Certification of vitamins and carotenoids in SRM 3280 multivitamin/multielement tablets.

A new multivitamin/multielement dietary supplement Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST), with certified and reference concentration values for 13 vitamins, 24 elements, and 2 carotenoids. The constituents have been measured by multiple analytical methods with data contributed by NIST and by collaborating laboratories. This effort included the first use of isotope dilution mass spectrometry for value assignment of both fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs). Excellent agreement was obtained among the methods, with relative expanded uncertainties for the certified concentration values typically ranging from <2% to 15% for vitamins.

Total selenium and selenomethionine in pharmaceutical yeast tablets: assessment of the state of the art of measurement capabilities through international intercomparison CCQM-P86.

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg(-1) SeMet) was provided. Both high-performance liquid chromatography (HPLC)-ICP-MS or gas chromatography (GC)-ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg(-1) (n = 13, with a standard deviation of 9.7 mg kg(-1)) and 561.5 mg kg(-1) (n = 11, with a standard deviation of 44.3 mg kg(-1)) with median values of 337.6 and 575.0 mg kg(-1) for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg(-1) Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.

Novel piperidinyloxy oxazolidinone antimicrobial agents.

Oxazolidinone antibacterial agents, where the N-substituted piperazinyl group of eperezolid was replaced with a N-substituted piperidinyloxy moiety, were synthesized and shown to be active against a variety of resistant and susceptible Gram-positive organisms. The effect of ring size, positional isomerism, and fluorine substitution on antibacterial activity was examined.

Inhibitors of bacterial two-component signalling systems.

Bacterial two-component regulatory systems (TCS) play a pivotal role in the process of infection. These signal transduction systems enable bacterial pathogens to mount an adaptive response and cope with diverse environmental stresses, including nutrient deprivation, antibiotic onslaught and phagocytosis. Interest in these systems as novel bacterial targets has been rekindled by the recent discovery of several essential systems in important Gram-positive and Gram-negative pathogens. Several series of TCS inhibitors derived from broad screening approaches have been reported in the literature, however, most appear to suffer from poor selectivity, excessive protein binding and/or limited bioavailability. Consequently, pharmaceutical chemists have turned to alternate strategies, such as the design of substrate-based inhibitors, the generation of combinatorial libraries and the isolation of natural products, to identify inhibitors with more desirable properties. Recent structural studies of the histidine protein kinase and response regulator proteins that constitute TCS may provide a foundation for a structure-based design approach to TCS inhibitors.

Bacteremic pneumonia due to multidrug-resistant pneumococci in 3 patients treated unsuccessfully with azithromycin and successfully with levofloxacin.

Three patients with bacteremic pneumonia caused by multidrug-resistant Streptococcus pneumoniae were treated unsuccessfully with azithromycin. One S. pneumoniae isolate carried a mef determinant for an efflux pump; a second isolate had an erm determinant. All 3 patients were successfully treated with levofloxacin, an antipneumococcal fluoroquinolone.

Seroprevalence of Kaposi's sarcoma-associated herpesvirus in healthy adults in Israel.

To determine if Kaposi's sarcoma-associated herpesvirus (KSHV) prevalence is correlated with the 9-fold difference in the incidence of classic Kaposi's sarcoma observed among Israeli Jewish populations, we conducted a cross-sectional KSHV seroprevalence survey in a population of 166 HIV-seronegative healthy subjects from the general population (26 women, 140 men). Eight individuals (4.8%) (all men) were seropositive for KSHV; differences between men and women were not statistically significant. If we consider the sensitivity and specificity of the assays, the corrected prevalence would be 6.1% (95% confidence interval 2.0-10.1). We noticed a non-statistically 5.5-fold difference between individuals above and below 40 years of age, but did not find an association with the incidence of classic KS among the Israeli Jewish sub-population, according to their origin. This suggests that KSHV is only necessary, albeit not sufficient, cause of classic Kaposi's sarcoma.

The diagnosis and management of ductal carcinoma in-situ of the breast.

The widespread utilization of screening mammography has produced a shift in the stage of breast cancer at diagnosis in the US: Currently, 12% to 15% of newly diagnosed breast cancer cases annually are ductal carcinoma in-situ (DCIS). The diagnosis is made, in at least 90% of patients, with mammography. Only about 10% of patients will have a palpable mass. The accurate characterization and visualization of calcifications typically requires magnification of mammographic imaging. The morphology of the calcifications is generally considered to be the most important factor in differentiating benign from malignant formations. Round and uniform shapes are more likely to be benign, while linear and heterogeneous morphologies are associated with DCIS. Following a complete mammographic work-up, most suspicious lesions are potential candidates for a stereotactic core needle biopsy. Ten percent to 50% of patients initially diagnosed with atypical ductal hyperplasia by needle biopsy have subsequently been surgically diagnosed with cancer near the biopsy site. Due to this relatively high incidence of co-existent carcinoma, a needle biopsy diagnosis of atypical ductal hyperplasia necessitates subsequent surgical excision. The most important change in our thinking about DCIS was from a monolithic view, conceiving of DCIS as a single disease highly likely to invade if left untreated, to the realization that DCIS represents a non-obligate precursor with a variable risk of progression, depending on a combination of factors, such as histology, lesion, size, and margin status. In discussing treatment options, patients should understand that local recurrence following total mastectomy is rare and that this is the procedure of choice for disease that cannot be adequately encompassed with a breast-conserving approach. If the patient and her surgeon are in agreement about proceeding with a breast-conserving approach, there needs to be a clear understanding of the incidence and implications of local recurrence. In all such discussions with newly diagnosed patients, however, it is essential to emphasize the excellent prognosis with this disease, irrespective of the surgical approach.

Post-source decay in the analysis of polystyrene by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Various secondary series are observed in matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra of polystyrene. The number and positions of the series depend on the choice of matrix and added cation. For a given treatment, series observed in linear mode are not necessarily observed in reflectron mode, and vice versa. Post-source decay analysis was used to determine that the secondary series arise at least in part from formation and decay of adducts of polystyrene with matrix species. There is some treatment-to-treatment variation, but adduct formation and decay were observed for all tested treatments. The multiplicity of secondary series makes it unclear whether post-source decay occurs for the main series (polystyrene + cation)+ ions under the conditions normally used for polystyrene analysis. Such ions do undergo post-source decay at laser fluences greater than normally used. Although only polystyrene was investigated in this work, other polymers may also produce adduct and decay series in MALDI analysis. Their presence can mask the presence of minor components in a sample, but at least as observed here, do not have a strong influence on molecular mass determinations.

Effectiveness of fluoroquinolones against gram-positive bacteria.

Fluoroquinolones are broad-spectrum and therapeutically effective antibacterial agents that have retained high activity against methicillin-susceptible Staphylococcus aureus (MSSA) and streptococci. Primary targets of these agents in Gram-positive bacteria are DNA topoisomerase and DNA gyrase. High-level resistance is associated with at least two mutations in either target, or combinations of at least two mutations in multiple targets, particularly affecting the ParC subunit of topoisomerase IV and the GyrA subunit of DNA gyrase. This resistance may be enhanced when combined with mutations that increase fluoroquinolone efflux. Data from fluoroquinolone-resistant clinical isolates suggest that as yet unidentified mutations may also be involved in clinical resistance.

Is experience with human immunodeficiency virus disease related to clinical practice? A survey of rural primary care physicians.

BACKGROUND: Human immunodeficiency virus (HIV) disease is spreading to the rural United States, and medical care is increasingly provided by local primary care physicians. A volume-outcome relationship might exist in HIV care. However, little is known about the HIV experience and practices of rural primary care physicians. OBJECTIVES: To estimate the HIV experience of rural primary care physicians, and to determine whether experience is associated with use of newer management strategies, confidence in care, and consultation needs. DESIGN: Telephone survey of a random sample of primary care physicians. SETTING: Primary care sites in nonmetropolitan California. PARTICIPANTS: One hundred twenty eligible primary care physicians in nonmetropolitan California, with 102 respondents (85.0%). MAIN OUTCOME MEASURES: Physicians' HIV experience, use of protease inhibitors and viral load tests, familiarity with vertical HIV transmission prophylaxis, confidence in HIV care, and consultation needs. RESULTS: Most physicians were low-volume providers of HIV care and had limited knowledge of newer management strategies. Experience with protease inhibitors and viral load tests was significantly related to number of recent patients with HIV; 25.0% of those with 1 to 3 patients but 75.0% of those with 4 or more patients had prescribed protease inhibitors (P = .01), whereas 20.8% of those with 1 to 3 patients but 83.3% of those with 4 or more patients had used a viral load test (P = .001). Only 59.8% of all respondents, but 100.0% of those with 4 or more patients, were familiar with vertical HIV transmission prophylaxis (P = .001). After adjustment for other characteristics, HIV experience remained significantly associated with use of newer management strategies (P = .01) and familiarity with vertical HIV transmission prophylaxis (P = .007). Physicians' confidence in HIV care increased with experience (P = .006), and consultation needs decreased (P = .006). CONCLUSIONS: Primary care physicians in rural California lacked in-depth experience with HIV disease. Experience was significantly associated with use of newer HIV management strategies, confidence, and consultation needs. Treating 4 or more patients with HIV or acquired immunodeficiency syndrome may be the threshold above which primary care physicians rapidly adopt new strategies and have confidence in their care.

Multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems.

Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 microM and MICs ranging from 0.5 to >16 microg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4x MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 microg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

Sentinel lymphadenectomy for breast cancer: experience with 180 consecutive patients: efficacy of filtered technetium 99m sulphur colloid with overnight migration time.

BACKGROUND: Axillary node status remains the most important prognostic indicator of survival in breast cancer patients. Only 25% to 35% of patients having standard level I/II axillary dissection have involved nodes, yet all accept the potential for morbidity after the operation. This study was conducted to assess whether status of the sentinel node(s) was an accurate predictor of the presence of metastatic disease in axillary or internal mammary nodes. STUDY DESIGN: In 180 patients, technetium 99m sulphur colloid was injected in a 4-quadrant peritumoral distribution. During the first phase of the study, 72 patients had sentinel node excision followed by a level I/II axillary dissection. During the second phase of the study, 108 patients had sentinel node excision and only those with positive nodes had completion axillary dissection. Nodes were examined after formalin fixation by taking 10 sections at 20-microm intervals and staining with hematoxylin-eosin. RESULTS: Sentinel nodes were found in 162 (90%) of 180 patients. The mean number of sentinel nodes examined was 3.1. Of the 162 patients with successful lymphatic mapping, positive sentinel nodes were found in 44 (27%). In 23 (66%) of 35 patients with positive sentinel nodes who had a completion level I/II axillary dissection, the sentinel nodes were the only positive nodes. The concurrent negative predictive value was 4% in the first 72 patients who had completion axillary dissection after sentinel node excision, and 2% for the entire series. With evolution of technique, identification of sentinel nodes with radiolabeled colloid was successful in 97% of the last 100 patients. CONCLUSIONS: Because the concurrent negative predictive value was low, sentinel node excision appeared to accurately identify node status, potentially avoiding the need for standard level I/II axillary dissection in sentinel node-negative patients.

Resolving the common clinical dilemmas of syphilis.

The diagnosis and treatment of syphilis can present difficult dilemmas. Serologic tests can be negative if they are performed at the stage when lesions are present, and the VDRL test can be negative in patients with late syphilis. Cerebrospinal fluid examination is not required in patients with primary or secondary disease and no neurologic signs or symptoms, but it may be warranted in patients with late latent syphilis or in whom the duration of infection is unknown. Patients with penicillin allergy can be treated with alternative regimens if they have primary or secondary syphilis. Penicillin is the only effective drug for neurosyphilis; oral desensitization should be accomplished before treatment of penicillin-allergic patients. Other dilemmas may be encountered in the treatment of patients who have concurrent human immunodeficiency virus infection.