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1.
Article in English | WPRIM | ID: wpr-887743

ABSTRACT

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (


Subject(s)
Adolescent , Adult , Child , Child, Preschool , Cytomegalovirus/genetics , Cytomegalovirus Infections/virology , DNA, Viral/analysis , Epstein-Barr Virus Infections/virology , Female , Herpesvirus 4, Human/genetics , Humans , Infant , Infant, Newborn , Male , Middle Aged , Nucleic Acid Amplification Techniques , Recombinases/genetics , Young Adult
2.
Article in English | WPRIM | ID: wpr-887737

ABSTRACT

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of


Subject(s)
Antitubercular Agents , Bacterial Proteins/genetics , Catalase/genetics , DNA, Bacterial/analysis , DNA-Directed RNA Polymerases/genetics , Drug Resistance, Multiple, Bacterial/genetics , Isoniazid , Molecular Diagnostic Techniques/methods , Mutation , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Amplification Techniques/methods , Oxidoreductases/genetics , Phenotype , Rifampin , Whole Genome Sequencing
3.
Rev. cuba. med. trop ; 72(2): e522, mayo.-ago. 2020. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1149912

ABSTRACT

Introducción: Los ensayos para cuantificar el ADN del virus de la hepatitis B (VHB) o carga viral son imprescindibles en el diagnóstico y en el seguimiento de los pacientes con hepatitis B crónica; de ahí que estén disponibles estuches diagnósticos para esta función. En el presente estudio se muestra la validación de SUMASIGNAL VHB (un paso), el cual es un sistema de reacción en cadena de la polimerasa en tiempo real (RCP-TR) para la cuantificación del genoma del VHB, propuesto por el Centro de Inmunoensayo. Objetivo: Evaluar el desempeño analítico de SUMASIGNAL VHB (un paso). Métodos: Se utilizó un panel de 80 muestras de suero bien caracterizadas y el Tercer Estándar Internacional de la OMS para las técnicas de amplificación de ácidos nucleicos del virus de la hepatitis B. Se determinaron las características del ensayo como especificidad clínica, especificidad analítica (reactividad cruzada), rango lineal o linealidad y exactitud, precisión intraensayo y comparación con un ensayo de referencia. Resultados: La especificidad analítica y clínica fue del 100 por ciento. Al evaluar la linealidad y exactitud con un estándar de referencia de la OMS, se obtuvo que la totalidad de las diferencias entre los Log10 del valor obtenido y el de referencia resultaron inferiores a 0,5 Log10 (r= 0,9977 y r2= 0,9954). Además, se obtuvieron bajos coeficientes de variación intraensayo. La evaluación comparativa con el estuche comercial Artus HBV RG PCR kit mostró una correlación fuerte (r= 0,8882). Conclusiones: SUMASIGNAL VHB (un paso) es un ensayo fácil de realizar manualmente, es rápido e incluye reactivos de extracción de ácidos nucleicos. Teniendo en cuenta la validez del método para el uso previsto, puede ser recomendado para su introducción en el diagnóstico, la vigilancia y la indicación de tratamiento en los pacientes con hepatitis B crónica(AU)


Introduction: Assays to quantify hepatitis B virus (HBV) DNA or viral load are indispensable for the diagnosis and follow-up of patients with chronic hepatitis B, hence the availability of diagnostic kits for this purpose. The present study deals with the validation of HBV SUMASIGNAL (one step), a real time polymerase chain reaction (RT-PCR) system for quantification of the HBV genome proposed by the Immunoassay Center. Objective: Evaluate the analytical performance of HBV SUMASIGNAL (one step). Methods: Use was made of a panel of 80 well characterized serum samples and the Third WHO International Standard for hepatitis B virus nucleic acid amplification techniques. Determination was performed of assay characteristics such as clinical specificity, analytical specificity (cross-reactivity), linear range or linearity and accuracy, intra-assay precision and comparison with a reference assay. Results: Analytical and clinical specificity was 100 percent. Evaluation of linearity and accuracy with a WHO reference standard revealed that all the differences between the log10 of the value obtained and the reference value were lower than 0.5 log10 (r= 0.9977 and r2= 0.9954). The intra-assay variation coefficients obtained were low. Comparative evaluation with the commercial Artus HBV RG PCR kit showed a strong correlation (r= 0.8882). Conclusions: The assay HBV SUMASIGNAL (one step) is easy to conduct manually, fast and includes reagents for nucleic acid extraction. Based on the validity of the method for the use in mind, it may be recommended for incorporation into the diagnosis, surveillance and treatment of patients with chronic hepatitis B(AU)


Subject(s)
Humans , Hepatitis B virus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Real-Time Polymerase Chain Reaction/methods , Validation Study
4.
Rev. chil. obstet. ginecol. (En línea) ; 85(2): 175-184, abr. 2020. tab
Article in Spanish | LILACS | ID: biblio-1115514

ABSTRACT

OBJETIVO: revisar los diferentes métodos de diagnóstico de la tricomoniasis vaginal disponibles hasta el presente. MATERIALES Y MÉTODOS: se revisó la bibliografía latinoamericano e internacional a través de los sitios electrónicos de Pub-Med y Scielo. RESULTADOS: la Tricomonas vaginalis es considera como la enfermedad de transmisión sexual no viral, curable más frecuente y prevalente en el mundo. Se revisan los diferentes de métodos para diagnosticar la presencia de la tricomonas vaginalis en pacientes femeninos con síntomas y signos de la infección producida por el protozoario flagelado. CONCLUSIONES: se revisaron los diferentes métodos de diagnostico de la infección producida por la Tricomonas vaginalis en pacientes femeninas, desde los clásicos hasta los más actuales que emplean alta tecnología.


OBJECTIVE: to review the different diagnostic methods of Trichomonas vaginal available at the present time. MATERIAL AND METHOD: it was reviewed the Latin-American and international bibliography using the Pub-Med and Scielo web sites. RESULTS: Trichomonas vaginalis is considered the most common and prevalent sexual transmitted disease curable and non-viral worldwide. It was reviewed the different methods to diagnose the presence of Trichomonas vaginalis in female patients with symptoms and signs of infection produces by the flagellate protozoa. CONCLUSION: Different methods of diagnosis of the infection produced by Trichomonas vaginalis, since the classics to the most current methods that use high technology, were reviewed.


Subject(s)
Humans , Female , Trichomonas Vaginitis/diagnosis , Trichomonas vaginalis , Vaginal Smears , Sexually Transmitted Diseases/diagnosis , Sensitivity and Specificity , Nucleic Acid Amplification Techniques , Culture Techniques , Antigens/analysis
5.
Int. j. odontostomatol. (Print) ; 14(1): 131-135, mar. 2020. tab, graf
Article in English | LILACS | ID: biblio-1056512

ABSTRACT

ABSTRACT: Oral Lichen planus (OLP) is one of the main inflammatory diseases of the oral mucosa that is considered as a potentially malignant disorder. The exact pathogenesis of OLP remains to be completely understood. However, presence of bacteria has been associated to the inflammatory response observed in OLP. Particularly, Helicobacter pylori a major etiological agent of gastrointestinal inflammatory diseases and risk factor for gastric cancer, has been associated to Lichen planus. Here we studied a group of Chilean patients if there is any association between the presence of Helicobacter pylori and the clinical manifestation of OLP. We found a significant difference between the patients positive for H. pylori and the age of OLP diagnosis, suggesting that oral H. pylori might induce the disease at an earlier age. However, we could not confirm a statistically significance between the presence of the bacteria and OLP.


RESUMEN: Liquen Plano Oral (LPO) es una enfermedad inflamatoria de la mucosa oral considerada como desorden potencialmente maligno. La patogénesis exacta de LPO es desconocida. Sin embargo, se ha asociado la presencia de bacterias como responsables de la inflamación observada en LPO. Particularmente, Helicobacter pylori (H. pylori), agente etiológico principal de enfermedades inflamatorias gastrointestinales y factor de riesgo de cáncer gástrico, ha sido asociado con LPO. Se estudió la posible asociación entre H. pylori y manifestaciones clínicas de LPO en un grupo de pacientes Chilenos. Se encontró diferencia significativa entre los pacientes positivos para H. pylori y la edad de diagnóstico de LPO, sugiriendo que H. pylori podría inducir la enfermedad a temprana edad. Sin embargo, no se pudo confirmar significancia estadística entre la presencia de esta bacteria y la presencia de displasia en LPO.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Helicobacter pylori/isolation & purification , Lichen Planus, Oral/physiopathology , Lichen Planus, Oral/microbiology , Mouth/microbiology , Saliva/microbiology , Chile , Gene Amplification , Statistics, Nonparametric , Nucleic Acid Amplification Techniques
6.
Adv Rheumatol ; 60: 42, 2020. tab
Article in English | LILACS | ID: biblio-1130805

ABSTRACT

Abstract Background: Human herpesviruses (HHVs) are responsible for a significant number of clinical manifestations in systemic lupus erythematous (SLE) patients. The aim of this study was to determine the frequency of active HHV infections in SLE patients and correlating them with disease activity. Methods: Serum samples were collected from 71 SLE patients and their DNAs were extracted and analyzed to detect HHV-DNA viruses using the nucleic acid amplification technique. Results: Fifteen out of the 71 (21.1%) patients tested positive for the HHV-DNA virus. Of them, 11/15 HHV-DNA-positive patients (73.3%) had SLE activity index (SLEDAI - Systemic Lupus Erythematosus Disease Activity Index) ≥8 (p = 0.0001). Active HCMV infection was the mostly frequently observed infection, occurring in 6/15 patients (40%). The frequencies of other active viral infections were 22% for HSV-1, 16.7% for HHV-7, and 5.5% for HSV-2. Viral coinfection (two or more viruses detected in the same sample) occurred in three patients (16.7%). Active HHV infections in SLE patients are more frequent in those with active SLE (≥8), who is at high risk of HHV reactivation and HCMV disease. Conclusion: Viral surveillance is important to identify active HHV infections that can cause clinical symptoms and other complication in SLE patients.


Subject(s)
Humans , Herpesviridae Infections/diagnosis , Nucleic Acid Amplification Techniques/instrumentation , Lupus Erythematosus, Systemic/physiopathology , Polymerase Chain Reaction/instrumentation , Coinfection
7.
Article in Chinese | WPRIM | ID: wpr-828566

ABSTRACT

OBJECTIVE@#To explore the feasibility of surgical treatment for cancer patients complicated with corona virus disease 2019 (COVID-19).@*METHODS@#The management and clinical outcome of a sigmoid cancer patient with COVID-19 were analyzed.@*RESULTS@#The inflammation indicators and fever of this patient were effectively controlled and the lung lesions remained stable after active anti-viral treatment, then the radical colorectomy was performed after the viral negative conversion for twice.@*CONCLUSIONS@#The case indicates that it may feasible to undergo radical tumor surgery for cancer patients with COVID-19 after the virus nucleic acid testing turns negative and more studies are needed to confirm this conclusion.


Subject(s)
Antiviral Agents , Therapeutic Uses , Betacoronavirus , Clinical Laboratory Techniques , Colonic Neoplasms , General Surgery , Virology , Coronavirus Infections , Diagnosis , Drug Therapy , Fever , Humans , Nucleic Acid Amplification Techniques , Pandemics , Pneumonia, Viral , Diagnosis , Drug Therapy
8.
Chinese Journal of Biotechnology ; (12): 1232-1240, 2020.
Article in Chinese | WPRIM | ID: wpr-826854

ABSTRACT

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.


Subject(s)
Base Sequence , Cloning, Molecular , Genetic Vectors , Genetics , Mutagenesis, Site-Directed , Methods , Nucleic Acid Amplification Techniques , Plasmids , Polymerase Chain Reaction
9.
Article in Chinese | WPRIM | ID: wpr-879921

ABSTRACT

OBJECTIVE@#To evaluate the effect of standardized health education on the sputum specimen collection rate for nucleic acid detection of coronavirus disease 2019 (COVID-19).@*METHODS@#Two hundred and twenty-seven patients in fever clinics and isolation wards of Sir Run Run Shaw Hospital of Zhejiang University and 307 migrant workers returning to 5 enterprises in Shanghai from February 3 to March 14, 2020 were enrolled in the study. Through clarifying the procedures of collecting sputum specimens, making graphic/video health education materials, standardizing the contents and methods of health education, we conducted education to the subjects. The subject expectorated spontaneously or with medical assistance. For patients, the number of sampling attempts and sputum acquisition times were documented before and after the implementation of the standardized expectoration method; for the returning migrant employees in the enterprises, only the number of collected samples after the implementation of the standardized expectoration method were recorded.@*RESULTS@#A total of 378 sputum samples were collected from 227 patients. The sputum sampling rates before and after the implementation of health education were 40.9%and 58.4%, respectively (@*CONCLUSIONS@#The education for standardized sputum sample collection method can effectively increase the sputum collection rate.


Subject(s)
Betacoronavirus/genetics , COVID-19 , China , Coronavirus Infections/diagnosis , Efficiency , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , SARS-CoV-2 , Specimen Handling/methods , Sputum , Time Factors
10.
Rev. Soc. Bras. Med. Trop ; 53: e20200104, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS, SES-SP | ID: biblio-1136907

ABSTRACT

Abstract INTRODUCTION: Gene-Xpert MTB RIF (Xpert) is based on nucleic acid amplification by real-time polymerase chain reaction, which allows for the identification of Mycobacterium tuberculosis and rifampin resistance. We describe the use of Xpert for extrapulmonary tuberculosis (EPTB) in children and adolescents. METHODS: A case series of two reference centers in Rio de Janeiro from 2014-2019. RESULTS: The final diagnosis of EPTB was established in 11/36 (31%) patients, with five cases detectable by Xpert. For lymph node evaluation (9/11), diagnosis by Xpert occurred in 5/9 patients, all with caseous aspects. CONCLUSIONS: Xpert can facilitate the rapid diagnosis of lymph node tuberculosis.


Subject(s)
Humans , Infant , Child, Preschool , Child , Adolescent , Tuberculosis , Mycobacterium tuberculosis , Rifampin , Nucleic Acid Amplification Techniques
11.
Braz. j. med. biol. res ; 53(11): e9056, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS | ID: biblio-1132484

ABSTRACT

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.


Subject(s)
Humans , Meningitis, Cryptococcal/diagnosis , Cryptococcus neoformans/genetics , Sequence Analysis, DNA , Oligonucleotide Array Sequence Analysis , Nucleic Acid Amplification Techniques
12.
Mem. Inst. Oswaldo Cruz ; 115: e200006, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135222

ABSTRACT

BACKGROUND Occult hepatitis B virus (HBV) - characterized by the absence of detectable HBsAg in the presence of HBV DNA - represents a potential threat for blood safety. OBJECTIVES This study was conducted with the aim to investigate the serological and molecular characterization of occult HBV infection (OBI) among blood donors in Mozambique. METHODS 1,502 blood donors were tested for HBsAg. All HBsAg-negative individuals were tested for HBV DNA. Antibodies against HBV core, surface and HBe antigen (anti-HBc, anti-HBs, HBeAg) were measured in HBV DNA positive individuals. FINDINGS 1435 serum samples were HBsAg negative and 16 positive for HBV DNA, 14 confirmed to have OBI, corresponding to a frequency of 0.98%. Of the 14 OBI infections identified, 13/14 (92.8%) were positive for anti-HBc, 4/14 (28.5%) for anti-HBs, and no samples were reactive for HBeAg. Of the 14 OBI cases, nine samples (64.2%) were sequenced for the S/P region. Eight samples (88.9%) belonged to genotype A1 and one (11.1%) to genotype E. One escape mutation (T123A) associated with OBI and various amino acid substitutions for genotype A1 and E were observed. MAIN CONCLUSIONS Our results show the importance of using nucleic acid amplification test to detect occult hepatitis B infection in blood donors in Mozambique.


Subject(s)
Humans , Male , Female , Adult , Blood Donors , Hepatitis B virus/isolation & purification , Hepatitis B virus/genetics , Nucleic Acid Amplification Techniques/methods , Hepatitis B/diagnosis , Hepatitis B Surface Antigens/genetics , Phylogeny , DNA, Viral , Polymerase Chain Reaction , Cross-Sectional Studies , Mozambique
13.
J. bras. pneumol ; 45(2): e20180128, 2019. tab, graf
Article in English | LILACS | ID: biblio-1002440

ABSTRACT

ABSTRACT Objective: To evaluate the rapid diagnosis of multidrug-resistant tuberculosis, by using a commercial line probe assay for rifampicin and isoniazid detection (LPA-plus), in the routine workflow of a tuberculosis reference laboratory. Methods: The LPA-plus was prospectively evaluated on 341 isolates concurrently submitted to the automated liquid drug susceptibility testing system. Results: Among 303 phenotypically valid results, none was genotypically rifampicin false-susceptible (13/13; 100% sensitivity). Two rifampicin-susceptible isolates harboured rpoB mutations (288/290; 99.3% specificity) which, however, were non-resistance-conferring mutations. LPA-plus missed three isoniazid-resistant isolates (23/26; 88.5% sensitivity) and detected all isoniazid-susceptible isolates (277/277; 100% specificity). Among the 38 (11%) invalid phenotypic results, LPA-plus identified 31 rifampicin- and isoniazid-susceptible isolates, one isoniazid-resistant and six as non-Mycobacterium tuberculosis complex. Conclusions: LPA-plus showed excellent agreement (≥91%) and accuracy (≥99%). Implementing LPA-plus in our setting can speed up the diagnosis of multidrug-resistant tuberculosis, yield a significantly higher number of valid results than phenotypic drug susceptibility testing and provide further information on the drug-resistance level.


RESUMO Objetivo: Avaliar o diagnóstico rápido de tuberculose multirresistente, utilizando um teste comercial de sondas em linha (LPA-plus), na rotina de um laboratório de referência de tuberculose. Métodos: O teste LPA-plus foi avaliado prospectivamente em 341 isolados simultaneamente submetidos ao teste de suscetibilidade aos antimicrobianos em meio líquido, pelo sistema automatizado. Resultados: Entre os 303 resultados fenotipicamente válidos, nenhum foi genotipicamente falso suscetível à rifampicina (13/13; 100% de sensibilidade). Dois isolados sensíveis à rifampicina apresentavam mutações no gene rpoB (288/290; especificidade de 99,3%), as quais, no entanto, não são associadas à resistência a rifampicina. O LPA-plus não identificou resistência à isoniazida em três isolados fenotipicamente resistentes (23/26; 88,5% de sensibilidade) e detectou todos os isolados sensíveis à isoniazida (277/277; especificidade de 100%). Entre os 38 (11%) resultados fenotípicos inválidos, o LPA-plus identificou 31 isolados sensíveis à rifampicina e à isoniazida, um resistente à isoniazida e seis como micobactérias não tuberculosas. Conclusões: O LPA-plus mostrou excelente concordância (≥91%) e acurácia (≥99%). Sua implementação pode acelerar o diagnóstico da tuberculose multirresistente, produzir número significativamente maior de resultados válidos do que o teste fenotípico de suscetibilidade aos antimicrobianos e fornecer informações adicionais sobre o nível de resistência aos fármacos.


Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Tuberculosis, Multidrug-Resistant/diagnosis , Molecular Diagnostic Techniques/methods , Phenotype , Rifampin/pharmacology , Time Factors , DNA, Bacterial , Microbial Sensitivity Tests , Prospective Studies , Reproducibility of Results , Sensitivity and Specificity , Tuberculosis, Multidrug-Resistant/microbiology , Nucleic Acid Amplification Techniques/methods , Early Diagnosis , Isoniazid/pharmacology , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/pharmacology
14.
J. bras. pneumol ; 45(2): e20170451, 2019. tab
Article in English | LILACS | ID: biblio-1040271

ABSTRACT

ABSTRACT Tuberculosis continues to be a major public health problem worldwide. The aim of the present study was to evaluate the accuracy of the Xpert MTB/RIF rapid molecular test for tuberculosis, using pulmonary samples obtained from patients treated at the Júlia Kubitschek Hospital, which is operated by the Hospital Foundation of the State of Minas Gerais, in the city of Belo Horizonte, Brazil. This was a retrospective study comparing the Xpert MTB/RIF test results with those of standard culture for Mycobacterium tuberculosis and phenotypic susceptibility tests. Although the Xpert MTB/RIF test showed high accuracy for the detection of M. tuberculosis and its resistance to rifampin, attention must be given to the clinical status of the patient, in relation to the test results, as well as to the limitations of molecular tests.


RESUMO A tuberculose permanece como um grave problema de saúde pública. O objetivo deste estudo foi avaliar a acurácia do teste rápido molecular Xpert MTB/RIF em amostras pulmonares no Hospital Júlia Kubitschek, Fundação Hospitalar do Estado de Minas Gerais, localizado em Belo Horizonte (MG). Trata-se de um estudo descritivo retrospectivo, considerando-se como método padrão a cultura para o bacilo da tuberculose e o teste de sensibilidade fenotípico. O teste Xpert MTB/RIF apresentou ótima acurácia para a detecção da tuberculose e resistência à rifampicina, mas é necessária a atenção a dados clínicos do paciente em relação ao resultado do exame e às limitações dos testes moleculares.


Subject(s)
Humans , Sputum/microbiology , Trachea/microbiology , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/microbiology , Bronchoalveolar Lavage Fluid/microbiology , Nucleic Acid Amplification Techniques/methods , Rifampin/pharmacology , DNA, Bacterial , Reproducibility of Results , Retrospective Studies , Sensitivity and Specificity , Molecular Diagnostic Techniques/methods , Drug Resistance, Bacterial/drug effects , Tertiary Care Centers , Antibiotics, Antitubercular/pharmacology , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/drug effects
15.
Braz. j. med. biol. res ; 52(3): e8186, 2019. tab, graf
Article in English | LILACS | ID: biblio-989465

ABSTRACT

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.


Subject(s)
Nucleic Acid Amplification Techniques/methods , Genes, Bacterial , Klebsiella pneumoniae/genetics , Plasmids/isolation & purification , Plasmids/genetics , Temperature , Time Factors , Polymerase Chain Reaction/methods , Reproducibility of Results , Sequence Analysis, DNA , DNA Primers/isolation & purification , DNA Primers/genetics , Limit of Detection , Klebsiella pneumoniae/isolation & purification
16.
Rev. Soc. Bras. Med. Trop ; 52: e20180432, 2019. tab
Article in English | LILACS | ID: biblio-1003137

ABSTRACT

Abstract By decreasing the pre-seroconversion window period, nucleic acid testing (NAT) has improved the safety of blood products and reduced the risk of transfusion-transmitted infections. Between 2011 and 2017, NAT determinations for approximately 898,202 donations were performed at Fundação Pró-Sangue/Hemocentro de São Paulo (FPS-HSP). Three seronegative HIV-viremic donations were detected. The NAT yield rate per million donations was 3.34 for HIV, and the acute HIV-1 infections detected are described, followed by a brief review of the situation in Brazil.


Subject(s)
Humans , Male , Adult , Blood Donors , DNA, Viral/blood , RNA, Viral/blood , HIV Infections/diagnosis , HIV-1/genetics , Nucleic Acid Amplification Techniques
17.
Article in Chinese | WPRIM | ID: wpr-781323

ABSTRACT

OBJECTIVE@#To explore the feasibility of high performance liquid chromatography (DHPLC) combined with multiple ligation-dependent probe amplification (MLPA) for the prenatal diagnosis of spinal muscular atrophy (SMA).@*METHODS@#Three families who had given birth to children with SMA type I were subjected to prenatal diagnosis. Peripheral blood samples were collected from the three couples, and 10 mL amniotic fluid was taken for each fetus through amniocentesis at 16-24 gestational week. Following DNA extraction, maternal contamination was excluded by STR analysis. Copy numbers of the SMN genes were detected by denaturing high performance liquid chromatography (DHPLC). Relative copy number of SMN1, SMN2 and reference genes was detected with a MLPA P021 assay kit.@*RESULTS@#The three couples were all found to harbor heterozygous deletion of exon 7 of the SMN1 gene by DHPLC. MLPA analysis also suggested that the three couples were all carriers of SMA mutations. The fetus of family 1 harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus had SMA. The fetus of family 2 also harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, while the copy number of SMN2 gene was normal, suggesting that the fetus was a SMA patient too. The fetus of family 3 harbored heterozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus was a carrier.@*CONCLUSION@#DHPLC can effectively screen carriers of SMA mutations. Combined DHPLC and MLPA can provide accurate diagnosis for fetuses with a high risk for SMA.


Subject(s)
Child , Chromatography, High Pressure Liquid , Female , Homozygote , Humans , Muscular Atrophy, Spinal , Diagnosis , Nucleic Acid Amplification Techniques , Pregnancy , Prenatal Diagnosis , Sequence Deletion , Survival of Motor Neuron 1 Protein , Genetics , Survival of Motor Neuron 2 Protein , Genetics
18.
Article in Korean | WPRIM | ID: wpr-759589

ABSTRACT

HBV core antibody and surface antibody test are currently conducted for those donors showing non-discriminated reactive (NDR) results on a nucleic acid amplification test (NAT) as a blood donor screening assay. It is necessary to investigate the relationship with HCV or HIV in the donors showing NDR results. From June 12th, 2012 to December 31st, 2018, 0.05% (9,020/17,798,461) donors showed NDR results on a NAT. Among the donors showing NDR results, 17 and 18 donors showed positive results on serological assay of HCV and HIV, respectively. 23 donors with NDR results showed positive results on the serological assay or NAT for HCV or HIV on the following donation. Further study and more accumulated data are required because it may be difficult to find the cause of NDR results by the current serological assay that is used for screening blood donors.


Subject(s)
Blood Donors , HIV , Humans , Mass Screening , Nucleic Acid Amplification Techniques , Tissue Donors
19.
Article in Korean | WPRIM | ID: wpr-759580

ABSTRACT

BACKGROUND: Since December 15 2017, donors showing a non-discriminated reactive (NDR) result in the nucleic acid amplification test (NAT) have been temporarily deferred and anti-HBc and anti-HBs assays as additional tests were performed. Donors with an anti-HBc reactive result and less than 100 IU/L of anti-HBs could not be released and can request a reentry test after more than six months. This study considered the effects of additional tests for NDR donors by analyzing the reentry test results in donors not released in the additional test. METHODS: This study examined the results of the additional test for NDR donors from January 2017 to September 2018 and the reentry test of the donors not released in the additional test. RESULTS: NAT was conducted on 4,706,051 blood donors over the period and 2,545 (0.05%) of them showed NDR. A total of 656 (25.8%) of the NDR donors were not released in the additional test. Among them, 246 donors requested a reentry test; 222 (90.2%) donors were not reentered, and 23 (10.4%) showed HBV NAT reactive results in the reentry test. Among the remaining 24 reentered donors, 2 donors (8.3%) showed anti-HBc nonreactive results in the reentry test and 22 donors (91.7%) showed higher than 100 IU/L of anti-HBs. CONCLUSION: The follow-up of NDR donors may be significant because some donors showed different results between screening test and reentry test. In addition the effectiveness of the introduction of additional tests for the NDR donors has been proved to be effective.


Subject(s)
Blood Donors , Follow-Up Studies , Humans , Mass Screening , Nucleic Acid Amplification Techniques , Tissue Donors
20.
Article in Korean | WPRIM | ID: wpr-759579

ABSTRACT

BACKGROUND: A nucleic acid amplification test was adopted to detect transfusion-transmitted infectious agents. In the case of HTLV, however, there was no internal control (IC) because the laboratory developed polymerase chain reaction (laboratory-developed PCR) was used. In this study, noncompetitive IC was constructed for the laboratory-developed PCR of HTLV and the effectiveness was compared with the competitive test that was constructed in a previous study. METHODS: As a competitive IC, plasmid DNA, including the primer recognition sequence for the amplification of the HTLV pX region, was constructed. As a noncompetitive IC, an additional primer was constructed for the amplification of the housekeeping gene, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The performance of the competitive and noncompetitive IC was verified and compared using 10 HTLV positive samples and 10 negative samples. In addition, the detection limits in the assay adopting competitive IC and noncompetitive IC were compared. RESULTS: In the case of competitive IC applications, all 10 positive samples were positive and all 10 negative samples were negative. In the case of noncompetitive IC applications, however, one positive sample was not detected. The detection limit of the assay using competitive IC was 100 pg and that of the assay using noncompetitive IC was 1 ng. CONCLUSION: Although the manufacturing processes is not required using noncompetitive IC, the adoption of competitive IC is more effective to ensure the assay results because the ability of detection of the assay adopting competitive IC was better than that using noncompetitive IC.


Subject(s)
DNA , Genes, Essential , Glyceraldehyde 3-Phosphate , Limit of Detection , Nucleic Acid Amplification Techniques , Oxidoreductases , Plasmids , Polymerase Chain Reaction
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