Nontuberculous mycobacteria, or NTM, are a sprawling group of more than 200 described species that live in soil, dust, air, and both natural and drinking water sources. Unlike their infamous relative Mycobacterium tuberculosis, they are not spread from person to person. Instead, people encounter them in the environment, and for most individuals exposure causes no harm. But in susceptible patients, including those with damaged lungs or weakened immune systems, NTM can establish persistent infections, most often in the lungs, and occasionally in lymph nodes, skin, and soft tissues. Their hydrophobic, lipid-rich cell walls help them stick to surfaces and shrug off disinfectants, which allows them to colonize water distribution systems, biofilms, and even healthcare settings.

Diagnosing NTM disease is notoriously difficult, particularly in low- and middle-income countries. Symptoms and radiological findings of pulmonary NTM infection can closely mimic tuberculosis, so patients may be misdiagnosed, treated for the wrong disease, or diagnosed late. Accurate identification of the infecting species matters because it distinguishes NTM from tuberculosis and guides appropriate therapy. Yet the highest-resolution tools, such as whole-genome sequencing and targeted gene sequencing, remain out of routine reach in many resource-limited laboratories because of cost, infrastructure, and bioinformatics demands.

The Ecuadorian team analyzed 249 isolates processed at the national reference laboratory during the study window, excluding 24 that could not be assigned a conclusive species-level identification. The remaining 225 isolates came from patients with clinical suspicion of mycobacterial infection, defined by a positive acid-fast bacilli smear, symptoms consistent with pulmonary or extrapulmonary mycobacterial disease, and a negative GeneXpert MTB/RIF result, which rules out tuberculosis. The researchers classified isolates by anatomical origin: pulmonary samples included sputum, bronchoalveolar lavage, bronchial lavage, bronchial aspirates, and gastric aspirates, while extrapulmonary samples ranged from urine and abscesses to lymph nodes, cerebrospinal fluid, and surgical wounds.

Two molecular methods underpinned the species identifications. For 133 isolates, the team used the GenoType Mycobacterium CM assay, a commercial line-probe assay that amplifies mycobacterial DNA and hybridizes it to membrane strips carrying species-specific probes, with banding patterns read against the manufacturer’s interpretation chart. The other 92 isolates were identified by PCR-restriction fragment length polymorphism analysis of the hsp65 gene, a lower-cost in-house technique in which a 441-base-pair fragment is amplified, digested with the restriction enzymes BstEII and HaeIII, and matched against reference fragment patterns in the public PRASITE database. PRA-hsp65 dominated the early years of the study, while the line-probe assay was adopted from 2022 onward; in 2021 both methods ran in parallel but on separate, non-overlapping subsets of isolates. Which method was used each year depended largely on the budget available from the Ministry of Public Health.

The results reveal a clear pulmonary predominance: 205 of the 225 isolates, or 91.11 percent, came from respiratory samples, and only 20 from extrapulmonary sites. Patients ranged from 1 to 92 years old, with a median age of 54 years, and men accounted for 57.78 percent of isolates overall. The male predominance was concentrated in the pulmonary group, where roughly 60 percent of patients were male, a pattern consistent with international reports linking pulmonary NTM disease to predisposing factors such as chronic obstructive pulmonary disease and smoking. The small extrapulmonary group, by contrast, showed a female predominance of about three to one, echoing surveillance data from the United States where skin and soft-tissue NTM infections are more frequently reported in women. Geographically, the picture was uneven: El Oro led with 48 isolates, followed by Pichincha with 40 and Los Ríos with 35, together accounting for nearly 55 percent of the total.

Across the six-year period, the laboratory identified 11 distinct NTM species: M. avium and M. intracellulare, both members of the M. avium complex, along with M. abscessus, M. fortuitum, M. chelonae, M. kansasii, M. szulgai, M. gordonae, M. flavescens, M. terrae, and M. scrofulaceum. Among pulmonary isolates, four species dominated: M. fortuitum at 19.51 percent, M. intracellulare at 18.05 percent, M. kansasii at 16.10 percent, and M. abscessus at 13.66 percent. In extrapulmonary isolates, M. fortuitum and M. abscessus topped the list at 30 and 25 percent respectively, consistent with the established role of rapidly growing mycobacteria in skin, soft-tissue, and lymph node infections, and mirroring a recent case series from neighboring Colombia. Fisher’s exact tests with Benjamini-Hochberg correction for multiple comparisons found no statistically significant differences in species distribution between pulmonary and extrapulmonary isolates, though the authors caution that only 20 extrapulmonary isolates limited the statistical power of that comparison.

The study’s most consequential finding may be what it did not find. Only 225 NTM isolates at a national reference laboratory over six years, in a country of about 17 million people, is remarkably few, and the authors argue this points to substantial underdiagnosis. Unlike tuberculosis, which is subject to mandatory notification in Ecuador, NTM infections are not routinely reported, and prior national data are so sparse that the new findings could not be placed in historical context. The geographic concentration in three provinces likely reflects referral patterns and unequal access to laboratory services rather than true regional variation in NTM burden, and the authors explicitly warn that the data should not be read as representative of national epidemiology.

The researchers also acknowledge technical limitations inherent to their methods. Both PRA-hsp65 and line-probe assays have lower discriminatory power than sequencing-based approaches, particularly for closely related taxa such as the M. abscessus complex, M. fortuitum and related species, the M. terrae complex, and certain M. kansasii variants, where band patterns may overlap or intraspecies polymorphisms may go undetected. Because no isolate was tested by both methods, the dataset cannot support any comparison of diagnostic performance between them. Detailed clinical records, imaging, and the full microbiological criteria required to confirm NTM disease under international guidelines were also unavailable, so the analysis treated the samples as isolates rather than confirmed cases of disease.

Even with those caveats, the study marks a milestone as the first longitudinal overview of NTM diversity at Ecuador’s national reference laboratory. The authors call for strengthening laboratory capacity and epidemiological surveillance, incorporating complementary species-identification methods such as Sanger sequencing of the hsp65, rpoB, or 16S-23S ITS regions, and adding pulmonary NTM disease to the list of notifiable conditions within national tuberculosis control programs. As NTM incidence rises globally and these opportunistic pathogens gain recognition as an emerging public health concern, the Ecuadorian experience illustrates a wider challenge for low- and middle-income countries: the organisms are everywhere, the diagnostic tools exist, but without surveillance systems and investment, the true scale of the problem remains invisible.

Subject of Research: Species distribution and underdiagnosis of nontuberculous mycobacteria in Ecuador

Article Title: Species distribution of nontuberculous mycobacteria identified at a national reference laboratory in Ecuador from 2019 to 2024

Article References: Franco-Sotomayor, G., Carvajal, E., Nicola, E., Cardenas Franco, C., Salcedo Maldonado, A. B., Orlando, S. A., & Garcia-Bereguiain, M. A. (2026). Species distribution of nontuberculous mycobacteria identified at a national reference laboratory in Ecuador from 2019 to 2024. Frontiers in Cellular and Infection Microbiology, 16, Article 1965771. https://doi.org/10.3389/fcimb.2026.1965771

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1965771

Keywords: nontuberculous mycobacteria, Ecuador, Mycobacterium abscessus, Mycobacterium fortuitum, pulmonary infection, line-probe assay, PRA-hsp65, molecular diagnostics, epidemiology, surveillance, tuberculosis mimics, public health