Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.973
Filtrar
1.
Hypertension ; 74(5): 1152-1159, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31564164

RESUMO

Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, P=6.58×10-24) was ANO4, a putative Ca2+-activated chloride channel. We have investigated the role of ANO4 in human adrenal, and whether it functions like the prototype anoctamin, ANO1. We evaluated ANO4 mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of ANO4 and ANO1 overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca2+-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of ANO4 in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; P=4.93×10-7). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. ANO4 overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca2+-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.


Assuntos
Aldosterona/biossíntese , Anoctaminas/genética , Regulação da Expressão Gênica , Hiperaldosteronismo/genética , Hipertensão/fisiopatologia , Transdução de Sinais/genética , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Análise de Variância , Comunicação Celular/genética , Proliferação de Células , Células Cultivadas , Imunofluorescência , Humanos , Hiperaldosteronismo/patologia , Hipertensão/etiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise Serial de Tecidos , Técnicas de Cultura de Tecidos , Transcriptoma/genética , Regulação para Cima , Zona Fasciculada/metabolismo , Zona Fasciculada/patologia , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia
2.
Arch Virol ; 164(12): 3095-3098, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31606853

RESUMO

Bovine alphaherpesvirus 2 (BoHV-2) is the etiologic agent of bovine mammillitis (BM) and pseudo-lumpy skin disease. BM is also important because its clinical presentation can be confused with foot-and-mouth disease (FMD), making it necessary to establish differential diagnoses and perform additional laboratory tests. The objective of this work was to use a validated real-time PCR assay to test for the presence of BoHV-2 in samples from cattle and buffalo with suspected vesicular disease in Brazil. The method could detect the virus at a concentration of 0.5 fg/µL and had 99.4% amplification efficiency, a repeatability error of only 4.1%, and good reproducibility with other reagents. No evidence of BoHV-2 causing vesicular disease in cattle and buffalo was found in this work. This study was able to validate a new methodology for detection of BoHV-2 and evaluate its usefulness for investigating outbreaks of vesicular disease Brazil. The importance of BoHV-2 in cases involving other clinical signs should still be studied using the qPCR developed in this work.


Assuntos
Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Brasil/epidemiologia , Búfalos/virologia , Bovinos , Doenças dos Bovinos/epidemiologia , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/classificação , Herpesvirus Bovino 1/genética
3.
J Med Microbiol ; 68(11): 1641-1648, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31526456

RESUMO

Introduction: Onychomycosis is a debilitating, difficult-to-treat nail fungal infection with increasing prevalence worldwide. The main etiological agents are dermatophytes, which are common causative pathogens in superficial fungal mycoses. Conventional detection methods such as fungal culture have low sensitivity and specificity and are time-consuming.Aim: The main objective of this study was to design, develop and validate a real-time probe-based multiplex qPCR assay for the detection of dermatophytes and Fusarium species.Methodology: The performance characteristics of the qPCR assays were evaluated. The multiplex qPCR assays targeted four genes (assay 1: pan-dermatophytes/Fusarium spp.; assay 2: Trichophyton rubrum/Microsporum spp.). Analytical validation was accomplished using 150 fungal isolates and clinical validation was done on 204 nail specimens. The performance parameters were compared against the gold standard (fungal culture) and expanded gold standard (culture in conjunction with sequencing).Results: Both the single-plex and multiplex qPCR assays performed well especially when compared against the expanded gold standard. Among the 204 tested nail specimens, the culture method showed that 125 (61.3 %) were infected with at least one organism, of which 40 yielded positive results for dermatophytes and Fusarium spp. These target organisms detected include 20 dermatophytes and 22 Fusarium spp. The developed qPCR assays demonstrated excellent limit of detection, efficiency, coefficient of determination, analytical and clinical sensitivity and specificity.Conclusion: The multiplex qPCR assays were reliable for the diagnosis of onychomycosis, with shorter turn-around time as compared to culture method. This aids in the planning of treatment strategies to achieve optimal therapeutic outcome.


Assuntos
Arthrodermataceae/isolamento & purificação , Dermatomicoses/microbiologia , Fusariose/microbiologia , Fusarium/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Arthrodermataceae/classificação , Arthrodermataceae/genética , Dermatomicoses/diagnóstico , Fusariose/diagnóstico , Fusarium/genética , Humanos , Sensibilidade e Especificidade
4.
BMC Infect Dis ; 19(1): 773, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484497

RESUMO

BACKGROUND: The etiology of acute liver failure (ALF) is often unknown and reported to be associated with herpesviruses in a number of cases. In this study, we examined for betaherpesviruses infections in patients with ALF of unknown etiology using a multiplex qPCR to Betaherpesviruses subfamily. METHODS: Liver explant and serum samples from 27 patients with ALF of unknown etiology were analyzed with the aid of multiplex qPCR to identify betaherpesviruses. All positive samples were sequenced to confirm herpes infection and liver enzyme levels evaluated. RESULTS: Betaherpesviruses infection was effectively detected using multiplex qPCR. Six (22%) HHV-6, one (3%) HCMV and two (7%) dual infections (one with HHV-7/HHV-6, and the other with HHV-7/ HCMV). Interestingly, HHV-7 was only detected in the presence of other betaherpesviruses. Sequencing information confirmed betaherpesviruses infection. High hepatic enzyme levels and INR values> 1.5 were determined in all betaherpesvirus-positive patients. CONCLUSIONS: Multiplex qPCR facilitated efficient quantification, indicating that differentiation between betaherpesviruses is possible with the sole use of real-time PCR. Liver explant and serum samples were positive for some betaherpesviruses, and coinfection of HHV-7 with HHV-6 and HCMV was additionally detected. Based on these results, we propose that ALF patients should be screened for the presence of betaherpesviruses.


Assuntos
Betaherpesvirinae/genética , Betaherpesvirinae/isolamento & purificação , Infecções por Herpesviridae/diagnóstico , Falência Hepática Aguda/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Adolescente , Adulto , Brasil/epidemiologia , Criança , DNA Viral/sangue , DNA Viral/isolamento & purificação , Diagnóstico Diferencial , Feminino , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Humanos , Incidência , Falência Hepática Aguda/epidemiologia , Falência Hepática Aguda/virologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto Jovem
5.
BMC Infect Dis ; 19(1): 778, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488066

RESUMO

BACKGROUND: A diagnostic method to simultaneously detect and discriminate porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) in clinical specimens is imperative for the differential diagnosis and monitoring and control of PCVs in the field. METHODS: Three primer pairs were designed and used to develop a multiplex PCR assay. And 286 samples from 8 farms in Hubei province were tested by the developed multiplex PCR assay to demonstrate the accuracy. RESULTS: Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/µL of PCV1, PCV2 OR PCV3. The results of the tissue samples detection showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and real-time PCR methods. CONCLUSIONS: The multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei province, Central China.


Assuntos
Infecções por Circoviridae/diagnóstico , Circovirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Animais , China , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Circovirus/classificação , Circovirus/genética , Diagnóstico Diferencial , Genes Virais , Incidência , Tipagem Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/epidemiologia , Virologia/métodos
6.
J Med Microbiol ; 68(10): 1455-1465, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31478826

RESUMO

Introduction. Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al. J . Med . Microbiol. 2016;65(12):1521-1527).Aims. We aimed to improve the quadruplex method by adding a 16S rRNA gene target as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays, thus avoiding the possibility of false-negative reporting.Methodology. Universal 16S rRNA gene primers and a probe were defined. The novel method was tested using 36 bacterial isolates and 17 clinical samples. Experimental robustness to temperature and reagent concentration variations was assessed.Results. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species Corynebacterium belfantii) from C. ulcerans and C. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness were demonstrated. The lower limit of detection for C. diphtheriae, C. ulcerans and tox targets was 1.86 genome copies per 5 µl reaction volume. The method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England - National Infection Service, London, UK).Conclusion. This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.


Assuntos
Infecções por Corynebacterium/diagnóstico , Corynebacterium/isolamento & purificação , Difteria/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Corynebacterium/classificação , Corynebacterium/genética , Infecções por Corynebacterium/microbiologia , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/isolamento & purificação , DNA Bacteriano/genética , Difteria/microbiologia , Toxina Diftérica/genética , Humanos , RNA Ribossômico 16S/genética
7.
BMC Infect Dis ; 19(1): 827, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547805

RESUMO

BACKGROUND: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. METHODS: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. RESULTS: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. CONCLUSIONS: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.


Assuntos
Azitromicina/farmacologia , Farmacorresistência Bacteriana/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Mycoplasma genitalium/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Antibacterianos/farmacologia , Humanos , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/microbiologia , Especificidade por Substrato
8.
Ann Hematol ; 98(10): 2347-2355, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31446458

RESUMO

Molecular measurable residual disease (MRD) monitoring based on real-time quantitative reverse transcription PCR (RT-qPCR) plays an important role in acute promyelocytic leukemia (APL) management, but the performance status of clinical reports is unknown. This study focuses on the specific elements in molecular MRD monitoring report and their impact on clinical decision-making. The participating laboratories were asked to submit real and formal clinical reports for mock samples panel with APL clinical case. The MRD-specific elements were analyzed and summarized. The significance of longitudinal MRD monitoring curve and the missing MRD-specific elements for clinical decision-making were assessed. MRD-specific elements were significantly missing in clinical reports. The element "testing results" existed great inconsistencies in the written form of testing items and data. The longitudinal MRD monitoring curve of false-negative or false-positive MRD result was obviously different from all-correct. It not only identified MRD time point of tissue sampling relative to treatment and ensured the reliability of the negative MRD results, but also gave MRD diagnosis, clinical interpretation, and further recommendation. Clinician-friendly reports with MRD-specific elements can better serve clinical practice. The correctly intuitive results and clinically important MRD-specific elements can provide a good description of the reliability and clinical significance of MRD results.


Assuntos
Registros Eletrônicos de Saúde , Leucemia Promielocítica Aguda , Reação em Cadeia da Polimerase em Tempo Real/métodos , Humanos , Leucemia Promielocítica Aguda/sangue , Leucemia Promielocítica Aguda/genética , Monitorização Fisiológica/métodos , Neoplasia Residual
9.
BMC Infect Dis ; 19(1): 753, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462296

RESUMO

BACKGROUND: Leprosy continues to be a health problem in endemic areas. More than 200,000 new cases of leprosy per year suggest that transmission of the disease is still ongoing, presumably as airborne infection through nasal droplets. Late diagnosis supports continued transmission and increases the individual risk for functional disabilities. Laboratory tools are considered beneficial to facilitate early detection and clinical assessment of cases. The aim of this study was to validate molecular tools allowing detection, quantification and assessment of viability of M. leprae from nasal swab samples which are easy to obtain without the need of any invasive procedures. METHODS: Validation of two real-time PCRs detecting M. leprae DNA (RLEP qPCR) and RNA (16S rRNA RT qPCR) was conducted on "must not detect"/"must detect" samples and 160 pre-treatment nasal swab samples from 20 clinically diagnosed multibacillary (MB) leprosy patients from Togo. RESULTS: Both assays were 100% M. leprae specific and showed analytical sensitivities of three templates each. Out of 20 clinically diagnosed MB leprosy patients, 15 (75.0%) had a positive RLEP qPCR result from nasal swab samples. The 16S rRNA RT qPCR detected viable bacilli in nasal swab samples of ten out of these 15 RLEP positive patients (66.7%). CONCLUSION: The combined RLEP/16S rRNA (RT) qPCR assay provides a sensitive and specific tool to determine the bacterial load and viability of M. leprae from nasal swab samples and is applicable for early diagnosis, monitoring treatment response and investigating the role of nasal carriage of M. leprae in human-to-human transmission through aerosol infection.


Assuntos
Hanseníase/microbiologia , Mycobacterium leprae/genética , Cavidade Nasal/microbiologia , RNA Ribossômico 16S , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , DNA Bacteriano/genética , Humanos , Hanseníase/diagnóstico , Hanseníase Multibacilar/diagnóstico , Hanseníase Multibacilar/microbiologia , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/patogenicidade , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Togo , Adulto Jovem
10.
Sci Total Environ ; 686: 1104-1112, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31412506

RESUMO

Identification of fecal contamination sources in surface water has become heavily dependent on quantitative PCR (qPCR) because this technique allows for the rapid enumeration of fecal indicator bacteria as well as the detection and quantification of fecal source-associated genetic markers in the environment. Identification of contamination sources in impaired waters is a prerequisite for developing best management practices to reduce future pollution. Proper management decisions rely on the quality and interpretation of qPCR data. In this study, we developed a method to determine analytical and process lower limits of detection (LLOD) and quantification (LLOQ) using two cattle-associated genetic markers targeting Bacteroidales. Analytical LLOD (ALLOD) for both CowM2 and CowM3 genetic markers in the qPCR assay were five gene copies per reaction. Using composite fecal DNA, the analytical LLOQ (ALLOQ) determined for CowM2 and CowM3 were 78 and 195 gene copies/reaction, respectively. When plasmid DNA was used, the ALLOQ for CowM2 and CowM3 were 46 and 20 gene copies/reaction, respectively. The process LLOD (PLLOD) for CowM2 and CowM3 were 0.4 and 0.02 mg feces/filter (wet weight), respectively. Using the standard deviation value of 0.25 as a cut-off point for LLOQ in regression analysis, the process LLOQ (PLLOQ) for CowM2 and CowM3 were 3.2 and 0.3 mg feces/filter, respectively. These results indicate that CowM3 exhibited superior performance characteristics compared with CowM2 for fecal samples collected from our geographical region. Moreover, the method for calculating LLOD and LLOQ developed here can be applied to other microbial source tracking studies.


Assuntos
Bacteroidetes/isolamento & purificação , Monitoramento Ambiental/métodos , Fezes/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Microbiologia da Água , Animais , Bactérias/isolamento & purificação , Bacteroidetes/química , Bacteroidetes/genética , Bovinos , Marcadores Genéticos , Limite de Detecção
11.
BMC Infect Dis ; 19(1): 680, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370795

RESUMO

BACKGROUND: A major challenge in dengue management in resource limited settings is the confirmation of diagnosis. Clinical features of dengue often overlap with other infections and molecular diagnostic tools are not readily accessible to clinicians at hospitals. In addition, the prediction of plasma leakage in dengue is also difficult. Hematocrit level and ultrasound scans (combined with clinical parameters) are helpful to detect plasma leakage once it has happened, not before. METHODS: Colombo Dengue Study (CDS) is a prospective cohort study of clinically suspected adult dengue patients recruited from the National hospital of Sri Lanka (within the first 3 days of fever) that aimed to a) identify clinical and basic laboratory test parameters to differentiate dengue from non-dengue fever, b) evaluate the comparative efficacy of loop-mediated isothermal amplification (LAMP) for dengue diagnosis (vs. NS1 antigen test and RT-qPCR) and c) identify early associations that are predictive of plasma leakage or severe dengue. The basic laboratory tests considered here included hematological parameters, serum biochemistry and inflammatory markers. RESULTS: Only 70% of clinically suspected patients were confirmed as having dengue by either the NS1 antigen test or RT-qPCR. On a Bayesian latent class model which assumes no "gold standard", LAMP performed equally or better than RT-qPCR and NS1 antigen test respectively. When confirmed dengue patients were compared with others, the earlier group had significantly lower lymphocyte counts and higher aspartate aminotransferase levels (AST) within the first 3 days of fever. Confirmed dengue patients with plasma leakage had a lower mean age and a higher median baseline AST level compared to those without plasma leakage (p < 0.05). CONCLUSION: Clinical suspicion overestimates the true number of dengue patients. RT-LAMP is a potentially useful low-cost diagnostic tool for dengue diagnosis. Confirmed dengue patients had significantly higher AST levels and lower lymphocyte counts in early disease compared to others. In confirmed dengue patients, younger age and a higher AST level in early infection were associated with subsequent plasma leakage.


Assuntos
Aspartato Aminotransferases/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Dengue Grave/diagnóstico , Dengue Grave/etiologia , Adulto , Teorema de Bayes , Biomarcadores/sangue , Estudos de Coortes , Dengue/diagnóstico , Vírus da Dengue/genética , Feminino , Febre/virologia , Humanos , Testes Imunológicos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Medição de Risco , Sensibilidade e Especificidade , Dengue Grave/sangue , Sri Lanka
12.
Malar J ; 18(1): 272, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399031

RESUMO

BACKGROUND: To assess the occurrence of Plasmodium ovale wallikeri and Plasmodium ovale curtisi species in travellers returning to Germany, two real-time PCR protocols for the detection and differentiation of the two P. ovale species were compared. Results of parasite differentiation were correlated with patient data. METHODS: Residual nucleic acid extractions from EDTA blood samples of patients with P. ovale spp. malaria, collected between 2010 and 2019 at the National Reference Centre for Tropical Pathogens in Germany, were subjected to further parasite discrimination in a retrospective assessment. All samples had been analysed by microscopy and by P. ovale spp.-specific real-time PCR without discrimination on species level. Two different real-time PCR protocols for species discrimination of P. o. curtisi and P. o. wallikeri were carried out. Results were correlated with patient data on gender, age, travel destination, thrombocyte count, and duration of parasite latency. RESULTS: Samples from 77 P. ovale spp. malaria patients were assessed, with a male:female ratio of about 2:1 and a median age of 30 years. Parasitaemia was low, ranging from few visible parasites up to 1% infected erythrocytes. Discriminative real-time PCRs revealed 41 cases of P. o. curtisi and 36 cases of P. o. wallikeri infections. Concordance of results by the two PCR approaches was 100%. Assessment of travel destinations confirmed co-existence of P. o. curtisi and P. o. wallikeri over a wide range of countries in sub-Saharan Africa. Latency periods for the two P. ovale species were similar, with median values of 56.0 days for P. o. curtisi and 58.0 days for P. o. wallikeri; likewise, there was no statistically significant difference in thrombocyte count with median values of 138.5/µL for patients with P. o. curtisi and 152.0/µL for P. o. wallikeri-infected patients. CONCLUSIONS: Two different real-time PCR protocols were found to be suitable for the discrimination of P. o. curtisi and P. o. wallikeri with only minor differences in sensitivity. Due to the overall low parasitaemia and the lack of differences in severity-related aspects like parasite latency periods or thrombocyte counts, this study supports the use of P. ovale spp. PCR without discrimination on species level to confirm the diagnosis and to inform clinical management of malaria in these patients.


Assuntos
Doenças Transmissíveis Importadas/diagnóstico , Malária/diagnóstico , Plasmodium ovale/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Doenças Transmissíveis Importadas/classificação , Doenças Transmissíveis Importadas/prevenção & controle , Estudos Transversais , Feminino , Alemanha , Humanos , Malária/classificação , Malária/prevenção & controle , Masculino , Pessoa de Meia-Idade , Plasmodium ovale/classificação , Plasmodium ovale/genética , Estudos Retrospectivos , Viagem , Adulto Jovem
13.
BMC Infect Dis ; 19(1): 701, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395014

RESUMO

BACKGROUND: In recent years, studies on the diagnostic accuracy of in-house real-time PCR (hRT-PCR) assay for the detection of Mycobacterium tuberculosis (Mtb) have been reported with unignorable discrepancies. To assess the overall accuracy of the hRT-PCR assay for Mtb diagnosis in different samples for individuals with active pulmonary and extra-pulmonary Mtb infection, a systematic review and meta-analysis were performed. METHODS: The PUBMED, EMBASE, Web of Science, and Cochrane databases were searched up to June 2017 for eligible studies that estimated diagnostic sensitivity and specificity with the hRT-PCR assay in respiratory and non-respiratory samples in pulmonary and extra-pulmonary Mtb infection patients, with Mtb culture as the reference standard. Bivariate random effect models were used to provide pooled estimation of diagnostic accuracy. Further, subgroup and meta-regression analyses were performed to explore sources of heterogeneity. The risk of bias was assessed by the QUADAS-2 tool. RESULTS: Of the 3589 candidate studies, 18 eligible studies met our inclusion criteria. Compared to Mtb culture data, the pooled sensitivity and specificity were 0.96 and 0.92, respectively. The diagnostic odds ratio (DOR) was 192.96 (95% CI 68.46, 543.90), and the area under the summary ROC curve (AUC) was 0.9791. There was significant heterogeneity in sensitivity and specificity among the enrolled studies (p < 0.001). The studies with high-quality assessment and application of respiratory specimen were associated with better accuracy. CONCLUSIONS: In low-income/high-burden settings, our results suggested that the hRT-PCR assay could be a useful test for the diagnosis of TB with high sensitivity and specificity.


Assuntos
Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose/diagnóstico , Humanos , Curva ROC , Sensibilidade e Especificidade , Tuberculose/genética , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/genética
14.
Mikrobiyol Bul ; 53(3): 285-296, 2019 Jul.
Artigo em Turco | MEDLINE | ID: mdl-31414630

RESUMO

BK virus (BKV) viral load quantification has a distinct role in the clinical control of BKV nephropathy and organ rejection among renal transplant recipients. In this study, it was aimed to compare BKV DNA measurement values performed with two different real-time polymerase chain reaction (PCR) methods and to determine BKV genotypes in renal transplant recipients. Totally, 150 clinical samples tested previously in two different laboratories (Lab-1 and Lab-2) from adult and pediatric renal transplantation patients were included in the study. Fifty plasma samples of 50 different patients from Lab-1, 50 plasma and 50 urine samples of 58 different patients from Lab-2 were included in the study. Viral nucleic acid extraction was performed with automatized systems in Lab-1 and Lab-2 (EZ1, Qiagen, Germany and MagNA Pure 96, Roche Diagnostics, Germany; respectively;). Real-time PCR procedure was carried out in Lab-1 with an amplification mixture of primer, probe sequences targeting VP-1 gene region using RotorGene (Qiagen, Germany) and in Lab-2 with an amplification mixture of primer, probe sequences targeting VP-2 gene region using ABI Prism 7500 (Applied Biosystems, USA). BKV genotyping was performed with multiplex PCR using primer, probe sequences for BKV genotypes I-IV. In both of the laboratories, 82 (54.6%) of the samples were found as positive, 37(24.6%) samples were found as negative and a moderate agreement was found between qualitative results of two real-time PCR methods (ƙ= 0.56, p<0.001). Median viral load values were 4.1 x 104 copies/ml (321-6 x 109) in Lab-1 and 3.3 x 105 copies/ml (224-8.3 x 1010) in Lab-2 for positive samples. According to the lineer regression analysis of quantitative results, moderate (R2= 0.52, p<0.001) and high (R2= 0.88, p<0.001) correlation was found for plasma (n= 52) and urine (n= 30) samples, respectively. Bland-Altman analysis yielded a mean difference of -0.58 log10 for all samples. For plasma samples mean difference was -0.29 log10, while it was -1.1 log10 for urine samples. In all samples, Lab-1 measurements were lower than Lab-2 measurements. A mean difference of -1.1 log10 indicated that the measurement values of Lab-2 were more higher than Lab-1 measurments with an average of 1.1 log10. Supporting this result, 71.9% of the samples had a measurement difference more than 0.5 log 10 and 29.2% of the samples had a measurement difference more than 1 log10. Only 28.1% of the samples were measured within clinically acceptable log difference range (less than 0.5 log10). BKV genotyping was performed only for 74 different patient samples with sufficient copy numbers and genotype I (81.7%), IV (15.5%), II (1.4%), I+IV (1.4%) were detected. When the results were compared; 66.6% (n= 12) of the genotype IV samples had more than 1 log10 and 83.3% of them had more than 0.5 log10 viral load measurement difference. Correlation and linear regression analyzes were insufficient for the comparison ofthe results of the two different tests. It will be appropriate for each center to monitor patients with the same test until the international BKV standard developed by the World Health Organization is optimized. The clinical correlation of the tests is limited to the currently used test. The result of incorrect BKV quantification affects the clinical decision. Measurements less than the actual value will lead to the development of BKV nephropathy, and higher measurements will lead to unnecessary allograft biopsy and unnecessary reduction of immunosuppression.


Assuntos
Vírus BK , Infecções por Polyomavirus , Reação em Cadeia da Polimerase em Tempo Real , Infecções Tumorais por Vírus , Adulto , Vírus BK/genética , Criança , DNA Viral , Genótipo , Alemanha , Humanos , Transplante de Rim , Infecções por Polyomavirus/diagnóstico , Infecções por Polyomavirus/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Transplantados , Infecções Tumorais por Vírus/diagnóstico , Infecções Tumorais por Vírus/virologia , Carga Viral
15.
Microbiol Res ; 228: 126305, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422234

RESUMO

Traditional culture-based enumeration methods were compared with the ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) technique to assess Bdellovibrio-and-like-organisms (BALOs) predator-prey interactions. Gram-negative [Pseudomonas spp. and Klebsiella pneumoniae (K. pneumoniae)] and Gram-positive [Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium)] organisms were employed as prey cells, while a Bdellovibrio bacteriovorus strain (PF13) was used as the predator. The co-culture experiments were also compared in diluted nutrient broth (DNB) and HEPES buffer. In both media, K. pneumoniae (maximum log reduction of 5.13) and Pseudomonas fluorescens (P. fluorescens) (maximum log reduction of 4.21) were sensitive to predation by B. bacteriovorus PF13 as their cell counts and gene copies were reduced during all the co-culture experiments, while the concentration of B. bacteriovorus PF13 increased. The concentration of B. bacteriovorus PF13 also increased in the presence of S. aureus (HEPES buffer) and E. faecium (DNB), indicating that the predator interacted with these Gram-positive prey in order to survive. Moreover, as no predator plaques were produced in the co-culture experiments with P. aeruginosa (DNB and HEPES buffer), S. aureus (DNB and HEPES buffer) and E. faecium (HEPES buffer), EMA-qPCR proved to be beneficial in monitoring the concentration of B. bacteriovorus. In conclusion, the cell counts and/or EMA-qPCR analysis for the HEPES buffer and DNB assays were successfully employed to monitor the predation of P. fluorescens and K. pneumoniae by B. bacteriovorus, while E. faecium was sensitive to predation in DNB and S. aureus was sensitive to predation in HEPES buffer.


Assuntos
Azidas , Fenômenos Fisiológicos Bacterianos , Interações Microbianas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Bactérias , Bdellovibrio bacteriovorus/fisiologia , Técnicas de Cocultura , Contagem de Colônia Microbiana , Enterococcus faecium/fisiologia , Klebsiella pneumoniae/fisiologia , Pseudomonas/fisiologia , Staphylococcus aureus/fisiologia , Águas Residuárias/microbiologia , Purificação da Água
16.
Food Chem ; 300: 125205, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31330372

RESUMO

For efficient extraction of amplifiable DNA from edible vegetable oils, we developed a novel DNA extraction approach based on the non-silica-based dipolar nanocomposites. The nanoparticle comprises a hydrophilic polymethyl methacrylate core with abundant capillaries, hydrophilic vesicles decorated with molecules having DNA affinity and a coating hydrophobic polystyrene layer. The nanoparticles are soluble in oil, adsorb the DNA from the aqueous phase and gave a high DNA recovery ratio. All DNA extracts from fully refined vegetable oil soybean, peanut, rapeseed, and cottonseed oils, including their blends, were sufficiently pure to be amplified by real-time PCR targeting the chloroplast ribulose-1,5-bisphosphate gene (rbcL), therefore, the species of origin and their ratios in mixed vegetable oils blended from two or three oil-species could be determined. These results indicate that the novel DNA isolation and real-time PCR kit is a simple, sensitive and efficient tool for the species identification and traceability in refined vegetable oils.


Assuntos
DNA de Plantas/isolamento & purificação , Nanopartículas/química , Óleos Vegetais/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Verduras/genética , Fracionamento Químico/métodos , Cloroplastos/genética , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Polimetil Metacrilato/química , Ribulosefosfatos/genética , Dióxido de Silício
17.
BMC Infect Dis ; 19(1): 616, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299916

RESUMO

BACKGROUND: The point mutations in 23S rRNA gene of Mycoplasma pneumoniae (M. pneumoniae) can lead to high-level resistance to macrolides. This study aimed to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions A2063G and A2064G of 23S rRNA gene. METHODS: We detected 178 pharyngeal swab specimens and calculated the proportions of resistant and sensitive quasispecies using ASPCR assays. ASPCR assays can detect down to 10 copies of 23S rRNA gene and achieved sensitivities of < 0.1% for A2063G and A2064G. We also compared the findings of ASPCR with the results of nested PCR with sequencing. RESULTS: Of 178 samples, 164 were found to have M. pneumoniae including 90.85% (149/164) samples with macrolide-resistant M. pneumoniae (MRMP) quasispecies by ASPCR, while 153 were found to be M. pneumoniae-positive including 71.90% (110/153) samples with MRMP quasispecies by nested PCR with sequencing. Of the 164 M. pneumoniae-positive samples, 61.59% (101/164) had the mixed population of wild-type and mutant M. pneumoniae, and 56.44% (57/101) of the latter contained the mutations at low frequency (≤50%). CONCLUSION: ASPCR indicated that sensitive and resistant quasispecies coexisted in most of the M. pneumoniae positive samples. The ASPCR was a highly sensitive, accurate and rapid method for detecting the macrolide resistance-associated mutations and it could provide earlier and more drug-resistant information for M. pneumoniae research and the clinical therapy.


Assuntos
Farmacorresistência Bacteriana/genética , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/diagnóstico , RNA Ribossômico 23S/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Antibacterianos/uso terapêutico , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Feminino , Humanos , Macrolídeos/uso terapêutico , Mycoplasma pneumoniae/isolamento & purificação , Faringe/microbiologia , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/microbiologia , Mutação Puntual , RNA Ribossômico 23S/genética , Reprodutibilidade dos Testes
18.
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
19.
BMC Infect Dis ; 19(1): 646, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324234

RESUMO

BACKGROUND: The aim of this study was to establish a set of assessment methods suitable for evaluating the complex indoor environment of hospital wards and to ascertain the composition of bacteria and microbial ecology of hospital wards. METHODS: Colony-forming units (CFUs), PM2.5 detection, real-time PCR, and adenosine triphosphate (ATP) bioluminescence assay were employed to evaluate the complexity of indoor air in 18 wards of nine departments in a hospital and two student dormitories in a university. Subsequently, the microbial samples were quantified and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Although the studied indices were relatively independent, the PM2.5 content was correlated with bacterial CFUs determined by passive sedimentation method, bacterial and fungal counts measured by real-time PCR, and ATP bioluminescence assay. The composition of microorganisms in the air of hospital wards differed from that in the air of student dormitories. The dominant genera in hospital wards were Staphylococcus (39.4%), Micrococcus (21.9%), Corynebacterium (11.7%), Kocuria (4.4%), Bacillus (2.9%), Streptococcus (1.6%), Moraxella (1.6%), and Enterococcus (1.3%), and the microbial ecology differed between Respiration Dept. III and other hospital departments. Additionally, 11.1 and 27.3% of bacteria in hospital wards and student dormitories were not identified, respectively. CONCLUSIONS: Assessment of environmental quality of hospital wards should be based on comprehensive analysis with multiple indicators. There may be imbalances in the microbial diversity in the hospital wards, therefore, monitoring of the environmental quality of hospitals is important in the prevention of nosocomial infections.


Assuntos
Microbiologia do Ar , Poluição do Ar em Ambientes Fechados/análise , Hospitais , Medições Luminescentes/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trifosfato de Adenosina/análise , Carga Bacteriana/métodos , China , Contagem de Colônia Microbiana/métodos , Humanos , Material Particulado/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus
20.
Artigo em Inglês | MEDLINE | ID: mdl-31305225

RESUMO

A reliable and sensitive identification method is required to tackle food adulteration mainly in meat production. We developed a dry reagent based ready-to-use single tube quadruplex PCR assay for accurate identification of chicken, mutton, beef and pork. The assay was found to be specific and reproducible. Thermo-stability studies of lyophilized PCR master mix were conducted at different temperature and time intervals, which revealed significant stability for 75 days at 4°C and for 60 days at 25°C. The developed assay was shown to be sensitive down to 16 pg DNA per reaction and the detection limit was found to be 0.01% (w/w) of each species. Furthermore, this method has been applied to the analysis of 68 commercial meat products and the results indicated that nine samples contained non-declared meat components. This dry reagent-based quadruplex PCR assay can be utilized to monitor various processed food products and also to maintain quality control in food industries mainly in the resource-limited settings.


Assuntos
Galinhas/genética , DNA/genética , Análise de Alimentos , Produtos da Carne/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ovinos/genética , Suínos/genética , Animais , Bovinos , Controle de Qualidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA