RESUMEN
We have developed a single nucleotide polymorphism (SNP) detection microarray for the human pathogenic yeast, Candida albicans, consisting of synthetic oligonucleotides bound to microscope slides. The array consists of multiple replicates of 79 SNPs, derived from 19 discrete loci located on all eight chromosomes. These loci include seven genes consisting of 57 SNPs that comprise a multi-locus sequence typing (MLST) consensus scheme for the species. The remaining 22 SNPs are from 12 additional loci located at intervals on the remaining chromosomes. In order to include highly informative polymorphisms from the MLST set on the array we performed a linkage analysis of major genotypes between the two pairs of MLST-linked genes. In addition, we performed a matched-set analysis for each SNP located within individual MLST loci. This analysis resulted in the reduction of informatively redundant mutations in the array for a large percentage of strains. We believe that a SNP array will be helpful in extending our knowledge of the epidemiology and genetics of C. albicans as a supplement to MLST typing.
Asunto(s)
Candida albicans/genética , Técnicas de Tipificación Micológica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Alelos , Secuencia de Bases , Candida albicans/clasificación , Cromosomas Fúngicos/genética , ADN de Hongos/genética , ADN Intergénico/genética , Ligamiento Genético , Humanos , Mutación , Recombinación Genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection. METHODS: Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens. RESULTS: Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1ß for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1ß, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines. CONCLUSIONS: Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.