Prospective Analysis of urINe LAM to Eliminate NTM Sputum Screening (PAINLESS) study: Rationale and trial design for testing urine lipoarabinomannan as a marker of NTM lung infection in cystic fibrosis

Abstract

Background: Routine screening for nontuberculous mycobacterial (NTM) lung disease is dependent on sputum cultures. This is particularly challenging in the cystic fibrosis (CF) population due to reduced sputum production and low culture sensitivity. Biomarkers of infection that do not rely on sputum may lead to earlier diagnosis, but validation trials require a unique prospective design.

Purpose: The rationale of this trial is to investigate the utility of urine lipoarabinomannan (LAM) as a test to identify people with CF with a new positive NTM culture. We hypothesize that urine LAM is a sensitive, non-invasive screening test with a high negative predictive value to identify individuals with a relatively low risk of having positive NTM sputum culture.

Study design: This is a prospective, single-center, non-randomized observational study in adults with CF, 3 years of negative NTM cultures, and no known history of NTM positive cultures. Patients are followed for two year-long observational periods with the primary endpoint being a positive NTM sputum culture within a year of a positive urine LAM result and a secondary endpoint of a positive NTM sputum culture within 3 years of a positive urine LAM result. Study implementation includes remote consent and sample collection to accommodate changes from the COVID-19 pandemic.

Conclusions: This report describes the study design of an observational study aimed at using a urine biomarker to assist in the diagnosis of NTM lung infection in pwCF. If successful, urine LAM could be used as an adjunct to traditional sputum cultures for routine NTM screening, and replace cultures in low-risk individuals unable to produce sputum.

Fig 1. PAINLESS schedule of events.

a Refer to PAINLESS Study Timeline (Figure 2). b On-study clinical follow-up visits will occur as clinically indicated in clinic, the hospital or at home. c End-of-study visit will be the first clinical visit occurring >  9 months after the start of Observational Year 2. d Study personnel will continue to review NTM culture results and clinical events recorded in the CFFPR for up to 5 years following end of study. e Samples may be obtained in clinic, the hospital or at home, either expectorated or sputum induction. If results from oropharyngeal swab or bronchoscopy are available, they will also be recorded. ^f Urine sample must be collected within 30 days of a sputum sample at the start of Year 1 and Year 2. End-of-study urine sample does not need to be linked to a sputum sample. ^g Standard clinical assessments will be obtained from CFF patient registry database. ^h Replacement urine for LAM assay if needed.

Fig 2. PAINLESS study design and timeline: Panel

A: Screening criteria requires no history of NTM positive cultures combined with at least 3 negative sputum cultures for NTM over the past 3 years. Panel B: At enrollment, the first urine sample for LAM analysis is collected and is within 30 days of a NTM sputum culture. It is anticipated that a positive urine LAM will be detected in a subset of subject, who will comprise a “high risk cohort” that is more likely to have a positive NTM sputum culture. Panel C: Over the course of the study, NTM sputum cultures will be obtained when available in the context of clinical care. Panel D: At the end of Year 1, a second urine will be collected for LAM analysis. It is anticipated that some subjects with negative LAM at enrollment will convert to positive at each follow-up interval, and likewise some subjects with positive LAM at enrollment will convert to negative. Panel E: At the end of the second year of observation a 3^rd urine sample will be collected for LAM analysis, as well as sputum for NTM culture (if available). Panel F: Results from future sputum NTM cultures will be monitored from the CFF Patient Registry for up to 5 years following the end of study procedures.

Fig 3. Representative GC/MS chromatograms showing the absence and/or presence of urinary LAM related to NTM-negativity and/or positivity.

A) D-Arabinose (D-ara) MS/MS method monitoring m/z 420.9-m/z 192.9): Four characteristic peaks of Internal Standard (¹³C₅-D-Arabinose; top panel); Sequentially (top to bottom), 309 and 308 NTM negative, D-ara negative; BP201 and 303 are positive, LAM positive, NTM positive. B) Tuberculostearic acid (TBSA) Single ion monitoring (SIM) at m/z 297.3: TBSA standard (C:19; top panel); Sequentially, 309 and 308 NTM negative, TBSA negative; BP201 and 303 are TBSA positive, LAM positive, NTM positive..