Assessment of treatment response by colony forming units, time to culture positivity and the molecular bacterial load assay compared in a mouse tuberculosis model

Gerjo J De Knegt,Laura Dickinson,Henry Pertinez,Dimitrios Evangelopoulos,Timothy D Mchugh,Irma A.J.M Bakker-Woudenberg,Gerry R Davies,Jurriaan E.M De Steenwinkel

doi:10.1016/j.tube.2017.05.002

Gerjo J De Knegt, Laura Dickinson + Show 6 more

Open Access

https://doi.org/10.1016/j.tube.2017.05.002

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Abstract

The aim of the study is to compare counting of colony forming units (CFU), the time to positivity (TTP) assay and the molecular bacterial load (MBL) assay, and explore whether the last assays can detect a subpopulation which is unable to grown on solid media. CFU counting, TTP and the MBL assay were used to determine the mycobacterial load in matched lung samples of a murine tuberculosis model. Mice were treated for 24 weeks with 4 treatment arms: isoniazid (H) - rifampicin (R) - pyrazinamide (Z), HRZ-Streptomycin (S), HRZ – ethambutol (E) or ZES.Inverse relationships were observed when comparing TPP with CFU or MBL. Positive associations were observed when comparing CFU with MBL. Description of the net elimination of bacteria was performed for CFU vs. time, MBL vs. time and 1/TTP vs. time and fitted by nonlinear regression. CFU vs. time and 1/TTP vs. time showed bi-phasic declines with the exception of HRZE. A similar rank order, based on the alpha slope, was found comparing CFU vs. time and TTP vs. time, respectively HRZE, HRZ, HRZS and ZES. In contrast, MBL vs. time showed a mono-phasic decline with a flat gradient of elimination and a different rank order respectively, ZES, HRZ, HRZE and HRZS. The correlations found between methods reflects the ability of each to discern the general mycobacterial load. Based on the description of net elimination, we conclude that the MBL assay can detect a subpopulation of Mycobacterium tuberculosis which is not detected by the CFU or TTP assays.

Highlights

Tuberculosis (TB) is a bacterial infection that affects more than ten million people globally every year, with 1.8 million deaths recorded in 2015 and is ranked as a leading cause of death from infectious diseases worldwide [1].Mycobacterial populations in TB patients are heterogeneous; several subpopulations of bacteria have been identified in the lungs, in different microenvironments [2]
One of the benefits of an animal model is the ability to study the total population of Mycobacterium tuberculosis in the lung, including the more hidden bacteria in granuloma like formations, whereas in sputum from TB patient only the secreted bacteria can be studied [20]
Animal studies from the past using colony forming units (CFU) counts as a parameter are maybe not predictive enough because it has been shown by Hu et al that CFU counts are not representative for the total population present in an in vitro systems as well as in the lungs of TB infected mice, using resuscitation promoting factors (RPFs) to resuscitate a persistent and non-growing population [11]

Summary

Introduction

Mycobacterial populations in TB patients are heterogeneous; several subpopulations of bacteria have been identified in the lungs, in different microenvironments [2]. Environmental stress and antibiotic pressure can drive Mycobacterium tuberculosis into a non-replicating state [3,4]. Term treatment is required to eradicate all these subpopulations of Mycobacterium tuberculosis [5]. In order to shorten the treatment duration, new drugs and novel combinations are essential and provide the best opportunity to eliminate all mycobacterial subpopulations. For the development of new drugs and regimens, it is important at an early stage to determine a drug's and regimen's potency to prevent relapse, which will determine the required treatment duration with this regimen [6]

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