Detection and quantification of differentially culturable tubercle bacteria in sputum from patients with tuberculosis

Melissa D. Chengalroyen, Germar M. Beukes, Bhavna G. Gordhan, Elizabeth M. Streicher, Gavin Churchyard, Richard Hafner, Robin Warren, Kennedy Otwombe, Neil Martinson, Bavesh D. Kana

Research output: Contribution to journalArticle

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 CFdependent 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 CFsupplemented 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 Rpfindependent 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.

Original languageEnglish (US)
Pages (from-to)1532-1540
Number of pages9
JournalAmerican Journal of Respiratory and Critical Care Medicine
Volume194
Issue number12
DOIs
StatePublished - Dec 15 2016

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Sputum
Tuberculosis
Bacteria
HIV-1
CD4 Lymphocyte Count
Mycobacterium
Resuscitation
Immunity
Biomarkers

Keywords

  • Culturability
  • HIV
  • Limiting dilution assay
  • Resuscitation-promoting factors
  • Tuberculosis

ASJC Scopus subject areas

  • Medicine(all)
  • Pulmonary and Respiratory Medicine
  • Critical Care and Intensive Care Medicine

Cite this

Chengalroyen, M. D., Beukes, G. M., Gordhan, B. G., Streicher, E. M., Churchyard, G., Hafner, R., ... Kana, B. D. (2016). Detection and quantification of differentially culturable tubercle bacteria in sputum from patients with tuberculosis. American Journal of Respiratory and Critical Care Medicine, 194(12), 1532-1540. https://doi.org/10.1164/rccm.201604-0769OC

Detection and quantification of differentially culturable tubercle bacteria in sputum from patients with tuberculosis. / Chengalroyen, Melissa D.; Beukes, Germar M.; Gordhan, Bhavna G.; Streicher, Elizabeth M.; Churchyard, Gavin; Hafner, Richard; Warren, Robin; Otwombe, Kennedy; Martinson, Neil; Kana, Bavesh D.

In: American Journal of Respiratory and Critical Care Medicine, Vol. 194, No. 12, 15.12.2016, p. 1532-1540.

Research output: Contribution to journalArticle

Chengalroyen, MD, Beukes, GM, Gordhan, BG, Streicher, EM, Churchyard, G, Hafner, R, Warren, R, Otwombe, K, Martinson, N & Kana, BD 2016, 'Detection and quantification of differentially culturable tubercle bacteria in sputum from patients with tuberculosis', American Journal of Respiratory and Critical Care Medicine, vol. 194, no. 12, pp. 1532-1540. https://doi.org/10.1164/rccm.201604-0769OC
Chengalroyen, Melissa D. ; Beukes, Germar M. ; Gordhan, Bhavna G. ; Streicher, Elizabeth M. ; Churchyard, Gavin ; Hafner, Richard ; Warren, Robin ; Otwombe, Kennedy ; Martinson, Neil ; Kana, Bavesh D. / Detection and quantification of differentially culturable tubercle bacteria in sputum from patients with tuberculosis. In: American Journal of Respiratory and Critical Care Medicine. 2016 ; Vol. 194, No. 12. pp. 1532-1540.
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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 CFdependent 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 CFsupplemented 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 Rpfindependent 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.",
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AU - Churchyard, Gavin

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