RESUMEN
Sampling the blood compartment by an invasive procedure such as phlebotomy is the most common approach used for diagnostic purposes. However, phlebotomy has several drawbacks including pain, vasovagal reactions, and anxiety. Therefore, alternative approaches should be tested to minimize patient's discomfort. Saliva is a reasonable compartment; when obtained, it generates little or no anxiety. We setup a multiplexed serology assay for detection of Toxoplasma gondii IgG and IgM, rubella IgG, and CMV IgG, in serum, whole blood, and saliva using novel plasmonic gold (pGOLD) chips. pGOLD test results in serum, whole blood, and saliva were compared with commercial kits test results in serum. One hundred twenty serum/saliva sets (Lyon) and 28 serum/whole blood/saliva sets (Nice) from France were tested. In serum and whole blood, sensitivity and specificity of multiplex T. gondii, CMV, and rubella IgG were 100% in pGOLD when compared to commercial test results in serum. In saliva, sensitivity and specificity for T. gondii and rubella IgG were 100%, and for CMV IgG, sensitivity and specificity were 92.9% and 100%, respectively, when compared to commercial test results in serum. We were also able to detect T. gondii IgM in saliva with sensitivity and specificity of 100% and 95.4%, respectively, when compared to serum test results. Serological testing by multiplex pGOLD assay for T. gondii, rubella, and CMV in saliva is reliable and likely to be more acceptable for systematic screening of pregnant women, newborn, and immunocompromised patients.
Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , Oro/química , Análisis por Matrices de Proteínas/normas , Rubéola (Sarampión Alemán)/diagnóstico , Saliva/inmunología , Pruebas Serológicas/normas , Toxoplasmosis/diagnóstico , Adolescente , Adulto , Anticuerpos Antiprotozoarios/análisis , Anticuerpos Antivirales/análisis , Antígenos de Protozoos/química , Antígenos Virales/química , Niño , Preescolar , Citomegalovirus/inmunología , Citomegalovirus/aislamiento & purificación , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Lactante , Recién Nacido , Persona de Mediana Edad , Virus de la Rubéola/inmunología , Virus de la Rubéola/aislamiento & purificación , Sensibilidad y Especificidad , Toxoplasma/inmunología , Toxoplasma/aislamiento & purificación , Adulto JovenRESUMEN
BACKGROUND: HSV-1 infection is very common worldwide and can be associated with severe medical conditions such as encephalitis and neonatal herpes. Current diagnosis relies heavily on molecular assays targeting the viral genome. One of the pitfalls of these assays is genetic variability, as viral polymorphisms located in the target region can impair the detection of HSV-1. OBJECTIVES: The aim of this study was to determine if genetic diversity of HSV-1 could account for equivocal assay results we obtained during routine diagnosis of HSV-1 in a genital specimen with the HSV-1 HSV-2 VZV R-gene® assay. STUDY DESIGN: The presence of HSV-1 in the genital swab studied was assessed using the HSV-1 HSV-2 VZV R-gene® qPCR assay and viral culture. Genetic variability of the patient's HSV-1 isolate was determined using molecular cloning, sequencing and comparison of the sequence of the isolate with those of previously published strains. RESULTS: We identified an HSV-1 isolate carrying a novel single-nucleotide polymorphism (SNP) located in the US7 gene from a genital swab. This SNP resulted in a missense substitution in the gI protein. It was responsible for the generation of an altered amplification curve when the genital specimen was checked for HSV-1 presence with the Argene HSV-1 HSV-2 VZV R-gene® assay, preventing unequivocal determination of the HSV-1 status of the sample analyzed. CONCLUSIONS: This study raises awareness of the risk of misdiagnosis of HSV-1 infections when "single-target" molecular assays are employed.
Asunto(s)
Variación Genética , Herpes Simple/diagnóstico , Herpesvirus Humano 1/genética , Polimorfismo de Nucleótido Simple , Proteínas Virales/genética , ADN Viral , Femenino , Herpes Genital/diagnóstico , Herpes Genital/virología , Herpes Simple/virología , Herpesvirus Humano 1/aislamiento & purificación , Humanos , Técnicas de Diagnóstico MolecularRESUMEN
Congenital cytomegalovirus (CMV) infection is the leading cause of sensoneurinal disability due to infectious congenital disease. The diagnosis of congenital CMV infection is based on the search of CMV in the urine within the first two weeks of life. Viral culture of urine is the gold standard. However, the PCR is highly sensitive and faster. It is becoming an alternative choice. The objective of this study is the validation of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine. Repeatability, reproducibility, detection limit and inter-sample contamination were evaluated. Urine samples from patients (n=141) were collected and analyzed simultaneously in culture and PCR in order to assess the correlation of these two methods. The sensitivity and specificity of PCR were also calculated. The Abbott RealTime CMV PCR in urine is an automated and sensitive method (detection limit 200 UI/mL). Fidelity is very good (standard deviation of repeatability: 0.08 to 0.15 LogUI/mL and reproducibility 0.18 LogUI/mL). We can note a good correlation between culture and Abbott RealTime CMV PCR (kappa 96%). When considering rapid culture as reference, real-time PCR was highly sensitive (100%) and specific (98.2%). The real-time PCR by Abbott RealTime CMV with m2000 is optimal for CMV detection in urine.
Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Urinálisis , Automatización de Laboratorios , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/congénito , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/orina , Humanos , Recién Nacido , Tamizaje Neonatal/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Urinálisis/instrumentación , Urinálisis/métodos , Urinálisis/normas , Virología/instrumentación , Virología/métodosRESUMEN
Here, we report an epidemiological and entomological investigation of a cluster of cases of spotted fever group (SFG) rickettsiosis occurring in southern France. A family of 3 (husband, wife, and their son) presented with symptoms compatible with SFG rickettsiosis. For 2 patients, serum samples presented increased levels of IgM and IgG for SFG Rickettsia. The patients' home was investigated, and Rhipicephalus sanguineus ticks were collected from the floor from behind the furniture. Of 22 ticks collected, 20 tested positive for Rickettsia. As Rh. sanguineus serves as a vector for both Rickettsia conorii and Ri. massiliae in southern France, all Rh. sanguineus isolates were tested by real-time PCR and conventional PCR to detect the 2 species. Nine ticks tested positive for Ri. conorii subsp. caspia (marking the first documentation of this subspecies in France), 7 tested positive for Ri. massiliae, and 4 tested positive for both rickettsiae. This study is the first report of coinfection of Rh. sanguineus ticks with Ri. conorii and Ri. massiliae in southern France.
Asunto(s)
Fiebre Botonosa/epidemiología , Salud de la Familia , Rhipicephalus sanguineus/microbiología , Rickettsia/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/sangre , Fiebre Botonosa/transmisión , Análisis por Conglomerados , ADN Bacteriano/genética , Femenino , Francia/epidemiología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Reacción en Cadena de la Polimerasa , Rickettsia/clasificación , Rickettsia/genética , Población UrbanaRESUMEN
We previously isolated human papillomavirus 83 (HPV83m) from a cervical smear. Sequence analysis of E6 and E7 proteins highlighted five mutations located in the second putative zinc-finger region of E6 (E6m), an important domain for protein-protein or protein-DNA interactions. Here, we show that E6m of HPV83m can trigger human primary cell proliferation and anchorage-independent growth properties, similarly to E6 of HPV16, a high-risk HPV (HR-HPV). Interestingly, we demonstrate that, in contrast to E6 of HPV16, E6m corrupts neither p53 stability nor telomerase activity, but acts as a specific modulator of the transcriptional machinery. By studying E6m reversion mutants, we confirmed the importance of the second zinc-finger domain in triggering the observed upregulation of cell growth and of the transcriptional machinery. Reversion of these mutations in E6m (to yield strain E6r) fully abolished the oncogenic potential of E6m, transforming the phenotype of E6 from a high-risk to a low-risk phenotype. Importantly, our data define the importance of a cluster of mutations in the second zinc finger of E6m in increasing the oncogenic potential of HPV83.
Asunto(s)
Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/genética , Papillomaviridae/patogenicidad , Displasia del Cuello del Útero/virología , Proliferación Celular , Células Cultivadas , ADN Viral/química , ADN Viral/genética , Células Epiteliales/fisiología , Células Epiteliales/virología , Humanos , Papillomaviridae/aislamiento & purificación , Mutación Puntual , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Dedos de ZincRESUMEN
Reticulated platelets are young platelets containing mRNA. They reflect the rate of thrombopoesis. The aim of this study was to evaluate the reliability of the percentage of reticulated platelets (IPF%) as a diagnostic test for thrombocytopenia pathogenesis. IPF% was measured using XE 2100 Sysmex. An IPF% reference range in 52 healthy individuals was established as 1-4.5% with a median 2.2%. In all the 13 patients with idiopathic thrombocytopenic purpura IPF% was increased (median 11.8, range 5.3-54.3%). Only 7 out of 18 patients with disseminated intravascular coagulation had high IPF% (median 5.4%, range 2.9-14.1%). Surprisingly, IPF% was increased in 17 out of 22 patients with acute leukaemia (median 9.7%, range 0.9-41.9%). In CIVD, IPF% values correlated with the severity of the illness. Increased values in acute leukaemia could not be explained by non specific staining but by delayed maturation of reticulated platelets. A high IPF% does not substantiate hyperdestructive thrombocytopenia but a diagnosis of idiopathic thrombocytopenic purpura should be questioned if IPF% is not raised.