Development and validation of a liquid chromatography coupled to tandem mass spectrometry method for the monitoring of temsavir plasma concentrations in people living with HIV.

A majority of people living with HIV (PLWH) now have access to HIV treatment with high antiviral potency and favorable tolerability profile. However, in some treatment experienced PLWH viral strains resistant to major current classes of antiretrovirals have emerged, usually due to periods with continued virus replication in the presence of failing drug regimens and thus selection pressure. In such context, new treatment options are therefore needed. Fostemsavir (RUKOBIA®) is the prodrug of temsavir, a first-in-class oral attachment inhibitor approved for the treatment of heavily treatment-experienced adults with multidrug-resistant HIV-1 infection. In this case RUKOBIA® is part of a complex regimen of antiretroviral drugs, often in addition to other drugs for chronic co-morbidities (e.g., heart disease, diabetes mellitus, hepatic and renal impairment, etc). In such a multi-drug regimen context, therapeutic drug monitoring (TDM) of temsavir can be necessary to exclude or adjust for relevant drug-drug interactions. A highly selective assay by liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS) was therefore developed for the quantification of temsavir in human plasma. A convenient sample preparation using protein precipitation with acetonitrile followed by supernatant dilution was carried out. Temsavir and fostemsavir were separated in less than 2 min using a multi-step UPLC gradient, thus ensuring adequate quantification of temsavir. The assay for the quantification of temsavir was extensively validated over the large range of clinically relevant concentrations from 1 to 10,000 ng/mL, in accordance with international bioanalytical method guidelines. The method achieves excellent performance in terms of trueness (99.7 - 105.3%), repeatability and intermediate precision (both from 1.6% to 5.8%). This LC-MS/MS method is now part of the routine analyses of the Laboratory of the Service of Clinical Pharmacology of Lausanne (CHUV), Switzerland, as an integrated part of our general TDM Service for antiretrovirals

Role of previous hospitalization in clinically-significant MRSA infection among HIV-infected inpatients: results of a case-control study

<p>Abstract</p> <p>Background</p> <p>HIV-infected subjects have high incidence rates of <it>Staphylococcus aureus </it>infections, with both methicillin-susceptible and methicillin-resistant (MRSA) strains. Possible explanations could include the high burden of colonization, the behavioral risk factors, and the frequent exposures to health care facilities of HIV-infected patients. The purpose of the study was to assess the risk factors for clinically- significant methicillin-resistant <it>Staphylococcus aureus </it>(CS-MRSA) infections in HIV-infected patients admitted to Infectious Diseases Units.</p> <p>Methods</p> <p>From January 1, 2002 to December 31, 2005, we conducted a retrospective case-control (1:2) study. We identified all the cases of CS-MRSA infections in HIV-infected patients admitted to the National Institute for Infectious Diseases (INMI) "Lazzaro Spallanzani" in the 4-year study period. A conditional logistic regression model was used to identify risk factors for CS-MRSA infection.</p> <p>Results</p> <p>We found 27 CS-MRSA infections, i.e. 0.9 CS-MRSA infections per 100 HIV-infected individuals cared for in our Institute. At multivariate analysis, independent predictors of CS-MRSA infection were cumulative hospital stay, invasive procedures in the previous year, and low CD4 cell count. Particularly, the risk for CS-MRSA increased by 14% per an increase of 5 days hospitalization in the previous year. Finally, we identified a low frequency of community-acquired MRSA infections (only 1 of 27; 3.7%) among HIV-infected patients.</p> <p>Conclusion</p> <p>Clinicians should be aware of the risk for CS-MRSA infection in the clinical management of HIV-infected patients, especially in those patients with a low CD4 cell count, longer previous hospital stay, and previous invasive procedures.</p

Inclusive V0V^0 Production Cross Sections from 920 GeV Fixed Target Proton-Nucleus Collisions

Inclusive differential cross sections $d\sigma_{pA}/dx_F$ and $d\sigma_{pA}/dp_t^2$ for the production of \kzeros, \lambdazero, and \antilambda particles are measured at HERA in proton-induced reactions on C, Al, Ti, and W targets. The incident beam energy is 920 GeV, corresponding to $\sqrt {s} = 41.6$ GeV in the proton-nucleon system. The ratios of differential cross sections \rklpa and \rllpa are measured to be $6.2\pm 0.5$ and $0.66\pm 0.07$, respectively, for \xf $\approx-0.06$. No significant dependence upon the target material is observed. Within errors, the slopes of the transverse momentum distributions $d\sigma_{pA}/dp_t^2$ also show no significant dependence upon the target material. The dependence of the extrapolated total cross sections $\sigma_{pA}$ on the atomic mass $A$ of the target material is discussed, and the deduced cross sections per nucleon $\sigma_{pN}$ are compared with results obtained at other energies.Comment: 17 pages, 7 figures, 5 table

Methicillin-Resistant Staphylococcus aureus Infection and Hospitalization in High-Risk Patients in the Year following Detection

Many studies have evaluated methicillin-resistant Staphylococcus aureus (MRSA) infections during single hospitalizations and subsequent readmissions to the same institution. None have assessed the comprehensive burden of MRSA infection in the period after hospital discharge while accounting for healthcare utilization across institutions.We conducted a retrospective cohort study of adult patients insured by Harvard Pilgrim Health Care who were newly-detected to harbor MRSA between January 1991 and December 2003 at a tertiary care medical center. We evaluated all MRSA-attributable infections associated with hospitalization in the year following new detection, regardless of hospital location. Data were collected on comorbidities, healthcare utilization, mortality and MRSA outcomes. Of 591 newly-detected MRSA carriers, 23% were colonized and 77% were infected upon detection. In the year following detection, 196 (33%) patients developed 317 discrete and unrelated MRSA infections. The most common infections were pneumonia (34%), soft tissue (27%), and primary bloodstream (18%) infections. Infections occurred a median of 56 days post-detection. Of all infections, 26% involved bacteremia, and 17% caused MRSA-attributable death. During the admission where MRSA was newly-detected, 14% (82/576) developed subsequent infection. Of those surviving to discharge, 24% (114/482) developed post-discharge infections in the year following detection. Half (99/185, 54%) of post-discharge infections caused readmission, and most (104/185, 55%) occurred over 90 days post-discharge.In high-risk tertiary care patients, newly-detected MRSA carriage confers large risks of infection and substantial attributable mortality in the year following acquisition. Most infections occur post-discharge, and 18% of infections associated with readmission occurred in hospitals other than the one where MRSA was newly-detected. Despite gains in reducing MRSA infections during hospitalization, the risk of MRSA infection among critically and chronically ill carriers persists after discharge and warrants targeted prevention strategies

Increased prevalence of methicillin-resistant Staphylococcus aureus nasal colonization in household contacts of children with community acquired disease

<p>Abstract</p> <p>Background</p> <p>To measure Methicillin-resistant <it>Staphylococcus aureus </it>(MRSA) nasal colonization prevalence in household contacts of children with current community associated (CA)-MRSA infections (study group) in comparison with a group of household contacts of children without suspected <it>Staphylococcus aureus </it>infection (a control group).</p> <p>Methods</p> <p>This is a cross sectional study. Cultures of the anterior nares were taken. Relatedness of isolated strains was tested using pulse field gel electrophoresis (PFGE).</p> <p>Results</p> <p>The prevalence of MRSA colonization in the study group was significantly higher than in the control group (18/77 (23%) vs 3/77 (3.9%); p ≤ 0.001). The prevalence of SA colonization was 28/77 (36%) in the study group and 16/77 (21%) in the control group (p = 0.032). The prevalence of SA nasal colonization among patients was 6/24 (25%); one with methicillin-susceptible <it>S. aureus </it>(MSSA) and 5 with MRSA. In the study (patient) group, 14/24 (58%) families had at least one household member who was colonized with MRSA compared to 2/29 (6.9%) in the control group (p = 0.001). Of 69 total isolates tested by PFGE, 40 (58%) were related to USA300. Panton-Valetine leukocidin (PVL) genes were detected in 30/52 (58%) tested isolates. Among the families with ≥1 contact colonized with MRSA, similar PFGE profiles were found between the index patient and a contact in 10/14 families.</p> <p>Conclusions</p> <p>Prevalence of asymptomatic nasal carriage of MRSA is higher among household contacts of patients with CA-MRSA disease than control group. Decolonizing such carriers may help prevent recurrent CA-MRSA infections.</p

Staphylococcal Toxic Shock Syndrome 2000–2006: Epidemiology, Clinical Features, and Molecular Characteristics

Circulating strains of Staphylococcus aureus (SA) have changed in the last 30 years including the emergence of community-associated methicillin-resistant SA (MRSA). A report suggested staphylococcal toxic shock syndrome (TSS) was increasing over 2000-2003. The last population-based assessment of TSS was 1986.Population-based active surveillance for TSS meeting the CDC definition using ICD-9 codes was conducted in the Minneapolis-St. Paul area (population 2,642,056) from 2000-2006. Medical records of potential cases were reviewed for case criteria, antimicrobial susceptibility, risk factors, and outcome. Superantigen PCR testing and PFGE were performed on available isolates from probable and confirmed cases.Of 7,491 hospitalizations that received one of the ICD-9 study codes, 61 TSS cases (33 menstrual, 28 non-menstrual) were identified. The average annual incidence per 100,000 of all, menstrual, and non-menstrual TSS was 0.52 (95% CI, 0.32-0.77), 0.69 (0.39-1.16), and 0.32 (0.12-0.67), respectively. Women 13-24 years had the highest incidence at 1.41 (0.63-2.61). No increase in incidence was observed from 2000-2006. MRSA was isolated in 1 menstrual and 3 non-menstrual cases (7% of TSS cases); 1 isolate was USA400. The superantigen gene tst-1 was identified in 20 (80%) of isolates and was more common in menstrual compared to non-menstrual isolates (89% vs. 50%, p = 0.07). Superantigen genes sea, seb and sec were found more frequently among non-menstrual compared to menstrual isolates [100% vs 25% (p = 0.4), 60% vs 0% (p<0.01), and 25% vs 13% (p = 0.5), respectively].TSS incidence remained stable across our surveillance period of 2000-2006 and compared to past population-based estimates in the 1980s. MRSA accounted for a small percentage of TSS cases. tst-1 continues to be the superantigen associated with the majority of menstrual cases. The CDC case definition identifies the most severe cases and has been consistently used but likely results in a substantial underestimation of the total TSS disease burden

Molecular Typing and Phenotype Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Blood in Taiwan

BACKGROUND: Staphylococcus aureus causes a variety of severe infections such as bacteremia and sepsis. At present, 60-80% of S. aureus isolates from Taiwan are methicillin resistant (MRSA). It has been shown that certain MRSA clones circulate worldwide. The goals of this study were to identify MRSA clones in Taiwan and to correlate the molecular types of isolates with their phenotypes. METHODS: A total of 157 MRSA isolates from bacteremic patients were collected from nine medical centers. They were typed based on polymorphisms in agr, SCCmec, MLST, spa, and dru. Phenotypes characterized included Panton-Valentine leucocidin (pvl), inducible macrolide-lincosamide-streptogramin B resistance (MLSBi), vancomycin (VA) and daptomycin (DAP) minimal inhibitory concentrations (MIC), and superantigenic toxin gene profiles. Difference between two consecutive samples was determined by Mann-Whitney-U test, and difference between two categorical variables was determined by Fisher's exact test. RESULTS: Four major MRSA clone complexes CC1, CC5, CC8, and CC59 were found, including 4 CC1, 9 CC5, 111 CC8, and 28 CC59 isolates. These clones had the following molecular types: CC1: SCCmecIV and ST573; CC5: SCCmecII and ST5; CC8: SCCmecIII, ST239, and ST241, and CC59: SCCmecIV, SCCmecV(T), ST59, and ST338. The toxin gene profiles of these clones were CC1: sec-seg-(sei)-sell-selm-(seln)-selo; CC5: sec-seg-sei-sell-selm-(seln)-selp-tst1; CC8: sea-selk-selq, and CC59: seb-selk-selq. Most isolates with SCCmecV(T), ST59, spat437, and dru11 types were pvl(+) (13 isolates), while multidrug resistance (≥4 antimicrobials) were associated with SCCmecIII, ST239, spa t037, agrI, and dru14 (119 isolates) (p<0.001). One hundred and twenty four isolates with the following molecular types had higher VA MIC: SCCmecII and SCCmecIII; ST5, ST239, and ST241; spa t002, t037, and t421; dru4, dru10, dru12, dru13, and dru14 (p<0.05). No particular molecular types were found to be associated with MLSBi phenotype. CONCLUSIONS: Four major MRSA clone complexes were found in Taiwan. Further studies are needed to delineate the evolution of MRSA isolates

Capsaicin Protects Mice from Community-Associated Methicillin-Resistant Staphylococcus aureus Pneumonia

BACKGROUND: α-toxin is one of the major virulence factors secreted by most Staphylococcus aureus strains, which played a central role in the pathogenesis of S. aureus pneumonia. The aim of this study was to investigate the impact of capsaicin on the production of α-toxin by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA 300 and to further assess its performance in the treatment of CA-MRSA pneumonia in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: The in vitro effects of capsaicin on α-toxin production by S. aureus USA 300 were determined using hemolysis, western blot, and real-time RT-PCR assays. The influence of capsaicin on the α-toxin-mediated injury of human alveolar epithelial cells was determined using viability and cytotoxicity assays. Mice were infected intranasally with S. aureus USA300; the in vivo protective effects of capsaicin against S. aureus pneumonia were assessed by monitoring the mortality, histopathological changes and cytokine levels. Low concentrations of capsaicin substantially decreased the production of α-toxin by S. aureus USA 300 without affecting the bacterial viability. The addition of capsaicin prevented α-toxin-mediated human alveolar cell (A549) injury in co-culture with S. aureus. Furthermore, the in vivo experiments indicated that capsaicin protected mice from CA-MRSA pneumonia caused by strain USA 300. CONCLUSIONS/SIGNIFICANCE: Capsaicin inhibits the production of α-toxin by CA-MRSA strain USA 300 in vitro and protects mice from CA-MRSA pneumonia in vivo. However, the results need further confirmation with other CA-MRSA lineages. This study supports the views of anti-virulence as a new antibacterial approach for chemotherapy

Global Changes in Staphylococcus aureus Gene Expression in Human Blood

Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LACΔhlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LACΔhlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LACΔhlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence