Detection and Quantification of Differentially Culturable Tubercle Bacteria in Sputum from Patients with Tuberculosis - PubMed (original) (raw)

Detection and Quantification of Differentially Culturable Tubercle Bacteria in Sputum from Patients with Tuberculosis

Melissa D Chengalroyen et al. Am J Respir Crit Care Med. 2016.

Abstract

Rationale: Recent studies suggest that baseline tuberculous sputum comprises a mixture of routinely culturable and differentially culturable tubercle bacteria (DCTB). The latter seems to be drug tolerant and dependent on resuscitation-promoting factors (Rpfs).

Objectives: To further explore this, we assessed sputum from patients with tuberculosis for DCTB and studied the impact of exogenous culture filtrate (CF) supplementation ex vivo.

Methods: Sputum samples from adults with tuberculosis and HIV-1 and adults with no HIV-1 were used for most probable number (MPN) assays supplemented with CF and Rpf-deficient CF, to detect CF-dependent and Rpf-independent DCTB, respectively.

Measurements and main results: In 110 individuals, 19.1% harbored CF-dependent DCTB and no Rpf-independent DCTB. Furthermore, 11.8% yielded Rpf-independent DCTB with no CF-dependent DCTB. In addition, 53.6% displayed both CF-dependent and Rpf-independent DCTB, 1.8% carried CF-independent DCTB, and 13.6% had no DCTB. Sputum from individuals without HIV-1 yielded higher CF-supplemented MPN counts compared with counterparts with HIV-1. Furthermore, individuals with HIV-1 with CD4 counts greater than 200 cells/mm3 displayed higher CF-supplemented MPN counts compared with participants with HIV-1 with CD4 counts less than 200 cells/mm3. CF supplementation allowed for detection of mycobacteria in 34 patients with no culturable bacteria on solid media. Additionally, the use of CF enhanced detection of sputum smear-negative individuals.

Conclusions: These observations demonstrate a novel Rpf-independent DCTB population in sputum and reveal that reduced host immunity is associated with lower prevalence of CF-responsive bacteria. Quantification of DCTB in standard TB diagnosis would be beneficial because these organisms provide a putative biomarker to monitor treatment response and risk of disease recurrence.

Keywords: HIV; culturability; limiting dilution assay; resuscitation-promoting factors; tuberculosis.

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Figures

Figure 1.

Figure 1.

Participant disposition flow chart. (A) Most probable number (MPN) assays were set up with culture filtrate (CF) and resuscitation-promoting factor (Rpf)− CF supplementation of growth media. CF was isolated from wild type Mycobacterium tuberculosis and Rpf− CF from a quintuple rpf_gene-knockout mutant, allowing for the detection of Rpf-dependent or Rpf-independent differentially culturable tubercle bacteria (DCTB), respectively. To control for the CF effect, MPN assays with no CF supplementation were also performed, which allowed for detection of CF-independent DCTB. (B) A total of 156 patients were analyzed in this study. These included individuals that had strong clinical indication for TB disease either through a positive smear or positive GeneXpert result. Of these, 46 patients were excluded because of lack of culture-confirmed_M. tuberculosis infection detected by MGIT, MPN, and/or CFU. Patients were also deemed ineligible if they were on antibiotic treatment or infected with drug-resistant strains. Of the 110 eligible patients, 21 and 13 had CF-dependent and Rpf-independent DCTB, respectively, whereas 59 patients had mixed DCTB populations. Sputum from 15 patients did not harbor DCTB, and two patients carried CF-independent DCTB. (C) A glossary of terms used in the figure. *Mixtures of CF-dependent DCTB and Rpf-independent DCTB populations. †In some cases, the data for laboratory diagnosis (GeneXpert, MGIT, HAIN, and smear status) were not available. HAIN = Hain Lifesciences Line Probe Assay Genotype MTBDR plus; MGIT = mycobacteria growth indicator tube.

Figure 2.

Figure 2.

Distribution of differentially culturable tubercle bacteria (DCTB) in a cross-sectional group of patients with tuberculosis. Shown on the_y-axis_ are individual patients with their relative proportions of DCTB (given as the quantum of resuscitatable bacteria, reported as the resuscitation index [RI = MPN/CFU]) on the_x-axis_. Culture filtrate (CF)-dependent DCTB, calculated as the log(CF+ MPN/CFU), is reflected in red. If the calculation of CF-dependent or Rpf-independent DCTB yielded a negative value, this was adjusted to 0 because only the growth stimulatory effects of CF/Rpf− CF were considered. Rpf-independent DCTB, calculated as the log(Rpf− MPN/CFU), is reflected in blue. CF-independent DCTB, calculated as the log(MPN No CF/CFU), is shown in_green_. In cases where the CFU was zero, a value of 1 was used to reflect the absence of culturable bacteria, which indicates that the entire population detected in the MPN assay constituted DCTB. The combined_colored bars_ reflect patients with both CF-dependent and independent DCTB populations and absence of bars indicated no detectable DCTB. Inset depicts log CFU counts in samples with no detectable DCTB population. MPN = most probable number; Rpf = resuscitation-promoting factors.

Figure 3.

Figure 3.

Measures of bacterial load stratified by HIV-1 infection status or CD4 T-cell counts. (A) Scatterplot depicting bacterial load distributions in HIV infected/uninfected individuals. (B) Scatterplot depicting bacterial load distributions in individuals with high versus low CD4 T-cell counts. Error bars represent medians and interquartile ranges. To determine statistical significance, the Mann–Whitney _U_test was used with a 95% confidence interval. CF+ MPN (red), Rpf− MPN (blue), MPN no CF (green). Two participants had missing CD4 counts. In all categories the CF+ MPN/Rpf− MPN yielded higher bacterial counts when compared with the MPN no CF (P < 0.0001). *Significant with a 95% confidence interval. CF = culture filtrate; MPN = most probable number; ND = no bacterial growth detected; Rpf = resuscitation-promoting factors.

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