bWe developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. M ycobacterium tuberculosis infects one-third of the world's population, with approximately nine million new cases and two million deaths attributable to the disease each year (1). Early case detection and rapid treatment are considered the most effective control strategies to reduce M. tuberculosis transmission (2), especially in cases involving multidrug-resistant (MDR) or extensively drug-resistant (XDR) M. tuberculosis. Culture-based methods remain the gold standard for diagnosing drug-resistant M. tuberculosis but can take several weeks or months to complete. Thus, nucleic acid-based drug susceptibility tests are becoming increasingly attractive as diagnostic tools in order to initiate individualized, patient-appropriate treatment in a timely manner.Technologies such as Cepheid's GeneXpert and Hain line probe assays reduce the time to diagnosis for many tuberculosis (TB) patients, provide a rapid read-out indicating resistance to rifampin or selected mutations conferring resistance to other firstor second-line drugs, and illustrate the potential to deploy molecular tests closer to the point of need (3). However, the number of known genes and mutations conferring resistance to first-and second-line drugs greatly exceeds the multiplexing capacity of these platforms, which may limit their clinical efficacy in the treatment and control of multidrug-resistant or extensively drug-resistant TB. Planar and suspension microarrays are well suited to address the multiple-gene, multiple-mutation challenge of diagnosing drug-resistant TB (4-12), but clinical adoption of microarray technology is hampered by poor reproducibility (13-15), complex workflows, and/or extensive user subjectivity and involvement in image and data analysis (16). In order to translate microarrays into efficacious TB diagnostics at the point of need, it is therefore necessary to simplify user interaction with the technology while retaining the ability to detect multiple genes and multiple mutations in a timely manner. The objectives of this study were to develop a gel element microarray test for MDR TB at a level of coverage surpassing what is currently available with WHO-endorsed molecular platforms, estimate the analytical specificity of the test on M. tuberculosis isolates of known genotype and phenotype, and compare the performance of the test to that of conventional drug susceptibility testing as a precursor to integrating the method into an entirely closed-amplicon consumable (17, 18) and sample-to-answer system. MATERIALS AND METHODSIsolates and positive control...
Dermatophytoses account for nearly a quarter of all fungal infections worldwide. These difficult to treat infections of the skin, hair, and nails, are growing more resistant to conventional antifungal treatments, and when treatable, often require prolonged therapeutic regimens. For centuries, essential oils have been used to treat a variety of ailments. In this study, we evaluated the clinical effects in vitro of 65 essential oils and 21 essential oil blends against various clinical species/strains of dermatophytes from two primary genera, Microsporum and Trichophyton. Our aim: To determine the overall activity of a wide range of essential oils against a number of clinical strains of dermatophytes. For all assays, 16 clinically derived species/strains of dermatophytes were used. The activity of each essential oil was assessed using a modified disk-diffusion assay over a period of 21 days of incubation vs. standard antifungal drugs. Subsequently, we determined the minimum inhibitory dilution possible for the most potent essential oils and performed combination testing to determine if synergy could be demonstrated with sub-inhibitory concentrations. We also assessed the effect of repeated vs. single applications. Of all the essential oils tested, cassia, cilantro, cinnamon, thyme, and oregano were the most potent along with one blend, DDR Prime; all genera/species tested were completely inhibited for 21 days following a single application. Many of the other oils tested exhibited temporal differences in activity where significant inhibition was observed ≤10 days of incubation which declined by day 21. Synergistic combinations were achieved with oregano and cilantro, cassia, or cinnamon bark; rose and cassia were also synergistic. Repeat application maintained complete inhibition for citronella, lemon myrtle, and litsea out to 21 days, but not lemon grass or On Guard. More study is necessary to understand the ways essential oils inhibit the growth of dermatophytes. Comprehensive research aimed at understanding the mechanism of action of essential oils and their components may provide the basis for a natural alternative to topical antifungal drugs. Such research could be envisioned to target optimal combinations and determine the timing between applications to provide for maximum inhibition of recurrence or growth.
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