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1.
J Med Microbiol ; 68(11): 1622-1628, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31596198

RESUMO

Introduction. Nosocomial transmission of Mycobacterium tuberculosis is an important health issue and the detection of tuberculosis (TB) cases is the main tool for controlling this disease.Aim. We aimed to assess the possible occurrence of nosocomial transmission of M. tuberculosis in a reference hospital for HIV/AIDS patients and evaluate both the performance of the Xpert MTB/RIF (Xpert) platform and drug resistance profiles.Methodology. We evaluated the performance of the Xpert platform. Samples that tested positive on the BACTEC MGIT 320 (MGIT320) platform were submitted for genotyping and drug susceptibility testing.Results. In this study, pulmonary and extrapulmonary samples from 407 patients were evaluated, and among these, 15.5 % were diagnosed with TB by the MGIT320 platform, with a TB/HIV coinfection rate of 52.4 %. The Xpert platform gave positive results for TB for 11 samples with negative results on the MGIT320 platform. In the genotyping results, 53.3 % of the strains clustered; of these strains, half were in two of the four clusters formed, and the patients had visited the hospital on the same day. Drug resistance was observed in 11.7 % of the strains.Conclusion. Putative nosocomial transmission of M. tuberculosis was detected, showing that genotyping is a powerful approach for understanding the dynamics of M. tuberculosis transmission, especially in a high-burden TB and HIV landscape.


Assuntos
Infecções por HIV/complicações , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/microbiologia , Síndrome de Imunodeficiência Adquirida/complicações , Antibióticos Antituberculose/farmacologia , Técnicas de Laboratório Clínico , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/microbiologia , Estudos Transversais , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Filogenia , Tuberculose , Tuberculose Pulmonar/diagnóstico
2.
Pan Afr Med J ; 33: 110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31489088

RESUMO

Introduction: The World Health Organization endorsed (2010) the use of Xpert MTB/RIF and countries are shifting from smear microscopy (smear)-based to Xpert MTB/RIF-based tuberculosis (TB) diagnostic algorithms. As with smear, sputum quality may predict the likelihood of obtaining a bacteriologically-confirmed TB when using Xpert MTB/RIF. Methods: From 08/12-11/2014, all people living with HIV were recruited at 22 clinics. For patients screened positive using the four TB symptoms their sputa were tested by Xpert MTB/RIF and smear. Laboratorians assessed and recorded sputum appearance and volume. The yield of bacteriologically-positive sputum evaluated using Xpert MTB/RIF and smear, likelihood-ratios were calculated. Results: Among 6,041 patients enrolled 2,296 were presumptive TB, 1,305 (56.8%) had > 1 sputa collected and 644/1,305 (49.3%) had both Xpert MTB/RIF and smear results. Since >1 sputa collected from 644 patients 954 sputa were tested by Xpert MTB/RIF and smear. Bacteriologically-positive sputum was two-fold higher with Xpert MTB/RIF 11.4% versus smear 5.3%, p < 0.001. Sputum appearance and quantity were not predictive of bacteriologically-positive results, except volume of 2ml to < 3ml, tested by Xpert MTB/RIF LR+= 1.26 (95% CI, 1.05-1.50). Conclusion: Xpert MTB/RIF test yield to bacteriologically-positive sputum was superior to smear. Sputum quality and quantity, however, were not consistently predictive of bacteriologically-positive results by Xpert MTB/RIF or smear.


Assuntos
Técnicas Bacteriológicas/métodos , Microscopia/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Adulto , Feminino , Seguimentos , Humanos , Masculino , Técnicas de Diagnóstico Molecular/métodos , Estudos Prospectivos , Tuberculose/epidemiologia
3.
Presse Med ; 48(7-8 Pt 1): 816-824, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31439443

RESUMO

Diagnosis of mature B cell malignancies is highly multidisciplinary. Biological tools provide diagnostic, prognostic and theranostic information. Biological hematology allows considering mature B cell diseases from two perspectives : cellular and molecular approaches. Cytomorphology and flow cytometry are tools from cell hematology. Conventional cytogenetics, FISH and molecular biology are tools from molecular hematology. NGS is a new technique that could dramatically change diagnostic and therapeutic management of B cell malignancies in the near future. Integration of clinical, pathological and biological data allows for personalized management of these diseases.


Assuntos
Técnicas de Laboratório Clínico/métodos , Leucemia de Células B/diagnóstico , Linfoma de Células B/diagnóstico , Integração de Sistemas , Hibridização Genômica Comparativa/métodos , Citodiagnóstico/métodos , Análise Citogenética/métodos , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunofenotipagem/métodos , Hibridização in Situ Fluorescente/métodos , Leucemia de Células B/genética , Leucemia de Células B/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Técnicas de Diagnóstico Molecular/métodos , Imagem Multimodal/métodos , Imagem Multimodal/tendências
4.
Presse Med ; 48(7-8 Pt 1): 832-841, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31444019

RESUMO

Lymphoplasmocytic lymphona with monoclonal lgM, rare. Median age at diagnosis 70 years old, frail population. Heterogenous clinic presentation. Molecular diagnosis with MYD88. Treatment required for symptomatic WM patients only. 1st line therapy: DRC. Input of targeted therapies (ibrutinib) for frail patients, maintenance effect.


Assuntos
Macroglobulinemia de Waldenstrom , Idade de Início , Idoso , Análise Mutacional de DNA/métodos , Idoso Fragilizado , Humanos , Técnicas de Diagnóstico Molecular/métodos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Fator 88 de Diferenciação Mieloide/genética , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/epidemiologia , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/terapia
5.
Arch Virol ; 164(10): 2581-2584, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31359148

RESUMO

Maize chlorotic mottle virus (MCMV), an important quarantine virus, causes lethal necrosis in maize when coinfected with a potyvirid, which is seriously threatening the production of maize worldwide. In this study, recombinase polymerase amplification (RPA), a novel isothermal DNA amplification and detection technique, was developed to detect MCMV in maize crops. A pair of specific primers was designed based on the conserved sequences of the MCMV coat protein region. The RT-RPA assay was carried out as an isothermal reaction at 38 °C that was complete within 30 min, and no cross-reactivity was detected with other viruses infecting maize in China. The limit of detection of the RT-RPA assay was tenfold lower than that of ordinary RT-PCR. Moreover, this method was successfully applied to test field-collected samples. The newly developed RT-RPA assay offers a reliable, sensitive and efficient method for rapid detection of MCMV in maize in equipment-limited diagnostic laboratories and on-site facilities.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Tombusviridae/isolamento & purificação , Proteínas do Capsídeo/genética , China , Primers do DNA/genética , Sensibilidade e Especificidade , Temperatura Ambiente , Fatores de Tempo , Tombusviridae/classificação , Tombusviridae/genética
6.
J Cancer Res Clin Oncol ; 145(8): 1977-1986, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31309300

RESUMO

CONTEXT: Parathyroid carcinoma (PC) is a rare endocrine malignancy with no approved systemic therapies for unresectable locally invasive or distant metastatic disease. Understanding the molecular changes in advanced PC can provide better understanding of this disease and potentially help directing targeted therapy. OBJECTIVE: To evaluate tumor-specific genetic changes using next-generation sequencing (NGS) panels. DESIGN: All patients with advanced PC were tested for hot-spot panels using NGS panels including a 50-gene panel, a 409-gene panel if the standard 50-gene panel (Ion Torrent, Life Technology) was negative or a FoundationOne panel. SETTING: The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. PATIENTS OR OTHER PARTICIPANTS: 11 patients with advanced PC were selected to undergo molecular testing. MAIN OUTCOME MEASURE(S): Genetic profiles of advanced PC. RESULTS: Among the 11 patients, 4 patients had the 50-gene panel only, 6 had 409-gene panel after a negative 50-gene panel and 1 had FoundationOne. One patient who had 50-gene panel only also had his metastatic site (esophagus) of his tumor tested with FoundationOne. The most common mutations identified were in the PI3 K (PIK3CA, TSC1 and ATM) (4/11 patients) and TP53 (3/11) pathways. Genes not previously reported to be mutated in PC included: SDHA, TERT promoter and DICER1. Actionable mutations were found in 54% (6/11) of the patients. CONCLUSIONS: Mutational profiling using NGS panels in advanced PC has yielded important potentially targetable genetic alterations. Larger studies are needed to identify commonly mutated genes in advanced PC patients. Development of novel therapies targeting these cellular pathways should be considered.


Assuntos
Carcinoma/genética , Perfilação da Expressão Gênica , Técnicas de Diagnóstico Molecular/métodos , Monitorização Fisiológica/métodos , Neoplasias das Paratireoides/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/diagnóstico , Carcinoma/patologia , Carcinoma/terapia , Análise Mutacional de DNA/métodos , Progressão da Doença , Feminino , Seguimentos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/tendências , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Neoplasias das Paratireoides/diagnóstico , Neoplasias das Paratireoides/patologia , Neoplasias das Paratireoides/terapia
7.
J Med Microbiol ; 68(9): 1324-1329, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31355739

RESUMO

Purpose. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK).Methodology. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK.Results. There was no difference in the performance of both primer sets for all HSV-1 dilutions (P=0.83) using a CIM or between a CIM and a swab (P=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (P=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (P=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (P<0.001).Conclusions. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.


Assuntos
Córnea/virologia , Herpes Simples/diagnóstico , Herpesvirus Humano 1/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Primers do DNA/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
8.
Zhonghua Fu Chan Ke Za Zhi ; 54(5): 307-311, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31154711

RESUMO

Objective: To evaluate the feasibility of the BioPerfectus multiplex real time (BMRT) HPV assay for self-sample cervical cancer screening. Methods: Eight hundreds and thirty-nine self-collected and physician-obtained DNA samples from the Shenzhen cervical cancer screening trial Ⅳ(SHENCCAST-Ⅳ) study collected samples for cervical cancer screening during June 2013 to September 2014 were detected by BMRT HPV assay to evaluate the screening efficacy. Results: A total of the 839 women who were screened, 804 with complete BMRT HPV data was included in the study, and average age was (46±7) years. Of the 804 women, the positive rates of 14 high-risk HPV genotypes (including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 subtype) of self-sample and physician-obtained samples were 12.2% (98/804) and 12.8% (103/804), respectively (χ(2)=0.14, P=0.71). Self-collected samples with HPV-positive had significantly more cells (median 19 901.0) than physician-obtained samples (median 1 778.4), and there was statistically significant difference (Z=-7.61, P<0.01). The degree of agreement between self-sample and physician-obtained samples of HPV 16, HPV 18 and other 12 high risk HPV genotype was 99.8%, 100.0% and 96.1%, respectively. And the consistent Kappa value was 0.95, 1.00 and 0.81, respectively. Of 804 samples, there were 6 cervical intraepithelial neoplasia (CIN)Ⅱ(+) cases. There were no missed CINⅡ(+) cases by BMRT HPV assay. Conclusion: BMRT HPV assay is feasible for self-sample cervical cancer screening.


Assuntos
Neoplasia Intraepitelial Cervical/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Autoexame/métodos , Manejo de Espécimes/métodos , Neoplasias do Colo do Útero/diagnóstico , Virologia/métodos , Adolescente , Adulto , Idoso , Neoplasia Intraepitelial Cervical/virologia , DNA Viral , Detecção Precoce de Câncer , Feminino , Humanos , Programas de Rastreamento , Pessoa de Meia-Idade , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Sensibilidade e Especificidade , Esfregaço Vaginal , Adulto Jovem
9.
APMIS ; 127(9): 627-634, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31225920

RESUMO

Sexually transmitted infections (STIs) remain major public health problems globally. Appropriate laboratory diagnosis of STIs is rare in Ukraine. We investigated the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) using the US FDA-approved Aptima Combo 2 and Aptima TV assays and compared the results with the conventional routine diagnostic tests (CDTs) in Ukraine. Urogenital swabs from consecutive mostly symptomatic females (n = 296) and males (n = 159) were examined. The prevalences were as follows: 10% (n = 47) of TV, 5.3% (n = 24) of CT and 1.5% (n = 7) of NG. The specificity of some CDTs was high, for example, 100% for NG culture, TV IgG ELISA, CT IgM ELISA and CT microscopy, but lower for other CDTs, that is, from 44% to 99.8%. The sensitivity of all CDTs was suboptimal, that is, 71% (n = 5) for NG microscopy, 57% (n = 4) for NG culture, 53% (n = 8) for CT IgG ELISA, 33% (n = 1) for TV IgG ELISA, 28% (n = 13) for TV microscopy, 25% (n = 1) for CT IgA ELISA, 20% (n = 3) for CT IgM ELISA and 0% (n = 0) for CT microscopy. The prevalences of particularly TV and CT were high, but substantial also for NG, in Ternopil, Ukraine. The sensitivities of all CDTs were low, and widespread implementation of validated, quality-assured and cost-effective molecular diagnostic STI tests in Ukraine is imperative.


Assuntos
Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/epidemiologia , Gonorreia/diagnóstico , Gonorreia/epidemiologia , Vaginite por Trichomonas/diagnóstico , Vaginite por Trichomonas/epidemiologia , Adolescente , Adulto , Chlamydia trachomatis/isolamento & purificação , Estudos Transversais , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Neisseria gonorrhoeae/isolamento & purificação , Prevalência , Sensibilidade e Especificidade , Doenças Sexualmente Transmissíveis/diagnóstico , Doenças Sexualmente Transmissíveis/epidemiologia , Trichomonas vaginalis/isolamento & purificação , Ucrânia/epidemiologia , Estados Unidos , United States Food and Drug Administration , Adulto Jovem
10.
J Med Microbiol ; 68(8): 1204-1210, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31184572

RESUMO

INTRODUCTION: In recent years, metagenomic next-generation sequencing (mNGS) has become widely used in medical microbiology to detect pathogen infection. AIM: We aimed to assess the diagnostic performance of mNGS of cerebrospinal fluid (CSF) for prediction of cryptococcal meningitis (CM). METHODOLOGY: A comparative evaluation of mNGS (performed on CSF samples) and conventional methods, including India ink staining, culture for fungi and cryptococcal-antigen (CrAg) detection by enzyme immunoassay, was performed on 12 consecutive non-HIV-infected patients with chronic or subacute CM. RESULTS: India ink staining and culture of the CSF were positive for Cryptococcus in 83.33 % (10/12) of the samples; 100 % (11/11) were positive via CrAg EIA. The mNGS results of the CSF identified DNA sequences corresponding to Cryptococcus in 75 % of samples (9/12). However, the DNA of both C. neoformans s.l. and C. gattii s.l. was detected concurrently in 33.33 % (4/12). CONCLUSION: mNGS is helpful for identifying Cryptococcus species. The application of mNGS, together with India ink staining, culture methods, and CrAg, may significantly improve the diagnostic precision in CM, thereby informing choice of appropriate antifungal treatment courses.


Assuntos
Cryptococcus gattii/genética , Cryptococcus neoformans/genética , Meningite Criptocócica/diagnóstico , Meningite Criptocócica/microbiologia , Metagenômica , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Idoso , Coinfecção/diagnóstico , Cryptococcus gattii/isolamento & purificação , Cryptococcus neoformans/isolamento & purificação , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Masculino , Meningite Criptocócica/líquido cefalorraquidiano , Técnicas Microbiológicas , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Análise de Sequência de DNA
11.
J Med Microbiol ; 68(8): 1219-1226, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31237534

RESUMO

PURPOSE: The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles. METHODOLOGY: A total of 38 positive BC bottles were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. DNA was extracted from each aliquot using an extraction protocol that combined glass bead beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole-genome sequencing was performed on up to 12 samples through a single MinION flow cell. RESULTS: All GN bacteria identifications made by MinION sequencing for 30 min using the What's In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum ß-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes. CONCLUSION: We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.


Assuntos
Bacteriemia/microbiologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , beta-Lactamases/genética , Antibacterianos/farmacologia , DNA Bacteriano/genética , Genoma Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Nanoporos , Reação em Cadeia da Polimerase/normas , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/normas , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , Fatores de Tempo
12.
PLoS Negl Trop Dis ; 13(6): e0007400, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181059

RESUMO

BACKGROUND: Early detection of Mycobacterium leprae is a key strategy for disrupting the transmission chain of leprosy and preventing the potential onset of physical disabilities. Clinical diagnosis is essential, but some of the presented symptoms may go unnoticed, even by specialists. In areas of greater endemicity, serological and molecular tests have been performed and analyzed separately for the follow-up of household contacts, who are at high risk of developing the disease. The accuracy of these tests is still debated, and it is necessary to make them more reliable, especially for the identification of cases of leprosy between contacts. We proposed an integrated analysis of molecular and serological methods using artificial intelligence by the random forest (RF) algorithm to better diagnose and predict new cases of leprosy. METHODS: The study was developed in Governador Valadares, Brazil, a hyperendemic region for leprosy. A longitudinal study was performed, including new cases diagnosed in 2011 and their respective household contacts, who were followed in 2011, 2012, and 2016. All contacts were diligently evaluated by clinicians from Reference Center for Endemic Diseases (CREDEN-PES) before being classified as asymptomatic. Samples of slit skin smears (SSS) from the earlobe of the patients and household contacts were collected for quantitative polymerase chain reaction (qPCR) of 16S rRNA, and peripheral blood samples were collected for ELISA assays to detect LID-1 and ND-O-LID. RESULTS: The statistical analysis of the tests revealed sensitivity for anti-LID-1 (63.2%), anti-ND-O-LID (57.9%), qPCR SSS (36.8%), and smear microscopy (30.2%). However, the use of RF allowed for an expressive increase in sensitivity in the diagnosis of multibacillary leprosy (90.5%) and especially paucibacillary leprosy (70.6%). It is important to report that the specificity was 92.5%. CONCLUSION: The proposed model using RF allows for the diagnosis of leprosy with high sensitivity and specificity and the early identification of new cases among household contacts.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Características da Família , Saúde da Família , Hanseníase/diagnóstico , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/sangue , Inteligência Artificial , Brasil , Criança , Pré-Escolar , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Adulto Jovem
13.
PLoS Negl Trop Dis ; 13(6): e0007363, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206520

RESUMO

Soil-transmitted helminths (STH) are a major cause of morbidity in tropical developing countries with a global infection prevalence of more than one billion people and disease burden of around 3.4 million disability adjusted life years. Infection prevalence directly correlates to inadequate sanitation, impoverished conditions and limited access to public health systems. Underestimation of infection prevalence using traditional microscopy-based diagnostic techniques is common, specifically in populations with access to benzimidazole mass treatment programs and a predominance of low intensity infections. In this study, we developed a multiplexed-tandem qPCR (MT-PCR) tool to identify and quantify STH eggs in stool samples. We have assessed this assay by measuring infection prevalence and intensity in field samples of two cohorts of participants from Timor-Leste and Cambodia, which were collected as part of earlier epidemiological studies. MT-PCR diagnostic parameters were compared to a previously published multiplexed qPCR for STH detection. The MT-PCR assay agreed strongly with qPCR data and showed a diagnostic specificity of 99.60-100.00% (sensitivity of 83.33-100.00%) compared to qPCR and kappa agreement exceeding 0.85 in all tests. In addition, the MT-PCR has the added advantage of distinguishing Ancylostoma spp. species, namely Ancylostoma duodenale and Ancylostoma ceylanicum. This semi-automated platform uses a standardized, manufactured reagent kit, shows excellent run-to-run consistency/repeatability and supports high-throughput detection and quantitation at a moderate cost.


Assuntos
Fezes/parasitologia , Helmintíase/diagnóstico , Helmintos/isolamento & purificação , Enteropatias Parasitárias/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Camboja , Criança , Pré-Escolar , Feminino , Helmintos/classificação , Helmintos/genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Timor-Leste , Adulto Jovem
14.
PLoS Negl Trop Dis ; 13(6): e0007480, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31158221

RESUMO

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas.


Assuntos
Febre de Chikungunya/diagnóstico , Vírus Chikungunya/isolamento & purificação , Técnicas de Genotipagem/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Análise de Sequência de DNA/métodos , Brasil , Febre de Chikungunya/virologia , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Dessecação , Humanos , Transcrição Reversa , Temperatura Ambiente
15.
Biosensors (Basel) ; 9(2)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117262

RESUMO

Infectious diseases still pose an omnipresent threat to global and public health, especially in many countries and rural areas of cities. Underlying reasons of such serious maladies can be summarized as the paucity of appropriate analysis methods and subsequent treatment strategies due to the limited access of centralized and equipped health care facilities for diagnosis. Biosensors hold great impact to turn our current analytical methods into diagnostic strategies by restructuring their sensing module for the detection of biomolecules, especially nano-sized objects such as protein biomarkers and viruses. Unquestionably, current sensing platforms require continuous updates to address growing challenges in the diagnosis of viruses as viruses change quickly and spread largely from person-to-person, indicating the urgency of early diagnosis. Some of the challenges can be classified in biological barriers (specificity, low number of targets, and biological matrices) and technological limitations (detection limit, linear dynamic range, stability, and reliability), as well as economical aspects that limit their implementation into resource-scarce settings. In this review, the principle and types of biosensors and their applications in the diagnosis of distinct infectious diseases were comprehensively explained. The deployment of current biosensors into resource-scarce settings is further discussed for virus detection by elaborating the pros and cons of existing methods as a conclusion and future perspective.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas de Diagnóstico Molecular/métodos , Viroses/diagnóstico , Animais , Técnicas Eletroquímicas/métodos , Humanos , Vírus/química , Vírus/isolamento & purificação
16.
Biosensors (Basel) ; 9(2)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137893

RESUMO

Detection of the breast cancer cells is important for early diagnosis of the cancer. We applied thickness shear mode acoustics method (TSM) for detection of SK-BR-3 breast cancer cells using DNA aptamers specific to HER2 positive membrane receptors. The biotinylated aptamers were immobilized at the neutravidin layer chemisorbed at gold surface of TSM transducer. Addition of the cells resulted in decrease of resonant frequency, fs, and in increase of motional resistance, Rm. Using gold nanoparticles (AuNPs), modified by aptamers it was possible improving the limit of detection (LOD) that reached 550 cells/mL, while without amplification the sensitivity of the detection of SK-BR-3 cells was 1574 cells/mL. HER2 negative cell line MDA-MB-231 did not resulted in significant changes of fs. The viability studies demonstrated that cells are stable at experimental conditions used during at least 8 h. AuNPs were not toxic on the cells up to concentration of 1 µg/mL.


Assuntos
Acústica/instrumentação , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Neoplasias da Mama/patologia , Receptor ErbB-2/análise , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Ouro/química , Humanos , Nanopartículas Metálicas/química , Técnicas de Diagnóstico Molecular/métodos , Receptor ErbB-2/metabolismo
17.
Dis Markers ; 2019: 5232780, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31089394

RESUMO

Background: Combination of multiple biomarkers was an effective strategy to improve sensitivity in cancer diagnosis and screening. However, the performance of the combination of methylated SEPT9 and SDC2 for detection of colorectal cancer (CRC) has yet to be reported. Methods: A new qPCR-based assay combining the detection of methylated SEPT9 and SDC2 was used. Methylation statuses of SEPT9 and SDC2 were examined in 19 sets of cancer tissues and paired adjacent tissues and further evaluated with 225 serum samples, including 111 CRC patients and 114 no evidence of disease individuals. Results: SEPT9 and SDC2 methylation levels were higher in 94.7% and 100.0% of cancer tissues than in their paired adjacent tissues. The sensitivities for detecting CRC by SEPT9 methylation alone and SDC2 methylation alone were 73.0% (95% CI: 63.6-80.8%) and 71.2% (95% CI: 61.8-79.2%), respectively, with the same specificity of 95.6% (95% CI: 89.6-98.4%). However, when SEPT9 methylation was combined with SDC2 methylation to detect CRC, the sensitivity was improved to 86.5% (95% CI: 78.4-92.0%) with a specificity of 92.1% (95% CI: 85.1-96.1%). Conclusion: The combination of methylated SEPT9 and SDC2 detection in serum has the potential to be a noninvasive strategy for CRC screening.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Técnicas de Diagnóstico Molecular/métodos , Septinas/sangue , Sindecana-2/sangue , Idoso , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Septinas/genética , Sindecana-2/genética
18.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067725

RESUMO

New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods, and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders.


Assuntos
Biomarcadores Tumorais/genética , Leucemia Mieloide Aguda/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Humanos , Leucemia Mieloide Aguda/terapia , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/normas
19.
Korean J Parasitol ; 57(2): 207-211, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31104416

RESUMO

Anisakiasis is a zoonotic disease induced by anisakid nematodes, and endoscopic inspection is used for a diagnosis or remedy for it. Anisakis simplex, Anisakis physeteris, and Pseudoterranova decipiens had been reported to be the major species causing human infections, particularly, in Japan. However, in Korea, recent studies strongly suggested that Anisakis pegreffii is the major species of human infections. To support this suggestion, we collected anisakid larvae (n=20) from 20 human patients who were undergone gastrointestinal endoscopy at a health check-up center in Korea, and molecular identification was performed on the larvae using PCR-RFLP analysis and gene sequencing of rDNA ITS regions and mtDNA cox2. In addition, anisakid larvae (n=53) collected from the sea eel (Astroconger myriaster) were also examined for comparison with those extracted from humans. The results showed that all human samples (100%) were identified as A. pegreffii, whereas 90.7% of the samples from the sea eel were A. pegreffii with the remaining 9.3% being Hysterothylacium aduncum. Our study confirmed that A. pegreffii is the predominant species causing human anisakiasis in Korea, and this seems to be due to the predominance of this larval type in the fish (sea eels) popularly consumed by the Korean people. The possibility of human infection with H. aduncum in Korea is also suggested.


Assuntos
Anisaquíase/diagnóstico , Anisakis/classificação , Anisakis/isolamento & purificação , Endoscopia Gastrointestinal/métodos , Técnicas de Diagnóstico Molecular/métodos , Adulto , Idoso , Animais , Anisaquíase/veterinária , Anisakis/genética , Ciclo-Oxigenase 2/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Enguias/parasitologia , Feminino , Doenças dos Peixes/parasitologia , Humanos , Larva/classificação , Larva/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , República da Coreia , Análise de Sequência de DNA
20.
Int J Mol Sci ; 20(9)2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064083

RESUMO

Avian infectious bronchitis virus (IBV) causes considerable economic losses in the poultry industry worldwide, including Taiwan. IBV is among the most important pathogens in chickens, and it spreads rapidly among flocks. In addition to dozens of known serotypes, new viral variants have emerged due to the viral evolution and antigenic variation in IBVs. Therefore, the development of a sensitive, specific, and easily performed assay is crucial for the rapid detection and surveillance of IBV infections. A rapid and simple immunochromatographic strip (ICS) was developed in this study by employing monoclonal antibodies against spike and nucleocapsid proteins of IBV as the tracer and the capture antibody. The ICS showed high specificity in detecting IBV antigens, including several IBV genotypes and novel variants, as opposed to three other common avian respiratory viruses. The detection limit of the strip reached 104.4 50% embryo-infective dose. Moreover, in the experimental chicken model, the strip test demonstrated consistency in detecting IBV with RT-PCR gene detection. Taken together, this antigen detection strip has the potential to serve as an on-farm rapid test for IBV; therefore, it may facilitate surveillance and control of the disease.


Assuntos
Infecções por Coronavirus/diagnóstico , Vírus da Bronquite Infecciosa/imunologia , Técnicas de Diagnóstico Molecular/veterinária , Doenças das Aves Domésticas/diagnóstico , Animais , Antígenos Virais/imunologia , Galinhas , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Feminino , Imunoensaio/métodos , Imunoensaio/normas , Imunoensaio/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Doenças das Aves Domésticas/virologia , Fitas Reagentes/normas
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