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14 pages, 1494 KiB

Open AccessArticle

Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development

by Dimitrios Evangelopoulos, Carolyn M. Shoen, Isobella Honeyborne, Simon Clark, Ann Williams, Galina V. Mukamolova, Michael H. Cynamon and Timothy D. McHugh

Microorganisms 2022, 10(2), 460; https://doi.org/10.3390/microorganisms10020460 - 17 Feb 2022

Cited by 3 | Viewed by 2813

Abstract

Background: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism’s slow growth in culture media. The existence of differentially culturable populations of M.tuberculosis may [...] Read more.

Background: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism’s slow growth in culture media. The existence of differentially culturable populations of M.tuberculosis may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. Objectives: We report the adaptation of our robust, culture-free assay utilising 16S ribosomal RNA, developed for sputum, to enumerate the number of bacteria present in animal tissues as a tool to improve the read-outs in preclinical drug efficacy studies. Methods: Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of M. tuberculosis and an internal RNA control. Tissues were homogenised, total RNA extracted, and enumeration performed using RT-qPCR. We then evaluated the utility of the assay, in comparison to bacterial counts estimated using growth assays on solid and liquid media, to accurately inform bacterial load in tissues from M. tuberculosis-infected mice before and during treatment with a panel of drug combinations. Results: When tested on lung tissues derived from infected mice, the MBL assay produced comparable results to the bacterial counts in solid culture (colony forming units: CFU). Notably, under specific drug treatments, the MBL assay was able to detect a significantly higher number of M. tuberculosis compared to CFU, likely indicating the presence of bacteria that were unable to produce colonies in solid-based culture. Additionally, growth recovery in liquid media using the most probable number (MPN) assay was able to account for the discrepancy between the MBL assay and CFU number, suggesting that the MBL assay detects differentially culturable sub-populations of M. tuberculosis. Conclusions: The MBL assay can enumerate the bacterial load in animal tissues in real time without the need to wait for extended periods for cultures to grow. The readout correlates well with CFUs. Importantly, we have shown that the MBL is able to measure specific populations of bacteria not cultured on solid agar. The adaptation of this assay for preclinical studies has the potential to decrease the readout time of data acquisition from animal experiments and could represent a valuable tool for tuberculosis drug discovery and development. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

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11 pages, 560 KiB

Open AccessArticle

Pancreatin-Cetyl Pyridinium Chloride Digestion and Decontamination Method; A Novel, Sensitive, Cost-Effective Method for Culturing Mycobacterium tuberculosis

by Pottathil Shinu, Anroop B. Nair, Snawar Hussain, Mohamed A. Morsy and Wafaa E. Soliman

Microorganisms 2021, 9(10), 2025; https://doi.org/10.3390/microorganisms9102025 - 24 Sep 2021

Cited by 1 | Viewed by 2276

Abstract

The present study evaluated the performance of newly developed pancreatin-cetylpyridinium chloride (pancreatin-CPC) digestion and decontamination method (DDM) with N-acetyl L-Cysteine-sodium hydroxide (NALC-NaOH) DDM for isolation of Mycobacteria from clinically suspected pulmonary tuberculosis (PTB) patients. For the study, sputum samples (n = 613) [...] Read more.

The present study evaluated the performance of newly developed pancreatin-cetylpyridinium chloride (pancreatin-CPC) digestion and decontamination method (DDM) with N-acetyl L-Cysteine-sodium hydroxide (NALC-NaOH) DDM for isolation of Mycobacteria from clinically suspected pulmonary tuberculosis (PTB) patients. For the study, sputum samples (n = 613) obtained from clinically suspected PTB cases were subjected to direct microscopy, pretreatment with NALC-NaOH DDM (reference method), and pancreatin-CPC DDM followed by culture, and the data were analyzed. The direct microscopy illustrated diagnostic accuracies of 60.4% (sensitivity), 99.77% (specificity), 98.9% (positive predictive value) and 88.3% (negative predictive value), respectively (against culture) for the detection of Mycobacterial species. The pancreatin-CPC DDM showed competitive diagnostic accuracies (against NALC-NaOH DDM) of 99.32% (sensitivity), 94.07% (specificity), 85.05% (positive predictive value), and 99.76% (negative predictive value), respectively, for the isolation of Mycobacterial species. In conclusion, pancreatin-CPC DMM was a highly sensitive, technically simple, and cost-effective method, suggesting its competence to substitute the currently used NALC-NaOH DDM. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

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8 pages, 893 KiB

Open AccessCommunication

Decontamination Strategies Used for AFB Culture Significantly Reduce the Viability of Mycobacterium abscessus Complex in Sputum Samples from Patients with Cystic Fibrosis

by Dominic Stephenson, Audrey Perry, Andrew Nelson, Ali E. Robb, Matthew F. Thomas, Stephen J. Bourke, John D. Perry and Amanda L. Jones

Microorganisms 2021, 9(8), 1597; https://doi.org/10.3390/microorganisms9081597 - 27 Jul 2021

Cited by 12 | Viewed by 3198

Abstract

Nontuberculous mycobacteria are important respiratory pathogens in patients with cystic fibrosis (CF). For diagnosis, international guidelines recommend culture of sputum that has been decontaminated via chemical treatment. Fifty-six sputum samples from 32 patients known to be previously colonized or infected with NTM were [...] Read more.

Nontuberculous mycobacteria are important respiratory pathogens in patients with cystic fibrosis (CF). For diagnosis, international guidelines recommend culture of sputum that has been decontaminated via chemical treatment. Fifty-six sputum samples from 32 patients known to be previously colonized or infected with NTM were subdivided, and the aliquots were subjected to six different decontamination strategies, followed by quantitative culture for NTM. Thirty sputum samples contained Mycobacterium abscessus complex (MABSC) and 11 contained Mycobacterium avium complex (MAC). Decontamination strategies included treatment with N-acetyl L-cysteine with 2% sodium hydroxide (NALC-NaOH), 4% NaOH, 1% chlorhexidine, 0.5 N sulfuric acid, 5% oxalic acid, double decontamination with NALC-NaOH, followed by 5% oxalic acid, and saline (0.85%) as a control. The samples were also cultured directly with no treatment. Treatment with NALC-NaOH resulted in an average reduction in colony count of 87% for MABSC when compared with direct culture. NaOH at 4% caused a 98.3% average reduction in colony count. All treatments that included NaOH resulted in colony counts that were statistically lower than those obtained from direct culture or the saline-treated control (p < 0.05). Standard treatments using sulfuric or oxalic acids were less deleterious, but still resulted in an average reduction in colony count of at least 30%. The viability of MAC was much less affected by most decontamination treatments. In conclusion, the viability of MABSC was severely compromised by standard decontamination regimens. This supports recent evidence showing that optimal recovery of MABSC is achieved by culture on an appropriate selective agar without decontamination of sputum samples. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

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Review

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17 pages, 486 KiB

Open AccessReview

Pre-Clinical Tools for Predicting Drug Efficacy in Treatment of Tuberculosis

by Hasmik Margaryan, Dimitrios D. Evangelopoulos, Leticia Muraro Wildner and Timothy D. McHugh

Microorganisms 2022, 10(3), 514; https://doi.org/10.3390/microorganisms10030514 - 26 Feb 2022

Cited by 4 | Viewed by 2669

Abstract

Combination therapy has, to some extent, been successful in limiting the emergence of drug-resistant tuberculosis. Drug combinations achieve this advantage by simultaneously acting on different targets and metabolic pathways. Additionally, drug combination therapies are shown to shorten the duration of therapy for tuberculosis. [...] Read more.

Combination therapy has, to some extent, been successful in limiting the emergence of drug-resistant tuberculosis. Drug combinations achieve this advantage by simultaneously acting on different targets and metabolic pathways. Additionally, drug combination therapies are shown to shorten the duration of therapy for tuberculosis. As new drugs are being developed, to overcome the challenge of finding new and effective drug combinations, systems biology commonly uses approaches that analyse mycobacterial cellular processes. These approaches identify the regulatory networks, metabolic pathways, and signaling programs associated with M. tuberculosis infection and survival. Different preclinical models that assess anti-tuberculosis drug activity are available, but the combination of models that is most predictive of clinical treatment efficacy remains unclear. In this structured literature review, we appraise the options to accelerate the TB drug development pipeline through the evaluation of preclinical testing assays of drug combinations. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

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14 pages, 319 KiB

Open AccessReview

Application of Bacteriophages for Mycobacterial Infections, from Diagnosis to Treatment

by Christopher G. Shield, Benjamin M. C. Swift, Timothy D. McHugh, Rebekah M. Dedrick, Graham F. Hatfull and Giovanni Satta

Microorganisms 2021, 9(11), 2366; https://doi.org/10.3390/microorganisms9112366 - 16 Nov 2021

Cited by 25 | Viewed by 4791

Abstract

Mycobacterium tuberculosis and other non-tuberculous mycobacteria are responsible for a variety of different infections affecting millions of patients worldwide. Their diagnosis is often problematic and delayed until late in the course of disease, requiring a high index of suspicion and the combined efforts [...] Read more.

Mycobacterium tuberculosis and other non-tuberculous mycobacteria are responsible for a variety of different infections affecting millions of patients worldwide. Their diagnosis is often problematic and delayed until late in the course of disease, requiring a high index of suspicion and the combined efforts of clinical and laboratory colleagues. Molecular methods, such as PCR platforms, are available, but expensive, and with limited sensitivity in the case of paucibacillary disease. Treatment of mycobacterial infections is also challenging, typically requiring months of multiple and combined antibiotics, with associated side effects and toxicities. The presence of innate and acquired drug resistance further complicates the picture, with dramatic cases without effective treatment options. Bacteriophages (viruses that infect bacteria) have been used for decades in Eastern Europe for the treatment of common bacterial infections, but there is limited clinical experience of their use in mycobacterial infections. More recently, bacteriophages’ clinical utility has been re-visited and their use has been successfully demonstrated both as diagnostic and treatment options. This review will focus specifically on how mycobacteriophages have been used recently in the diagnosis and treatment of different mycobacterial infections, as potential emerging technologies, and as an alternative treatment option. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

15 pages, 974 KiB

Open AccessReview

Diabetes-Associated Susceptibility to Tuberculosis: Contribution of Hyperglycemia vs. Dyslipidemia

by Minh Dao Ngo, Stacey Bartlett and Katharina Ronacher

Microorganisms 2021, 9(11), 2282; https://doi.org/10.3390/microorganisms9112282 - 2 Nov 2021

Cited by 22 | Viewed by 8195

Abstract

Diabetes is a major risk factor for tuberculosis (TB). Diabetes increases the risk of the progression from latent tuberculosis infection (LTBI) to active pulmonary TB and TB patients with diabetes are at greater risk of more severe disease and adverse TB treatment outcomes [...] Read more.

Diabetes is a major risk factor for tuberculosis (TB). Diabetes increases the risk of the progression from latent tuberculosis infection (LTBI) to active pulmonary TB and TB patients with diabetes are at greater risk of more severe disease and adverse TB treatment outcomes compared to TB patients without co-morbidities. Diabetes is a complex disease, characterised not only by hyperglycemia but also by various forms of dyslipidemia. However, the relative contribution of these underlying metabolic factors to increased susceptibility to TB are poorly understood. This review summarises our current knowledge on the epidemiology and clinical manifestation of TB and diabetes comorbidity. We subsequently dissect the relative contributions of body mass index, hyperglycemia, elevated cholesterol and triglycerides on TB disease severity and treatment outcomes. Lastly, we discuss the impact of selected glucose and cholesterol-lowering treatments frequently used in the management of diabetes on TB treatment outcomes. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

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15 pages, 813 KiB

Open AccessReview

Whole Genome Sequencing in the Management of Non-Tuberculous Mycobacterial Infections

by Matúš Dohál, Igor Porvazník, Ivan Solovič and Juraj Mokrý

Microorganisms 2021, 9(11), 2237; https://doi.org/10.3390/microorganisms9112237 - 27 Oct 2021

Cited by 18 | Viewed by 3957

Abstract

Infections caused by non-tuberculous mycobacteria (NTM) have been a public health problem in recent decades and contribute significantly to the clinical and economic burden globally. The diagnosis of infections is difficult and time-consuming and, in addition, the conventional diagnostics tests do not have [...] Read more.

Infections caused by non-tuberculous mycobacteria (NTM) have been a public health problem in recent decades and contribute significantly to the clinical and economic burden globally. The diagnosis of infections is difficult and time-consuming and, in addition, the conventional diagnostics tests do not have sufficient discrimination power in species identification due to cross-reactions and not fully specific probes. However, technological advances have been made and the whole genome sequencing (WGS) method has been shown to be an essential part of routine diagnostics in clinical mycobacteriology laboratories. The use of this technology has contributed to the characterization of new species of mycobacteria, as well as the identification of gene mutations encoding resistance and virulence factors. Sequencing data also allowed to track global outbreaks of nosocomial NTM infections caused by M. abscessus complex and M. chimaera. To highlight the utility of WGS, we summarize recent scientific studies on WGS as a tool suitable for the management of NTM-induced infections in clinical practice. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

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16 pages, 846 KiB

Open AccessReview

Personalised Medicine for Tuberculosis and Non-Tuberculous Mycobacterial Pulmonary Disease

by Kartik Kumar and Onn Min Kon

Microorganisms 2021, 9(11), 2220; https://doi.org/10.3390/microorganisms9112220 - 26 Oct 2021

Cited by 8 | Viewed by 3437

Abstract

Personalised medicine, in which clinical management is individualised to the genotypic and phenotypic data of patients, offers a promising means by which to enhance outcomes in the management of mycobacterial pulmonary infections. In this review, we provide an overview of how personalised medicine [...] Read more.

Personalised medicine, in which clinical management is individualised to the genotypic and phenotypic data of patients, offers a promising means by which to enhance outcomes in the management of mycobacterial pulmonary infections. In this review, we provide an overview of how personalised medicine approaches may be utilised to identify patients at risk of developing tuberculosis (TB) or non-tuberculous mycobacterial pulmonary disease (NTM-PD), diagnose these conditions and guide effective treatment strategies. Despite recent technological and therapeutic advances, TB and NTM-PD remain challenging conditions to diagnose and treat. Studies have identified a range of genetic and immune factors that predispose patients to pulmonary mycobacterial infections. Molecular tests such as nucleic acid amplification assays and next generation sequencing provide a rapid means by which to identify mycobacterial isolates and their antibiotic resistance profiles, thus guiding selection of appropriate antimicrobials. Host-directed therapies and therapeutic drug monitoring offer ways of tailoring management to the clinical needs of patients at an individualised level. Biomarkers may hold promise in differentiating between latent and active TB, as well as in predicting mycobacterial disease progression and response to treatment. Full article

(This article belongs to the Special Issue Mycobacterial Infections: Diagnostics, Biomarkers and Treatment)

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