Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease–A pilot study

Paige K. Marty, Balaji Pathakumari, Thomas M. Cox,Virginia P. Van Keulen, Courtney L. Erskine, Maleeha Shah, Mounika Vadiyala, Pedro Arias-Sanchez, Snigdha Karnakoti, Kelly M. Pennington, Elitza S. Theel, Cecilia S. Lindestam Arlehamn, Tobias Peikert, Patricio Escalante


Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22).


Nontuberculous mycobacteria (NTM) pathogens are causing more disease burden than tuberculosis (TB) in several regions of the world. The annual incidence and prevalence of NTM disease from 2008 to 2015 increased from 3.13 to 4.73 and 6.78 to 11.70 per 100 000/year respectively in the United States [1]. NTM lung disease (NTM-LD) is the most common presentation accounting for 80–90% of all cases [2]. 

Materials and methods

The study was approved by the Mayo Clinic Institutional Review Board (IRB)(IRB#:09–003253). All study participants signed a written informed consent and were enrolled in Mayo Clinic, Rochester, Minnesota between August 2017 and November 2021. All the methods were carried out in accordance with relevant guidelines and regulations after obtaining approval and recommendations from the Mayo Clinic IRB. This pilot study was part of a prospective, single-center, laboratory technician-blinded larger study to investigate new immune biomarkers in tuberculosis, which includes patients with NTM-LD as control subjects


Ninety-two individuals were screened for study eligibility and 52 were excluded, including 44 subjects with LTBI (Fig 1). We enrolled 22 control subjects and 18 patients of NTM-LD, including 8 patients with progressive disease and 10 patients with nonprogressive NTM-LD that did not require antimicrobial therapy (Tables 1, 2)


The diagnosis of NTM-LD can be delayed or missed as some patients exhibit non-specific symptoms for several months or years before the diagnosis is made, and for some patients, diagnosis comes after the disease has progressed substantially [17, 18]. Our study findings suggest that a blood-based multiparametric IFN-γ ELISpot


Our pilot study results suggest that PPD-specific CD25+CD134+ upregulation in T-cells is a potential blood biomarker to accurately diagnose NTM-LD.

Citation: Marty PK, Pathakumari B, Cox TM, Van Keulen VP, Erskine CL, Shah M, et al. (2024) Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease–A pilot study. PLoS ONE 19(4): e0301659.

Editor: Mao-Shui Wang, Shandong Public Health Clinical Center: Shandong Provincial Chest Hospital, CHINA

Received: October 3, 2023; Accepted: March 20, 2024; Published: April 19, 2024

Copyright: © 2024 Marty et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data and its Supporting Information files are within the manuscript.

Funding: This work was supported by the Lucile Nelson Career Development Award in Pulmonary Research. Part of this work was supported by the CHEST Foundation (K.M.P., P.E.), and the National Institute of Allergy and Infectious Diseases at the National Institutes of Health [AI141591 to P.E.]. Part of this project was also supported by Grant Number UL1 TR000135 from the National Center for Advancing Translational Sciences (NCATS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: P.E. and T.P., and their institution have filed two patent applications related to immunodiagnostic laboratory methodologies for latent tuberculosis infection (Patent numbers: 9678071 and 10401360), which are not included in this manuscript. To date, there has been no income or royalties associated with those filed patent applications. This does not alter our adherence to PLOS ONE policies on sharing data and materials. PE participated in a short-term advisory scientific board for DiaSorin Molecular in 2020, which was outside the scope of the submitted manuscript, and honorarium was paid to Mayo Clinic. E.S.T serves as a consultant for Roche Diagnostics (Basel, Switzerland), Euroimmun US (Mountain Lakes, NJ, USA), and Seriummune Inc. (Goleta, CA, USA) on topics outside the scope of this manuscript. P.E., T.P., and E.S.T. have no other conflicts to declare. P.K.M., B.P., T.M.C., V.P.V, C.L.E., M.S., M.V., P.A.S., S.K., K.M.P., and C. S. L. A. have no conflicts to declare.





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