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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