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1.
Trials ; 22(1): 680, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620213

RESUMO

BACKGROUND: Hospital-acquired and ventilator-associated pneumonias (HAP and VAP) are common in critical care and can be life-threatening. Rapid microbiological diagnostics, linked to an algorithm to translate their results into antibiotic choices, could simultaneously improve patient outcomes and antimicrobial stewardship. METHODS: The INHALE Randomised Controlled Trial is a multi-centre, parallel study exploring the potential of the BioFire FilmArray molecular diagnostic to guide antibiotic treatment of HAP/VAP in intensive care units (ICU); it identifies pathogens and key antibiotic resistance in around 90 min. The comparator is standard care whereby the patient receives empirical antibiotics until microbiological culture results become available, typically after 48-72 h. Adult and paediatric ICU patients are eligible if they are about to receive antibiotics for a suspected lower respiratory infection (including HAP/VAP) for the first time or a change in antibiotic because of a deteriorating clinical condition. Breathing spontaneously or intubated, they must have been hospitalised for 48 h or more. Patients are randomised 1:1 to receive either antibiotics guided by the FilmArray molecular diagnostic and its trial-based prescribing algorithm or standard care, meaning empirical antibiotics based on local policy, adapted subsequently based upon local microbiology culture results. Co-primary outcomes are (i) non-inferiority in clinical cure of pneumonia at 14 days post-randomisation and (ii) superiority in antimicrobial stewardship at 24 h post-randomisation (defined as % of patients on active and proportionate antibiotics). Secondary outcomes include further stewardship reviews; length of ICU stay; co-morbidity indicators, including septic shock, change in sequential organ failure assessment scores, and secondary pneumonias; ventilator-free days; adverse events over 21 days; all-cause mortality; and total antibiotic usage. Both cost-effectiveness of the molecular diagnostic-guided therapy and behavioural aspects determining antibiotic prescribing are being explored. A sample size of 552 will be required to detect clinically significant results with 90% power and 5% significance for the co-primary outcomes. DISCUSSION: This trial will test whether the potential merits of rapid molecular diagnostics for pathogen and resistance detection in HAP/VAP are realised in patient outcomes and/or improved antibiotic stewardship. TRIAL REGISTRATION: ISRCTN Registry ISRCTN16483855 . Retrospectively registered on 15 July 2019.


Assuntos
Gestão de Antimicrobianos , Pneumonia Associada à Ventilação Mecânica , Adulto , Criança , Cuidados Críticos , Hospitais , Humanos , Estudos Multicêntricos como Assunto , Patologia Molecular , Pneumonia Associada à Ventilação Mecânica/diagnóstico , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto , Reino Unido
2.
Front Cell Infect Microbiol ; 11: 715821, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34650933

RESUMO

Infections caused by multidrug-resistant Gram-negative organisms have become a global threat. Such infections can be very difficult to treat, especially when they are caused by carbapenemase-producing organisms (CPO). Since infections caused by CPO tend to have worse outcomes than non-CPO infections, it is important to identify the type of carbapenemase present in the isolate or at least the Ambler Class (i.e., A, B, or D), to optimize therapy. Many of the newer beta-lactam/beta-lactamase inhibitor combinations are not active against organisms carrying Class B metallo-enzymes, so differentiating organisms with Class A or D carbapenemases from those with Class B enzymes rapidly is critical. Using molecular tests to detect and differentiate carbapenem-resistance genes (CRG) in bacterial isolates provides fast and actionable results, but utilization of these tests globally appears to be low. Detecting CRG directly in positive blood culture bottles or in syndromic panels coupled with bacterial identification are helpful when results are positive, however, even negative results can provide guidance for anti-infective therapy for key organism-drug combinations when linked to local epidemiology. This perspective will focus on the reluctance of laboratories to use molecular tests as aids to developing therapeutic strategies for infections caused by carbapenem-resistant organisms and how to overcome that reluctance.


Assuntos
Carbapenêmicos , Patologia Molecular , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
3.
Urologe A ; 60(10): 1349-1358, 2021 Oct.
Artigo em Alemão | MEDLINE | ID: mdl-34550396

RESUMO

In the future, precision medicine with agents targeting specific genetic alterations will play an important role in bladder cancer. This includes both single genetic alterations (e.g. FGFR3) and gene panel analyses in patients with no further therapeutic options, rare cancer subtypes or unusual clinical courses. These molecular analyses can be carried out on formalin-fixed paraffin-embedded tumor samples and the results should be discussed in interdisciplinary molecular tumor boards in order to either recommend approved targeted therapies or suggest patients for molecular-based clinical trials, compassionate use programs or off-label use of drugs. The remuneration of molecular diagnostics is largely well-represented for the outpatient sector in Germany; however, the covering of treatment costs must currently be approved by the health insurances.


Assuntos
Neoplasias da Bexiga Urinária , Testes Genéticos , Humanos , Mutação , Patologia Molecular , Medicina de Precisão , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/terapia
4.
Lung Cancer ; 160: 118-126, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34500194

RESUMO

Epidermal growth factor receptor (EGFR) mutation testing in advanced non-small-cell lung cancer (NSCLC) has evolved rapidly over the past decade, largely triggered by the introduction of the targeted EGFR tyrosine kinase inhibitors (TKIs). Initially used to detect common EGFR mutations and determine the most appropriate first-line therapy at diagnosis, testing methodologies have expanded to test for multiple mutations at multiple time points throughout the disease course. Here we review the current mutation testing approaches, including types of biopsies, and the available assays commonly used in the clinic. Specific application of these approaches in advanced NSCLC, including current guideline recommendations, and potential future developments are discussed.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutação , Patologia Molecular , Tecnologia
5.
Rev Soc Bras Med Trop ; 54: e01822021, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495256

RESUMO

INTRODUCTION: Visceral leishmaniasis (VL) is an important zoonosis in Brazil. Previous identification of parasitized dogs can also help prevent the disease in humans, even in non-endemic areas of the country. The Brazilian Ministry of Health recommends diagnosis in dogs using a DPP® (rapid test) as a screening test and an immunoenzymatic assay (ELISA) as a confirmatory test (DPP®+ELISA), and culling infected dogs as a legal control measure. However, the accuracy of these serological tests has been questioned. METHODS: VL in dogs was investigated in a non-endemic area of the São Paulo state for three consecutive years, and the performances of different diagnostic tests were compared. RESULTS: A total of 331 dog samples were collected in 2015, 373 in 2016, and 347 in 2017. The seroprevalence by DPP®+ELISA was 3.3, 3.2, and 0.3%, respectively, and by indirect immunofluorescence assay (IFA), it was 3.0, 5.6, and 5.5%, respectively. ELISA confirmed 18.4% of DPP® positive samples. The concordance between the IFA and DPP® was 83.9%. The concordance between IFA and DPP®+ELISA was 92.9%. A molecular diagnostic test (PCR) was performed in 63.2% of the seropositive samples, all of which were negative. CONCLUSIONS: In non-endemic areas, diagnostic tests in dogs should be carefully evaluated to avoid false results.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Anticorpos Antiprotozoários , Brasil/epidemiologia , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/veterinária , Patologia Molecular , Estudos Soroepidemiológicos
6.
Nat Commun ; 12(1): 5216, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471137

RESUMO

Bacterial biosensors, or bactosensors, are promising agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we show how novel, clinically relevant sensing modalities can be introduced into bactosensors in a modular fashion. To do so, we have leveraged a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We apply EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improve the sensitivity and lower the limit-of-detection of the sensing module by directed evolution. We then engineer a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.


Assuntos
Bactérias/metabolismo , Biomarcadores/metabolismo , Técnicas Biossensoriais , Proteínas de Transporte/metabolismo , Patologia Molecular/métodos , Bactérias/genética , Ácidos e Sais Biliares/sangue , Técnicas Biossensoriais/métodos , Proteínas de Transporte/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Transplante de Fígado , Engenharia Metabólica/métodos , Sensibilidade e Especificidade , Alinhamento de Sequência , Vibrio , Vibrioses/diagnóstico
7.
Anal Methods ; 13(34): 3744-3763, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34473144

RESUMO

As the COVID-19 pandemic continues to escalate globally and acquires new mutations, accurate diagnostic technologies continue to play a vital role in controlling and understanding the epidemiology of this disease. A plethora of technologies have enabled the diagnosis of individuals, informed clinical management, aided population-wide screening to determine transmission rates and identified cases within the wider community and high-risk settings. This review explores the application of molecular diagnostics technologies in controlling the spread of COVID-19, and the key factors that affect the sensitivity and specificity of the tests used.


Assuntos
COVID-19 , Humanos , Pandemias , Patologia Molecular , SARS-CoV-2 , Sensibilidade e Especificidade
9.
Talanta ; 235: 122797, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517655

RESUMO

As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18-25 °C) for 1-2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.


Assuntos
COVID-19 , Microfluídica , Humanos , Patologia Molecular , Reação em Cadeia da Polimerase , SARS-CoV-2 , Temperatura
11.
Hautarzt ; 72(9): 751-759, 2021 Sep.
Artigo em Alemão | MEDLINE | ID: mdl-34383107

RESUMO

The basis of allergen immunotherapy (AIT) is the diagnosis of the eliciting allergen sources, which is a challenge, especially in the case of multiple sensitizations. Molecular allergy diagnostics can be of special help, since detection of "marker allergens", usually important major allergens, allows to distinguish between primary sensitization and cross-reactions. Thus, the indication and extract selection for AIT can be facilitated. While molecular diagnosis is particularly useful for double-sensitized hymenoptera venom and polysensitized pollen allergic patients, the benefit is probably lower in case of house dust mite allergy.


Assuntos
Alérgenos , Hipersensibilidade , Dessensibilização Imunológica , Humanos , Hipersensibilidade/diagnóstico , Hipersensibilidade/terapia , Imunoglobulina E , Patologia Molecular
12.
BMJ Glob Health ; 6(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34429298

RESUMO

BACKGROUND: Early access to diagnosis is crucial for effective management of any disease including tuberculosis (TB). We investigated the barriers and opportunities to maximise uptake and utilisation of molecular diagnostics in routine healthcare settings. METHODS: Using the implementation of WHO approved TB diagnostics, Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) and Line Probe Assay (LPA) as a benchmark, we evaluated the barriers and how they could be unlocked to maximise uptake and utilisation of molecular diagnostics. RESULTS: Health officers representing 190 districts/counties participated in the survey across Kenya, Tanzania and Uganda. The survey findings were corroborated by 145 healthcare facility (HCF) audits and 11 policy-maker engagement workshops. Xpert MTB/RIF coverage was 66%, falling behind microscopy and clinical diagnosis by 33% and 1%, respectively. Stratified by HCF type, Xpert MTB/RIF implementation was 56%, 96% and 95% at district, regional and national referral hospital levels. LPA coverage was 4%, 3% below culture across the three countries. Out of 111 HCFs with Xpert MTB/RIF, 37 (33%) used it to full capacity, performing ≥8 tests per day of which 51% of these were level five (zonal consultant and national referral) HCFs. Likewise, 75% of LPA was available at level five HCFs. Underutilisation of Xpert MTB/RIF and LPA was mainly attributed to inadequate-utilities, 26% and human resource, 22%. Underfinancing was the main reason underlying failure to acquire molecular diagnostics. Second to underfinancing was lack of awareness with 33% healthcare administrators and 49% practitioners were unaware of LPA as TB diagnostic. Creation of a national health tax and decentralising its management was proposed by policy-makers as a booster of domestic financing needed to increase access to diagnostics. CONCLUSION: Our findings suggest higher uptake and utilisation of molecular diagnostics at tertiary level HCFs contrary to the WHO recommendation. Country-led solutions are crucial for unlocking barriers to increase access to diagnostics.


Assuntos
Mycobacterium tuberculosis , Tuberculose Pulmonar , Humanos , Mycobacterium tuberculosis/genética , Patologia Molecular , Rifampina , Sensibilidade e Especificidade
13.
Am J Trop Med Hyg ; 105(3): 627-629, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34339384

RESUMO

Environmental Mycobacterium ulcerans causes a disabling skin disease called Buruli ulcer. Recent studies completed the knowledge of the evolving geographic extension and epidemiology of Buruli ulcer in West Africa, where Côte d'Ivoire is reporting the highest number of cases. We report seven polymerase chain reaction-documented patients in Burkina Faso, a neighboring country of Côte d'Ivoire, where previously Buruli ulcer cases were confirmed primarily using clinical arguments.


Assuntos
Úlcera de Buruli/diagnóstico , Mycobacterium ulcerans/genética , Adolescente , Adulto , Burkina Faso/epidemiologia , Úlcera de Buruli/epidemiologia , Úlcera de Buruli/transmissão , Costa do Marfim , Transmissão de Doença Infecciosa , Doenças Endêmicas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Patologia Molecular , Reação em Cadeia da Polimerase em Tempo Real , População Rural , Adulto Jovem
14.
ACS Biomater Sci Eng ; 7(9): 4669-4676, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34437802

RESUMO

The COVID-19 pandemic has exposed the dependence of diagnostic laboratories on a handful of large corporations with market monopolies on the worldwide supply of reagents, consumables, and hardware for molecular diagnostics. Global shortages of key consumables for RT-qPCR detection of SARS-CoV-2 RNA have impaired the ability to run essential, routine diagnostic services. Here, we describe a workflow for rapid detection of SARS-CoV-2 RNA in upper respiratory samples including nasal swabs and saliva, utilizing low-cost equipment and readily accessible reagents. Using repurposed Creality3D Ender-3 three-dimensional (3D) printers, we built a semiautomated paramagnetic bead RNA extraction platform. The hardware for the system was built for $300 USD, and the material cost per reaction was $1 USD. Named the Ender VX500, instrument performance when paired with RT-qPCR for SARS-CoV-2 detection in nasal and saliva specimens was two virus copies per microliter. There was a high-performance agreement (assessed using 458 COVID-19 nasal swab specimens) with the Aptima SARS-CoV-2 assay run on the Hologic Panther, a commercial automated RNA extraction and detection platform. Inter- and intrainstrument precision was excellent (coefficients of variation (CoV) of 1.10 and 0.66-1.32%, respectively) across four instruments. The platform is scalable with throughput ranging from 23 specimens on a single instrument run by one user in 50 min to 364 specimens on four instruments run by four users in 190 min. Step-by-step instructions and protocols for building and running the Ender VX500 have been made available without restriction.


Assuntos
COVID-19 , Humanos , Pandemias , Patologia Molecular , RNA Viral/genética , SARS-CoV-2
15.
Zhonghua Bing Li Xue Za Zhi ; 50(7): 701-703, 2021 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-34405601
16.
Pathologe ; 42(5): 464-471, 2021 Sep.
Artigo em Alemão | MEDLINE | ID: mdl-34402977

RESUMO

Ductal adenocarcinoma is the most common tumor of the pancreas. Although relatively rare, it poses one of the greatest oncological challenges because of its poor prognosis, which has so far only slightly improved. Progress has been made in the more precise classification and standardization of the morphological assessment. In the current WHO classification, prognostically relevant subtypes are clearly delimited among themselves and from ductal adenocarcinoma not otherwise specified (NOS). In the recent TNM classification, a size-based T­category was introduced. Diagnostically, the histological assessment of the resection specimen is relatively easy; on the other hand, assessment of the fine-needle biopsy from the primary tumor or a liver metastasis is still difficult. The molecular stratification of ductal adenocarcinoma and the other pancreatic neoplasms has made great progress. This not only defined the genetics of tumor entities, but also identified the prognosis and biology of tumor groups on the basis of RNA expression patterns. The range of treatment could be expanded by targeted molecular therapies (especially for patients with BRCA1/2 germline mutations, NTRK- or NRG1-fusions, or oncogenic BRAF and PIK3CA mutations as well as tumors with microsatellite instability (MSI)), even if targeted therapies are currently only available for a minority of patients (<10%).


Assuntos
Neoplasias da Mama , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/genética , Feminino , Humanos , Pâncreas , Neoplasias Pancreáticas/genética , Patologia Molecular , Prognóstico
17.
Oncol. (Guayaquil) ; 31(2): 93-103, 31 de agosto 2021.
Artigo em Espanhol | LILACS | ID: biblio-1284421

RESUMO

Introducción: El carcinoma basocelular (CBC)es una de las neoplasias más comunes de la piel. En nuestro país, por su localización, representa una entidad patológica de gran importancia, por la radiación ultravioleta elevada, que es inversamente proporcional a la latitud geográfica en la que nos encontramos en Ecuador. El objetivo del presente trabajo es revisar las características claves que distinguen al Carcinoma basocelular, y actualizar los conocimientos, incluyendo la evidencia disponible en hallazgos histopatológicos, marcadores de inmunohistoquímica y metástasis en esta patología


Introduction: Basal cell carcinoma BCC is one of the most common skin neoplasms. In our country, because of its location, it represents a pathological entity of great importance, due to the high ultraviolet radiation, that is inversely proportional to the geographical latitude we are in Ecuador. This paper objective is to review the key features that distinguish basal cellcarcinoma, and update knowledge, including the available evidence on histopathological findings, immunohistochemical markers and metastasis in this pathology.


Introdução: Carcinoma basocelular O CBC é uma das neoplasias cutâneas mais comuns. Em nosso país, por sua localização, representa uma entidade patológica de grande importância, devido à alta radiação ultravioleta, que é inversamente proporcional à latitude geográfica em que nos encontramos no Equador. O objetivo deste artigo é revisar as principais características que distinguem o carcinoma basocelular e atualizar o conhecimento, incluindo as evidências disponíveis sobre achados histopatológicos, marcadores imunohistoquímicos e metástases nessa patologia.


Assuntos
Humanos , Adulto , Imuno-Histoquímica , Carcinoma Basocelular , Neoplasias Cutâneas , Patologia Molecular , Metástase Neoplásica
18.
Front Immunol ; 12: 648881, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34276646

RESUMO

Background: Diagnosis of antiphospholipid syndrome (APS) is based on the positivity of laboratory criteria antiphospholipid antibodies (aPLs). Test results for aPLs could be contradictory among different detection methods as well as commercial manufacturers. This study aimed to assess and compare the diagnostic and analytic performances of four commercial assays prevalently used in China. Methods: A total of 313 patients including 100 patients diagnosed with primary APS, 52 with APS secondary to SLE, 71 with SLE, and 90 health controls were recruited. Serum IgG, IgM, and IgA for aCL, and aß2GPI antibodies were detected with two ELISA and two CLIA systems, and test system with the best diagnostic value was explored of its correlation with key clinical features. Results: CLIA by YHLO Biotech Co. was considered as the system with the best predictive power, where 58.55 and 57.89% of APS patients were positive for aCL or aß2GPI for at least one antibody (IgG or IgM or IgA). Overall, CLIA showed better performance characteristics than traditional ELISA test systems. Conclusion: CLIA was considered as a better platform for aPL detection in APS diagnosis. A combination of other detection platforms could assist in differential diagnosis as well as in identifying high-risk patients.


Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Patologia Molecular/métodos , Kit de Reagentes para Diagnóstico , Adulto , Anticorpos Anticardiolipina/imunologia , Síndrome Antifosfolipídica/diagnóstico , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Patologia Molecular/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto Jovem , beta 2-Glicoproteína I/imunologia
19.
Mol Immunol ; 137: 57-66, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34216999

RESUMO

Patients with inborn errors of immunity (IEI) present with a heterogeneous clinical and immunological phenotype, therefore a correct molecular diagnosis is crucial for the classification and subsequent therapeutic management. On the other hand, IEI are a group of rare congenital diseases with highly diverse features and, in most cases, an as yet unknown genetic etiology. Next generation sequencing has facilitated genetic examinations of rare inherited disorders during the recent years, thus allowing a suitable molecular diagnosis in the IEI patients. This review aimed to investigate the current findings about these techniques in the field of IEI, suggesting an efficient stepwise approach to molecular diagnosis of inborn errors of immunity.


Assuntos
Doenças Genéticas Inatas/genética , Doenças do Sistema Imunitário/genética , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Patologia Molecular , Fenótipo
20.
Am J Trop Med Hyg ; 105(3): 660-669, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34270450

RESUMO

The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.


Assuntos
Leishmania braziliensis/genética , Leishmania guyanensis/genética , Leishmaniose/parasitologia , Proteínas de Protozoários/genética , DNA de Protozoário/análise , Humanos , Leishmania/genética , Leishmaniose/diagnóstico , Patologia Molecular/métodos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sequenciamento Completo do Genoma
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