Assessment of differentially culturable tubercle bacteria assays for the detection of tuberculosis infection in asymptomatic household contacts and the implications for intra-household transmission: a longitudinal cohort study.
BACKGROUND: Conventional culture methods for tuberculosis diagnosis miss differentially culturable tubercle bacteria (DCTB), which grow only in liquid assays supplemented with growth-enhancing factors. This limitation, combined with inadequate contact tracing and screening, often fails to identify asymptomatic individuals, with live bacilli detectable by enhanced culture methods. This shortfall results in undiagnosed reservoirs of bacteria, potentially fuelling ongoing transmission. In this study, we aimed to investigate whether DCTB assays provide greater sensitivity by detecting more Mycobacterium tuberculosis infections than conventional culture and whether this enhanced detection improves the resolution of intrahousehold transmission mapping. In addition, we sought to evaluate whether DCTB populations can progress to conventional culture positivity, thereby highlighting their clinical and epidemiological relevance. METHODS: In this prospective observational longitudinal cohort study, drug-susceptible or rifampicin-resistant tuberculosis index participants aged 12 years or older, were recruited from primary healthcare clinics from two South African districts. Inclusion criteria were informed consent, Xpert MTB/RIF Ultra-positive results, tuberculosis symptoms (>2 weeks), provision of baseline samples, at least one consenting household contact, and documented HIV status. Household contacts of the index patients and control households were also recruited. Sputum specimens were collected at baseline and 2, 4, 8, 12, and 16 months from the index participants and household contacts. Samples were analysed by conventional mycobacterial growth indicator tube (MGIT) culture, and colony-forming unit assays to identify viable bacteria. Enhanced culture to detect DCTB involved serial dilution of sputum in liquid culture, supplemented with M tuberculosis culture filtrate as a source of growth stimulatory factors. Whole-genome sequencing (WGS) of cultured isolates was performed to trace household transmission. FINDINGS: Between June 1, 2020, and Feb 6, 2024, 293 index participants (183 [62%] male), 701 household contacts (453 [65%] female), and 122 control participants (67 [55%] female) were enrolled. At baseline, 249 (85%) of 293 index participants and 110 (16%) of 701 household contact sputum samples were positive for M tuberculosis by MGIT conventional culture. For baseline MGIT-negative specimens, DCTB assays detected M tuberculosis in an additional 21 (7%) of 293 index participants and 26 (4%) of 701 household contacts. Over 16 months of follow-up, DCTB assays identified 61 (8·7%) of 701 additional tuberculosis-positive household contacts not detected by conventional culture. WGS-guided transmission mapping using conventional culture identified transmission in 16 (15%) of 104 households, whereas DCTB assays detected an additional 19 (18%) of 104 transmission events. No evidence of intrahousehold transmission was found in the remaining 69 (66%) of 104 tuberculosis-positive households. Over the 16-month follow-up period, conventional culture identified 233 positive household contacts, of which 195 (84%) were asymptomatic. DCTB assays detected an additional 94 cases of M tuberculosis positivity in household contacts, of which 79 (84%) were asymptomatic. In control households, tuberculosis prevalence at baseline was two (2%) of 122, with an additional three (3%) of 122 identified during follow-up. INTERPRETATION: DCTB assays provide substantial value by detecting asymptomatic individuals missed by conventional culture, revealing a potentially important reservoir of subclinical infection, which could sustain transmission. In addition, DCTB detection uncovers transmission linkages missed by conventional culture, providing a more comprehensive understanding of M tuberculosis transmission dynamics and highlighting the need to incorporate enhanced culture methods into diagnostic and surveillance strategies, to strengthen early case identification and tuberculosis control efforts. FUNDING: National Institutes of Health.