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CAP Home > CAP Reference Resources and Publications > CAP TODAY > CAP Today Archive 2003 > June 2003 Clinical Abstracts
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  Clinical Abstracts

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cap today

June 2003

Epidemiology of the hepatitis E virus
Hepatitis E virus, an RNA virus transmitted enterically, is a major cause of hepatitis in many areas of the world. HEV infection during outbreaks is primarily transmitted through contaminated water. Veterinarians and persons working with pigs are also at greater risk for HEV infection. The disease is typically self-limited but may be associated with severe complications, particularly in pregnant women (case-fatality rate, approximately 20 percent). North America and Europe have traditionally been considered nonendemic for HEV, although the seroprevalence rate ranges from one to five percent. The authors of this study examined the level of infection in regions where HEV is considered nonendemic by analyzing the excreted virus in the urban sewage of various geographic areas. Hepatitis A virus was also analyzed. Urban sewage samples were collected at the entry of water treatment plants in Barcelona, Spain; Patras, Greece; Washington, DC; Nancy, France; and Umeå, Sweden. Serum samples were obtained from 13 human patients who had tested positive for IgG anti-HEV antibody and 73 healthy pigs (swine fecal samples were also tested) in Spain. Enzyme immunoassay, reverse transcription (RT)-PCR tests, and viral sequencing were performed. Twenty of 46 urban sewage samples collected in Barcelona tested positive for HEV. One of five samples from Washington, DC, and one of four samples from Nancy, France, also tested positive for HEV RNA. HAV RNA was detected in samples from all four countries. The 13 human patients tested negative for HEV RNA, and IgG anti-HEV was undetectable in seven of the 13 (53.8 percent). Ten (13.7 percent) of the swine serum samples were positive for anti-HEV IgG. HEV was detected in pig fecal samples and was amplified by RT-PCR, including one new swine HEV strain. The authors concluded that HEV may be more prevalent than previously thought in industrialized countries and that endemic HEV infections are likely present in Europe and the United States. Furthermore, animals may serve as a reservoir for HEV.

Clemente-Casares P, Pina S, Buti M, et al. Hepatitis E virus epidemiology in industrialized countries. Emerg Infect Dis. 2003;9:448–454.

Correspondence: Rosina Girones, Dept. of Microbiology, Faculty of Biology, University of Barcelona, Avd. Diagonal 645, 08028 Barcelona, Spain; rgirones@ub.edu.

Using the LightCycler to detect smallpox virus DNA
Polymerase chain reaction for detecting smallpox and differentiating viruses within the genus Orthopoxvirus have been labor-intensive conventional PCR assays using consensus or sequence-specific primers and subsequent digestion of amplicons with several restriction endonucleases followed by gel electrophoresis. More recently, homemade PCR amplification detection systems based on oligonucleotide microchip technology have been used to perform an assay in six hours. The authors described an even more rapid three-hour real-time assay for nucleic acid extraction and amplification based on use of the Roche LightCycler instrument. This assay detects smallpox virus and differentiates the DNA of this agent from the DNA of the other viruses classified within the genus Orthopoxvirus. The authors used a 300-bp plasmid fragment of the hemagglutinin gene as the target DNA to develop a rapid real-time LightCycler PCR assay for laboratory detection of smallpox. They used based pair mismatches in the 204-bp amplicon to discriminate cowpox virus, monkeypox virus, and vaccinia virus, including the Dryvax vaccine strain, from smallpox using melting curve analysis. The analytical sensitivity of this method was five to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus. The DNA of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.

Espy MJ, Cockerill FR, Meyer RF, et al. Detection of smallpox virus DNA by LightCycler PCR. J Clin Microbiol. 2002;40: 1985–1988.

Reprints: Thomas F. Smith, Division of Clinical Microbiology, Mayo Clinic, 200 First St., SW, Rochester, MN 55905; tfsmith@mayo.edu.

Using the 13C-urea breath test in childhood H. pylori infection
In children and adults, the microorganism Helicobacter pylori is thought to play an important role in the pathogenesis of chronic gastritis and peptic ulcer disease, especially duodenal ulcer. The organism has also been cited as a possible risk factor for common pediatric conditions, such as iron-deficiency anemia and atopic dermatitis. Tests for the organism include biopsy, histology, urease test, and microbiologic culture. These tests, however, are invasive and may show false-negative results in some patients because of sampling error and because colonization of the organism is sometimes patchy in the stomach. On the other hand, noninvasive tests like serology have been found to be less reliable in children than adults, and the stool antigen test is still being studied for its clinical usefulness in children. The 13C-urea breath test (UBT), developed recently, has demonstrated a fairly high sensitivity and specificity for diagnosing active H. pylori infection in adults. Though some studies have suggested that the UBT shows diagnostic accuracy in children with the infection, published guidelines for managing H. pylori infection in children have not unequivocally recommended its use. This is partly because the optimum cutoff value remains to be established in this population. This is largely due to the limited number of patients in biopsy-based studies of the test. Further, in the 13C-UBT, expiratory 13CO2 is usually measured by isotope ratio mass spectroscopy, but this is an expensive method. More recently, a nondispersive infrared spectrometry method was used, which is inexpensive. The authors compared these two methods for determining 13CO2. They examined 232 Japanese children, aged two to 16 years, who underwent upper GI endoscopy and gastric biopsies. From a final study population of 220 children, they identified 131 cases of gastritis, 15 of gastric ulcer, 72 of duodenal ulcer, and two of combined ulcer. Histology, urease test, and culture were performed on biopsies. Breath samples for the 13C-UBT were obtained at baseline and 20 minutes after ingesting 13C-urea (tagged). Based on the biopsy tests, a cutoff value was determined using a receiver operating characteristic curve. In 26 of the children (seven infected and 19 noninfected), paired breath samples were measured using nondispersive infrared spectrometry and isotope ratio mass spectrometry. Biopsy tests indicated that 40 percent (89) of the children were infected with H. pylori and 60 percent (131) were not infected. There were no statistical differences in the mean change in 13C values at 20 minutes between male and female H. pylori infected and noninfected patients. The best cutoff was defined by ROC analysis as 3.5 percent. The overall sensitivity and specificity at this cutoff were 97.8 percent and 98.5 percent, respectively. There was a close correlation between the results obtained with isotope ratio mass spectrometry and the NDIRS methods. The authors concluded that 13C-UBT with a cutoff value of 3.5 percent is an accurate method for diagnosing H. pylori infection in children.

Kato S, Ozawa K, Konno M, et al. Diagnostic accuracy of the 13C-urea breath test for childhood Helicobacter pylori infection: a multicenter Japanese study. Am J Gastroenterol. 2002;97:1668–1673.

Reprints: Dr. Seiichi Kato, Dept. of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

Limitations of D-dimer testing in suspected venous thromboembolism
D-dimer testing is often used to evaluate patients suspected of having venous thromboembolism. False-positive D-dimer tests can be seen in a variety of diseases, such as pneumonia and malignancy. False-negative D-dimer tests also can be seen in some patients, including those receiving anticoagulant therapy. The authors of this prospective study examined the efficacy of D-dimer testing by four methods in unselected, hospitalized patients undergoing radiologic evaluation for possible venous thromboembolism. Blood samples for D-dimer testing were obtained within 24 hours of radiologic study (venous ultrasound of any body site, ventilation-perfusion lung scans, helical computed tomography of the chest, and angiography of the deep veins or pulmonary arteries). The four D-dimer assays were performed using enzyme-linked immunosorbent assay, plasma agglutination, whole blood agglutination, and immunoturbidimetric methods. C-reactive protein levels were also determined. Of 250 patients enrolled in the study, 203 patients were evaluated. Forty-five patients (22 percent) had thrombosis by radiographic imaging (pulmonary embolism—21 patients, deep vein thrombosis alone—20 patients, both conditions—four patients). The sensitivity of the four assays varied from 60 to 96 percent, and the specificity varied from 13 to 59 percent. The specificity of all four assays was lower in patients who had been hospitalized for at least four days and with increasing age of the patient and increasing C-reactive protein levels. Nineteen (42 percent) of the 45 patients with thrombosis had a negative D-dimer assay by at least one method. There were no significant differences identified between patients with only true-positive D-dimer tests and those with one or more false-negative tests. The C-reactive protein level in patients with thrombosis as compared to those without thrombosis was not statistically different (P=0.78). D-dimer results were not used as a criterion to withhold anticoagulation in these patients. The authors concluded that D-dimer testing has limited clinical utility because of its poor specificity and that the D-dimer test is less reliable for excluding venous thromboembolism in unselected inpatients than in symptomatic outpatients. The authors also suggested that D-dimer ELISA testing not be considered a first-line diagnostic test in inpatients suspected of having venous thromboembolism.

Brotman DJ, Segal JB, Jani JT, et al. Limitations of D-dimer testing in unselected inpatients with suspected venous thromboembolism. Am J Med. 2003;114:276–282.

Correspondence: Dr. Daniel J. Brotman, Dept. of General Internal Medicine, E13, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195; brotmad@ccf.org.

European standardization of ISI calibration of POC coagulation monitors
Reliable estimation and standardization of International Sensitivity Index calibration is required for whole blood point-of-care testing prothrombin time monitors to conform to the World Health Organization prothrombin standardization scheme. Multicenter calibration is needed to provide reliable ISI according to the WHO guidelines. If a POC testing monitoring system is regarded as a secondary standard, then a minimum of two centers is necessary for this comparison. It remains to be established whether the WHO recommendations, designed for conventional PT testing, are appropriate for whole blood POC PT monitors. The European Concerted Action on Anticoagulation (ECAA), an initiative funded by the European Community, reported on multicenter calibration of whole blood POC testing monitors at 10 centers. The findings showed that calibrations of two types of POC testing monitoring systems—the CoaguChek Mini and TAS PT-NC—were less precise than calibrations of conventional PT systems. The authors performed a statistical simulation on the basis of the ECAA calibration exercise to determine the minimum number of centers required for reliable ISI and International Normalized Ratio determinations using the two POC testing whole blood PT monitoring systems. They found that with both systems, the range of ISI and INR deviation increased as the number of centers involved was reduced. For the CoaguChek Mini, at least five centers were needed for satisfactory INR deviation in 95 percent of calibrations. With the TAS PT-NC, three centers gave satisfactory INR results at this level. Overall, the number of centers required for multicenter calibration of these two systems was greater than the two originally proposed by the WHO guidelines for conventional PT testing.

Poller L, Keown M, Chauhan N, et al. European Concerted Action on Anticoagulation (ECAA). Minimum number of centres for reliable International Sensitivity Index calibration of CoaguChek and TAS point-of-care whole blood monitors. Thromb Res. 2002;107:61–66.

Reprints: L. Poller, ECAA Central Facility, School of Biological Sciences, University of Manchester, 3.239 Stopford Bldg., Manchester, M13 9PT, United Kingdom; ecaa@man.ac.uk.

Comparing fingerstick to venipuncture for measles, rubella antibody testing
For performing large-population antibody studies, the use of blood samples obtained by fingerstick offers certain advantages over venipuncture, such as lower cost and convenience. Only a few studies, however, have examined the use of fingerstick testing for antibody levels. The authors conducted a study in which they compared fingerstick samples and venipuncture specimens obtained simultaneously from healthy adult subjects. Blood specimens were obtained from 20 healthy adult subjects (age range, 20 to 50 years) who were thought to be immune by vaccination or disease. Samples from each subject were collected by standard methodology at the same encounter. Commercially available measles and rubella antibody assays were performed. Excellent correlation between serum collected from fingerstick and from venipuncture was observed for measles and rubella assays (correlation coefficients, 0.98 for measles and 0.83 for rubella). Immunity based on fingerstick serum also agreed with immunity based on venipuncture serum for measles and rubella. The authors concluded that the collection and assay of fingerstick specimens can be accurate, feasible, and reliable when compared to venipuncture.

Pinsky NA, Loepfe TR, Jacobson RM, et al. Comparison of fingerstick versus venipuncture for antibody testing of measles and rubella. Scand J Infect Dis. 2003;35:107–109.

Correspondence: Dr. G.A. Poland, Mayo Vaccine Research Group, Mayo Clinic and Foundation, 611 C Guggenheim Bldg., 200 First St., SW, Rochester, MN 55905; poland.gregory@mayo.edu.

Determining von Willebrand factor activity using a collagen-binding assay
Quantitative or qualitative abnormalities of the large plasma protein known as von Willebrand factor (vWf) give rise to the inherited bleeding disorder von Willebrand disease (vWD). The acquired form of vWD is associated with several types of underlying conditions, including autoimmune and neoplastic diseases. It is necessary to determine the concentration and properties of vWf in the diagnosis of vWD. For many years, assays for ristocetin cofactor or ristocetin cofactor activity— based on the ability of the tested plasma to aggregate fresh or conserved human platelets in the presence of ristocetin—were used for functional determination of vWf activity. An alternative to this approach, developed in 1986, involves a microplate procedure based on the amount of vWf bound to immobilized collagen. The assay depends on coating the incubation with collagen, incubating a sample of diluted plasma, incubating with conjugate anti-vWf, and incubating with substrate. Many researchers have reported problems with coating, thus high concentrations of collagen and coating incubation times of 48 hours or longer typically are recommended. The type of collagen and its source also appear to be important factors and have a significant influence on the binding ability of vWf. The authors studied the optimal collagen source and coating conditions in the determination of vWf activity in the collagen-binding assay. They found that modification of coating conditions and use of an alkaline buffer permit the use of relatively low concentrations of collagen—5 to 10 µg/mL—and allow the test to be completed in one day. The authors concluded that the procedure is useful in the diagnosis of vWD and allows researchers to distinguish between disease types I and II.

Paczuski R. Determination of von Willebrand factor activity with collagen-binding assay and diagnosis of von Willebrand disease: effect of collagen source and coating conditions. J Lab Clin Med. 2002;140: 250–254.

Reprints: Ryszard Paczuski, Dept. of Pathophysiology, Ludwik Rydygier Medical University, UI. M. Sklodowskiej-Curie 9, 85-094 Bydgoszcz, Poland.

Preserving urine flow cytometry and microscopy samples
Urine particles that have not been preserved need to be examined within one hour after voiding if stored at ambient temperature or within four hours if refrigerated to avoid lysis of cellular material. Traditionally, 500 mL/L ethanol is used to preserve uroepithelial cells for cytology. And 20 g/L polyethylene glycol has been added to the ethanol fixative (Saccomanno’s fixative) to improve preservation results. Other approaches have used boric acid or boric acid in combination with formic acid or other stabilizing media. The authors investigated ways to preserve urine particles for automated analysis with the Sysmex UF-100 flow cytometer and for visual microscopy. They preserved specimens by refrigeration at 4°C without preservatives (procedure one); in a lyophilized solution intended to preserve specimens for bacterial culture (procedure two); in 10 mL/L of formalin and 0.15 mol/L NaCl (procedure three); in 80 mL/L ethanol with 20 g/L of polyethylene glycol (procedure four); and by storage at 20°C without preservatives (procedure five). They used test strip measurements to select specimens positive for leukocyte esterase, hemoglobin, albumin, or nitrite. They performed urinalysis for 106 consecutive strip-positive specimens on the UF-100 and by phase-contrast microscopy. They performed automated analysis on arrival in the morning, on the same day in the afternoon, and after one and three days. Visual microscopy was performed on arrival and three days later. The first three methods—refrigeration, lyophilization, and formalin—gave urine bacterial counts that were well preserved, with a false-positive rate of zero to 3.4 percent at day three versus 28 percent without preservation. Erythrocytes tended to be poorly preserved for three days. After one day, fair preservation was seen with lyophilization, compared with less favorable preservation with simple refrigeration. Leukocytes were well preserved by all five procedures examined using the acidic adult urines investigated. Counts of casts and large epithelial cells were increased artifactually in formalin. Lyophilization performed as well as refrigeration for specimens analyzed by visual microscopy. The authors concluded that urine specimens from adults could be stabilized at room temperature for automated particle analysis and visual microscopy in several ways.

Kouri T, Vuotari L, Pohjavaara S, et al. Preservation of urine for flow cytometric and visual microscopic testing. Clin Chem. 2002;48:900–905.

Reprints: Timo Kouri, Oulu University Hospital, Laboratory Administration, P.O. Box 50, FIN-90029, OYS, Finland; timo.kouri@ ppshp.fi.

Clinical pathology abstracts editors

Michael Bissell, MD, PhD, MPH, professor and director of clinical services and vice chair, Department of Pathology, Ohio State University Medical Center, Columbus.

Ronald Domen, MD, professor of pathology, medicine, and humanities, Penn State University College of Medicine, Hershey, Pa.

   
 

 

 

   
 
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