Research Article: Lipoarabinomannan in sputum to detect bacterial load and treatment response in patients with pulmonary tuberculosis: Analytic validation and evaluation in two cohorts

Date Published: April 12, 2019

Publisher: Public Library of Science

Author(s): Masanori Kawasaki, Carmenchu Echiverri, Lawrence Raymond, Elizabeth Cadena, Evelyn Reside, Maria Tarcela Gler, Tetsuya Oda, Ryuta Ito, Ryo Higashiyama, Kiyonori Katsuragi, Yongge Liu, Mark Hatherill

Abstract: BackgroundLipoarabinomannan (LAM) is a major antigen of Mycobacterium tuberculosis (MTB). In this report, we evaluated the ability of a novel immunoassay to measure concentrations of LAM in sputum as a biomarker of bacterial load prior to and during treatment in pulmonary tuberculosis (TB) patients.Methods and findingsPhage display technology was used to isolate monoclonal antibodies binding to epitopes unique in LAM from MTB and slow-growing nontuberculous mycobacteria (NTM). Using these antibodies, a sandwich enzyme-linked immunosorbent assay (LAM-ELISA) was developed to quantitate LAM concentration. The LAM-ELISA had a lower limit of quantification of 15 pg/mL LAM, corresponding to 121 colony-forming units (CFUs)/mL of MTB strain H37Rv. It detected slow-growing NTMs but without cross-reacting to common oral bacteria. Two clinical studies were performed between the years 2013 and 2016 in Manila, Philippines, in patients without known human immunodeficiency virus (HIV) coinfection. In a case-control cohort diagnostic study, sputum specimens were collected from 308 patients (aged 17-69 years; 62% male) diagnosed as having pulmonary TB diseases or non-TB diseases, but who could expectorate sputum, and were then evaluated by smear microscopy, BACTEC MGIT 960 Mycobacterial Detection System (MGIT) and Lowenstein-Jensen (LJ) culture, and LAM-ELISA. Some sputum specimens were also examined by Xpert MTB/RIF. The LAM-ELISA detected all smear- and MTB-culture–positive samples (n = 70) and 50% (n = 29) of smear-negative but culture-positive samples (n = 58) (versus 79.3%; 46 positive cases by the Xpert MTB/RIF), but none from non-TB patients (n = 56). Among both LAM and MGIT MTB-culture-positive samples, log10-transformed LAM concentration and MGIT time to detection (TTD) showed a good inverse relationship (r = −0.803, p < 0.0001). In a prospective longitudinal cohort study, 40 drug-susceptible pulmonary TB patients (aged 18-69 years; 60% male) were enrolled during the first 56 days of the standard 4-drug therapy. Declines in sputum LAM concentrations correlated with increases of MGIT TTD in individual patients. There was a 1.29 log10 decrease of sputum LAM concentration, corresponding to an increase of 221 hours for MGIT TTD during the first 14 days of treatment, a treatment duration often used in early bactericidal activity (EBA) trials. Major limitations of this study include a relatively small number of patients, treatment duration up to only 56 days, lack of quantitative sputum culture CFU count data, and no examination of the correlation of sputum LAM to clinical cure.ConclusionsThese results indicate that the LAM-ELISA can determine LAM concentration in sputum, and sputum LAM measured by the assay may be used as a biomarker of bacterial load prior to and during TB treatment. Additional studies are needed to examine the predictive value of this novel biomarker on treatment outcomes.

Partial Text: Tuberculosis (TB), caused by infection with M. tuberculosis (MTB), now ranks alongside human immunodeficiency virus (HIV) as a leading cause of death worldwide because of infection [1]. In contrast to the treatment management of viral diseases such as HIV and hepatitis C, in which real-time viral load tests have significantly improved disease management and treatment outcomes [2,3], current tools to measure MTB load are limited by either low sensitivity and low quantitative characteristics (sputum smear microscopy) or requiring up to two months to obtain results (sputum culture) [4]. Knowledge of bacterial load prior to and during treatment is important for the determination of infectiousness and, most importantly, whether a patient is responding to treatment. The lack of a real-time test to monitor treatment response impedes the improvement of current programmatic TB treatment, under which TB patients are treated largely in a standardized approach with fixed-duration-based regimens [5,6]. Inevitably, good responders are treated longer than necessary by drugs associated with significant adverse reactions, resulting in wasting resources (economic and human), while poor responders are not identified early, resulting in infection of others and potential development of drug resistance. Additionally, the lack of a real-time test to determine bacterial load limits the ability to conduct adaptive trial designs in TB drug development because culture results are the only accepted surrogate marker for efficacy [7].

We evaluated the performance of a new immunoassay, the LAM-ELISA, to quantitate LAM concentration in preclinical studies and on sputum specimens obtained from two patient cohorts in Manila, Philippines. In preclinical studies, the LAM-ELISA demonstrated an LLoQ of 15 pg/mL LAM. It detected slow-growing NTMs but without cross-reacting to common oral bacteria. In clinical studies, sputum LAM concentrations correlated with bacterial burden determined by culture prior to treatment in pulmonary TB patients. Further, changes of sputum LAM concentration during TB treatment correlated with those of bacterial burden measured by culture. These results suggest sputum LAM concentration measured by the LAM-ELISA may have the potential as a biomarker of bacterial load prior to and during treatment.



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