RESUMO
High-throughput centralized testing for tuberculosis (TB) and drug resistance is important, but comparative data are limited. In this retrospective cross-sectional study, participants were recruited from Johannesburg, South Africa, and Tbilisi, Georgia. The index tests, Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), were performed on specimens stored frozen for an extended period of time (beyond manufacturer-validated specifications) and compared to paired Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF (Xpert) results obtained with fresh specimens. The detection reference standard was the Mycobacterium tuberculosis complex culture, and for resistance detection, it was phenotypic drug susceptibility testing. The median age of 474 participants was 39 (interquartile range [IQR], 31 to 51) years. On decontaminated sputum, Xpert Ultra had a sensitivity of 91%, compared to 77% for RT-MTB, with a difference of +14% (95% confidence interval [CI], +9.2 to +21%; 18/127). On raw sputum, Xpert Ultra exhibited a sensitivity of 89% and Xpert one of 88%, compared to 80% for RT-MTB, exhibiting differences of +10% (95% CI, +3.3 to +18%; 9/93) and +8.6% (95% CI, +2.4 to +17%; 8/93), respectively. Specificity was ≥98% for all tests. All three tests showed high sensitivity and specificity for detection of rifampin resistance. Abbott assays may have lower sensitivity than Xpert and Xpert Ultra for TB detection but similar performance for detection of resistance. The differences in TB detection may be attributable to differences in testing of frozen (Abbott) versus fresh (Xpert) samples. Studies in compliance with manufacturer's instructions are required to compare performance. IMPORTANCE In 2019, 10 million people fell ill with tuberculosis (TB), of whom 1.4 million died. There are few comparative studies of diagnostic assays, particularly those aiming to be used in high-throughput laboratories. One such assay is the Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), which uses the m2000 platform already in use in many settings for HIV load testing and allows the diagnosis of TB and resistance to two first-line drugs, rifampin and isoniazid. Our study compared the RT-MTB and RT-MTB RIF/INH to the WHO-recommended Xpert MTB/RIF Ultra and Xpert MTB/RIF. The study is the largest comparative study to date and was performed independent of the manufacturer. The study results suggest that the Abbott RealTime MTB may have a lower sensitivity, but the study may have placed the Abbott test at a disadvantage by using frozen samples and comparing the results to those for fresh samples for the Xpert.
Assuntos
Antituberculosos/farmacologia , Testes Diagnósticos de Rotina/métodos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/farmacologia , Tuberculose Pulmonar/diagnóstico , Adulto , Estudos Transversais , Farmacorresistência Bacteriana , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Estudos Retrospectivos , África do Sul , Escarro/microbiologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
BACKGROUND: In 2004, Ontario delisted routine eye examinations for people aged 20-64 years, potentially encouraging patients seeking eye care to visit government-insured primary care providers (PCPs) rather than optometrists whose services had been deinsured. We investigated if utilization of PCP services for nonrefractive eye conditions increased after 2004 among Ontarians who were affected by the delisting. METHODS: We conducted a comparative analysis of the utilization of PCP services for nonrefractive eye conditions in Ontario using administrative data from 2000 to 2014. We included participants without a visit to government-insured optometrists or ophthalmologists in the year before the study year; we excluded participants with existing diabetes. Changes in utilization before and after delisting were statistically assessed using segmented regression analysis in subgroups stratified by age, sex, rurality and neighbourhood income. RESULTS: A significant increase in utilization of PCP services for nonrefractive ocular diagnoses after 2004 was documented among people affected by the delisting: 17.8% (95% confidence interval [CI] 17.0% to 18.7%) for people aged 20-39 years and 11.6% (95% CI 10.6% to 12.5%) for people aged 40-64 years. This corresponds to an increase in the number of patients who visited PCPs for nonrefractive ocular diagnoses of 10 690 (95% CI 321 to 21 059) for people aged 20-39 years and 20 682 (95% CI -94 to 41 457) for people aged 40-64 years. Among people aged 65 years and older (an age group not affected by the delisting), utilization of PCP services for nonrefractive ocular diagnoses was stable (p = 0.95) throughout the study period. Changes in utilization of PCP services for nonocular diagnoses were nonsignificant among people aged 0-19, 40-64 and 65 years and older. INTERPRETATION: After delisting, utilization of the services of government-funded PCPs for nonrefractive ocular diagnoses significantly increased among Ontarians affected by the delisting. The impact on ocular outcomes and the cost-effectiveness of increased use of PCPs for ocular management warrants further investigation and policy-makers' consideration.
Assuntos
Testes Diagnósticos de Rotina/métodos , Oftalmopatias/diagnóstico , Optometria/métodos , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Atenção Primária à Saúde , Testes Visuais/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Oftalmopatias/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Cobertura do Seguro , Masculino , Pessoa de Meia-Idade , Programas Nacionais de Saúde , Ontário/epidemiologia , Oftalmologistas , Optometristas , Adulto JovemAssuntos
Alérgenos/imunologia , Testes Diagnósticos de Rotina/estatística & dados numéricos , Testes Diagnósticos de Rotina/normas , Mau Uso de Serviços de Saúde/estatística & dados numéricos , Imunoensaio/estatística & dados numéricos , Imunoensaio/normas , Revisão da Utilização de Seguros , Estudos Transversais , Testes Diagnósticos de Rotina/métodos , Humanos , Imunoensaio/métodos , Imunoglobulina E/imunologia , Programas Nacionais de Saúde , Washington/epidemiologiaRESUMO
BACKGROUND: Cardiomyopathic manifestations induced by continuous blood transfusion are the leading cause of death among patients with thalassemia major (TM). Despite introduction of chelation therapy, heart failure after cardiomyopathic manifestations is still a major threat to patients. METHODS: We performed a search of relevant English-language literature, retrieving publications from the PubMed database and the Google Scholar search engine (2005-2018). We used "thalassemia major", "cardiomyopathy", "iron overload", "cardiac magnetic resonance T2" "chelation therapy", and "iron burden" as keywords. RESULTS: The results of the studies we found suggest that cardiac hepcidin is a major regulator of iron homeostasis in cardiac tissue. Unlike previous assumptions, the heart appears to have a limited regeneration capability, originating from a small population of hypoxic cardiomyocytes. CONCLUSIONS: Oxygen levels determine cardiomyocyte gene-expression patterns. Upregulation of cardiac hepcidin in hypoxia preserves cardiomyocytes from forming out of reactive oxygen species catalyzed by free cellular iron in cardiomyocytes. Using the limited regeneration capacity of cardiac cells and gaining further understanding of the cellular aspects of cardiomyopathic manifestations may help health care professionals to develop new therapeutic strategies.
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Cardiomiopatias/diagnóstico , Cardiomiopatias/terapia , Terapia por Quelação/métodos , Testes Diagnósticos de Rotina/métodos , Gerenciamento Clínico , Sobrecarga de Ferro/complicações , Talassemia/complicações , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Humanos , Talassemia/terapiaRESUMO
BACKGROUND: There is an increased interest in using digital refractometers to indirectly assess colostrum quality of dairy cattle, but knowledge on diagnostic accuracy for Norwegian Red dairy cows is lacking. Recent research has indicated a profound variability in the colostrum quality among dairy cows and herds in Norway. The aim of this study was to evaluate the diagnostic test sensitivity and specificity of a digital refractometer (Brix refractometer) at different cut-offs in Brix% for detection of colostrum of high quality (> 50 g/L) defined by the gold standard single radial immunodiffusion (IgG g/L). Furthermore, we aimed to identify possible associations between selected herd and cow-level management factors and colostrum IgG-levels in Norwegian Red dairy cows. RESULTS: Median colostrum IgG level across 167 cows from 19 herds was 35.0 g/L, ranging from 5 to 129 g/L. Mean Brix% (± SD) was 19.7 ± 4.12%, ranging from 10.1 to 30.5. Most samples (72.5%) had inferior quality as compared to the international standard of 50 g/L. Brix% and IgG in colostrum were strongly correlated (r = 0.71, P < 0.001). A Brix cut-off of 22%, which is currently recommended, yielded a sensitivity of (95% CI) 69.4% (54.6-81.7) and a specificity of 83.1% (75.0-89.3) for identifying colostrum with high quality (> 50 g/L). The only factor found to be associated with low colostrum quality was parity. Specifically, cows in the second parity were found to produce colostrum with low quality compared to cows in parities four and later. CONCLUSIONS: The agreement between colostrum IgG and Brix% is good. However, the diagnostic test evaluation indicates suboptimal performance in identifying high vs. low colostrum quality in this population, possibly related to a high proportion of the samples with < 50 g/L IgG. The only factor found to be associated with low colostrum quality was parity. Specifically, cows in the second parity were found to produce colostrum with lower quality. Future research should investigate colostrum and serum IgG levels which best prevent calf illness under Norwegian conditions.
Assuntos
Colostro/fisiologia , Indústria de Laticínios , Testes Diagnósticos de Rotina/veterinária , Imunoglobulina G/análise , Refratometria/veterinária , Animais , Bovinos , Indústria de Laticínios/métodos , Testes Diagnósticos de Rotina/métodos , Feminino , Refratometria/instrumentação , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Preventive chemotherapy (PC) with benzimidazole drugs is the backbone of soil-transmitted helminth (STH) control programs. Over the past decade, drug coverage has increased and with it, the possibility of developing anthelmintic resistance. It is therefore of utmost importance to monitor drug efficacy. Currently, a variety of novel diagnostic methods are available, but it remains unclear whether they can be used to monitor drug efficacy. In this study, we compared the efficacy of albendazole (ALB) measured by different diagnostic methods in a head-to-head comparison to the recommended single Kato-Katz. METHODS: An ALB efficacy trial was performed in 3 different STH-endemic countries (Ethiopia, Lao PDR and Tanzania), each with a different PC-history. During these trials, stool samples were evaluated with Kato-Katz (single and duplicate), Mini-FLOTAC, FECPAKG2, and qPCR. The reduction rate in mean eggs per gram of stool (ERR) and mean genome equivalents / ml of DNA extract (GERR) were calculated to estimate drug efficacy. PRINCIPAL FINDINGS AND CONCLUSIONS: The results of the efficacy trials showed that none of the evaluated diagnostic methods could provide reduction rates that were equivalent to a single Kato-Katz for all STH. However, despite differences in clinical sensitivity and egg counts, they agreed in classifying efficacy according to World Health Organization (WHO) guidelines. This demonstrates that diagnostic methods for assessing drug efficacy should be validated with their intended-use in mind and that other factors like user-friendliness and costs will likely be important factors in driving the choice of diagnostics. In addition, ALB efficacy against STH infections was lower in sites with a longer history of PC. Yet, further research is needed to identify factors that contribute to this finding and to verify whether reduced efficacy can be associated with mutations in the ß-tubulin gene that have previously been linked to anthelmintic resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.
Assuntos
Albendazol/uso terapêutico , Anti-Helmínticos/uso terapêutico , Helmintíase/diagnóstico , Helmintíase/tratamento farmacológico , Solo/parasitologia , Administração Oral , Albendazol/administração & dosagem , Animais , Brasil , Criança , Testes Diagnósticos de Rotina/métodos , Etiópia , Fezes/parasitologia , Feminino , Helmintos/genética , Humanos , Laos , Masculino , Contagem de Ovos de Parasitas/métodos , Sensibilidade e Especificidade , Tanzânia , Tubulina (Proteína)/genética , Organização Mundial da SaúdeRESUMO
INTRODUCTION: Jaundice caused by hyperbilirubinaemia is a physiological phenomenon in the neonatal period. However, severe hyperbilirubinaemia, when left untreated, may cause kernicterus, a severe condition resulting in lifelong neurological disabilities. Although commonly applied, visual inspection is ineffective in identifying severe hyperbilirubinaemia. We aim to investigate whether among babies cared for in primary care: (1) transcutaneous bilirubin (TcB) screening can help reduce severe hyperbilirubinaemia and (2) primary care-based (versus hospital-based) phototherapy can help reduce hospital admissions. METHODS AND ANALYSIS: A factorial stepped-wedge cluster randomised controlled trial will be conducted in seven Dutch primary care birth centres (PCBC). Neonates born after 35 weeks of gestation and cared for at a participating PCBC for at least 2 days within the first week of life are eligible, provided they have not received phototherapy before. According to the stepped-wedge design, following a phase of 'usual care' (visual assessment and selective total serum bilirubin (TSB) quantification), either daily TcB measurement or, if indicated, phototherapy in the PCBC will be implemented (phase II). In phase III, both interventions will be evaluated in each PCBC. We aim to include 5500 neonates over 3 years.Primary outcomes are assessed at 14 days of life: (1) the proportion of neonates having experienced severe hyperbilirubinaemia (for the TcB screening intervention), defined as a TSB above the mean of the phototherapy and the exchange transfusion threshold and (2) the proportion of neonates having required hospital admission for hyperbilirubinaemia treatment (for the phototherapy intervention in primary care). ETHICS AND DISSEMINATION: This study has been approved by the Medical Research Ethics Committee of the Erasmus MC Rotterdam, the Netherlands (MEC-2017-473). Written parental informed consent will be obtained. Results from this study will be published in peer-reviewed journals and presented at (inter)national meetings. TRIAL REGISTRATION NUMBER: NTR7187.
Assuntos
Bilirrubina/análise , Hiperbilirrubinemia Neonatal/diagnóstico , Icterícia Neonatal/prevenção & controle , Monitorização Fisiológica/métodos , Triagem Neonatal/métodos , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Hiperbilirrubinemia Neonatal/prevenção & controle , Lactente , Recém-Nascido , Masculino , Projetos de PesquisaRESUMO
Ocular pythiosis is the second most common form of human pythiosis, and the rates of evisceration/enucleation in Thailand are 55-79%. This prospective study was conducted to evaluate treatment outcomes of the combination therapy protocol and the potential use of serum (1â3)-ß-glucan (BG) and Pythium insidiosum-specific antibody (Pi-Ab) as an aid to diagnosis and monitoring of ocular pythiosis. Thirty patients were enrolled in the study and 14 (non-globe salvage) required evisceration/enucleation. The globe salvage group was significantly younger, and first ocular surgeries were performed significantly sooner than in the non-globe salvage group. Serum BG and Pi-Ab levels were similar among the 2 groups over time. In vitro susceptibility testing of antifungal agents revealed relatively high minimum inhibitory concentrations and lack of synergistic effect. Serum BG and Pi-Ab would not be useful in diagnosis and monitoring of ocular pythiosis. Until effective antimicrobial agents are discovered, ocular surgeries are still the mainstay therapy in Thailand.
Assuntos
Antifúngicos/administração & dosagem , Antígenos de Fungos/administração & dosagem , Terapia Combinada/métodos , Infecções Oculares Fúngicas/terapia , Fatores Imunológicos/administração & dosagem , Pitiose/terapia , Pythium/efeitos dos fármacos , Adulto , Anticorpos Antifúngicos/sangue , Testes Diagnósticos de Rotina/métodos , Infecções Oculares Fúngicas/diagnóstico , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Estudos Prospectivos , Proteoglicanas , Pitiose/diagnóstico , Pythium/isolamento & purificação , Tailândia , Resultado do Tratamento , Adulto Jovem , beta-Glucanas/sangueRESUMO
Treatment of suspected infections in critically ill patients requires the timely initiation of appropriate antimicrobials and rapid de-escalation of unnecessary broad-spectrum coverage. New advances in rapid diagnostic tests can now offer earlier detection of pathogen and potential resistance mechanisms within hours of initial culture growth. These technologies, combined with pharmacist antimicrobial stewardship efforts, may result in shorten time to adequate coverage or earlier de-escalation of unnecessary broad spectrum antimicrobials, which could improve patient outcomes and lower overall treatment cost. Furthermore, de-escalation of antimicrobials may lead to decreased emergence of resistant organisms and adverse events associated with antimicrobials. Clinical pharmacists should be aware of new rapid diagnostic tests, including their application, clinical evidence, and limitations, in order to implement the most appropriate clinical treatment strategy when patients have positive cultures. This review will focus on commercially available rapid diagnostic tests for infections that are routinely encountered by critically ill patients, including gram-positive and gram-negative bacterial blood stream infections, Candida, and Clostridioides difficile.
Assuntos
Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/tratamento farmacológico , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/normas , Unidades de Terapia Intensiva/normas , Antibacterianos/uso terapêutico , Gestão de Antimicrobianos , Bacteriemia/diagnóstico , Estado Terminal , Infecção Hospitalar/diagnóstico , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Strategies are needed to improve time to optimal therapy in patients with bloodstream infections (BSI) due to resistant Gram-negative (GN) pathogens. Accelerate Pheno (ACC) can provide antimicrobial susceptibility results within 7 h of a positive culture and may more rapidly optimize therapy. The primary objective of this study was to evaluate the hypothetical impact of ACC on time to effective therapy (TTET) and time to definitive therapy (TTDT) among patients with BSI due to resistant GN pathogens. ACC was performed on resistant GN BSI isolates, and results were not available to clinicians in real time. A potential benefit of having ACC on TTET or TTDT was determined if modifications to antimicrobial regimens could have been made sooner with ACC. Comparisons on the impact of ACC in the presence or absence of testing by the Verigene Gram-negative blood culture test (Verigene GN-BC) were performed. Sixty-one patients with resistant GN BSI were evaluated. The median actual TTET and TTDT in the cohort were 25.9 h (interquartile range [IQR], 18.5, 42.1) and 47.6 h (IQR, 24.9, 79.6), respectively. Almost half of the patients had potential improvement in TTET and/or TTDT with ACC. In patients who would have had a benefit the median potential decreases in TTET and TTDT were 16.6 h (IQR, 5.5 to 30.6) and 29.8 h (IQR, 13.6 to 43), respectively. The largest potential improvements were seen in patients for whom Verigene results were not available. In conclusion, among patients with resistant GN BSI in a setting where other rapid diagnostic technologies are utilized, ACC results could have further improved TTET and TTDT.
Assuntos
Bacteriemia/tratamento farmacológico , Testes Diagnósticos de Rotina/métodos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Testes de Sensibilidade Microbiana/métodos , Tempo para o Tratamento , Gestão de Antimicrobianos/métodos , Hemocultura , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
OBJECTIVES: Triazole resistance in Aspergillus spp. is emerging and complicates prophylaxis and treatment of invasive aspergillosis (IA) worldwide. New polymerase chain reaction (PCR) tests on broncho-alveolar lavage (BAL) fluid allow for detection of triazole resistance at a genetic level, which has opened up new possibilities for targeted therapy. In the absence of clinical trials, a modelling study delivers estimates of the added value of resistance detection with PCR, and which empiric therapy would be optimal when local resistance rates are known. DESIGN: A decision-analytic modelling study was performed based on epidemiological data of IA, extended with estimated dynamics of resistance rates and treatment effectiveness. Six clinical strategies were compared that differ in use of PCR diagnostics (used vs not used) and in empiric therapeutic choice in case of unknown triazole susceptibility: voriconazole, liposomal amphotericin B (LAmB) or both. Outcome measures were proportion of correct treatment, survival and serious adverse events. RESULTS: Implementing aspergillus PCR tests was projected to result in residual treatment-susceptibility mismatches of <5% for a triazole resistance rate up to 20% (using voriconazole). Empiric LAmB outperformed voriconazole at resistance rates >5-20%, depending on PCR use and estimated survival benefits of voriconazole over LAmB. Combination therapy of voriconazole and LAmB performed best at all resistance rates, but the advantage over the other strategies should be weighed against the expected increased number of drug-related serious adverse events. The advantage of combination therapy over LAmB monotherapy became smaller at higher triazole resistance rates. CONCLUSIONS: Introduction of current aspergillus PCR tests on BAL fluid is an effective way to increase the proportion of patients that receive targeted therapy for IA. The results indicate that close monitoring of background resistance rates and adverse drug events are important to attain the potential benefits of LAmB. The choice of strategy ultimately depends on the probability of triazole resistance, the availability of PCR and individual patient characteristics.
Assuntos
Antifúngicos/uso terapêutico , Testes Diagnósticos de Rotina/métodos , Gerenciamento Clínico , Farmacorresistência Fúngica , Doenças Hematológicas/complicações , Aspergilose Pulmonar Invasiva/diagnóstico , Triazóis/uso terapêutico , Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Aspergillus/isolamento & purificação , Líquido da Lavagem Broncoalveolar/microbiologia , Simulação por Computador , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Humanos , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Testes de Sensibilidade Microbiana/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Resultado do Tratamento , Triazóis/farmacologiaRESUMO
Twenty-eight warmblood mares were monitored during their late pregnancy in the Teaching Hospital of Ghent University. The reliability of two commercial assays (enzyme immunoassay and glutaraldehyde coagulation test) used for determining the IgG concentrations of their newborn foals was tested. Mammary secretions were examined at the time of foaling (T0), and then 4 (T1) and 8 (T2) hours after foaling by refractometry and electrophoresis. The foals' blood IgG levels were measured at T1 and T2 as a routine clinical diagnostic examination using two different commercial test kits (SNAP Foal Ig and Gamma-Check E) and T0, T1 and T2 samples were stored (at -18 °C) for immunoglobulin (Ig) determination by electrophoresis. Differences between the results of refractometry and electrophoresis occurred in 27.8% of the colostrum analyses. Some serum IgG could be detected immediately post partum (T0) in 75% of the foals, and 42.82% of the newborn foals acquired a serum concentration of more than 800 mg/dl IgG within 8 h of birth. Compared to the electrophoresis, the glutaraldehyde test scored better (85%) than the enzyme immunoassay (74%), although both are accurate and safe to use since they clearly distinguish between safe and unsafe IgG concentrations.
Assuntos
Testes de Coagulação Sanguínea/veterinária , Testes Diagnósticos de Rotina/veterinária , Eletroforese/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Refratometria/veterinária , Animais , Animais Recém-Nascidos , Testes de Coagulação Sanguínea/métodos , Colostro/química , Testes Diagnósticos de Rotina/métodos , Eletroforese/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Glutaral/química , Cavalos , Imunoglobulina G/sangue , Refratometria/métodos , Reprodutibilidade dos TestesRESUMO
Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagentâ¯+â¯RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15â¯day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus.
Assuntos
Ascomicetos/genética , Calmodulina/genética , DNA Complementar , Malus/microbiologia , Biologia Molecular/métodos , RNA Fúngico/isolamento & purificação , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , DNA Fúngico/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Regulação Fúngica da Expressão Gênica , Índia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 28S/genéticaRESUMO
Patients often seek opinions from allergists regarding unconventional testing for adverse reactions to foods. These tests include flow cytometry to measure the change in white blood cell volumes after incubation with foods, measurement of serum IgG or IgG4 antibodies directed against foods, intradermal provocation-neutralization with food allergens, hair analysis, electrodermal testing, and applied kinesiology. In some cases, although the laboratory methods may be valid, there are no studies showing correlation with disease. In other cases, blinded, controlled studies have shown a lack of reproducibility and a lack of correlation with disease. Most of the tests lack biologic plausibility. By understanding the methodology of these tests and the lack of evidence supporting their utility, allergists can provide knowledgeable, evidence-based information to patients who inquire about them.
Assuntos
Testes Diagnósticos de Rotina/métodos , Hipersensibilidade Alimentar/diagnóstico , Testes Imunológicos/métodos , Resposta Galvânica da Pele , Cabelo/química , Humanos , Imunoglobulina G/imunologia , Cinesiologia AplicadaRESUMO
PURPOSE: ULISSE is the only study that prospectively assessed the efficiency of a standardized strategy, compared to an open strategy for the etiologic diagnosis of uveitis. Our aim was to evaluate the diagnostic yield of the tests prescribed in the ULISSE study to clarify their relevance. METHODS: ULISSE is a non-inferiority, prospective, multicenter and cluster randomized study. The standardized strategy is a two-steps strategy: in the first step, common standard tests were performed, and in the second step, tests were guided by the clinical and anatomic type of uveitis. We reported the relevance of the diagnostic tests used in the standardized strategy, as well as the profitability of the tests that were prescribed to more than twenty patients in each group. Based on diagnostic criteria, either an ophthalmologist, or an internist, established the profitability of a test by considering whether the test lead to a diagnosis or not. RESULTS: Among the 676 patients included (standardized 303; open 373), a diagnosis was made for 152 (50.4%) in the standardized group and 203 (54.4%) in the open group. The most common entities were HLA-B27 associated uveitis (22%), spondyloarthritis (11%), sarcoidosis (18%), tuberculosis (10.7%) and herpes virus infections (8.5%). Among the first step's systematic tests, tuberculin skin test was the most contributive investigation (17.1%), followed by chest X-ray (8.4%), C reactive protein and ESR (6.6% and 5.1%), complete blood count (2.2%) and VDRL (2.0%). The second step's most often contributive tests were: HLA B27 (56.3%), chest-CT (30.3%) and angiotensin converting enzyme (ACE) (16.5%). HLA B27 and ACE were significantly more contributive in the standardized group than in the open group. Immunological tests were never contributive. Among the free investigations, or among the investigations guided by clinical or paraclinical findings, the most often contributive tests were: Quantiferon® (24%), electrophoresis of serum protein (7.8%) and sacroiliac imagery (46.4%). Intracellular serologies (1.7%), serum calcium (2.1%) and hepatic tests (3.3%) were exceptionally contributive. Among the third intention tests, labial salivary gland biopsies were contributive in 17.9% of cases, but the profitability of other invasive investigations (anterior chamber tap, vitrectomy, bronchoscopy and lumbar puncture) or specialized imagery (18F-FDG PET, Brain MRI) could not be determined since these test were rarely performed. CONCLUSION: Only a few diagnostic tests are useful for the etiological assessment of uveitis. They are often cheap, simple, more often guided by the clinical findings, and lead to an etiological diagnosis in most patients. On the other hand, some tests are never or exceptionally contributive, such as immunological tests or intracellular serologies. Further studies are required to evaluate the profitability of third intention imagery and invasive investigations.
Assuntos
Testes Diagnósticos de Rotina/métodos , Uveíte/diagnóstico , Uveíte/etiologia , Adulto , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Uveíte/patologiaRESUMO
Antimicrobial resistance is the most pressing medical challenge of the past decade. At the front line are clinical laboratories, which are responsible for accurately reporting antimicrobial susceptibility test (AST) results to clinicians and public health authorities. The ability of the laboratory to detect resistance has been hampered by several factors. In 2016, the 21st Century Cures Act was signed into law, marking an important step toward resolving many regulatory dilemmas that hampered development and updates to commercial AST systems (cASTs). We describe the pathway and history of U.S. regulation of cASTs and outline both the rewards and unmet needs possible from the 21st Century Cures Act.
Assuntos
Antibacterianos/uso terapêutico , Testes Diagnósticos de Rotina/métodos , Política de Saúde/história , Política de Saúde/legislação & jurisprudência , Testes de Sensibilidade Microbiana/métodos , Testes Diagnósticos de Rotina/normas , História do Século XXI , Humanos , Testes de Sensibilidade Microbiana/normas , Estados UnidosRESUMO
BACKGROUND: Private sector drug shops are an important source of malaria treatment in Africa, yet diagnosis without parasitological testing is common among these providers. Accurate rapid diagnostic tests for malaria (mRDTs) require limited training and present an opportunity to increase access to correct diagnosis. The present study was a cost-effectiveness analysis of the introduction of mRDTs in Ugandan drug shops. METHODS: Drug shop vendors were trained to perform and sell subsidised mRDTs and artemisinin-based combination therapies (ACTs) in the intervention arm while vendors offered ACTs following presumptive diagnosis of malaria in the control arm. The effect on the proportion of customers with fever 'appropriately treated of malaria with ACT' was captured during a randomised trial in drug shops in Mukono District, Uganda. Health sector costs included: training of drug shop vendors, community sensitisation, supervision and provision of mRDTs and ACTs to drug shops. Household costs of treatment-seeking were captured in a representative sample of drug shop customers. FINDINGS: The introduction of mRDTs in drug shops was associated with a large improvement of diagnosis and treatment of malaria, resulting in low incremental costs for the health sector at US$0.55 per patient appropriately treated of malaria. High expenditure on non-ACT drugs by households contributed to higher incremental societal costs of US$3.83. Sensitivity analysis showed that mRDTs would become less cost-effective compared to presumptive diagnosis with increasing malaria prevalence and lower adherence to negative mRDT results. CONCLUSION: mRDTs in drug shops improved the targeting of ACTs to malaria patients and are likely to be considered cost-effective compared to presumptive diagnosis, although the increased costs borne by households when the test result is negative are a concern.
Assuntos
Antimaláricos/normas , Artemisininas/normas , Febre/tratamento farmacológico , Malária/tratamento farmacológico , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Comércio/normas , Análise Custo-Benefício , Testes Diagnósticos de Rotina/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Febre/diagnóstico , Febre/parasitologia , Humanos , Malária/epidemiologia , Malária/parasitologia , Setor Privado/normas , UgandaRESUMO
DNAzymes are catalytically active DNA molecules that are obtained via in vitro selection. RNA-cleaving DNAzymes have attracted significant attention for both therapeutic and diagnostic applications due to their excellent programmability, stability, and activity. They can be designed to cleave a specific mRNA to down-regulate gene expression. At the same time, DNAzymes can sense a broad range of analytes. By combining these two functions, theranostic DNAzymes are obtained. This review summarizes the progress of DNAzyme for theranostic applications. First, in vitro selection of DNAzymes is briefly introduced, and some representative DNAzymes related to biological applications are summarized. Then, the applications of DNAzyme for RNA cleaving are reviewed. DNAzymes have been used to cleave RNA for treating various diseases, such as viral infection, cancer, inflammation and atherosclerosis. Several formulations have entered clinical trials. Next, the use of DNAzymes for detecting metal ions, small molecules and nucleic acids related to disease diagnosis is summarized. Finally, the theranostic applications of DNAzyme are reviewed. The challenges to be addressed include poor DNAzyme activity under biological conditions, mRNA accessibility, delivery, and quantification of gene expression. Possible solutions to overcome these challenges are discussed, and future directions of the field are speculated.
Assuntos
DNA Catalítico/metabolismo , RNA/metabolismo , Nanomedicina Teranóstica/métodos , Animais , Terapia Biológica/métodos , Pesquisa Biomédica/tendências , Testes Diagnósticos de Rotina/métodos , HumanosRESUMO
Tuberculosis (TB) has recently surpassed HIV as the primary infectious disease killer worldwide, but the two diseases continue to display lethal synergy. The burden of TB is disproportionately borne by people living with HIV, particularly where HIV and poverty coexist. The impact of these diseases on one another is bidirectional, with HIV increasing risk of TB infection and disease progression and TB slowing CD4 recovery and increasing progression to AIDS and death among the HIV infected. Both antiretroviral therapy (ART) and latent TB infection (LTBI) treatment mitigate the impact of coinfection, and ART is now recommended for HIV-infected patients independent of CD4 count. LTBI screening should be performed for all HIV-positive people at the time of diagnosis, when their CD4 count rises above 200, and yearly if there is repeated exposure. Tuberculin skin tests (TSTs) may perform better with serial testing than interferon gamma release assays (IGRAs). Any patient with HIV and a TST induration of ≥5 mm should be evaluated for active TB disease and treated for LTBI if active disease is ruled out. Because HIV impairs multiple aspects of immune function, progressive HIV is associated with lower rates of cavitary pulmonary TB and higher rates of disseminated and extrapulmonary disease, so a high index of suspicion is important, and sputum should be obtained for evaluation even if chest radiographs are negative. TB diagnosis is similar in patients with and without TB, relying on smear, culture, and nucleic acid amplification tests, which are the initial tests of choice. TSTs and IGRAs should not be used in the evaluation of active TB disease since these tests are often negative with active disease. Though not always performed in resource-limited settings, drug susceptibility testing should be performed on all TB isolates from HIV-positive patients. Urine lipoarabinomannan testing may also be helpful in HIV-positive patients with disseminated disease. Treatment of TB in HIV-infected patients is similar to that of TB in HIV-negative patients except that daily therapy is required for all coinfected patients, vitamin B6 supplementation should be given to all coinfected patients receiving isoniazid to reduce peripheral neuropathy, and specific attention needs to be paid to drug-drug interactions between rifamycins and many classes of antiretrovirals. In patients requiring ART that contains ritonavir or cobicistat, this can be managed by the use of rifabutin at 150 mg daily in place of rifampin. For newly diagnosed coinfected patients, mortality is lower if treatment is provided in parallel, rather than serially, with treatment initiation within 2 weeks preferred for those with CD4 counts of <50 and within 8 to 12 weeks for those with higher CD4 counts. When TB immune reconstitution inflammatory syndrome occurs, patients can often be treated symptomatically with nonsteroidal anti-inflammatory drugs, but a minority will benefit from steroids. Generally, patients who do not have space-occupying lesions such as occurs in TB meningitis do not require cessation of therapy.