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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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