Nontuberculous mycobacteria testing and culture positivity in the United States.

BACKGROUND: Nontuberculous mycobacteria (NTM) are environmental bacteria which may cause chronic lung disease. The prevalence of NTM pulmonary infection and disease has been increasing in the United States and globally. The predominant clinically relevant species of NTM in the United States are Mycobacterium avium complex (MAC) species and Mycobacterium abscessus. With the development of rapid species identification methods for NTM (e.g. PCR probes), more testing for NTM is being conducted through commercial labs, such as Laboratory Corporation of America (Labcorp), which provides deidentified real-time testing data to the Centers for Disease Control (CDC) pursuant to a data sharing agreement. Because NTM lung infections are not reportable in most states, other data sources are key to understanding NTM testing patterns, positivity rates, and species distributions to track infection trends and identify clinical care needs. METHODS: We obtained national Labcorp data for the period January 2019 through mid-April 2022. We subset the data to only respiratory samples sent for Acid Fast Bacilli (AFB) cultures. NTM positive results were defined as those which identified an NTM species and are not Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium gordonae. RESULTS: Overall, 112,528 respiratory samples were sent for AFB testing during the study period; 26.3% were from the Southeast U.S., identified as HSS Region IV in the Labcorp dataset, and 23.0% were from the Pacific and South Pacific region (Region IX). The culture positive prevalence ranged from 20.2% in the Southeast to 9.2% in the East North Central region (Region V). In the Southeast US, M. abscessus prevalence was 4.0%. For MAC, the highest prevalence was observed in the Mountain region (Region VII) (13.5%) and the lowest proportion was in the East South Central region (7.3%, Region III). Among positive tests, the proportion which was MAC varied from 61.8% to 88.9% and was highest in the Northeast U.S. The proportion of positive samples which were M. abscessus ranged from 3.8% to 19.7% and was highest in the Southeast. CONCLUSIONS: The Southeastern region of the U.S. has the highest rate of culture positivity in Labcorp tests for total NTM and, of all positive tests, the highest proportion of M. abscessus. These estimates may underrepresent the true number of M. abscessus infections because M. absesscus-specific probes are not commercially available and not all NTM testing in the United States is done by Labcorp. Analysis of real-time testing data from commercial laboratories may provide insights into risk factors for NTM culture positivity in 'hotspot' areas.

Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses.

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Prospective healthcare-associated links in transmission of nontuberculous mycobacteria among people with cystic fibrosis (pHALT NTM) study: Rationale and study design.

BACKGROUND: Healthcare-associated acquisition and transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been described, and remains a concern for both patients and providers. This report describes the design of a prospective observational study utilizing the standardized epidemiologic investigation toolkit for healthcare-associated links in transmission of NTM among pwCF. METHODS: This is a parallel multi-site study of pwCF who have infections with respiratory NTM isolates and receive healthcare within a common CF Care Center. Participants have a history of one or more NTM positive airway cultures and have been identified as having NTM infections suggestive of a possible outbreak within a single Center, based on NTM isolate genomic analysis. Participants are enrolled in the study over a 3-year period. Primary endpoints are identification of shared healthcare-associated source(s) among pwCF in a Center, identification of healthcare environmental dust and water biofilm NTM isolates that are genetically highly-related to respiratory isolates, and identification of common home of residence watersheds among pwCF infected with clustered isolates. Secondary endpoints include characterization of healthcare-associated transmission and/or acquisition modes and settings as well as description of incidence and prevalence of healthcare-associated environmental NTM species/subspecies by geographical region. DISCUSSION: We hypothesize that genetically highly-related isolates of NTM among pwCF cared for at the same Center may arise from healthcare sources including patient-to-patient transmission and/or acquisition from health-care environmental dust and/or water biofilms. This study design utilizes a published, standardized, evidence-based epidemiologic toolkit to facilitate confidential, independent healthcare-associated NTM outbreak investigations within CF Care Centers. This study will facilitate real-time, rapid detection and mitigation of healthcare-associated NTM outbreaks to reduce NTM risk, inform infection prevention and control guidelines, and characterize the prevalence and origin of NTM outbreaks from healthcare-associated patient-to-patient transmission and/or environmental acquisition. This study will systematically characterize human disease causing NTM isolates from serial collection of healthcare environmental dust and water biofilms and define the most common healthcare environmental sources harboring NTM biofilms. TRIAL REGISTRATION: ClinicalTrials.gov NCT05686837.

The risk of pulmonary NTM infections and water-quality constituents among persons with cystic fibrosis in the United States, 2010-2019.

Rationale: The prevalence of nontuberculous mycobacterial (NTM) pulmonary disease varies geographically in the United States. Previous studies indicate that the presence of certain water-quality constituents in source water increases NTM infection risk. Objective: To identify water-quality constituents that influence the risk of NTM pulmonary infection in persons with cystic fibrosis in the United States. Methods: We conducted a population-based case-control study using NTM incidence data collected from the Cystic Fibrosis Foundation Patient Registry during 2010-2019. We linked patient zip code to the county and associated patient county of residence with surface water data extracted from the Water Quality Portal. We used logistic regression models to estimate the odds of NTM infection as a function of water-quality constituents. We modeled two outcomes: pulmonary infection due to Mycobacterium avium complex (MAC) and Mycobacterium abscessus species. Results: We identified 484 MAC cases, 222 M. abscessus cases and 2816 NTM-negative cystic fibrosis controls resident in 11 states. In multivariable models, we found that for every 1-standardized unit increase in the log concentration of sulfate and vanadium in surface water at the county level, the odds of infection increased by 39% and 21%, respectively, among persons with cystic fibrosis with MAC compared with cystic fibrosis-NTM-negative controls. When modeling M. abscessus as the dependent variable, every 1-standardized unit increase in the log concentration of molybdenum increased the odds of infection by 36%. Conclusions: These findings suggest that naturally occurring and anthropogenic water-quality constituents may influence the NTM abundance in water sources that supply municipal water systems, thereby increasing MAC and M. abscessus infection risk.

Incidence of nontuberculous mycobacteria infections among persons with cystic fibrosis in the United States (2010-2019).

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous, environmental bacteria that can cause chronic lung disease. Persons with cystic fibrosis (pwCF) are at high risk for NTM. Approximately 1 in 5 pwCF in the United States (U.S.) is affected by pathogenic NTM species, and incidence rates of NTM have been increasing among pwCF as well as in the general population. Prevalence of NTM pulmonary infections (PI) varies widely across the United States because of geographic variation in environmental exposures. This study will present updated region-level incidence of NTM infections in the cystic fibrosis (CF) population in the U.S. METHODS: We used the Cystic Fibrosis Foundation Patient Registry (CFFPR) data for the period 2010 through 2019. Our study population comprised persons with CF ≥ 12 years of age who had been tested for NTM PI. We included only registry participants with NTM culture results. We defined incident cases as persons with one positive mycobacterial culture preceded by ≥ two negative mycobacterial cultures. We defined non-cases as persons with ≥ two negative mycobacterial cultures. We estimated average annual NTM PI incidence by region. Using quasi-Poisson models, we calculated annual percent change in incidence by region. RESULTS: We identified 3,771 incident NTM infections. Of these cases, 1,816 (48.2%) were Mycobacterium avium complex (MAC) infections and 960 (25.5%) were Mycobacterium abscessus infections. The average annual incidence of NTM PI among pwCF in the U.S. was 58.0 cases per 1,000 persons. The Northeast had the highest incidence of MAC (33.5/1,000 persons tested) and the South had the highest incidence of M. abscessus (20.3/1,000 persons tested). From 2010 to 2019, the annual incidence of total NTM PI increased significantly by 3.5% per year in the U.S. CONCLUSIONS: NTM PI incidence is increasing among pwCF. Identifying high risk areas and increasing trends is important for allocating public health and clinical resources as well as evaluating interventions.

Molecular Epidemiologic Investigation of Mycobacterium intracellulare Subspecies chimaera Lung Infections at an Adult Cystic Fibrosis Program.

Rationale: Outbreaks of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) have been reported at CF centers with conflicting conclusions. The occurrence of NTM at the UVMC (University of Vermont Medical Center) adult CF program was investigated. Objectives: Use the HALT NTM (Healthcare-associated Links in Transmission of NTM) toolkit to investigate the healthcare-associated transmission and/or acquisition of NTM among pwCF having genetically similar NTM isolates. Methods: Whole genome sequencing of NTM isolates from 23 pwCF was conducted to identify genetically similar NTM isolate clusters (30 or fewer single-nucleotide polymorphism differences). The epidemiological investigation, comparison of respiratory and healthcare environmental isolates, and home residence watershed mapping were analyzed. Results: Whole genome sequencing analysis revealed two clusters of NTM isolates (Mycobacterium avium and M. intracellulare ssp. chimaera) among pwCF. The epidemiologic investigation demonstrated opportunities for healthcare-associated transmission within both clusters. Healthcare environmental M. avium isolates revealed no genetic similarity to respiratory isolates. However, M. intracellulare ssp. chimaera respiratory isolates revealed greater genetic similarity to a hospital water biofilm isolate than to each other. Neither cluster had all subjects residing in the same watershed. Conclusions: This study suggests the healthcare-associated transmission of M. avium among pwCF is unlikely at UVMC but supports the healthcare-associated environmental acquisition of M. intracellulare ssp. chimaera. The presence of genetically similar isolates alone is insufficient to confirm healthcare-associated transmission and/or acquisition. The HALT NTM toolkit standardizes outbreak investigation with genetic analysis, epidemiologic investigation, healthcare environmental sampling, and home of residence watershed identification to test the frequency and nature of healthcare-associated NTM transmission among pwCF.

Environmental risk of nontuberculous mycobacterial infection: Strategies for advancing methodology.

The National Institute of Allergy and Infectious Diseases organized a symposium in June 2022, to facilitate discussion of the environmental risks for nontuberculous mycobacteria exposure and disease. The expert researchers presented recent studies and identified numerous research gaps. This report summarizes the discussion and identifies six major areas of future research related to culture-based and culture independent laboratory methods, alternate culture media and culturing conditions, frameworks for standardized laboratory methods, improved environmental sampling strategies, validation of exposure measures, and availability of high-quality spatiotemporal data.

Methods of isolation and identification of nontuberculous mycobacteria from environmental samples: A scoping review.

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment. Some species of NTM are pathogenic and cause lung disease in susceptible persons. Epidemiologic studies of environmental NTM infection risk rely on both culture-dependent and culture-independent techniques for NTM isolation and identification. In this review, we summarized current methods used to isolate and identify NTM from the environment. We searched PubMed, Embase, Scopus, Web of Science: Core Collection, and Global Health (CAB Direct) for peer-reviewed studies from the last 12 years. We identified 1685 unique citations and 110 studies met our inclusion and exclusion criteria. Approximately half (55%) of the studies identified in this review used a combination of culture-independent and culture-dependent methods. The most common environmental substrate analyzed was water (n = 90). Identification of current, common methods for the isolation and identification of NTM from environmental samples may contribute to the development of standard methodological practices in the future. The choice of isolation method is based on the research question, environment, and species. A summary of common methods may contribute to the development of standard practices for isolation and identification of NTM from environmental samples, which may lead to more robust and comparable results.

Detecting clusters of high nontuberculous mycobacteria infection risk for persons with cystic fibrosis - An analysis of U.S. counties.

Nontuberculous mycobacteria are ubiquitous environmental bacteria that frequently cause disease in persons with cystic fibrosis (pwCF). The risks for NTM infection vary geographically. Detection of high-risk areas is important for focusing prevention efforts. In this study, we apply five cluster detection methods to identify counties with high NTM infection risk. Four clusters were detected by at least three of the five methods, including twenty-five counties in five states. The geographic area and number of counties in each cluster depended upon the detection method used. Identifying these clusters supports future studies of environmental predictors of infection and will inform control and prevention efforts.

Vanadium in groundwater aquifers increases the risk of MAC pulmonary infection in O'ahu, Hawai'i.

Hawai'i has the highest prevalence of nontuberculous mycobacterial (NTM) pulmonary disease in the United States. Previous studies indicate that certain trace metals in surface water increase the risk of NTM infection. Objective: To identify whether trace metals influence the risk of NTM infection in O'ahu, Hawai'i. Methods: A population-based ecologic cohort study was conducted using NTM infection incidence data from patients enrolled at Kaiser Permanente Hawai'i during 2005-2019. We obtained sociodemographic, microbiologic, and geocoded residential data for all Kaiser Permanente Hawai'i beneficiaries. To estimate the risk of NTM pulmonary infection from exposure to groundwater constituents, we obtained groundwater data from three data sources: (1) Water Quality Portal; (2) the Hawai'i Department of Health; and (3) Brigham Young University, Department of Geological Science faculty. Data were aggregated by an aquifer and were associated with the corresponding beneficiary aquifer of residence. We used Poisson regression models with backward elimination to generate models for NTM infection risk as a function of groundwater constituents. We modeled two outcomes: Mycobacterium avium complex (MAC) species and Mycobacterium abscessus group species. Results: For every 1-unit increase in the log concentration of vanadium in groundwater at the aquifer level, infection risk increased by 22% among MAC patients. We did not observe significant associations between water-quality constituents and infection risk among M. abscessus patients. Conclusions: Concentrations of vanadium in groundwater were associated with MAC pulmonary infection in O'ahu, Hawai'i. These findings provide evidence that naturally occurring trace metals influence the presence of NTM in water sources that supply municipal water systems.

Nontuberculous Mycobacteria Infection Risk and Trace Metals in Surface Water: A Population-based Ecologic Epidemiologic Study in Oregon.

Rationale: Nontuberculous mycobacteria (NTM) are ubiquitous environmental bacteria, and some pathogenic species cause lung disease. Environmental factors contribute to increased NTM abundance, with higher potential for exposure and infection. Objectives: To identify water-quality constituents that influence the risk of NTM infection in Oregon. Methods: We conducted a population-based cohort study using patient incidence data from the Oregon statewide NTM laboratory data collected as part of a public health surveillance project from 2007 through 2012. To estimate the risk of NTM pulmonary infection (PI) from exposure to water constituents, we extracted water-quality data from the Water Quality Portal and associated these data with corresponding patient county of residence. Using generalized linear models, we modeled two outcomes: Mycobacterium avium complex species PI and Mycobacterium abscessus group species PI. Results: For every 1-unit increase in the log concentration of vanadium in surface water, infection risk increased by 49% among persons with Mycobacterium avium complex PI. Among those with Mycobacterium abscessus PI, we observed that for every 1-unit increase in the log concentration of molybdenum in surface water, infection risk increased by 41%. The highest risk of infection due to Mycobacterium abscessus group infection was concentrated in counties within the Northwestern region of Oregon. High infection risk associated with Mycobacterium avium complex species did not show any geographic pattern. Conclusions: Concentrations of the trace metals molybdenum and vanadium in surface water sources were associated with NTM infection in Oregon. These findings may help identify regions at higher risk of NTM infection to guide risk reduction strategies.

Nontuberculous mycobacterial infection and environmental molybdenum in persons with cystic fibrosis: a case-control study in Colorado.

RATIONALE: Nontuberculous mycobacteria (NTM) are ubiquitous environmental bacteria that may cause chronic lung disease and are one of the most difficult-to-treat infections among persons with cystic fibrosis (pwCF). Environmental factors likely contribute to increased NTM densities, with higher potential for exposure and infection. OBJECTIVE: To identify water-quality constituents that influence odds of NTM infection among pwCF in Colorado. METHODS: We conducted a population-based nested case-control study using patient data from the Colorado CF Center NTM database. We associated data from pwCF and water-quality data extracted from the Water Quality Portal to estimate odds of NTM infection. Using Bayesian generalized linear models with binomial-distributed discrete responses, we modeled three separate outcomes; any NTM infection, infections due to Mycobacterium avium complex species, and infections due to M. abscessus group species. RESULTS: We observed a consistent association with molybdenum in the source water and M. abscessus group species infection among pwCF in all models. For every 1-unit increase in the log concentration of molybdenum in surface water, the odds of infection for those with M. abscessus group species compared to those who were NTM culture-negative increased by 79%. The odds of M. abscessus group infection varied by county; the counties with the highest probability of infection are located along the major rivers. CONCLUSIONS: We have identified molybdenum in the source water as the most predictive factor of M. abscessus group infection among pwCF in Colorado. This finding will help inform patients at risk for NTM of their relative risks in residing within specific regions.

Environmental predictors of pulmonary nontuberculous mycobacteria (NTM) sputum positivity among persons with cystic fibrosis in the state of Florida.

Nontuberculous mycobacteria (NTM) are opportunistic human pathogens that are commonly found in soil and water, and exposure to these organisms may cause pulmonary nontuberculous mycobacterial disease. Persons with cystic fibrosis (CF) are at high risk for developing pulmonary NTM infections, and studies have shown that prolonged exposure to certain environments can increase the risk of pulmonary NTM. It is therefore important to determine the risk associated with different geographic areas. Using annualized registry data obtained from the Cystic Fibrosis Foundation Patient Registry for 2010 through 2017, we conducted a geospatial analysis of NTM infections among persons with CF in Florida. A Bernoulli model in SaTScan was used to identify clustering of ZIP codes with higher than expected numbers of NTM culture positive individuals. Generalized linear mixed models with a binomial distribution were used to test the association of environmental variables and NTM culture positivity. We identified a significant cluster of M. abscessus and predictors of NTM sputum positivity, including annual precipitation and soil mineral levels.

Estimating the optimal population upper bound for scan methods in retrospective disease surveillance.

Correctly and quickly identifying disease patterns and clusters is a vital aspect of public health and epidemiology so that disease outbreaks can be mitigated as effectively as possible. The circular scan method is one of the most commonly used methods for detecting disease outbreaks and clusters in retrospective and prospective disease surveillance. The circular scan method requires a population upper bound in order to construct the set of candidate zones to be scanned, which is usually set to 50% of the total population. The performance of the circular scan method is affected by the choice of the population upper bound, and choosing an upper bound different from the default value can improve the method's performance. Recently, the Gini coefficient based on the Lorenz curve, which was originally used in economics, was proposed to determine a better population upper bound. We present the elbow method, a new method for choosing the population upper bound, which seeks to address some of the limitations of the Gini-based method while improving the performance of the circular scan method over the default value. To evaluate the performance of the proposed approach, we evaluate the sensitivity and positive predictive value of the circular scan method for publicly-available benchmark data for the default value, the Gini coefficient method, and the elbow method.

Nontuberculous Mycobacterial Disease and Molybdenum in Colorado Watersheds.

Nontuberculous mycobacteria (NTM) are environmental bacteria that may cause chronic lung disease. Environmental factors that favor NTM growth likely increase the risk of NTM exposure within specific environments. We aimed to identify water-quality constituents (Al, As, Cd, Ca, Cu, Fe, Pb, Mg, Mn, Mo, Ni, K, Se, Na, Zn, and pH) associated with NTM disease across Colorado watersheds. We conducted a geospatial, ecological study, associating data from patients with NTM disease treated at National Jewish Health and water-quality data from the Water Quality Portal. Water-quality constituents associated with disease risk were identified using generalized linear models with Poisson-distributed discrete responses. We observed a highly robust association between molybdenum (Mo) in the source water and disease risk. For every 1- unit increase in the log concentration of molybdenum in the source water, disease risk increased by 17.0%. We also observed a statistically significant association between calcium (Ca) in the source water and disease risk. The risk of NTM varied by watershed and was associated with watershed-specific water-quality constituents. These findings may inform mitigation strategies to decrease the overall risk of exposure.

The Associations Between Clinical Respiratory Outcomes and Ambient Wildfire Smoke Exposure Among Pediatric Asthma Patients at National Jewish Health, 2012-2015.

Wildfires are a growing threat in the United States. At a population level, exposure to ambient wildfire smoke is known to be associated with severe asthma outcomes such as hospitalizations. However, little work has been done on subacute clinical asthma outcomes, especially in sensitive populations. This study retrospectively investigated associations between ambient wildfire smoke exposure and measures of lung function and asthma control, Forced Expiratory Volume in 1 Second (FEV1) and the Asthma Control Test (ACT) and Children's Asthma Control Test (CACT) test scores, during nonurgent clinic visits. The study population consisted of pediatric asthma patients (ages 4-21; n = 1,404 for FEV1 and n = 395 for ACT/CACT) at National Jewish Health, a respiratory referral hospital in Denver, Colorado, and therefore represents a more severe asthma phenotype than the general pediatric asthma population. Wildfire smoke-related PM2.5 at patients' residential ZIP codes was characterized using satellite-derived smoke polygons from NOAA's Hazard Mapping System combined with kriging of ground-based U.S. EPA monitors. Mixed effect models were used to estimate associations between clinical outcomes and smoke PM2.5 exposure, controlling for known risk factors and confounders. Among older children aged 12-21 we found that wildfire PM2.5 was associated with lower FEV1 the next day but higher FEV1 the day after. We found no associations between wildfire PM2.5 and FEV1 in younger children or between wildfire PM2.5 and asthma control measured by the ACT/CACT in all ages. We speculate that rescue medication usage by older children may decrease respiratory symptoms caused by wildfire smoke.

The Rise and Fall and Rise of Linkage Analysis as a Technique for Finding and Characterizing Inherited Influences on Disease Expression.

For many years, family-based studies using linkage analysis represented the primary approach for identifying disease genes. This strategy is responsible for the identification of the greatest number of genes proven to cause human disease. However, technical advancements in next generation sequencing and high throughput genotyping, coupled with the apparent simplicity of association testing, led to the rejection of family-based studies and of linkage analysis. At present, genetic association methods, using case-control comparisons, have become the exclusive approach for detecting disease-related genes, particularly those underlying common, complex diseases. In this chapter, we present a historical overview of linkage analysis, including a description of how the approach works, as well as its strengths and weaknesses. We discuss how the transition from family-based studies to population comparison association studies led to a critical loss of information with respect to genetic etiology and inheritance, and we present historical and contemporary examples of linkage analysis "success stories" in identifying genes contributing to the development of human disease. Currently, linkage analysis is re-emerging as a useful approach for identifying disease genes, determining genetic parameters, and resolving genetic heterogeneity. We posit that the combination of linkage analysis, association testing, and high throughput sequencing provides a powerful approach for identifying disease-causing genes.

A Geospatial Epidemiologic Analysis of Nontuberculous Mycobacterial Infection: An Ecological Study in Colorado.

RATIONALE: Nontuberculous mycobacteria (NTM) are ubiquitous environmental microorganisms. Infection is thought to result primarily from exposure to soil and/or water sources. NTM disease prevalence varies greatly by geographic region, but the geospatial factors influencing this variation remain unclear. OBJECTIVES: To identify sociodemographic and environmental ecological risk factors associated with NTM infection and disease in Colorado. METHODS: We conducted an ecological study, combining data from patients with a diagnosis of NTM disease from National Jewish Health's electronic medical record database and ZIP code-level sociodemographic and environmental exposure data obtained from the U.S. Geological Survey, the U.S. Department of Agriculture, and the U.S. Census Bureau. We used spatial scan methods to identify high-risk clusters of NTM disease in Colorado. Ecological risk factors for disease were assessed using Bayesian generalized linear models assuming Poisson-distributed discrete responses (case counts by ZIP code) with the log link function. RESULTS: We identified two statistically significant high-risk clusters of disease. The primary cluster included ZIP codes in urban regions of Denver and Aurora, as well as regions south of Denver, on the east side of the Continental Divide. The secondary cluster was located on the west side of the Continental Divide in rural and mountainous regions. After adjustment for sociodemographic, drive time, and soil variables, we identified three watershed areas with relative risks of 12.2, 4.6, and 4.2 for slowly growing NTM infections compared with the mean disease risk for all watersheds in Colorado. This study population carries with it inherent limitations that may introduce bias. The lack of complete capture of NTM cases in Colorado may be related to factors such as disease severity, education and income levels, and insurance status. CONCLUSIONS: Our findings provide evidence that water derived from particular watersheds may be an important source of NTM exposure in Colorado. The watershed with the greatest risk of NTM disease contains the Dillon Reservoir. This reservoir is also the main water supply for major cities located in the two watersheds with the second and third highest disease risk in the state, suggesting an important possible source of infection.