College of American Pathologists
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  Clinical Abstracts






December 2007

Michael Bissell, MD, PhD

Neisseria meningitidis antibiotic susceptibility
Measuring testosterone using mass spectrometry
Effects of astrocyte-derived transforming growth factor-beta on estradiol
Value of serial B-type natriuretic peptides in acute coronary syndrome

bullet Neisseria meningitidis antibiotic susceptibility

Neisseria meningitides is a leading cause of bacterial meningitis and severe sepsis in the United States, other industrialized countries of the Americas and Europe, and the developing world. It is also a major cause of periodic epidemics in sub-Saharan Africa and areas of the Middle East. Resistance to some antimicrobial agents used as therapy for invasive infections or prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. The authors examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. The isolates were recovered between 1917 and 2004 and represented all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute's broth microdilution method using Mueller-Hinton lysed horse blood broth. A subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by broth and agar methods. Growth in broth was enhanced by CO2 incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79% to 100% essential agreement), and MICs also generally agreed closely (92% to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two atmospheres. Elevated penicillin and ampicillin MICs (>0.12 μg/mL and >0.25 μg/mL, respectively) occurred in 14.3 percent and 8.6 percent of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced -lactamase. Elevated tetracycline and doxycycline, but not minocycline, MICs were associated with efflux-mediated resistance encoded by tet(B) in 13 strains. Resistance to sulfisoxazole in 21.7 percent of strains and to trimethoprim-sulfamethoxazole in 21 percent resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene. Two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP. Two strains had reduced quinolone susceptibility due to mutations of gyrA. The determination of the susceptibilities of a large group of meningococcal strains, including strains with characterized resistance mechanisms, to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.

Jorgensen JH, Crawford SA, Fiebelkorn KR. Susceptibility of Neisseria meningitides to 16 antimicrobial agents and characterization of resistance mechanisms affecting some agents. J Clin Microbiol. 2005;43:3162-3171.

Reprints: James H. Jorgensen, Dept. of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900;

bullet Measuring testosterone using mass spectrometry

Measuring testosterone in serum or plasma is essential for investigating androgenic status and monitoring stimulatory, suppressive, or replacement therapy in children and adults of both sexes. In the late 1960s and 1970s, radioimmunoassay (RIA) methods became available for this purpose. Early methods incorporated solvent extraction and chromatographic purification before measurement by competitive RIA. These methods were well validated but, because of their complexity, were confined to specialist units. To address the rapidly increasing number of requests for testosterone measurements, high-throughput assays became necessary. They were first developed by modifying existing methods by discarding the chromatographic separation and, eventually, the solvent extraction step. Commercial nonextraction, nonisotopic methods became widely available as manual techniques in the 1980s and 1990s and, with the further development of excess reagent nonequilibrium immunoassays, as fully automated immunoassay platforms. This methodology has led to many complex analytes, including testosterone, being introduced to the repertoire of routine biochemistry laboratory tests, particularly because sample-volume requirements are low and throughput high. However, the omission of preimmunoassay purification steps has led to problems of anomalous high or low results. Therefore, the authors conducted a study to compare the results obtained by the stable isotope-dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) method with those obtained from their in-house solvent-extraction RIA. The authors used ID/LC-MS/MS to measure testosterone in plasma and serum. The sample volume was 50 μL in duplicate; preparation and analysis were carried out in a single tube; and a batch of 192 tubes was analyzed in 17.5 hours. Intra- and interassay imprecision was less than 15 percent in the range of 0.3 to 49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625 to 20 nmol/L was 96 percent (coefficient of variation, 12%; n=26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with the authors' extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L). However, they were 0.35 for samples from men containing less than 3 nmol/L testosterone and 0.77 for samples from women containing more than 8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak. The authors concluded that the ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.

Cawood ML, Field HP, Ford CG, et al. Testosterone measurement by isotope-dilution liquid chromatography-tandem mass spectrometry: validation of a method for routine clinical practice. Clin Chem. 2005;51:1472-1479.

Reprints: Marion L. Cawood, SAS Centre for Steroid Hormones (Leeds), Dept. of Clinical Biochemistry & Immunology, Leeds General Infirmary, Great George St., Leeds LS1 3EX, United Kingdom;

bullet Effects of astrocyte-derived transforming growth factor-beta on estradiol

The ovarian steroid hormone 17 -estradiol exerts a wide array of actions in the central nervous system. Although most work has focused on the hypothalamic control of reproduction, recent evidence suggests that 17 -estradiol (E2) may also influence diverse processes, such as the regulation of synaptic plasticity and neuroprotection. Although potentially beneficial, E2 possesses distinct limitations, including an increased risk of breast and uterine cancers, which may decrease its clinical utility. Because of such negative side effects, there is growing interest in the development and potential therapeutic use of SERMs (selective estrogen receptor [ER] modulators). SERMs are a class of steroidal or nonsteroidal drugs that may exert E2-like agonistic or antagonistic effects depending on the tissue type, ER isoforms present in the cell, and coactivators/corepressors recruited to the receptor-DNA complex. Tamoxifen (TMX), a first-generation SERM prescribed to approximately 700,000 women, is employed as an ER antagonist in treating E2-dependent breast cancer. However, TMX is also neuroprotective of ischemic stroke and methamphetamine toxicity in animal models and promotes synaptic plasticity in the hippocampus in a manner similar to E2, suggesting an agonistic function of TMX, and potentially other SERMs, in the brain. In contrast, antagonistic effects of TMX are reported in some parts of the CNS, implying the potential for cell type and regional specificity. Despite the well-documented role of E2 in neuroprotection and synaptic plasticity, the cellular and molecular mechanisms underlying these actions remain poorly understood. Astrocytes, the most abundant cell type in the brain, influence many of these same functions and thus may represent a mediator of estrogen action. The authors conducted a study to examine the regulatory effect and underlying cell signaling mechanisms of E2-induced release of neurotropic growth factors from primary rat cortical astrocyte cultures. The results revealed that E2 (0.5, 1, and 10 nM) and TMX (1 μM) increased the expression and release of the neuroprotective cytokines TGF- and TGF- 2 (TGF- ) from cortical astrocytes. The stimulatory effect of E2 was attenuated by the estrogen receptor antagonist ICI182,780, suggesting ER dependency. The effect of E2 also appeared to involve mediation by the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway, because E2 rapidly induced Akt phosphorylation, and pharmacological or molecular inhibition of the PI3K/Akt pathway prevented E2-induced release of TGF- . Additionally, the membrane-impermeant conjugate E2-BSA stimulated the release of TGF- , suggesting the potential involvement of a membrane-bound ER. Finally, E2, TMX, and E2-BSA were shown to protect neuronal-astrocyte cocultures from camptothecin-induced neuronal cell death, effects that were attenuated by ICI182,780, Akt inhibition, or TGF- immunoneutralization. The authors concluded that, as a whole, these studies suggest that E2 induction of TGF- release from cortical astrocytes could provide a mechanism of neuroprotection. They also show that E2 stimulation of TGF- expression and release from astrocytes occurs via an ER-dependent mechanism involving mediation by the PI3K/Akt signaling pathway.

Dhandapani KM, Wade FM, Mahesh VB, et al. Astrocyte-derived transforming growth factor- mediates the neuroprotective effects of 17 -estradiol: involvement of nonclassical genomic signaling pathways. Endocrinology. 2005;146:2749-2759.

Reprints: Dr. Darrell W. Brann, Institute of Molecular Medicine and Genetics, Program in Development Neurobiology, 1120 15th St., Medical College of Georgia, Augusta, GA 30912;

bullet Value of serial B-type natriuretic peptides in acute coronary syndrome

B-type natriuretic peptide is released from cardiac myocytes in response to increased wall stress and is elevated in settings in which myocardial ischemia occurs. It is released during ischemia provoked by exercise testing and after acute myocardial infarction, with some patients exhibiting a late increase, possibly related to adverse remodeling. When measured at presentation, up to seven days after the onset of acute coronary syndrome (ACS), blood concentrations of B-type natriuretic peptide (BNP) and N-amino terminal fragment of the prohormone BNP (NT-proBNP) are strongly associated with short- and long-term risk of death and new congestive heart failure (CHF). These data have led to the approval of BNP and NT-proBNP levels for risk stratification in ACS and to the proposal that serial determinations during followup may be useful for additional prognostic assessment and monitoring response to therapy. However, few studies have evaluated serial measurements of BNP during extended followup of patients after ACS. Therefore, the authors conducted a study to determine whether concentrations of BNP at study entry (prior to hospital discharge for ACS) and at outpatient followup at four and 12 months are associated with subsequent clinical outcomes. The authors investigated the prognostic value of BNP and changes in its concentration in a prospective observational substudy of 4,497 patients with non-ST-elevation or ST-elevation ACS who were enrolled in phase Z of the A to Z trial. The trial was conducted in 322 acute care hospitals in 41 countries between 1999 and 2003. The main outcome measure was death from any cause or new onset of CHF through two years. Levels of BNP were available for 4,266 patients at study entry, 3,618 patients at four months, and 2,966 patients at 12 months. The authors reported 230 deaths and 163 incident cases of CHF during followup. Adjusting for age, gender, index event, renal function, hypertension, prior heart failure, and diabetes, elevated levels of BNP (greater than 80 pg/mL) were associated with subsequent death or new CHF when measured at study entry (111 [21%] versus 246 [7%]; adjusted hazard ratio, 2.5; 95% confidence interval, 2.0-3.3), at four months (34 [19%] versus 125 [4%]; adjusted hazard ratio, 3.9; 95% confidence interval, 2.6-6.0), and at 12 months (19 [11%] versus 37 [1%]; adjusted hazard ratio, 4.7; 95% confidence interval, 2.5-8.9). Patients with newly elevated levels of BNP at four months were at increased risk of death or new CHF (10 [15%] versus 105 [3%]; hazard ratio, 4.5; 95% confidence interval, 2.3-8.6). Patients with elevated levels of BNP at study entry and with BNP levels lower than 80 pg/mL at four months tended to have only modestly increased risk (hazard ratio, 1.7; 95% confidence interval, 1.0-2.9) compared with patients with BNP levels lower than 80 pg/mL at both followup visits. The authors concluded that serial determinations of BNP levels during outpatient followup after ACS predict the risk of death or new CHF. Changes in BNP levels over time are associated with long-term clinical outcomes and may provide a basis for enhanced clinical decisionmaking in patients after the onset of ACS.

Morrow DA, de Lemos JA, Blazing MA, et al. Prognostic value of serial B-type natriuretic peptide testing during follow-up of patients with unstable coronary artery disease. JAMA. 2005;294:2866-2871.

Reprints: Dr. David A. Morrow, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115;

Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.