Simultaneous measurement of 16S-rRNA and pre-16S-rRNA as a strategy to monitor clinical tuberculosis

Evelin Dombay; Wilber Sabiiti; Daniela Alferes de Lima Headley; M. Bonifác Légrády; Nina van Campen; Sanne Zweijpfennig; Martin J. Boeree; Derek J. Sloan; Stephen H. Gillespie

doi:10.3389/frabi.2026.1760862

Simultaneous measurement of 16S-rRNA and pre-16S-rRNA as a strategy to monitor clinical tuberculosis

Evelin Dombay
, Wilber Sabiiti
, Daniela Alferes de Lima Headley
, M. Bonifác Légrády
, Nina van Campen
, Sanne Zweijpfennig
, Martin J. Boeree
, Derek J. Sloan
, Stephen H. Gillespie^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Background: Culture-based biomarkers of TB treatment response monitoring, e.g., Mycobacterial Growth Indicatory Tube (MGIT), are compromised when bacteria enter a non-replicating persister phase limiting the measurement of antibiotic efficacy and resistance. Understanding how antibiotic exposure to antibiotics alters bacterial physiology could help develop more effective TB therapies. We developed a novel assay with simultaneous measurement of 16S rRNA (bacterial burden) and its precursor, pre-16S rRNA (metabolic activity), and tested it on samples from patients in a trial of optimised-dose rifampicin.

Methods: We developed a multiplex reverse transcriptase quantitative PCR assay (RT-qPCR) to measure relative gene expression of pre-16S rRNA and 16S rRNA in pre-treatment (control) and sequential samples from patients in the Phase II HIGHRIF2 (NCT00760149) clinical trial. We constructed a mathematical model to assess changes in pre-16S gene expression relative to 16S rRNA over time, facilitating the comparison of rifampicin doses’ efficacy.

Findings: In a retrospective study of 19 patients, pre-16S rRNA and 16S rRNA decreased steadily during the initial 36 days of treatment. This was evidenced by the rising cycle threshold (Cq) values slope 0.404 and 0.212, respectively, however, pre-16S rRNA decreased significantly quicker (P<0.0001). The changes in the relative gene expression of pre-16S rRNA during treatment fitted a double exponential decay curve (R² = 0.996). According to this model, 1200 mg RIF-containing therapy exerted the most potent and rapid impact on pre-16S rRNA expression (Maximum suppression (R_min)=1.694, T (time) =9.78 days), and also resulted in the swiftest daily reduction in bacterial load (–0.072 log₁₀ CFU ml^-1/day).

Interpretation: The pre-16S rRNA and 16S rRNA gene expression multiplex PCR reported here provides an easy to use and rapid marker of drug efficacy and has potential to assess the efficacy of existing or novel drug combinations.

Original language	English
Article number	1760862
Number of pages	12
Journal	Frontiers in Antibiotics
Volume	5
DOIs	https://doi.org/10.3389/frabi.2026.1760862
Publication status	Published - 8 Apr 2026

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Clinical trials
Diagnostics
PCR
Treatment monitoring
Tuberculosis

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@article{a82c468b8fe84edb920270e7b60967ff,

title = "Simultaneous measurement of 16S-rRNA and pre-16S-rRNA as a strategy to monitor clinical tuberculosis",

abstract = "Background: Culture-based biomarkers of TB treatment response monitoring, e.g., Mycobacterial Growth Indicatory Tube (MGIT), are compromised when bacteria enter a non-replicating persister phase limiting the measurement of antibiotic efficacy and resistance. Understanding how antibiotic exposure to antibiotics alters bacterial physiology could help develop more effective TB therapies. We developed a novel assay with simultaneous measurement of 16S rRNA (bacterial burden) and its precursor, pre-16S rRNA (metabolic activity), and tested it on samples from patients in a trial of optimised-dose rifampicin. Methods: We developed a multiplex reverse transcriptase quantitative PCR assay (RT-qPCR) to measure relative gene expression of pre-16S rRNA and 16S rRNA in pre-treatment (control) and sequential samples from patients in the Phase II HIGHRIF2 (NCT00760149) clinical trial. We constructed a mathematical model to assess changes in pre-16S gene expression relative to 16S rRNA over time, facilitating the comparison of rifampicin doses{\textquoteright} efficacy. Findings: In a retrospective study of 19 patients, pre-16S rRNA and 16S rRNA decreased steadily during the initial 36 days of treatment. This was evidenced by the rising cycle threshold (Cq) values slope 0.404 and 0.212, respectively, however, pre-16S rRNA decreased significantly quicker (P<0.0001). The changes in the relative gene expression of pre-16S rRNA during treatment fitted a double exponential decay curve (R2 = 0.996). According to this model, 1200 mg RIF-containing therapy exerted the most potent and rapid impact on pre-16S rRNA expression (Maximum suppression (Rmin)=1.694, T (time) =9.78 days), and also resulted in the swiftest daily reduction in bacterial load (–0.072 log10 CFU ml-1/day). Interpretation: The pre-16S rRNA and 16S rRNA gene expression multiplex PCR reported here provides an easy to use and rapid marker of drug efficacy and has potential to assess the efficacy of existing or novel drug combinations.",

keywords = "Clinical trials, Diagnostics, PCR, Treatment monitoring, Tuberculosis",

author = "Evelin Dombay and Wilber Sabiiti and \{Alferes de Lima Headley\}, Daniela and L{\'e}gr{\'a}dy, \{M. Bonif{\'a}c\} and \{van Campen\}, Nina and Sanne Zweijpfennig and Boeree, \{Martin J.\} and Sloan, \{Derek J.\} and Gillespie, \{Stephen H.\}",

note = "Funding: This study was funded by European \& Developing Countries Clinical Trials Partnership (EDCTP) through the Pan-African Consortium for the Evaluation of Antituberculosis Antibiotics (PanACEA), grant agreement reference number TRIA2015-1102.",

year = "2026",

month = apr,

day = "8",

doi = "10.3389/frabi.2026.1760862",

language = "English",

volume = "5",

journal = "Frontiers in Antibiotics",

issn = "2813-2467",

publisher = "Frontiers Media SA",

}

TY - JOUR

T1 - Simultaneous measurement of 16S-rRNA and pre-16S-rRNA as a strategy to monitor clinical tuberculosis

AU - Dombay, Evelin

AU - Sabiiti, Wilber

AU - Alferes de Lima Headley, Daniela

AU - Légrády, M. Bonifác

AU - van Campen, Nina

AU - Zweijpfennig, Sanne

AU - Boeree, Martin J.

AU - Sloan, Derek J.

AU - Gillespie, Stephen H.

N1 - Funding: This study was funded by European & Developing Countries Clinical Trials Partnership (EDCTP) through the Pan-African Consortium for the Evaluation of Antituberculosis Antibiotics (PanACEA), grant agreement reference number TRIA2015-1102.

PY - 2026/4/8

Y1 - 2026/4/8

N2 - Background: Culture-based biomarkers of TB treatment response monitoring, e.g., Mycobacterial Growth Indicatory Tube (MGIT), are compromised when bacteria enter a non-replicating persister phase limiting the measurement of antibiotic efficacy and resistance. Understanding how antibiotic exposure to antibiotics alters bacterial physiology could help develop more effective TB therapies. We developed a novel assay with simultaneous measurement of 16S rRNA (bacterial burden) and its precursor, pre-16S rRNA (metabolic activity), and tested it on samples from patients in a trial of optimised-dose rifampicin. Methods: We developed a multiplex reverse transcriptase quantitative PCR assay (RT-qPCR) to measure relative gene expression of pre-16S rRNA and 16S rRNA in pre-treatment (control) and sequential samples from patients in the Phase II HIGHRIF2 (NCT00760149) clinical trial. We constructed a mathematical model to assess changes in pre-16S gene expression relative to 16S rRNA over time, facilitating the comparison of rifampicin doses’ efficacy. Findings: In a retrospective study of 19 patients, pre-16S rRNA and 16S rRNA decreased steadily during the initial 36 days of treatment. This was evidenced by the rising cycle threshold (Cq) values slope 0.404 and 0.212, respectively, however, pre-16S rRNA decreased significantly quicker (P<0.0001). The changes in the relative gene expression of pre-16S rRNA during treatment fitted a double exponential decay curve (R2 = 0.996). According to this model, 1200 mg RIF-containing therapy exerted the most potent and rapid impact on pre-16S rRNA expression (Maximum suppression (Rmin)=1.694, T (time) =9.78 days), and also resulted in the swiftest daily reduction in bacterial load (–0.072 log10 CFU ml-1/day). Interpretation: The pre-16S rRNA and 16S rRNA gene expression multiplex PCR reported here provides an easy to use and rapid marker of drug efficacy and has potential to assess the efficacy of existing or novel drug combinations.

AB - Background: Culture-based biomarkers of TB treatment response monitoring, e.g., Mycobacterial Growth Indicatory Tube (MGIT), are compromised when bacteria enter a non-replicating persister phase limiting the measurement of antibiotic efficacy and resistance. Understanding how antibiotic exposure to antibiotics alters bacterial physiology could help develop more effective TB therapies. We developed a novel assay with simultaneous measurement of 16S rRNA (bacterial burden) and its precursor, pre-16S rRNA (metabolic activity), and tested it on samples from patients in a trial of optimised-dose rifampicin. Methods: We developed a multiplex reverse transcriptase quantitative PCR assay (RT-qPCR) to measure relative gene expression of pre-16S rRNA and 16S rRNA in pre-treatment (control) and sequential samples from patients in the Phase II HIGHRIF2 (NCT00760149) clinical trial. We constructed a mathematical model to assess changes in pre-16S gene expression relative to 16S rRNA over time, facilitating the comparison of rifampicin doses’ efficacy. Findings: In a retrospective study of 19 patients, pre-16S rRNA and 16S rRNA decreased steadily during the initial 36 days of treatment. This was evidenced by the rising cycle threshold (Cq) values slope 0.404 and 0.212, respectively, however, pre-16S rRNA decreased significantly quicker (P<0.0001). The changes in the relative gene expression of pre-16S rRNA during treatment fitted a double exponential decay curve (R2 = 0.996). According to this model, 1200 mg RIF-containing therapy exerted the most potent and rapid impact on pre-16S rRNA expression (Maximum suppression (Rmin)=1.694, T (time) =9.78 days), and also resulted in the swiftest daily reduction in bacterial load (–0.072 log10 CFU ml-1/day). Interpretation: The pre-16S rRNA and 16S rRNA gene expression multiplex PCR reported here provides an easy to use and rapid marker of drug efficacy and has potential to assess the efficacy of existing or novel drug combinations.

KW - Clinical trials

KW - Diagnostics

KW - PCR

KW - Treatment monitoring

KW - Tuberculosis

U2 - 10.3389/frabi.2026.1760862

DO - 10.3389/frabi.2026.1760862

M3 - Article

SN - 2813-2467

VL - 5

JO - Frontiers in Antibiotics

JF - Frontiers in Antibiotics

M1 - 1760862

ER -

Simultaneous measurement of 16S-rRNA and pre-16S-rRNA as a strategy to monitor clinical tuberculosis

Abstract

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