Serum Cell-Free DNA-based Detection of Mycobacterium avium Complex Infection

Abstract

Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.

Keywords: Mycobacterium avium complex; clustered regularly interspaced short palindromic repeats; molecular diagnostics; pulmonary disease.

Figure 1.

Study participants. (A and B) Clinical information and archived serum from patients with Mycobacterium avium complex (MAC) pulmonary disease (PD) and control subjects without nontuberculous mycobacterial (NTM) disease enrolled in the Oregon Health & Science University (OHSU) Northwest NTM Biobank (A) and patients with MAC PD/disseminated MAC seen at the NIH Clinical Center and non-NTM control subjects enrolled in the OHSU Northwest NTM Biobank (B) were respectively used to generate a discovery and a validation cohort to assess CRISPR MAC assay diagnostic performance. All MAC PD cases met American Thoracic Society/European Respiratory Society/European Society of Clinical Microbiology and Infectious Diseases/Infectious Diseases Society of America disease criteria (6) and included a subgroup of individuals who initiated MAC-directed treatment. Non-NTM control patients had diagnoses of bronchiectasis or COPD but had no histories of NTM PD or NTM-positive respiratory cultures. A subset of OHSU biobank patients with MAC PD with follow-up samples after treatment initiation were evaluated to analyze the serum MAC cfDNA response to treatment. cfDNA = cell-free DNA; COPD = chronic obstructive pulmonary disease; CRISPR = clustered regularly interspaced short palindromic repeats; Rx = medication.

Figure 2.

Clustered regularly interspaced short palindromic repeats (CRISPR) Mycobacterium avium complex (MAC) diagnostic assay performance. (A) Culture, smear, and MAC cell-free DNA (cfDNA) data for 71 individuals with MAC pulmonary disease (PD) before treatment initiation. (B–E) CRISPR MAC signal detected in (B) pretreatment sera of 15 control subjects and 71 individuals with MAC PD, (C) individuals infected with M. avium or M. intracellulare, (D) smear-positive and smear-negative individuals, and (E) patients with MAC PD with and without cavitary disease. (F) Culture and MAC cfDNA results for the MAC PD/disseminated MAC, disease control (nontuberculous mycobacterial culture negative with chronic obstructive pulmonary disease [COPD] and/or bronchiectasis), and healthy control groups. (G) CRISPR MAC signal detected in pretreatment sera of the MAC PD, disseminated MAC, disease control, and healthy control groups. (H and I) CRISPR MAC signal differences between (H) healthy control subjects and disease control subjects with COPD or bronchiectasis and (I) patients with MAC PD with M. avium and M. intracellulare infections (median with interquartile range). P values were calculated using two-sided Wilcoxon rank sum tests. For cfDNA, (+), (++), and (+++) indicate low to high CRISPR MAC signal tertiles. Graphs depict mean ± SD of three technical replicates for each sample; the red dashed line indicates the threshold for a positive CRISPR MAC signal. a.u. = arbitrary units; unk = unknown.

Figure 3.

Clustered regularly interspaced short palindromic repeats (CRISPR) Mycobacterium avium complex (MAC) assay monitors treatment response. (A and B) Culture, smear, and MAC cell-free DNA (cfDNA) results (A) and changes in serum CRISPR MAC signal (B) for MAC PD medication (Rx) cohort subjects at the indicated treatment intervals. (C) Change in CRISPR MAC signal and respiratory symptom score (RSS) results for MAC PD Rx cohort subjects at 3 months after enrollment; the circled data point indicates data from a patient with both bronchiectasis and COPD but who revealed culture conversion, showing an RSS decrease despite a marked decrease in CRISPR MAC signal. (D) Culture, smear, and MAC cfDNA results for patients with MAC PD who did and did not respond to treatment within a 12-month follow-up interval, as determined by culture conversion. (E and F) Changes of MAC cfDNA concentrations in (E) responders and (F) nonresponders before and after 12-month treatment. P values were calculated using two-sided Wilcoxon signed rank tests. For cfDNA, (+), (++), and (+++) indicate low to high CRISPR MAC signal tertiles. Graphs depict mean ± SD of three technical replicates for each sample; the red dashed line indicates the threshold for a positive CRISPR MAC signal. a.u. = arbitrary units.