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October 2003

Effect of ethnicity on reference ranges
While clinical chemists generally are aware of the need to consider race or ethnicity as a possible variable on which to subdivide an interval when setting up reference ranges, the effect of ethnicity on various analyte values has not been systematically studied. Data from the National Health and Nutrition Examination Survey, however, makes such a study possible. The Third National Health and Nutrition Examination Survey (NHANES III) contains data from 33,994 people age two months and older who participated in the survey. Whether to use separate reference intervals has been addressed by Harris and Boyd. Their recommendation, which is used by the NCCLS, is that two groups should be combined into a single group unless their means or standard deviations, or both, exceed appropriate predetermined thresholds. The authors used individuals who were known to be healthy as a gold standard and then determined how adding ethnicity affected these estimators. They used a previously described statistical outlier detection scheme to reduce the effect of atypical observations on these estimators. Taking advantage of the large numbers of patients in the NHANES III survey and previously described analytical procedures, the authors concluded that using the Harris and Boyd criteria indicates that separate reference intervals are warranted among the three ethnic groups considered: nonHispanic whites, nonHispanic blacks, and Mexican-Americans.

Horn PS, Pesce AJ. Effect of ethnicity on reference intervals. Clin Chem. 2002;48: 1802–1804.

Reprints: Paul S. Horn, Dept. of Mathematical Sciences, University of Cincinnati, Cincinnati, OH 45221-0025; paul.horn@uc.edu

Growing Trichomonas vaginalis on modified Columbia agar
The gold standard for culture of the parasitic protozoan Trichomonas vaginalis is broth culture. Diagnosis is based on direct microscopy, often using the saline wet-mount preparation to observe motile organisms. Various liquid culture media have been developed for broth culture of this organism; however, identifying positive samples typically requires frequent microscopic observations for up to seven days. The authors evaluated the suitability of a solid medium cultivation technique in the routine microbiology lab. They carried out two studies to evaluate modified Columbia agar (MCA) for isolating T. vaginalis from clinical samples. The first study looked at 889 vaginal samples and compared isolation on MCA to that on a liquid medium. Sixty-three of the samples were positive for T. vaginalis, of which MCA identified 62 (98.4 percent) and broth identified 58 (92.1 percent). In the second study, trichomoniasis was diagnosed within the scope of a screening program for 39,585 men and women by culture on MCA and direct microscopy. Culture on MCA detected 199 (98.5 percent) and Gram staining detected 163 (80.7 percent) of 202 positive specimens. Wet-mount preparations used for symptomatic patients identified 103 (92.8 percent) of 111 cases. The authors concluded that culture of T. vaginalis from clinical samples on MCA is highly sensitive and reliable and saves time.

Stary A, Kuchinka-Koch A, Teodorowicz L. Detection of Trichomonas vaginalis on modified Columbia agar in the routine laboratory. J Clin Microbiol. 2002;40:3277–3280.

Reprints: Angelika Stary, Outpatients’ Center for Diagnosis of Infectious Venerodermatological Diseases, Franz-Jonas-Platz 8/2/3, A-1210 Vienna, Austria; angelika.stary@univie.ac.at

Bacterial quinolone mediated by plasmids
Bacterial resistance to quinolone antibiotics arises by mutations in the chromosomal genes for type II topoisomerases, the targets of quinolone action, and by changes in expression of efflux pumps and porins that control the accumulation of the agents inside the bacterial cell. Transmissible resistance was recently discovered on a plasmid from a resistant clinical isolate. This plasmid augments resistance to nalidixic acid, ciprofloxacin, and other fluoroquinolones 4- to 8-fold and supplements resistance due to gyrA, gyrB, and porin or efflux pump mutations. The authors reported the cloning of Qnr, a novel plasmid-encoded quinolone resistance gene, the amplification and purification of Qnr, its gene product, and the demonstration that Qnr directly protects Escherichia coli DNA gyrase from quinolone inhibition. The gene product Qnr was a 218 amino-acid protein belonging to the pentapeptide repeat family and shared sequence homology with the immunity protein McbG, which is thought to protect DNA gyrase from the action of microcin B17. The authors tested the ability of Qnr to reverse the inhibition of gyrase activity by quinolones. Gyrase protection was proportional to the concentration of Qnr-His6 and inversely proportional to the concentration of ciprofloxacin. The protective activity of Qnr-His6 was lost by boiling the protein and involved neither quinolone activation nor independent gyrase activity. The exact mechanism of how Qnr protects DNA gyrase and the prevalence of this resistance mechanism in clinical isolates remains to be determined.

Tran JH, Jacoby GA. Mechanism of plasmid-mediated quinolone resistance. Proc Natl Acad Sci. 2002;99: 5638–5642.

Reprints: George A. Jacoby, Lahey Clinic, 41 Mall Rd., Burlington, MA 01805; george.a. jacoby@ lahey.org

Measurement of holo-transcobalamin
The majority of vitamin B12, or total cobalamin, in plasma is bound to two carrier proteins—transcobalamin (TC) and haptocorrin (HC). TC is essential for transporting cobalamin from the intestine to most cells of the body. Cobalamin deficiency eventually develops in patients lacking this protein. TC carries a third of the circulating cobalamin (holo-transcobalamin), but the major portion of this protein is present in its unsaturated form (apo-TC). Recently, improved measurements specifically for the holo-TC fraction have been developed, but there is some discussion about whether these measurements can help in the elucidation of cobalamin metabolism. The authors studied the effect of oral vitamin B12 treatment on fluctuations in plasma total cobalamin and its binding proteins in patients who were not suffering from vitamin B12 deficiency. They studied 88 patients from 38 to 80 years old who were undergoing coronary angiography. These subjects were randomized into four groups: daily oral treatment with vitamin B12 (0.4 mg), folic acid (0.8 mg), and vitamin B 6 (40 mg); treatment with vitamin B12 and folic acid; treatment with vitamin B6; and placebo. Blood was obtained from each group of patients before treatment and at three, 14, 28, and 84 days thereafter. In the patients treated with vitamin B12, the maximum change in concentrations was already observed after three days for total TC (–16 percent), holo-TC (54 percent), and TC saturation (82 percent). Holo-HC (20 percent) and plasma-TC (28 percent) showed an initial burst but had increased further at 84 days. All of these changes were highly significant compared with the changes in the control group, (P<0.0001). The authors concluded that holo-TC can be regarded as an early marker of changes in cobalamin homeostasis.

Nexo E, Hvas AM, Bleie Ø, et al. Holo-transcobalamin is an early marker of changes in cobalamin homeostasis. A randomized placebo-controlled study. Clin Chem. 2002;48: 1768–1771.

Reprints: Ebba Nexo, Dept. of Clinical Biochemistry, AKH, Aarhus University Hospital, Norrebrogade 44, DK 8000 Aarhus C. Denmark; E.Nexo@dadlnet.dk