Creating a virtual slide map from sputum smear images for region-of-interest localisation in automated microscopy

Includes abstract.Includes bibliographical references (leaves 140-144).Automated microscopy for the detection of tuberculosis (TB) in sputum smears seeks to address the strain on technicians in busy TB laboratories and to achieve faster diagnosis in countries with a heavy TB burden. As a step in the development of an automated microscope, the project described here was concerned with microscope auto-positioning; this primarily involves generating a point of reference on a slide, which can be used to automatically bring desired fields on the slide to the field-of-view of the microscope for re-examination. The study was carried out using a conventional microscope and Ziehl- Neelsen (ZN) stained sputum smear slides. All images were captured at 40x magnification. A digital replication, the virtual slide map, of an actual slide was constructed by combining the manually acquired images of the different fields of the slide. The geometric hashing scheme was found to be suitable for auto-stitching a large number of images (over 300 images) to form a virtual slide map. An object recognition algorithm, which was also based on the geometric hashing technique, was used to localise a query image (the current field-of-view) on the virtual slide map. This localised field-of-view then served as the point of reference. The true positive (correct localisation of a query image on the virtual slide map) rate achieved by the algorithm was above 88% even for noisy query images captured at slide orientations up to 26°. The image registration error, computed as the average mean square error, was less than 14 pixel2 (corresponding to 1.02 μm2 and 0.001% error in an image measuring 1030 x 1300 pixels) corresponding to a root mean square registration error of 3.7 pixels. Superior image registration accuracy was obtained at the expense of time using the scale invariant feature transform (SIFT), with a image registration error of 1 pixel2 (0.07 μm2). The object recognition algorithm is inherently robust to changes in slide orientation and placement, which are likely to occur in practice as it is impossible to place the slide in exactly the same position on the microscope at different times. Moreover, the algorithm showed high tolerance to illumination changes and robustness to noise

Network analysis of Diagnostic Medical Device Development for Infectious Diseases Prevalent in South Africa

Infectious diseases are a major health concern in South Africa and many other developing countries. The local development of medical devices for infectious diseases in such settings, utilizing the local knowledge base, has the potential to improve the accuracy of and access to diagnoses and to lead to the devices being more context-appropriate and affordable. The aim of this project was to examine the landscape of diagnostic medical device development targeting infectious diseases prevalent in South Africa for the period 2000-2016, particularly with regard to collaboration between institutions in different sectors and the contributions of different collaborators. Such knowledge would be beneficial to future technological and policy developments aimed at improving access to diagnostic services and treatment in the South African context. Collaboration across four sectors was considered: university, hospital, industry and science councils and facilities. A bibliometric analysis was performed, and publications documenting medical device development for diagnosis of infectious diseases were extracted. Co-authorship of the journal and conference articles was used as a proxy for collaboration across organisations. Affiliation data extracted from the publications were used to generate a collaboration network. Netdraw, a network visualisation tool, was used to visualize the network, and network metrics such as degree centrality, betweenness centrality and closeness centrality, as well as group density measures, were produced using UCINET software. The collaboration network and the network metrics were used to determine which organisations have collaborated and which collaborators have played the most active and influential roles in diagnostic device development. The university sector was found to make the largest contribution to the development of diagnostic medical devices in South Africa, and also played a key role in transmitting information throughout the network due to its high frequency of connections to other organisations. The most prevalent type of inter-sectoral collaboration was between universities and science councils and facilities, while universities were found to collaborate most amongst themselves with regard to intrasectoral collaboration. Foreign organisations played a prominent role in diagnostic device development between 2012 and 2016. Tuberculosis was the most prevalent infectious disease in diagnostic device development research, and computer-aided detection was a common feature of research on diagnostic device development

Nanomedicines for the treatment of tuberculosis

Mycobacterium tuberculosis (Mtb) is the bacterium responsible for the human disease tuberculosis (TB). This study aimed to develop a rapid, low cost assay for screening the anti-mycobacterial properties of new therapies. This assay employed a Green Fluorescent Protein reporter strain of Mycobacteruim avium subspecies paratuberculosis (Map), a pathogenic species causing paratuberculosis in ruminants that can be considered a model for Mtb. Mycobacterial growth over time was monitored by fluorescence, testing new potential therapies including metal and drug nanoparticles (NPs), over a range of concentrations for up to 7 days. The new Map-based assay was sufficiently sensitive to distinguish between the toxicity of different metal/metal oxide NPs, ranked: Ag>Cu(II)O>ZnO. Solid drug NPs (SDNs) of TB antibiotics (rifampicin, isoniazid and pyrazinamide) were compared to conventional antibiotics. SDNs of rifampicin were found to be 100 times more toxic to Map than the conventional antibiotics. Fluorescence microscopy revealed the uptake of SDNs by infected macrophages with possible co-localisation with Map. Pilot data supports the ability of the SDN to kill the intracellular Map. These results support the benefits of nanomedicine and suggest that drug doses could be reduced, if delivered as a nano-formulation

Tuberculosis

Data are rapidly accumulating from all over the world regarding the efficacy of standardized treatment regimens for drug-sensitive, drug-resistant TB and latent TB infection. While we are facing the menace of multi drug-resistant TB [MDR-TB], extensively drug-resistant tuberculosis [XDR­ TB] has emerged threatening to undermine global efforts at TB control. Hence we have included chapters to cover all aspects of the diagnosis and management of MDR TB. This book will cover all these developments in great detail. With the widespread availability of internet globally various standard web resources available on TB have also been included so that the readers may get the comprehensive and updated guidelines from these resources. The changing clinical presentation of TB, advances in laboratory, imaging diagnostic modalities, therapeutic measures and emergence of MDR TB all suggest a pressing need to have a updated book on TB. Furthermore, while all physicians encounter the TB disease in their clinical practice, there have been a lot of controversies and misconceptions over various issues for the diagnosis and management of TB. Paucity of a well referenced, updated, standard book of TB has prompted us to undertake this venture sharing the clinical experience of global experts of TB. Our book contains chapters on epidemiology, immune-pathology, diagnosis, treatment and latest advances for TB, highlighting the global perspective of tuberculosis. World-wide resurgence of MDR TB indicates that the battle against this foe of mankind will continue in the coming years. TB still remains to be a research priority of paramount importance from medical, social and financial aspects and we have attempted to highlight all the aspects for the treatment of TB. We believe that this book will serve as a practical guide for the diagnosis and management of TB for practicing physicians (especially pulmonologists and internists) and all those who are involved in the management of TB

Non-viral delivery strategies to guide therapeutic nucleic acids through cellular barriers

Genetische aandoeningen worden hoofdzakelijk veroorzaakt door de afwezigheid van essentiële of aanwezigheid van slecht functionerende eiwitten. Bijgevolg zijn deze aandoeningen het ideale doelwit voor gentherapie. Bij taaislijmziekte bijvoorbeeld kan de genetische afwijking behandeld worden door toevoeging van het gecorrigeerde ‘cystic fibrosis transmembrane conductance regulator’ (CFTR-) eiwit. Sommige kankers en virale infecties daarentegen kunnen behandeld worden door specifieke onderdrukking van respectievelijk kankerverwekkende of voor het virus levensnoodzakelijke eiwitten. Om het gewenste eiwit toe te voegen kan gebruik gemaakt worden van plasmide DNA (pDNA) dat codeert voor dit eiwit. De onderdrukking van de expressie van een specifiek eiwit kan verkregen worden door ‘short interfering’ RNA (siRNA-) moleculen aan de doelwitcellen toe te dienen. SiRNA-moleculen veroorzaken immers een sequentiespecifieke afbraak van boodschapper RNA (mRNA) en bijgevolg ook onderdrukking van de expressie van het overeenkomstige eiwit. Beide synthetische nucleïnezuren kunnen maar tot therapeutische successen leiden indien ze tot in het cytoplasma (in geval van siRNA) of tot in de kern (in geval van pDNA) van de doelwitcellen geraken. Bovendien moeten de nucleïnezuren intact hun doelwit bereiken, aangezien dit noodzakelijk is voor het behoud van hun biologische activiteit. Geavanceerde afgiftesystemen, viraal of niet-viraal, zijn dan ook noodzakelijk en vormen één van de belangrijkste onderzoeksonderwerpen binnen gentherapie

The DosRST two-component system of Mycobacterium tuberculosis: Characterizing the activation mechanism of DosR response regulator as a potential target for novel antimycobacterial drugs

La tuberculosi, la malaltia infecciosa causada per Mycobacterium tuberculosis, es un problema de salut global que provoca aproximadament 2 milions de morts anuals. Un terç de la població mundial es troba crònicament infectada amb Mycobacterium tuberculosis però no mostra símptomes clínics encara que té un risc d’un 10% de desenvolupar la malaltia, la qual cosa representa un reservori incontrolable de tuberculosi. En aquesta condició asimptomàtica, coneguda com a tuberculosi latent, Mycobacterium tuberculosis es localitza en lesions granulomatoses a l’hoste i és resistent als medicaments antimicobacterians existents en l’actualitat. En bacteris, els sistemes reguladors de dos components son un conjunt de proteïnes implicades en l’adaptació a canvis en l’entorn del microorganisme. Un sistema de dos components típic consta d’una histidina quinasa unida a membrana que té un paper essencial com a sensor dels canvis ambientals i un regulador transcripcional citosòlic que exerceix la seva funció controlant l’expressió de gens diana. Aquest parell de proteïnes funciona com un interruptor molecular que controla diferents respostes adaptatives a canvis a l’ambient cel•lular. Mycobacterium tuberculosis té 11 sistemes de dos components complets. El sistema DosRST, composat per un regulador transcripcional, DosR, i dues histidines quinases, DosS i DosT, té un paper estel•lar en l’adaptació de Mycobacterium tuberculosis a la tuberculosi latent. DosS i DosT s’autofosforilen en residus conservats d’histidina i ambdues proteïnes transfereixen aquesta un unitat de fosfat al residu d’àcid aspàrtic en posició 54 del regulador DosR. La fosforilació d’Asp54 és un interruptor que activa DosR i incrementa la seva afinitat per als promotors dels gens que regula. Les treonines 198 i 205 de DosR tenen un paper crucial en la dimerització de DosR i en la seva unió al DNA. Les dinàmiques moleculars realitzades amb la proteïna salvatge DosR i amb versions mutants mostren diferències notables en la formació del dímer actiu. També mostren una reducció o abolició completa de les interaccions proteïna-DNA a causa de les repulsions generades pels residus mutants carregats negativament. Encara més, les substitucions en Thr198 i Thr205 tenen un important efecte en la fosforilació química i enzimàtica de DosR així com també en la seva defosforilació catalitzada per DosS. El sistema de dos components DosRST és una bona diana per al desenvolupament de nous compostos amb activitat antimicobacteriana contra formes dorments de Mycobacterium tuberculosis. S’ha iniciat un programa de recerca dirigit al desenvolupament d’inhibidors específics de la proteïna reguladors DosR, usant com a punt de partida compostos comercials estructuralment relacionats amb un derivat fenilcumarínic que ha estat descrit com a molècula que interfereix en la interacció DosR-DNA.La tuberculosis, la enfermedad infecciosa causada por Mycobacterium tuberculosis, es un problema de salud global que provoca aproximadamente 2 millones de muertes anuales. Un tercio de la población mundial se encuentra crónicamente infectada con Mycobacterium tuberculosis pero no muestra síntomas clínicos aunque tiene un riesgo de un 10% de desarrollar la enfermedad, lo que representa un reservorio incontrolable de tuberculosis. En esta condición asintomática, conocida como tuberculosis latente, Mycobacterium tuberculosis se localiza en lesiones granulomatosas en el huésped y es resistente a los medicamentos antimicobacterianos existentes en la actualidad. En bacterias, los sistemas regulatorios de dos componentes son un conjunto de proteínas implicadas en la adaptación a cambios en el entorno del microorganismo. Un sistema de dos componentes típico consta de una histidina quinasa unida a membrana que tiene un papel esencial como sensor de los cambios ambientales y un regulador transcripcional citosólico que ejerce su función controlando la expresión de genes diana. Este par de proteínas funciona como un interruptor molecular que controla distintas respuestas adaptativas a cambios en el ambiente celular. Mycobacterium tuberculosis tiene 11 sistemas de dos componentes completos. El sistema DosRST, compuesto por un regulador transcripcional, DosR, y dos histidinas quinasas, DosS y DosT, juega un papel estelar en la adaptación de Mycobacterium tuberculosis a la tuberculosis latente. DosS y DosT se autofosforilan en residuos conservados de histidina y ambas proteínas transfieren esta unidad de fosfato al residuo de ácido aspártico en posición 54 del regulador DosR. La fosforilación de Asp54 es un interruptor que activa a DosR e incrementa su afinidad por los promotores de los genes que regula. Las treoninas 198 y 205 de DosR juegan un papel crucial en la dimerización de DosR y en su unión al DNA. Las dinámicas moleculares realizadas con la proteína salvaje DosR y con versiones mutantes muestran diferencias notables en la formación del dímero activo. También muestran una reducción o abolición completa de las interacciones proteína-DNA a causa de las repulsiones generadas por los residuos mutantes cargados negativamente. Más aún, las sustituciones en Thr198 y Thr205 tienen un importante efecto en la fosforilación química y enzimática de DosR así como también en su defosforilaición catalizada por DosS. El sistema de dos componentes DosRST es una buena diana para el desarrollo de nuevos compuestos con actividad antimicobacteriana contra formas durmientes de Mycobacterium tuberculosis. Se ha iniciado un programa de investigación dirigido al desarrollo de inhibidores específicos de la proteína reguladora DosR, usando como punto de partida compuestos comerciales estructuralmente relacionados con un derivado fenilcumarínico que ha sido descrito como molécula que interfiere en la interacción DosR-DNA.Tuberculosis, the infectious disease caused by Mycobacterium tuberculosis, is a global health problem with approximately two million deaths annually. One-third of the world population is chronically infected with Mycobacterium tuberculosis but do not show clinical symptoms although there is a 10% risk to development active disease, representing an uncontrollable reservoir of tuberculosis. In this asymptomatic condition, referred to as latent tuberculosis, Mycobacterium tuberculosis is located within granulomatous lesions in the host and is resistant to currently available antimycobacterial drugs. Two-component regulatory systems in bacteria are a major class of signal transduction proteins involved in adaptation to environmental changes. Typical system contains a membrane-bound histidine kinase that plays a crucial role in sensing environmental stimuli, and a cytosolic response regulator. This pair of proteins functions as a molecular switch that controls diverse adaptive environmental responses. Mycobacterium tuberculosis has eleven complete two-component systems. The DosRST system, composed of a response regulator, DosR, and two histidine kinases, DosS and DosT, plays a key role in Mycobacterium tuberculosis adaptation to latent tuberculosis. DosS and DosT autophosphorylate at conserved histidine residues and both proteins transfer this phosphor moiety to aspartic acid residue 54 of DosR. The phosphorylation of Asp54 serves as a switch to activate DosR and to increase the affinity for its cognate DNA promoters. Threonines 198 and 205 of DosR play a crucial role in DosR dimerization and DNA binding. The molecular dynamics with wild type and mutant version of DosR show different stability in the formation of the active DosR dimer. They also show the reduction or the abolishment of protein-DNA interactions because of the repulsions generated by negatively charged mutated residues. Moreover, substitutions in threonines 198 and 205 of DosR have a relevant effect on the chemical and enzymatic phosphorylation of DosR and on its DosS-catalysed dephosphorylation. The DosRST two-component system is a good target for the development of novel antimycobacterial drugs against dormant forms of M. tuberculosis. A structure-based discovery programme of inhibitors of DosR response regulator has been initiated with commercially available compounds showing a certain degree of similarity with a phenylcoumarin derivative previously described as DosR-DNA interfering molecule

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated