from Bangladesh TB patients. One hundred and thirty two paired samples were tested. RESULTS: The RT MTB RIF/INH inhibition rate was 0%. One hundred and twenty-two paired samples had results above the assay limit of detection and were analyzed by comparing with results from phenotypic drug sensitivity testing, GeneXpert MTB/RIF (Xpert), and MTBDR plus (Hain). RT MTB RIF/INH results were in good agreement with those of GeneXpert and Hain. CONCLUSION: The ability of this assay to detect RIF and INH resistance may contribute to the global control of multidrug resistant tuberculosis.

Analytical and clinical performance characteristics of the Abbott RealTime MTB RIF/INH Resistance, an assay for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis in pulmonary specimens.

Clinical management of drug-resistant tuberculosis patients continues to present significant challenges to global health. To tackle these challenges, the Abbott RealTime MTB RIF/INH Resistance assay was developed to accelerate the diagnosis of rifampicin and/or isoniazid resistant tuberculosis to within a day. This article summarizes the performance of the Abbott RealTime MTB RIF/INH Resistance assay; including reliability, analytical sensitivity, and clinical sensitivity/specificity as compared to Cepheid GeneXpert MTB/RIF version 1.0 and Hain MTBDRplus version 2.0. The limit of detection (LOD) of the Abbott RealTime MTB RIF/INH Resistance assay was determined to be 32 colony forming units/milliliter (cfu/mL) using the Mycobacterium tuberculosis (MTB) strain H37Rv cell line. For rifampicin resistance detection, the Abbott RealTime MTB RIF/INH Resistance assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Cepheid GeneXpert MTB/RIF. For isoniazid resistance detection, the assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Hain MTBDRplus. The performance data presented herein demonstrate that the Abbott RealTime MTB RIF/INH Resistance assay is a sensitive, robust, and reliable test for realtime simultaneous detection of first line anti-tuberculosis antibiotics rifampicin and isoniazid in patient specimens.

Interaction of nitric oxide with catalase: structural and kinetic analysis.

We present the structures of bovine catalase in its native form and complexed with ammonia and nitric oxide, obtained by X-ray crystallography. Using the NO generator 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, we were able to generate sufficiently high NO concentrations within the catalase crystals that substantial occupation was observed despite a high dissociation rate. Nitric oxide seems to be slightly bent from the heme normal that may indicate some iron(II) character in the formally ferric catalase. Microspectrophotometric investigations inline with the synchrotron X-ray beam reveal photoreduction of the central heme iron. In the cases of the native and ammonia-complexed catalase, reduction is accompanied by a relaxation phase. This is likely not the case for the catalase NO complex. The kinetics of binding of NO to catalase were investigated using NO photolyzed from N,N'-bis(carboxymethyl)-N,N'-dinitroso-p-phenylenediamine using an assay that combines catalase with myoglobin binding kinetics. The off rate is 1.5 s(-1). Implications for catalase function are discussed.

Techniques for investigating hydroxylamine disproportionation by hydroxylamine oxidoreductases.

Hydroxylamine, an important intermediate in ammonia oxidation by ammonia oxidizing bacteria (AOB), is inherently unstable with respect to disproportionation. The process is slow in neutral solutions, but could potentially be catalyzed by enzymes such as the hydroxylamine oxidoreductases, which normally catalyze the oxidation of ammonia to nitrite in the AOB. Disproportionation could be physiologically important to some AOB under microaerobic conditions, and could also confound in vitro analyses if it occurs and is not taken into consideration. This chapter presents methods for detecting ammonia, nitric oxide, nitrite, nitrous oxide, and isotopically labeled dinitrogen, which are the most thermodynamically favored products of hydroxylamine disproportionation.

Enzymatic interconversion of ammonia and nitrite: the right tool for the job.

Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea normally catalyzes oxidation of NH(2)OH to NO(2)(-). This paper reports experiments in which HAO was thermodynamically poised to catalyze reduction of NO(2)(-) to NH(4)(+). HAO was found to catalyze the reduction of NO(2)(-) by methyl viologen monocation radical (MV(red)), displaying a hyperbolic dependence on NO(2)(-) concentration, with a k(cat1) of 6.8 ± 0.3 s(-1) and a K(m1) of 7.6 ± 0.9 mM. HAO also catalyzed the reduction of NH(2)OH by MV(red), with a hyperbolic dependence on NH(2)OH concentration, and a k(cat2) of 245 ± 3 s(-1) and a K(m2) of 6.8 ± 0.2 mM (k(cat1) and k(cat2) reflect the maximum number of electrons transferred from MV(red) per second). We had previously demonstrated that HAO catalyzes the reduction of NO by MV(red) to yield first NH(2)OH and then NH(4)(+). Thus, overall, HAO is seen to act like a cytochrome c nitrite reductase, which catalyzes the six-electron reduction of NO(2)(-) to NH(4)(+) by MV(red). In the presence of Ru(NH(3))(2+) (Ru(II)) and Ru(NH(3))(3+) (Ru(III)) at ratios exceeding 200:1, HAO exhibited no detectable Ru(II)-NO(2)(-) oxidoreductase activity, though such activity is thermodynamically favored. On the other hand, HAO could still catalyze the oxidation of NH(2)OH to NO by Ru(III) under these conditions. The oxidative process exhibited a hyperbolic dependence on NH(2)OH concentration, with a k(cat3) of 98 ± 3 s(-1) and a K(m3) of 5.2 ± 0.8 µM. Finally, HAO was found not to catalyze the disproportionation of NH(2)OH, despite the thermodynamic favorability of such a process, and the apparent opportunity presented by the HAO structure. Mechanisms are proposed to explain all the kinetic data.

Kinetic and product distribution analysis of NO* reductase activity in Nitrosomonas europaea hydroxylamine oxidoreductase.

Hydroxylamine oxidoreductase (HAO) from the ammonia-oxidizing bacterium Nitrosomonas europaea normally catalyzes the four-electron oxidation of hydroxylamine to nitrite, which is the second step in ammonia-dependent respiration. Here we show that, in the presence of methyl viologen monocation radical (MV(red)), HAO can catalyze the reduction of nitric oxide to ammonia. The process is analogous to that catalyzed by cytochrome c nitrite reductase, an enzyme found in some bacteria that use nitrite as a terminal electron acceptor during anaerobic respiration. The availability of a reduction pathway to ammonia is an important factor to consider when designing in vitro studies of HAO, and may also have some physiological relevance. The reduction of nitric oxide to ammonia proceeds in two kinetically distinct steps: nitric oxide is first reduced to hydroxylamine, and then hydroxylamine is reduced to ammonia at a tenfold slower rate. The second step was investigated independently in solutions initially containing hydroxylamine, MV(red), and HAO. Both steps show first-order dependence on nitric oxide and HAO concentrations, and zero-order dependence on MV(red) concentration. The rate constants governing each reduction step were found to have values of (4.7 +/- 0.3) x 10(5) and (2.06 +/- 0.04) x 10(4) M(-1) s(-1), respectively. A second reduction pathway, with second-order dependence on nitric oxide, may become available as the concentration of nitric oxide is increased. Such a pathway might lead to production of nitrous oxide. We estimate a maximum value of (1.5 +/- 0.05) x 10(10) M(-2) s(-1) for the rate constant of the alternative pathway, which is small and suggests that the pathway is not physiologically important.

Laser photoinitiated nitrosylation of 3-electron reduced Nm europaea hydroxylamine oxidoreductase: kinetic and thermodynamic properties of the nitrosylated enzyme.

Hydroxylamine-cytochrome c554 oxidoreductase (HAO) catalyzes the 4-e(-) oxidation of NH(2)OH to NO(2)(-) by cytochrome c554. The electrons are transferred from NH(2)OH to a 5-coordinate heme known as P(460), the active site of HAO. From P(460), c-type hemes transport the electrons through the enzyme to a remote solvent-exposed c-heme, where cyt c554 reduction occurs. When 3-60 microM NO* are photogenerated by laser flash photolysis of N,N'-bis-(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine, in a solution containing approximately 1 microM HAO prereduced by 3 e(-)/subunit, the HAO c-heme pool is subsequently oxidized by up to 1 e(-)/HAO subunit. The reaction rate for HAO oxidation shows first-order dependence on [HAO], and zero-order dependence on [NO*] (k(obs) = 1250 +/- 150 s(-)(1)). However, the total HAO oxidized shows hyperbolic dependence on [NO*]. We suggest that NO* first binds reversibly to P(460) giving a {Fe(NO)}(6) moiety. Intramolecular electron transfer (IET) from the c-heme pool then reduces P(460) to {Fe(NO)}.(7) The overall binding constant (K) for formation of {Fe(NO)}(7) from free NO* and 3-e(-) reduced HAO was measured at (7.7 +/- 0.6) x10(4) M(-1). This value is larger than that for typical ferriheme proteins ( approximately 10(4) M(-1)), but much smaller than that for the corresponding ferroheme proteins ( approximately 10(11) M(-1)). The final product generated by nitrosylating 3-e(-) reduced HAO is believed to be the same species obtained by adding NH(2)OH to the fully oxidized enzyme. The experiments described herein suggest that when NH(2)OH and HAO first react, only two of the NH(2)OH electrons end up in the c-heme pool. The other two remain at P(460) as part of an {Fe(NO)}(7) moiety. These results are discussed in relation to earlier studies that investigated the effect of putting fully oxidized and fully reduced HAO under 1 atm of NO*.