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1.
Int J Mol Sci ; 25(5)2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38474308

RESUMO

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host-pathogen interactions with implications for transmission and evolution.


Assuntos
Vírus Bluetongue , Ceratopogonidae , Chironomidae , Coinfecção , Animais , Temperatura , Vírus Bluetongue/genética , Sorogrupo
2.
Clin Infect Dis ; 73(7): e2342-e2349, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-32975577

RESUMO

BACKGROUND: Post-treatment Lyme disease symptoms/syndrome (PTLDS) occurs in approximately 10% of patients with Lyme disease following antibiotic treatment. Biomarkers or specific clinical symptoms to identify patients with PTLDS do not currently exist and the PTLDS classification is based on the report of persistent, subjective symptoms for ≥6 months following antibiotic treatment for Lyme disease. METHODS: Untargeted liquid chromatography-mass spectrometry metabolomics was used to determine longitudinal metabolic responses and biosignatures in PTLDS and clinically cured non-PTLDS Lyme patients. Evaluation of biosignatures included (1) defining altered classes of metabolites, (2) elastic net regularization to define metabolites that most strongly defined PTLDS and non-PTLDS patients at different time points, (3) changes in the longitudinal abundance of metabolites, and (4) linear discriminant analysis to evaluate robustness in a second patient cohort. RESULTS: This study determined that observable metabolic differences exist between PTLDS and non-PTLDS patients at multiple time points. The metabolites with differential abundance included those from glycerophospholipid, bile acid, and acylcarnitine metabolism. Distinct longitudinal patterns of metabolite abundance indicated a greater metabolic variability in PTLDS versus non-PTLDS patients. Small numbers of metabolites (6 to 40) could be used to define PTLDS versus non-PTLDS patients at defined time points, and the findings were validated in a second cohort of PTLDS and non-PTLDS patients. CONCLUSIONS: These data provide evidence that an objective metabolite-based measurement can distinguish patients with PTLDS and help understand the underlying biochemistry of PTLDS.


Assuntos
Doença de Lyme , Síndrome Pós-Lyme , Antibacterianos/uso terapêutico , Cromatografia Líquida , Estudos de Coortes , Humanos , Doença de Lyme/diagnóstico , Doença de Lyme/tratamento farmacológico , Síndrome Pós-Lyme/tratamento farmacológico
3.
Antimicrob Agents Chemother ; 65(11): e0058321, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34370580

RESUMO

Multiple drug discovery initiatives for tuberculosis are currently ongoing to identify and develop new potent drugs with novel targets in order to shorten treatment duration. One of the drug classes with a new mode of action is DprE1 inhibitors targeting an essential process in cell wall synthesis of Mycobacterium tuberculosis. In this investigation, three DprE1 inhibitors currently in clinical trials, TBA-7371, PBTZ169, and OPC-167832, were evaluated side-by-side as single agents in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon tuberculosis infection. The goal was to confirm the efficacy of the DprE1 inhibitors in a mouse tuberculosis model with advanced pulmonary pathology and perform comprehensive analysis of plasma, lung, and lesion-centric drug levels to establish pharmacokinetic-pharmacodynamic (PK-PD) parameters predicting efficacy at the site of infection. Results showed significant efficacy for all three DprE1 inhibitors in the C3HeB/FeJ mouse model after 2 months of treatment. Superior efficacy was observed for OPC-167832 even at low-dose levels, which can be attributed to its low MIC, favorable distribution, and sustained retention above the MIC throughout the dosing interval in caseous necrotic lesions, where the majority of bacteria reside in C3HeB/FeJ mice. These results support further progression of the three drug candidates through clinical development for tuberculosis treatment.


Assuntos
Mycobacterium tuberculosis , Tiazinas , Tuberculose , Animais , Camundongos , Camundongos Endogâmicos C3H , Piperazinas , Tuberculose/tratamento farmacológico
4.
J Proteome Res ; 19(2): 610-623, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31821002

RESUMO

Lyme disease is a tick-borne bacterial illness that occurs in areas of North America, Europe, and Asia. Early infection typically presents as generalized symptoms with an erythema migrans (EM) skin lesion. Dissemination of the pathogen Borrelia burgdorferi can result in multiple EM skin lesions or in extracutaneous manifestations such as Lyme neuroborreliosis. Metabolic biosignatures of patients with early Lyme disease can potentially provide diagnostic targets as well as highlight metabolic pathways that contribute to pathogenesis. Sera from well-characterized patients diagnosed with either early localized Lyme disease (ELL) or early disseminated Lyme disease (EDL), plus healthy controls (HC), from the United States were analyzed by liquid chromatography-mass spectrometry (LC-MS). Comparative analyses were performed between ELL, or EDL, or ELL combined with EDL, and the HC to develop biosignatures present in early Lyme disease. A direct comparison between ELL and EDL was also performed to develop a biosignature for stages of early Lyme disease. Metabolic pathway analysis and chemical identification of metabolites with LC-tandem mass spectrometry (LC-MS/MS) demonstrated alterations of eicosanoid, bile acid, sphingolipid, glycerophospholipid, and acylcarnitine metabolic pathways during early Lyme disease. These metabolic alterations were confirmed using a separate set of serum samples for validation. The findings demonstrated that infection of humans with B. burgdorferi alters defined metabolic pathways that are associated with inflammatory responses, liver function, lipid metabolism, and mitochondrial function. Additionally, the data provide evidence that metabolic pathways can be used to mark the progression of early Lyme disease.


Assuntos
Doença de Lyme , Neuroborreliose de Lyme , Ásia , Cromatografia Líquida , Europa (Continente) , Humanos , Doença de Lyme/diagnóstico , Espectrometria de Massas em Tandem
5.
J Clin Microbiol ; 58(6)2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32245831

RESUMO

Identification of biomarkers for latent Mycobacterium tuberculosis infection and risk of progression to tuberculosis (TB) disease are needed to better identify individuals to target for preventive therapy, predict disease risk, and potentially predict preventive therapy efficacy. Our group developed multiple reaction monitoring mass spectrometry (MRM-MS) assays that detected M. tuberculosis peptides in serum extracellular vesicles from TB patients. We subsequently optimized this MRM-MS assay to selectively identify 40 M. tuberculosis peptides from 19 proteins that most commonly copurify with serum vesicles of patients with TB. Here, we used this technology to evaluate if M. tuberculosis peptides can also be detected in individuals with latent TB infection (LTBI). Serum extracellular vesicles from 74 individuals presumed to have latent M. tuberculosis infection (LTBI) based on close contact with a household member with TB or a recent tuberculin skin test (TST) conversion were included in this study. Twenty-nine samples from individuals with no evidence of TB infection by TST and no known exposure to TB were used as controls to establish a threshold to account for nonspecific/background signal. We identified at least one of the 40 M. tuberculosis peptides in 70 (95%) individuals with LTBI. A single peptide from the glutamine synthetase (GlnA1) enzyme was identified in 61/74 (82%) individuals with LTBI, suggesting peptides from M. tuberculosis proteins involved in nitrogen metabolism might be candidates for pathogen-specific biomarkers for detection of LTBI. The detection of M. tuberculosis peptides in serum extracellular vesicles from persons with LTBI represents a potential advance in the diagnosis of LTBI.


Assuntos
Vesículas Extracelulares , Tuberculose Latente , Mycobacterium tuberculosis , Humanos , Tuberculose Latente/diagnóstico , Peptídeos , Teste Tuberculínico
6.
Int Psychogeriatr ; 30(6): 867-880, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-28462764

RESUMO

ABSTRACTBackground:In this paper, we report progress on "Neighborhoods: our people, our places" an international study about how people living with dementia interact with their neighborhoods. The ideas of social health and citizenship are drawn upon to contextualize the data and make a case for recognizing and understanding the strengths and agency of people with dementia. In particular, we address the lived experience of the environment as a route to better understanding the capabilities, capacities, and competencies of people living with dementia. In doing this, our aim is to demonstrate the contribution of social engagement and environmental support to social health. METHODS: The study aims to "map" local spaces and networks across three field sites (Manchester, Central Scotland and Linkoping, Sweden). It employs a mix of qualitative and participatory approaches that include mobile and visual methods intended to create knowledge that will inform the design and piloting of a neighborhood-based intervention. RESULTS: Our research shows that the neighborhood plays an active role in the lives of people with dementia, setting limits, and constraints but also offering significant opportunities, encompassing forms of help and support as yet rarely discussed in the field of dementia studies. The paper presents new and distinctive insights into the relationship between neighborhoods and everyday life for people with dementia that have important implications for the debate on social health and policy concerning dementia friendly communities. CONCLUSION: We end by reflecting on the messages for policy and practice that are beginning to emerge from this on-going study.


Assuntos
Demência/psicologia , Relações Interpessoais , Características de Residência , Adulto , Idoso , Idoso de 80 Anos ou mais , Demência/terapia , Feminino , Humanos , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Escócia , Suécia
7.
Viruses ; 16(2)2024 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-38400016

RESUMO

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct-cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution.


Assuntos
Vírus Bluetongue , Ceratopogonidae , Coinfecção , Animais , Sorogrupo , Vírus Bluetongue/genética , Coinfecção/veterinária , Genótipo
8.
Viruses ; 16(1)2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38257841

RESUMO

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.


Assuntos
Coronavirus Felino , Peritonite Infecciosa Felina , Gatos , Animais , Proteômica , Apresentação de Antígeno , Apoptose , Oligonucleotídeos , Proteínas Sanguíneas
9.
bioRxiv ; 2023 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-36747638

RESUMO

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure. Author Summary: WNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

10.
Pathogens ; 12(6)2023 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-37375457

RESUMO

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

11.
ACS Infect Dis ; 9(8): 1458-1469, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37428112

RESUMO

Intra-household contacts (HCs) of leprosy patients are at increased risk of infection by Mycobacterium leprae and about ∼5-10% will develop active disease. A prognostic tool to identify HCs with the greatest risk of progressing to active disease would enhance early leprosy diagnosis and optimize prophylactic intervention. Previous metabolomics studies suggest that host lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are potential biomarkers for leprosy. In this study, we investigated retrospective sera of leprosy HCs by liquid chromatography-mass spectrometry and enzyme-linked immunoassay to determine whether circulating levels of ω-3 and ω-6 PUFA metabolites were altered in HCs that developed leprosy (HCDL) in comparison to those that did not (HCNDL). Sera were collected from HCs at the time of index case diagnosis and before clinical signs/symptoms of leprosy. Our findings showed that HCDL sera exhibited a distinct metabolic profile in comparison to HCDNL. Specifically, arachidonic acid, leukotriene B4, 11-hydroxyeicosatetraenoic acid, prostaglandin D2, and lipoxin A4 were elevated in HCDL. In contrast, prostaglandin E2 levels were reduced in HCDL. The ω-3 PUFAs, docosahexaenoic acid, eicosapentaenoic acid, and the docosahexaenoic acid-derived resolvin D1 and maresin-1 were also elevated in HCDL individuals compared to HCNDL. Principal component analyses provided further evidence that lipid mediators could serve as an early biomarker for progression to active leprosy. A logistic model identified resolvin D1 and D2, and prostaglandin D2 as having the greatest potential for early detection of HCs that will manifest leprosy.


Assuntos
Ácidos Graxos Ômega-3 , Hanseníase , Humanos , Ácidos Docosa-Hexaenoicos , Mycobacterium leprae/metabolismo , Estudos Retrospectivos , Ácidos Graxos Insaturados/metabolismo , Hanseníase/diagnóstico , Prostaglandinas , Biomarcadores
12.
Hum Reprod ; 27(5): 1292-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22422778

RESUMO

BACKGROUND: This study aimed to calculate costs and health-related quality of life of women with endometriosis-associated symptoms treated in referral centres. METHODS: A prospective, multi-centre, questionnaire-based survey measured costs and quality of life in ambulatory care and in 12 tertiary care centres in 10 countries. The study enrolled women with a diagnosis of endometriosis and with at least one centre-specific contact related to endometriosis-associated symptoms in 2008. The main outcome measures were health care costs, costs of productivity loss, total costs and quality-adjusted life years. Predictors of costs were identified using regression analysis. RESULTS: Data analysis of 909 women demonstrated that the average annual total cost per woman was €9579 (95% confidence interval €8559-€10 599). Costs of productivity loss of €6298 per woman were double the health care costs of €3113 per woman. Health care costs were mainly due to surgery (29%), monitoring tests (19%) and hospitalization (18%) and physician visits (16%). Endometriosis-associated symptoms generated 0.809 quality-adjusted life years per woman. Decreased quality of life was the most important predictor of direct health care and total costs. Costs were greater with increasing severity of endometriosis, presence of pelvic pain, presence of infertility and a higher number of years since diagnosis. CONCLUSIONS: Our study invited women to report resource use based on endometriosis-associated symptoms only, rather than drawing on a control population of women without endometriosis. Our study showed that the economic burden associated with endometriosis treated in referral centres is high and is similar to other chronic diseases (diabetes, Crohn's disease, rheumatoid arthritis). It arises predominantly from productivity loss, and is predicted by decreased quality of life.


Assuntos
Endometriose/economia , Custos de Cuidados de Saúde/estatística & dados numéricos , Qualidade de Vida , Adulto , Assistência Ambulatorial , Efeitos Psicossociais da Doença , Endometriose/complicações , Feminino , Humanos , Infertilidade Feminina/complicações , Dor Pélvica/complicações , Estudos Prospectivos , Anos de Vida Ajustados por Qualidade de Vida , Análise de Regressão , Centros de Atenção Terciária
13.
PLoS One ; 17(10): e0275838, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36219600

RESUMO

The World Health Organization (WHO) emphasizes that tuberculosis (TB) in children and adolescents is often overlooked by healthcare providers and difficult to diagnose. As childhood TB cases rise, finding a diagnostic high in sensitivity and specificity is critical. In this study 91 urine samples from children aged 1-10 years were analyzed for tuberculostearic acid (TBSA) by gas chromatography/mass spectrometry (GC/MS) and capture ELISA (C-ELISA). In C-ELISA the CS35/A194-01 antibody performed very poorly with both curve-based and model-based cutoffs. The area under the ROC curve (AUC) of the CS35 OD450 values was only 0.60. Replacing the capture antibody with BJ76 gave a better performance in both sensitivity and specificity (AUC = 0.95). When these samples were analyzed by GC/MS, 41 classified as 'probable/possible' for TB were distinctly TBSA positive with ten samples having <3 ng/mL LAM. However, from the 50 samples with 'unlikely' TB classification, 36 were negative but 7 had >3 ng/mL and were designated as LAM positive. This experimental assay assessment study signifies that i) the antibody pair CS35/A194-01 that has been successful for adult active TB diagnosis is not adequate when LAM level is low as in pediatric TB; ii) no one mAb appears to recognize all TB-specific LAM epitopes.


Assuntos
Infecções por HIV , Mycobacterium tuberculosis , Tuberculose , Adolescente , Adulto , Anticorpos , Criança , Epitopos , Humanos , Limite de Detecção , Lipopolissacarídeos , Sensibilidade e Especificidade , Tuberculose/diagnóstico
14.
Sci Rep ; 12(1): 1478, 2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35087163

RESUMO

We provide a pipeline for data preprocessing, biomarker selection, and classification of liquid chromatography-mass spectrometry (LCMS) serum samples to generate a prospective diagnostic test for Lyme disease. We utilize tools of machine learning (ML), e.g., sparse support vector machines (SSVM), iterative feature removal (IFR), and k-fold feature ranking to select several biomarkers and build a discriminant model for Lyme disease. We report a 98.13% test balanced success rate (BSR) of our model based on a sequestered test set of LCMS serum samples. The methodology employed is general and can be readily adapted to other LCMS, or metabolomics, data sets.


Assuntos
Doença de Lyme/diagnóstico , Metabolômica/métodos , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão/métodos , Conjuntos de Dados como Assunto , Voluntários Saudáveis , Humanos , Doença de Lyme/sangue , Espectrometria de Massas/métodos , Máquina de Vetores de Suporte
15.
J Microbiol Biol Educ ; 22(3)2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34970390

RESUMO

It is critical for science, technology, engineering, and mathematics (STEM) students to develop competencies in science communication, including science writing. However, it can be difficult for instructors and departments to assess the quality of their students' science writing. Many published science writing rubrics are specific to certain genres like lab reports. We thus developed a Universal Science Writing Rubric (USWR) that is usable regardless of the genre or audience of science writing. This tool enables students, instructors, and departments to assess science writing written to lay or scientific audiences, focusing on important rhetorical concerns like science content and interpretation rather than simply surface features like grammar. We demonstrate the use of our USWR on various life science lab reports, scientific review articles, grant proposals, and news articles, showing that the USWR is sensitive enough to highlight statistically significant differences between groups of student writing samples and valid enough to produce results that echo published and anecdotal observations of STEM student science writing skills. Thus, the USWR is a useful tool for assessment of STEM student science writing that is widely applicable in the classroom and laboratory.

16.
Sci Rep ; 11(1): 2922, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536495

RESUMO

Our study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 µL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97-100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates-tuberculostearic acid (TBSA) and D-arabinose (D-ara)-were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.


Assuntos
Lipopolissacarídeos/urina , Mycobacterium tuberculosis/isolamento & purificação , Manejo de Espécimes/métodos , Tuberculose/diagnóstico , Adulto , Criança , Estudos de Coortes , Estudos de Viabilidade , Humanos , Testes Imunológicos/métodos , Limite de Detecção , Espectrometria de Massas , Peru , Escarro/microbiologia , Tuberculose/microbiologia , Tuberculose/urina
17.
PLoS One ; 16(9): e0257615, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34547058

RESUMO

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 µg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.


Assuntos
Biomarcadores/urina , Endopeptidase K/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Tuberculose/diagnóstico , Endopeptidase K/química , Ensaio de Imunoadsorção Enzimática/instrumentação , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Lipopolissacarídeos/urina , Papel , Temperatura
18.
Oncolytic Virother ; 9: 17-29, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548076

RESUMO

PURPOSE: Rhabdomyosarcomas (RMS) are difficult tumors to treat with conventional therapies. Publications indicate that oncolytic virotherapy (OV) could benefit cancer patients with tumors that are refractory to conventional treatments. It is believed that the efficacy of OV can be enhanced when used in combination with other treatments. This study evaluated the response of mice with aggressive alveolar RMS (ARMS) allografts to treatment with an OV [recombinant myxoma virus (MYXVΔserp2)] in combination with a Janus kinase (JAK) inhibitor (oclacitinib). Oclacitinib is known to inhibit JAK1 and JAK2 cell signaling pathways, which should limit the antiviral Type I interferon response. However, oclacitinib does not inhibit immune pathways that promote antigen presentation, which help stimulate an anti-cancer immune response. MATERIALS AND METHODS: To determine if MYXVΔserp2 and oclacitinib could improve outcomes in animals with ARMS, nude mice were inoculated subcutaneously with murine ARMS cells to establish tumors. Immune responses, tumor growth, and clinical signs in mice treated with combination therapy were compared to mice given placebo therapy and mice treated with OV alone. RESULTS: Combination therapy was safe; no viral DNA was detected in off-target organs, only within tumors. As predicted, viral DNA was detected in tumors of mice given oclacitinib and MYXVΔserp2 for a longer time period than mice treated with OV alone. Although tumor growth rates and median survival times were not significantly different between groups, clinical signs were less severe in mice treated with OV. CONCLUSION: Our data indicate that MYXVΔserp2 treatment benefits mice with ARMS by reducing clinical signs of disease and improving quality of life.

19.
J Cyst Fibros ; 19(5): 801-807, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32624408

RESUMO

BACKGROUND: Individuals with Cystic fibrosis (CF) are the most vulnerable population for pulmonary infection with nontuberculous mycobacteria (NTM). Screening, diagnosis, and assessment of treatment response currently depend on traditional culture techniques, but sputum analysis for NTM in CF is challenging, and associated with a low sensitivity. The cell wall lipoarabinomannan (LAM), a lipoglycan found in all mycobacterial species, and has been validated as a biomarker in urine for active Mycobacterium tuberculosis infection. METHODS: Urine from a CF cohort (n = 44) well-characterized for NTM infection status by airway cultures was analyzed for LAM by gas chromatography/mass spectrometry. All subjects with positive sputum cultures for NTM had varying amounts of LAM in their urine. No LAM was detected in subjects who never had a positive culture (14/45). One individual initially classified as NTM sputum negative subsequently developed NTM disease 657 days after the initial urine LAM testing. Repeat urine LAM testing turned positive, correlating to her positive NTM status. Subjects infected with subspecies of M. abscessus had greater LAM quantities than those infected with M. avium complex (MAC). There was no correlation with disease activity or treatment status and LAM quantity. A TB Capture ELISA using anti-LAM antibodies demonstrated very poor sensitivity in identifying individuals with positive NTM sputum cultures. CONCLUSION: These findings support the conclusion that urine LAM related to NTM infection may be a useful screening test to determine patients at low risk for having a positive NTM sputum culture, as part of a lifetime screening strategy in the CF population.


Assuntos
Fibrose Cística/complicações , Fibrose Cística/urina , Lipopolissacarídeos/urina , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/urina , Adolescente , Adulto , Biomarcadores/urina , Criança , Estudos de Coortes , Fibrose Cística/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Valor Preditivo dos Testes , Escarro/microbiologia , Adulto Jovem
20.
Commun Biol ; 3(1): 518, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948809

RESUMO

Competition between viruses and Wolbachia for host lipids is a proposed mechanism of Wolbachia-mediated virus blocking in insects. Yet, the metabolomic interaction between virus and symbiont within the mosquito has not been clearly defined. We compare the lipid profiles of Aedes aegypti mosquitoes bearing mono- or dual-infections of the Wolbachia wMel strain and dengue virus serotype 3 (DENV3). We found metabolic signatures of infection-induced intracellular events but little evidence to support direct competition between Wolbachia and virus for host lipids. Lipid profiles of dual-infected mosquitoes resemble those of DENV3 mono-infected mosquitoes, suggesting virus-driven modulation dominates over that of Wolbachia. Interestingly, knockdown of key metabolic enzymes suggests cardiolipins are host factors for DENV3 and Wolbachia replication. These findings define the Wolbachia-DENV3 metabolic interaction as indirectly antagonistic, rather than directly competitive, and reveal new research avenues with respect to mosquito × virus interactions at the molecular level.


Assuntos
Aedes/metabolismo , Vírus da Dengue/genética , Metabolismo dos Lipídeos/genética , Wolbachia/genética , Aedes/microbiologia , Aedes/patogenicidade , Aedes/virologia , Animais , Dengue/genética , Dengue/metabolismo , Dengue/microbiologia , Dengue/virologia , Vírus da Dengue/metabolismo , Vírus da Dengue/patogenicidade , Humanos , Insetos Vetores/genética , Insetos Vetores/microbiologia , Insetos Vetores/virologia , Controle Biológico de Vetores , Replicação Viral/genética , Wolbachia/metabolismo , Wolbachia/patogenicidade
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