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September 2003
Measuring
human trefoil factor 3 by ELISA
Trefoil factors have an unusual, compact structure and are extremely
stable toward proteolytic digestion, acid, and heat degradation.
They are expressed in several tissues of the body but are most pronounced
in the gastrointestinal tract. Under normal circumstances, TFF1
and TFF2 are primarily located in the stomach, and TFF3 is present
predominantly in the mucus cells of the small and large intestine.
Upregulation of the expression of all three TFF members in the gastrointestinal
tract occurs at the site of damage in such conditions as inflammatory
bowel disease and peptic ulcer. In addition, trefoil peptides are
aberrantly secreted by a variety of tumors. The trefoil peptides
appear to be involved in mucosal defense and restitution in the
GI tract. The authors undertook a study to define the physiologic
and potential diagnostic values of TFF3 by developing an enzyme-linked
immunosorbent assay for it. The assay had a detection limit of 3.0
pmol/L and an imprecision of five to nine percent for mean concentrations
of 13 to 65 pmol/L. This corresponds to serum concentrations of
65 to 330 pmol/L. The authors found no cross-reaction between this
assay and human TFF1 and TFF2. Neither food intake nor menstrual
cycle changes influenced the values of TFF3 significantly. The 95
percent reference interval for the compound in serum from healthy
blood donors was 95 to 250 pmol/L and did not vary with age and
varied only slightly with gender. TFF3 was increased in serum from
patients with inflammation or ulceration of the upper GI tract,
or both. In serial measurements of serum from three patients with
severe exacerbation of chronic inflammatory bowel disease restricted
to the colon, normal concentrations and only minor variations during
treatment and tapering were observed.
Vestergaard
EM, Poulsen SS, Grønbaek H, et al. Development and evaluation
of an ELISA for human trefoil factor 3. Clin Chem. 2002;48:1689–1695.
Reprints:
Else Marie Vestergaard, Dept. of Clinical Biochemistry, Aarhus University
Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark; else.marie.vestergaard@
dadlnet.dk
Detecting
anthrax by PCR
Bacillus anthracis,
which causes anthrax in humans and animals, is easily cultivated in
its vegetative, or bacillary, form using conventional growth media,
and sporulation can be achieved using nutrient-deficient media. A
spore size of less than 5 µm can be easily inhaled into the
lower respiratory tract in humans. The hardy nature of these spores,
plus the ease of cultivation and the high mortality associated with
inhalation anthrax, makes this organism a potential biological warfare
agent and weapon of mass destruction. To ensure that patients infected
with anthrax are treated quickly, tests that rapidly identify the
organism in culture, clinical specimens, or environmental samples
are necessary. Culture is the gold standard for identification, but
it may require 24 to 48 hours or more and requires specialized testing,
such as a direct fluorescent antibody staining of the capsule and
cell wall polysaccharide and lysis of colonies by gamma phage. These
confirmatory tests generally are not available in clinical microbiology
labs, except at higher level public health laboratories. Virulent
isolates of B. anthracis contain two plasmids, pX01 and pX02,
that have unique virulence gene targets that allow B. anthracis
to be rapidly identified by PCR. Several conventional PCR assays for
identifying these organisms have been described. These assays are,
however, time consuming and labor intensive. The authors developed
a new real-time PCR detection assay that uses the Roche LightCycler
instrument. This assay uses PCR primers and probes designed to identify
gene sequences specific for plasmids pX01 and pX02. The assays can
be completed in less than an hour. The gene encoding the protective
antigen (pagA) was detected in 29 of 29 virulent B. anthracis
strains, and the gene encoding the capsular protein B (capB)
was detected in 28 of 29 of the same strains. Three avirulent strains
containing only pX01 or pX02, and therefore only the pagA
or capB genes, could be detected and differentiated from
virulent strains. The results were negative for 57 bacterial strains
representing a broad range of organisms, including Bacillus
species other than anthracis and other non-Bacillus
species. The analytical sensitivity achieved by the assay was one
copy per microliter of sample. The authors concluded that the LightCycler
method appears to be suitable for rapidly identifying cultured isolates
of B. anthracis, but additional clinical studies are necessary
to determine if it is useful for rapidly identifying B. anthracis
directly from human specimens.
Bell
CA, Uhl JR, Hadfield TL, et al. Detection of Bacillus anthracis
DNA by LightCycler PCR. J Clin Microbiol. 2002; 40:2897–2902.
Reprints: Franklin
R. Cockerill III, Mayo Clinic, Division of Microbiology, 200 First
St. SW, Rochester, MN 55905; cockerill.
franklin@mayo.edu
Effectiveness
of three urinary Legionnaires’ disease antigen tests
Legionella pneumophila is the organism responsible for
more than 90 percent of Legionnaires’ disease cases in the
United States. Legionella species are responsible for one
to five percent of cases of community-acquired pneumonia. Legionnaires’
disease cannot be distinguished clinically or radiographically from
pneumonias caused by other microbial pathogens. The basis for laboratory
diagnosis of Legionnaires’ disease is culture, serologic testing,
and antigen detection in urine. Cultural isolation of Legionella
from respiratory secretions is a fairly insensitive diagnostic test
and requires at least three days of incubation before a positive
result can be generated. Seroconversion, on the other hand, has
high sensitivity and specificity, but it is of limited clinical
value since it can take up to nine weeks for patients to develop
detectable antibodies. Urinary antigen testing is reasonably sensitive
and highly specific and provides rapid results. Enzyme immunoassay
and immunochromatographic tests vary markedly, depending on differences
in patient characteristics, the serogroup of the organism with which
the patients are infected, the time of urine collection in the course
of the illness, and whether the urine is concentrated before testing.
The authors assessed the value of urinary antigen testing for Legionella
during a large outbreak that occurred in the Netherlands in 1999.
They tested two enzyme immunoassays—Binax and Biotest—and
one immunochromatographic assay—Binax Now. They used concentrated
urine specimens from the 188 cases of Legionnaires’ disease
that occurred in the Netherlands outbreak in 1999. They calculated
sensitivity using positive culture or seroconversion, or both, as
the gold standard in the outbreak-related patients with radiographically
confirmed pneumonia who fulfilled the epidemiological criteria.
The Binax EIA showed an overall sensitivity of 69 percent; the Biotest
EIA showed a sensitivity of 71 percent; and the Binax Now assay
showed a sensitivity of 72 percent. When concentrated urine specimens
were used, the overall sensitivities increased to 79, 74, and 81
percent, respectively. Analyzing the data by multiple logistic regression
analysis with backward elimination, the authors found a statistically
significant association between clinical severity and test sensitivity
for all tests. The test sensitivities for patients with mild Legionnaires’
disease ranged from 40 to 53 percent, whereas the test sensitivities
for patients with more severe disease who needed immediate special
medical care ranged from 88 to 100 percent. The authors concluded
that mild pneumonia may be underdiagnosed if urine antigen tests
alone are used.
Yzerman
EPF, den Boer JW, Lettinga KD, et al. Sensitivity of three urinary
antigen tests associated with clinical severity in a large outbreak
of Legionnaires’ disease in the Netherlands. J Clin Microbiol.
2002;40: 3232–3236.
Reprints:
Ed P.F. Yzerman, Regional Laboratory of Public Health Haarlem, Boerhaavelaan
26, 2035 RC Haarlem, Netherlands; e.yzerman@streeklabhaarlem.nl
Antibiotic
resistance in Staphylococcus isolates from fecal samples of healthy
kids
Staphylococci can be important causes of human infections, but they
are also found as nonpathogenic microorganisms in the human gastrointestinal
tract. The use of antibiotics can be a selection factor for antibiotic
resistance in the intestinal environment. A number of studies have
reported on the resistance mechanisms in Staphylococcus
isolates, but little data are available on the antibiotic resistance
phenotypes or the mechanisms of resistance in nonpathogenic intestinal
isolates. The authors analyzed the antibiotic resistance phenotypes
and mechanisms of resistance in Staphylococcus isolates
recovered from feces samples of healthy children. They collected
50 fecal specimens from 50 healthy children (ages seven to 23 months;
25 boys and 25 girls). The samples were obtained from nurseries
in Spain from November 2000 to January 2001. Thirty-nine Staphylococcus
isolates with different mechanisms of resistance were recovered
from these children and tested for their susceptibility to 17 antibiotics.
The percentages of resistance of the staphylococci to some antibiotics
were: penicillin, 87 percent; erythromycin, 64 percent; tobramycin,
36 percent; tetracycline, 20.5 percent; kanamycin, 15.4 percent;
and gentamicin, 13 percent. The mecA gene was detected
in nine coagulase-negative staphylococci. The authors concluded
that the rates of antibiotic resistance in staphylococcal isolates
recovered from the feces of healthy children are high. A followup
study should be conducted to analyze more extensively the resistance
mechanisms in fecal nonpathogenic bacteria.
Dominguez E,
Zarazaga M, Torres C. Antibiotic resistance in Staphylococcus isolates
obtained from fecal samples of healthy children. J Clin Microbiol.
2002;40: 2638–2641.
Reprints: Carmen
Torres, Area de Bioquímica y Biología Molecular, Universidad
de La Rioja, Madre de Dios, 51, 26006 Logroño, Spain; carmen.torres@daa.unirioja.es
Confirmatory
test for toxoplasma serology in pregnant women
False-positive or erroneously true-positive IgM test results for
toxoplasma in pregnancy can be misleading and result in unnecessary
abortions. Consequently, the FDA has recommended that positive IgM
results undergo confirmatory testing. Confirmatory tests may include
those for detecting IgG, IgM, IgA, and IgE antibodies, otherwise
known as the toxoplasma serological profile. This profile has proved
useful in differentiating between recently acquired and distant
infections. A number of tests have recently become available for
the avidity of toxoplasma IgG antibodies in an effort to differentiate
between recently acquired and distant infections. The functional
affinity of specific IgG antibodies is initially low after primary
antigenic challenge and increases during subsequent weeks and months
by antigen-driven B-cell selection. The authors have reported results
of testing for the avidity of IgG in pregnant women during the first
12 weeks of gestation using LabSystems’ IgG avidity enzyme
immunoassay method. They found that with this method, a high avidity
tends to exclude the possibility that the infection occurred during
the previous 12 weeks. The VIDAS high-IgG-avidity test, available
in Europe, is designed to rule out the possibility that infection
occurred during the prior four months. The authors undertook a study
to evaluate the role of the VIDAS Toxo-IgG avidity test and the
toxoplasma serological profile for confirmatory testing of pregnant
women during the first 16 weeks of gestation. Sera from 132 women
in the first 16 weeks of pregnancy were chosen because at least
one test in the toxoplasma serologic profile suggested or was equivocal
for a recently acquired infection. High avidity antibodies were
demonstrated in 75 percent of 99 sera positive with the IgM ELISA
and 31.3 percent of 16 sera with acute toxoplasma serological profile
results. A significant percentage of sera with equivocal results
with the IgM ELISA or profile also had high-avidity test results.
Of 39 women for whom treatment with spiramycin had been suggested
to attempt to prevent congenital transmission, 19 (48.7 percent)
had high-avidity antibodies. The authors concluded that these findings
highlight the value of the VIDAS IgG avidity kit when used in combination
with the toxoplasma serological profile to exclude recent infection.
Montoya JG,
Liesenfeld O, Kinney S. VIDAS test for avidity of Toxoplasma-specific
immunoglobulin G for confirmatory testing of pregnant women. J
Clin Microbiol. 2002; 40:2504–2508.
Reprints: Jose
G. Montoya, Research Institute, Palo Alto Medical Foundation, Ames
Building, 795 El Camino Real, Palo Alto, CA 94301; gilberto@leland.stanford.edu
Determining
gender using maternal serum HCG levels
Fetal gender has been shown to have a significant influence on maternal
serum levels of human chorionic gonadotropin (MSHCG). Third trimester
MSHCG is higher in women carrying a female fetus than in those carrying
a male. Recent studies have found that maternal serum free b-HCG
is also significantly higher in the late first trimester—10
to 14 weeks gestation—in women carrying female fetuses. The
reason for the gender-related difference has remained elusive. The
authors hypothesized that if gender-related differences in MSHCG
could be demonstrated prior to establishing fetal hypothalamic-hypophyseal-gonadal
axis, they then may be attributed to the differential expression
of genes by the trophoblast. The authors determined whether the
gender-related difference in MSHCG can be detected as early as week
three post-fertilization, the time that MSHCG is usually first measured.
They studied subjects in the in vitro fertilization setting because
it provides precise dating of gestational age and early sonography
for the number of gestational sacs. The study included 347 in vitro
fertilization cycles from 335 patients and only pregnancies with
a single implanted embryo that resulted in a single live birth of
known gender. MSHCG was measured on days 14 through 20 post-fertilization.
Levels were expressed as gestational age-corrected multiples of
the median (MoMS). The MoMS for MSHCG were compared according to
fetal gender, and the levels were 18.5 percent higher in week three
post-fertilization in the presence of a female fetus (P
<0.0002). The authors concluded that because fetal gender-related
difference in MSHCG could be demonstrated as early as week three
post-fertilization, the difference may be attributed to placental
factors and not to the effects of the fetal-hypothalamic-hypophyseal-gonadal
axis.
Yaron Y, Lehavi
O, Orr-Urtreger A, et al. Maternal serum HCG is higher in the presence
of a female fetus as early as week 3 post-fertilization. Hum
Reprod. 2002;17: 485–489.
Reprints: Yuval
Yaron, Prenatal Diagnosis Unit, Genetic Institute, Sourasky Medical
Center, 6 Weizmann St., Tel Aviv, 64239, Israel; yyaron@tasmc.health.gov.il
Biochemical
elements associated with Chlamydia pneumoniae seropositivity in
community-dwelling subjects
Chlamydia pneumoniae infection has been linked to coronary
atherosclerosis. Endothelial cells for which activation is linked
to atherosclerosis appear to be the target of C. pneumoniae
infection in vivo. Infection of human endothelial cells with C.
pneumoniae results in stimulation of a wide variety of cytokines,
adhesion molecules, chemokines, and proteins with procoagulant activity.
Enhanced expression of endothelial adhesion molecules by C.
pneumoniae infection is associated with rolling, adhesion,
and transmigration of leukocytes and monocytes. There are circulating
forms of adhesion molecules, such as monocyte chemoattractant protein-1
(MCP-1), although the origin of these molecules is not clear. One
possible source is shedding from the surface of endothelium of macrophages.
The authors hypothesized that chronic, persistent C. pneumoniae
constantly stimulates the expression of adhesion molecules and chemokines,
which could be reflected by increases in the plasma concentrations
of these substances. They tested this hypothesis by evaluating the
association between seropositivity for C. pneumoniae infection
and plasma concentrations of soluble intercellular molecule-1 (ICAM-1)
and vascular cellular adhesion molecule-1 (VCAM-1), as well as MCP-1
in healthy community-dwelling residents. They investigated 200 residents
who were free from cardiovascular disease and were not taking medication.
They examined plasma levels of IgA and IgG antibodies to C. pneumoniae
and measured these by enzyme-linked immunosorbent assay. Subjects
were divided into three groups according to the indices of antibodies:
C. pneumoniae seronegative (n=57); C. pneumoniae intermediate
(n=81); and C. pneumoniae seropositive (n=62). The plasma
concentrations of the soluble forms of ICAM-1, VCAM-1, and MCP-1
were determined by ELISA. Plasma concentrations of ICAM-1 and VCAM-1
were significantly different among the C. pneumoniae seronegative,
intermediate, and seropositive groups, respectively. However, the
MCP-1 was not significantly different among the three groups. Stepwise
regression analysis showed that plasma concentration of ICAM-1 was
significantly associated with C. pneumoniae seropositivity,
independent of any other known risk factors for atherosclerosis
and carotid intima-media thickness. The authors concluded that C.
pneumoniae seropositivity is associated with higher plasma
concentrations of soluble forms of adhesion molecules in the general
population.
Kohara K, Tabara
Y, Yamamoto Y, et al. Chlamydia pneumoniae seropositivity
is associated with increased plasma levels of soluble cellular adhesion
molecules in community-dwelling subjects: the Shimanami Health Promoting
Program (J-SHIPP) study. Stroke. 2002;33:1474–1479.
Reprints: Dr.
Katsuhiko Kohara, Dept. of Geriatric Medicine, Ehime University
School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun,
Ehime 791-0295, Japan;
koharak@ m.ehime-u.ac.jp
Real-time
RT-PCR assays for tumor markers in primary breast cancer
The plasminogen activation system and matrix metalloproteinases
play a key role in the degradation of basement membrane and extracellular
matrix in tissue remodeling, cancer cell invasion, and metastasis.
Urokinase-type plasminogen activator (uPA) is a serine protease
that catalyzes the conversion of plasminogen to plasmin, an active
enzyme that is able to degrade various extracellular matrix proteins
and activate matrix metalloproteinases (MMPs) and growth factors.
Plasminogen activator inhibitor type-1 (PAI-1) is a multifaceted
proteolytic inhibitor that not only functions as a fibrinolytic
inhibitor but also plays an important role in signal transduction,
cell adherence, and cell migration. PAI-1 may be a key factor in
tumor invasion and metastasis. PAI-1 and uPA have been linked to
poor prognosis in several cancers, including breast cancer. Conversely,
MMPs are involved in several pathologic processes, including tumor
invasion, in which degradation of the extracellular matrix is a
key event. MMP activities are regulated by tissue inhibitors of
metalloproteinases (TIMPs). Four members of this family have been
identified, among which TIMP-1 acts against all members of the collagenase,
stromelysin, and gelatinase classes of MMPs. The authors developed
and used a novel quantitative real-time RT-PCR assay for specific
quantitation of uPA, PAI-1, and TIMP-1 mRNA in 54 breast cancer
tissues. They amplified gene fragments in the LightCycler real-time
PCR system using gene-specific primers and SYBR GreenI. The results
were normalized to b-actin mRNA. They also quantified antigen and
functional concentrations of these compounds. The intra- and interassays
variabilities for mRNA quantification showed mean standard deviations
for the crossing point of 0.12 and 0.15 cycles, respectively. The
mRNA and antigen concentrations of PAI-1, uPA, and TIMP-1, and the
functional concentrations of PAI-1 and uPA, increased with tumor
severity. This increase was statistically significant for PAI-1,
uPA, and TIMP-1 mRNA antigen concentrations and for uPA functional
concentrations. Node-positive patients demonstrated significantly
higher concentrations of PAI-1, uPA, and TIMP-1 mRNA and antigen
concentrations than those who were node negative.
Castello R,
Estelles A, Vazquez C, et al. Quantitative real-time reverse transcription-PCR
assay for urokinase plasminogen activator, plasminogen activator
inhibitor type I, and tissue metalloproteinase inhibitor type I
gene expressions in primary breast cancer. Clin Chem. 2002;48:1288–1295.
Reprints: Amparo
Estelles, Hospital Universitario La Fe, Centro de Investigación,
Avda. Campanar 21, 46009 Valencia, Spain; estelles_amp@gva.esn
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. bruene@rhrk.uni-kl.de
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