Over one-third of African-American MGUS and multiple myeloma patients are carriers of hyperphosphorylated paratarg-7, an autosomal dominantly inherited risk factor for MGUS/MM.

As hyperphosphorylated paratarg-7 (pP-7) carrier state was shown to be the first molecularly defined autosomal dominantly inherited risk factor for monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma (MM) in a European population, the prevalence of pP-7 carrier state among African-Americans who have a significantly higher incidence of MGUS/MM is of interest. We therefore determined pP-7 carrier state and paraproteins with specificity for P-7 in African-American, European and Japanese patients with MGUS/MM and healthy controls. By isoelectric focusing and ELISA, a paratarg-7-specific paraprotein and the associated pP-7 carrier state was observed in 30/81 (37.0%) African-American, 42/252 (16.7%) European and 7/176 (4.0%) Japanese MGUS/MM patients (p < 0.001). A pP-7 carrier state was found in 11/100 (11.0%) African-American, 8/550 (1.5%) European and 1/278 (0.4%) Japanese healthy controls (p < 0.001), resulting in an odds ratio for MGUS/MM of 4.8 (p < 0.001) among African-American, 13.6 among European (p < 0.001) and 11.5 (p = 0.023) among Japanese carriers of pP-7. We conclude that pP-7 carriers are most prevalent among African-Americans, but a pP-7 carrier state is the strongest molecularly defined single risk factor for MGUS/MM known to date in all three ethnic groups. The high prevalence of pP-7 carriers among African-American patients emphasizes a predominant role of this genetic factor in the pathogenesis of these diseases. The large number of pP7 African-American patients and controls should facilitate the identification of the SNP or mutation underlying the pP-7 carrier state.

Antifungal susceptibility testing of Candida isolates from the Candida surveillance study.

Candida species are a common cause of nosocomial bloodstream infections. Recent surveillance has shown an increase in the relative proportion of infections caused by Candida glabrata, which has reduced susceptibility to fluconazole. We undertook sentinel surveillance with antifungal susceptibility testing to monitor the trends in the proportions of various Candida species causing invasive disease. Forty-one institutions participated in the Candida Surveillance Study. All isolates were submitted to a central laboratory for identification and susceptibility testing. Susceptibility testing was performed in compliance with CLSI guidelines using a custom, broth dilution, microtiter system. There were 5,900 isolates submitted for identification and antifungal susceptibility testing. The distribution of species was as follows: C. albicans, 2,567 (43.5%) isolates; C. glabrata, 1,464 (24.8%) isolates; C. parapsilosis, 1,048 (17.8%) isolates; C. tropicalis, 527 (8.9%) isolates; C. krusei, 109 (1.9%) isolates; C. lusitaniae, 76 (1.3%) isolates; and other Candida species, 109 (1.9%) isolates. Resistance to fluconazole occurred in 1.2% of C. albicans isolates, 5.9% of C. glabrata isolates, 0.3% of C. parapsilosis isolates, and 0.4% of C. tropicalis isolates. Resistance to fluconazole was highly predictive of resistance to voriconazole. Resistance to echinocandins was rarely found, occurring in only 0.2% of all isolates. The rate of fluconazole susceptibility increased significantly from 87.5% in 2005 to 97.4% in 2007. The proportion of cases of disease caused by various Candida species did not change appreciably between 2004 and 2007, and the rate of antifungal susceptibility was high.

Real-time PCR quantitation of subtype C HIV DNA in a Zambian discordant couple cohort.

To study the viral sequence diversity that is characteristic of HIV infection, PCR amplification and sequencing of viral genes is an essential step. However, a limitation of traditional PCR methods is that one viral target may be preferentially amplified over another when multiple sequences are present. This presents a particular problem when conclusions about diversity are made from one or only a few PCRs. One way to avoid resampling is to perform a large number of PCR amplifications on a single template; however, this requires that extensive dilution series be carried out on each patient sample to identify the appropriate concentration of input DNA. Here we describe the development and implementation of a quantitative real-time PCR (qPCR) method that detects a short sequence in gag and is optimized to detect subtype C HIV sequences. The standard curve was externally validated using two chronically infected cell lines carrying a known number of HIV copies per genome, and this assay yielded reproducible and accurate measurements on patient DNA samples over a wide range of input targets. The qPCR assay results were consistent with those obtained by the traditional limiting dilution method yet entailed only a fraction of the time and reagents required for the latter. This robust and quantitative real-time assay can be used to ensure that each viral sequence obtained through PCR represents a single template for studies in which the diversity of the entire population must be accurately portrayed, and can readily be applied to other research settings and viral subtypes.

Prognostic significance of leukopenia at the time of diagnosis in acute myeloid leukemia.

We investigated the clinical significance of leukopenia at the time of diagnosis in a cohort of 225 patients with newly diagnosed acute myeloid leukemia (AML) at a single institution. Leukocyte count was treated as a continuous variable and, using a receiver operating characteristic curve (ROC), a cutoff of 3,600/µL had the best sensitivity and specificity for remission (complete remission [CR]), relapse-free survival [RFS], and overall survival [OS]). In a multivariable model, leukopenia at diagnosis had no effects on CR (hazard ratio [HR] = 2.02; confidence interval [CI], 0.9-4.3; P = .07), RFS (HR = 0.93; CI, 0.5-1.5; P = .8), or OS (HR = 1.05; CI, 0.7-1.5; P = .7). No differential expression of cell surface molecules (CD34, c-Kit, CXCR4, PECAM, VLA2, VLA-, VLA4, VLA5, and FLT3) was observed on simultaneously obtained marrow and blood blasts in the high- vs. low-leukocyte groups. We conclude that leukopenia at diagnosis carries no prognostic significance in AML.

A general approach to the analysis of errors and failure modes in the base-calling function in automated fluorescent DNA sequencing.

We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.