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1.
Mol Ther Nucleic Acids ; 25: 237-250, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34458008

RESUMO

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

2.
Theranostics ; 11(17): 8430-8447, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34373751

RESUMO

Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate. Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions. Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting. Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.

3.
Dev Comp Immunol ; 121: 104080, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33781781

RESUMO

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9+ cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research.

4.
Front Immunol ; 11: 2009, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903433

RESUMO

Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates the feed of farm animals. Pigs with their monogastric digestive system are in particular sensitive to DON-contaminated feed. At high concentrations, DON causes acute toxic effects, whereas lower concentrations lead to more subtle changes in the metabolism. This applies in particular to the immune system, for which immunosuppressive but also immunostimulatory phenomena have been described. Research in human and rodent cell lines indicates that this may be partially explained by a binding of DON to the ribosome and subsequent influences on cell signaling molecules like mitogen-activated protein kinases. However, a detailed understanding of the influence of DON on functional traits of porcine immune cells is still lacking. In this study, we investigated the influence of DON on transcription factor expression and cytokine production within CD4+, CD8+, and γδ T cells in vitro. At a DON concentration, that already negatively affects proliferation after Concanavalin A stimulation (0.8 µM) an increase of T-bet expression in CD4+ and CD8+ T cells was observed. This increase in T-bet expression coincided with elevated levels of IFN-γ and TNF-α producing T-cell populations. Increases in T-bet expression and cytokine production were found in proliferating and non-proliferating T cells, although increases were more prominent in proliferating cell subsets. Differently, IL-17A production by CD4+ T cells was not influenced by DON. In addition, frequencies of regulatory T cells and their expression of Foxp3 were not affected. In γδ T cells, GATA-3 expression was slightly reduced by DON, whereas T-bet levels were only slightly modulated and hence IFN-γ, TNF-α, or IL-17A production were not affected. Our results show for the single-cell level that DON has the capacity to modulate the expression of transcription factors and related cytokines. In particular, they suggest that for CD4+ and CD8+ T cells, DON can drive T-cell differentiation into a pro-inflammatory type-1 direction, probably depending on the already prevailing cytokine milieu. This could have beneficial or detrimental effects in ongoing immune responses to infection or vaccination.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fator de Transcrição GATA3/metabolismo , Micotoxinas/toxicidade , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/imunologia , Tricotecenos/toxicidade , Ração Animal , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Contaminação de Alimentos , Fusarium , Fator de Transcrição GATA3/genética , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Suínos , Proteínas com Domínio T/genética , Regulação para Cima
5.
J Invest Dermatol ; 140(10): 1985-1993.e5, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32142798

RESUMO

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.


Assuntos
Sistemas CRISPR-Cas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Edição de Genes , Células Cultivadas , Reparo do DNA por Junção de Extremidades , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Queratinócitos/metabolismo , Mutação , Ribonucleoproteínas/farmacologia
6.
Pediatr Infect Dis J ; 39(7): 591-597, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32091501

RESUMO

BACKGROUND: Infants are a target population for new tuberculosis (TB) vaccines. TB incidence estimates are needed to guide the design of trials. To determine the TB incidence and cohort retention among young children using comprehensive diagnostic methods in a high burden area. METHODS: Infants 0-42 days were enrolled. Through 4 monthly follow-up and unscheduled (sick) visits up to the age of 2 years, infants with presumptive TB based on a history of contact, TB symptoms or pre-determined hospitalization criteria were admitted to a case verification ward. Two induced sputa and gastric aspirates were collected for culture and GeneXpert. Mantoux and HIV tests were done. Clinical management was based on the Keith Edwards score. Cases were classified into microbiologically confirmed or radiologic, diagnosed by blinded expert assessment. Cox regression was used to identify risk factors for incident TB and study retention. RESULTS: Of 2900 infants enrolled, 927 (32%) developed presumptive TB, 737/927 (80%) were investigated. Sixty-nine TB cases were diagnosed (bacteriologic and radiologic). All TB incidence was 2/100 person-years of observation (pyo) (95% CI: 1.65-2.65). Nine were bacteriologic cases, incidence 0.3/100 pyo. The radiologic TB incidence was 1.82/100 pyo. Bacteriologic TB was associated with infant HIV infection, higher Keith Edwards scores. Completeness of 4-month vaccinations and HIV infection were positively associated with retention. CONCLUSIONS: TB incidence was high. An all TB endpoint would require a sample size of a few thousand children, but tens of thousands, when limited to bacteriologic TB.


Assuntos
Programas de Rastreamento , Tuberculose/epidemiologia , Estudos de Coortes , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Quênia/epidemiologia , Masculino , Modelos de Riscos Proporcionais , Fatores de Risco , Escarro/microbiologia , Teste Tuberculínico , Tuberculose/diagnóstico , Tuberculose/microbiologia
7.
PLoS One ; 15(1): e0225710, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31917797

RESUMO

OBJECTIVE: To better understand factors contributing to underutilization of laboratory services for health care delivery in sub-Saharan Africa, we conducted a study in Senegalese Antenatal Care clinics (ANC) and laboratories to determine the extent of underutilization, contributing factors, and bottlenecks in the cascade of care from first ANC visit, test uptake, to availability of test results and appropriate clinical management. METHODS: At 16 health facilities, pregnant women attending for their first ANC visit were consecutively recruited and information was prospectively collected on the request, execution, results and clinical management of seven nationally recommended laboratory screening tests for normal pregnancy: hemoglobin concentration (Hb), syphilis serology, HIV serology, determination of proteinuria (PU), determination of blood group and Rhesus factor, Emmel test to detect sickle cell disease, and glycaemia. Health facility staff were interviewed on human resource capacity, management of the ANC and the laboratory, and availability and use of guidelines. RESULTS: Of 1246 ANC attendants, 400 (32%) had complete results. Completeness varied between facilities from 0-99%. In multilevel logistic regression analysis of women nested in facilities, complete uptake was lower if women started ANC later in pregnancy; very low in rural ANC attendants who ever delivered compared to urban primigravidae (OR 0.064; 95%CI 0.00-0.52); and higher if the facility routinely recommended all seven tests. In the cascade from test request to clinical management, the most frequent bottleneck was non-execution of requested tests, while unavailability of results for executed test was uncommon (<2%). Overall, of 525 abnormal test results 97(18%) had a record of adequate clinical management. CONCLUSION: Our study illustrates challenges to test uptake even when laboratory testing capacity is in place, with large differences between facilities, and underscores the importance of management, policy, and the importance of considering local context in order to improve service delivery to expectant mothers.


Assuntos
Técnicas de Laboratório Clínico/estatística & dados numéricos , Cuidado Pré-Natal/estatística & dados numéricos , Adolescente , Adulto , Feminino , Acesso aos Serviços de Saúde/estatística & dados numéricos , Humanos , Masculino , Gravidez , Senegal , Adulto Jovem
8.
BMC Infect Dis ; 19(1): 682, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375068

RESUMO

BACKGROUND: Adolescents are a prime target group for tuberculosis (TB) vaccine trials that include prevention of infection (POI). The BCG vaccine is given at birth and does not prevent TB infection. TB infection, a critical endpoint for POI vaccine trials would need to be documented to estimate sample sizes in target populations. METHODS: Adolescents aged 12-18 years of age were enrolled in an area under continuous demographic surveillance. A tuberculin skin test (TST) survey was conducted as part of a study on TB prevalence and incidence. All adolescents got TSTs at enrolment and returned after 72 h for reading. A TST of ≥10 mm if HIV negative or ≥ 5 mm if HIV positive, was considered positive. RESULTS: Of 4808 adolescents returning for TST readings (96% of those enrolled), mean age was 14.4 (SD 1.9), 4518(94%) were enrolled in school and 21(0.4%) gave a previous history of tuberculosis. Among adolescents with TST reactivity, the mean TST induration was 13.2 mm (SD 5.4). The overall prevalence of latent TB infection was 1544/4808 (32.1, 95% CI 29.2-35.1) with a corresponding annual risk of TB infection (ARTI) of 2.6% (95% CI 2.2-3.1). Risk factors for a positive TST included being male (OR 1.3, 95% CI 1.2,1.5), history of having a household TB contact (OR 1.5, 95% CI 1.2,1.8), having a BCG scar (OR 1.5,95% CI 1.2,1.8), living in a rural area (OR 1.4, 95% CI 1.1,1.9), and being out of school (OR 1.8, 95% CI 1.4,2.3). CONCLUSION: We conclude that the high TB transmission rates we found in this study, suggest that adolescents in this region may be an appropriate target group for TB vaccine trials including TB vaccine trials aiming to prevent infection.


Assuntos
Tuberculose/epidemiologia , Adolescente , Vacina BCG/uso terapêutico , Criança , Feminino , Humanos , Quênia/epidemiologia , Tuberculose Latente/diagnóstico , Tuberculose Latente/epidemiologia , Masculino , Mycobacterium tuberculosis/patogenicidade , Prevalência , Fatores de Risco , Instituições Acadêmicas , Teste Tuberculínico , Tuberculose/diagnóstico
9.
Pediatr Infect Dis J ; 37(1): 10-15, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28719498

RESUMO

SETTING: Siaya County, with the highest tuberculosis notification rates in Kenya. OBJECTIVES: To determine the incidence of active tuberculosis and 1-year cohort retention in 12-18-year-old adolescents, in preparation for phase III tuberculosis vaccine trials. METHODS: Adolescents were enrolled and followed up for 1-2 years to determine tuberculosis incidence. Adolescents with a positive tuberculin skin test, history of cohabitation with a tuberculosis case or at least 1 tuberculosis symptom received clinical and sputum examination and a chest radiograph. Definite tuberculosis cases were bacteriologically confirmed and clinical cases diagnosed by a clinician based on a suggestive chest radiograph and having clinical symptoms. Risk factors were explored using Poisson regression. RESULTS: Among 4934 adolescents without tuberculosis at baseline, 26 tuberculosis cases were identified during follow-up with a corresponding incidence density of 4.4 [95% confidence interval (CI): 3.0-6.4] events per 1000 person-years of observation, 12 definite tuberculosis cases; incidence density of 2.0 (95% CI: 0.9-3.1). Having previous tuberculosis (rate ratio: 12.5; CI: 1.8-100) and presence of tuberculin skin test conversion (rate ratio: 3.4; CI: 1.5-7.7) were significantly associated with higher risk of tuberculosis. Overall (4086/4925), 83.0% of adolescents were retained in the study after 1 year of follow-up. Being female, older, out of school and being orphaned were significant risk factors for loss to follow-up. CONCLUSION: The tuberculosis incidence in adolescents will help inform future tuberculosis vaccine trial sample size calculations for this setting. The predictive factors for tuberculosis and retention can be further explored in future trials.


Assuntos
Projetos de Pesquisa Epidemiológica , Tuberculose/epidemiologia , Adolescente , Criança , Feminino , Seguimentos , Humanos , Incidência , Quênia/epidemiologia , Perda de Seguimento , Masculino , Fatores de Risco , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose
10.
PLoS One ; 10(5): e0126065, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25996389

RESUMO

BACKGROUND: Xpert MTB/RIF, the first automated molecular test for tuberculosis, is transforming the diagnostic landscape in high-burden settings. This study assessed the impact of up-front Xpert MTB/RIF testing on detection of pulmonary tuberculosis (PTB) and rifampicin-resistant PTB (DR-TB) cases in India. METHODS: This demonstration study was implemented in 18 sub-district level TB programme units (TUs) in India in diverse geographic and demographic settings covering a population of 8.8 million. A baseline phase in 14 TUs captured programmatic baseline data, and an intervention phase in 18 TUs had Xpert MTB/RIF offered to all presumptive TB patients. We estimated changes in detection of TB and DR-TB, the former using binomial regression models to adjust for clustering and covariates. RESULTS: In the 14 study TUs, which participated in both phases, 10,675 and 70,556 presumptive TB patients were enrolled in the baseline and intervention phase, respectively, and 1,532 (14.4%) and 14,299 (20.3%) bacteriologically confirmed PTB cases were detected. The implementation of Xpert MTB/RIF was associated with increases in both notification rates of bacteriologically confirmed TB cases (adjusted incidence rate ratio [aIRR] 1.39; CI 1.18-1.64), and proportion of bacteriological confirmed TB cases among presumptive TB cases (adjusted risk ratio (aRR) 1.33; CI 1.6-1.52). Compared with the baseline strategy of selective drug-susceptibility testing only for PTB cases at high risk of drug-resistant TB, Xpert MTB/RIF implementation increased rifampicin resistant TB case detection by over fivefold. Among, 2765 rifampicin resistance cases detected, 1055 were retested with conventional drug susceptibility testing (DST). Positive predictive value (PPV) of rifampicin resistance detected by Xpert MTB/RIF was 94.7% (CI 91.3-98.1), in comparison to conventional DST. CONCLUSION: Introduction of Xpert MTB/RIF as initial diagnostic test for TB in public health facilities significantly increased case-notification rates of all bacteriologically confirmed TB by 39% and rifampicin-resistant TB case notification by fivefold.


Assuntos
Técnicas de Diagnóstico Molecular , Vigilância em Saúde Pública , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/epidemiologia , Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Feminino , Geografia Médica , Humanos , Índia/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico
11.
Int J Infect Dis ; 35: 11-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25770911

RESUMO

OBJECTIVE: The aim of this study was to determine the prevalence of tuberculosis (TB) in adolescents in western Kenya. METHODS: A cohort study of 5004 adolescents aged 12-18 years was conducted. Adolescents were screened for prevalent TB using clinical criteria, history of TB contact, and a Mantoux test. Cases of suspected TB were investigated through two sputum examinations (microscopy and liquid culture) and chest radiography. RESULTS: Out of 5004 adolescents enrolled, 1960 (39.2%) were identified with suspected TB, including 1544 with a positive Mantoux (prevalence 1544/4808, 32.1%), 515 with symptoms suggestive of TB (10.3%), and 144 (2.9%) with household TB contact. Sixteen culture-confirmed (definite) and 18 probable pulmonary TB (PTB) cases were identified, reflecting a prevalence estimate of 3.2/1000 (definite) and 6.8/1000 all PTB, respectively. Only one smear-positive case was detected. The case notification rate among 12-18-year-old adolescents for all TB was 101/100000, yielding a patient diagnostic rate of 0.13 (95% confidence interval 0.03-3.7) cases detected per person-year for all TB. CONCLUSION: The prevalence of PTB among adolescents is high, with the majority of cases not detected routinely. Innovative active case finding including the wider use of Xpert MTB/RIF is needed to detect smear-negative TB among adolescents.


Assuntos
Tuberculose Pulmonar/epidemiologia , Adolescente , Criança , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Controle de Infecções , Quênia/epidemiologia , Masculino , Prevalência , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/prevenção & controle
12.
BMC Infect Dis ; 14: 532, 2014 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-25326816

RESUMO

BACKGROUND: To inform the choice of an appropriate screening and diagnostic algorithm for tuberculosis (TB) screening initiatives in different epidemiological settings, we compare algorithms composed of currently available methods. METHODS: Of twelve algorithms composed of screening for symptoms (prolonged cough or any TB symptom) and/or chest radiography abnormalities, and either sputum-smear microscopy (SSM) or Xpert MTB/RIF (XP) as confirmatory test we model algorithm outcomes and summarize the yield, number needed to screen (NNS) and positive predictive value (PPV) for different levels of TB prevalence. RESULTS: Screening for prolonged cough has low yield, 22% if confirmatory testing is by SSM and 32% if XP, and a high NNS, exceeding 1000 if TB prevalence is ≤0.5%. Due to low specificity the PPV of screening for any TB symptom followed by SSM is less than 50%, even if TB prevalence is 2%. CXR screening for TB abnormalities followed by XP has the highest case detection (87%) and lowest NNS, but is resource intensive. CXR as a second screen for symptom screen positives improves efficiency. CONCLUSIONS: The ideal algorithm does not exist. The choice will be setting specific, for which this study provides guidance. Generally an algorithm composed of CXR screening followed by confirmatory testing with XP can achieve the lowest NNS and highest PPV, and is the least amenable to setting-specific variation. However resource requirements for tests and equipment may be prohibitive in some settings and a reason to opt for symptom screening and SSM. To better inform disease control programs we need empirical data to confirm the modeled yield, cost-effectiveness studies, transmission models and a better screening test.


Assuntos
Tuberculose Pulmonar/diagnóstico , Efeitos Psicossociais da Doença , Humanos , Programas de Rastreamento/métodos , Sensibilidade e Especificidade
13.
PLoS One ; 8(12): e82786, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24367555

RESUMO

BACKGROUND: High costs are a limitation to scaling up the Xpert MTB/RIF assay (Xpert) for the diagnosis of tuberculosis in resource-constrained settings. A triaging strategy in which a sensitive but not necessarily highly specific rapid test is used to select patients for Xpert may result in a more affordable diagnostic algorithm. To inform the selection and development of particular diagnostics as a triage test we explored combinations of sensitivity, specificity and cost at which a hypothetical triage test will improve affordability of the Xpert assay. METHODS: In a decision analytical model parameterized for Uganda, India and South Africa, we compared a diagnostic algorithm in which a cohort of patients with presumptive TB received Xpert to a triage algorithm whereby only those with a positive triage test were tested by Xpert. FINDINGS: A triage test with sensitivity equal to Xpert, 75% specificity, and costs of US$5 per patient tested reduced total diagnostic costs by 42% in the Uganda setting, and by 34% and 39% respectively in the India and South Africa settings. When exploring triage algorithms with lower sensitivity, the use of an example triage test with 95% sensitivity relative to Xpert, 75% specificity and test costs $5 resulted in similar cost reduction, and was cost-effective by the WHO willingness-to-pay threshold compared to Xpert for all in Uganda, but not in India and South Africa. The gain in affordability of the examined triage algorithms increased with decreasing prevalence of tuberculosis among the cohort. CONCLUSIONS: A triage test strategy could potentially improve the affordability of Xpert for TB diagnosis, particularly in low-income countries and with enhanced case-finding. Tests and markers with lower accuracy than desired of a diagnostic test may fall within the ranges of sensitivity, specificity and cost required for triage tests and be developed as such.


Assuntos
Triagem/economia , Tuberculose/diagnóstico , Algoritmos , Análise Custo-Benefício , Técnicas de Apoio para a Decisão , Humanos , Índia , África do Sul , Uganda
14.
PLoS One ; 8(4): e61162, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23634206

RESUMO

BACKGROUND: The findings of a prevalence survey conducted in western Kenya, in a population with 14.9% HIV prevalence suggested inadequate case finding. We found a high burden of infectious and largely undiagnosed pulmonary tuberculosis (PTB), that a quarter of the prevalent cases had not yet sought care, and a low case detection rate. OBJECTIVE AND METHODS: We aimed to identify factors associated with inadequate case finding among adults with PTB in this population by comparing characteristics of 194 PTB patients diagnosed in a health facility after self-report, i.e., through passive case detection, with 88 patients identified through active case detection during the prevalence survey. We examined associations between method of case detection and patient characteristics, including HIV-status, socio-demographic variables and disease severity in univariable and multivariable logistic regression analyses. FINDINGS: HIV-infection was associated with faster passive case detection in univariable analysis (crude OR 3.5, 95% confidence interval (CI) 2.0-5.9), but in multivariable logistic regression this was largely explained by the presence of cough, illness and clinically diagnosed smear-negative TB (adjusted OR (aOR) HIV 1.8, 95% CI 0.85-3.7). Among the HIV-uninfected passive case detection was less successful in older patients aOR 0.76, 95%CI 0.60-0.97 per 10 years increase), and women (aOR 0.27, 95%CI 0.10-0.73). Reported current or past alcohol use reduced passive case detection in both groups (0.42, 95% CI 0.23-0.79). Among smear-positive patients median durations of cough were 4.0 and 6.9 months in HIV-infected and uninfected patients, respectively. CONCLUSION: HIV-uninfected patients with infectious TB who were older, female, relatively less ill, or had a cough of a shorter duration were less likely found through passive case detection. In addition to intensified case finding in HIV-infected persons, increasing the suspicion of TB among HIV-uninfected women and the elderly are needed to improve TB case detection in Kenya.


Assuntos
Programas de Rastreamento/métodos , População Rural/estatística & dados numéricos , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/epidemiologia , Adolescente , Adulto , Coleta de Dados , Reações Falso-Negativas , Feminino , Infecções por HIV/complicações , Pessoal de Saúde/estatística & dados numéricos , Humanos , Quênia/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Tuberculose Pulmonar/complicações , Adulto Jovem
15.
PLoS One ; 7(7): e38691, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22792158

RESUMO

BACKGROUND: We conducted a tuberculosis (TB) prevalence survey and evaluated the screening methods used in our survey, to assess if screening in TB prevalence surveys could be simplified, and to assess the accuracy of screening algorithms that may be applicable for active case finding. METHODS: All participants with a positive screen on either a symptom questionnaire, chest radiography (CXR) and/or sputum smear microscopy submitted sputum for culture. HIV status was obtained from prevalent cases. We estimated the accuracy of modified screening strategies with bacteriologically confirmed TB as the gold standard, and compared these with other survey reports. We also assessed whether sequential rather than parallel application of symptom, CXR and HIV screening would substantially reduce the number of participants requiring CXR and/or sputum culture. RESULTS: Presence of any abnormality on CXR had 94% (95%CI 88-98) sensitivity (92% in HIV-infected and 100% in HIV-uninfected) and 73% (95%CI 68-77) specificity. Symptom screening combinations had significantly lower sensitivity than CXR except for 'any TB symptom' which had 90% (95%CI 84-95) sensitivity (96% in HIV-infected and 82% in HIV-uninfected) and 32% (95%CI 30-34) specificity. Smear microscopy did not yield additional suspects, thus the combined symptom/CXR screen applied in the survey had 100% (95%CI 97-100) sensitivity. Specificity was 65% (95%CI 61-68). Sequential application of first a symptom screen for 'any symptom', followed by CXR-evaluation and different suspect criteria depending on HIV status would result in the largest reduction of the need for CXR and sputum culture, approximately 36%, but would underestimate prevalence by 11%. CONCLUSION: CXR screening alone had higher accuracy compared to symptom screening alone. Combined CXR and symptom screening had the highest sensitivity and remains important for suspect identification in TB prevalence surveys in settings where bacteriological sputum examination of all participants is not feasible.


Assuntos
Programas de Rastreamento , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Coinfecção , Infecções por HIV/diagnóstico , Humanos , Quênia , Radiografia Pulmonar de Massa , Prevalência , Sensibilidade e Especificidade , Inquéritos e Questionários , Tuberculose/prevenção & controle
16.
BMC Public Health ; 11: 515, 2011 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-21714895

RESUMO

BACKGROUND: The two issues mostly affecting the success of tuberculosis (TB) control programmes are delay in presentation and non-adherence to treatment. It is important to understand the factors that contribute to these issues, particularly in resource limited settings, where rates of tuberculosis are high. The objective of this study is to assess health-seeking behaviour and health care experiences among persons with pulmonary tuberculosis, and identify the reasons patients might not complete their treatment. METHODS: We performed qualitative one-on-one in-depth interviews with pulmonary tuberculosis patients in nine health facilities in rural western Kenya. Thirty-one patients, 18 women and 13 men, participated in the study. All reside in an area of western Kenya with a Health and Demographic Surveillance System (HDSS). They had attended treatment for up to 4 weeks on scheduled TB clinic days in September and October 2005.The nine sites all provide diagnostic and treatment services. Eight of the facilities were public (3 hospitals and 5 health centres) and one was a mission health centre. RESULTS: Most patients initially self-treated with herbal remedies or drugs purchased from kiosks or pharmacies before seeking professional care. The reported time from initial symptoms to TB diagnosis ranged from 3 weeks to 9 years. Misinterpretation of early symptoms and financial constraints were the most common reasons reported for the delay.We also explored potential reasons that patients might discontinue their treatment before completing it. Reasons included being unaware of the duration of TB treatment, stopping treatment once symptoms subsided, and lack of family support. CONCLUSIONS: This qualitative study highlighted important challenges to TB control in rural western Kenya, and provided useful information that was further validated in a quantitative study in the same area.


Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Aceitação pelo Paciente de Cuidados de Saúde , Cooperação do Paciente , Tuberculose/tratamento farmacológico , Adolescente , Adulto , Feminino , Humanos , Entrevistas como Assunto , Quênia , Masculino , Pessoa de Meia-Idade , População Rural , Adulto Jovem
17.
Am J Respir Crit Care Med ; 183(9): 1245-53, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21239690

RESUMO

RATIONALE: Limited information exists on the prevalence of tuberculosis and adequacy of case finding in African populations with high rates of HIV. OBJECTIVES: To estimate the prevalence of bacteriologically confirmed pulmonary tuberculosis (PTB) and the fraction attributable to HIV, and to evaluate case detection. METHODS: Residents aged 15 years and older, from 40 randomly sampled clusters, provided two sputum samples for microscopy; those with chest radiograph abnormalities or symptoms suggestive of PTB provided one additional sputum sample for culture. MEASUREMENTS AND MAIN RESULTS: PTB was defined by a culture positive for Mycobacterium tuberculosis or two positive smears. Persons with PTB were offered HIV testing and interviewed on care-seeking behavior. We estimated the population-attributable fraction of HIV on prevalent and notified PTB, the patient diagnostic rate, and case detection rate using provincial TB notification data. Among 20,566 participants, 123 had PTB. TB prevalence was 6.0/1,000 (95% confidence interval, 4.6-7.4) for all PTB and 2.5/1,000 (1.6-3.4) for smear-positive PTB. Of 101 prevalent TB cases tested, 52 (51%) were HIV infected, and 58 (64%) of 91 cases who were not on treatment and were interviewed had not sought care. Forty-eight percent of prevalent and 65% of notified PTB cases were attributable to HIV. For smear-positive and smear-negative PTB combined, the patient diagnostic rate was 1.4 cases detected per person-year among HIV-infected persons having PTB and 0.6 for those who were HIV uninfected, corresponding to case detection rates of 56 and 65%, respectively. CONCLUSIONS: Undiagnosed PTB is common in this community. TB case finding needs improvement, for instance through intensified case finding with mobile smear microscopy services, rigorous HIV testing, and improved diagnosis of smear-negative TB.


Assuntos
Tuberculose Pulmonar/epidemiologia , Adolescente , Adulto , Causalidade , Análise por Conglomerados , Comorbidade , Feminino , Infecções por HIV/epidemiologia , Humanos , Quênia/epidemiologia , Masculino , Mycobacterium tuberculosis , Prevalência , População Rural/estatística & dados numéricos , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Adulto Jovem
18.
J Med Microbiol ; 57(Pt 3): 304-309, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18287292

RESUMO

The aim of this study was to compare conventional 16S rRNA gene PCR, real-time 16S rRNA gene PCR and real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR as detection methods for M. genitalium infection. The study also determined the prevalence of M. genitalium in male and female patients attending a sexually transmitted infections clinic in a rural area in the west of Sweden. First void urine (FVU) and/or urethral swabs were collected from 381 men, and FVU and/or cervical swabs and/or urethral swabs were collected from 298 women. A total of 213 specimens were used in the PCR comparative study: 98 consecutively sampled specimens from patients enrolled in the prevalence study, 36 consecutively sampled specimens from patients with symptoms of urethritis and 79 specimens from patients positive for M. genitalium by real-time MgPa gene PCR in the prevalence study. A true-positive M. genitalium DNA specimen was defined as either a specimen positive in any two PCR assays or a specimen whose PCR product was verified by DNA sequencing. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %), respectively. In the PCR comparative study, M. genitalium DNA was detected in 61/76 (80.3 %) of true-positive specimens by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Real-time MgPa gene PCR thus had higher sensitivity compared with conventional 16S rRNA gene PCR and had considerably increased sensitivity compared with real-time 16S rRNA gene PCR for detection of M. genitalium DNA. Real-time MgPa gene PCR is well suited for the clinical diagnosis of M. genitalium.


Assuntos
Colo do Útero/microbiologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Uretra/microbiologia , Urina/microbiologia , Adesinas Bacterianas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/classificação , Mycoplasma genitalium/genética , Prevalência , RNA Ribossômico 16S/genética , População Rural , Manejo de Espécimes/métodos , Suécia/epidemiologia
19.
J Acquir Immune Defic Syndr ; 40(3): 344-9, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16249710

RESUMO

OBJECTIVES: To improve uptake in a program to prevent mother-to-child HIV transmission and describe lessons relevant for prevention of mother-to-child transmission programs in resource-poor settings. METHODS: Implementation of a pilot project that evaluates approaches to increase program uptake at health facility level at New Nyanza Provincial General Hospital, a public hospital in western Kenya, an area with high HIV prevalence. Client flow was revised to integrate counseling, HIV testing, and dispensing of single-dose nevirapine into routine antenatal services. The number of facilities providing PMCT services was expanded to increase district-wide coverage. Main outcome measures were uptake of counseling, HIV testing, nevirapine, and estimated program impact. RESULTS: Uptake of counseling and testing improved from 55 to 68% (P < 0.001), nevirapine uptake from 57% to 70% (P < 0.001), and estimated program impact from 15% to 23% (P = 0.03). Aggregate reports compare well with computer-entered data. CONCLUSION: Addressing institutional factors can improve uptake, but expected program impact remains low for several reasons, including relatively low efficacy of the intervention and missed opportunities in the labor room.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Países em Desenvolvimento , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Hospitais Públicos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Nevirapina/uso terapêutico , Complicações Infecciosas na Gravidez/prevenção & controle , Avaliação de Programas e Projetos de Saúde , Aconselhamento , Feminino , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Hospitais Gerais , Humanos , Quênia , Projetos Piloto , Gravidez
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