INTRODUCTION:Sarcoidosis is a heterogeneous granulomatous disease with highly variable clinical trajectories, yet no validated biomarkers exist to distinguish progressive sarcoidosis (P-sarcoidosis) from non-progressive disease (NP-sarcoidosis). This lack of tractable biomarkers limits early risk stratification and impedes therapeutic decision-making. Preliminary data from our group suggest that P-sarcoidosis and NP-sarcoidosis may be differentiated by blood-derived and peripheral blood mononuclear cell (PBMC)-derived molecular signatures, as well as ex vivo granuloma biogenesis in response to putative disease-causing antigens. This protocol describes a multi-omic study aimed at identifying mechanistically grounded, clinically translatable biomarkers that distinguish P-sarcoidosis from NP-sarcoidosis. METHODS AND ANALYSIS:We will perform an integrative proteomic and transcriptomic analysis across three biological compartments: ex vivo granuloma model, PBMCs and plasma. Participants with clinically adjudicated P-sarcoidosis or NP-sarcoidosis will provide blood samples for multi-omic profiling. P-sarcoidosis versus NP-sarcoidosis phenotype will be assessed based on changes in spirometry, diffusing capacity for carbon monoxide, chest radiography and need for treatment for pulmonary symptoms. Patient-reported outcomes will also be recorded. Data-driven computational approaches will be used to identify molecular pathways associated with granuloma formation and disease persistence and to develop a classifier that distinguishes P-sarcoidosis from NP-sarcoidosis. Rigorous internal validation, feature-selection procedures and statistical controls for high-dimensional data will be applied. Candidate biomarkers emerging from multi-compartment integration will be prioritised based on biological coherence, reproducibility and clinical feasibility. ETHICS AND DISSEMINATION:The study protocol has been approved by the Biomedical Research Alliance of New York, serving as a single Institutional Review Board (IRB) for the project (IRB # 23-02-503), as well as at National Jewish Health (IRB# HS-4091), University of Minnesota (STUDY00020121/SITE00002051) and The Ohio State University (IRB# 2023X0140). All participants will provide informed consent prior to enrolment. Results will be disseminated through peer-reviewed publications, scientific conferences and presentations to patients and advocacy groups. De-identified datasets and analytic workflows will be shared in accordance with institutional policies and data-sharing agreements.

ABSTRACT The genus Mycobacterium is highly diverse, and in addition to Mycobacterium tuberculosis, it includes many other pathogenic and non-pathogenic taxa which are collectively referred to as the nontuberculous mycobacteria. Although a subset of nontuberculous mycobacteria are of known clinical significance, the specific traits that dictate pathogenicity remain largely undetermined. We hypothesized that pathogenic nontuberculous mycobacteria are likely to have optimal growth at low pH (~pH 4.5–5.5) and at temperatures near that of the human body (~37°C), enabling survival within human tissues. To test this hypothesis, we measured the growth preferences of 20 strains of nontuberculous mycobacteria, spanning the breadth of diversity across the genus, using cellular activity as a proxy for growth in vitro. Pathogens tended to share preferences for optimal growth at lower pH and higher temperature conditions, while those of limited or no clinical significance generally had growth optima outside a range that would be conducive to effective growth within the human body. We then used our in vitro measurements of pH preferences to determine if we could predict the environmental distributions of nontuberculous mycobacteria, as determined from a cultivation-independent survey of 143 soils across the United States. We show that the measured in vitro pH preferences effectively predicted the distributions of nontuberculous mycobacteria in soil. Our work highlights the broad range in pH and temperature preferences across the genus, demonstrates the utility of these measurements for differentiating human pathogens from non-pathogens, and describes how measured growth preferences can be used to predict the environmental distributions of nontuberculous mycobacteria.IMPORTANCEThe genus Mycobacterium includes Mycobacterium tuberculosis as well as over 200 taxa collectively referred to as the nontuberculous mycobacteria (NTM). Although most nontuberculous mycobacteria have limited clinical significance, a handful of species within the genus are responsible for a growing number of chronic infections worldwide—yet the traits that underlie their pathogenicity remain largely undetermined. We measured the growth preferences of 20 strains of nontuberculous mycobacteria in vitro, finding that NTM pathogens tended to share preferences for optimal growth at lower pH and higher temperature (similar to conditions encountered in a human host), while those of no clinical significance generally had growth optima outside the conditions of the human body. We then used our in vitro measurements to show that we could predict the distributions of nontuberculous mycobacteria in soil, an important environmental reservoir and potential source of exposure and subsequent infection. Our work highlights not only the broad range in pH and temperature preferences across the genus, but the utility of measuring these traits for understanding NTM pathogenicity and ultimately, advancing our understanding of the ecology of nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) are environmental microorganisms for which large, systematic studies of niche diversity are lacking. We performed a semi-longitudinal state-wide sampling campaign (2015-2019) for environmental NTM across Hawai'i. A volunteer network collected 2,334 water biofilms, soil, and dust samples from built (n = 1,946) and natural (n = 388) sites. Of these, 541 contained culturable NTM (23%) and per island hotspots identified. Of 74 NTM species recovered, the most prevalent rapid growing mycobacteria (RGM) were Mycobacterium porcinum, Mycobacterium chelonae, and Mycobacterium abscessus. Mycobacterium intracellulare subsp. chimaera was the most frequently isolated slow growing mycobacteria (SGM). Our longitudinal analyses indicate widespread colonization of diverse niches by species less associated with lung infections such as M. chelonae. In contrast, household water biofilms tended to be reliable niches for M. abscessus and M. avium complex species colonization across the 5-year study. Analysis of 590 deidentified lung samples from Hawai'i and other Pacific Islands revealed M. chimaera as the most frequently isolated species (40%, 238/590). Phylogenetic analysis of environmental and lung M. abscessus suggests most cluster within dominant circulating clone 1 (DCC1). Contrastingly, most Hawai'i and other Pacific Island M. chimaera were distinct from previously studied European isolates, leading to the identification of two novel clusters of phylogenetically related strains we have termed Pacific Island Circulating Cluster 1 and 2 (PCC1, PCC2). PCC1 consists exclusively of Hawai'i/Pacific Island isolates, while PCC2 was enriched by lung samples and was mostly collected from Hawai'i/Pacific Islands. These data reveal the genetic diversity, ecological niches, and potential reservoirs of NTM in varied Hawai'i ecosystems.IMPORTANCENearly one in four environmental Hawai'i samples tested positive for any NTM species, with hotspots often overlapping population centers. Recovery of any NTM species occurred just as often from natural settings as homes and public buildings, highlighting exposures as a normal part of life. Soil was the most common reservoir for NTM colonization, but we distinguish NTM species pertinent to lung disease that were far more likely to be found in water biofilms, such as showerheads and kitchen sinks. No single species dominated the environment; yet, the type of NTM found in water systems closely mirrored those recovered from patients' lung samples. Genetic analyses revealed that Hawai'i harbors distinct, locally circulating strains, including lineages not linked to known hospital outbreaks. Together, these findings improve our understanding of where precarious exposures can occur and inform public health strategies to reduce exposures by highlighting niches that are common hotspots for NTM colonization.

Background Cystic Fibrosis (CF) Centers worldwide have reported healthcare-associated outbreaks of nontuberculous mycobacteria (NTM). We report a retrospective investigation of shared Mycobacterium abscessus strains among people with cystic fibrosis (pwCF) receiving care at Dell Children’s/Ascension combined Pediatric and Adult CF Program (DCMC). Methods Whole genome sequencing (WGS) was used to identify genetically similar isolates among 167 NTM isolates from 57 pwCF. Epidemiological investigation, respiratory and environmental isolate comparisons, and watershed mapping were performed. Results WGS analysis revealed four M. abscessus clusters, two ssp. abscessus and two ssp. massiliense. One subject was infected with two distinct clustered M. abscessus (ssp. abscessus and ssp. massiliense). Epidemiologic investigation demonstrated opportunities for healthcare-associated transmission within all clusters. Two ssp. massiliense subject pairs had healthcare overlaps and high genomic relatedness, including one cohabitating sibling pair. M. abscessus recovered from DCMC revealed genetic similarity to a respiratory isolate from one patient who was never exposed to the hospital environment. Conclusions We identified shared M. abscessus strains via genomic analysis among pwCF at DCMC. None of the clustered patient isolates matched hospital environmental isolates at the genomic level. One hospital environmental isolate had genomic similarity to a respiratory isolate of M. abscessus, but the epidemiologic investigation revealed no evidence of subject exposure to the hospital setting. One ssp. massiliense subject pair had the same level of pangenome relatedness as the sibling pair and epidemiological investigation revealed overlap in the clinic, supporting healthcare-associated person-to-person transmission among the pair within a cluster. One pwCF had polyclonal clustered infections, suggesting multiple environmental sources of acquisition outside the healthcare environment.

Pulmonary nontuberculous mycobacteria (NTM) disease is an emerging public health challenge that is especially problematic in people with cystic fibrosis (CF). Effective treatment depends on accurate species and subspecies identification and antimicrobial susceptibility status. We evaluated the GenoType NTM-DR VER 1.0 assay using biobanked NTM isolates with whole genome sequence (WGS) data and control isolates (total n=285). Species and subspecies detection sensitivity and specificity were 100% for all species and subspecies except for two subspecies of M. intracellulare, that demonstrated a small degree of discrepant identification between M. intracellulare subspecies intracellulare and subspecies chimaera. All antimicrobial resistance markers were identified with 100% sensitivity and specificity.We conclude that the GenoType NTM-DR assay offers a rapid and accurate option for identifying the most frequently encountered pathogenic NTM taxa and drug resistance markers.SUPPORT: Colorado CF Research Development Program and Colorado CF National Resource Centers funded by the Cystic Fibrosis Foundation, NJH Advanced Diagnostics Laboratories, Colorado Advanced Industries Accelerator Grant

Rationale Nontuberculous mycobacteria (NTM) has been reported to be transmitted between people with cystic fibrosis (CF) attending CF centres. A suspected Mycobacterium abscessus outbreak was investigated at the University of Texas Southwestern (UTSW) Adult CF Program using a combination of pathogen genomic sequencing and epidemiologic methods. The objectives of the present study were to apply the Healthcare-Associated Links in Transmission of NTM (HALT NTM) study to investigate the occurrence of potential healthcare-associated transmission and/or acquisition of NTM among people with CF infected with genetically similar NTM isolates. Methods Whole-genome sequencing of respiratory M. abscessus isolates from 50 people with CF receiving care at UTSW was performed to identify genetically similar isolates. Epidemiologic investigation, comparison of respiratory and environmental isolates, and home residence watershed mapping were studied. Measurements and main results Whole-genome sequencing analysis demonstrated seven clusters of genetically similar M. abscessus (four ssp . abscessus and three ssp. massiliense ). Epidemiologic investigation revealed potential opportunities for healthcare-associated transmission within three of these clusters. Healthcare environmental sampling did not recover M. abscessus , but did recover four human disease-causing species of NTM. No subjects having clustered infections lived in the same home residence watershed. Some subjects were infected with more than one M. abscessus genotype, both within and outside of the dominant circulating clones. Conclusions Healthcare-associated person-to-person transmission of M. abscessus appears to be rare at this centre. However, polyclonal infections of M. abscessus species and subspecies, not originating from the endemic hospital environment, suggest multiple shared modes of acquisition outside the healthcare setting.

Mycobacterium abscessus is an intrinsically drug-resistant, rapidly growing, nontuberculous mycobacterium; extrapulmonary infections have been reported in association with medical tourism (1). During November-December 2022, two Colorado hospitals (hospitals A and B) treated patient A, a Colorado woman aged 30-39 years, for M. abscessus meningitis. In October 2022, she had received intrathecal donor embryonic stem cell injections in Baja California, Mexico to treat multiple sclerosis and subsequently experienced headaches and fevers, consistent with meningitis. Her cerebrospinal fluid revealed neutrophilic pleocytosis and grew M. abscessus in culture at hospital A. Hospital A's physicians consulted hospital B's infectious diseases (ID) physicians to co-manage this patient (2).

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.

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that can cause chronic lung disease. Within the U.S., Hawai'i shows the highest prevalence rates of NTM lung infections. Here, we investigated a potential role for active volcanism at the Kīlauea Volcano located on Hawai'i Island in promoting NTM growth and diversity. We recovered NTM that are known to cause lung disease from plumbing biofilms and soils collected from the Kīlauea environment. We also discovered viable Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium intracellulare subsp. chimaera on volcanic ash collected during the 2018 Kīlauea eruption. Analysis of soil samples showed that NTM prevalence is positively associated with bulk content of phosphorus, sulfur, and total organic carbon. In growth assays, we showed that phosphorus utilization is essential for proliferation of Kīlauea-derived NTM, and demonstrate that NTM cultured with volcanic ash adhere to ash surfaces and remain viable. Ambient dust collected on O'ahu concurrent with the 2018 eruption contained abundant fresh volcanic glass, suggestive of inter-island ash transport. Phylogenomic analyses using whole genome sequencing revealed that Kīlauea-derived NTM are genetically similar to respiratory isolates identified on other Hawaiian Islands. Consequently, we posit that volcanic eruptions could redistribute environmental microorganisms over large scales. While additional studies are needed to confirm a direct role of ash in NTM dispersal, our results suggest that volcanic particulates harbor and can redistribute NTM and should therefore be studied as a fomite for these burgeoning, environmentally acquired respiratory infections.

a targeted next-generation sequencing (NGS) assay for the identification of Mycobacterium species and subspecies from clinical specimens. The assay targets ~400 bp of the hsp 65 gene for identification of Mycobacterium species, together with portions of 18 additional genes, including rpo B

MOTIVATION Short-read whole genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences, and sequencing bias reduce the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias, and GC content. RESULTS Reference based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality (MQ) filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness, and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems, and more generally for WGS applications in other organisms. AVAILABILITY All relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Nontuberculous mycobacteria (NTM) are ubiquitous environmental opportunistic pathogens that can cause chronic lung disease. Within the United States, Hawai'i has the highest incidence of NTM lung disease, though the precise reasons are yet to be fully elucidated. One possibility is the high prevalence of NTM in the Hawai'i environment acting as a potential reservoir for opportunistic NTM infections. Through our previous initiatives to collect and characterize NTM in Hawai'i, community scientists of Hawai'i have collected thousands of environmental samples for sequencing. Here, these community scientists were invited for the first time into a high school lab in O'ahu for a genomic sequencing workshop, where participants sequenced four of the collected isolate genomic samples using the Oxford Nanopore Technologies MinION sequencer. Participants generated high quality long read data that when combined with short read Illumina data yielded complete bacterial genomic assemblies suitable for in-depth analysis. The gene annotation analysis identified a suite of genes that might help NTM thrive in the Hawai'i environment. Further, we found evidence of co-occurring methylobacteria, revealed from the sequencing data, suggesting that in some cases methylobacteria and NTM may coexist in the same niche, challenging previously accepted paradigms. The sequencing efforts presented here generated novel insights regarding the potential survival strategies and microbial interactions of NTM in the geographic hot spot of Hawai'i. We highlight the contributions of community scientists and present an activity that can be reimplemented as a workshop or classroom activity by other research groups to engage their local communities.

Nontuberculous mycobacterial (NTM) infections are a concern to people with cystic fibrosis (pwCF) due to the challenges in treatment and questions of infection acquisition. An evidence-based, standardized approach is used to investigate healthcare-associated NTM outbreaks.

Hibernation is a natural model of extreme physiology in a mammal. Throughout winter, small hibernators repeatedly undergo rapid, dramatic swings in body temperature, perfusion, and oxygen delivery. To gain insight into the molecular mechanisms that support homeostasis despite the numerous challenges posed by this dynamic physiology, we collected 13-lined ground squirrel adrenal glands from at least five individuals representing six key timepoints across the year using body temperature telemetry. Differentially expressed genes were identified using RNA-seq, revealing both strong seasonal and torpor-arousal cycle effects on gene expression. Two novel findings emerge from this study. First, transcripts encoding multiple genes involved in steroidogenesis decreased seasonally. Taken together with morphometric analyses, the data are consistent with preservation of mineralocorticoids but suppression of glucocorticoid and androgen output throughout winter hibernation. Second, a temporally orchestrated, serial gene expression program unfolds across the brief arousal periods. This program initiates during early rewarming with the transient activation of a set of immediate early response (IER) genes, comprised of both transcription factors and the RNA degradation proteins that assure their rapid turnover. This pulse in turn activates a cellular stress response program to restore proteostasis comprised of protein turnover, synthesis, and folding machinery. These and other data support a general model for gene expression across the torpor-arousal cycle that is facilitated in synchrony with whole body temperature shifts; induction of the immediate early response upon rewarming activates a proteostasis program followed by a restored tissue-specific gene expression profile enabling renewal, repair, and survival of the torpid state.NEW & NOTEWORTHY This pioneer study of adrenal gland gene expression dynamics in hibernating ground squirrels leverages the power of RNA-seq on multiple precisely timed samples to demonstrate: 1) steroidogenesis is seasonally reorganized to preserve aldosterone at the expense of glucocorticoids and androgens throughout winter hibernation; 2) a serial gene expression program unfolds during each short arousal whereby immediate early response genes induce the gene expression machinery that restores proteostasis and the cell-specific expression profile before torpor reentry.

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.

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.