Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status

Abstract

Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.

Keywords: aerosol sampling; subclinical; tuberculosis.

Fig. 1.

Presentation of symptoms at baseline. (A) Percentage of participants with one or more self-reported symptoms. (B) Distribution of the number of symptoms per individual by diagnostic group; the median symptom number for groups A, B, and C was 3, 2, and 2, respectively.

Fig. 2.

The detection of Mtb bacilli in bioaerosol samples from all three diagnostic groups at baseline. (A) Counts of putative Mtb with medians 9, 4.5, and 8 (represented by the thick black bars) for groups A, B, and C, respectively. No difference was found between the groups with Wilcoxon rank-sum testing after Bonferroni correction for multiple comparisons. (B) Percentage Mtb bioaerosol positive samples per diagnostic group. Owing to contamination of the slides, four samples were uninterpretable; these are not included in the denominator of the prevalence proportions.

Fig. 3.

The detection of Mtb bioaerosols decreased similarly in all three groups over 6 mo. (A) The percentage of bioaerosol samples that were positive for Mtb. The det.nominator is all visits yielding an interpretable sample. Thirteen samples were uninterpretable; these are not included in the denominator of the prevalence proportions. The error bars represent the SEM. Fisher’s exact tests performed on each visit found no significant differences. (B) Secular trends in the number of Mtb detected in bioaerosol samples of each diagnostic group. Lines connect the samples from the same participant at each successive visit.

Fig. 4.

Kaplan–Meier plots showing proportion with symptom resolution over time. The first visit at which each symptom is not reported is considered the time to resolution. Individuals reporting symptoms at the final visit are censored at the time of this visit. (A) shows the time to resolution of self-reported persistent cough, (B) fever, (C) subjective weight loss, and (D) night sweats. All plots compare TB patients on treatment (groups A and B) and those individuals not diagnosed with TB and therefore not treated (group C). If a symptom recurred after resolution, this was not included. The shaded areas represent 95% CI. Log-rank test P-values are given in the Top Right corner.

Fig. 5.

Kaplan–Meier plots showing time to Mtb bacillary clearance from collected bioaerosol. The first visit at which Mtb is not detected in the sample is considered the time to clearance. Individuals with detectable Mtb at the final visit are censored at the time of this visit. (A) All participants. (B) TB patients on treatment (groups A and B) are separated from those individuals not diagnosed with TB and therefore not treated (group C). (C) Comparison of time to clearance between those with a previous history of TB and those without. (D) Participants separated according to HIV status. The shaded areas represent 95% CI. Log-rank test P-values are given in the Top Right corner for panels B–D. PLHIV, person living with HIV.

Fig. 6.

Kaplan–Meier plots showing the proportion of individuals with any TB symptom (persistent cough, fever, weight loss, and night sweats) against days since first sampling. The first visit without reported symptoms is considered the time to clearance. Participants with symptoms at the final visit are censored at the time of this visit. (A) All participants. (B) Participants separated into TB patients on treatment (groups A and B) and those not diagnosed with TB and therefore not treated (group C). (C) Comparison of participants with a previous history of TB and without. (D) Comparison of participants according to HIV status. The shaded areas represent 95% CI. Log-rank test P-values are given in the Top Right corner for panels B–D. PLHIV, person living with HIV.

Fig. 7.

Examining the change in DMN-trehalose phenotype and Mtb cell length through time. A comparison of (A) polarity index and (B) Mtb cell length between treatment groups at each visit. Polarity index is defined as the fluorescence intensity in arbitrary fluorescence units (AFU) of the brighter pole divided by the fluorescence intensity at the midcell. A Wilcoxon rank-sum test was performed.