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Leptospirosis is a common and underdiagnosed zoonosis. Two rapid assays for serological diagnosis of acute leptospirosis in diagnostic laboratories, the immunoglobulin M (IgM)-dipstick assay and the indirect hemagglutination assay (IHA), were evaluated and compared with standard assays. Sera were examined from 104 patients admitted to a hospital for investigation in a leptospirosis diagnostic protocol. Specimens for serology were taken on days 1 and 4 of the patients' hospital stay. Antibodies were detected using an IgM-enzyme-linked immunosorbent assay (ELISA), microscopic agglutination test (MAT), an IgM-dipstick assay, and an IHA. Fifty-one patients were found to have leptospirosis. The sensitivity of the IgM-dipstick assay was 98%, its specificity was 90.6%, its positive predictive value was 90.9%, and its negative predictive value was 98%. The sensitivity of the IHA was 92.2%, its specificity was 94.4%, its positive predictive value was 95.9%, and its negative predictive value was 92.7%. The standard IgM-ELISA and MAT, were positive in the first samples tested from 67 and 55% of the cases, respectively, and the rapid IgM-dipstick assay and IHA were positive in 71 and 49%, respectively, in the first sample tested. Both rapid assays are highly sensitive and specific. Neither requires specialized equipment, and both are suitable for use in diagnostic laboratories.

Since the publication of the "Three-color supplement to the NIAID/DAIDS Guideline for Flow Cytometric Immunophenotyping" in 1996 (1), significant scientific and technological advances in the development and production of reagents, instrumentation, and software have increased the use of multicolor flow cytometry in both research and clinical laboratories. With the increased adoption of three and four-color flow cytometry as the preferred methodology in determining patients' CD4 and CD8 T-cell counts, it has become apparent that a gating strategy that integrates the bright CD45 cells reduces interlaboratory and intralaboratory variability. Traditionally, a lymphocyte gate for immunophenotyping is derived from a bivariate frequency distribution histogram that includes 90°side scatter (SSC) and forward light scatter (FSC) frequency patterns. This type of histogram configuration is called a homogenous gating protocol (2). The advantage of the combination of bright CD45 fluorescence and light scatter, a heterogeneous gating protocol, was first reported in 1993 (3). Over the past few years, it has been determined that this alternative approach provides a more reproducible and accurate lymphocyte gate (4 -6). This heterogeneous method will be referred to as the CD45 gating method.The purpose of this article is to update the 1996 NIAID/ DAIDS Guideline and include the use of the CD45 gating method to minimize measurement variability with multicolor flow cytometry for the enumeration of T-cell subsets. Some information is provided about the advantages of four-color flow cytometry and the use of single-platform bead-based technology for determining absolute and percentage of lymphocytes. The addition of integrated fluorosphere counting provides a single-platform protocol that facilitates the simultaneous determination of both absolute and percentage of lymphocyte subsets. The specifications and recommendations were developed for use in laboratories supporting clinical trials and epidemiolog-

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Segments of the ospA gene of Borrelia burgdorferi were amplified by the polymerase chain reaction (PCR). Oligonucleotide primers used in the reaction flank a 309-base-pair segment within the ospA gene. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis or dot hybridization with a 32P-labeled probe. This segment was amplified in all strains of B. burgdorferi tested, but it was not detected in other bacterial species. An additional primer pair which has a broad specificity for conserved 16S rRNA sequences that are present in eubacteria amplified a 215-base-pair fragment in all organisms tested. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. We were able to detect 10 organisms per ml of blood or urine, using PCR with dot hybridization detection. DNA extraction is not required for sample preparation. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 animals, one blood specimen showed reactivity in the PCR.