A survey of β-lactamase-producing Haemophilus influenzae an evaluation of 5750 isolates

Previous studies have reported that 15%–34% of Haemophilus influenzae produce β-lactamase. A surveillance program was developed by SmithKline Beecham Clinical Laboratories to determine the current percentage of β-lactamase-producing H. influenzae from five selected geographic locations in the United States. In 1993, results of 5750 isolates from specimens submitted to five reference clinical laboratories were evaluated. Data were collected from 29 states and the District of Columbia. The percentages of β-lactamase-producing H. influenzae was 33% and ranged from 22%–40% for the individual states.     

Characterization of the inoculum effect with Haemophilus influenzae and β-lactams

An inoculum effect is defined as a four-fold or greater increase in MIC with an increase in bacterial inocula. Haemophilus influenzae was tested for an inoculum effect with ampicillin, cefuroxime, and amoxicillin/clavulanate using the standard initial inocula (5 × 10⁵ CFU/mL) and a higher initial inocula (1 × 10⁷ CFU/mL). An inoculum effect was observed with both β-lactamase (TEM-1, ROB-1) positive and β-lactamase negative strains of H. influenzae when MICs were determined based on turbidity. MICs based on viable cell counts however, demonstrated that only β-lactamase positive strains of H. influenzae produced an inoculum effect. These observations suggest that MICs determined based on turbidity, using high initial inocula, are not reliable when examining the inoculum effect in H. influenzae. The magnitude of the inoculum effect with β-lactamase positive strains was β-lactam dependent (ampicillin > amoxicillin/clavulanate > cefuroxime). β-lactam kill-curves confirmed the aforementioned results. Addition of the β-lactamase inhibitor clavulanate completely reversed the inoculum effect in β-lactamase (TEM-1 and ROB-1) positive strains of H. influenzae with all β-lactams tested. Introduction of the β-lactamase gene TEM-1 on plasmid vector pLS88 into a β-lactamase negative strain of H. influenzae (Rd) produced an inoculum effect based on viable cell counts. In conclusion, our results suggest that the β-lactam inoculum effect demonstrated by H. influenzae is the result of β-lactamase production and is poorly assessed by turbidity.     

Determination of urinary free cortisol by liquid chromatography‐tandem mass spectrometry

Measurement of urinary free cortisol is clinically important in the diagnosis of Cushing's syndrome. While liquid chromatography (LC) with UV detection provides much better specificity than immunologic methods, certain drugs cause interference. Detection by mass spectrometry (MS) is a potentially superior method. Our analysis utilizes 1?mL urine spiked with 6‐α‐methylprednisolone as internal standard. The samples were extracted with dichlormethane and the extract was washed, evaporated to dryness and analyzed by LC‐MS/MS operating in the negative mode after separation on a reversed‐phase C18 column. The calibration curves for analysis of urinary cortisol exhibited consistent linearity and reproducibility in the range of 10–400?nmol/L. Inter‐assay CVs were 4.0–7.6% at mean concentrations of 21–153?nmol/L. The detection limit was 1?nmol/L (signal‐to‐noise ratio=3). The mean recovery of cortisol added to urine ranged from 67% to 87% and that of the internal standard from 71% to 76%. The regression equation for the LC‐MS/MS (x) and HPLC (y) methods was: y=1.095x+8.0 (r=0.996; n=111). Drugs known to interfere with UV detection did not cause problems here. The sensitivity and specificity of the MS/MS method for urinary free cortisol offer advantages over HPLC with UV detection by eliminating drug interference. The higher equipment costs in comparison with HPLC methods using UV detection are balanced by higher throughput, thanks to shorter chromatographic run times.     

Comparison of three commercial calibrators for alpha-tocopherol using liquid chromatography–tandem mass spectrometry

Objectives

Alpha-tocopherol is the predominant form of vitamin E in plasma and is routinely measured to assess vitamin E status. Agreement between vitamin E assays is essential to provide consistent result interpretation. Lack of agreement among calibrators is potentially a significant obstacle to method harmonization. The aim of this study was to ascertain the agreement between commercial secondary calibrators for analysis of serum/plasma alpha-tocopherol using two methods of isotope dilution liquid chromatography–tandem mass spectrometry (LC–MSMS).

Design and methods

Three commercial single level calibrators (Bio-Rad Laboratories, Chromsystems Diagnostics and RECIPE) were prepared in quintuplicate in conjunction with an in-house calibrator set for alpha-tocopherol. Samples were analyzed by two methods using an Agilent-6490 LC–MSMS.

Results

The linearity of both methods ranged from at least 4 to 70 μmol/L. The expanded within run imprecision of both LC–MSMS methods was ± 6% at 95.4% confidence interval across the assay range. The percentage observed difference for the commercial calibrators was calculated from the observed mean against the given value of the calibrator: Bio-Rad (bias + 1.4% in both methods); Chromsystems (bias + 5.4% [first method] and + 5.0% [second method]); and RECIPE (bias − 8.9% [first method] and − 9.8% [second method]).

Conclusions

Results demonstrate an overall discordance between the commercial calibrators that is greater than the assay uncertainty. It is observed that the greatest deviation of the three calibrators is traceable to a different standard reference material. This lack of harmonization means that results from different laboratories may not be comparable.     

Determination of β4-galactosyltransferase-7 activity using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Objectives

Patients with Ehlers-Danlos syndrome were described to contain reduced activities of β4-galactosyltransferase-7 (β4Gal-T7). Therefore, measurement of β4Gal-T7 activity can help to characterize defects in proteoglycan biosynthesis in patients with connective tissue diseases.

Design and methods

We developed a sensitive and specific method to assay β4Gal-T7 which is based on the transfer of galactose from UDP-galactose to the synthetic peptide Bio-BIK-F-Xyl.

Results

Calibration curves exhibited consistent linearity in the range of 10-2000 μg/L Bio-BIK-F-Xyl-Gal, corresponding to a β4Gal-T7 activity of 3.5-659 μU/L. The limit of detection and the lower limit of quantification were 3.70 μg/L (1.22 μU/L) and 4.50 μg/L Bio-BIK-F-Xyl-Gal (1.48 μU/L β4Gal-T7 activity), respectively. Interassay imprecision (CV) was 8.1-13.1% in the range from 15.9 to 659 μU/L, and mean recovery was 85.3% (range 61.7-106.3%).

Conclusions

This sensitive, robust and interference-free LC-MS/MS assay allows an accurate determination of β4Gal-T7 activity in human body fluids.     

Moraxella catarrhalis outer membrane vesicles carry β-lactamase and promote survival of Streptococcus pneumoniae and Haemophilus influenzae by inactivating amoxicillin

Moraxella catarrhalis is a common pathogen found in children with upper respiratory tract infections and in patients with chronic obstructive pulmonary disease during exacerbations. The bacterial species is often isolated together with Streptococcus pneumoniae and Haemophilus influenzae. Outer membrane vesicles (OMVs) are released by M. catarrhalis and contain phospholipids, adhesins, and immunomodulatory compounds such as lipooligosaccharide. We have recently shown that M. catarrhalis OMVs exist in patients upon nasopharyngeal colonization. As virtually all M. catarrhalis isolates are β-lactamase positive, the goal of this study was to investigate whether M. catarrhalis OMVs carry β-lactamase and to analyze if OMV consequently can prevent amoxicillin-induced killing. Recombinant β-lactamase was produced and antibodies were raised in rabbits. Transmission electron microscopy, flow cytometry, and Western blotting verified that OMVs carried β-lactamase. Moreover, enzyme assays revealed that M. catarrhalis OMVs contained active β-lactamase. OMVs (25 μg/ml) incubated with amoxicillin for 1 h completely hydrolyzed amoxicillin at concentrations up to 2.5 μg/ml. In functional experiments, preincubation of amoxicillin (10× MIC) with M. catarrhalis OMVs fully rescued amoxicillin-susceptible M. catarrhalis, S. pneumoniae, and type b or nontypeable H. influenzae from β-lactam-induced killing. Our results suggest that the presence of amoxicillin-resistant M. catarrhalis originating from β-lactamase-containing OMVs may pave the way for respiratory pathogens that by definition are susceptible to β-lactam antibiotics.     

High frequency of β-lactamase-negative, ampicillin-resistant strains of Haemophilus influenzae in patients with chronic bronchitis in Japan

In Japan, the increasing incidence of β-lactum-resistant Haemophilus influenzae infections is of growing concern. We retrospectively studied whether the prevalence of β-lactamase-negative ampicillin-resistant strains of H. influenzae was influenced by chronic lung diseases. H. influenzae isolates, obtained from patients who were diagnosed with acute or chronic bronchitis, or acute exacerbation of chronic bronchitis in 2005, were studied. In addition to susceptibility testing, polymerase chain reaction (PCR) was performed for the detection of TEM-1 β-lactamase, and Asn526-Lys and Ser385-Thr amino acid substitutions in the ftsI gene encoding penicillin-binding protein-3 (PBP-3). The minimum inhibitory concentration values of β-lactams were found to be increased in isolates from patients with chronic bronchitis who had been repeatedly administered antibiotics. Genetic analysis using PCR suggested that this might be associated with a high frequency of β-lactamase-negative strains with mutations in PBP-3. The presence of β-lactum-resistant strains needs to be considered for patients with chronic bronchitis in whom H. influenzae is isolated as a causative pathogen.     

Performance of plasma free metanephrines measured by liquid chromatography–tandem mass spectrometry in the diagnosis of pheochromocytoma

BackgroundPlasma free metanephrines have proved a highly sensitive biochemical test for the diagnosis of pheochromocytoma. We have developed and validated a simple, LC–MS/MS method to determine plasma metanephrines and compared the diagnostic efficacy of the method with an enzyme immunoassay procedure in 151 patients, 38 with histologically confirmed pheochromocytoma.MethodsOff-line solid phase extraction in a 96-well plate format was used to isolate metanephrines from 100-μL of plasma, followed by rapid separation with hydrophilic interaction chromatography. Mass spectrometry detection was performed in multiple-reaction monitoring mode using a tandem quadrupole mass spectrometer with positive electrospray ionization.ResultsDetection limits were < 0.1 nmol/l with method linearity up to 23.0 nmol/L for normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT). Method comparison with an automated LC–MS/MS yielded Deming regression slopes of r = 0.94 for NMN, r = 0.98 for MN and r = 0.94 for 3-MT. Method comparison with enzyme immunoassay revealed regression slope of r = 1.28 (NMN) and 1.25 (MN) with values approximately 25% lower than LC–MS/MS. Plasma metanephrines by LC–MS/MS identified all 38 patients with phaeochromocytoma compared with 36 cases by immunoassay.ConclusionsPlasma metanephrines measured by LC–MS/MS are a reliable and sensitive test for the biochemical detection of pheochromocytoma.     

Determination of free tetrahydrocortisol and tetrahydrocortisone ratio in urine by liquid chromatography‐tandem mass spectrometry

Objective. Measurement of urinary free tetrahydrocortisol and tetrahydrocortisone ratio (allo‐THF+THF)/THE is clinically important in the diagnosis of hypertension caused by congenital absence of 11β‐hydroxysteroid dehydrogenase type 2 (apparent mineralocorticoid excess, AME) or inhibition of the enzyme after licorice ingestion. Although gas chromatography‐mass spectrometry (GC‐MS) provides reliable results, it requires derivatization and is lengthy and time‐consuming. The purpose of this study was to demonstrate that detection by liquid chromatography‐mass spectrometry (LC‐MS) is a potentially superior method. Material and methods. The analysis utilizes 1 mL urine. The samples were extracted with solid‐phase extraction (SPE) using ethyl acetate as eluent. The extract was evaporated to dryness, and allo‐tetrahydrocortisol (allo‐THF), THF and THE concentrations were analyzed by LC‐MS/MS operating in the negative mode after separation on a reversed‐phase column. The calibration curves exhibited consistent linearity and reproducibility in the range of 7.5–120 nmol/L. Interassay CVs were 7.0–10 % at mean ratios of (allo‐THF+THF)/THE of 0.54–1.9. The detection limit of the analytes was 0.4–0.8 nmol/L (signal‐to‐noise ratio = 3). The mean recovery of the three analytes ranged from 88 to 95 %. The regression equation for the free ratio using the LC‐MS/MS (x) method and the total ratio using the GC‐MS (y) method was: y = 0.30x+0.91 (r = 0.61; n = 25). Conclusions. The sensitivity and specificity of the LC‐MS/MS method offer an advantage over GC‐MS by eliminating derivatization. The high costs of equipment are balanced by higher through‐put, owing also to shorter chromatographic run times.     

Simultaneous detection of 19 drugs of abuse on dried urine spot by liquid chromatography–tandem mass spectrometry

Background

The lack of specificity of immunoassays for drugs of abuse testing (DAT), and concerns over its cost in conjunction with reflex confirmatory tests prompted us to investigate the combinatorial use of dried urine spot (DUS) and LC–MS/MS as an alternative.

Methods

The method development and validation were performed in accordance with the guidelines published by FDA and CLSI.

Results

In this study we established and validated the precision, accuracy, and linearity of our DUS–LC–MS/MS method, and assessed the recovery, interference, and carryover as well. The linearity check for all 19 analytes demonstrated slopes between 0.94 and 1.04, and R² always greater than 0.99. Between-batch CV for QC at 4 difference levels ranged from 1.1% to 10%, where CV of LLOQ ranged from 1.2% to 12.8% and CV of ULOQ ranged from 0.8% to 5.1%. A concordance study with patient specimens between our method and GC–MS demonstrated 80.8% to 100% agreement. Stability of DUS specimens was assessed up to 30 days and the measured concentrations ranged from 94% to 114% of the 100 ng/mL urine calibrator used for this assessment.

Conclusions

We established and validated a DUS–LC–MS/MS method for DAT that conforms to the guidelines dictated by FDA, CLSI, and SAMHSA. While our method with high sensitivity and specificity provides an alternative diagnostic utility to EMIT immunoassays, it also offers superior solutions in specimen transportation, preservation, and storage. The benefits of our method are apparent in reducing turnaround time and test costs that result in better patient care.     

Quantitative analysis of powdered caffeine products purchased from the Internet using liquid chromatography–quadrupole time-of-flight mass spectrometry

Context: Powdered caffeine is sold on the Internet as a supplement. Severe toxicity and fatalities have been reported with use, but it is unclear if this toxicity was due to excessive dosing, mislabeled products, or adulterant stimulants. Our objective was to analyze the contents of commercially available powdered caffeine products in order to assess product purity and presence of additional ingredients, contaminants, or adulterants which may contribute to toxicity. Methods: A sample of nine powdered caffeine products was purchased from the Internet. Two sample replicates of each caffeine product were analyzed. Liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was used to identify and quantify substances in the purchased products and purity of the compounds were calculated. Results: Comparison of actual mass versus labeled mass of caffeine demonstrated a mean purity of 88.25% (SD 13.41%) and median purity of 90.1%. The products studied contained 1.6–5.3?g per teaspoon. Labeling on these products had limited instructions regarding how to measure the recommended dose. Conclusions: Powdered caffeine products that are readily available on the Internet contained relatively pure caffeine with no additional detected stimulants. High purity, small serving size, and lack of clear dosing instructions may place users at risk of toxicity.     

Molecular Organization of Small Plasmids Bearing blaTEM-1 and Conferring Resistance to β-Lactams in Haemophilus influenzae

TEM-1 is the dominant β-lactamase of Haemophilus influenzae and can be located on small plasmids. Three distinct plasmids with sizes from 4,304 to 5,646 nucleotides (nt) were characterized: pA1606, pA1209, and pPN223. In addition to TEM-1 and a replication enzyme of the Rep 3 superfamily, pA1606 carries a Tn3 resolvase gene and pA1606 and pA1209 carry an open reading frame (ORF) similar to a plasmid recombination enzyme gene described in Gram-positive bacteria. The plasmids transformed strain Rd to the ampicillin-resistant phenotype.     

High-resolution ultrasound imaging of human skin in vivo by using three-dimensional ultrasound microscopy

Observing the morphology of human skin is important in the diagnosis of skin cancer and inflammation and in the assessment of skin aging. High-frequency ultrasound imaging provides high spatial resolution of the deep layers of the skin, which cannot be visualized by optical methods. The objectives of the present study were to develop a three-dimensional (3-D) ultrasound microscope and to observe the morphology of normal human skin in vivo. A concave polyvinylidene fluoride transducer with a central frequency of 120 MHz was excited using an electric pulse generated by semiconductor switching. The transducer was scanned two-dimensionally by using two linear motors on the region-of-interest and the ultrasonic reflection was digitized with 2-GHz sampling. Consecutive B-mode images perpendicular to the skin surface were reconstructed to generate multiplanar reconstructed images and 3-D volume-rendering images clearly showing microstructures such as sebaceous glands and hair follicles. The 3-D ultrasound microscope could be used to successfully image the morphology of human skin noninvasively and may provide important information on skin structure.     

A rapid and fully-automated method for the quantitation of tricyclic antidepressants in serum using turbulent-flow liquid chromatography–tandem mass spectrometry

BackgroundWe describe a fully-automated turbulent-flow liquid chromatography–tandem mass spectrometry method for the detection of tricyclic antidepressant drugs (amitriptyline, desipramine, imipramine, and nortriptyline) in serum.MethodsHuman serum and an internal standard were injected directly onto a Cyclone-P online solid-phase extraction (SPE) column (0.5 × 50 mm). Following removal of serum proteins and other components the analytes were transferred to a Hypersil Gold C-18 (50 × 3 mm) analytical column. Elution occurred with a gradient of water and acetonitrile each with 0.1% formic acid. Analytes were ionized and detected over a 3.5 min analysis time by electrospray-ionization mass spectrometry with selected reaction monitoring (SRM). Matrix effects were well-characterized and carryover, precision, linearity, recovery and limits of detection and quantitation were evaluated.ResultsThe simple and complex precision CVs for all compounds were ≤ 16%. The limits of detection and quantitation for all drugs were ≤ 3 ng/ml and < 20 ng/ml, respectively. Recoveries were between 97 and 114%. Slopes for method comparison plots were all > 0.96. Proficiency testing materials had values within 2 SDI of peer group means for all drugs.ConclusionBased on validation data, this is a specific, sensitive fully-automated method for rapid quantitation of tricyclic antidepressants in serum.