Project I. Azole resistance in Candida glabrata? CgPDR1 a major contributing factor in fluconazole resistance arising during therapy? ? Azole resistance frequently develops during prolonged treatment in Candida glabrata, a yeast with intrinsically low susceptibility to azoles. We have shown in our previous study that one of the resistance mechanisms was increased expression of the transcriptional regulator gene, CgPDR1, which upregulated the expression of CgCDR1, a multidrug transporter gene. Five putative functional domains (DNA-binding, inhibitory, fungal-specific transcriptional factor, nuclear localization, and activation) were identified in the CgPdr1p based on its similarity to Saccharomyces cerevisiae Pdr1p. To date, we have analyzed a total of 10 pairs clinical susceptible and resistant isolates. Comparison of the CgPDR1 promoter and ORF deduced amino acid sequences within each pair revealed that each of the 10 resistant isolates has acquired a different single mutation. CgPDR1 from one susceptible and ten resistant isolates were introduced into a mutated cgpdr1 locus of an azole-susceptible laboratory strain via an integrative transformation. Fluconazole MIC analysis showed that the ten mutants containing CgPDR1 open reading frames of resistant isolates had an increase in azole resistance compared to the laboratory wild-type strain and to the mutant which contained the CgPDR1 from the susceptible isolate. This confirmed that the CgPDR1 mutations in the ten resistant strains accounted for their fluconazole resistance. In addition, Northern hybridization and real time PCR were used to analyze the effect of the altered CgPdr1p on the expression of CgPDR1 and the CgPDR1 target gene, CgCDR1. The latter gene codes for the major azole drug efflux pump. The gene expression data indicated that the expression of CgPDR1 and CgCDR1 were upregulated in all ten strains harboring the CgPDR1 from the resistant isolates compared to the strain harboring the CgPDR1 from the susceptible isolates as well as the laboratory wild-type strain. Six out of the total ten mutations occurred in the inhibitory domain while one in the activation domain and one in the fungal-specific transcriptional factor domain. Importantly, two mutations (N764I, R772I) occurred in an area of undefined function, which is in the vicinity of the nuclear localization domain. We concluded that CgPDR1 is a major contributing factor in clinical azole resistance and this region of undefined function is significant in the function of CgPdr1p and azole resistance.? ? ? The two putative Pleiotropic Drug Response Elements (PDREs) in the CgPDR1 promoter are not essential for the expression of CgPDR1? ? We postulated that the transcriptional regulator, CgPDR1, may autoregulate itself, based on the above findings. Autoregulation could be direct or mediated by other genes interacting with CgPDR1 as a target. If autoregulation were direct, the PDREs upstream of CgPDR1 should be similar to those of other CgPDR1 gene targets. Based on homology with Saccharomyces cerevisiae, two putative CgPDR1 PDREs were identified upstream of CgPDR1. Two test whether these PDREs were responsible for CgPDR1 expression, the CgPDR1 promoter was fused to a LacZ reporter. Three reporter constructs with various lengths of CgPDR1 promoter, 1.4kb, 1.1kb, and 0.5kb respectively, were introduced into Candida glabrata. The beta-galactosidase activity assay revealed that the strain carried the reporter construct with the 0.5kb promoter had diminished beta-galactosidae activity compared to the two strains carrying the longer promoter region. Two putative PDREs were found at -557 to -550 (TCCGTGGA) and -701 to -694 away from the initiation codon ATG. Mutations (TAAGTGGA) were introduced into the two putative PDREs to investigate whether the PDRE(s) were important in the function of the CgPDR1 promoter. Surprisingly, mutations in either or both of the two putative PDRE did not abolish the beta-galactosidae activity. The data indicated that even though the region between 0.5kb and -1.1kb contains important sequences for CgPDR1 promoter activity but the two putative PDREs were not essential for the CgPDR1 expression. ? ? Project II Cryptococcosis in patients with idiopathic CD4 lymphocytopenia (ICL)? Analysis of data obtained from a prospective study of patients with ICL: 39 patients were followed for an average of 5 years after diagnosis. Cryptococcal and non tuberculous mycobacterial infections accounted for the majority of presenting opportunistic infections. There was a pattern of increased activation and turnover in CD4 but not CD8 T lymphocytes, manifested by increased HLA-DR and KI67 expression and increased ex vivo bromodeoxyuridine (BrdU) uptake. The level of CD4 activation and turnover were positively correlated with each other but negatively correlated with the level of CD4 counts. Thirty two (81.6%) patients had low CD4 numbers throughout the follow up period and 7 returned to normal CD4 numbers after an average of 31 months. The follow up was significant for manifestations of human papilloma virus (HPV) infections, dermatomal varicella zoster virus (VZV) infections and autoimmune diseases although more severe opportunistic infections were seen in 4 patients. Three died from various causes and three from ICL related opportunistic infections (Epstein bar virus-related lymphoproliferative disease, progressive multifocal leukoencephalopathy and mycobacterium avium complex infection). The majority had a benign clinical course. A low CD8 count at disease presentation was identified as predictor of poor outcome? ? Project III Voriconazole toxicity? As a part of an ongoing prospective study of voriconazole blood levels and toxicity, we investigated hallucinations after treatment of 66 patients with voriconazole. We reported eight patients (12.1%) with hematologic malignancies who developed hallucinations after initiation of voriconazole. In seven, hallucinations appeared in the first day of the intravenous formulation. All patients had visual hallucinations, 5 also had auditory. Three of them had also visual changes such as blurred vision and photopsia. The theme of hallucinations seemed to be unique for each patient but was only frightening or disturbing in three. All patients remained oriented, alert and able to recognize their hallucinations as unreal. Voriconazole was discontinued in five of the eight patients due to the severity of symptoms and in two hallucinations disappeared when intravenous therapy was changed to oral. The voriconazole blood level on the day of drug discontinuation or just before a change to the oral form in five of the patients was 6.38, 7.66, 5.67, 5.24 and 1.97g/ml, most of them within the top 19% of voriconazole level measurements (or >5g/ml) reported in previous studies. No medications causing hallucinations or increasing voriconazole levels were identified.
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