Erin Kelley

Triple Combination Antiviral Drug (TCAD) Composed of Amantadine, Oseltamivir, and Ribavirin Impedes the Selection of Drug-Resistant Influenza A Virus

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.

Rapid quantification of single-nucleotide mutations in mixed influenza A viral populations using allele-specific mixture analysis

Monitoring antiviral resistance in influenza is critical to public health epidemiology and pandemic preparedness activities. Effective monitoring requires methods to detect low-level resistance and to monitor the change in resistance as a function of time and drug treatment. Resistance-conferring single-nucleotide mutations in influenza virus are ideal targets for such methods. In the present study, fives sets of paired TaqMan allele-specific PCR (ASPCR) assays were developed and validated for quantitative single-nucleotide polymorphism (SNP) analysis. This novel method using Delta Ct is termed allele-specific mixture analysis (ASMA) or FluASMA. The FluASMA assays target L26F, V27A, A30T, and S31N mutations in the A/Albany/1/98 (H3N2) M2 gene and H275Y mutation in the A/New Caledonia/20/99 (H1N1) NA gene and have a limit of quantification of 0.25-0.50% mutant. The error for % mutant estimation was less than 10% in all FluASMA assays, with intra-run Delta Ct coefficient of variance (CoV) at <or=2% and inter-run Delta Ct CoV at <or=5%. Results from the current study demonstrate that FluASMA is a highly sensitive and quantitative SNP analysis method, even for minor mutant components (<1%).

Comparison of TaqMan PCR Assays for Detection of the Melioidosis Agent Burkholderia pseudomallei in Clinical Specimens

ABSTRACT 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.

Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organisms 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.

Rapid and robust phylotyping of spa t003, a dominant MRSA clone in Luxembourg and other European countries

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.

Direct detection of Coccidioides from Arizona soils using CocciENV, a highly sensitive and specific real-time PCR assay

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.

Real-time PCR assays for genotyping of Cryptococcus gattii in North America

BackgroundCryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen.MethodsWe developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human.ResultsValidation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA.ConclusionsThe (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.