In high tuberculosis (TB) burden settings HIV-infection confers markedly higher risk of TB infection and TB disease [
1]. In general, pulmonary TB in persons living with HIV (PLH) presents with less cavitation and lower bacillary loads in sputum [
2‐
6] and is therefore more likely to be below the detection limit of smear microscopy than in immunocompetent individuals [
7]. Inclusion criteria for clinical trials of novel agents or regimens against TB often require smear-positive disease to assure baseline positive
Mycobacterium tuberculosis cultures in samples taken prior to initiation of TB treatment to ensure measurable treatment responses from a high baseline [
8,
9]. Smear-negative PLH are therefore underrepresented in studies of novel treatments for pulmonary TB [
10‐
13].
Four semi-quantitative methods are used to infer bacillary load - the concentration of mycobacteria - in a biological sample [
14‐
20]. Smear grading on Ziehl-Nielsen or auramine stained smears directly detects bacteria by light or fluorescent microscopy [
14]. The Xpert MTB/RIF assay (Cepheid Innovation, Sunnyvale, CA) is based on nucleic acid amplification technology (NAAT) [
15]. It has a reported limit of detection (LOD) of as low as 112.6 CFU/ ml (colony forming units per millilitre) and is increasingly replacing smear microscopy as the primary diagnostic measure on sputum from individuals with presumptive pulmonary TB [
16‐
18]. Subsequently, the Xpert MTB/RIF-Ultra assay has become available that has a reported LOD of 15.6 CFU/ml [
19]. The Xpert’s cycle threshold (Ct or Cq) reflects bacillary load as an inverse measure, where low values represent high bacillary numbers. The fluorescent signal produced from the NAAT emerges sooner if more DNA is present and in practice, those with
M. tuberculosis detected results have Ct values from 15 to 28. In the automated Mycobacterial Growth Indicator Tube (MGIT) liquid culture system (Becton, Dickinson, Franklin Lakes, NJ), the time-to-positive (TTP) signal occurs sooner when bacillary load is high [
20]. Positive results are detected from as low as 3 days with high loads but the system can flag positive to up to 42 days, after which it is considered negative. Bacterial cell counts on solid media provide numeric read outs and viable bacillary load can be quantified accurately. Cultures - although providing evidence of mycobacterial viability, which smear microscopy and Xpert are unable to do - may not be of immediate value because positive results for bacterial cell counts take at least 3 weeks for bacteria to form colonies, and MGIT culture can take up to 42 days for a positive result [
14] by which time decisions about initiating TB treatment likely have already been made. Withholding treatment in patients potentially eligible for a clinical trial for the culture results would be unacceptable.
In this study we followed a cohort of adult PLH with pulmonary TB, both smear-negative and smear-positive, and subjected their serial sputum specimens whilst on therapy to these four semi-quantitative methods to characterise their initial, and extended mycobacterial responses to TB treatment. Our overall objective was to assess if HIV-infected, smear-negative patients with pulmonary TB have demonstrable bacillary responses to therapy and could be included in clinical trials of novel treatment regimens.