Specific human gene expression in response to infection is an effective marker for diagnosis of latent and active tuberculosis

• Published in: Scientific reports 2024-11, Vol.14 (1), p.26884-15, Article 26884
• Main Authors: Nakiboneka, Ritah, Walbaum, Natasha, Musisi, Emmanuel, Nevels, Michael, Nyirenda, Tonney, Nliwasa, Marriott, Msefula, Chisomo L., Sloan, Derek, Sabiiti, Wilber
• Format: Article
• Language: English
• Subjects: 631/337/2019; 692/4017; Active tuberculosis; Adult; Biomarkers; Diagnosis; DNA microarrays; DNA probes; Female; Gene expression; Host gene expression; Humanities and Social Sciences; Humans; Infections; Krueppel-like factor; Latent tuberculosis; Latent Tuberculosis - diagnosis; Latent Tuberculosis - genetics; Latent Tuberculosis - microbiology; Literature reviews; Lung diseases; Male; multidisciplinary; Mycobacterium tuberculosis - genetics; Polymerase chain reaction; Respiratory diseases; Reverse transcriptase-quantitative PCR; RNA-directed DNA polymerase; Science; Science (multidisciplinary); Sensitivity analysis; Tuberculosis; Tuberculosis - diagnosis; Tuberculosis - genetics; Tuberculosis - microbiology; Tuberculosis, Pulmonary - diagnosis; Tuberculosis, Pulmonary - genetics; Tuberculosis, Pulmonary - microbiology
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-77164-5

RNA sequencing and microarray analysis revealed transcriptional markers expressed in whole blood can differentiate active pulmonary TB (ATB) from other respiratory diseases (ORDs), and latent TB infection (LTBI) from healthy controls (HC). Here we describe a streamlined reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay that could be applied at near point-of-care for diagnosing and distinguishing ATB from ORDs and LTBI from HC. A literature review was undertaken to identify the most plausible host-gene markers (HGM) of TB infection. Primers, and dual labelled hydrolysis probes were designed and analytically evaluated for accuracy in an in-vitro model of infection using a lung fibroblast cell-line. Best performing genes were multiplexed into panels of 2–4 targets and taken forward for clinical evaluation. Mycobacteria Growth Indicator Tube and QuantiFERON-TB Gold Plus were used as reference tests for ATB and LTBI respectively. A total of 16 HGM were selected and incorporated into five multiplex RT-qPCR panels. PCR assay efficiency of all evaluated targets was ≥ 90% with a median analytical sensitivity of 292 copies/µl [IQR: 215.0-358.3 copies/µl], and a median limit of quantification of 61.7 copies/µl [IQR: 29.4-176.3 copies/µl]. Clinically, ATB was characterised by higher gene expression than ORDs, while LTBI was associated with lower gene expression than HC, Kruskal-Wallis p