Plasma extracellular vesicle proteins as promising noninvasive biomarkers for diagnosis of idiopathic pulmonary fibrosis.

High-resolution computed tomography (HRCT) imaging is critical for diagnostic evaluation of Idiopathic Pulmonary Fibrosis (IPF). However, several other interstitial lung diseases (ILDs) often exhibit radiologic pattern similar to IPF on HRCT making the diagnosis of the disease difficult. Therefore, biomarkers that distinguish IPF from other ILDs can be a valuable aid in diagnosis. Using mass spectrometry, we performed proteomic analysis of plasma extracellular vesicles (EVs) in patients diagnosed with IPF, chronic hypersensitivity pneumonitis, nonspecific interstitial pneumonitis, and healthy subjects. A five-protein signature was identified by lasso regression and was validated in an independent cohort using ELISA. The five-protein signature derived from mass spectrometry data showed an area under the receiver operating characteristic curve of 0.915 (95%CI: 0.819-1.011) and 0.958 (95%CI: 0.882-1.034) for differentiating IPF from other ILDs and from healthy subjects, respectively. Stepwise backwards elimination yielded a model with 3 and 2 proteins for discriminating IPF from other ILDs and healthy subjects, respectively, without compromising diagnostic accuracy. In summary, we discovered and validated EV protein biomarkers for differential diagnosis of IPF in independent cohorts. Interestingly, the biomarker panel could also distinguish IPF and healthy subjects with high accuracy. The biomarkers need to be evaluated in large prospective cohorts to establish their clinical utility.

Realistic biomarkers from plasma extracellular vesicles for detection of beryllium exposure.

PURPOSE: Exposures related to beryllium (Be) are an enduring concern among workers in the nuclear weapons and other high-tech industries, calling for regular and rigorous biological monitoring. Conventional biomonitoring of Be in urine is not informative of cumulative exposure nor health outcomes. Biomarkers of exposure to Be based on non-invasive biomonitoring could help refine disease risk assessment. In a cohort of workers with Be exposure, we employed blood plasma extracellular vesicles (EVs) to discover novel biomarkers of exposure to Be. METHODS: EVs were isolated from plasma using size-exclusion chromatography and subjected to mass spectrometry-based proteomics. A protein-based classifier was developed using LASSO regression and validated by ELISA. RESULTS: We discovered a dual biomarker signature comprising zymogen granule protein 16B and putative protein FAM10A4 that differentiated between Be-exposed and -unexposed subjects. ELISA-based quantification of the biomarkers in an independent cohort of samples confirmed higher expression of the signature in the Be-exposed group, displaying high predictive accuracy (AUROC = 0.919). Furthermore, the biomarkers efficiently discriminated high- and low-exposure groups (AUROC = 0.749). CONCLUSIONS: This is the first report of EV biomarkers associated with Be exposure and exposure levels. The biomarkers could be implemented in resource-limited settings for Be exposure assessment.

Comparison of Two Commercial Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Systems for Identification of Nontuberculous Mycobacteria.

OBJECTIVES: This multicenter study's aim was to assess the performance of two commercially available matrix-assisted laser desorption/ionization time of flight mass spectrometry systems in identifying a challenge collection of clinically relevant nontuberculous mycobacteria (NTM). METHODS: NTM clinical isolates (n = 244) belonging to 23 species/subspecies were identified by gene sequencing and analyzed using Bruker Biotyper with Mycobacterial Library v5.0.0 and bioMérieux VITEK MS with v3.0 database. RESULTS: Using the Bruker or bioMérieux systems, 92% and 95% of NTM strains, respectively, were identified at least to the complex/group level; 62% and 57%, respectively, were identified to the highest taxonomic level. Differentiation between members of Mycobacterium abscessus, M fortuitum, M mucogenicum, M avium, and M terrae complexes/groups was problematic for both systems, as was identification of M chelonae for the Bruker system. CONCLUSIONS: Both systems identified most NTM isolates to the group/complex level, and many to the highest taxonomic level. Performance was comparable.

Variation among human, veterinary and environmental Mycobacterium chelonae-abscessus complex isolates observed using core genome phylogenomic analysis, targeted gene comparison, and anti-microbial susceptibility patterns.

Mycobacterium chelonae is a member of the Mycobacterium chelonae-abscessus complex and a cause of opportunistic disease in fish, reptiles, birds, and mammals including humans. Isolates in the complex are often difficult to identify and have differing antimicrobial susceptibilities. Thirty-one previously identified rapidly-growing, non-tuberculous Mycobacterium sp. isolates cultured from biofilms, fish, reptiles, mammals, including humans, and three ATCC reference strains were evaluated with nine M. chelonae-abscessus complex whole genome sequences from GenBank by phylogenomic analysis, targeted gene comparisons, and in-vitro antimicrobial susceptibility patterns to assess strain variation among isolates from different sources. Results revealed minimal genetic variation among the M. chelonae strains. However, the core genomic alignment and SNP pattern of the complete 16S rRNA sequence clearly separated the turtle type strain ATCC 35752T from the clinical isolates and human reference strain "M. chelonae chemovar niacinogenes" ATCC 19237, providing evidence of two distinct subspecies. Concatenation of the partial rpoB (752 bp) and complete hsp65 (1,626 bp) sequence produced the same species/subspecies delineations as the core phylogeny. Partial rpoB and hsp65 sequences identified all the clinical isolates to the appropriate species level when respective cut-offs of 98% and 98.4% identity to the M. chelonae type strain ATCC 35752T were employed. The human strain, ATCC19237, was the most representative strain for the evaluated human, veterinary, and environmental strains. Additionally, two isolates were identified as Mycobacterium saopaulense, its first identification in a non-fish or non-human host.

Mycobacterium avium in Community and Household Water, Suburban Philadelphia, Pennsylvania, USA, 2010-2012.

Attention to environmental sources of Mycobacterium avium complex (MAC) infection is a vital component of disease prevention and control. We investigated MAC colonization of household plumbing in suburban Philadelphia, Pennsylvania, USA. We used variable-number tandem-repeat genotyping and whole-genome sequencing with core genome single-nucleotide variant analysis to compare M. avium from household plumbing biofilms with M. avium isolates from patient respiratory specimens. M. avium was recovered from 30 (81.1%) of 37 households, including 19 (90.5%) of 21 M. avium patient households. For 11 (52.4%) of 21 patients with M. avium disease, isolates recovered from their respiratory and household samples were of the same genotype. Within the same community, 18 (85.7%) of 21 M. avium respiratory isolates genotypically matched household plumbing isolates. Six predominant genotypes were recovered across multiple households and respiratory specimens. M. avium colonizing municipal water and household plumbing may be a substantial source of MAC pulmonary infection.

Mycobacterium avium pseudo-outbreak associated with an outpatient bronchoscopy clinic: Lessons for reprocessing.

We identified a pseudo-outbreak of Mycobacterium avium in an outpatient bronchoscopy clinic following an increase in clinic procedure volume. We terminated the pseudo-outbreak by increasing the frequency of automated endoscope reprocessors (AER) filter changes from quarterly to monthly. Filter changing schedules should depend on use rather than fixed time intervals.

Mycobacterium talmoniae, a Potential Pulmonary Pathogen Isolated from Multiple Patients with Bronchiectasis in the United States, Including the First Case of Clinical Disease in a Patient with Cystic Fibrosis.

We characterize three respiratory isolates of the recently described species Mycobacterium talmoniae recovered in Texas, Louisiana, and Massachusetts, including the first case of disease in a patient with underlying cystic fibrosis. The three isolates had a 100% match to M. talmoniae NE-TNMC-100812T by complete 16S rRNA, rpoB region V, and hsp65 gene sequencing. Core genomic comparisons between one isolate and the type strain revealed an average nucleotide identity of 99.8%. The isolates were susceptible to clarithromycin, amikacin, and rifabutin, while resistance was observed for tetracyclines, ciprofloxacin, and linezolid. M. talmoniae should be added to the list of potential pulmonary pathogens, including in the setting of cystic fibrosis.

Mycobacterium decipiens sp. nov., a new species closely related to the Mycobacterium tuberculosis complex.

Two mycobacterial strains with close similarity to the Mycobacterium tuberculosis complex (MTBC) were isolated from cutaneous lesions of patients in the USA and Italy. At the phenotypic level, similarities to the MTBC included slow growth rate, rough morphotype of the unpigmented colonies and nearly identical high-performance liquid chromatography profiles of mycolic acids. In contrast to the MTBC, the strains were niacin- and nitrate-negative, and catalase-positive both at 68 °C and in semi-quantitative tests. The clinical isolates were more closely related to M. tuberculosis than to any other known mycobacterium and scored positive with commercial DNA probes (Hologic AccuProbe M. tuberculosis). Both average nucleotide identity and genome-to-genome distance suggested the strains are different from the MTBC. Therefore, given the distinguishing phenotypic and genomic-scale differences, we submit that the strains belong to a new species we have named Mycobacteriumdecipiens with type strain TBL 1200985T (=ATCC TSD-117T=DSM 105360T).

Performance of Vitek MS v3.0 for Identification of Mycobacterium Species from Patient Samples by Use of Automated Liquid Medium Systems.

The accuracy and robustness of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system was evaluated by identifying mycobacteria from automated liquid-medium systems using patient samples. This is the first report to demonstrate that proteins within the liquid medium, its supplements, and decontamination reagents for nonsterile patient samples do not generate misidentification or false-positive results by use of the Vitek MS v3.0 system. Prior to testing with patient samples, a seeded-culture study was conducted to challenge the accuracy of the Vitek MS system at identifying mycobacteria from liquid medium by mimicking a clinical workflow. Seventy-seven Mycobacterium strains representing 21 species, seeded in simulated sputum, were decontaminated, inoculated into BacT/Alert MP liquid culture medium, incubated until positivity, and identified using the Vitek MS system. A total of 383 liquid cultures were tested, of which 379 (99%) were identified correctly to the species/complex/group level, 4 (1%) gave a "no-identification" result, and no misidentifications were observed. Following the simulated-sputum study, a total of 73 smear-positive liquid-medium cultures detected using BD BBL MGIT and VersaTREK Myco liquid media were identified by the Vitek MS system. Sixty-four cultures (87.7%) were correctly identified to the species/complex/group level; 7 (9.6%) resulted in no identification; and 2 (2.7%) were misidentified at the species level. These results indicate that the Vitek MS v3.0 system is an accurate tool for routine diagnostics of Mycobacterium species isolated from liquid cultures.

Evaluation of the Vitek MS v3.0 Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry System for Identification of Mycobacterium and Nocardia Species.

This multicenter study was designed to assess the accuracy and reproducibility of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system for identification of Mycobacterium and Nocardia species compared to DNA sequencing. A total of 963 clinical isolates representing 51 taxa were evaluated. In all, 663 isolates were correctly identified to the species level (69%), with another 231 (24%) correctly identified to the complex or group level. Fifty-five isolates (6%) could not be identified despite repeat testing. All of the tuberculous mycobacteria (45/45; 100%) and most of the nontuberculous mycobacteria (569/606; 94%) were correctly identified at least to the group or complex level. However, not all species or subspecies within the M. tuberculosis, M. abscessus, and M. avium complexes and within the M. fortuitum and M. mucogenicum groups could be differentiated. Among the 312 Nocardia isolates tested, 236 (76%) were correctly identified to the species level, with an additional 44 (14%) correctly identified to the complex level. Species within the N. nova and N. transvalensis complexes could not always be differentiated. Eleven percent of the isolates (103/963) underwent repeat testing in order to get a final result. Identification of a representative set of Mycobacterium and Nocardia species was highly reproducible, with 297 of 300 (99%) replicates correctly identified using multiple kit lots, instruments, analysts, and sites. These findings demonstrate that the system is robust and has utility for the routine identification of mycobacteria and Nocardia in clinical practice.

Mycobacterium grossiae sp. nov., a rapidly growing, scotochromogenic species isolated from human clinical respiratory and blood culture specimens.

A previously undescribed, rapidly growing, scotochromogenic species of the genus Mycobacterium (represented by strains PB739T and GK) was isolated from two clinical sources - the sputum of a 76-year-old patient with severe chronic obstructive pulmonary disease, history of tuberculosis exposure and Mycobacterium avium complex isolated years prior; and the blood of a 15-year-old male with B-cell acute lymphoblastic leukaemia status post bone marrow transplant. The isolates grew as dark orange colonies at 25-37 °C after 5 days, sharing features in common with other closely related species. Analysis of the complete 16S rRNA gene sequence (1492 bp) of strain PB739T demonstrated that the isolate shared 98.8 % relatedness with Mycobacterium wolinskyi. Partial 429 bp hsp65 and 744 bp rpoB region V sequence analyses revealed that the sequences of the novel isolate shared 94.8 and 92.1 % similarity with those of Mycobacterium neoaurum and Mycobacterium aurum, respectively. Biochemical profiling, antimicrobial susceptibility testing, HPLC/gas-liquid chromatography analyses and multilocus sequence typing support the taxonomic status of these isolates (PB739T and GK) as representatives of a novel species. Both isolates were susceptible to the Clinical and Laboratory Standards Institute recommended antimicrobials for susceptibility testing of rapidly growing mycobacteria including amikacin, ciprofloxacin, moxifloxacin, doxycycline/minocycline, imipenem, linezolid, clarithromycin and trimethropin/sulfamethoxazole. Both isolates PB739T and GK showed intermediate susceptibility to cefoxitin. We propose the name Mycobacterium grossiae sp. nov. for this novel species and have deposited the type strain in the DSMZ and CIP culture collections. The type strain is PB739T (=DSM 104744T=CIP 111318T).

Two-Phase Hospital-Associated Outbreak of Mycobacterium abscessus: Investigation and Mitigation.

BACKGROUND: Nontuberculous mycobacteria (NTM) commonly colonize municipal water supplies and cause healthcare-associated outbreaks. We investigated a biphasic outbreak of Mycobacterium abscessus at a tertiary care hospital. METHODS: Case patients had recent hospital exposure and laboratory-confirmed colonization or infection with M. abscessus from January 2013 through December 2015. We conducted a multidisciplinary epidemiologic, field, and laboratory investigation. RESULTS: The incidence rate of M. abscessus increased from 0.7 cases per 10000 patient-days during the baseline period (January 2013-July 2013) to 3.0 cases per 10000 patient-days during phase 1 of the outbreak (August 2013-May 2014) (incidence rate ratio, 4.6 [95% confidence interval, 2.3-8.8]; P < .001). Thirty-six of 71 (51%) phase 1 cases were lung transplant patients with positive respiratory cultures. We eliminated tap water exposure to the aerodigestive tract among high-risk patients, and the incidence rate decreased to baseline. Twelve of 24 (50%) phase 2 (December 2014-June 2015) cases occurred in cardiac surgery patients with invasive infections. Phase 2 resolved after we implemented an intensified disinfection protocol and used sterile water for heater-cooler units of cardiopulmonary bypass machines. Molecular fingerprinting of clinical isolates identified 2 clonal strains of M. abscessus; 1 clone was isolated from water sources at a new hospital addition. We made several water engineering interventions to improve water flow and increase disinfectant levels. CONCLUSIONS: We investigated and mitigated a 2-phase clonal outbreak of M. abscessus linked to hospital tap water. Healthcare facilities with endemic NTM should consider similar tap water avoidance and engineering strategies to decrease risk of NTM infection.

Emergence of mmpT5 Variants during Bedaquiline Treatment of Mycobacterium intracellulare Lung Disease.

Bedaquiline (BDQ), a diarylquinoline antibiotic that targets ATP synthase, is effective for the treatment of Mycobacterium tuberculosis infections that no longer respond to conventional drugs. While investigating the off-label use of BDQ as salvage therapy, seven of 13 patients with Mycobacterium intracellulare lung disease had an initial microbiological response and then relapsed. Whole-genome comparison of pretreatment and relapse isolates of M. intracellulare uncovered mutations in a previously uncharacterized locus, mmpT5 Preliminary analysis suggested similarities between mmpT5 and the mmpR5 locus, which is associated with low-level BDQ resistance in M. tuberculosis Both genes encode transcriptional regulators and are adjacent to orthologs of the mmpS5-mmpL5 drug efflux operon. However, MmpT5 belongs to the TetR superfamily, whereas MmpR5 is a MarR family protein. Targeted sequencing uncovered nonsynonymous mmpT5 mutations in isolates from all seven relapse cases, including two pretreatment isolates. In contrast, only two relapse patient isolates had nonsynonymous changes in ATP synthase subunit c (atpE), the primary target of BDQ. Susceptibility testing indicated that mmpT5 mutations are associated with modest 2- to 8-fold increases in MICs for BDQ and clofazimine, whereas one atpE mutant exhibited a 50-fold increase in MIC for BDQ. Bedaquiline shows potential for the treatment of M. intracellulare lung disease, but optimization of treatment regimens is required to prevent the emergence of mmpT5 variants and microbiological relapse.

Mycobacterium arupense, Mycobacterium heraklionense, and a Newly Proposed Species, "Mycobacterium virginiense" sp. nov., but Not Mycobacterium nonchromogenicum, as Species of the Mycobacterium terrae Complex Causing Tenosynovitis and Osteomyelitis.

Mycobacterium terrae complex has been recognized as a cause of tenosynovitis, with M. terrae and Mycobacterium nonchromogenicum reported as the primary etiologic pathogens. The molecular taxonomy of the M. terrae complex causing tenosynovitis has not been established despite approximately 50 previously reported cases. We evaluated 26 isolates of the M. terrae complex associated with tenosynovitis or osteomyelitis recovered between 1984 and 2014 from 13 states, including 5 isolates reported in 1991 as M. nonchromogenicum by nonmolecular methods. The isolates belonged to three validated species, one new proposed species, and two novel related strains. The majority of isolates (20/26, or 77%) belonged to two recently described species: Mycobacterium arupense (10 isolates, or 38%) and Mycobacterium heraklionense (10 isolates, or 38%). Three isolates (12%) had 100% sequence identity to each other by 16S rRNA and 99.3 to 100% identity by rpoB gene region V sequencing and represent a previously undescribed species within the M. terrae complex. There were no isolates of M. terrae or M. nonchromogenicum, including among the five isolates reported in 1991. The 26 isolates were susceptible to clarithromycin (100%), rifabutin (100%), ethambutol (92%), and sulfamethoxazole or trimethoprim-sulfamethoxazole (70%). The current study suggests that M. arupense, M. heraklionense, and a newly proposed species ("M. virginiense" sp. nov.; proposed type strain MO-233 [DSM 100883, CIP 110918]) within the M. terrae complex are the major causes of tenosynovitis and osteomyelitis in the United States, with little change over 20 years. Species identification within this complex requires sequencing methods.

Variable-Number Tandem-Repeat Analysis of Respiratory and Household Water Biofilm Isolates of "Mycobacterium avium subsp. hominissuis" with Establishment of a PCR Database.

"Mycobacterium aviumsubsp.hominissuis" is an important cause of pulmonary disease. It is acquired from environmental sources, but there is no methodology for large population studies. We evaluated the potential of variable-number tandem-repeat (VNTR) analysis. Clinical and household biofilmM. aviumisolates underwent molecular identification. Testing for IS901was done to separateM. aviumsubsp.aviumfromM. aviumsubsp.hominissuis VNTR types were defined using VNTR loci, and subtyping was performed using 3'hsp65and internal transcribed spacer (ITS) sequencing. Forty-nine VNTR types and eight subtypes ofM. aviumsubsp.hominissuis(IS901negative) were identified among 416 isolates ofM. aviumfrom 121 patients and 80 biofilm sites. Of those types, 67% were found only among patient isolates, 11% only among household water isolates, and 23% among both. Of 13 VNTR types that included ≥4 patients, the majority (61.5%) represented geographic clustering (same city). Most VNTR types with multiple patients belonged to the same 3'hsp65sequence code (sequevar). A total of 44 isolates belonging to fourM. aviumsubsp.hominissuisVNTR types (8%), including three with the rare Mav-F ITS sequence and 0/8 subspecies, produced amplicons with IS901PCR primers. By sequencing, all 44 amplicons were not IS901but ISMav6, which was recently observed in Japan but had not been previously described among U.S. isolates. VNTR analysis ofM. aviumsubsp.hominissuisisolates is easier and faster than pulsed-field gel electrophoresis. Seven VNTR loci separated 417 isolates into 49 types. No isolates ofM. aviumsubsp.aviumwere identified. The distributions of the VNTR copy numbers, the allelic diversity, and the low prevalence of ISMav6 differed from the findings for respiratory isolates reported from Japan.

Nocardia thailandica Pulmonary Nocardiosis in a Post-Solid Organ Transplant Patient.

Nocardia thailandica is a rare pathogen related to Nocardia asteroides, Nocardia neocaledoniensis, and Nocardia caishijiensis that, since its original description in 2004, has only been reported to cause wound and ocular infections in humans. We report a case of pulmonary nocardiosis caused by Nocardia thailandica in a 66-year-old solid organ transplant patient from Connecticut, which was identified at the molecular taxonomic level by secA1 analysis, 16S rRNA gene sequencing, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To our knowledge, this is the first reported case of N. thailandica in the United States and the first report of pulmonary infection by this pathogen in the literature.

Utility of sequencing the erm(41) gene in isolates of Mycobacterium abscessus subsp. abscessus with low and intermediate clarithromycin MICs.

The erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of ≥8 µg/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of ≤8 µg/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of ≥16 µg/ml, including ATCC 19977T, revealed 10 sequevars. The sequence in ATCC 19977T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 µg/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of ≤2 (80%), 4 (10%), and 8 (10%) µg/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from ≤2 to ≤4 µg/ml and that isolates with an MIC of 8 µg/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.