New and Improved Diagnostics for Detection of Drug-Resistant Pulmonary Tuberculosis.

PURPOSE OF REVIEW: Tuberculosis (TB) remains a global emergency and continues to kill 1.7 million people globally each year. Drug-resistant TB is now well established throughout the world and most TB patients are not being screened for drug resistance due to lack of laboratory resources and rapid accurate point-of-care tests. Accurate and rapid diagnosis of TB and drug-resistant TB is of paramount importance in establishing appropriate clinical management and infection control measures. During the past decade, there have been significant advances in diagnostic technologies for TB and drug-resistant TB. The purpose of this article is to review the current data, recommendations and evidence base for these tests. RECENT FINDINGS: Second-line drug susceptibility testing (DST) is complex and expensive. Automated liquid culture systems and molecular line probe assays are recommended by the WHO as the current 'gold standard' for first-line DST. Liquid culture DST for aminoglycosides, polypeptides and fluoroquinolones has been shown to have relatively good reliability and reproducibility for diagnosis of extensively drug-resistant TB; however, DST for other second-line drugs (ethionamide, prothionamide, cycloserine, terizidone, para-aminosalicylic acid, clofazimine, amoxicillin-clavulanate, clarithromycin, linezolid) is not recommended. Automated liquid culture systems are currently recommended by the WHO as the 'gold standard' for second-line DST. SUMMARY: In this review, we describe the phenotypic and genotypic methods currently available for the diagnosis of TB and drug-resistant forms of Mycobacterium tuberculosis and discuss future prospects for TB diagnostics. Current technologies for the detection of drug resistant M. tuberculosis vary greatly in terms of turnaround time, cost and complexity. Ultimately, the 'holy grail' diagnostic for TB must fulfil all technical specifications for a good point-of-care test, screen for drug resistance concurrently and be adaptable to the various health system levels and to countries with diverse economic status and TB burden. © 2011 Lippincott Williams & Wilkins, Inc

Foreign Object Detection and Localization in Chest X-rays using Deep Learning

Pulmonary abnormalities, such as Tuberculosis (TB), Asthma and/or Chronic obstructive are global threats. Nearly 1.6 million died from TB alone according to the WHO (World Health Organization) report 2019. Computer scientists together with medical experts have designed and reported automated screening systems for chest X-ray (CXR) images. However, most of the research works did not consider detecting foreign objects, such as buttons, coins, ring, pins, bone pieces and other medical devices (e.g. pacemaker) all together that can hinder the performance of automatic screening system. The circle-like foreign objects, such as coins are often confused with nodules, which is one of the primary indicators of Tuberculosis. Thus, in an automated screening process foreign objects need to be separated. Unlike the previous works, we will employ deep learning models, such as Faster R-CNN (Faster Region Proposal Convolutional Neural Network), to detect almost all kinds of foreign objects in CXR images. This research is mainly focused on the detection of foreign objects that are of almost all shapes, sizes and texture in CXR using convolutional neural network. Instead of relying on handcrafted features, we now let machine to find distinguished features to achieve an error as low as possible (technically, 10^-4). We also localize their spatial position in CXR, so that the further process of screening can be advanced and at the same time misdiagnosis and confusion can be eliminated

Tuberculosis diagnostics and biomarkers: needs, challenges, recent advances, and opportunities

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics

Mobile Phone Based Clinical Microscopy for Global Health Applications

Light microscopy provides a simple, cost-effective, and vital method for the diagnosis and screening of hematologic and infectious diseases. In many regions of the world, however, the required equipment is either unavailable or insufficiently portable, and operators may not possess adequate training to make full use of the images obtained. Counterintuitively, these same regions are often well served by mobile phone networks, suggesting the possibility of leveraging portable, camera-enabled mobile phones for diagnostic imaging and telemedicine. Toward this end we have built a mobile phone-mounted light microscope and demonstrated its potential for clinical use by imaging P. falciparum-infected and sickle red blood cells in brightfield and M. tuberculosis-infected sputum samples in fluorescence with LED excitation. In all cases resolution exceeded that necessary to detect blood cell and microorganism morphology, and with the tuberculosis samples we took further advantage of the digitized images to demonstrate automated bacillus counting via image analysis software. We expect such a telemedicine system for global healthcare via mobile phone – offering inexpensive brightfield and fluorescence microscopy integrated with automated image analysis – to provide an important tool for disease diagnosis and screening, particularly in the developing world and rural areas where laboratory facilities are scarce but mobile phone infrastructure is extensive

Home-based tuberculosis contact investigation in Uganda: a household randomised trial.

IntroductionThe World Health Organization (WHO) recommends household tuberculosis (TB) contact investigation in low-income countries, but most contacts do not complete a full clinical and laboratory evaluation.MethodsWe performed a randomised trial of home-based, SMS-facilitated, household TB contact investigation in Kampala, Uganda. Community health workers (CHWs) visited homes of index patients with pulmonary TB to screen household contacts for TB. Entire households were randomly allocated to clinic (standard-of-care) or home (intervention) evaluation. In the intervention arm, CHWs offered HIV testing to adults; collected sputum from symptomatic contacts and persons living with HIV (PLWHs) if ≥5 years; and transported sputum for microbiologic testing. CHWs referred PLWHs, children <5 years, and anyone unable to complete sputum testing to clinic. Sputum testing results and/or follow-up instructions were returned by automated SMS texts. The primary outcome was completion of a full TB evaluation within 14 days; secondary outcomes were TB and HIV diagnoses and treatments among screened contacts.ResultsThere were 471 contacts of 190 index patients allocated to the intervention and 448 contacts of 182 index patients allocated to the standard-of-care. CHWs identified 190/471 (40%) intervention and 213/448 (48%) standard-of-care contacts requiring TB evaluation. In the intervention arm, CHWs obtained sputum from 35/91 (39%) of sputum-eligible contacts and SMSs were sent to 95/190 (50%). Completion of TB evaluation in the intervention and standard-of-care arms at 14 days (14% versus 15%; difference -1%, 95% CI -9% to 7%, p=0.81) and yields of confirmed TB (1.5% versus 1.1%, p=0.62) and new HIV (2.0% versus 1.8%, p=0.90) diagnoses were similar.ConclusionsHome-based, SMS-facilitated evaluation did not improve completion or yield of household TB contact investigation, likely due to challenges delivering the intervention components

A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

PMCID: PMC3617142This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Enhanced heterogeneity of rpoB in Mycobacterium tuberculosis found at low pH.

OBJECTIVES: The aim of this study was to gain an insight into the molecular mechanisms of the evolution of rifampicin resistance in response to controlled changes in the environment. METHODS: We determined the proportion of rpoB mutants in the chemostat culture and characterized the sequence of mutations found in the rifampicin resistance-determining region of rpoB in a steady-state chemostat at pH 7.0 and 6.2. RESULTS: The overall proportion of rpoB mutants of strain H37Rv remained constant for 37 days at pH 7.0, ranging between 3.6 x 10(-8) and 8.9 x 10(-8); however, the spectrum of mutations varied. The most commonly detected mutation, serine to leucine mutation at codon 531 (S531L), increased from 40% to 89%, while other mutations (S531W, H526Y, H526D, H526R, S522L and D516V) decreased over the 37 day sampling period. Changing the pH from 7.0 to 6.2 did not significantly alter the overall proportion of mutants, but resulted in a decrease in the percentage of strains harbouring S531L (from 89% to 50%) accompanied by an increase in the range of different mutations from 4 to 12. CONCLUSIONS: The data confirm that the fitness of strains with the S531L mutation is greater than that of strains containing other mutations. We also conclude that at low pH the environment is permissive for a wider spectrum of mutations, which may provide opportunities for a successful mutant to survive

Evaluation of the burden of unsuspected pulmonary tuberculosis and co-morbidity with non-communicable diseases in sputum producing adult inpatients

A high burden of tuberculosis (TB) occurs in sub-Saharan African countries and many cases of active TB and drug-resistant TB remain undiagnosed. Tertiary care hospitals provide an opportunity to study TB co-morbidity with non-communicable and other communicable diseases (NCDs/CDs). We evaluated the burden of undiagnosed pulmonary TB and multi-drug resistant TB in adult inpatients, regardless of their primary admission diagnosis, in a tertiary referral centre. In this prospective study, newly admitted adult inpatients able to produce sputum at the University Teaching Hospital, Lusaka, Zambia, were screened for pulmonary TB using fluorescent smear microscopy and automated liquid culture. The burden of pulmonary TB, unsuspected TB, TB co-morbidity with NCDs and CDs was determined. Sputum was analysed from 900 inpatients (70.6% HIV infected) 277 (30.8%) non-TB suspects, 286 (31.8%) TB suspects and 337 (37.4%) were already receiving TB treatment. 202/900 (22.4%) of patients had culture confirmed TB. TB co-morbidity was detected in 20/275 (7.3%) NCD patients, significantly associated with diabetes (P = 0.006, OR 6.571, 95%CI: 1.706-25.3). 27/202 (13.4%) TB cases were unsuspected. There were 18 confirmed cases of MDR-TB, 5 of which were unsuspected. A large burden of unsuspected pulmonary TB co-morbidity exists in inpatients with NCDs and other CDs. Pro-active sputum screening of all inpatients in tertiary referral centres in high TB endemic countries is recommended. The scale of the problem of undiagnosed MDR-TB in inpatients requires further study

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

A Novel Scoring Based Distributed Protein Docking Application to Improve Enrichment

Molecular docking is a computational technique which predicts the binding energy and the preferred binding mode of a ligand to a protein target. Virtual screening is a tool which uses docking to investigate large chemical libraries to identify ligands that bind favorably to a protein target. We have developed a novel scoring based distributed protein docking application to improve enrichment in virtual screening. The application addresses the issue of time and cost of screening in contrast to conventional systematic parallel virtual screening methods in two ways. Firstly, it automates the process of creating and launching multiple independent dockings on a high performance computing cluster. Secondly, it uses a N˙ aive Bayes scoring function to calculate binding energy of un-docked ligands to identify and preferentially dock (Autodock predicted) better binders. The application was tested on four proteins using a library of 10,573 ligands. In all the experiments, (i). 200 of the 1000 best binders are identified after docking only 14% of the chemical library, (ii). 9 or 10 best-binders are identified after docking only 19% of the chemical library, and (iii). no significant enrichment is observed after docking 70% of the chemical library. The results show significant increase in enrichment of potential drug leads in early rounds of virtual screening