There is growing concern that vancomycin has diminished activity for methicillin-resistant Staphylococcus aureus ( Despite its sustained in vitro microbiologic inhibitory activity, clinicians now question the continued utility of vancomycin for methicillin-resistant Staphylococcus aureus (MRSA) infections (18,23). Within the past 5 years, multiple reports have described MRSA strains with vancomycin MICs at the high end of the CLSI susceptibility range (MICs of 2 mg/liter) (6,18,22). Data suggest that vancomycin has reduced activity against MRSA infections, with vancomycin MICs at the high end of the CLSI susceptibility range (6,11,16,19,20).At the Albany Medical Center Hospital (AMCH), a large proportion of the MRSA bloodstream isolates have vancomycin MICs at the high end of the CLSI susceptibility range. The MIC 50 and MIC 90 for the 76 MRSA bloodstream isolates (59 patients) obtained by using the Etest method and recovered between

Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.

Mixed infections with seasonal influenza A virus strains are a common occurrence and an important source of genetic diversity. Prolonged viral shedding, as observed in immunocompromised individuals, can lead to mutational accumulation over extended periods. Recently, drug resistance was reported in immunosuppressed patients infected with the 2009 pandemic influenza A (H1N1) virus within a few days after oseltamivir treatment was initiated. To better understand the evolution and emergence of drug resistance in these circumstances, we used a deep sequencing approach to survey the viral population from an immunosuppressed patient infected with H1N1/2009 influenza and treated with neuraminidase inhibitors. This patient harbored 3 genetic variants from 2 phylogenetically distinct viral clades of pandemic H1N1/2009, strongly suggestive of mixed infection. Strikingly, one of these variants also developed drug resistance de novo in response to oseltamivir treatment. Immunocompromised individuals may, therefore, constitute an important source of genetic and phenotypic diversity, both through mixed infection and de novo mutation.

No abstract

SummaryT cell receptor (TCR)-recognizing regulatory cells, induced after vaccination with self-reactive T cells or TC1K peptides, have been shown to prevent autoimmunity. We have asked whether this regulation is involved in the maintenance of peripheral tolerance to myelin basic protein (MBP) in an autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). Antigen-induced EAE in (SJL X B10.PL)F1 mice is transient in that most animals recover permanently from the disease. Most of the initial encephalitogenic T cells recognize MBP Acl-9 and predominantly use the TCR V[38.2 gene segment. In mice recovering from MBP-induced EAE, regulatory CD4 + T cells (Treg) specific for a single immunodominant TCR peptide B5 (76-101) from framework region 3 of the V[38.2 chain, become primed. We have earlier shown that cloned B5-reactive Treg can specifically downregulate responses to Acl-9 and also protect mice from EAE. These CD4 Treg clones predominantly use the TCtL V[314 or VI33 gene segments. Here we have directly tested whether deletion/blocking of the Treg from the peripheral repertoire affects the spontaneous recovery from EAE. Treatment of F1 mice with appropriate V[3-specific monoclonal antibodies resulted in an increase in the severity and duration of the disease: even relapses were seen in one-third to one-half of the Treg-deleted mice. Interestingly, chronic disease in treated mice appears to be due to the presence of Acl-9-specific T cells. Thus, once self-tolerance to MBP is broken by immunization with the antigen in strong adjuvant, TCR peptide-specific CD4 Treg cells participate in reestablishing peripheral tolerance. Thus, a failure to generate Treg may be implicated in chronic autoimmune conditions.

In summary, these systems demonstrated excellent performance. Furthermore, each system has benefits which will ensure they will all have a niche in a clinical laboratory.

Patients with MRSA bloodstream infections in the ICU or with a history of vancomycin exposure should be considered at high risk of infection with strains for which vancomycin MICs are elevated. Appropriate and aggressive empirical therapy is required for these patients.

The therapeutic utility of a human immunodeficiency virus type 1 (HIV-1) protease inhibitor may depend on its intracellular concentration, which is a property of its uptake, metabolism, and/or efflux. Previous studies in our laboratory indicated that the addition of alpha 1 acid glycoprotein (alpha 1 AGP) to the medium markedly increased the amount of the drug required to limit infection in vitro. In this study, physiologically relevant concentrations of alpha 1 AGP and a radiolabeled inhibitor, A-80987, were used to determine both the uptake and activity of the agent in HIV-1-infected human peripheral blood mononuclear cells and cell lines. Both the uptake and efflux of 14C-labeled A-80987 were rapid (t1/2, < 5 min). Uptake of the drug was linearly dependent on the concentration but insensitive to the metabolic inhibitors KF, sodium cyanide, or CCCP (carbonyl cyanide m-chlorophenyl hydrazone). The amount of A-80987 which entered the cells was inversely proportional to the concentration of alpha 1 AGP (r2, 0.99) and directly proportional to the amount of extracellular non-protein-bound drug (r2, 0.99). Most importantly, the antiviral activity of the drug in HIV-1-infected peripheral blood mononuclear cells and MT-2 cells was directly related to the amount of intracellular A-80987. This study demonstrates that A-80987 binds to alpha 1 AGP, resulting in a free fraction below 10%. Cellular uptake of A-80987 is proportionally decreased in the presence of alpha 1 AGP, which results in less-than-expected antiviral activity. Importantly, we demonstrate for the first time that the inhibition of HIV protease is highly correlated with the amount of intracellular inhibitor.