Acute and chronic sleeping sickness are fatal neglected tropical diseases caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense respectively (members of the sub-genus Trypanozoon). Accurate diagnostics are needed to guide treatment since the symptoms of disease are non-specific and the drugs that are used for treatment are too toxic to be administered to unconfirmed cases. Tests need to be simple enough to confirm clinical diagnosis of sleeping sickness in poorly-resourced, peripheral health centres and for use as epidemiological tools to detect T. b. rhodesiense in the zoonotic reservoirs of infection. This study focuses upon LAMP (loop-mediated isothermal amplification) as a novel diagnostic for sleeping sickness that may serve to bridge the gap between the need for sensitive, specific molecular diagnostics on the one hand and ‘field-friendly’ diagnostics on the other. Here, two previously published LAMP assays for Trypanozoons were compared to classic PCR based methods for the diagnosis of Trypanozoon infection status in 428 cattle blood samples. The results did not support the use of LAMP as an improved system for surveillance of T. b. rhodesiense in the zoonotic cattle reservoir. T. b. rhodesiense and T. b. gambiense subspecies specific LAMP assays were evaluated against traditional reference subspecies specific PCR tests, using DNA purified from 86 cryopreserved trypanosome isolates. Novel LAMP assays for these subspecies were also designed and evaluated. Both the published and novel assays for T. b. rhodesiense (targeting different regions of the SRA gene) were sensitive, specific and reliable when applied to purified DNAs, but were less consistent on field samples. The novel T. b. gambiense LAMP (targeting TgsGP) was sensitive and specific but this was not the case for the published LAMP assay (targeting the 5.8S rRNA gene). However reliability may be less than optimal for LAMP TgsGP. Finally, simple endpoint readout methods for LAMP were evaluated. The colour change reagent hydroxynaphthol blue was identified as the best currently available method taking cost, ease of use and reliability into consideration. In 2009 the number of reported sleeping sickness cases fell below 10,000 for the first time in 50 years. Improved LAMP diagnostics could facilitate the diagnosis of sleeping sickness and support the continued fight against this neglected, but deadly disease.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Background: Respiratory Syncytial Virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and children worldwide. Early diagnosis of RSV infection is associated with shorter periods of hospitalisation and decreased mortality. Current point of care (PoC) tests for RSV is less sensitive than molecular methods. Reverse transcription loop-mediated isothermal amplification (RT-LAMP), is a novel method of nucleic acid detection which allows for rapid, robust amplification, and visual detection of infectious agents. Aim: The objective of this study was to develop a novel, rapid, and sensitive multiplex RSV RT-LAMP assay for PoC diagnosis of RSV A and B. Methods: Preparation of a quantitative RSV standard for assay optimisation was done using a rapid hypotonic burst recovery method of infective virus during sub-passaging, and a shell vial fluorescent focus assay for titration of culture-derived viral stock. We designed a single set of eight primers targeting the large polymerase gene of both RSV A and B, and developed a novel single-step multiplex RSV RT-LAMP assay, using an in-house reaction mix and the Rotor-Gene Q real-time thermocycler (Qiagen, Hilden, Germany). The metal ion indicator hydroxy naphtol blue (HNB) was added to the multiplex RSV RT-LAMP assay for visual detection of RSV. Results: The final optimised multiplex RSV RT-LAMP assay had an analytical detection sensitivity of <10 focus forming units (FFU) per reaction for both RSV A and B, with a mean time to positivity of 21.85 minutes (95% CI 19.2-24.5 minutes), compared to 90-120 minutes for conventional PCR. Evaluated against the Seeplex RV15 multiplex PCR (Seegene, Seoul, Korea) by testing 44 (22 RSV A/22 RSV B) nasopharyngeal specimens, the multiplex RSV RT-LAMP assay had a sensitivity of 100%, and a specificity of 100% when screened against nine common respiratory viruses. Visual detection of RSV using HNB as colorimetric reagent was equivalent to the analytical sensitivity (10 FFU/reaction) and specificity (100%) of the multiplex RSV RT-LAMP assay. Conclusion: Compared with conventional PCR, our novel single-step multiplex RSV RTLAMP assay had excellent sensitivity, specificity, and when combined with HNB dye could provide accurate visual diagnosis within 1 hour. We envisage that this multiplex RSV RTLAMP assay will be used for rapid and sensitive RSV detection at the PoC. / AFRIKAANSE OPSOMMING: Agtergrond: Respiratoriese Syncytial Virus (RSV) is die hoof oorsaak van erge laer lugweginfeksie in babas en kinders wêreldwyd. Vroeë diagnose van RSV infeksie word geassosieer met korter periodes van hospitalisasie en verlaagde mortaliteit. Huidige punt van sorg (PoC) toetse vir RSV is minder sensitief as molekulêre metodes. Omgekeerde transkripsie lus-gemedieerde isotermiese amplifisering (RT-LAMP), is 'n nuwe metode van nukleïensuur opsporing wat voorsiening maak vir vinnige, doeltreffende amplifisering, en visuele bevestiging van aansteeklike agente. Doel: Die doel van hierdie studie was om 'n nuwe, vinnige en sensitiewe multipleks RSV RTLAMP toets te ontwikkel wat PoC diagnose van RSV A en B in staat stel. Metodes: Voorbereiding van 'n kwantitatiewe RSV standaard vir toets optimisering is gedoen met behulp van 'n hipotoniese sel-lise metode van infektiewe virus tydens sub-kultuur, en 'n “shell-vial” kultuur en fluorosensie fokus toets vir titrasie van kultuur-geproduseerde virus voorraad. Ons het 'n enkele stel van agt inleiers ontwerp wat gebaseer is op die groot polimerase geen van beide RSV A en B, en 'n nuwe enkel-stap multipleks RSV RT-LAMP toets ontwikkel, met gebruik van 'n in-huis reaksie mengsel en die Rotor-Gene Q “real-time” thermocycler (Qiagen, Hilden, Duitsland). Die metaalioon aanwyser hidroksi naphtol blou (HNB) is bygevoeg in die multipleks RSV RT-LAMP toets vir visuele bevestiging van RSV. Resultate: Die finale geoptimiseerde multipleks RSV RT-LAMP toets het 'n analitiese sensitiwiteit van <10 fokus vormende eenhede (FFU) per reaksie vir beide RSV A en B gehad, met 'n gemiddelde tyd tot positiwiteit van 21.85 minute (95% CI 19.2-24.5 minute) , in vergelyking met 90-120 minute vir konvensionele PCR. Geëvalueer teen die Seeplex RV15 multipleks PCR (Seegene, Seoul, Korea) deur 44 (22 RSV A/22 RSV B) nasofaringeale monsters te toets, het die multipleks RSV RT-LAMP toets 'n sensitiwiteit van 100% getoon, en 'n spesifisiteit van 100% wanneer getoets teen nege algemene respiratoriese virusse. Visuele bevestiging van RSV met gebruik van HNB as kolorimetriese reagens was gelykstaande aan die analitiese sensitiwiteit (10 FFU/reaksie) en spesifisiteit (100%) van die multipleks RSV RT-LAMP toets. Gevolgtrekking: In vergelyking met konvensionele PCR, het ons nuwe enkel-stap multipleks RSV RT-LAMP toets uitstekende sensitiwiteit, spesifisiteit, en wanneer dit gekombineer word met HNB kleurstof kon dit akkurate visuele diagnose voorsien binne 1 uur. Ons verwag dat hierdie multipleks RSV RT-LAMP toets gebruik sal word vir vinnige en sensitiewe RSV bevestiging by die PoC.

<p><a>The hemotrophic mycoplasma<i>, Mycoplasma ovis</i>, is found in sheep and goats throughout the world. This pathogenic bacterium is capable of causing an acute, life-threatening infection as well as chronic or subclinical infections in these animals. The purposes of the present studies were to develop <i>M. ovis</i>-specific assays for detection of this hemoplasma, and to better understand infection dynamics within pregnant ewes and lambs. </a>The first study describes the development and validation of a SYBR^® Green quantitative PCR (qPCR) assay, which was subsequently used to determine the prevalence of <i>M. ovis</i> infection within a population of goats and to evaluate risk factors for infection. This highly sensitive and specific assay consistently detected as few as 10 copies of plasmid/reaction. Convenience-based sampling of 362 goats from 61 farms located in Indiana revealed a prevalence of infection of 18% (95% confidence interval (CI), 14% to 22%). Bacterial loads of <i>M. ovis</i> ranged from 1.05 x 10³ to 1.85 x 10⁵copies/mL of blood with a mean of 1.31 x 10⁴copies/mL of blood. The only risk factor associated with hemoplasma infection was the production use of the goat; dairy goats had a 3.3 fold increase compared with the prevalence in goats used for meat. This study not only demonstrates that <i>M. ovis</i> infection is common in goats in Indiana, but shows the variability of bacterial loads that can be found in chronically-infected animals. While sub-clinically infected goats may have a bacteremia, levels are characteristically less than 2.0 x 10⁵copies/mL.</p><p> The second project utilized a combination of cross-sectional and longitudinal studies to estimate the prevalence of <i>M. ovis</i> infection from a cohort of naturally-infected pregnant ewes, assess changes in their bacterial loads, and determine the incidence of <i>M. ovis</i> in lambs pre- and post-weaning. The prevalence of <i>M. ovis</i> infection in ewes was not found to be significantly different during pregnancy, and before and after weaning of the lambs, with prevalence estimates of 45% (95% CI, 23.1 – 68.5), 36% (95% CI, 17.9 – 57.4), and 44%, (95% CI, 24.4 – 65.1), respectively. Bacterial loads of the ewes from the cross-sectional study ranged from 10⁴to 10⁹copies/mL of blood, with the median bacterial load at 10⁵ copies/mL of blood. While higher bacterial loads are typical of an acute infection, none of the ewes in this study had overt clinical signs. The data suggest that <i>M. ovis</i> loads may be higher in pregnant sheep, particularly in ewes half-way through pregnancy. Most of the <i>M. ovis</i> infections in the study lambs were detected post-weaning which suggests that transplacental or transmammary infection of <i>M. ovis</i> are unlikely routes.</p><p> In the third study, a subset of <i>M. ovis</i> genes for use in a multi-locus sequence typing assay (MLST) were evaluated. Next-generation sequencing was performed to generate data from pooled DNA amplicons in order to identify single nucleotide polymorphisms (SNPs) of <i>M. ovis </i>from five genes. Evaluation of the quality and depths of coverage for the reads and SNPs indicated that the pooled DNA amplicons produced reads and SNPs having high quality and sufficient depth. This pooling technique is a cost-effective alternative to whole-genome sequencing. While the MLST has good discriminatory power and may be used to identify genetically distant and divergent clusters of <i>M. ovis</i> from different geographical origins, within a herd the discrimination power is low, which may hamper its usefulness in transmission studies. </p><p> The fourth and final study was the development of a loop-mediated isothermal amplification (LAMP) assay targeting the dnaK gene of <i>M. ovis</i>, with comparison of the assay to conventional PCR (cPCR). The metal ion indicator hydroxynaphthol blue (HNB) was added prior to the reaction, which allowed for visual detection of LAMP-positive samples as indicated by a color change from violet to sky blue. <i>Mycoplasma ovis</i> was consistently detected in 45 minutes with the LAMP assay at a reaction temperature of 64°C, with more infected sheep being detected than by cPCR. Therefore, the LAMP assay is fast and reliable in the detection of <i>M. ovis</i>. The developed LAMP assay may have applications in diagnostics, surveillance and disease management as well as prevalence studies. However, a more robust molecular technique is necessary for <i>M. ovis</i> isolate or stain discrimination to investigate transmission or disease spread in an outbreak.</p><p> </p><p> In conclusion, three new molecular tools for the detection of <i>M. ovis</i> in goats and sheep were developed as results of these studies. We have shown that the qPCR assay is an efficient tool for detection and quantification of <i>M. ovis</i> loads in blood from both of these species. On the other hand, the value of the LAMP assay is for reliable detection of infection (not quantification), especially in resource-limited situations. The five-locus MLST protocol developed herein, a typing assay based on the polymorphism of five gene sequences, is a laborious technique requiring DNA extraction, PCR amplification, purification and sequencing of target loci. The value of this technique is not as a routine diagnostic, but rather it may be used to better understand the genetic diversity of <i>M. ovis</i> and investigate strain variations. Most importantly, the scheme is sufficiently robust to allow direct genotyping of <i>M. ovis</i> in total blood DNA extracts without culture isolation. The MLST approach may prove useful as a tool for future investigations of transmission and disease spread. These studies have also expanded our understanding of the infection dynamics of <i>M. ovis</i> in pregnant sheep and lambs. It is shown herein that despite the high prevalence and sometimes high bacterial loads in pregnant ewes, <i>M. ovis</i> does not appear to be transmitted to the lambs in utero or during the perinatal period. The lambs become infected mostly after weaning; this may suggest a protective effect during the pre-weaning period and/or subsequent exposure/infection from their environment. </p><br>

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18898号 / 医博第4009号 / 新制||医||1009(附属図書館) / 31849 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木原 正博, 教授 中川 一路, 教授 一山 智 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00270/full / 付記する学位プログラム名：グローバル生存学大学院連携プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19619号 / 医博第4126号 / 新制||医||1015(附属図書館) / 32655 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 木原 正博, 教授 松林 公蔵 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

En ökad utbredning av vankomycinresistenta enterokocker (VRE) har setts i Sverige sedan 2007. Bakteriemi orsakad av VRE är mycket svårbehandlad, varför snabbare tillförlitlig resistensdiagnostik är betydelsefullt för att minska dödlighet, vårdtider, vårdkostnader och belastning på sjukvårdssystemet. På mikrobiologilaboratoriet, Region Jönköpings län (RJL), tar idag identifiering av fenotypisk vankomycinresistens vid optimala förhållanden 6 timmar, räknat från att enterokocker konstaterats växa i blodet. Resistensgenerna vanA och vanB, som bland andra orsakar vankomycinresistens hos enterokocker, kan genetiskt verifieras med loop-mediated isothermal amplification men tar idag upp till ett dygn då bakteriekolonier används som analysmaterial i arbetsrutinen på molekylärbiologilaboratoriet, RJL. Syftet med studien var att utvärdera bakteriepellet som analysmaterial för genetisk identifiering av vanA och vanB, på Genie® II Mk2 med eazyplex® VRE basic, hos enterokocker från positiva blododlingar. För att utvärdera bakteriepellet som analysmaterial analyserades isolat av Enterococcus faecium (n=17) och Enterococcus faecalis (n=5) från bakteriepellets tillverkade från simulerade positiva blododlingar med eazyplex® VRE basic på Genie® II Mk2, varpå resultaten jämfördes mot isolatens faktiska närvaro/frånvaro av vanA/vanB. Samstämmigheten av de uppmätta- och de förväntade resultaten var fullständig, vilket indikerar att bakteriepellet med hög tillförlitlighet kan användas som analysmaterial till eazyplex® VRE basic för att påvisa vanA och vanB hos enterokocker i blododlingar. / An increased prevalence of vancomycin-resistant enterococci (VRE) has been observed in Sweden since 2007. Treating bacteremia caused by VRE is difficult, which is why faster, and reliable resistance diagnostics are important. At the Microbiology laboratory, Region Jönköping County, the identification of phenotypic vancomycin resistance under optimal conditions takes 6 hours from when growth of enterococci in blood is determined. The genes vanA and vanB, which among others cause vancomycin resistance, can be genetically verified by loop-mediated isothermal amplification, but takes up to one day since bacterial colonies are used as analysis material. The aim of the study was to evaluate bacterial pellet as an analytical material for genetic identification of vanA and vanB, on Genie® II Mk2 with eazyplex® VRE basic, in enterococci from positive blood cultures. To evaluate the bacterial pellet, isolates of Enterococcus faecium (n=17) and Enterococcus faecalis (n=5) from bacterial pellets made from simulated positive blood cultures were analyzed with eazyplex® VRE basic on the Genie® II Mk2, and the results were compared to the actual presence/absence of vanA/vanB in the isolates. The complete coherence between the expected and measured results indicates that the bacterial pellet can be used as an analytical material for eazyplex® VRE basic.

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative

abstract: HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of HIV-1 RNA in blood. However, viral load assays are not widely available in sub-Saharan Africa and cost between 50-$139 USD per test on average where available. To address this problem, a mixed-methods approach was undertaken to design a novel and inexpensive viral load diagnostic for HIV-1 and to evaluate barriers to its adoption in a developing country. The assay was produced based on loop-mediated isothermal amplification (LAMP). Blood samples from twenty-one individuals were spiked with varying concentrations of HIV-1 RNA to evaluate the sensitivity and specificity of LAMP. Under isothermal conditions, LAMP was performed with an initial reverse-transcription step (RT-LAMP) and primers designed for HIV-1 subtype C. Each reaction generated up to a few billion copies of target DNA within an hour. Presence of target was detected through naked-eye observation of a fluorescent indicator and verified by DNA gel electrophoresis and real-time fluorescence. The assay successfully detected the presence of HIV in samples with a broad range of HIV RNA concentration, from over 120,000 copies/reaction to 120 copies/reaction. In order to better understand barriers to adoption of LAMP in developing countries, a feasibility study was undertaken in Tanzania, a low-income country facing significant problems in healthcare. Medical professionals in Northern Tanzania were surveyed for feedback regarding perspectives of current HIV assays, patient treatment strategies, availability of treatment, treatment priorities, HIV transmission, and barriers to adoption of the HIV-1 LAMP assay. The majority of medical providers surveyed indicated that the proposed LAMP assay is too expensive for their patient populations. Significant gender differences were observed in response to some survey questions. Female medical providers were more likely to cite stigma as a source problem of the HIV epidemic than male medical providers while males were more likely to cite lack of education as a source problem than female medical providers. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

<div>Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.</div><div><br></div><div>Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use.</div>