Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei . In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1- orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia -like fimbrial/ Burkholderia thailandensis -like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin.

Coccidioides immitis and Coccidioides posadasii are soil fungi endemic to desert regions of the southwestern United States, and the causative agents of valley fever, or coccidioidomycosis. Studies have shown that the distribution of Coccidioides in soils is sporadic and cannot be explained by soil characteristics alone, suggesting that biotic and other abiotic factors should be examined. However, tools to reliably and robustly screen the large number of soils needed to investigate these potential associations have not been available. Thus, we developed a real-time polymerase chain reaction (PCR) assay for testing environmental samples by modifying CocciDx, an assay validated for testing clinical specimens to facilitate coccidioidomycosis diagnosis. For this study, we collected soil samples from previously established locations of C. posadasii in Arizona and new locations in fall 2013 and spring 2014, and screened the extracted DNA with the new assay known as CocciEnv. To verify the presence of Coccidioides in soil using an alternate method, we employed next generation amplicon sequencing targeting the ITS2 region. Results show our modified assay, CocciEnv, is a rapid and robust method for detecting Coccidioides DNA in complex environmental samples. The ability to test a large number of soils for the presence of Coccidioides is a much-needed tool in the understanding of the ecology of the organism and epidemiology of the disease and will greatly improve our understanding of this human pathogen.

Widespread resistance among circulating influenza A strains to at least one of the anti-influenza drugs is a major public health concern. A triple combination antiviral drug (TCAD) regimen comprised of amantadine, oseltamivir, and ribavirin has been shown to have synergistic and broad spectrum activity against influenza A strains, including drug resistant strains. Here, we used mathematical modeling along with three different experimental approaches to understand the effects of single agents, double combinations, and the TCAD regimen on resistance in influenza in vitro, including: 1) serial passage at constant drug concentrations, 2) serial passage at escalating drug concentrations, and 3) evaluation of the contribution of each component of the TCAD regimen to the suppression of resistance. Consistent with the modeling which demonstrated that three drugs were required to suppress the emergence of resistance in influenza A, treatment with the TCAD regimen resulted in the sustained suppression of drug resistant viruses, whereas treatment with amantadine alone or the amantadine-oseltamivir double combination led to the rapid selection of resistant variants which comprised ∼100% of the population. Furthermore, the TCAD regimen imposed a high genetic barrier to resistance, requiring multiple mutations in order to escape the effects of all the drugs in the regimen. Finally, we demonstrate that each drug in the TCAD regimen made a significant contribution to the suppression of virus breakthrough and resistance at clinically achievable concentrations. Taken together, these data demonstrate that the TCAD regimen was superior to double combinations and single agents at suppressing resistance, and that three drugs at a minimum were required to impede the selection of drug resistant variants in influenza A virus. The use of mathematical modeling with multiple experimental designs and molecular readouts to evaluate and optimize combination drug regimens for the suppression of resistance may be broadly applicable to other infectious diseases.

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism's phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing. In addition, 29 publicly available whole genome sequences were included to create a single nucleotide polymorphism (SNP)-based phylogenetic tree encompassing 11 distinct lineages. All strains of a particular sequence type fell into the same clade with clear groupings of the major clonal complexes of CC8, CC5, CC30, CC45 and CC1. Using a novel analysis method, we plotted the homoplasy density and SNP density across the whole genome and found evidence of recombination throughout the entire chromosome, but when we examined individual clonal lineages we found very little recombination. However, when we analyzed three branches of multiple lineages, we saw intermediate and differing levels of recombination between them. These data demonstrate that in S. aureus, recombination occurs across major lineages that subsequently expand in a clonal manner. Estimated mutation rates for the CC8 and CC5 lineages were different from each other. While the CC8 lineage rate was similar to previous studies, the CC5 lineage was 100-fold greater. Fifty known virulence genes were screened in all genomes in silico to determine their distribution across major clades. Thirty-three genes were present variably across clades, most of which were not constrained by ancestry, indicating horizontal gene transfer or gene loss.

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ObjectiveWe examined the activity of an HIV-1 immunogen (Remune) on viral load, CD4 cells and HIV-1 speci®c immunity. MethodsPlasma and peripheral blood mononuclear cells were obtained in a prede®ned random subset of subjects (n = 252) from a multicentre, double-blind, adjuvant-controlled phase III clinical endpoint study. ResultsThe subjects treated with the HIV-1 immunogen had a signi®cantly greater decline in viral load at multiple time points (P < 0.05), a trend towards increased CD4+ T cell counts and signi®cantly enhanced HIV-1 speci®c immune responses as measured by HIV-1 lymphocyte proliferation (P < 0.001) compared to the adjuvant control group. Furthermore, in the HIV-1 immunogen treated group, enhanced HIV-1 speci®c lymphocyte proliferative immune responses were associated with decreased HIV-1 plasma RNA. ConclusionThese results suggest that, in a prede®ned, random subset of subjects, a bene®cial effect of the HIV-1 immunogen was observed on viral load, CD4+ T cells, and HIV-speci®c immunity. These differences were observed in a background of multiple drug therapies. Ongoing trials are evaluating the effect of the combination of this HIV-1 speci®c, immune-based therapy with potent antiviral drug therapy on virological outcomes. IntroductionRecent advances in HIV-1 antiviral drug therapy have had a signi®cant impact on AIDS morbidity and mortality in industrialized countries [1±3]. In North America and Europe antiviral drug`cocktails' (antiretroviral therapy), which include a protease inhibitor (PI), have become the standard of care [4]. Nevertheless, reservoirs of replication competent HIV-1 appear to be maintained despite chronic treatment with combination antiviral drug therapy [5,6]. This may, in part, explain some recent reports suggesting the occurrence of virologic failure in approximately 20± 40% of patients after 2 years of potent antiviral drug therapy [7,8] We examined the effect of HIV-1 speci®c, immunebased therapy in a large, multicentre, phase III, clinical endpoint study, which began in the spring of 1996, just prior to the approval of the newer more potent antiviral drug therapies such as PIs. With the anticipation that these newer agents would become part of the standard of care, subjects were allowed to take any combination of antiviral drug therapy and to switch combinations without restrictions during this trial. Asymptomatic HIV-1 infected subjects with CD4 cell counts of 300±549 cells/mL were randomized to receive the HIV-1 immunogen (Remune) or the adjuvant control (incomplete Freund's adjuvant; IFA) and followed for an average of 2.5 years with the primary endpoint being time to clinical progression or death. The trial was halted by an independent data safety monitoring board (DSMB) due to lack of signi®cant differences between the two treatment groups in terms of clinical endpoints, although signi®cant effects were observed on CD4+ T cell counts favouring the HIV-1 immunogen group (P < 0.05), as reported elsewhere [23]. Within this trial, a randomly selected, prede®ned subset...

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Backgroundspa typing is a common genotyping tool for methicillin-resistant Staphylococcus aureus (MRSA) in Europe. Given the high prevalence of dominant clones, spa-typing is proving to be limited in its ability to distinguish outbreak isolates from background isolates. New molecular tools need to be employed to improve subtyping of dominant local MRSA strains (e.g., spa type t003).MethodsPhylogenetically critical, or canonical, SNPs (can-SNPs) were identified as subtyping targets through sequence analysis of 40 MRSA whole genomes from Luxembourg. Real-time PCR assays were designed around target SNPs and validated using a repository of 240 previously sub-typed and epidemiologically characterized Luxembourg MRSA isolates, including 153 community and hospital isolates, 69 isolates from long term care (LTC) facilities, and 21 prospectively analyzed MRSA isolates. Selected isolates were also analyzed by whole genome SNP typing (WGST) for comparison to the SNP assays and other subtyping techniques.ResultsFourteen real-time PCR assays were developed and validated, including two assays to determine presence of spa t003 or t008. The other twelve assays successfully provided a high degree of resolution within the t003 subtype. WGST analysis of the LTC facility isolates provided greater resolution than other subtyping tools, identifying clusters indicative of ongoing transmission within LTC facilities.ConclusionscanSNP-based PCR assays are useful for local level MRSA phylotyping, especially in the presence of one or more dominant clones. The assays designed here can be easily adapted for investigating t003 MRSA strains in other regions in Western Europe. WGST provides substantially better resolution than other typing methods.