College of American Pathologists
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  Clinical Abstracts




May 2008

Michael Bissell, MD, PhD, MPH

Effects of alcohol wipes on blood alcohol level
Individualizing cyclosporine dosage in transplant patients
Microbial exposure and asthma predilection
Thalassemia in patients with microcytosis who had hemoglobinopathy assessed
Draft genome sequence of Trichomonas vaginalis
RovA: a pathogenetic factor for bubonic plague
The hemagglutinin from an H5N1 influenza virus

Effects of alcohol wipes on blood alcohol level Effects of alcohol wipes on blood alcohol level

Ethanol-based hand sanitizers are used in most health care facilities in the United States. Infection control personnel advocate the use of generous quantities of such sanitizers before and after contact with patients. This translates to using these products 10 to 25 times per hour, depending on the task being performed. Although it is assumed that little systemic absorption of ethanol occurs while using hand sanitizers, many alcohols are absorbed to varying degrees via the transdermal route. Because dermal absorption of alcohols has been demonstrated in a previous study, and ethanol intoxication by employees in the medical workplace is a potentially serious finding, it is of forensic and medicolegal importance to elucidate how frequent use of ethanol-based hand sanitizers affects serum blood ethanol levels. To investigate this, the authors studied five volunteers who frequently applied such sanitizers. The subjects applied 5 mL of a product (62% denatured ethyl alcohol manufactured by Kimberly—Clark) to both hands and rubbed until dry. This was repeated 50 times over four hours. Participants had their blood drawn before and after completing the study. Each participant was not exposed to alcohol during the 12 hours preceding the study. All of the subjects had an initial blood ethanol level of less than 5 mg/dL and all completed the four-hour study. No adverse reactions were noted during the study. Blood ethanol level on completing the 50 applications of hand sanitizer was less than 5 mg/dL in the five participants. The authors concluded that frequent use of ethanol-based hand sanitizers in accordance with their labeling does not raise serum blood ethanol levels.

Miller MA, Rosin A, Levsky ME, et al. Does the clinical use of ethanol-based hand sanitizer elevate blood alcohol levels? A prospective study. Am J Emerg Med. 2006;24:815–817.

Reprints: Dr. Michael A. Miller, C.R. Darnall Army Medical Center, 36000 Darnall Loop, Ft. Hood, TX 76544;
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Individualizing cyclosporine dosage in transplant patients Individualizing cyclosporine dosage in transplant patients

Cyclosporine has a narrow therapeutic index, and there is substantial evidence supporting a concentration effect/toxicity relationship. Cyclosporine is characterized by large pharmacokinetic variability, with a 12-fold inter-individual variability in absorption and a seven-fold variability in clearance. Although an association between rejection rates and cyclosporine area under the curve (AUC) has been established, considerable effort has been applied to finding a surrogate metric for cyclosporine AUC that is logistically feasible and effective. Limited sampling strategies and computer modeling tools using Bayesian techniques are well suited to addressing this problem and have been reported. Limited sampling strategies provide an estimate of cyclosporine AUC after collecting two or more blood samples. However, sampling strategies developed using stepwise regression approaches allow no flexibility in sample timing, are generally considered to be inconvenient for the patient, and may not predict AUC accurately. The authors conducted a study to develop and validate a Bayesian decision support tool for cyclosporine for stable heart transplant recipients who received metabolic inhibitors. The tool was developed using published pharmacokinetic information implemented using Abbott­base pharmacokinetic software. The model was evaluated in 47 stable heart transplant recipients who received concomitant administration of drugs known to inhibit cyclosporine metabolism—that is, diltiazem, ketoconazole, and a combination of diltiazem and ketoconazole. A three-point feedback strategy with samples collected at zero, one, and two hours after an oral cyclosporine dose was used to predict area under the concentration-time curve in the first 12 hours post-dose (AUC0-12). In group A, patients who received cyclosporine alone showed a good correlation between observed and model-predicted cyclosporine AUC0-12: r2=0.871; P<0.001; precision, 12 percent; accuracy, 13 percent. Furthermore, the Bayesian model provided acceptable predictions in patients who received cyclosporine with metabolic inhibitors. Group B (diltiazem) was r2=0.791; P<0.001; precision, 19 percent; accuracy, 22 percent. Group C (ketoconazole) was r2=0.761; P<0.001; precision, four percent; accuracy, 12 percent. Group D (diltiazem plus ketoconazole) was r2=0.818; P<0.001; precision, 14 percent; accuracy, 17 percent. The authors concluded that the Bayesian decision support tool described can predict cyclosporine AUC0-12 in a cohort of patients with variable cyclosporine absorption who received metabolic inhibitors. Bayesian modeling offers a number of advantages over single-point metrics that are used to adjust cyclosporine dose and may further refine cyclosporine therapy.

Ray JE, Keogh AM, McLachlan AJ. Decision support tool to individualize cyclosporine dose in stable, long-term heart transplant recipients receiving metabolic inhibitors: overcoming limitations of cyclosporine C2 monitoring. J Heart Lung Transplant. 2006; 25: 1223– 1229.

Reprints: John E. Ray, Dept. of Clinical Pharmacology and Toxicology, St. Vincent’s Hospital, Victoria St., Darlinghurst, Sydney, NSW 2010, Australia;
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Microbial exposure and asthma predilection Microbial exposure and asthma predilection

It has been suggested that early life exposures to microbial agents might prevent the development of atopy and asthma. Various studies have reported a reduced risk of atopy, hay fever, and asthma in farmers’ child­ren and adolescents with suspected high bacterial endotoxin exposures. Other studies in rural and nonrural environments reported a significant inverse association between indoor endotoxin levels and atopic sensitization, hay fever, and atopic asthma. It has also been demonstrated that atopy is associated with a genetic polymorphism for CD14, the endotoxin receptor on monocytes and other inflammatory cells. The latter, however, was not confirmed in other studies in different populations. Finally, animal studies have demonstrated that endotoxin might protect against atopy and asthma. The mechanisms underlying these protective effects are not clear but might be related to upregulation of TH1 and downregulation of TH2 lymphocyte immunity. The authors assessed the role of indoor endotoxin, (1-3)-β-D-glucans, and extracellular polysaccharides from the genera Penicillium and Aspergillus (EPS-Pen/Asp) on the development of atopy and asthma symptoms in the first four years of life of children with atopic mothers in the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) birth cohort study. They also assessed the same associations for crude house dust levels. Endotoxin, fungal (1-3)-β-D-glucans, EPS-Pen/Asp, and dust from living room floors were measured in subjects at three months of age. Serum IgE levels against common allergens were determined at one and four years, and questionnaire information about respiratory morbidity was collected annually. Microbial levels in mattresses were low and not associated with serum IgE levels, doctor-diagnosed asthma, and wheeze. Floor levels of biocontaminants and dust were inversely associated with doctor-diagnosed asthma, being most pronounced for endotoxin (odds ratio, 0.40; 95% confidence interval, 0.21–0.77) and EPS-Pen/Asp (OR, 0.42; 95% CI, 0.18–0.99). Mutual adjustment for other exposures did not significantly alter the results for endotoxin and only moderately affected the results for EPS-Pen/Asp. Persistent wheeze was also consistently less common in the high-exposure group, being significant only for EPS-Pen/Asp (OR, 0.37; 95% CI, 0.15–0.96). Transient wheeze and wheeze in the past 12 months were also reduced, but effects were smaller and not significant. Relationships with serum-specific IgE levels, which could only be assessed in 41 percent of the subjects at age four years, were less pronounced and were statistically significant only for EPS-Pen/Asp. The authors concluded that early exposure to common microbial contaminants, including fungal agents, might protect against asthma and constitute a novel target for prevention.

Douwes J, van Strien R, Doekes G, et al. Does early indoor microbial exposure reduce the risk of asthma? The Prevention and Incidence of Asthma and Mite Allergy birth cohort study. J Allergy Clin Immunol. 2006;117:1067–1073.

Reprints: Dr. Bert Brunekreef, Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80178, 3508TD, Utrecht, Netherlands;
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Thalassemia in patients with microcytosis who had hemoglobinopathy assessed Thalassemia in patients with microcytosis who had hemoglobinopathy assessed

Thalassemia syndromes are among the most common inherited abnorm­al­ities worldwide. Clinical symptoms vary from mild microcytic anemia in heterozygous thalassemia trait to life-threatening anemia in homozygous or compound heterozygous thalassemia major. Thalassemia is caused by genetic abnormalities of the <- or ®-globin genes of hemoglobin A, resulting in decreased production of the corresponding <- or ®-globin chains of the hemoglobin A molecule. Abnormalities fall into the broad categories of gene deletions that eliminate the whole gene, mutations within the gene that decrease globin production, and variant hemoglobin that is produced in decreased amounts. A-thalassemia, which is common in people who are Southeast Asian, African, Middle Eastern, and Mediterranean, is usually caused by a deletion of one or more of the four inherited ®-globin genes on chromosome 16 («/«). ®-thalassemia is common in people who are Mediterranean, Arab, Afri­can, Indian, and Southeast Asian. It usually results from mutations within the ®-globin gene that decrease ®-globin production. The two inherited ®-globin genes are found on chromosome 11. In Ontario, Canada, ®-thalassemia is easily detected through measurement of hemoglobin A2, but most laboratories do not do exhaustive DNA investigations for ®-thalassemia. Therefore, the prevalence of thalassemia in microcytic samples for hemoglobinopathy investigation in Ontario is unknown. To address this, the authors performed a prospective cohort study in which samples referred for hemoglobinopathy investigation were also evaluated for ®-thalassemia by DNA testing. Of 800 samples submitted, 664 were evaluable. Of the 664 patients represented, 163 (24.5%) were ®-thalassemia major carriers, 68 (10.2%) were hemoglobin Bart’s hydrops fetalis carriers, and 361 (54.4%) had some form of thalassemia. The authors concluded that microcytosis due to thalassemia is common in Ontario, and that major forms of thalassemia, including forms predisposing to hemoglobin Bart’s hydops fetalis and ®-thalassemia major, are frequent. This illustrates the importance of adequate prenatal and laboratory investigation for these abnormalities in Ontario and other similar multi-ethnic jurisdictions worldwide.

Lafferty JD, Barth DS, Sheridan BL, et al. Prevalence of thalassemia in patients with microcytosis referred for hemoglobinopathy investigation in Ontario: a prospective cohort study. Am J Clin Pathol. 2007; 127: 192– 195.

Reprints: Dr. Mark A. Crowther, Room L208, St. Joseph’s Hospital, 50 Charlton Ave. East, Hamilton, Canada L8N 4A6
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Draft genome sequence of Trichomonas <em>vaginalis</em> Draft genome sequence of Trichomonas vaginalis

Trichomonas vaginalis is a flagellated protist that causes trichomoniasis, a common but overlooked sexually transmitted human infection, with approximately 170 million cases occurring annually worldwide. The extracellular parasite resides in the urogenital tract and can cause vaginitis in women and urethritis and prostatitis in men. Acute infections are associated with pelvic inflammatory disease, increased risk of HIV-1 infection, and adverse pregnancy outcomes. T. vaginalis is a member of the parabasalid lineage of microaerophilic eukaryotes that lack mitochondria and peroxisomes but contain unusual organelles called hydrogenosomes. Although previously considered to be one of the earliest branching eukaryotic lineages, recent analyses leave the evolutionary relationship of parabasalids to other major eukaryotic groups unresolved. The authors reported the draft sequence of T. vaginalis, the first parabasalid genome to be described. The T. vaginalis genome sequence, generated using whole-genome shotgun methodology, contains 1.4 million shotgun reads assembled into 17,290 scaffolds at approximately 7.2∞ coverage. At least 65 percent of the T. vaginalis genome is repetitive. Despite several procedures developed to improve the assembly, the superabundance of repeats resulted in a highly fragmented sequence, preventing investigation of T. vaginalis genome architecture. The repeat sequences also hampered measurement of genome size, but the authors estimate it to be approximately 160 Mb. A core set of approximately 60,000 protein-coding genes was identified, endowing T. vaginalis with one of the highest coding capacities among eukaryotes. Introns were identified in 65 genes, including the approximately 20 previously documented. Transfer RNAs for all 20 amino acids were found, and approximately 250 ribosomal DNA units were identified on small contigs and localized to one of the six T. vaginalis chromosomes. Repeats and transposable elements comprise about two-thirds of the approximately 160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins, may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which supports a common evolutionary origin of this unusual organelle with mitochondria.

Carlton JM, Hirt RP, Silva JC, et al. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science. 2007;315:207–212.

Reprints: Jane M. Carlton, Institute for Genomic Research, 9712 Medical Research Drive, Rockville, MD 20850;
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RovA: a pathogenetic factor for bubonic plague RovA: a pathogenetic factor for bubonic plague

Yersinia pestis is the etiological agent of plague, an acute zoonotic infection that is often fatal in humans. Plague is primarily a disease afflicting rodent populations, and bacteria are usually transmitted to human hosts by the bite of an infected flea. Y. pestis can rapidly disseminate from a subcutaneous infection site into the lymphatic system and regional lymph nodes. The swelling of these infected lymph nodes into characteristic buboes is the classic symptom of bubonic plague. Infection can progress into the circulatory system, resulting in a systemic disease that can lead to colonization of a variety of tissues, including the lungs. Development of secondary pneumonic infection poses a significant health risk as Y. pestis becomes highly transmissible during patient coughing. Aerosolized bacteria can be easily inhaled, causing a primary pneumonic infection, a serious and rapidly fatal disease, in a new host. Systemic and pneumonic plague result in high mortality rates because of rapid proliferation of bacteria and quick onset of disease pathology. Y. pestis is a highly virulent pathogen because of its ability to escape the host immune system and rapidly proliferate within host tissues. The pathogenic species of Yersinia contain the transcriptional regulator RovA. In Y. pseudotuberculosis and Y. enterocolitica, RovA regulates expression of the invasion factor invasin (inv), which mediates translocation across the intestinal epithelium. A Y. enterocolitica rovA mutant has a significant decrease in virulence by LD50 analysis and an altered rate of dissemination compared with wild type or an inv mutant, suggesting that RovA regulates multiple virulence factors. The authors showed the involvement of RovA in the virulence of Y. pestis, which naturally lacks a functional inv gene. A Y. pestis ΔrovA mutant is attenuated approximately 80-fold by LD50 and is defective in dissemination/coloni­zation of spleens and lungs after subcutaneous inoculation. However, the ΔrovA mutant is only slightly attenuated when given via an intranasal or intraperitoneal route, indicating a more important role for RovA in bubonic plague than in pneumonic plague or systemic infection. Microarray analysis was used to define the RovA regulon. The psa locus was among the most highly downregulated loci in the ΔrovA mutant. A ΔpsaA mutant had a significant dissemination defect after subcutaneous infection but only slight attenuation by the pneumonic disease model, closely mimicking the virulence defect seen with the ΔrovA mutant. DNA-binding studies revealed that RovA specifically interacts with the psaE and psaA promoter regions, indicating a direct role for RovA in regulating this locus. Therefore, RovA appears to be a global transcription factor in Y. pestis and, through its regulatory influence on genes such as psaEFABC, contributes to the virulence of Y. pestis.

Cathelyn JS, Crosby SD, Lathem WW, et al. RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague. Proc Natl Acad Sci US. 2006;103:13514–13519.

Reprints: Virginia L. Miller, Depts. of Molecular Microbiology and Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110;
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The hemagglutinin from an H5N1 influenza virus The hemagglutinin from an H5N1 influenza virus

The H5N1 avian influenza virus, commonly called “bird flu,” is a highly contagious and deadly pathogen in poultry. Since late 2003, H5N1 has reached epizootic levels in domestic fowl in a number of Asian countries, including China, Vietnam, Thailand, Korea, Indonesia, Japan, and Cambodia, and has now spread to wild bird populations. More recently, the H5N1 virus has spread to infect bird populations across much of Europe and into Africa. However, its spread to the human population has so far been limited, with only 191 documented severe infections, but with a high mortality of 108 deaths in Indonesia, Vietnam, Thailand, Cambodia, China, Iraq, Turkey, Azerbaijan, and Egypt as of April 4, 2006. Of these, evidence suggests that direct bird-to-human transmission, although indirect transmission, perhaps through contaminated water supplies, cannot be ruled out. Of the three influenza pandemics of the last century, the 1957 (H2N2) and 1968 (H3N2) pandemic viruses were avian-human reassortments in which three and two of the eight avian gene segments, respectively, were reassorted into an already circulating human-adapted virus. The origin of the genes of the 1918 influenza virus (H1N1), which killed about 50 million people worldwide, is unknown. The extinct pandemic virus from 1918 has recently been reconstructed in the laboratory and was found to be highly virulent in mice and chicken embryos. With continued outbreaks of the H5N1 virus in poultry and wild birds, additional human cases are likely, and the potential for emergence of a human-adapted H5 virus, by reassortment or mutation, is a threat to public health worldwide. The hemagglutinin structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese H5N1 influenza virus, is more closely related to the 1918 and other human H1 hemagglutinins than to a 1997 duck H5 hemagglutinin. Glycan microarray analysis of this Viet04 hemagglutinin reveals an avian α2-3 sialic acid receptor binding preference. The introduction of mutations that can convert H1 serotype hemagglutinins to human α2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations that can convert avian H2 and H3 hemagglutinins to human receptor specificity, when inserted onto the Viet04 H5 hemagglutinin framework, permitted binding to a natural human α2-6 glycan. This suggests a path for this H5N1 virus to gain a foothold in the human population.

Stevens J, Blixt O, Tumpey TM, et al. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science. 2006; 312:404–410.

Reprints: James Stevens or Ian A. Wilson, Dept. of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037; or
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Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.