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1.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 1018-1022, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31484272

RESUMO

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.


Assuntos
Bactérias/genética , DNA Bacteriano/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Replicação de Sequência Autossustentável/métodos , Primers do DNA , Humanos , Sensibilidade e Especificidade
2.
Cochrane Database Syst Rev ; 9: CD009551, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31478559

RESUMO

BACKGROUND: This is an update of the original review published in the Cochrane Database of Systematic Reviews Issue 10, 2015.Invasive aspergillosis (IA) is the most common life-threatening opportunistic invasive mould infection in immunocompromised people. Early diagnosis of IA and prompt administration of appropriate antifungal treatment are critical to the survival of people with IA. Antifungal drugs can be given as prophylaxis or empirical therapy, instigated on the basis of a diagnostic strategy (the pre-emptive approach) or for treating established disease. Consequently, there is an urgent need for research into both new diagnostic tools and drug treatment strategies. Increasingly, newer methods such as polymerase chain reaction (PCR) to detect fungal nucleic acids are being investigated. OBJECTIVES: To provide an overall summary of the diagnostic accuracy of PCR-based tests on blood specimens for the diagnosis of IA in immunocompromised people. SEARCH METHODS: We searched MEDLINE (1946 to June 2015) and Embase (1980 to June 2015). We also searched LILACS, DARE, Health Technology Assessment, Web of Science and Scopus to June 2015. We checked the reference lists of all the studies identified by the above methods and contacted relevant authors and researchers in the field. For this review update we updated electronic searches of the Cochrane Central Register of Controlled Trials (CENTRAL; 2018, Issue 3) in the Cochrane Library; MEDLINE via Ovid (June 2015 to March week 2 2018); and Embase via Ovid (June 2015 to 2018 week 12). SELECTION CRITERIA: We included studies that: i) compared the results of blood PCR tests with the reference standard published by the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG); ii) reported data on false-positive, true-positive, false-negative and true-negative results of the diagnostic tests under investigation separately; and iii) evaluated the test(s) prospectively in cohorts of people from a relevant clinical population, defined as a group of individuals at high risk for invasive aspergillosis. Case-control and retrospective studies were excluded from the analysis. DATA COLLECTION AND ANALYSIS: Authors independently assessed quality and extracted data. For PCR assays, we evaluated the requirement for either one or two consecutive samples to be positive for diagnostic accuracy. We investigated heterogeneity by subgroup analyses. We plotted estimates of sensitivity and specificity from each study in receiver operating characteristics (ROC) space and constructed forest plots for visual examination of variation in test accuracy. We performed meta-analyses using the bivariate model to produce summary estimates of sensitivity and specificity. MAIN RESULTS: We included 29 primary studies (18 from the original review and 11 from this update), corresponding to 34 data sets, published between 2000 and 2018 in the meta-analyses, with a mean prevalence of proven or probable IA of 16.3 (median prevalence 11.1% , range 2.5% to 57.1%). Most patients had received chemotherapy for haematological malignancy or had undergone hematopoietic stem cell transplantation. Several PCR techniques were used among the included studies. The sensitivity and specificity of PCR for the diagnosis of IA varied according to the interpretative criteria used to define a test as positive. The summary estimates of sensitivity and specificity were 79.2% (95% confidence interval (CI) 71.0 to 85.5) and 79.6% (95% CI 69.9 to 86.6) for a single positive test result, and 59.6% (95% CI 40.7 to 76.0) and 95.1% (95% CI 87.0 to 98.2) for two consecutive positive test results. AUTHORS' CONCLUSIONS: PCR shows moderate diagnostic accuracy when used as screening tests for IA in high-risk patient groups. Importantly the sensitivity of the test confers a high negative predictive value (NPV) such that a negative test allows the diagnosis to be excluded. Consecutive positives show good specificity in diagnosis of IA and could be used to trigger radiological and other investigations or for pre-emptive therapy in the absence of specific radiological signs when the clinical suspicion of infection is high. When a single PCR positive test is used as the diagnostic criterion for IA in a population of 100 people with a disease prevalence of 16.3% (overall mean prevalence), three people with IA would be missed (sensitivity 79.2%, 20.8% false negatives), and 17 people would be unnecessarily treated or referred for further tests (specificity of 79.6%, 21.4% false positives). If we use the two positive test requirement in a population with the same disease prevalence, it would mean that nine IA people would be missed (sensitivity 59.6%, 40.4% false negatives) and four people would be unnecessarily treated or referred for further tests (specificity of 95.1%, 4.9% false positives). Like galactomannan, PCR has good NPV for excluding disease, but the low prevalence of disease limits the ability to rule in a diagnosis. As these biomarkers detect different markers of disease, combining them is likely to prove more useful.


Assuntos
Aspergilose/sangue , Aspergilose/diagnóstico , Hospedeiro Imunocomprometido , Infecções Oportunistas , Reação em Cadeia da Polimerase/métodos , Estudos de Casos e Controles , Humanos , Infecções Oportunistas/sangue , Infecções Oportunistas/diagnóstico , Valor Preditivo dos Testes , Sensibilidade e Especificidade
3.
Arch Virol ; 164(11): 2775-2781, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31401693

RESUMO

Diagnosis and epidemiological analysis of human parvovirus B19 (hB19V) infections are essential for disease management in severely ill patients. This study aimed to evaluate the performance of an optimized NS1-VP1u nested PCR for detection and sequencing of viruses in clinical samples using 224 clinical and five reference samples. PCR sensitivity, specificity, and positive and negative predictive values were perfect (100%). While phylogenetic analysis of a 615 bp-long fragment demonstrated that the viruses in all of the samples belonged to genotype 1, this study confirmed that this optimized PCR could detect all known hB19V with high performance.


Assuntos
Proteínas do Capsídeo/genética , Eritema Infeccioso/diagnóstico , Eritema Infeccioso/epidemiologia , Parvovirus B19 Humano/genética , Proteínas não Estruturais Virais/genética , DNA Viral/genética , Eritema Infeccioso/virologia , Humanos , Filogenia , Reação em Cadeia da Polimerase/métodos
4.
J Appl Oral Sci ; 27: e20180256, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365706

RESUMO

OBJECTIVE: The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. METHODOLOGY: Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). RESULTS: Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). CONCLUSIONS: The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.


Assuntos
DNA Ribossômico/isolamento & purificação , Cavidade Pulpar/microbiologia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Streptococcus/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , Humanos , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico/genética , Reprodutibilidade dos Testes , Tratamento do Canal Radicular/métodos , Streptococcus/genética
5.
Niger J Clin Pract ; 22(8): 1083-1090, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31417051

RESUMO

Aims: The aim of this study was to provide epidemiological data about the presence of Salmonella spp. and Shigella spp. in raw milk samples collected from different animals. Methods: A total of 231 raw milk samples from 48 cows, 65 goats, 65 sheep, and 53 donkeys were studied. The ISO 6579:2002 and ISO 21567:2004 methods, antimicrobial susceptibility tests, and serotyping were performed. Species and subspecies discriminations were made via matrix-assisted laser desorption/ionization-time of flight mass spectrometry. After DNA isolation from all samples, Salmonella spp. and Shigella spp. were detected using real-time polymerase chain reaction (PCR) kits. Results: Five samples (2.16%) showed positivity out of 231 raw milk samples for Salmonella spp., and 2 (0.87%) samples were detected to be positive by multiplex real-time PCR design. Conclusion: We found that raw milk samples were not free of Salmonella spp. and Shigella spp. and need to be tested routinely to avoid public health problems. Rapid and reliable real-time PCR method can be developed and used for this purposes instead of slow bacterial culture processes.


Assuntos
DNA Bacteriano/análise , Contaminação de Alimentos/análise , Leite/microbiologia , Reação em Cadeia da Polimerase/métodos , Salmonella/genética , Salmonella/isolamento & purificação , Shigella/genética , Shigella/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Equidae , Feminino , Cabras , Humanos , Salmonella/classificação , Sensibilidade e Especificidade , Ovinos , Shigella/classificação
6.
Cochrane Database Syst Rev ; 8: CD011871, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31425612

RESUMO

BACKGROUND: Early diagnosis of leptospirosis may contribute to the effectiveness of antimicrobial therapy and early outbreak recognition. Nucleic acid and antigen detection tests have the potential for early diagnosis of leptospirosis. With this systematic review, we assessed the sensitivity and specificity of nucleic acid and antigen detection tests. OBJECTIVES: To determine the diagnostic test accuracy of nucleic acid and antigen detection tests for the diagnosis of human symptomatic leptospirosis. SEARCH METHODS: We searched electronic databases including MEDLINE, Embase, the Cochrane Library, and regional databases from inception to 6 July 2018. We did not apply restrictions to language or time of publication. SELECTION CRITERIA: We included diagnostic cross-sectional studies and case-control studies of tests that made use of nucleic acid and antigen detection methods in people suspected of systemic leptospirosis. As reference standards, we considered the microscopic agglutination test alone (which detects antibodies against leptospirosis) or in a composite reference standard with culturing or other serological tests. Studies were excluded when the controls were healthy individuals or when there were insufficient data to calculate sensitivity and specificity. DATA COLLECTION AND ANALYSIS: At least two review authors independently extracted data from each study. We used the revised Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2) to assess risk of bias. We calculated study-specific values for sensitivity and specificity with 95% confidence intervals (CI) and pooled the results in a meta-analysis when appropriate. We used the bivariate model for index tests with one positivity threshold, and we used the hierarchical summary receiver operating characteristic model for index tests with multiple positivity thresholds. As possible sources of heterogeneity, we explored: timing of index test, disease prevalence, blood sample type, primers or target genes, and the real-time polymerase chain reaction (PCR) visualisation method. These were added as covariates to the meta-regression models. MAIN RESULTS: We included 41 studies evaluating nine index tests (conventional PCR (in short: PCR), real-time PCR, nested PCR, PCR performed twice, loop-mediated isothermal amplification, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, immunochromatography-based lateral flow assay, and dipstick assay) with 5981 participants (1834 with and 4147 without leptospirosis). Methodological quality criteria were often not reported, and the risk of bias of the reference standard was generally considered high. The applicability of findings was limited by the frequent use of frozen samples. We conducted meta-analyses for the PCR and the real-time PCR on blood products.The pooled sensitivity of the PCR was 70% (95% CI 37% to 90%) and the pooled specificity was 95% (95% CI 75% to 99%). When studies with a high risk of bias in the reference standard domain were excluded, the pooled sensitivity was 87% (95% CI 44% to 98%) and the pooled specificity was 97% (95% CI 60% to 100%). For the real-time PCR, we estimated a summary receiver operating characteristic curve. To illustrate, a point on the curve with 85% specificity had a sensitivity of 49% (95% CI 30% to 68%). Likewise, at 90% specificity, sensitivity was 40% (95% CI 24% to 59%) and at 95% specificity, sensitivity was 29% (95% CI 15% to 49%). The median specificity of real-time PCR on blood products was 92%. We did not formally compare the diagnostic test accuracy of PCR and real-time PCR, as direct comparison studies were lacking. Three of 15 studies analysing PCR on blood products reported the timing of sample collection in the studies included in the meta-analyses (range 1 to 7 days postonset of symptoms), and nine out of 16 studies analysing real-time PCR on blood products (range 1 to 19 days postonset of symptoms). In PCR studies, specificity was lower in settings with high leptospirosis prevalence. Other investigations of heterogeneity did not identify statistically significant associations. Two studies suggested that PCR and real-time PCR may be more sensitive on blood samples collected early in the disease stage. Results of other index tests were described narratively. AUTHORS' CONCLUSIONS: The validity of review findings are limited and should be interpreted with caution. There is a substantial between-study variability in the accuracy of PCR and real-time PCR, as well as a substantial variability in the prevalence of leptospirosis. Consequently, the position of PCR and real-time PCR in the clinical pathway depends on regional considerations such as disease prevalence, factors that are likely to influence accuracy, and downstream consequences of test results. There is insufficient evidence to conclude which of the nucleic acid and antigen detection tests is the most accurate. There is preliminary evidence that PCR and real-time PCR are more sensitive on blood samples collected early in the disease stage, but this needs to be confirmed in future studies.


Assuntos
Anticorpos Antibacterianos/imunologia , Leptospira/imunologia , Leptospirose/diagnóstico , Ácidos Nucleicos/sangue , Reação em Cadeia da Polimerase/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Leptospirose/sangue , Curva ROC , Sensibilidade e Especificidade
7.
Chem Commun (Camb) ; 55(58): 8466-8469, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31265022

RESUMO

We presented a branch migration based PCR in which a branch migration blocker was introduced to selectively reduce the amplification efficiency of the wild-type target and enrich the mutant-type target. The low-abundance mutations could be enriched and then detected by high resolution melting, Sanger sequencing or fluorescent DNA probe.


Assuntos
Análise Mutacional de DNA/métodos , DNA/análise , DNA/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Sondas de DNA/química , Sondas de DNA/genética , DNA Polimerase Dirigida por DNA/química , Fluorescência , Corantes Fluorescentes/química , Genes , Humanos , Limite de Detecção , Mutação , Hibridização de Ácido Nucleico
8.
J Agric Food Chem ; 67(29): 8268-8278, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31283221

RESUMO

Species authentication of meat and fish products is crucial to safeguard public health, economic investment, and religious sanctity. We developed a heptaplex polymerase chain reaction assay targeting short amplicon length (73-198 bp) for the simultaneous detection and differentiation of cow, buffalo, chicken, cat, dog, pig, and fish species in raw and processed food using species-specific primers targeting mitochondrial cytb, ND5, and 16s rRNA genes. Assay validation of adulterated and various heat-treated meatball matrices showed excellent stability and sensitivity under all processing conditions. The detection limit was 0.01-0.001 ng of DNA under pure states and 0.5% meat in meatball products. Buffalo was detected in 86.7% (13 out of 15) of tested commercial beef products, while chicken, pork, and fish products were found to be pure. The developed assay was efficient enough to detect target species simultaneously, even in highly degraded and processed food products at reduced time.


Assuntos
Contaminação de Alimentos/análise , Produtos da Carne/análise , Reação em Cadeia da Polimerase/métodos , Animais , Búfalos/genética , Gatos/genética , Bovinos/genética , Galinhas/genética , Cães/genética , Peixes/genética , Suínos/genética
9.
BMC Infect Dis ; 19(1): 598, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31288744

RESUMO

BACKGROUND: In Japan and other countries, the number of patients with syphilis is increasing year by year. Recently, the cases of the pulmonary involvement in patients with secondary syphilis have been reported. However, it is still undetermined how to obtain a desirable specimen for a diagnosis of the pulmonary involvement, and how to treat it if not cured. CASE PRESENTATION: A 34-year-old man presented with cough and swelling of the right inguinal nodes. A physical examination revealed erythematous papular rash over the palms, soles and abdomen. A 4 cm mass in the right lower lobe of the lung was detected on computed tomography. He was diagnosed as having secondary syphilis, because he was tested positive for the rapid plasma reagin and Treponema pallidum hemagglutination assay. Amoxycillin and probenecid were orally administered for 2 weeks. Subsequently, rash and serological markers were improved, however, the lung mass remained unchanged in size. Transbronchial biopsy (TBB) confirmed the pulmonary involvement of syphilis using polymerase chain reaction techniques (tpp47- and polA-PCR). Furthermore, following surgical resection revealed the lung mass to be an abscess. CONCLUSIONS: To our knowledge, this is the first surgically treated case of a lung abscess caused by syphilis, which was diagnosed by PCR techniques in TBB. This report could propose a useful diagnostic method for the pulmonary involvement of syphilis.


Assuntos
Reação em Cadeia da Polimerase/métodos , Sífilis/diagnóstico , Adulto , Brônquios/microbiologia , Brônquios/patologia , Proteína C-Reativa/análise , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Humanos , Masculino , Sífilis/microbiologia , Tomografia Computadorizada por Raios X , Treponema pallidum/genética , Treponema pallidum/isolamento & purificação
10.
Prev Vet Med ; 169: 104712, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31311647

RESUMO

Surra is a zoonotic disease caused by Trypanosoma evansi, affecting the health and production of the livestock significantly. There are several methods to diagnose this disease, which have different principles, sensitivity, and specificity. Among them, the serological techniques using T. evansi as antigen are powerful tools for its epidemiological surveillance. However, they are poorly used due to inefficient in vitro propagation of T. evansi, which requires the use of laboratory animals for antigen production. In the present study, whole cell lysate of T. brucei brucei propagated in vitro was used as an antigen for the detection of anti-T. evansi immunoglobulin G in cattle through an indirect-ELISA. Based on a total of 45 samples from non-infected and 45 samples from T. evansi infected cattle, the sensitivity and specificity were estimated as 100% and 97.7%, respectively. After the validation, serological and molecular surveys were carried out in 710 cattle samples from two endemic Colombian regions (Antioquia and Arauca departments) for T. evansi where molecular prevalences of ˜7.0% were detected through the year and sporadic outbreaks of T. vivax infections have been associated to low prevalence of this species (<1%). A total of 424 (59.7%) samples were positive by indirect-ELISA T. b. brucei, while PCR test for T. evansi and T. vivax, showed 49 (6.9%) and no positive samples, respectively. Interestingly, categories of animals aged>1 year, Bos taurus breed, and those raised under intensive farming system exhibited a higher seroprevalence to T. evansi (P < 0.05). The results displayed a new alternative for antibody detection anti-T. evansi in livestock, using parasites propagated in vitro as antigen, which presents the advantage of higher standardization potential, and avoid the use of live animal for antigen production. A larger availability of this ELISA will generate useful information for a better understanding of the epidemiologic aspects, as well as for the management and control of these diseases in Colombia. However, the ability of the test to detect and/or cross react with T. vivax infections remains to be investigated.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/veterinária , Reação em Cadeia da Polimerase/veterinária , Trypanosoma brucei brucei/isolamento & purificação , Animais , Bovinos , Colômbia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G , Gado , Reação em Cadeia da Polimerase/métodos
11.
Parasit Vectors ; 12(1): 360, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340841

RESUMO

BACKGROUND: Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37-42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1-10 target DNA copies in less than 20 minutes. METHODS: We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye. RESULTS: The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20-25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field. CONCLUSIONS: Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.


Assuntos
Alveolados/isolamento & purificação , Crassostrea/parasitologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Protozoárias em Animais/diagnóstico , Alveolados/genética , Animais , Primers do DNA/genética , DNA Intergênico/genética , Visualização de Dados , Eletroforese em Gel de Ágar , Reação em Cadeia da Polimerase/métodos , Infecções por Protozoários , Recombinases/genética , Sensibilidade e Especificidade
12.
BMC Infect Dis ; 19(1): 574, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31269905

RESUMO

BACKGROUND: Blood donor plasma samples were detected by the Ultrio Plus NAT system for HBV, HCV and HIV-1 in Shenzhen blood center, China. Reactive samples underwent further discriminatory testing of a single virus by the same methodology. A large number of cases of non-discriminated reactive (NDR) donors were found, leaving potential risk of transmitting HBV if not deferrals. This study identified those non-discriminated samples. METHODS: The NDR plasma samples from blood donation screening were detected and classified by additional molecular and serological tests. Molecular characterizations of DNA+ NDR were determined by sequencing analysis. RESULTS: A number of 259 (0.21%) NDR plasma samples from screening of 123,280 eligible blood donors were detected, which presented a higher rate (91.1%) of anti-HBc reactivity and nearly half (46.7%) of HBV DNA+ that classified as occult HBV infection (OBI). Most OBI strains were wild-type HBV, but some substitutions V168A, S174 N, V177A, Q129R/L/H, G145A/R in S region of genotype B (OBIB) and T47K/V/A, P49H/L, Q101R/H/K, S174 N, L175S, V177A, T118 M/R/K, G145R/A/K/E, R160K/N in S region of genotype C (OBIC) strains were identified in high frequency. CONCLUSION: Nearly half of NDR blood samples were identified as OBI, in which a number of important mutations were detected. NDR donation might have potential risk for HBV transmission, but need to be further investigated.


Assuntos
Doadores de Sangue , Vírus da Hepatite B/genética , Hepatite B/virologia , Adulto , China , Feminino , Genótipo , HIV-1/genética , Hepacivirus/genética , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase/métodos , Testes Sorológicos/métodos
13.
BMC Genomics ; 20(Suppl 7): 536, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31291895

RESUMO

BACKGROUND: Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study; ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. The sequences of BC-ROI combinations present in the libraries may be either known a priori or not. In the latter case, it is necessary to identify these combinations before performing functional experiments. Typically, this is done by PCR amplification of the BC-ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. However, chimeric DNA molecules formed on templates with identical spacer fragment during the amplification process may substantially hamper the identification of genuine BC-ROI combinations, and as a result lower the performance of the assays. RESULTS: To identify settings that minimize formation of chimeric products we tested a number of PCR amplification parameters, such as conventional and emulsion types of PCR, one- or two-round amplification strategies, amount of DNA template, number of PCR cycles, and the duration of the extension step. Using specific MPRA libraries as templates, we found that the two-round amplification of the BC-ROI regions with a very low initial template amount, an elongated extension step, and a specific number of PCR cycles result in as low as 0.30 and 0.32% of chimeric products for emulsion and conventional PCR approaches, respectively. CONCLUSIONS: We have identified PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries. In addition, we found that there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings.


Assuntos
DNA/genética , Biblioteca Gênica , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Primers do DNA , Sequenciamento de Nucleotídeos em Larga Escala , Moldes Genéticos
14.
Medicine (Baltimore) ; 98(29): e15992, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31335667

RESUMO

Patients with tuberculous pleurisy often remain undiagnosed even after blind thoracentesis and closed pleural biopsy (PB). In this study, we assessed the value of computed tomography (CT)-guided core needle biopsy of pleural lesion and evaluated the diagnostic accuracy of polymerase chain reaction (PCR)/staining for acid-fast bacilli (AFB) in suspicious tuberculous pleurisy undiagnosed in blind thoracentesis.Patients with exudative pleural effusion (PE) without specific etiology after blind thoracentesis and closed PB were enrolled in this study. PB specimens were obtained through CT-guided core needle biopsy of pleural lesion, then underwent PCR, AFB, histopathological examination, and some routine tests. Diagnostic values were evaluated through sensitivity, specificity, negative predictive value, positive predictive value, and accuracy.A total of 261 participants (TB group: 241, non-TB group: 20) were recruited. In this cohort, the sensitivity, specificity, and accuracy were 56.0%, 95.0%, and 59.0%, respectively for PCR, whereas 57.3%, 95.0%, and 60.2%, respectively for AFB. Their parallel test achieved an improved sensitivity (76.8%) and accuracy (77.8%), with a slight decrease in specificity (90.0%). In histopathological examination, granuloma was the most common finding in TB group (88.4%, 213/241), but also observed in non-TB group (10.0%, 2/20). In addition, pleural lymphocyte percentage in TB group was significantly higher than that of non-TB group (92% vs 61%, respectively; P = .003). However, no significant differences were found for other biomarkers.CT-guided core needle PB is essential for patients with exudative PE but undiagnosed after blind thoracentesis. Combining with PCR and AFB, it strongly improves the diagnosis of tuberculous pleurisy.


Assuntos
Biópsia Guiada por Imagem/métodos , Mycobacterium tuberculosis/isolamento & purificação , Pleura , Derrame Pleural/diagnóstico , Tuberculose Pleural/diagnóstico , Biópsia com Agulha de Grande Calibre/métodos , Confiabilidade dos Dados , Diagnóstico Diferencial , Erros de Diagnóstico/prevenção & controle , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Pleura/microbiologia , Pleura/patologia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , Reprodutibilidade dos Testes , Toracentese/métodos , Tomografia Computadorizada por Raios X/métodos , Tuberculose Pleural/microbiologia
15.
BMC Infect Dis ; 19(1): 589, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277586

RESUMO

BACKGROUD: Early diagnosis of gastric tuberculosis is often challenging because the disease is very rare and its clinical manifestation is nonspecific and misleading. To raise the awareness and emphasize early diagnosis of gastric tuberculosis, we present a case of gastric tuberculosis secondary to pleural and pulmonary tuberculosis. CASE PRESENTATION: A 26-year-old woman complained gastric pain for 1 month but showed no other symptoms, who had no previous exposure to tuberculosis.Gastric stromal tumor was originally suspected. However, the pathology of her gastroscopic biopsy of the gastric lesion showed granulomatous lesions and caseating necrosis. Gene sequencing of the biopsy specimen identified deoxyribonucleic acid fragment of Mycobacterium tuberculosis. Chest computed tomography scan revealed nodular shadows in the lesser curvature soft tissue of the stomach, patchy densities and calcified nodular shadows in the upper right lung, bilateral pleural thickening, and calcified pleural nodules. Thus, the diagnosis was gastric tuberculosis secondary to pulmonary and pleural tuberculosis. The patient was hospitalized and treated with the antituberculosis therapy for 1 week. After discharged from the hospital, the patient continued routine antituberculosis therapy for 18 months and was follow-up was normal.Literature search found 22 cases of gastric tuberculosis reported from 2000 to 2016. Review of the 22 cases suggested that polymerase chain reaction has been increasingly used in the recent years in addition to the conventional histopathological and bacteriological approaches. CONCLUSION: Clinical presentation of gastric tuberculosis is not specific.When granuloma or caseation is detected on biopsy in patients who are suspected of having gastric malignancy or acid peptic diseases, polymerase chain reaction for Mycobacterium tuberculosis could be used as an available and sensitive diagnostic test in addition to pathology, acid-fast bacilli smear staining and culture.


Assuntos
Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Tuberculose Gastrointestinal/diagnóstico , Adulto , Antituberculosos/uso terapêutico , Biópsia , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pulmão/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Gastrointestinal/tratamento farmacológico , Tuberculose Pleural/diagnóstico , Tuberculose Pleural/tratamento farmacológico , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico
16.
BMC Infect Dis ; 19(1): 623, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307404

RESUMO

BACKGROUND: Human leishmaniasis is one of the major parasitic diseases with worldwide distribution. Sri Lanka is a recently established focus of leishmaniasis caused by a variant Leishmania donovani. Early case detection and management is a main approach identified for L. donovani control in the regional leishmaniasis elimination drive. Usefulness of light microscopy and in-vitro culture are limited in chronic, atypical or treated lesions though timely and accurate detection of all light microscopy/in-vitro culture negative cases of all forms of leishmaniasis is necessary for treatment. Timely treatment is important to minimize risk for death in visceral disease and undesired sequelae of long standing infection and illness on both patients and community. We described a 100% sensitive, Leishmania spp. specific modified version of a nested PCR (Mo-STNPCR) that also minimizes carry over and cross contaminations while facilitate investigation of light microscopy and in-vitro culture negative clinically suggestive cases of leishmaniasis. METHODS: Leishmania DNA was amplified using previously published P221: 5'-GGTTCCTTTCCTGATTTACG-3' and P332: 5'-GGCCGGTAAAGGCCGAATAG-3'outer primers followed by a nested reaction using P223: 5'-TCCCATCGCAACCTCGGTT-3' and P333: 5'-AAGCGGGCGCGGTGCTG-3' inner primers that by passes the requirement of tube handling between the two steps of the conventional nested PCR. Leishmania DNA was detected in a range of infected tissue material. Infected material from patients with cutaneous leishmaniasis (n = 30), visceral leishmaniasis (n = 10) and from a control group including patients with non-leishmanial skin diseases (n = 10), other systemic diseases (n = 10) and healthy individuals (n = 10) were examined with Mo-STNPCR. Results were further compared with those of light microscopy and in-vitro culture. RESULTS: Mo-STNPCR method was 100% sensitive and 100% specific for diagnosis of leishmaniasis. Light microscopy and in-vitro culture were positive in 75.0% (n = 30/40) and 72.5% (n = 29/40) samples respectively where combined results of them gave 87.5% (n = 35/40) sensitivity. Mo-STNPCR did not cross react with control samples. Furthermore, Mo-STNPCR reduces the risk of cross-contaminations and carry over contaminations since the full reaction is carried out without opening the tubes. Per patient cost was calculated as 22 USD while the same was 3 and 6 USD for light microscopy and in-vitro culture respectively. CONCLUSION: Mo-STNPCR method is a useful tool in detecting leishmaniasis in minority of cases that go undetected by first line investigations.


Assuntos
Leishmania donovani/genética , Leishmaniose/diagnóstico , Sequência de Bases , Primers do DNA/metabolismo , DNA de Protozoário/isolamento & purificação , DNA de Protozoário/metabolismo , Humanos , Leishmania donovani/isolamento & purificação , Leishmaniose/parasitologia , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Alinhamento de Sequência
17.
World J Microbiol Biotechnol ; 35(7): 104, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31236765

RESUMO

Endophytic bacterial diversity in plants presents the level of interaction between culturable and non-culturable endophytic bacteria, thereby providing an appropriate insight into the endophytic environment. This study was conducted to determine the trend of culturable and non-culturable endophytic bacteria at two different sites encompassing four consecutive growth stages. For culturable endophytic bacteria, isolation was carried out using the dilution plate technique, and the obtained colonies were compared using PCR-restriction fragment length polymorphism (RFLP). Different RFLP-types were identified to their nearest neighbour using 16S rRNA sequencing. The non-culturable endophytic bacterial diversity was obtained by next generation sequencing. Results suggested a similar trend among the culturable and non-culturable bacteria for observed operational taxonomic units and diversity indices. It is noticeable that the endophytic bacteria inhabiting in stage 1 disappeared, and instead, different endophytic bacteria appeared. Moreover, the temporal persistence of certain culturable and non-culturable bacteria was also observed. In conclusion, the endophytic bacterial diversity in cucumber initially increased with the plant growth and then decreased at a later stage. Furthermore, it was suggested that plants regulate the number and diversity of endophytes throughout the lifecycle of plants.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biodiversidade , Cucumis sativus/microbiologia , Endófitos/classificação , Endófitos/isolamento & purificação , Microbiota , Bactérias/genética , Cucumis sativus/crescimento & desenvolvimento , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Endófitos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Microbiota/genética , Filogenia , Raízes de Plantas/microbiologia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética
18.
J Med Microbiol ; 68(7): 1003-1011, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31172912

RESUMO

PURPOSE: The present study aimed to establish pretreatment protocols as well as real-time and droplet digital polymerase chain reaction (PCR) methodologies to detect and quantify Erysipelothrix rhusiopathiae (ER) DNA in blood samples from infected chickens, as tools for routine diagnostics and monitoring of experimental infections. Chicken blood is a problematic matrix for PCR analysis because nucleated erythrocytes contribute large amounts of host DNA that inhibit amplification. METHODOLOGY: Using artificially spiked samples of fresh chicken blood, as well as blood samples from three experimental infection studies, the performance of pretreatment protocols, including choice of blood stabilization agent, centrifugation speeds and Ficoll gradient separation, was evaluated. The results were compared with those from traditional culture-based protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Results/Key findings. Simple preparations producing cell-free samples performed well on artificial spike-in samples, providing high sensitivity. However, performance was poor in clinical samples or artificial samples where the bacteria were incubated for 4 h or more in fresh blood prior to DNA extraction. In these samples, a Ficoll separation protocol that creates samples rich in lymphocytes, monocytes and thrombocytes prior to DNA extraction was far more effective. CONCLUSIONS: Our results indicate that ER bacteria undergo rapid phagocytosis in chicken blood and that analysis of a blood fraction enriched for phagocytic cells is necessary for reliable detection and quantification. The presented results explain the poor performance of PCR detection reported in previously published experimental ER infection studies, and the proposed solutions are likely to have broader implications for PCR-based veterinary diagnostics in non-mammalian host species such as poultry and fish.


Assuntos
Galinhas/microbiologia , DNA Bacteriano/genética , Infecções por Erysipelothrix/microbiologia , Erysipelothrix/genética , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/microbiologia , Animais , Erysipelothrix/isolamento & purificação , Infecções por Erysipelothrix/diagnóstico , Eritrócitos/citologia , Eritrócitos/microbiologia , Reação em Cadeia da Polimerase/métodos
19.
Anal Bioanal Chem ; 411(20): 5297-5307, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161322

RESUMO

The design and fabrication of a continuous-flow µPCR device with very short amplification time and low power consumption are presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9-m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the µPCR device in the established PCB industry. The µPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering the presented µPCR one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Detailed numerical calculations of the DNA residence time distributions, within an acceptable temperature range for denaturation, annealing, and extension, performed for the first time in the literature, provide useful information regarding the actual on-chip PCR protocol and justify the maximum volumetric flow rate for successful DNA amplification. The calculations indicate that the shortest amplification time is achieved when the device is operated at its enzyme kinetic limit (i.e., extension rate). Graphical abstract.


Assuntos
DNA/química , Dispositivos Lab-On-A-Chip , Manufaturas , Bifenilos Policlorados/química , Reação em Cadeia da Polimerase/métodos
20.
BMC Infect Dis ; 19(1): 495, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164085

RESUMO

BACKGROUND: There is currently no research on the diagnostic value of metagenomic next-generation sequencing (mNGS) for a single pathogens in CSF. The aim of this study was to analyse the value of mNGS for identifying Streptococcus pneumoniae (S. pneumoniae) in paediatric bacterial meningitis. METHODS: Bacterial meningitis (BM) cases from October 23, 2014, to December 31, 2016, and December 1, 2017, to July 31, 2018 at Beijing Children's Hospital were reviewed. Clinical features and pathogens were analysed. RESULTS: We diagnosed 135 patients with BM in this study. A total of 43 S. pneumoniae were identified by combination methods. 26/135 (19.3%) patients had positive results in S. pneumoniae by blood and/or cerebrospinal fluid (CSF) culture. Alere BinaxNow®Streptococcus pneumoniae Antigen test was positive in 35/135(25.9%) cases. 32/135 (23.7%) S. pneumoniae were identified by mNGS. Six CSF samples were identified as S. pneumoniae only by mNGS technology. Taking culture as the gold standard, the sensitivity and specificity of mNGS for diagnosing S. pneumoniae meningitis were 73.1 and 88.1%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of diagnosing S. pneumoniae meningitis by mNGS were 59.4 and 93.2%, respectively. When comparison between mNGS and combined tests (culture and Alere BinaxNow®Streptococcus pneumoniae Antigen test), the sensitivity and specificity of mNGS for S. pneumoniae identification were 70.3 and 93.9%, the PPV and NPV in the identification of S. pneumoniae by mNGS were 81.4 and 89.3%, respectively. The difference in number of unique reads of S. pneumoniaein from CSF sample (< 14 days onset) and CSF sample (> 14 days from onset) was statistically significant (170.5 VS. 13, P = 0.019). The difference in the collected time of CSF for culture and mNGS was statistically significant (4 days VS. 14 days, P < 0.001). CONCLUSIONS: mNGS has high sensitivity and specificity for S. pneumoniae identification. The pathogen load (number of unique reads) of S. pneumonia is related to the CSF collection time. mNGS was less affected than culture by the use of antibiotics before CSF collection.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Meningites Bacterianas/diagnóstico , Metagenômica/métodos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Adolescente , Fatores Etários , Antígenos de Bactérias/análise , Antígenos de Bactérias/sangue , Antígenos de Bactérias/líquido cefalorraquidiano , Antígenos de Bactérias/genética , Criança , Pré-Escolar , Testes Diagnósticos de Rotina , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Meningites Bacterianas/sangue , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/microbiologia , Pediatria/métodos , Reação em Cadeia da Polimerase/métodos , Valor Preditivo dos Testes , Sensibilidade e Especificidade
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