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1.
Chem Commun (Camb) ; 55(83): 12463-12466, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31576854

RESUMO

Herein, an ultrasensitive electrochemical biosensor is proposed for the quantification of the Flu A virus biomarker DNA (fDNA), and is based on loop-mediated isothermal amplification-generated hydrogen ions (LAMP-H+) which induce the formation of the dimer i-motif structure (DiMS) for signal transduction, coupled with exonuclease III (ExoIII)-assisted DNA walking for signal dual-amplification.


Assuntos
Técnicas Biossensoriais , DNA Viral/análise , Técnicas Eletroquímicas , Vírus da Influenza A/química , Técnicas de Amplificação de Ácido Nucleico , Prótons , Biomarcadores/análise , Dimerização , Íons/química
2.
Klin Lab Diagn ; 64(9): 565-570, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31610110

RESUMO

To analyze the method HBV covalent-closed circular DNA quantitative determination in liver puncture biopsies and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Republic of Uzbekistan. For quantitative analysis of HBV covalently closed circular DNA in a biopsy material a method was developed based on real-time PCR using TaqMan probes for the target fragment and for the endogenous reference gene, based on the detecting ccc HBV DNA method of Pollicino T. et al. When quantifying ccc DNA HBV in liver tissue of 18 moderately HBV activity with HBV DNA PCR positive results patients and 16 inactive HBsAg carriers, the ccc DNA HBV content was significantly different between groups (p<0.034) and in terms 1 copy of the ß-globin gene among moderate activity HBV patients amounted to 1.71±1.32 copies/cell, and for inactive HBsAg carriers 0.15±0.14 copies/cell. In the group of patients with severe liver fibrosis and cirrhosis, the amount of ccc DNA HBV in liver tissue in patients with HBV averaged 2.5±0.4 copies/cell, in patients with HBV + D on average 0.7±0.25 copies/cell, in patients with HCV + HBV co-infection 0.45±0.07 copies/cell, in patients with a preliminary diagnosis of chronic hepatitis C hepatitis, on average 0.12±0.04 copies/cell, in patients with cryptogenic hepatitis 0.2± 0.05 copies/cell. A significant difference was shown between the group of patients with chronic hepatitis B with marked fibrosis and cirrhosis of the liver with other patients groups, except for the group of 18 moderate activity chronic hepatitis B patients. The values of Student's t-test when compared with other groups were respectively: for patients with a HCV preliminary diagnosis t=5,92 p<0,05 f = 19, patients with cryptogenic hepatitis t=5,71 p<0,05 f = 18, with «inactive HBsAg carriage¼ t=5,55 p<0,05 f = 29, with HCV + HBV co-infection t=5,05 p<0,05 f = 15 and HBV + D co-infection t=3,82 p<0,05 f = 17. The covalently closed circular DNA HBV quantitative assessment method in liver puncture biopsies allows identifying HBsAgnegative chronic viral hepatitis B forms and also reflects the virus replication activity, which, in turn, makes it possible to assume further disease progression and evaluate the antiviral therapy effectiveness.


Assuntos
DNA Circular/análise , DNA Viral/análise , Hepatite B Crônica/diagnóstico , Biópsia por Agulha , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Humanos , Fígado , Federação Russa
3.
Medicine (Baltimore) ; 98(42): e17590, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31626130

RESUMO

To date, a small number of studies concerning the effects and safety of tenofovir disoproxil fumarate (TDF) in Chinese individuals were conducted. In this study, we aimed to assess the antiviral effects and nephrotoxicity of TDF in Chinese patients with chronic hepatitis B virus (HBV) infection.Patients with chronic HBV infection were prospectively recruited and TDF treatment was given for 96 weeks. HBV serologic markers, HBV DNA, creatinine and phosphorus were collected.Fifty-seven treatment-naïve and 48 treatment-experienced patients were recruited. Irrespective of the prior treatment history, more than 95% of patients achieved virological response during 96 weeks treatment with TDF. Estimated glomerular filtration rate (eGFR) significantly declined in the first year of treatment in patients with chronic hepatitis B or younger age (<65 years old) (both P < .05), while that was not achieved in patients with liver cirrhosis or older age (≥65 years old) (both P > .05). For patients who were treatment-naïve or treated previously with adefovir dipivoxil, eGFR declined at the 48th week; however, eGFR was partially recovered at the 96th week. Furthermore, multivariable analysis showed that basal eGFR <90 mL/min/1.73 m (P = .001; odds ratio: 4.821; 95% confidence interval: 1.904-12.206) is the only independent risk factor for eGFR <90 mL/min/1.73 m at the 96th week.TDF has potent antiviral effect in both treatment-naïve and treatment-experienced patients.


Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B Crônica/tratamento farmacológico , Tenofovir/uso terapêutico , Carga Viral , Adulto , Antivirais/uso terapêutico , China/epidemiologia , DNA Viral/análise , Feminino , Seguimentos , Antígenos E da Hepatite B/análise , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Humanos , Incidência , Masculino , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento
4.
Microbiol Res ; 228: 126300, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422230

RESUMO

Two morphologically different bacteriophages were isolated from the river and soil samples from various locations of Maharashtra, India against the phytopathogen Pseudomonas sp. that was recently reported to cause a new bacterial blight of pomegranate. Both the phages belonged to the order Caudovirales representing the families Siphoviridae (vB_Psp.S_PRɸL2) and Myoviridae (vB_Psp.M_SSɸL8). The multiplicity of infection ranged from 0.01 to 0.1, phage adsorption rate from 39% to 66%, latent period from 10 to 20 min with a burst size of 24-85 phage particles per infected host cell. The genome size of phages PRɸL2 and SSɸL8 was approximately 25.403 kb and 29.877 kb respectively. Restriction digestion pattern of phage genomic DNA was carried out for phage PRɸL2, Eco RI resulted in two bands and Hind III resulted in three bands while for phage SSɸL8, both Eco RI and Hind III each resulted in three bands. SDS-PAGE protein profile showed six bands for PRɸL2 and nine bands for SSɸL8 of different proteins. Phages showed high pH stability over a range of 4-9, temperature stability over a range of 4-50 °C and UV radiation showed a reduction up to 89.36% for PRɸL2 and 96% for SSɸL8. In short, the present research work discusses for the first time in-detailed characterization of phages of a phytopathogen Pseudomonas sp. from Maharashtra, India, which can be further efficiently used for biological control of the causative agent of a new bacterial blight disease of pomegranate.


Assuntos
Doenças das Plantas/microbiologia , Fagos de Pseudomonas/classificação , Fagos de Pseudomonas/isolamento & purificação , Pseudomonas/virologia , Punicaceae/microbiologia , Caudovirales/classificação , Caudovirales/genética , Caudovirales/isolamento & purificação , Caudovirales/ultraestrutura , DNA Viral/análise , Especificidade de Hospedeiro , Concentração de Íons de Hidrogênio , Índia , Viabilidade Microbiana , Myoviridae/classificação , Myoviridae/genética , Myoviridae/isolamento & purificação , Myoviridae/ultraestrutura , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/ultraestrutura , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Siphoviridae/ultraestrutura , Temperatura Ambiente , Raios Ultravioleta/efeitos adversos , Proteínas Virais/análise
5.
Arch Virol ; 164(11): 2683-2690, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428915

RESUMO

Current antiviral therapies against hepatitis B virus (HBV) infections, such as treatment with nucleos(t)ide analogs (NAs) and interferon alpha, can significantly lower HBV DNA titers, eventually to undetectable levels. However, it is still difficult to completely eliminate the stable template of HBV, the covalently closed circular DNA (cccDNA), and this contributes to viral rebound when treatment is discontinued. HBV pregenomic RNA (pgRNA), which was recently found to be present in the enveloped mature HBV viral particle in blood, is tentatively regarded, with still accumulating clinical evidence, as a novel bona fide virological marker reflecting the amount and status of cccDNA when serum HBV DNA becomes undetectable. HBV pgRNA and DNA share almost identical sequences, and it is therefore difficult to differentiate pgRNA from viral DNA using normal PCR methods. To exclude interference from viral DNA, methods for measuring pgRNA usually require a selective DNA degradation step, which is complicated and time-consuming and also compromises the accuracy of detection. In this study, we developed a simplified quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with improved accuracy achieved by probing the polyA tail of pgRNA. Using clinical serum samples, we observed that not all patients share the same 3' sequence, suggesting slight differences between HBV strains in the way they end transcription. We then designed and evaluated a universal primer and probe set for distinguishing HBV pgRNA from HBV DNA. Our results demonstrated that a one-step qRT-PCR assay could selectively amplify HBV pgRNA from a mixture of HBV RNA and DNA, which is valuable for clinical applications.


Assuntos
Vírus da Hepatite B/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , DNA Viral/análise , DNA Viral/genética , Hepatite B/virologia , Humanos , RNA Viral/análise , RNA Viral/genética
6.
Arch Virol ; 164(11): 2707-2713, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31456085

RESUMO

There is increasing evidence that the fecal indicator bacteria that are routinely used for testing water quality are inadequate for ensuring protection of the public health. Pepper mild mottle virus (PMMoV) has recently been suggested as an alternative indicator of human fecal contamination in water; however, in Egypt there are no data available about its occurrence and concentration in aquatic environment. The concentration of PMMoV in the influent and effluent of three wastewater treatment plants was measured using qRT-PCR over a period of one year and compared to that of human adenovirus (HAdV), which is considered an indicator for human fecal contamination. PMMoV was detected in ~ 94% of the influent samples and 78% of the effluent samples, with concentrations ranging from 3.9 × 104 to 3.3 × 108 genome copies/l (GC/l) in the influent and 3.9 × 104 to 1.2 × 107 GC/l in the effluent. Similarly, HAdV was identified in 88% and 78% of the influent and effluent samples, respectively. The HAdV concentration ranged between 1.5 × 104 and 1.5 × 107 GC/l for the influent and 2.6 × 104 and 4.4 × 106 GC/l for the effluent. No significant difference was found between the removal ratio of PMMoV and HAdV. Viral reduction of 0.2-1.9 log10 and 0.2- 2.3 log10 by the treatment process was observed for PMMoV and HAdV, respectively. Both viruses showed no clear seasonality. Our data support the use of PMMoV as a fecal indicator of wastewater contamination and a process indicator for the performance of the treatment process.


Assuntos
Adenovírus Humanos/isolamento & purificação , Tobamovirus/isolamento & purificação , Águas Residuárias/virologia , Poluição da Água/análise , Purificação da Água , Adenovírus Humanos/genética , DNA Viral/análise , Egito , Fezes/virologia , Humanos , Tobamovirus/genética , Microbiologia da Água
7.
Gut ; 68(12): 2206-2213, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31462554

RESUMO

OBJECTIVE: Although most patients with chronic hepatitis B (CHB) reach effective virological suppression with long-term nucleos(t)ide analogues (NA) therapy, some might not need to continue treatment for life. In this randomised, controlled, phase IV trial, we evaluated off-therapy outcomes in patients after discontinuing long-term NA therapy. DESIGN: Patients who had received NA therapy for ≥1 year and achieved virological suppression (hepatitis B e antigen (HBeAg) seroconversion combined with undetectable hepatitis B virus (HBV) DNA ≥12 months in HBeAg-positive patients or undetectable HBV DNA ≥36 months in HBeAg-negative patients) were randomised 2:1 to stop or continue NA therapy for 72 weeks. Sustained disease remission (HBeAg negative, HBV DNA <2000 IU/mL and normal alanine aminotransferase (ALT)) was evaluated at 72 weeks after stopping NA therapy. RESULTS: Among 67 enrolled patients, sustained disease remission was observed in 13/45 (29%) stop versus 18/22 (82%) continue patients. Hepatitis B surface antigen (HBsAg) loss occurred in two patients (one in each group). The median HBsAg decline from randomisation to week 72 was similar in both groups (0.2 (0.0-0.4) vs 0.1 (0.0-0.2) log IU/mL in stop vs continue patients). Among patients who stopped, 15/45 (33%) had virological or biochemical relapse and 17/45 (38%) were retreated according to predefined criteria. A total of 11/18 (61%) pretreatment HBeAg-positive versus 6/27 (22%) HBeAg-negative patients required retreatment (p=0.01). Fourteen (31%) patients developed ALT >10× upper limit of normal (ULN) and another 7 (16%) had ALT >5× ULN. No patients experienced liver decompensation or died. CONCLUSION: The findings of this prospective study suggest limited benefit of stopping NA therapy in chronic hepatitis B. TRIAL REGISTRATION NUMBER: NCT01911156.


Assuntos
Hepatite B Crônica/tratamento farmacológico , Nucleosídeos/análogos & derivados , DNA Viral/análise , Feminino , Seguimentos , Antígenos de Superfície da Hepatite B/análise , Antígenos E da Hepatite B/análise , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Nucleosídeos/uso terapêutico , Estudos Prospectivos , Resultado do Tratamento
8.
Medicine (Baltimore) ; 98(32): e16671, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31393366

RESUMO

RATIONALE: Neurological complications of varicella-zoster virus (VZV) infection include cerebral infarction, meningoencephalitis, segmental sensory disturbance, facial nerve palsy, and myelitis. Chronic myelitis is rarely reported. Diagnosis of VZV infection can be confirmed by elevated anti-VZV immunoglobulin G (IgG) antibody or detection of VZV DNA in the cerebrospinal fluid (CSF), the former reported to be superior. The detection rate of VZV DNA is generally thought to decrease with time after the onset of the condition. The utility of VZV DNA polymerase chain reaction (PCR) is thus thought to be limited to the acute phase of the disease. The presence of skin lesions also helps to render a diagnosis; however, cases of zoster sine herpete (ZSH), the occurrence of segmental symptoms without skin lesions, renders the diagnosis of VZV infection more difficult. Antiviral drugs, such as acyclovir, are the treatment of choice to resolve VZV infections. PATIENT CONCERNS: A 65-year-old Japanese man felt heaviness and a throbbing pain on the ulnar side of the right forearm. He was previously diagnosed with cervical spondylosis, and received nonsteroidal anti-inflammatory drugs with little improvement. Contrast cervical magnetic resonance imaging showed a swelling and an increased signal intensity of the spinal cord, and an enhancing lesion, all of which were suggestive of myelitis. DIAGNOSIS: We found no evidence for diagnoses of sarcoidosis, Behçet disease, multiple sclerosis, or neuromyelitis optica spectrum disorder. The CSF analysis revealed an elevation of the total protein concentration and that the patient was positive for VZV DNA, while anti-VZV IgG was not elevated. The patient was therefore diagnosed with ZSH myelitis. INTERVENTIONS: We administered acyclovir and valaciclovir as the first therapy. At the time of recurrence, we used high-dose acyclovir, vidarabine, and high-dose methylprednisolone pulse therapy. OUTCOMES: The patient's dysesthetic pain in the right upper limb improved following the first antiviral therapy. Two months later, he suffered a recurrence, but the second therapy significantly relieved his symptoms. LESSONS: VZV infection should be regarded as an important differential diagnosis of chronic myelitis. VZV DNA PCR should be performed even in the chronic phase of the condition to introduce the possibility of antiviral therapy as a treatment option.


Assuntos
Mielite/etiologia , Zoster Sine Herpete/complicações , Zoster Sine Herpete/diagnóstico , Idoso , Antivirais/uso terapêutico , DNA Viral/análise , Antebraço , Herpesvirus Humano 3/isolamento & purificação , Humanos , Imagem por Ressonância Magnética , Masculino , Mielite/diagnóstico por imagem , Reação em Cadeia da Polimerase , Recidiva , Zoster Sine Herpete/líquido cefalorraquidiano , Zoster Sine Herpete/tratamento farmacológico
9.
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
10.
Rev Soc Bras Med Trop ; 52: e20180457, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31271616

RESUMO

INTRODUCTION: We defined the cut-off values of the antigenemia and cytomegalovirus (CMV) DNA tests in HIV/AIDS patients to identify CMV disease. METHODS: A total of 97 samples from 68 patients with and without CMV disease were analyzed by viral DNA detection and antigenemia assay. RESULTS: Qualitative and quantitative results significantly differed between assays. The cut-off values for the antigenemia and qPCR assays were 1.5 positive cells/200,000 leukocytes and 3.715 log/mL, respectively. CONCLUSIONS: Antigenemia and qPCR are suitable for monitoring CMV disease in HIV patients, however, the threshold values should be determined within the centers where the patients are monitored.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/isolamento & purificação , DNA Viral/análise , Infecções Oportunistas Relacionadas com a AIDS/sangue , Antígenos Virais/sangue , Brasil/epidemiologia , Citomegalovirus/genética , Infecções por Citomegalovirus/sangue , DNA Viral/sangue , Humanos , Valor Preditivo dos Testes , Estudos Prospectivos , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Carga Viral
11.
BMC Vet Res ; 15(1): 226, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277650

RESUMO

BACKGROUND: Canine lobular orbital adenomas are benign tumors that arise from orbital glandular tissue and extend into the orbit, conjunctiva, and third eyelid. Surgical excision is challenging and recurrence rates are high following excision alone. Enucleation and exenteration reduces the likelihood of recurrence, but is a radical therapeutic option for an otherwise visual and comfortable eye. Human papillomavirus causes 4.5% of worldwide cancers in people and has been identified in up to 23% of benign salivary gland tumors. To date, the etiology of canine lobular orbital adenomas has not been established and it is reasonable to consider canine papillomaviruses as an associative agent with benign glandular tumors in dogs. Identification of the underlying etiology of these tumors may help establish treatment or preventative measures. The purpose of this study was to evaluate conjunctival and orbital tissue of phenotypically normal dogs and tissue from canine lobular orbital adenomas for the presence of papillomavirus DNA. RESULTS: Thirty seven canine lobular orbital adenoma samples (36 formalin fixed paraffin embedded (FFPE) tissue samples from 33 dogs and one freshly collected sample) were evaluated via polymerase chain reaction for the presence of papillomavirus DNA. Conjunctival tissue samples, from 10 dogs with normal ocular examinations, excised immediately following euthanasia, were used as phenotypically normal controls. Three FFPE and one freshly collected tissue samples previously confirmed to be positive for papillomavirus DNA were used as positive controls. PCR products verified positive controls. Papillomavirus DNA was not detected in fresh conjunctival tissue of the phenotypically normal control dogs or in samples of fresh or FFPE canine lobular orbital adenoma tissue. CONCLUSIONS: An association between papillomavirus and the development of canine lobular orbital adenomas is unlikely. Further research is needed to evaluate if other viruses play a role in the pathogenesis of canine lobular orbital adenomas.


Assuntos
Adenoma/veterinária , Doenças do Cão/virologia , Neoplasias Orbitárias/veterinária , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/veterinária , Adenoma/patologia , Adenoma/virologia , Animais , Túnica Conjuntiva/virologia , DNA Viral/análise , Doenças do Cão/patologia , Cães , Feminino , Masculino , Neoplasias Orbitárias/patologia , Neoplasias Orbitárias/virologia , Papillomaviridae/genética , Infecções por Papillomavirus/patologia
12.
Anal Chim Acta ; 1077: 297-304, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31307722

RESUMO

In this study, we designed a fluorescence enhancement strategy based on silver nanoparticle (AgNP) aggregates for the detection of hepatitis B virus DNA sequences. AgNPs were functioned with recognition probes (Cy3-probe) and hybrid probes (Oligomer-A and Oligomer-B). The presence of target DNA mediated the formation of sandwich complexes between the immobilized capture probes and the functionalized AgNPs, which was followed by hybridization-induced formation of AgNP aggregates. The fluorescent intensity could be extremely amplified by both the increasing number of fluorophores and metal enhanced fluorescence (MEF) effect. Under optimal conditions, this method achieved a detection limit of 50 fM which was 1560-fold lower than that of un-enhanced fluorescent assays. It was illustrated that the HBV DNA concentrations ranging from 100 fM to 10 nM had a good log-linear correlation with the corresponding fluorescent intensity (R = 0.991). Moreover, this method had high specificity both for distinguishing single-base mismatches and identifying target DNA under the interference of genomic DNA. This fluorescent microarray had high-throughput analytical potential and could apply to many other disease diagnoses.


Assuntos
DNA Viral/análise , Vírus da Hepatite B/genética , Nanopartículas Metálicas/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prata/química , Sondas de DNA , DNA Viral/genética , Limite de Detecção , Hibridização de Ácido Nucleico
13.
Microbiol Immunol ; 63(10): 407-412, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31342547

RESUMO

Loop-mediated isothermal amplification (LAMP) assays are used to detect diverse pathogens. Initially, LAMP amplicons were detected using electrophoresis; later, real-time monitoring based on turbidity was developed to overcome the problem of contamination with environmental DNA. Recently, real-time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. Here, methods of detecting LAMP amplicons are reviewed.


Assuntos
Infecções Bacterianas/microbiologia , DNA Bacteriano/análise , DNA de Protozoário/análise , DNA Viral/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Protozoários/parasitologia , Viroses/virologia , Fluorescência , Nefelometria e Turbidimetria/métodos , Reprodutibilidade dos Testes
15.
Ceska Gynekol ; 84(2): 84-92, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31238677

RESUMO

OBJECTIVES: The aim of the EXPL-HPV-002 study is to evaluate the integration of 14 high-risk HPV as a biomarker of the severity and the progression of cervical lesions. Such a „triage biomarker“ would help to reduce the number of unnecessary colposcopies, to avoid over-treatment of lesions that spontaneously regress and to better target the lesions requiring treatment. DESIGN: EXPL-HPV-002 is a prospective, open-label, single arm, GCP study conducted at 2 clinical sites in the Czech Republic. SETTINGS: Investigations centers: Private Gynecology Center, Brno; Gynecological and Obstetrical Clinic, Brno; Genotyping central lab: NRL for Papillomaviruses and polyomaviruses, IHBT, Prague; Histology Central reading: Aeskulab Pathology, Prague; Molecular combing HPV test: Genomic Vision, Bagneux. METHODS: From June 2016 to May 2018, 688 patients aged 25-65, referred to colposcopy after an abnormal Pap-smear, were enrolled in the study. Among them 60% were found HPV high-risk. The study is divided in two phases: 1. a cross-sectional phase using data collected at first visit (colposcopy images ± histology, pap-smear for HPV genotyping and molecular combing) to study the association between HPV integration status versus colposcopy and histology grades; 2. a longitudinal phase using data collected in follow-up visits: cytology at 6, 18 and 30 months and colposcopy ± histology at 12, 24 and 36 months. A pap-smear collected at 12, 24 and 36 months allows to perform genotyping and molecular combing. HPV integration status is analyzed in comparison with the evolution of lesions, viral clearance and HPV genotype. HPV genotyping and molecular combing were performed on pap-smear samples in central laboratories. Histology data were reviewed by central reading. RESULTS: The transversal phase of the study is achieved and shows that the HPV integration into the human DNA, monitored by molecular combing, can significantly differentiate normal subjects from women with cervical lesions or cancer. CONCLUSION: HPV integration into the host genome, monitored by Genomic Visions technology, is a reliable diagnostic biomarker that will greatly help clinicians to improve their medical decision tree.


Assuntos
Colposcopia , DNA Viral/análise , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/prevenção & controle , Esfregaço Vaginal , Adulto , Idoso , Estudos Transversais , República Tcheca , Sondas de DNA de HPV , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Gravidez , Estudos Prospectivos , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia
16.
Niger Postgrad Med J ; 26(2): 80-86, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31187746

RESUMO

Background: Hepatitis B virus (HBV) is hyperendemic in Nigeria. Available literature reveal genotype E as being predominant in West Africa. This study aimed at identifying the current pattern and prevalent genotypes of HBV in Zaria, Nigeria. Materials and Methods: Four millilitre of blood was collected in ethylenediaminetetraacetic acid-container from each of 165 HBV surface antigen-positive participants recruited purposively from the gastroenterology clinic from May to August, 2017. Plasma was separated and frozen at -20°C till analysis. Multiplex-nested polymerase chain reaction using type-specific primers was used to identify the various HBV genotypes. Results: Median (and interquartile range) age of the participants was 31.0 (25.5-39.0) years, with males constituting 107 (64.8%). Majority (83.6%) of the samples analysed were HBV-DNA-positive with 82.6% of the HBV-DNA-positive samples being mixed genotype infections. Irrespective of mode of occurrence, five HBV genotypes were identified with HBV/E (97.1%) being the most predominant, followed by HBV/B (82.6%), HBV/A (24.6%), then HBV/C (17.4%), while HBV/D (0.7%) was the least prevalent. Conclusion: In most (99.1%) of the mixed-infection were a combination of genotype E, the predominant genotype, with other genotypes predominantly genotype B. HBV genotypes E, B, A, C and D are the prevalent genotypes in Zaria, Nigeria, as they occur in single genotype and in mixed-genotypes pattern.


Assuntos
DNA Viral/análise , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B/epidemiologia , Genes Virais/genética , Genótipo , Hepatite B/virologia , Vírus da Hepatite B/genética , Humanos , Masculino , Nigéria/epidemiologia , Reação em Cadeia da Polimerase , Prevalência
17.
Vet Microbiol ; 234: 72-76, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213274

RESUMO

Adenoviruses are a relatively common infection of reptiles globally and are most often reported in captive central bearded dragons (Pogona vitticeps). We report the first evidence of adenoviruses in bearded dragons in their native habitat in Australia. Oral-cloacal swabs and blood samples were collected from 48 free-ranging bearded dragons from four study populations: western bearded dragons (P. minor minor) from Western Australia (n = 4), central bearded dragons (P. vitticeps) from central Australia (n = 2) and western New South Wales (NSW) (n = 29), and coastal bearded dragons (P. barbata) from south-east Queensland (n = 13). Samples were tested for the presence of adenoviruses using a broadly reactive (pan-adenovirus) PCR and a PCR specific for agamid adenovirus-1. Agamid adenovirus-1 was detected in swabs from eight of the dragons from western NSW and one of the coastal bearded dragons. Lizard atadenovirus A was detected in one of the dragons from western NSW. Adenoviruses were not detected in any blood sample. All bearded dragons, except one, were apparently healthy and so finding these adenoviruses in these animals is consistent with bearded dragons being natural hosts for these viruses.


Assuntos
Adenoviridae/isolamento & purificação , Lagartos/virologia , Adenoviridae/genética , Animais , Atadenovirus/isolamento & purificação , DNA Viral/análise , New South Wales , Reação em Cadeia da Polimerase , Queensland
18.
Microb Pathog ; 135: 103566, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31252065

RESUMO

BACKGROUND: Breast cancer is one of the most common cancers in the world particularly among Iranian women. Bovine leukemia virus (BLV) is an enzootic, exogenous, and oncogenic retrovirus that causes B-cell leukosis in 1-5% of infected cattle. The current study aimed at evaluating the correlation between BLV infection and breast cancer in an Iranian population. MATERIALS AND TECHNIQUES: A total of 400 samples including 200 breast cancer-suspected tissue samples and 200 blood samples of women without breast cancer, were collected from July 2017 to October 2018 from women referred to two general hospitals in Qom Province, Iran. The nested PCR technique was performed to determine the presence of tax and gag gene of BLV in the collected samples. RESULTS: Out of 200 breast cancer-suspected tissue samples, 172 samples were malignant in terms of pathology. Other samples were reported as non-malignant and non-tumor. Based on nested PCR technique, tax and gag genes of BLV were detected in 30% and 8% of breast cancer-suspected tissue samples, respectively. The frequency of BLV in blood samples collected from women without breast cancer was 16.5% (33/200). CONCLUSION: It seems that human breast cancer and BLV infection in cattle could be associated using nested PCR technique.


Assuntos
Sangue/virologia , Mama/virologia , Infecções por Deltaretrovirus/virologia , Vírus da Leucemia Bovina/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Mama/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/virologia , DNA Viral/análise , Infecções por Deltaretrovirus/epidemiologia , Feminino , Genes gag , Genes pX , Humanos , Irã (Geográfico)/epidemiologia , Vírus da Leucemia Bovina/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
19.
Cornea ; 38(9): 1185-1188, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31205162

RESUMO

PURPOSE: To report a case of severe bilateral necrotizing herpes simplex keratitis (HSK) in an immunocompetent patient, with genotyping of the underlying herpes simplex virus 1 (HSV-1). METHODS: Genetic analyses of HSV-1 in tear samples were performed with polymerase chain reaction-based restriction fragment length polymorphism, targeting the viral genes unique short (US)2, US4 (glycoprotein G), and US7 (glycoprotein I). RESULTS: A 64-year-old woman with no history of atopy or immune disorders manifested bilateral keratitis with geographic ulcer. Her initial visual acuity was 20/1000 (OD) and 20/20 (OS). Polymerase chain reaction testing of a tear sample revealed the presence of HSV-1 in both eyes, and the patient was diagnosed with bilateral HSK. Both eyes progressed to necrotizing keratitis during the treatment course. Continuous intensive treatment, at first with acyclovir ointment and oral valacyclovir and later with steroid eye drops for stromal keratitis, finally improved the patient's condition. However, after 2 years, her visual acuity was limited to 20/250 (OD) and 20/60 (OS) because of corneal opacity from scarring. We found that the strain in the current case had a genotype combination of C/A/B (for US2/US4/US7), a known pattern in Japan, in both eyes. CONCLUSIONS: We successfully performed an unprecedented genetic analysis of an HSV-1 strain isolated from a case of bilateral necrotizing HSK in an immunocompetent patient. The association of the HSV-1 genotype with the clinical manifestation remains unclear, calling for more data from new cases, especially from different geographic regions.


Assuntos
Herpesvirus Humano 1/genética , Ceratite Herpética/virologia , DNA Viral/análise , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição
20.
Indian J Ophthalmol ; 67(7): 1040-1046, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31238404

RESUMO

Purpose: To determine the presence of herpes simplex virus and varicella zoster virus (HSV 1 and 2, VZV) in the cornea of normal subjects by multiplex real time quantitative (qPCR) assay and evaluate its utility in the diagnosis of viral keratitis. Methods: Corneal epithelial cells from 33 eyes of 22 patients undergoing photorefractive keratectomy surgery (controls) and 50 corneal scrapings from 50 patients with suspected HSV keratitis were analyzed for the presence of HSV1 by conventional PCR and for presence of HSV1 and 2 and/or VZV by multiplex real-time PCR. Corneal scrapings of patients were also tested for HSV1 antigen by immunofluorescence assay (IFA). The results were compared and clinical records reviewed. Results: HSV1 and VZV DNA were detected in 8/33 controls (mean-14.3 ± 7.96, range: 3-29.1 copies/mL) and 2/33 controls (mean-10.7 ± 10.9, range 3-18.5 copies/ml) respectively. HSV2 was not detected in any of the controls. Copy numbers above the mean + 1SD of controls were considered significant for viral load in patient samples. Significantly higher number of corneal scrapings (39/50, 78%) from patients were positive for HSV1 (1.2 × 106 copies/mL ± 3.7 × 106 copies/mL) by real time qPCR compared to IFA (11/48, 23%, P value 0.0001) and conventional PCR (20/50, 40%, P value 0.0002). Double infection with HSV-1 (1.5 × 107 copies/ml) and HSV-2 (3.57 × 104 copies/ml) in one case and VZV infection (1.03 × 102 copies/ml) in another was also detected by the multiplex real-time PCR. Conclusion: Multiplex real-time PCR reliably detects HSV1 and 2 and VZV DNA and is ideal for the diagnosis of HSV and VZV keratitis in an ocular microbiology laboratory.


Assuntos
Infecções Oculares Virais/diagnóstico , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Herpesvirus Humano 3/genética , Ceratite Herpética/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecção pelo Vírus da Varicela-Zoster/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Córnea/patologia , Córnea/virologia , DNA Viral/análise , Infecções Oculares Virais/epidemiologia , Infecções Oculares Virais/virologia , Feminino , Seguimentos , Herpes Simples/diagnóstico , Herpes Simples/epidemiologia , Herpes Simples/virologia , Humanos , Índia/epidemiologia , Lactente , Ceratite Herpética/epidemiologia , Ceratite Herpética/virologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Infecção pelo Vírus da Varicela-Zoster/epidemiologia , Infecção pelo Vírus da Varicela-Zoster/virologia , Adulto Jovem
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