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Fluconazole Resistance Analysis in Candida Glabrata Isolates from OPC Patients
OOOOE Volume 110, Number 2 Results: 269 subjects (67% male) with 378 oral lesions were enrolled across low (132, 49%), high (97, 36%) and known cancer (40, 15%) risk classification groups. Mean age at enrollment was 53.7⫾13.2 years. Individual technique performance (sensitivity (CI)/specificity (CI)) for detecting OPML was: CE: 87 (0.78,0.93) / 71 (0.65, 0.77); VL: 58 (0.47, 0.68) / 40 (0.33, 0.46); TB: 80 (0.75, 0.85) / 53 (0.47, 0.59); and BB: 80 (0.75, 0.85) / 81 (0.76, 0.86). Examination of test combinations using logistic regression models and score statistic revealed significantly improved detection with the combination of CE⫹BB, modest improvement with CE⫹TB, and no improvement with CE⫹VL. Conclusions: CE alone results in a significant number of false negatives for OPML. CE performance is significantly improved with the addition of BB and somewhat improved with the addition of TB. However, the indications for BB and TB use, based upon clinical risk assessment, must be considered before test selection. Finally, CE, BB, and TB all require visual identification of the lesion which compromises the opportunity for earliest detection of OPML. Techniques to detect at-risk patients or identify lesions not visible during standard clinical examination could further improve early OPML detection. Funding: NIDCR/NIH: U54 DE14257 and NYUCI CCSG NIH/CI: P30 CA16087
Fluconazole Resistance Analysis in Candida Glabrata Isolates from OPC Patients Steven A. Mayen, Nathan P. Wiederhold, Tom A. Samuel, William R. Kirkpatrick, Thomas F. Patterson, Spencer W. Redding. University of Texas Health Science Center at San Antonio School of Dentistry University of Texas Health Science Center at San Antonio School of Medicine University of Texas at Austin College of Pharmacy South Texas Veterans Health Care System Objectives: Oropharyngeal candidiasis (OPC) caused by antifungal resistant yeast remains a significant clinical problem. Gain-of-function mutations within the gene encoding the transcription factor Pdr1p, which regulates the transcription of efflux pumps, have been reported to cause resistance to fluconazole in C. glabrata. Our objective was to assess the incidence of these mutations within clinical isolates obtained from patients with OPC Methods: 21 C. glabrata clinical isolates previously shown to have reduced susceptibility to fluconazole by CHROMagarbased testing were subcultured on Sabouraud dextrose agar plates. The minimum inhibitory concentrations were measured using fluconazole Etest strips. Isolates were grown to mid-logarithmic phase and DNA was extracted using a commercially available kit and segments of the PDR1 gene were amplified by PCR. Dideoxy sequencing was then performed and the sequence results were analyzed for the presence of point mutations within the PDR1 gene leading to an amino acid changes within Pdr1p. Results: Of the 21 C. glabrata clinical isolates tested, 8 were found to have a novel point mutation in PDR1 leading to an amino acid substitution within Pdr1p. Each of these isolates was susceptible dose-dependent (16 – 32 mcg/mL) or resistant (ⱖ 64 mcg/mL) to fluconazole. The most common point mutation (6 of 8 isolates) was a G727A mutation resulting in an aspartic acid to asparagine amino acid substitution at codon 243.
e35 Conclusions: These results demonstrate that point mutations within the transcription factor gene PDR1 are associated with reduced fluconazole susceptibility in clinical C. glabrata isolates from patients with OPC. Further studies are ongoing to assess the expression levels of PDR1 and the expression of the efflux genes that Pdr1p regulates. This work was partially supported by NIDCR Grant DE-18096 Funding: NIDCR Grant DE-18096
Salivary Analytes Indicative of Chronic Adult Periodontitis. JL Ebersole1, JL Schuster2; Jason Stevens1; R Kryscio3, Mark V. Thomas2, CS Miller4. 1 Center for Oral Health Research, University of Kentucky, 2 Division of Periodontology, Department of Oral Health Practice, University of Kentucky College of Dentistry, 3Department of Biostatistics, University of Kentucky College of Public Health, and 4Division of Oral Diagnosis and Oral Medicine, Department of Oral Health Practice, University of Kentucky College of Dentistry. Objectives: Saliva contains a large number of biomolecules, some of which have putative diagnostic utility. A potential problem with the use of biomolecules in diagnosis is the perception of day-to-day fluctuation due to within-subject variability in saliva collection and characteristics. This study documented these variations in orally healthy adult subjects related to six salivary analytes and evaluated the levels of these analytes in saliva of chronic adult periodontitis (CAP) patients compared to this variation, ie. normal range. Methods: Unstimulated whole saliva (5 ml) was collected every two to three days for 6 occasions from 30 subjects in good oral and systemic health. Additionally, whole saliva was collected from 50 patients with CAP, at baseline prior to entry into a longitudinal therapeutic study. Concentrations of IL-1, IL-6, MMP-8, PGE2, TNF␣ and albumin were examined and compared between these groups. Results: The results demonstrated variations in each of the analytes in the 180 (30 x 6) saliva samples from the normal subjects. Of interest was that a pattern of these inflammatory analytes were elevated in multiple saliva specimens from the same “normal subject”. Using the range of values in the normal samples, we constructed thresholds defining the “normal range” for each analyte to determine if they provided any discriminatory power for patients with periodontitis. No differences were noted for PGE2, TNF-␣ or albumin. IL-1 showed a positive predictive value (PPV) for CAP of 82.0% and negative predictive value (NPV) of 76.7% (threshold 20.43 pg/mL). MMP-8 demonstrated a PPV of 74.0% and NPV of 83.3% (threshold 128.18 pg/mL). Conclusions: These results addressed two issues, at least for this group of analytes. First, the concept that variation in targeted analytes in saliva from normal subjects is so large that the utility of saliva as a diagnostic fluid is suspect is fundamentally erroneous. Second, selected analytes that represent general host inflammatory/innate responses are indicative of CAP, and profiles of these analytes could have value as a diagnostic tool for oral disease at the public health, general dentist, and even periodontal specialist levels of patient care. Supported by NIH/NIDCR grant U01 DE RR020145.
Sleep Dysfunction and Burning Mouth Syndrome. Nita Chainani-Wu, Chaitra Bhat, Erin Madden, Sol Silverman Jr.
Fluconazole Resistance Analysis in Candida Glabrata Isolates from OPC Patients Steven A. Mayen, Nathan P. Wiederhold, Tom A. Samuel, William R. Kirkpatrick, Thomas F. Patterson, Spencer W. Redding. University of Texas Health Science Center at San Antonio School of Dentistry University of Texas Health Science Center at San Antonio School of Medicine University of Texas at Austin College of Pharmacy South Texas Veterans Health Care System Objectives: Oropharyngeal candidiasis (OPC) caused by antifungal resistant yeast remains a significant clinical problem. Gain-of-function mutations within the gene encoding the transcription factor Pdr1p, which regulates the transcription of efflux pumps, have been reported to cause resistance to fluconazole in C. glabrata. Our objective was to assess the incidence of these mutations within clinical isolates obtained from patients with OPC Methods: 21 C. glabrata clinical isolates previously shown to have reduced susceptibility to fluconazole by CHROMagarbased testing were subcultured on Sabouraud dextrose agar plates. The minimum inhibitory concentrations were measured using fluconazole Etest strips. Isolates were grown to mid-logarithmic phase and DNA was extracted using a commercially available kit and segments of the PDR1 gene were amplified by PCR. Dideoxy sequencing was then performed and the sequence results were analyzed for the presence of point mutations within the PDR1 gene leading to an amino acid changes within Pdr1p. Results: Of the 21 C. glabrata clinical isolates tested, 8 were found to have a novel point mutation in PDR1 leading to an amino acid substitution within Pdr1p. Each of these isolates was susceptible dose-dependent (16 – 32 mcg/mL) or resistant (ⱖ 64 mcg/mL) to fluconazole. The most common point mutation (6 of 8 isolates) was a G727A mutation resulting in an aspartic acid to asparagine amino acid substitution at codon 243.
e35 Conclusions: These results demonstrate that point mutations within the transcription factor gene PDR1 are associated with reduced fluconazole susceptibility in clinical C. glabrata isolates from patients with OPC. Further studies are ongoing to assess the expression levels of PDR1 and the expression of the efflux genes that Pdr1p regulates. This work was partially supported by NIDCR Grant DE-18096 Funding: NIDCR Grant DE-18096
Salivary Analytes Indicative of Chronic Adult Periodontitis. JL Ebersole1, JL Schuster2; Jason Stevens1; R Kryscio3, Mark V. Thomas2, CS Miller4. 1 Center for Oral Health Research, University of Kentucky, 2 Division of Periodontology, Department of Oral Health Practice, University of Kentucky College of Dentistry, 3Department of Biostatistics, University of Kentucky College of Public Health, and 4Division of Oral Diagnosis and Oral Medicine, Department of Oral Health Practice, University of Kentucky College of Dentistry. Objectives: Saliva contains a large number of biomolecules, some of which have putative diagnostic utility. A potential problem with the use of biomolecules in diagnosis is the perception of day-to-day fluctuation due to within-subject variability in saliva collection and characteristics. This study documented these variations in orally healthy adult subjects related to six salivary analytes and evaluated the levels of these analytes in saliva of chronic adult periodontitis (CAP) patients compared to this variation, ie. normal range. Methods: Unstimulated whole saliva (5 ml) was collected every two to three days for 6 occasions from 30 subjects in good oral and systemic health. Additionally, whole saliva was collected from 50 patients with CAP, at baseline prior to entry into a longitudinal therapeutic study. Concentrations of IL-1, IL-6, MMP-8, PGE2, TNF␣ and albumin were examined and compared between these groups. Results: The results demonstrated variations in each of the analytes in the 180 (30 x 6) saliva samples from the normal subjects. Of interest was that a pattern of these inflammatory analytes were elevated in multiple saliva specimens from the same “normal subject”. Using the range of values in the normal samples, we constructed thresholds defining the “normal range” for each analyte to determine if they provided any discriminatory power for patients with periodontitis. No differences were noted for PGE2, TNF-␣ or albumin. IL-1 showed a positive predictive value (PPV) for CAP of 82.0% and negative predictive value (NPV) of 76.7% (threshold 20.43 pg/mL). MMP-8 demonstrated a PPV of 74.0% and NPV of 83.3% (threshold 128.18 pg/mL). Conclusions: These results addressed two issues, at least for this group of analytes. First, the concept that variation in targeted analytes in saliva from normal subjects is so large that the utility of saliva as a diagnostic fluid is suspect is fundamentally erroneous. Second, selected analytes that represent general host inflammatory/innate responses are indicative of CAP, and profiles of these analytes could have value as a diagnostic tool for oral disease at the public health, general dentist, and even periodontal specialist levels of patient care. Supported by NIH/NIDCR grant U01 DE RR020145.
Sleep Dysfunction and Burning Mouth Syndrome. Nita Chainani-Wu, Chaitra Bhat, Erin Madden, Sol Silverman Jr.