Sputum cultures are the gold standard for tuberculosis (TB) diagnosis and treatment monitoring. However, cultures in MGIT liquid media are susceptible to microbial contamination, often rendering them uninterpretable. Research has shown that maintaining strict cold chains and supervised sample collection can reduce contamination rates, but few longitudinal studies with weekly sampling have explored this. Here we evaluated whether (1) the time between specimen collection and laboratory processing and (2) unsupervised specimen collection are associated with contamination rates. Additionally, we estimated contamination rates over the first 12 weeks of treatment and assessed clinical and behavioral predictors of contamination. We collected 3155 sputum specimens from 301 participants undergoing TB treatment. Contamination was lowest (12.3%) at treatment initiation, increased over the first few weeks, and stabilized around 30% from week 8 onwards. Samples collected without supervision were more likely to be contaminated at treatment initiation (p=0.048) and over the 12 weeks (p=0.028). We observed an inverse relationship between smear grade and contamination risk throughout the sampling period. These findings underscore the importance of supervised sputum collection to reduce contamination and provide ways to enhance the clinical and research value of weekly cultures, particularly those collected later in treatment. This is especially relevant for community-collected specimens used in monitoring treatment response.

The authors have declared no competing interest.

This study was funded by the United States National Institute of Allergy and Infectious Diseases (FAIN: R01AI119037)

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethical approval was granted by: Boston Medical Center Institutional Review Board (H-34970) South African Medical Research Council (EC011-5/2016) Health Research Ethics Committees Stellenbosch University Health Research Ethics Committees (N17/04/039 RECIP_MRC_EC011-5/2016).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors