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.

Prospective evaluation of nontuberculous mycobacteria disease in cystic fibrosis: The design of the PREDICT study.

BACKGROUND: Nontuberculous mycobacteria (NTM) are an important cause of airway infections in people with cystic fibrosis (pwCF). Isolation of NTM from respiratory specimens of pwCF do not mandate treatment in the absence of clinical and radiologic features of NTM pulmonary disease (NTM-PD), as some pwCF clear the infection without treatment and others do not appear to progress to NTM-PD despite persistent infection. An evidence-based protocol to standardize diagnosis of NTM-PD is needed to systematically identify pwCF who may benefit from treatment. METHODS: In this multicenter observational study, eligible pwCF who are 6 years of age and older and who have had a recent positive NTM culture are systematically evaluated for NTM-PD. Participants are identified based on positive NTM culture results obtained during routine clinical care and following enrollment are evaluated for NTM-PD and CF-related comorbidities. Participants are followed in PREDICT until they meet NTM-PD diagnostic criteria and are ready to initiate NTM treatment, or until study termination. Active participants who have not met these criteria are re-consented every 5 years to enable long-term participation. RESULTS: The primary endpoint will summarize the proportion of participants who meet the NTM-PD diagnosis definition. The time from enrollment to NTM-PD diagnosis will be derived from Kaplan-Meier estimates. CONCLUSION: A prospective protocol to identify NTM-PD in pwCF will test if this standardized approach defines a cohort with signs and symptoms associated with NTM-PD, to assist with clinical decision making and to build a framework for future therapeutic trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02073409.

Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus.

Mycobacterium abscessus, a species of nontuberculous mycobacteria (NTM), is an opportunistic pathogen that is readily cleared by healthy lungs but can cause pulmonary infections in people with chronic airway diseases. Although knowledge pertaining to molecular mechanisms of host defense against NTM is increasing, macrophage receptors that recognize M. abscessus remain poorly defined. Dectin-1, a C-type lectin receptor identified as a fungal receptor, has been shown to be a pathogen recognition receptor (PRR) for both M. tuberculosis and NTM. To better understand the role of Dectin-1 in host defense against M. abscessus, we tested whether blocking Dectin-1 impaired the uptake of M. abscessus by human macrophages, and we compared M. abscessus pulmonary infection in Dectin-1-deficient and wild-type mice. Blocking antibody for Dectin-1 did not reduce macrophage phagocytosis of M. abscessus, but did reduce the ingestion of the fungal antigen zymosan. Laminarin, a glucan that blocks Dectin-1 and other PRRs, caused decreased phagocytosis of both M. abscessus and zymosan. Dectin-1-/- mice exhibited no defects in the control of M. abscessus infection, and no differences were detected in immune cell populations between wild type and Dectin-1-/- mice. These data demonstrate that murine defense against M. abscessus pulmonary infection, as well as ingestion of M. abscessus by human macrophages, can occur independent of Dectin-1. Thus, additional PRR(s) recognized by laminarin participate in macrophage phagocytosis of M. abscessus.

Divergent host innate immune response to the smooth-to-rough M. abscessus adaptation to chronic infection.

Mycobacterium abscessus is a nontuberculous mycobacterium emerging as a significant pathogen for individuals with chronic lung disease, including cystic fibrosis and chronic obstructive pulmonary disease. Current therapeutics have poor efficacy. New strategies of bacterial control based on host defenses are appealing, but anti-mycobacterial immune mechanisms are poorly understood and are complicated by the appearance of smooth and rough morphotypes with distinct host responses. We explored the role of the complement system in the clearance of M. abscessus morphotypes by neutrophils, an abundant cell in these infections. M. abscessus opsonized with plasma from healthy individuals promoted greater killing by neutrophils compared to opsonization in heat-inactivated plasma. Rough clinical isolates were more resistant to complement but were still efficiently killed. Complement C3 associated strongly with the smooth morphotype while mannose-binding lectin 2 was associated with the rough morphotype. M. abscessus killing was dependent on C3, but not on C1q or Factor B; furthermore, competition of mannose-binding lectin 2 binding with mannan or N-acetyl-glucosamine during opsonization did not inhibit killing. These data suggest that M. abscessus does not canonically activate complement through the classical, alternative, or lectin pathways. Complement-mediated killing was dependent on IgG and IgM for smooth and on IgG for rough M. abscessus. Both morphotypes were recognized by Complement Receptor 3 (CD11b), but not CR1 (CD35), and in a carbohydrate- and calcium-dependent manner. These data suggest the smooth-to-rough adaptation changes complement recognition of M. abscessus and that complement is an important factor for M. abscessus infection.

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.

Deficient Complement Opsonization Impairs Mycobacterium avium Killing by Neutrophils in Cystic Fibrosis.

Nontuberculous mycobacteria (NTM), including Mycobacterium avium, are clinically important pathogens in cystic fibrosis (CF). The innate immune response to M. avium remains incompletely understood. We evaluated the role of complement opsonization in neutrophil-mediated killing of M. avium. Killing assays were performed using neutrophils from healthy donors (HDs) and persons with CF (pwCF). Clinical isolates of M. avium were opsonized with plasma from HDs or pwCF, which was intact or heat-treated to inactivate complement. HD neutrophils had killing activity against M. avium opsonized with intact HD plasma and killing was significantly reduced when M. avium was opsonized with heat-inactivated HD plasma. When opsonized with HD plasma, CF neutrophils had killing activity against M. avium that was not different than HD neutrophils. When opsonized with intact plasma from pwCF, HD neutrophil killing of M. avium was significantly reduced. Opsonization of M. avium with C3-depleted serum or IgM-depleted plasma resulted in significantly reduced killing. Plasma C3 levels were elevated in pwCF with NTM infection compared to pwCF without NTM infection. These studies demonstrate that human neutrophils efficiently kill M. avium when opsonized in the presence of plasma factors from HD that include C3 and IgM. Killing efficiency is significantly lower when the bacteria are opsonized with plasma from pwCF. This indicates a novel role for opsonization in neutrophil killing of M. avium and a deficiency in complement opsonization as a mechanism of impaired M. avium killing in CF. IMPORTANCE Mycobacterium avium is a member of a group of bacterial species termed nontuberculous mycobacteria (NTM) that cause lung disease in certain populations, including persons with cystic fibrosis (CF). NTM infections are challenging to diagnose and can be even more difficult to treat. This study investigated how the immune system responds to M. avium infection in CF. We found that neutrophils, the most abundant immune cell in the lungs in CF, can effectively kill M. avium in individuals both with and without CF. Another component of the immune response called the complement system is also required for this process. Levels of complement proteins are altered in persons with CF who have a history of NTM compared to those without a history of NTM infection. These results add to our understanding of how the immune system responds to M. avium, which can help pave the way toward better diagnostic and treatment strategies.

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease.

BACKGROUND: Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. METHODS: Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. RESULTS: No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. CONCLUSIONS: Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.

Culture independent markers of nontuberculous mycobacterial (NTM) lung infection and disease in the cystic fibrosis airway.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that affect a relatively small but significant portion of the people with cystic fibrosis (CF), and may cause increased morbidity and mortality in this population. Cultures from the airway are the only test currently in clinical use for detecting NTM. Culture techniques used in clinical laboratories are insensitive and poorly suited for population screening or to follow progression of disease or treatment response. The lack of sensitive and quantitative markers of NTM in the airway impedes patient care and clinical trial design, and has limited our understanding of patterns of acquisition, latency and pathogenesis of disease. Culture-independent markers of NTM infection have the potential to overcome many of the limitations of standard NTM cultures, especially the very slow growth, inability to quantitate bacterial burden, and low sensitivity due to required decontamination procedures. A range of markers have been identified in sputum, saliva, breath, blood, urine, as well as radiographic studies. Proposed markers to detect presence of NTM or transition to NTM disease include bacterial cell wall products and DNA, as well as markers of host immune response such as immunoglobulins and the gene expression of circulating leukocytes. In all cases the sensitivity of culture-independent markers is greater than standard cultures; however, most do not discriminate between various NTM species. Thus, each marker may be best suited for a specific clinical application, or combined with other markers and traditional cultures to improve diagnosis and monitoring of treatment response.

Nontuberculous Mycobacterial Infections in Cystic Fibrosis.

Nontuberculous mycobacteria (NTM) are important pathogens, with a longitudinal prevalence of up to 20% within the cystic fibrosis (CF) population. Diagnosis of NTM pulmonary disease in people with CF (pwCF) is challenging, as a majority have NTM infection that is transient or indolent, without evidence of clinical consequence. In addition, the radiographic and clinical manifestations of chronic coinfections with typical CF pathogens can overlap those of NTM, making diagnosis difficult. Comprehensive care of pwCF must be optimized to assess the true clinical impact of NTM and to improve response to treatment. Treatment requires prolonged, multidrug therapy that varies depending on NTM species, resistance pattern, and extent of disease. With a widespread use of highly effective modulator therapy (HEMT), clinical signs and symptoms of NTM disease may be less apparent, and sensitivity of sputum cultures further reduced. The development of a disease-specific approach to the diagnosis and treatment of NTM infection in pwCF is a research priority, as a lifelong strategy is needed for this high-risk population.

Specificity of Immunoglobulin Response to Nontuberculous Mycobacteria Infection in People with Cystic Fibrosis.

Nontuberculous mycobacteria (NTM) infections are increasingly prevalent in chronic lung diseases, including cystic fibrosis (CF). Mycobacterium abscessus is of particular concern due to relatively greater virulence and intrinsic antimicrobial resistance. Airway culture identification, the standard method for detecting pulmonary infection, is hindered by low sensitivity, long culture times, and reliance on sputum production or lavage. A culture-independent test for detecting NTM infection could complement, or replace, sputum culture, which is becoming more difficult to obtain with reduced sputum production by people with CF (pwCF) on highly effective modulator therapy. We describe an assay for the detection of plasma anti-M. abscessus antibodies of pwCF to antigens from M. abscessus lysates. Anti-M. abscessus IgG and IgA, but not IgM, discriminated with high specificity subjects infected with M. abscessus from those infected by M. avium complex, and from those with distant or no NTM infections. The IgG3 subclass predominated with minor contributions by other subclasses. Both aqueous and organic soluble antigens were recognized by plasma IgG. A validation cohort measuring IgG and IgG3 identified M. abscessus positive subjects, and elevated IgG was sustained over several years. These studies show the benefit of M. abscessus cell lysates to detect plasma IgG of subjects with CF and M. abscessus infections. Subclass analysis suggests that IgG3 is the predominant subtype in these subjects with chronic bacterial infections suggesting a defect in class maturation. Serodiagnosis could be useful to monitor M. abscessus group infections in chronic lung disease as an adjunct or alternative to culture. IMPORTANCE Lung infections with nontuberculous mycobacteria (NTM), and particularly Mycobacterium abscessus, a pathogen with high antibiotic resistance, are of great concern due to poor clinical outcomes and challenging detection in people with cystic fibrosis and other diseases. Standard detection methods are insensitive and increasingly difficult. We describe the measurement of NTM-specific antibodies from plasma to identify subjects infected with M. abscessus. The assay is sensitive and provides information on the immune response to NTM infections. This assay could be used to help identify subjects with NTM pulmonary infections and track disease progression, either alone or in conjunction with other tests.

Blood mRNA biomarkers distinguish variable systemic and sputum inflammation at treatment initiation of inhaled antibiotics in cystic fibrosis: A prospective non-randomized trial.

Inhaled antibiotics control chronic airway infection and maintain respiratory health in cystic fibrosis (CF). Given variation in patient responses to inhaled antibiotics, the ability to identify distinct responder phenotypes would facilitate the delivery of personalized care. Previously, a 10-gene panel was identified, measured directly from blood leukocytes, which predicted host response to intravenous antibiotic treatment during pulmonary exacerbations. In the current study, we tested whether the same panel predicted clinical response in subjects receiving a month of inhaled antibiotic therapy with aztreonam lysine (AZLI; Cayston®). A small cohort of CF subjects infected with Pseudomonas aeruginosa were enrolled at baseline health, prior to initiating one month's treatment with AZLI using the Altera® nebulizer system. Eighteen CF subjects underwent blood leukocyte gene panel measurements, sputum quantitative microbiology, spirometry, and C-reactive protein (CRP) measurement prior to onset and at completion of 4 weeks of AZLI therapy. Mean absolute improvement in percent predicted Forced Expiratory Volume in one second (ppFEV1) was 3%. Significant reductions in sputum bacterial colony counts were detected with treatment. CRP increased following treatment. While single genes within the panel did not change significantly following treatment, the analysis of multigene panel data demonstrated that HCA112 gene predicted ppFEV1 improvement. Hierarchical clustering based on gene expression yielded two distinctive molecular clusters before and after AZLI therapy. In conclusion, peripheral blood leukocyte genes quantifying inflammation are associated with responses to inhaled antibiotic therapy. Molecular quantification of systemic inflammation may indicate subgroups of CF subjects with variations in underlying inflammation and with variable clinical responses to inhaled antibiotics. Given the size limitation of the study, larger studies are needed in order to evaluate whether molecular measures may add precision to the determination of infectious and inflammatory outcomes following courses of inhaled antimicrobial therapies. Clinical Trials.gov Identifier: NCT01736839.

Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

Breath biomarkers associated withnontuberculosis mycobacteriadisease status in persons with cystic fibrosis: a pilot study.

Pulmonary infections caused by mycobacteria cause significant mortality and morbidity in the human population. Diagnosing mycobacterial infections is challenging. An infection can lead to active disease or remain indolent with little clinical consequence. In patients with pulmonarynontuberculosis mycobacteria(PNTM) identification of infection and diagnosis of disease can take months to years. Our previous studies showed the potential diagnostic power of volatile molecules in the exhaled breath samples to detect active pulmonaryM. tuberculosisinfection. Herein, we demonstrate the ability to detect the disease status of PNTM in the breath of persons with cystic fibrosis (PwCF). We putatively identified 17 volatile molecules that could discriminate between active-NTM disease (n= 6), indolent patients (n= 3), and those patients who have never cultured an NTM (n= 2). The results suggest that further confirmation of the breath biomarkers as a non-invasive and culture-independent tool for diagnosis of NTM disease in a larger cohort of PwCF is warranted.

Therapeutic efficacy of antimalarial drugs targeting DosRS signaling in Mycobacterium abscessus.

A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis, is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS. OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.

Investigating Nontuberculous Mycobacteria Transmission at the Colorado Adult Cystic Fibrosis Program.

Rationale: Healthcare-associated transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been investigated at CF centers worldwide, with conflicting conclusions. We investigated transmission at the Colorado Adult CF Program. Objectives: To systematically investigate healthcare-associated transmission and/or acquisition of NTM to determine similarity among respiratory and environmental isolates, and to compare home residence watershed mapping among pwCF having genetically similar NTM isolates. Methods: Whole-genome sequencing of NTM isolates from 80 pwCF was conducted to identify genetically similar isolate clusters (⩽30 SNP differences). Epidemiology, comparison of respiratory and environmental isolates, and home residence watershed mapping were analyzed. Measurements and Main Results: Whole-genome sequencing analysis revealed 11 clusters of NTM [6 Mycobacterium abscessus subspecies (ssp.) abscessus, 1 M. abscessus ssp. massiliense, 2 Mycobacterium avium, and 2 Mycobacterium intracellulare] among pwCF. Epidemiologic investigation demonstrated opportunities for healthcare-associated transmission in two M. abscessus and two M. avium clusters. Respiratory and healthcare environmental isolate comparisons revealed no genetic similarity. Individuals comprising one M. abscessus cluster, with no plausible healthcare-associated transmission, resided in the same watershed. Conclusions: This study suggests healthcare-associated transmission of M. abscessus is rare and includes a report of potential healthcare-associated transmission of M. avium among pwCF. One M. abscessus cluster possibly had common acquisition arising from residing in the same watershed. The presence of genetically similar isolates is insufficient to demonstrate healthcare-associated NTM transmission. Standardizing epidemiologic investigation, combined with environmental sampling and watershed analysis, will improve understanding of the frequency and nature of healthcare-associated NTM transmission among pwCF.

Genomic Analysis of a Hospital-Associated Outbreak of Mycobacterium abscessus: Implications on Transmission.

Whole-genome sequencing (WGS) has recently been used to investigate acquisition of Mycobacterium abscessus. Investigators have reached conflicting conclusions about the meaning of genetic distances for interpretation of person-to-person transmission. Existing genomic studies were limited by a lack of WGS from environmental M. abscessus isolates. In this study, we retrospectively analyzed the core and accessory genomes of 26 M. abscessus subsp. abscessus isolates collected over 7 years. Clinical isolates (n = 22) were obtained from a large hospital-associated outbreak of M. abscessus subsp. abscessus, the outbreak hospital before or after the outbreak, a neighboring hospital, and two outside laboratories. Environmental M. abscessus subsp. abscessus isolates (n = 4) were obtained from outbreak hospital water outlets. Phylogenomic analysis of study isolates revealed three clades with pairwise genetic distances ranging from 0 to 135 single-nucleotide polymorphisms (SNPs). Compared to a reference environmental outbreak isolate, all seven clinical outbreak isolates and the remaining three environmental isolates had highly similar core and accessory genomes, differing by up to 7 SNPs and a median of 1.6% accessory genes, respectively. Although genomic comparisons of 15 nonoutbreak clinical isolates revealed greater heterogeneity, five (33%) isolates had fewer than 20 SNPs compared to the reference environmental isolate, including two unrelated outside laboratory isolates with less than 4% accessory genome variation. Detailed genomic comparisons confirmed environmental acquisition of outbreak isolates of M. abscessus subsp. abscessus. SNP distances alone, however, did not clearly differentiate the mechanism of acquisition of outbreak versus nonoutbreak isolates. We conclude that successful investigation of M. abscessus subsp. abscessus clusters requires molecular and epidemiologic components, ideally complemented by environmental sampling.

Testing the effects of combining azithromycin with inhaled tobramycin for P. aeruginosa in cystic fibrosis: a randomised, controlled clinical trial.

RATIONALE: Inhaled tobramycin and oral azithromycin are common chronic therapies in people with cystic fibrosis and Pseudomonas aeruginosa airway infection. Some studies have shown that azithromycin can reduce the ability of tobramycin to kill P. aeruginosa. This trial was done to test the effects of combining azithromycin with inhaled tobramycin on clinical and microbiological outcomes in people already using inhaled tobramycin. We theorised that those randomised to placebo (no azithromycin) would have greater improvement in forced expiratory volume in one second (FEV1) and greater reduction in P. aeruginosa sputum in response to tobramycin. METHODS: A 6-week prospective, randomised, placebo-controlled, double-blind trial testing oral azithromycin versus placebo combined with clinically prescribed inhaled tobramycin in individuals with cystic fibrosis and P. aeruginosa airway infection. RESULTS: Over a 6-week period, including 4 weeks of inhaled tobramycin, the relative change in FEV1 did not statistically significantly differ between groups (azithromycin (n=56) minus placebo (n=52) difference: 3.44%; 95% CI: -0.48 to 7.35; p=0.085). Differences in secondary clinical outcomes, including patient-reported symptom scores, weight and need for additional antibiotics, did not significantly differ. Among the 29 azithromycin and 35 placebo participants providing paired sputum samples, the 6-week change in P. aeruginosa density differed in favour of the placebo group (difference: 0.75 log10 CFU/mL; 95% CI: 0.03 to 1.47; p=0.043). CONCLUSIONS: Despite having greater reduction in P. aeruginosa density in participants able to provide sputum samples, participants randomised to placebo with inhaled tobramycin did not experience significantly greater improvements in lung function or other clinical outcomes compared with those randomised to azithromycin with tobramycin.

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.

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

BACKGROUND: Healthcare-associated transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been reported and is of increasing concern. No standardized epidemiologic investigation tool has been published for healthcare-associated NTM outbreak investigations. This report describes the design of an ongoing observational study to standardize the approach to NTM outbreak investigation among pwCF. METHODS: This is a parallel multi-site study of pwCF within a single Center who have respiratory NTM isolates identified as being highly-similar. Participants have a history of positive airway cultures for NTM, receive care within a single Center, and have been identified as part of a possible outbreak based on genomic analysis of NTM isolates. Participants are enrolled in the study over a 3-year period. Primary endpoints are identification of a shared healthcare-associated encounter(s) among patients in a Center and identification of environmental isolates that are genetically highly-similar to respiratory isolates recovered from pwCF. Secondary endpoints include characterization of potential transmission modes and settings, as well as incidence and prevalence of healthcare-associated environmental NTM species/subspecies by geographical region. DISCUSSION: We hypothesize that genetically highly-similar strains of NTM among pwCF cared for at the same Center may arise from healthcare sources including patient-to-patient transmission and/or acquisition from environmental sources. This novel study design will establish a standardized, evidence-based epidemiologic investigation tool for healthcare-associated NTM outbreak investigation within CF Care Centers, will broaden the scope of independent outbreak investigations and demonstrate the frequency and nature of healthcare-associated NTM transmission in CF Care Centers nationwide. Furthermore, it will provide valuable insights into modeling risk factors associated with healthcare-associated NTM transmission and better inform future infection prevention and control guidelines. This study will systematically characterize clinically-relevant NTM isolates of CF healthcare environmental dust and water biofilms and set the stage to describe the most common environmental sources within the healthcare setting harboring clinically-relevant NTM isolates. TRIAL REGISTRATION: ClinicalTrials.gov NCT04024423. Date of registry July 18, 2019.