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1.
Vet Parasitol ; 273: 97-104, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31473450

RESUMO

This study aimed to investigate the presence of the natural transovarial transmission of tick-borne pathogens in unfed larvae obtained from engorged female ticks from domestic animals in Turkey. Larvae (n = 4530, 151 pools) obtained from 75 engorged female ticks and female carcasses were screened for the presence of certain tick-borne pathogens by PCR. The presence of transovarial transmission of Babesia occultans was detected in Hyalomma marginatum and Hy. excavatum, while Ba. ovis in Rhipicephalus bursa. Theileria annulata was detected only in Hy. excavatum and Rh. turanicus female carcasses, but not in their examined progenies. Additionally, Rickettsia aeschlimannii and Rickettsia raoultii were detected in Hy. marginatum and Dermacentor marginatus females, respectively, and all their examined larvae. Besides, Ri. slovaca was detected in a De. marginatus female carcass and its one of two examined larvae pools. The presence of mixed Ba. occultans and Ri. aeschlimannii infection was also determined in an Hy. marginatum female and its larvae. This is the first demonstration of transovarial transmission of Ba. occultans in naturally infected Hy. excavatum. These data suggested that Hy. excavatum may act as vector in the natural cycle of Ba. occultans.


Assuntos
Infecções Bacterianas/veterinária , Infecções Protozoárias em Animais/transmissão , Doenças Transmitidas por Carrapatos/veterinária , Carrapatos/microbiologia , Carrapatos/parasitologia , Animais , Animais Domésticos/microbiologia , Animais Domésticos/parasitologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/transmissão , DNA Bacteriano/genética , DNA de Protozoário/genética , Feminino , Larva/microbiologia , Larva/parasitologia , Ovário/microbiologia , Ovário/parasitologia , Reação em Cadeia da Polimerase , Infecções Protozoárias em Animais/parasitologia , Doenças Transmitidas por Carrapatos/microbiologia , Doenças Transmitidas por Carrapatos/parasitologia , Doenças Transmitidas por Carrapatos/transmissão , Turquia
2.
Braz J Med Biol Res ; 52(9): e8224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482975

RESUMO

Leishmaniasis is a neglected disease that affects a large part of the world population. Knowing the sand fly fauna of a region is of fundamental importance for guiding health surveillance actions related to the prevention and control of leishmaniasis. A total of 86 specimens of sand flies (60 females and 26 males) were collected. Using the classification proposed by Galati (2003), the following species were identified: Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1920), Evandromyia cortelezzi (Brethes, 1923), Ev. sallesi (Galvão & Coutinho, 1939), Nyssomyia whitmani (Atunes & Coutinho, 1939), Psathyromyia lutziana (Costa Lima, 1932), Ev. lenti (Mangabeira, 1938), Brumptomyia sp. (França and Parrot, 1921), and Pressatia sp. (Mangabeira, 1942). Using PCR with internal transcribed spacer target to identify infected sand flies, five Lu. longipalpis females were infected with Leishmania spp. Despite the small number of specimens collected, considerable species diversity was found in the study area.


Assuntos
Insetos Vetores/classificação , Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Psychodidae/classificação , Psychodidae/parasitologia , Animais , Brasil , DNA Espaçador Ribossômico/genética , Feminino , Leishmania/genética , Leishmaniose/transmissão , Masculino , Reação em Cadeia da Polimerase , RNA de Protozoário/genética
3.
Rev Soc Bras Med Trop ; 52: e20190243, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508786

RESUMO

INTRODUCTION: In recent decades, the prevalence of carbapenem-resistant Acinetobacter isolates has increased, and the production of oxacillinase (OXA)-type carbapenemases is the main mechanism underlying resistance. We evaluated OXA production from 114 Acinetobacter isolates collected between March and December 2013 from different clinical specimens of patients in two hospitals (Hospital 1 [n = 61] and Hospital 2 [n = 53]) located in Niterói, Rio de Janeiro, Brazil. We also evaluated the genetic diversity of OXA-producing isolates. METHODS: All the isolates were identified through the automated system Vitek II and matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS as belonging to the A. baumannii-A. calcoaceticuscomplex. Antimicrobial susceptibility profiles were verified through agar diffusion tests. The presence of OXA-encoding genes was confirmed by PCR. The genetic diversity of isolates positive for carbapenemase production was analyzed through pulsed-field gel electrophoresis. RESULTS: There was a high rate of resistance to carbapenems in the isolates (imipenem: 96%; meropenem: 92%) from both hospitals. Moreover, a high percentage (95.6%) of OXA-23-positive isolates was observed for both hospitals, indicating that this was the main mechanism of carbapenem-resistance among the studied population. In addition, most isolates (96.5%) were positive for bla OXA-51. A high genetic diversity and a few major genotypes were found among the OXA-23-positive isolates analyzed. Only intra-hospital dissemination was observed. CONCLUSIONS: The elevated dissemination of bla OXA-23-like observed among Acinetobacter isolates from both the studied hospitals highlights the need for continuous epidemiological surveillance in these institutions.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter/enzimologia , beta-Lactamases/biossíntese , Acinetobacter/efeitos dos fármacos , Acinetobacter/genética , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Brasil , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Hospitais Gerais , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , beta-Lactamases/efeitos dos fármacos
4.
Rev Soc Bras Med Trop ; 52: e20190304, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508787

RESUMO

INTRODUCTION: Human parvovirus B19 (B19V) is a common pathogen, which on infection causes variety of clinical conditions from benign self-limiting exanthematous disease and other similar pathologies to fetal death. METHODS: We collected 341 serum samples between the first and fourth day after the onset of symptoms from all patients suspected of dengue fever who were attended at Regional Hospital of Tefé. Initially, patients were screened for malaria by blood smear test and negative samples were sent to Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) situated in Manaus (AM) for dengue testing using semi-nested multiplex PCR. Further, we investigated 44 malaria and dengue-negative samples of children for B19V DNA by nested-PCR. Positive samples were analyzed by BLAST against entire public non-redundant nucleotide database and genotyped by phylogenetic analyses using neighbor-joining clustering method. RESULTS: Eight samples (18.2%) were found to be PCR positive. Fever, headache, ocular pain, and/or muscle pain were reported as the most frequent symptoms by the patients and none were diagnosed with rash at the time of sample collection. Phylogenetic analysis of major capsid protein 2 (VP2) and VP3 coding region showed high similarity with B19V genotype 1. CONCLUSIONS: Our results reveal the spread of B19V genotype 1 in Tefé. Moreover, our results emphasize the significance of laboratorial differential diagnosis using molecular techniques in patients with acute febrile, and thereby aid the health surveillance system in improving patient care even in the remote areas of Amazon.


Assuntos
DNA Viral/sangue , Dengue/diagnóstico , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase , Adulto Jovem
5.
Fortschr Neurol Psychiatr ; 87(9): 499-502, 2019 Sep.
Artigo em Alemão | MEDLINE | ID: mdl-31519025

RESUMO

Strains of methicillin-resistant Staphylococcus aureus (MRSA) are of major economic and health-related importance to early neurological and neurosurgical rehabilitation. It is crucial to identify MRSA-carriers as soon as possible upon admission in order to prevent transmissions and to initiate contact precautions and decolonization. The present study focuses on validity of a polymerase chain reaction (PCR) test to identify MRSA genetic material from nasopharyngeal samples (BD MAX MRSA XT, BD Diagnostics, Heidelberg, Germany) of early neurological and neurosurgical rehabilitation patients. PCR-results were compared to gold standard (culture). In 2013, 66 patients were tested using PCR and incubation within one week after admission. Sensitivity of PCR was 84.6 %, specificity 86.6 %. Positive predictive value (PPV) was only 61.1 %, while negative predictive value was as high as 95.8 %. In 39 cases, PCR and subsequent culture were done within one day, leading to a sensitivity of 100 % and a specificity of 90.3 %. In this subgroup, PPV was 72.7 %, NPV 100 %. The results from the study suggest that incubation should quickly follow a positive PCR finding (within 24 hours) in order to verify MRSA colonization. High NPV (95.8 resp. 100 %) indicate that PCR negative patients very likely are not colonized with MRSA. A positive PCR test is less reliable (due to false positive results) and should be followed by incubation in due course in order to avoid unnecessary contact precautions.


Assuntos
Programas de Rastreamento/normas , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Reabilitação Neurológica , Reação em Cadeia da Polimerase/normas , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Alemanha , Humanos , Sensibilidade e Especificidade
6.
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
7.
Z Gastroenterol ; 57(9): 1067-1076, 2019 Sep.
Artigo em Alemão | MEDLINE | ID: mdl-31525799

RESUMO

Intestinal tuberculosis is an infectious disease of the extrapulmonary manifestation with the Mycobacteria tuberculosis complex. In developed countries, this disease is rarely seen. The clinical features are heterogeneous and unspecific. Furthermore, intestinal tuberculosis poses diagnostic challenges. Regarding intestinal tuberculosis the Ziehl-Neelsen staining for acid-fast bacillus, PCR examination and culture methods show only poor sensitivity and specificity. In this case series, we present three patients suffering from intestinal tuberculosis, who were diagnosed and treated successfully. Furthermore, we review the literature about the pitfalls of the diagnostic approaches and the treatment options of intestinal tuberculosis.


Assuntos
Tuberculose Gastrointestinal/diagnóstico , Biópsia , Colonoscopia , Corantes , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Coloração e Rotulagem , Tuberculose Gastrointestinal/patologia
8.
Rev Inst Med Trop Sao Paulo ; 61: e52, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531630

RESUMO

In 2018, Bhutan reported 54 cases of malaria, of which six were indigenous, 14 introduced and 34 imported. Considering the continuous reduction in the number of indigenous cases, Bhutan plans to eliminate malaria by 2025 under the Bhutan Malaria Elimination Strategy. The study was conducted to assess the presence of asymptomatic plasmodial infection in both, Bhutanese population living in malaria-risk areas and in migrant workers to guide the elimination strategies. A cross-sectional study was conducted from April to May 2016 in 750 Bhutanese people and 473 migrant workers. Plasmodium falciparum and Plasmodium vivax infections were investigated by using a rapid diagnostic test (RDT) and the polymerase chain reaction (PCR). Prevalence of asymptomatic plasmodial infection based on PCR was 0.27% (95% CI: 0.05-1.07%) among Bhutanese people with a mean age of 43 years old. The proportions of males and females were 45% and 55%, respectively. Among migrant workers, the prevalence of asymptomatic plasmodial infection was 0.42% (95% CI: 0.07-1.69%) with a mean age of 30 years old. The majority of migrant workers were from the neighboring Indian State of West Bengal (57.51%), followed by Assam (12.26%). RDT in both study groups did not detect any plasmodial infection. The presence of a low prevalence of asymptomatic plasmodial infection indicates that the current elimination strategies and interventions are effective.


Assuntos
Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Infecções Assintomáticas , Butão/epidemiologia , Estudos Transversais , Feminino , Humanos , Índia , Malária Falciparum/epidemiologia , Malária Falciparum/prevenção & controle , Malária Vivax/epidemiologia , Malária Vivax/prevenção & controle , Masculino , Reação em Cadeia da Polimerase , Prevalência , Migrantes
9.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1787-1796, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559759

RESUMO

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.


Assuntos
Biocatálise , Quitina/análogos & derivados , Quitinases , Concentração de Íons de Hidrogênio , Reação em Cadeia da Polimerase
10.
Rinsho Ketsueki ; 60(8): 903-909, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31484887

RESUMO

A 78-year-old man with anemia (Hb 9.6 g/dl) and elevated serum immunoglobulin M (IgM 3,577 mg/dl) levels was referred to our hospital. Bone marrow aspiration yielded a dry tap, and bone marrow biopsy revealed the infiltration of CD20 positive lymphoplasmacytic lymphoma cells and myelofibrosis. The patient was diagnosed with Waldenström's macroglobulinemia complicated with myelofibrosis. TGF-ß plasma concentration was elevated. Further, after chemotherapy with bendamustine and rituximab, remission of both Waldenström's macroglobulinemia and myelofibrosis was achieved, and TGF-ß levels normalized. MYD88 L265P mutation was detected using highly sensitive digital PCR, which compared with currently used direct PCR product sequencing, has a superior sensitivity. The use of digital PCR has additional advantages toward MYD88 L265P detection, particularly when the available amount of sample DNA is limited owing to myelofibrosis.


Assuntos
Fator 88 de Diferenciação Mieloide/genética , Mielofibrose Primária , Macroglobulinemia de Waldenstrom , Idoso , Humanos , Imunoglobulina M , Masculino , Mutação , Reação em Cadeia da Polimerase , Mielofibrose Primária/complicações , Macroglobulinemia de Waldenstrom/complicações , Macroglobulinemia de Waldenstrom/genética
11.
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
12.
J Assoc Physicians India ; 67(9): 96, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31561703
13.
Zhonghua Shao Shang Za Zhi ; 35(8): 587-594, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31474038

RESUMO

Objective: To explore the preliminary application effect of real-time fluorescence recombinase polymerase amplification (RPA) in the detection of Candida albicans. Methods: (1) Candida albicans standard strain and negative control bacteria of Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Candida glabrata standard strains of respectively 1 mL were collected and their DNA were extracted by yeast/bacterial genomic kit. The specificity of polymerase chain reaction (PCR), real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. (2) One Candida albicans standard strain and one negative control bacteria of Candida glabrata standard strain were collected, resuscitated, and counted. Candida albicans was diluted 10 times to 1×10(7) to 1×10(1) colony-forming unit (CFU)/mL. The DNA of the two bacteria were extracted as experiment (1). The sensitivity of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. The number of cycles for amplification curve to reach the threshold in real-time fluorescent quantitative PCR, and time of appearance of specific amplification curve in real-time fluorescence RPA were recorded and compared with the results in PCR. The detection limit and rate of the above-mentioned 3 methods in detecting Candida albicans were analyzed, and the correlation between concentration of Candida albicans in real-time fluorescence RPA and detection time was analyzed. (3) One standard strain of Candida albicans was collected, and the DNA was extracted as experiment (1) and detected by PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA. The total detection time of the above-mentioned 3 methods was recorded, respectively. (4) The DNA of 31 clinical samples of suspected Candida albicans infection and 1 clinical sample of Candida albicans collected from cotton swab were extracted, PCR and real-time fluorescence RPA were carried out, and the positive detection rates of the above-mentioned methods were calculated. The DNA of the clinical samples with positive results in both PCR and real-time fluorescence RPA were extracted by yeast/bacterial genomic kit, chelex-100 boiling method, and repeatedly freeze-thawing with liquid nitrogen method, and real-time fluorescence RPA and PCR were carried out. The negative control bacteria was Candida glabrata in real-time fluorescence RPA, while negative control bacteria in PCR were the same as experiment (1). The positive results in PCR and real-time fluorescence RPA were observed and time for amplification curve to reach the fluorescence threshold in real-time fluorescence RPA was recorded, respectively. Data were processed with linear correlation analysis and t test. Results: (1) Three methods showed positive results in detecting standard strain of Candida albicans, and none of the 5 negative control bacteria showed positive results. (2) As the concentration of bacterial solution of Candida albicans decreased, the number of cycles for the amplification curve to reach the threshold increased in real-time fluorescent quantitative PCR, the time for appearance of specific amplification curve prolonged in real-time fluorescence RPA, and brightness of the gel strip weakened in PCR. None of the negative control bacteria in the above-mentioned 3 detection methods showed corresponding positive results. The detection limit of Candida albicans in real-time fluorescence RPA, PCR, and real-time fluorescent quantitative PCR was 1×10(1) CFU/mL. There was a significant negative correlation between the concentration of Candida albicans and the detection time in real-time fluorescence RPA (r=-0.95, P<0.01). The positive detection rates of PCR and real-time fluorescent quantitative PCR for Candida albicans of 1×10(1) to 1×10(7) CFU/mL were 100%. The positive detection rate of real-time fluorescence RPA for Candida albicans of 1×10(1) CFU/mL was 78%, and the positive detection rate of real-time fluorescence RPA for Candida albicans of 1×10(2) to 1×10(7) CFU/mL was 100%. (3) The total time of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA detection for Candida albicans was 133, 93, and 35 min, respectively. (4) The positive detection rate of real-time fluorescence RPA for 31 clinical samples of suspected Candida albicans infection was 32.26% (10/31), which was slightly lower than 35.48% (11/31) of PCR. Eleven clinical samples showed positive results both in real-time fluorescence RPA and PCR detection. No positive result was observed in the negative control bacteria detected both by real-time fluorescence RPA and PCR. The DNA was extracted by yeast/bacterial genomic extraction kit and chelex-100 boiling method for real-time fluorescence RPA detection. The time for the amplification curve to reach the threshold was (438±13) and (462±12) s, respectively, which was close (t=1.32, P>0.05). The DNA was extracted by repeatedly freeze-thawing with liquid nitrogen method for real-time fluorescence RPA, and the time for the amplification curve to reach the threshold in real-time fluorescence RPA was (584±15) s, which was significantly longer than that in the other 2 methods (t=7.55, 6.39, P<0.01). Conclusions: Real-time fluorescence RPA has advantages of rapid detection, simple operation, high sensitivity, and good specificity in detecting Candida albicans, which is worthy of clinical application.


Assuntos
Candida albicans/isolamento & purificação , Candidíase/diagnóstico , Reação em Cadeia da Polimerase , Humanos , Recombinases , Sensibilidade e Especificidade
14.
Acta Virol ; 63(3): 270-277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507192

RESUMO

Orf, also called contagious ecthyma or contagious pustular dermatitis, is a significant zoonotic disease that primarily affects goat and sheep globally. Currently, the infection by orf virus (ORFV) has been observed in different host species worldwide, including China. Here, a suspected outbreak of orf infection in a goat farm in Anhui Province in 2018 was investigated. Through PCR, electron microscopy, and cell culture techniques, we confirmed that the outbreak was caused by ORFV. Consequently, the orf virus strain was named the AH/LA/2018 strain. The amplified and sequenced ORFV011 (B2L) and ORFV059 (F1L) genes were used to construct phylogenetic trees to elucidate the genetic characteristics of the ORFV and the molecular epidemiology of orf. The present study is the first systematic evolution analysis of the ORFV strain isolated in Anhui Province. The results of this study will be helpful to better understand the characteristics of ORFV, to help prevent and control the transmission of ORFV at an early stage in China. Keywords: Anhui Province; goat; orf virus; phylogenetic analysis.


Assuntos
Ectima Contagioso , Vírus do Orf , Filogenia , Animais , Células Cultivadas , China/epidemiologia , Ectima Contagioso/virologia , Genes Virais/genética , Doenças das Cabras/epidemiologia , Doenças das Cabras/virologia , Cabras , Microscopia Eletrônica de Transmissão , Vírus do Orf/classificação , Vírus do Orf/ultraestrutura , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/virologia
15.
Zhonghua Fu Chan Ke Za Zhi ; 54(9): 601-607, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31550776

RESUMO

Objective: To explore the pathways of preeclampsia by investigating different effects of pravastatin (Pra) on and soluble FMS tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) in different preeclampsia (PE)-like mouse models. Methods: C57BL/6J mice were randomly subcutaneously injected with N-nitro-L-arginine methyl ester (L-NAME) or intraperitoneally injected with lipopolysaccharide (LPS) as PE-like mouse model, saline as normal pregnancy control (Con) respectively, daily at gestational 7-18 days. Pra was given daily at gestational 8-18 days in each model group and the mice were divided into Pra (L-NAME+Pra, LPS+Pra, Con+Pra) and saline (L-NAME+NS, LPS+NS, Con+NS) groups. Liver,placental tissue and blood of pregnant mice were collected on the 18th day of pregnancy. The levels of VEGF, PlGF and sFlt-1 in the liver, placenta and serum of mice in each group were compared by western blot, ELISA and real-time fluorescence quantitative PCR (RT-PCR). Results: (1) ELISA: Serum VEGF (205.70±3.43, 154.60±2.31) and PlGF (131.5±3.75, 101.50±4.31) levels were significantly increased in L-NAME+Pra group compared with L-NAME+NS group (all P<0.05). Serum VEGF (202.30±4.90, 144.50±6.71) and PlGF (121.50±3.86, 95.41±4.08) levels were significantly higher in LPS+Pra group than those in LPS+NS group (all P<0.05). Serum sFlt-1 level in LPS+Pra group was significantly lower than that in LPS+NS group (3.01±0.50, 776.60±80.06), serum sFlt-1 level in L-NAME+Pra group was significantly lower than that in L-NAME+NS group (2.60±0.06, 583.70±9.83; all P<0.05). (2) Western blot: the expression levels of PlGF (1.344±0.118, 0.664±0.143) and VEGF (1.34±0.12, 0.66±0.14) in the liver of mice in the L-NAME+Pra group were significantly higher than those in the L-NAME+NS group (all P<0.05), but the expression levels of PlGF and VEGF in the placenta of L-NAME+Pra group were not significantly different from those of L-NAME+NS group (all P>0.05). The expression levels of PlGF and VEGF in placenta and liver of pregnant mice in LPS+Pra group were not significantly different from those in LPS+N group (all P>0.05). (3) RT-PCR: the mRNA expression of PlGF and VEGF in placenta and liver of L-NAME+Pra group were not significantly different from those in L-NAME+NS group (all P>0.05). The mRNA expression levels of PlGF and VEGF in placenta and liver of LPS+Pra group were not significantly different from those of LPS+NS group (all P>0.05). Conclusions: Pra has different regulatory effects on vascular endothelial function in different PE-like models. It reveals that different pathogenesis and pathways exist in different PE-like changes.


Assuntos
Fator de Crescimento Placentário/efeitos dos fármacos , Pravastatina/uso terapêutico , Pré-Eclâmpsia/tratamento farmacológico , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Animais , Anticolesterolemiantes/farmacologia , Biomarcadores/sangue , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Placenta , Fator de Crescimento Placentário/sangue , Reação em Cadeia da Polimerase , Pravastatina/farmacologia , Pré-Eclâmpsia/sangue , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/sangue , Fator A de Crescimento do Endotélio Vascular/sangue
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 905-909, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515787

RESUMO

OBJECTIVE: To detect mutation of LBR gene in a pedigree affected with Pelger-Huёt anomaly (PHA) and to explore its clinical characteristics. METHODS: Genomic DNA was extracted from the pedigree and healthy controls. The 14 exons of the LBR gene were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified in other family members and 100 healthy controls. Polyphen-2 and SIFT software were used to predict the effect of the mutation, and Swiss-model software was used to simulate the protein structure. RESULTS: Three patients were found to carry a c.893G>A mutation in exon 8 of the LBR gene, which resulted in substitution of the 298th amino acid residue glycine by glutamic acid (p.Gly298Glu). The same mutation was not found in healthy family members and 100 healthy controls. The mutation was predicted to be damaging. Bioinformatic simulation showed the mutation has altered the 3D structure of the LBR protein. CONCLUSION: The c.893G>A (p.Gly298Glu) mutation in the LBR gene probably underlies the PHA in this pedigree and has enriched the spectrum of LBR gene mutations.


Assuntos
Anomalia de Pelger-Huët/genética , Receptores Citoplasmáticos e Nucleares/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Éxons , Humanos , Mutação , Linhagem , Reação em Cadeia da Polimerase
17.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 930-934, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515793

RESUMO

OBJECTIVE: To detect potential mutations of HEXB gene in an infant with Sandhoff disease (SD). METHODS: Genomic DNA was extracted from peripheral blood sample of the infant. All coding exons (exons 1 to 14) and splicing sites of the HEXB gene were subjected to PCR amplification and direct sequencing.PubMed Protein BLAST system was employed to analyze cross-species conservation of the mutant amino acid. PubMed BLAST CD-search was performed to identify functional domains destroyed by thecandidate mutations. Impact of the mutations was analyzed with software including PolyPhen-2, Mutation Taster and SIFT. Whole-exome sequencing was carried out to identify additional mutations. RESULTS: The infant was found to carry compound heterozygous mutations c.1652G>A(p.Cys551Tyr) and c.1389C>G (p.Tyr463*) of the HEXB gene. The c.1389C>G (p.Tyr463*) mutation may lead to destruction of two functional domains in ß subunit of the Hex protein. The c.1652G>A(p.Cys551Tyr) mutation, unreported previously,was predicted to be probably damaging by Bioinformatic analysis. CONCLUSION: Compound heterozygous mutations c.1652G>A(p.Cys551Tyr) and c.1389C>G (p.Tyr463*) in the HEXB gene probably underlie the disease in this patient.


Assuntos
Doença de Sandhoff/genética , Cadeia beta da beta-Hexosaminidase/genética , Análise Mutacional de DNA , Éxons , Heterozigoto , Humanos , Lactente , Mutação , Reação em Cadeia da Polimerase
18.
Medicine (Baltimore) ; 98(35): e16993, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464949

RESUMO

RATIONALE: Parvovirus B19 (PV) infection is usually symptomless and can cause benign, short-lived conditions. Anemia associated with PRCA is the most representative hematologic manifestation, but neutropenia and thrombocytopenia have been rarely reported. PATIENT CONCERNS: Three patients were admitted to the hospital with neutropenia and thrombocytopenia. The accompanying symptoms were fever, myalgia, rash, or arthralgia, and all patients were previously healthy. DIAGNOSIS: Patients were positive for PV PCR and diagnosed with PV infection. Before the diagnosis of PV infection, 2 patients underwent BM study and almost absence of erythroid progenitor cells in BM aspiration were a clue for the PV infection. Other BM findings were hypocellular marrow and a few hemophagocytic histiocytes. INTERVENTIONS: Patients received supportive care with follow-up of CBC. OUTCOMES: All 3 patients spontaneously recovered from neutropenia and thrombocytopenia within 3 weeks without severe complications. LESSONS: The evaluation of PV infection should be considered in situations where there is neutropenia and thrombocytopenia in healthy individuals even without anemia as a differential diagnosis.


Assuntos
Neutropenia/complicações , Infecções por Parvoviridae/complicações , Infecções por Parvoviridae/diagnóstico , Trombocitopenia/complicações , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano , Reação em Cadeia da Polimerase
19.
Medicine (Baltimore) ; 98(35): e17001, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464954

RESUMO

RATIONALE: Acute retinal necrosis (ARN), which is characterized by peripheral necrotizing retinitis, severe retinal arteritis, and progressive inflammatory reaction in the vitreous and anterior chambers, has been reported in cases with herpes simplex encephalitis (HSE). It is a relatively rare complication secondary to HSE. However, cases presented with viral encephalitis following ARN were seldom reported. PATIENT CONCERNS: A 43-year-old immunocompetent male patient manifested the aforesaid reverse situation. He developed HSE following 3-day systemic steroid therapy for abrupt ocular pain and rapidly decreased visual acuity, which was later diagnosed as ARN. Polymerase chain reaction (PCR) analysis of vitreous specimen verified herpes simplex virus-1 (HSV-1) infection. DIAGNOSIS: HSE associated with ARN. INTERVENTIONS: The patient was treated with intravenous acyclovir (500 mg every 8 h) for 21 days. A pulse of intravenous methylprednisolone, 500 mg/d for 5 days was given as an anti-inflammatory therapy, followed by prednisone taper. OUTCOMES: The patient's neurological symptoms got improved very soon after the therapy, but his vision acuity remained no perception of light in both eyes. LESSONS: The present case indicates that ARN can also be a risk factor for HSE. Once ARN was suspected, corticosteroid should be applied with caution and in combination with antiviral treatment to avoid progressive duplication of virus and its spread to the brain.


Assuntos
Encefalite por Herpes Simples/complicações , Herpesvirus Humano 1 , Síndrome de Necrose Retiniana Aguda/complicações , Aciclovir/uso terapêutico , Adulto , Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , DNA Viral/análise , Encefalite por Herpes Simples/diagnóstico , Encefalite por Herpes Simples/tratamento farmacológico , Humanos , Hospedeiro Imunocomprometido , Masculino , Metilprednisolona/uso terapêutico , Reação em Cadeia da Polimerase , Síndrome de Necrose Retiniana Aguda/diagnóstico , Síndrome de Necrose Retiniana Aguda/tratamento farmacológico
20.
Sud Med Ekspert ; 62(4): 19-21, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31407701

RESUMO

The amelogenin gene encodes dental enamel protein and is present in humans in two forms - AMELX and AMELY, located on the X- and Y-chromosomes, respectively. This rare case depicts a partial deletion of the AMELY gene. In the Into-Stil LLC laboratory, we performed the genetic testing of the DNA samples extracted from buccal epithelial cells of the alleged father and the disputed child (a boy). Genotyping was carried out using COrDIS Plus ('Gordis', Russian Federation) and AmpFLSTR Identifiler Direct PCR Amplification ('Applied Biosystems', USA) Kits. Our findings have demonstrated that both the alleged father and the disputed child lacked the fragments corresponding with the AMELY gene. Using both STR-systems, we detected, in the disputed child's genome, the allele formally identical to the allele in the genome of the alleged father. Further analysis using the COrDYS ('Gordis', Russian Federation) kit allowed us to detect the amplified fragments corresponding with all the STR loci of Y chromosome, except DYS576 and DYS449, which confirmed that both studied individuals belonged to male biological sex.


Assuntos
Amelogenina/genética , Deleção Cromossômica , Cromossomos Humanos Y/genética , Técnicas de Genotipagem , Paternidade , Criança , Humanos , Masculino , Reação em Cadeia da Polimerase , Federação Russa
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