The challenges and opportunities in tuberculosis control, from Texas to Afghanistan

Tuberculosis is one of the most ancient human pathogens. Very early in the history of humans, tuberculosis spread throughout the world and currently infects one third of the world’s population1. Tuberculosis, both as a pathogen and a disease state, is fascinating in its own right. Considering the global public health significance of tuberculosis, which infects one third of the world’s population and kills over 1.5 million people per year, tuberculosis remains a threat and demands further research. For these reasons, my undergraduate research has largely centered on tuberculosis. Chapter 1 gives a broad overview of tuberculosis, applicable but not specific to my research. Each subsequent chapter focuses on a research project, with relevant additional background on tuberculosis. Chapter 2 is based on an internship at the Texas Department of State Health Services analyzing reasons for delayed completion of tuberculosis therapy for patients whose therapy initiated in Texas in 2006. Chapter 3 examines the potential for molecular methods of tuberculosis in low-resource setting, building off of my experience using mutations in the rpoB gene as a surrogate marker of rifampin resistance in Afghanistan. Chapter 4 briefly expands upon further work done in the lab of Dr. Andrew Ellington at the University of Texas at Austin under the supervision of Xi Chen, developing non-enzymatic methods of nucleic acid detection to indicate the presence of tuberculosis and other pathogens.Biological Sciences, School o

Evaluation of a Novel Inaba Cholera Conjugate Vaccine in Mice

Vibrio cholerae is a non-invasive Gram-negative enteric pathogen that causes cholera, a severe dehydrating diarrheal illness of humans. Cholera is responsible for substantial morbidity and mortality in both endemic and epidemic settings. Current oral killed vaccines do not provide protection that lasts as long as natural cholera infection, and current cholera vaccines have greatly reduced efficacy in children, the population most affected by cholera in endemic areas. Protection against cholera appears to be mediated by immune responses that target the O-specific polysaccharide (OSP) of V. cholerae. Here we report analysis of the immunogenicity and protective efficacy in mice of a cholera conjugate vaccine containing V. cholerae O1 Inaba OSP conjugated to a recombinant immunogenic fragment of tetanus toxoid heavy chain (rTTHc). OSP-rTTHc induced prominent anti-OSP responses in these animals. Serum from vaccinated mice also provide protection in the infant mouse model of cholera infection. Our results suggest that a cholera conjugate might have development potential for evaluation in humans

Evaluation in Mice of a Conjugate Vaccine for Cholera Made from Vibrio cholerae O1 (Ogawa) O-Specific Polysaccharide

Background: Protective immunity against cholera is serogroup specific. Serogroup specificity in Vibrio cholerae is determined by the O-specific polysaccharide (OSP) of lipopolysaccharide (LPS). Generally, polysaccharides are poorly immunogenic, especially in young children. Methodology Here we report the evaluation in mice of a conjugate vaccine for cholera (OSP:TThc) made from V. cholerae O1 Ogawa O-Specific Polysaccharide–core (OSP) and recombinant tetanus toxoid heavy chain fragment (TThc). We immunized mice intramuscularly on days 0, 21, and 42 with OSP:TThc or OSP only, with or without dmLT, a non-toxigenic immunoadjuvant derived from heat labile toxin of Escherichia coli. Principal Findings We detected significant serum IgG antibody responses targeting OSP following a single immunization in mice receiving OSP:TThc with or without adjuvant. Anti-LPS IgG responses were detected following a second immunization in these cohorts. No anti-OSP or anti-LPS IgG responses were detected at any time in animals receiving un-conjugated OSP with or without immunoadjuvant, and in animals receiving immunoadjuvant alone. Responses were highest following immunization with adjuvant. Serum anti-OSP IgM responses were detected in mice receiving OSP:TThc with or without immunoadjuvant, and in mice receiving unconjugated OSP. Serum anti-LPS IgM and vibriocidal responses were detected in all vaccine cohorts except in mice receiving immunoadjuvant alone. No significant IgA anti-OSP or anti-LPS responses developed in any group. Administration of OSP:TThc and adjuvant also induced memory B cell responses targeting OSP and resulted in 95% protective efficacy in a mouse lethality cholera challenge model. Conclusion: We describe a protectively immunogenic cholera conjugate in mice. Development of a cholera conjugate vaccine could assist in inducing long-term protective immunity, especially in young children who respond poorly to polysaccharide antigens

Feasibility of achieving the 2025 WHO global tuberculosis targets in South Africa, China, and India: a combined analysis of 11 mathematical models.

BACKGROUND: The post-2015 End TB Strategy proposes targets of 50% reduction in tuberculosis incidence and 75% reduction in mortality from tuberculosis by 2025. We aimed to assess whether these targets are feasible in three high-burden countries with contrasting epidemiology and previous programmatic achievements. METHODS: 11 independently developed mathematical models of tuberculosis transmission projected the epidemiological impact of currently available tuberculosis interventions for prevention, diagnosis, and treatment in China, India, and South Africa. Models were calibrated with data on tuberculosis incidence and mortality in 2012. Representatives from national tuberculosis programmes and the advocacy community provided distinct country-specific intervention scenarios, which included screening for symptoms, active case finding, and preventive therapy. FINDINGS: Aggressive scale-up of any single intervention scenario could not achieve the post-2015 End TB Strategy targets in any country. However, the models projected that, in the South Africa national tuberculosis programme scenario, a combination of continuous isoniazid preventive therapy for individuals on antiretroviral therapy, expanded facility-based screening for symptoms of tuberculosis at health centres, and improved tuberculosis care could achieve a 55% reduction in incidence (range 31-62%) and a 72% reduction in mortality (range 64-82%) compared with 2015 levels. For India, and particularly for China, full scale-up of all interventions in tuberculosis-programme performance fell short of the 2025 targets, despite preventing a cumulative 3·4 million cases. The advocacy scenarios illustrated the high impact of detecting and treating latent tuberculosis. INTERPRETATION: Major reductions in tuberculosis burden seem possible with current interventions. However, additional interventions, adapted to country-specific tuberculosis epidemiology and health systems, are needed to reach the post-2015 End TB Strategy targets at country level. FUNDING: Bill and Melinda Gates Foundation

Cost-effectiveness and resource implications of aggressive action on tuberculosis in China, India, and South Africa: a combined analysis of nine models.

BACKGROUND: The post-2015 End TB Strategy sets global targets of reducing tuberculosis incidence by 50% and mortality by 75% by 2025. We aimed to assess resource requirements and cost-effectiveness of strategies to achieve these targets in China, India, and South Africa. METHODS: We examined intervention scenarios developed in consultation with country stakeholders, which scaled up existing interventions to high but feasible coverage by 2025. Nine independent modelling groups collaborated to estimate policy outcomes, and we estimated the cost of each scenario by synthesising service use estimates, empirical cost data, and expert opinion on implementation strategies. We estimated health effects (ie, disability-adjusted life-years averted) and resource implications for 2016-35, including patient-incurred costs. To assess resource requirements and cost-effectiveness, we compared scenarios with a base case representing continued current practice. FINDINGS: Incremental tuberculosis service costs differed by scenario and country, and in some cases they more than doubled existing funding needs. In general, expansion of tuberculosis services substantially reduced patient-incurred costs and, in India and China, produced net cost savings for most interventions under a societal perspective. In all three countries, expansion of access to care produced substantial health gains. Compared with current practice and conventional cost-effectiveness thresholds, most intervention approaches seemed highly cost-effective. INTERPRETATION: Expansion of tuberculosis services seems cost-effective for high-burden countries and could generate substantial health and economic benefits for patients, although substantial new funding would be required. Further work to determine the optimal intervention mix for each country is necessary. FUNDING: Bill & Melinda Gates Foundation

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

Grace Eckhoff, Vlad codrea, and Andrew D. Ellington are with the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA -- Andrew D. Ellington and Xi Chen are with the Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USAThe programmability and replicability of RNA and DNA have respectively enabled the design and selection of a number of allosteric ribozymes and deoxyribozymes. These catalysts have been adapted to function as signal transducers in biosensors and biochemical reaction networks both in vitro and in vivo. However, allosteric control of nucleic acid catalysts is currently limited by the fact that one molecule of effector (input) generally regulates at most one molecule of ribozyme or deoxyribozyme (output). In consequence, allosteric control is usually inefficient when the concentration of input molecules is low. In contrast, catalytic regulation of protein enzymes, as in protein phosphorylation cascades, generally allows one input molecule (e.g., one kinase molecule) to regulate multiple output molecules (e.g., kinase substrates). Achieving such catalytic signal amplification would also be of great utility for nucleic acid circuits. Here we show that allosteric regulation of nucleic acid enzymes can be coupled to signal amplification in an entropy-driven DNA circuit. In this circuit, kinetically trapped DNA logic gates are triggered by a specific sequence, and upon execution generate a peroxidase deoxyribozyme that converts a colorless substrate (ABTS) into a green product (ABTS•+). This scheme provides a new paradigm for the design of enzyme-free biosensors for point-of-care diagnostics.Institute for Cellular and Molecular BiologyChemistryBiochemistr

Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism.

International audienceThe human APOBEC3 (A3A-A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome

The trochlea is medialized by total knee arthroplasty: an intraoperative assessment in 61 patients.

Contains fulltext : 52742.pdf (publisher's version ) (Open Access)BACKGROUND: A medialization of the femoral component in a total knee arthroplasty (TKA) causes abnormal patellar tracking, which could result in patellar instability, pain, wear, and failure. Previous reports defined medialization in relation to the neutral position of the femoral component, but omitted to compare it to the anatomical position of the trochlea. We assessed intraoperatively whether there is a systematic error of the position of the prosthetic groove relative to the anatomical trochlea. MATERIAL AND METHODS: A special instrument was developed to measure consecutively the mediolateral position of the anatomical trochlea and the mediolateral position of the prosthetic groove. 3 experienced knee surgeons determined the mediolateral error of the prosthetic groove in primary TKAs in 61 patients. RESULTS: There was a significant medial error of the prosthetic groove relative to the preoperative position of the trochlea, with a mean medial error of 2.5 mm (SD 3.3) INTERPRETATION: Our findings indicate that the trochlea is medialized by TKA. Because a conscious medialization of the femoral component in a TKA produces abnormal patellar tracking patterns, further investigations will be needed to analyze the clinical consequences of this medialization of the trochlea

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh