College of American Pathologists
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  Clinical Abstracts






May 2007

Michael Bissell, MD, PhD

Typing of Bacillus anthracis
Interferon production in HIV infection
Performance of free light chain assays
B-type natriuretic peptide in patent ductus arteriosus in premature infants

bullet Typing of Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, is a genetically homogeneous and recently emerged pathogen, which complicates efforts to subtype it. In 1995, Henderson, et al., detected the first molecular difference among B. anthracis strains. The following year, Andersen, et al., demonstrated that the variation was due to a 12-nucleotide variable-number tandem repeat (VNTR) named vrrA. The molecular basis for the variation was due to differences in the number of repeated units, and five different allelic forms could be detected. An expansion of this strategy that further advanced the subtyping of B. anthracis involved analyzing multiple VNTR loci. Multiple-locus VNTR analysis (MLVA) typing systems use PCR amplification and fragment sizing to detect length polymorphisms in several VNTR regions. MLVA has proven to be a powerful tool for distinguishing among B. anthracis isolates and has been used to examine bioterrorism events and naturally occurring anthrax outbreaks. Keim, et al., used MLVA to examine a worldwide collection of B. anthracis isolates in the year 2000 and described 89 unique genotypes. Additional studies have used MLVA to examine regional patterns of anthrax diversity; these include studies of the genetic diversity of B. anthracis isolates in France and Poland. In Italy, anthrax is generally a sporadic disease that occurs, with a few exceptions, in the central and southern regions and on the major islands, where it almost exclusively affects animals at pasture. Since the late 1950s, the anthrax situation in Italy has improved as a result of vaccination campaigns with an attenuated live vaccine composed of spores in one percent saponin (Carbosap). This nationwide control system also includes disease outbreak surveillance. During the past five years, the Anthrax Reference Institute of Italy has been recovering B. anthracis isolates from national outbreaks, including archived specimens from historical outbreaks. However, the genetic composition of B. anthracis in Italy has not been studied adequately. The authors recovered 64 isolates of B. anthracis that are archived as spores at the maximum security laboratory of the Anthrax Reference Institute of Italy. They used MLVA to type the 64 B. anthracis isolates. MLVA revealed 10 unique genotypes; nine of these genotypes and the majority of isolates (63/64) belonged to the previously described genetic cluster A1.a. Within the A1.a isolates, two previously described genotypes—G1 and G3—which differ by a single mutation in the pX01 locus, account for the majority of isolates in Italy (53/63). The authors concluded that the low diversity of B. anthracis genotypes in Italy suggests a single, dominant historical introduction, followed by limited, localized differentiation.

Fasanella A, Van Ert M, Altamura SA, et al. Molecular diversity of Bacillus anthracis in Italy. J Clin Microbiol. 2005;43:3398–3401.

Reprints: Antonio Fasanella, Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata-Anthrax Reference Institute of Italy, Foggia 71100, Italy;

bullet Interferon production in HIV infection

Innate immunity is the first line of defense against HIV infection. Type I interferons (IFNs) are important players in innate immunity because of their antiviral activity against HIV and because they enhance T-cell stimulation. Two main types of leukocytes are involved in type I IFN production. Monocytes produce IFN in response to Sendai virus and other enveloped viruses. In HIV infection, their IFN production decreases late and is not correlated with opportunistic infections. Natural IFN-producing cells are approximately 50 times less frequent in peripheral blood than are monocytes, but they can produce 100 times more IFN per cell. They produce type I IFN in vitro in response to a broad range of enveloped viruses, including HIV, and to naked viruses complexed with antibodies. These cells have been identified as plasmacytoid dendritic cells. HIV, itself, induces IFN secretion from plasmacytoid dendritic cells, not from myeloid dendritic cells. Natural IFN-producing cells are progressively lost during HIV infection in association with progression to disease and the occurrence of opportunistic infections and Kaposi’s sarcoma. Circulating plasmacytoid and myeloid dendritic cell counts, measured by flow cytometry ex vivo and by rare-event analysis, are decreased in HIV chronic infection. Several studies have found a correlation between circulating plasmacytoid dendritic cell counts and IFN production from peripheral blood mononuclear cells (PBMCs) in vitro and an inverse correlation with disease progression. However, IFN production in vitro has never been tested during primary infection. The authors longitudinally studied 26 patients during the primary stage of HIV infection. Fifteen patients received highly active antiretroviral therapy (HAART) for 12 months. At the time of inclusion in the cohort, median type I IFN production in response to herpes simplex virus type 1 stimulation was dramatically impaired in PBMCs from HIV-infected patients compared with that in PBMCs from 31 uninfected donors (180 versus 800 IU/mL; P<0.0001). Median circulating plasmacytoid dendritic cell counts were also significantly decreased (7,300 versus 13,500 cells/mL; P=0.001). Twelve months later, IFN production returned to normal. The data suggested that HAART may help in recovering IFN production by plasmacytoid dendritic cells. The authors concluded that these data underline the potential for early antiretroviral treatment and IFN-a treatment to enhance viral control in a larger proportion of patients during the critical stage of primary infection.

Kamga I, Kahi S, Develioglu L, et al. Type I interferon production is profoundly and transiently impaired in primary HIV-1 infection. J Infect Dis. 2005;192:303–310.

Reprints: Dr. Anne Hosmalin, Dept. of Immunology, Institut Cochin, INSERM U 567, 27 rue du Fg St-Jacques, Bat G. Roussy, 8è étage, 75014 Paris, France;

bullet Performance of free light chain assays

A quantitative nephelometric assay for free light chains is available as a commercial test. The assay measures kappa and lambda light chains that circulate as light chain monomers or dimers and are not bound to immunoglobulin heavy chain. Quantification of the kappa and lambda free light chains and calculation of the free light chain kappa/lambda ratio have been reported to be sensitive and specific for detecting excess monoclonal free light chains. The authors recommended a diagnostic range for the free light chain kappa/lambda ratio that included 100 percent of a 282-sample reference population to maximize the diagnostic specificity and minimize false-positive results. Retrospective studies using stored serum from populations of patients with nonsecretory multiple myeloma (NSMM), primary systemic amyloidosis, light chain deposition disease (LCDD), and light chain multiple myeloma (LCMM) have documented the sensitivity of these assays and established their use as a complement to immunofixation electrophoresis. In addition to its diagnostic use in the free light chain diseases, the assay is used to monitor disease course in amyloidosis, LCDD, NSMM, and LCMM, in which there may be a band detected on immunofixation electrophoresis that cannot be quantified by protein electrophoresis. In 2003, the authors performed free light chain assays on 1,020 samples from Mayo Clinic patients. These patients were seen predominantly by clinicians in the Mayo Clinic’s division of hematology. To assess the performance of the free light chain assay in routine clinical laboratory practice, the authors reviewed the diagnoses and free light chain results for these 1,020 patients. All free light chain clinical test results generated in 2003 were abstracted from the laboratory information system. Diagnoses were obtained from the dysproteinemia database and patient medical history. The majority of these patients (88%) had bone marrow-derived monoclonal plasma cell disorders. The 121 patients who did not have monoclonal gammopathy had free light chain kappa/lambda ratios within the range of values obtained for a reference population in the authors’ laboratory. Among the patients with monoclonal gammopathies were patients with multiple myeloma (330), amyloidosis (269), monoclonal gammopathy of undetermined significance (114), smoldering multiple myeloma (72), plasmacytoma (22), NSMM (20), macroglobulinemia (nine), LCDD (seven), and a variety of other plasma cell disorders. Among the 110 amyloidosis patients who had not been treated previously and who had a free light chain assay performed within 120 days of diagnosis, the free light chain kappa/lambda ratio was positive in 91 percent compared with 69 percent for serum immunofixation electrophoresis and 83 percent for urine immunofixation electrophoresis. The combination of serum immunofixation electrophoresis and serum free light chain assay detected an abnormal result in 99 percent (109 of 110) of patients with amyloidosis. The authors concluded that this analysis of clinical laboratory data is consistent with results from published retrospective validation studies.

Katzmann JA, Abraham RS, Dispenzieri A, et al. Diagnostic performance of quantitative k and l free light chain assays in clinical practice. Clin Chem. 2005;51:878–881.

Reprints: Jerry A. Katzmann, Dept. of Laboratory Medicine and Pathology, Hilton 210, Mayo Clinic, Rochester, MN 55905;

bullet B-type natriuretic peptide in patent ductus arteriosus in premature infants

Patent ductus arteriosus is a cause of significant comorbidity in premature infants. More than 80 percent of premature infants born weighing less than 750 g have a persistent patent ductus arteriosus (PDA) beyond the third day. Aorticopulmonary shunting at the PDA is the main cause of congestive heart failure (CHF) in neonates. Excessive pulmonary blood flow results in increased ventilatory dependency and contributes to bronchopulmonary dysplasia. Additionally, feeding difficulty, necrotizing enterocolitis, and intracranial hemorrhage may result from diastolic runoff from the systemic circulation. Plasma B-type natriuretic peptide (BNP) is secreted by ventricular myocytes in response to pressure or volume overload of the cardiac ventricles. BNP regulates extracellular fluid volume and blood pressure by natriuresis, diuresis, vasodilation, and antagonism of the renin-angiotensin-aldosterone system. BNP is present in small amounts in healthy people and in increased amounts in those with cardiac disease. BNP is of diagnostic and prognostic value in assessing CHF in adults and is used as a bedside screen for CHF, to rapidly differentiate between cardiac and pulmonary causes of respiratory difficulty, and as a screening tool for ventricular hypertrophy, ventricular diastolic dysfunction, transplant rejection, and risk for sudden death in adult CHF patients. In term neonates, BNP levels peak just after birth and decline during the first three months of life. Bioimmunoassay of plasma BNP has been correlated with the magnitude of PDA shunts in preterm infants. BNP levels can be measured rapidly at the bedside using an FDA-approved device that uses 0.25 mL of arterial, venous, or capillary blood. This system is used as a screen for CHF in adults. The authors conducted a study to determine whether the rapid bedside determination of BNP level could be used to predict the presence and magnitude of PDA in preterm neonates. For the study, newborn infants admitted to the neonatal intensive care unit had paired echocardiography and BNP measurements at enrollment and every four to five days. Twenty neonates (gestational age, approximately 28.6 weeks; birth weight, approximately 1,161 g) had 81 paired echocardiography and BNP determinations. BNP ranged from 5 to 3,900 pg/mL. Fifty-six of 81 echocardiograms showed PDA. Significant correlations were found between BNP and ductal size and degree of shunting. Correlation was greater in infants more than two days old. The authors found that BNP greater than 300 pg/mL predicted significant PDA, whereas BNP of less than 105 pg/mL predicted absence of significant PDA. The authors concluded that bedside measurement of BNP correlates with magnitude of PDA in premature newborns, particularly beyond day two, and may be useful in guiding diagnostic and management strategies.

Flynn PA, Da Graca RL, Auld PAM, et al. The use of a bedside assay for plasma B-type natriuretic peptide as a biomarker in the management of patent ductus arteriosus in premature neonates. J Pediatr. 2005;147:38–42.

Reprints: Dr. Patrick A. Flynn, Division of Pediatric Cardiology, F-695B, Weill-Cornell Center of New York-Presbyterian Hospital, 525 E. 68th St., New York, NY 10021;

Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.