RESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) is one of the most common causes of respiratory tract infections among young children and the elderly. Timely and accurate diagnosis of respiratory tract infections improves patient care and minimizes unnecessary prescriptions of antibiotics. We sought to develop a rapid nucleic acid tests for the detection of RSV within minutes, while retaining the high sensitivity achieved with RT-PCR. METHODS: We developed and evaluated a reverse transcription isothermal nucleic acid amplification method, reverse transcription strand invasion based amplification (RT-SIBA), for the rapid detection of RSV. RESULTS: The developed RT-SIBA assay showed good sensitivity by detecting as few as 10 copies of RSV RNA within 20 min compared with reverse transcription polymerase chain reaction, which took approximately 2 h. The performance of the RT-SIBA RSV assay was further investigated using nasopharyngeal swab specimens. The RT-SIBA assay had a sensitivity of 100% (25/25) and a specificity of 100% (15/15). CONCLUSION: RT-SIBA did not require highly purified RNA for the rapid detection of RSV and was therefore compatible with rapid specimen processing methods. This reduces the complexity of specimen preparation and further shortens the total amount of time needed to detect RSV in clinical specimens. The developed RT-SIBA assay for RSV could be a useful tool for prompt management of this infection.
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Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Humanos , Nasofaringe/virología , Técnicas de Amplificación de Ácido Nucleico , ARN Viral/análisis , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/genética , Sensibilidad y EspecificidadRESUMEN
An RT-qPCR targeting EBOV-L including the preceding RNA extraction protocol were set up and evaluated.
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Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Humanos , ARN Viral/genéticaRESUMEN
OBJECTIVE: Microbiologic causes of facial palsy in children were investigated. STUDY DESIGN: Prospective clinical study. SETTING: Tertiary referral center. PATIENTS: Forty-six children aged 0 to 16 years with peripheral facial palsy. INTERVENTIONS: Paired serum samples and cerebrospinal fluid were tested to find indications of microbes associated with facial palsy. The microbes tested were herpes simplex virus 1 and 2, varicella-zoster virus, human herpesvirus-6, Mycoplasma pneumoniae, Borrelia burgdorferi, influenza A and B virus, picorna, cytomegalovirus, parainfluenza virus, respiratory syncytial virus, coxsackie B5 virus, adenovirus, and enterovirus, Chlamydia psittaci, and Toxoplasma gondii. Besides the routine tests in clinical practice, serum and cerebrospinal fluid samples were tested with a highly sensitive microarray assay for DNA of herpes simplex virus 1 and 2; human herpes virus 6A, 6B, and 7; Epstein-Barr virus, cytomegalovirus, and varicella zoster virus. RESULTS: Incidence for facial palsy was 8.6/100,000/children/year. Cause was highly plausible in 67% and probable in an additional 11% of cases. Borrelia burgdorferi caused facial palsy in 14 patients (30%), varicella zoster virus in 5 (11%) (one with concomitant adenovirus), influenza A in 3 (6%), herpes simplex virus 1 in 2 (4%) (one with concomitant enterovirus), otitis media in 2 (4%), and human herpesvirus 6 in 2 (4%). Mycoplasma pneumoniae, neurofibromatosis, and neonatal age facial palsy affected 1 child (2%) each. CONCLUSION: Microbiologic etiology association of pediatric facial palsy could frequently be confirmed. Borreliosis was the single most common cause; hence, cerebrospinal fluid sampling is recommended for all pediatric cases in endemic areas. Varicella zoster virus accounted for 11% of the cases, being the second most common factor.
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Parálisis de Bell/microbiología , Parálisis de Bell/virología , Enfermedad Aguda , Adolescente , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/líquido cefalorraquídeo , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/líquido cefalorraquídeo , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/microbiología , Borrelia/inmunología , Niño , Preescolar , Femenino , Humanos , Lactante , Recuento de Leucocitos , Neuroborreliosis de Lyme/clasificación , Neuroborreliosis de Lyme/microbiología , Masculino , Estudios Prospectivos , Garrapatas , Virosis/complicaciones , Virosis/virologíaRESUMEN
Microarrays are widely used in high-throughput DNA and RNA hybridization tests and recently adopted to protein and small molecule interaction studies in basic research and diagnostics. Parallel detection of serum antibodies and antigens has several potential applications in epidemiologic research, vaccine development, and in the diagnosis of allergies, autoimmunity, and infectious diseases. This study demonstrates an immobilization method for immunoassay-based microarray in conventional 96-well polystyrene plates for a serologic diagnostic method combined with quantitative C-reactive protein (CRP) assay. A synthetic peptide (HIV-1), a recombinant protein (Puumala hantavirus nucleocapsid), and purified virus preparations (Sindbis and adenoviruses) were used as antigens for virus-specific antibody detection and monoclonal anti-CRP antibody for antigen detection. The microarray was based on conventional enzyme immunoassays and densitometry from photographed results. Peptide and recombinant antigens functioned well, while whole virus antigens gave discrepant results in 1 out of 23 samples from the reference method, tested with human sera with various antibody responses. The CRP results were in concordance in the concentration range 0.5-150 mg/L with 2 commercially available CRP assays: ReaScan rapid test (R(2) = 0.9975) and Cobas 6000 analyzer (R(2) =0.9595). The results indicate that microtiter plates provide a promising platform for further development of microarrays for parallel antibody and antigen detection.
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Anticuerpos Antivirales/sangre , Proteína C-Reactiva/análisis , Técnicas para Inmunoenzimas/métodos , Análisis por Micromatrices/métodos , Humanos , Técnicas para Inmunoenzimas/instrumentación , Modelos Lineales , Análisis por Micromatrices/instrumentaciónRESUMEN
Rapid diagnosis is critical to minimize morbidity and mortality associated with infections of the central nervous system (CNS). In this study, we evaluated the performance of a multiplex polymerase chain reaction (PCR) and microarray-based method, Prove-it™ Herpes, in a routine clinical laboratory setting for the diagnostics of 7 herpesviruses in viral CNS infections. Cerebrospinal fluid samples (n = 495), which had arrived for diagnostics in the 5 participating laboratories, were analyzed for herpesvirus DNA both by the current PCR-based method of the laboratory and by the microarray assay. The sensitivity and specificity for the microarray assay were 93% and 99%, respectively. The microarray assay was considered as a rapid and robust diagnostic platform that was easily implemented into the laboratory workflow. The broad herpesvirus coverage and the small sample volume required by the assay could benefit the diagnostics and thus the treatment of life-threatening infections of the CNS, especially among immunocompromised patients.
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Técnicas de Laboratorio Clínico/métodos , Encefalitis por Herpes Simple/diagnóstico , Análisis por Micromatrices/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Virología/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Líquido Cefalorraquídeo/virología , Niño , Preescolar , ADN Viral/genética , ADN Viral/aislamiento & purificación , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Adulto JovenRESUMEN
The seroprevalence of human herpesviruses is high and reactivations occur frequently. A microarray was designed and tested for the detection of IgG and IgM antibodies for Puumala hantavirus (PUUV) and IgG antibodies against four herpesviruses. Initially, a microarray platform was set up using an unrelated in-house antigen, PUUV recombinant nucleocapsid protein, to optimize the protocol for the detection of antibodies. Detection of the four herpesviruses was set up in a microarray using the recombinant proteins of herpes simplex virus (HSV) glycoprotein G1 and G2, varicella-zoster virus (VZV) glycoprotein E, and cytomegalovirus (CMV) pp150 phosphoprotein. The results of the PUUV panel were in good agreement with the PUUV IgG immunofluorescent assay and IgM enzyme immunoassay (EIA). Seropositive and negative clinical reference panels were tested for herpesviruses by the serological microarray, and the results were compared to those of individual EIAs used for standard diagnostic purposes. The serologic microarray for HSV, VZV and CMV antibody detection gave good specificities for IgG. However, sensitivities of the assay varied depending on the herpesvirus detected. The serological microarray showed potential for screening purposes. The microarray based analyses were easy to perform, and HSV-1, HSV-2, VZV, and CMV antibodies could be detected on the same microarray.
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Anticuerpos Antivirales/sangre , Citomegalovirus/inmunología , Infecciones por Herpesviridae/diagnóstico , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 2/inmunología , Análisis por Matrices de Proteínas/métodos , Varicellovirus/inmunología , Antígenos Virales , Orthohantavirus/inmunología , Infecciones por Hantavirus/diagnóstico , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: To study characteristics of Melkersson-Rosenthal syndrome (MRS) patients with facial palsy (FP) and differences in patients treated at the Departments of Otorhinolaryngology and Dermatology. METHODS: Clinical picture of MRS was studied from patient charts at two departments. Patients with FP received a questionnaire and were examined. Tissue biopsies were searched for non-necrotizing granulomatous infiltrations typical of MRS and blood DNA for UNC-93B1 gene mutations predisposing to herpesvirus infection. RESULTS: At the Department of Otorhinolaryngology, all 18 MRS patients had FP, 9 the triad form. Two patients revealed non-necrotizing granulomatous infiltrations during acute edema episodes; another two had association with uveitis. Edema was rarely persistent and did not dominate the clinical picture. No UNC-93B1 mutations were found. At the Department of Dermatology, 2 patients had triad MRS and 15 had monosymptomatic granulomatous cheilitis with persistent edema and typical MRS histology. CONCLUSION: The clinical picture of MRS with FP differed from the current knowledge of edema-dominated MRS. More studies focusing on MRS with FP would broaden our understanding of the syndrome.
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Síndrome de Melkersson-Rosenthal/diagnóstico , Adulto , Anciano , Biopsia , ADN/genética , Diagnóstico Diferencial , Estudios de Seguimiento , Humanos , Síndrome de Melkersson-Rosenthal/etiología , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Encuestas y CuestionariosRESUMEN
We report triallelic patterns in several short tandem repeat (STR) loci revealed by routine paternity testing using the commercial AMPFlSTR Profiler and AMPFlSTR SGMplus kits. One case where the TPOX-locus (2p25.3) produced three peaks from the blood sample of a child was analysed further. Quantitative polymerase chain reaction (QPCR) and STR typing of the DNAs of the family trio revealed a large (>1.59 Mb) duplication flanking the TPOX-locus in chromosome 2 in both the mother and child. The implications of such genetic anomalies for paternity testing are discussed.