1) Host Pathogenesis Previous work characterizing patients with idiopathic bronchiectasis and chronic pulmonary nontuberculous mycobacteria (PNTM) infection described a unique phenotype of tall, asthenic, post-menopausal women with body morphotype features such as scoliosis, pectus excavatum, and cardiac valvular abnormalities that overlapped with Marfan syndrome and related disorders. We described overlapping features of known genetic disorders of mucociliary clearance,cystic fibrosis (CF) or primary ciliary dyskinesia (PCD). We previously described familial clustering of this phenotype with 13% of 109 probands having at least one relative with PNTM, and additional 11% with at least one other family member having bronchiectasis unassociated with diagnosed PNTM, and 19% reporting scoliosis, 6% mitral valve prolapse and 6% pectus excavatum in other family members. Using this well characterized cohort, we analyzed whole exome sequencing on 15 PNTM affected and 18 PNTM unaffected members (many of whom had other characteristic body morphotypic traits) of 9 families and 54 sporadic PNTM patients. After selecting for variants occurring at a population frequency of less than 2% and predicted to be deleterious (nonsynonymous, splicing, stop-gain, stop-loss, and start-loss), we used a candidate gene analysis approach selecting genes known to be associated with overlapping conditions: CF (n=1 gene), PCD or putative genes with roles in ciliagenesis or affecting ciliary beat frequency (n=40), connective tissue disorders (n=24), and genes associated with Mendelian Suceptibility to Mycobacterial Disease and related pathways (n=17). A significantly higher proportion of PNTM patients than population controls had variants in 1 or more of these 4 gene categories. PNTM affected and unaffected family members had similar variant frequencies for all categories except the immune related genes where only the PNTM affected family members had a higher frequency of variants than population controls. The results of this study support a hypothesis that PNTM disease associated with idiopathic bronchiectasis is a multigenic disorder with combinations of variants across related disease categories with likely environmental factors relating to NTM exposure contributing to pathogenesis. 2) Environmental exposure Previous work on the population distribution of PNTM in the US with higher prevalence in the Southeastern US, along the West coast, and certain states such as Hawaii have suggested possible environmental influences that relate to NTM exposure on PNTM pathogenesis. There remains controversy as to whether these risks are predominantly atmospheric with regional variation in general opportunities for exposure or related to personal habits which may increase exposure. The high prevalence of PNTM among patients with CF and the detailed longitudinal data collection in a national registry which captures over 90% of the US CF population make this an ideal target group to explore these risks. We analyzed data from the CF Foundation Patient Registry (CFFPR) from 2010-2012 to assess the prevalence of PNTM and potential risk factors for disease. Looking at patients over the age of 12, 58% had mycobacterial cultures reported and of these 14% were positive for NTM. Significant regional variation was noted with higher prevalence in the Southeast and West and geospatial clustering was noted in certain states such as Wisconsin, Arizona, Florida, and Maryland. Higher saturated vapor pressure was associated with an increased prevalence of NTM (odds ratio = 1.06; 95% confidence interval = 1.02-1.10). We found similar results in a case-control analysis of previously collected data from a national, multisite nested cohort study of PNTM in CF patients, incident PNTM cases and age and sex matched culture negative controls were administered a standardized soil and water exposure questionnaire. Commonly implicated personal activities in NTM exposure such as showering or use of municipal water sources were equally high in both case and control groups. Conversely, as we have reported in other studies, average annual atmospheric water vapor content was significantly predictive of center prevalence (P = 0.0019), with R(2) = 0.40 suggesting that regional variation in disease prevalence may related more to environmental conditions favoring mycobacterial growth and survival than to personal behaviors which increase exposure. Because NTM exposure in these high prevalent areas is similar among persons with and without PNTM, genetic susceptibility beyond the CF gene may play a role in disease pathogenesis. 3) Microbial pathogenesis Mycobacterium abscessus has been associated with significant lung disease and increased morbidity and mortality especially in the setting of CF. The relationship between this microbe and the host has varied significantly from a relatively innocuous relationship without evidence of apparent adverse effect of the clinical course of CF lung disease to rapid clinical decline leading to death. We assessed potential phenotypic and genomic characteristics of serial M. abscessus isolates obtained from CF and nonCF bronchiectasis patients during periods of relative clinical stability and during periods of rapid clinical decline to see if virulence and/or drug resistance factors could be identified. As with prior studies a change in phenotypic colony morphology from smooth to rough correlated with prolonged infection and clinical decline. Specific changes in fatty acid metabolism were characterized by MALDI-TOF mass spectrometry. Sequencing of the 8 genes in the GPL locus of rough colony isolates showed that 77% had deleterious mutations in mps1 or mps2 and 13% had previously unreported mutations in mmpL4a and mmpS4. Later isolates displayed accumulation of triacylglycerol and reduced expression of fadD23, a key gene involved in biosynthesis of complex lipids, sometimes preceding rough colony onset. These studies suggest that clonal diversification and a shift in lipid metabolism including the loss of GPL occur during chronic lung infection with M. abscessus. Detection of these changes in fatty acid metabolism by MALDI-TOF may be a rapid way of heralding subsequent clinical decline. 4) Drug development The treatment of PNTM disease requires prolonged, multi-drug, poorly tolerated regimens of limited efficacy. Amikacin has been shown to be highly active against many species of NTM but its effective use is limited by significant toxicity. Strategies have been considered to reduce toxicity and potentially increase efficacy by altering route of administration or modifying formulation of the drug to more specifically target areas of lung disease. We reported a retrospective analysis of the use of aerosolized off-the-shelf amikacin (for intravenous use) in a cohort of patients from our natural history study with treatment refractory M. avium complex or M. abscessus group lung infections which resulted in sustained negative cultures in 25% and reduced toxicity relative to intravenous amikacin. This study also suggested reduction in colony counts may be a more sensitive indicator of drug efficacy. In a separate analysis of a much larger cohort of patients with treatment naive M. avium complex, we showed strong predictive value of early reduction of colony counts as an indicator of long term conversion to negative cultures and correlation with both symptomatic and radiographic improvement. We used early data from these studies to design a multi-site, first in target-disease, randomized placebo controlled trial followed by open label continuation phase study of liposomal encapsulated amikacin for inhalation. This now completed study showed significant benefit and provided important outcome measure and sample size requirements for future treatment trials.
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