RESUMEN
AIM: This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis. METHODS: Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis (cerebral or gestational forms); group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA). RESULTS: Concentrations of serum-derived EVs from toxoplasmosis patients (mean: 2.4 x 1010 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs. Serum-derived EVs from group II (cerebral and gestational toxoplasmosis) up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p. CONCLUSION: These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis.
Asunto(s)
Complicaciones Parasitarias del Embarazo/sangre , Complicaciones Parasitarias del Embarazo/líquido cefalorraquídeo , Toxoplasmosis Cerebral/sangre , Toxoplasmosis Cerebral/líquido cefalorraquídeo , Toxoplasmosis/complicaciones , Micropartículas Derivadas de Células/genética , Micropartículas Derivadas de Células/patología , Exosomas/genética , Exosomas/patología , Vesículas Extracelulares/genética , Vesículas Extracelulares/patología , Femenino , Expresión Génica , Infecciones por VIH/sangre , Infecciones por VIH/líquido cefalorraquídeo , Infecciones por VIH/complicaciones , Voluntarios Sanos , Humanos , MicroARNs/sangre , MicroARNs/líquido cefalorraquídeo , MicroARNs/genética , Microscopía Electrónica de Transmisión , Embarazo , Complicaciones Parasitarias del Embarazo/genética , Toxoplasmosis/sangre , Toxoplasmosis/líquido cefalorraquídeo , Toxoplasmosis Cerebral/genéticaRESUMEN
BACKGROUND: Highly active antiretroviral therapy (HAART) restores the inflammatory immune response in AIDS patients and it may unmask previous subclinical infections or paradoxically exacerbate symptoms of opportunistic infections. Up to 25% of patients receiving HAART develop immune reconstitution inflammatory syndrome (IRIS). We describe six patients with IRIS central nervous system (CNSIRIS) manifestations emphasizing the relevance of CSF cultures and neuroimaging in early diagnosis and management. METHODS: Patients with CNSIRIS were identified among hospitalized HIV-infected patients that started HAART from January 2002 through December 2007 at a referral neurological center in Mexico. RESULTS: One-hundred and forty-two HIV-infected patients with neurological signs were hospitalized, 64 of which had received HAART, and six (9.3%) developed CNSIRIS. Five patients were male. Two cases of tuberculosis, two of cryptococcosis, one of brain toxoplasmosis, and one possible PML case were found. IRIS onset occurred within 12 weeks of HAART in five patients. Anti-infective therapy was continued. In one case, HAART was temporarily suspended. In long-term follow-up the clinical condition improved in all patients. CONCLUSIONS: CNSIRIS associated to opportunistic infections appeared in 9% of patients receiving HAART. Interestingly, no cases of malignancy or neoplasm IRIS-related were found. Frequent clinical assessment and neuroimaging studies supported diagnosis and treatment. Risk factors were similar to those found in other series.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Terapia Antirretroviral Altamente Activa/efectos adversos , Enfermedades del Sistema Nervioso Central/terapia , Síndrome Inflamatorio de Reconstitución Inmune/terapia , Infecciones Oportunistas Relacionadas con el SIDA/epidemiología , Infecciones Oportunistas Relacionadas con el SIDA/terapia , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Adulto , Antibacterianos/uso terapéutico , Antifúngicos/uso terapéutico , Recuento de Linfocito CD4 , Enfermedades del Sistema Nervioso Central/etiología , Criptococosis/líquido cefalorraquídeo , Criptococosis/etiología , Criptococosis/microbiología , Cryptococcus neoformans , Femenino , Humanos , Síndrome Inflamatorio de Reconstitución Inmune/etiología , Leucoencefalopatía Multifocal Progresiva/líquido cefalorraquídeo , Leucoencefalopatía Multifocal Progresiva/etiología , Imagen por Resonancia Magnética , Masculino , México , Mycobacterium tuberculosis , Neuroimagen , Estudios Retrospectivos , Toxoplasmosis/líquido cefalorraquídeo , Toxoplasmosis/etiología , Resultado del Tratamiento , Tuberculosis/líquido cefalorraquídeo , Tuberculosis/etiología , Tuberculosis/microbiologíaRESUMEN
Toxoplasmic encephalitis (TE) is one of the most common opportunistic infections in immunocompromised patients. In Cuba, despite the highly active antiretroviral therapy, TE is still the most important cause of cerebral mass lesions in patients infected with the human immunodeficiency virus (HIV). The detection of Toxoplasma gondii by PCR may facilitate the diagnosis and follow-up of TE in acquired immunodeficiency syndrome (AIDS) patients by direct identification of parasite DNA in clinical samples. The aim of the present study was to evaluate a rapid PCR method using the B1 gene to detect T. gondii in cerebrospinal fluid (CSF) samples from patients with suspected TE(AU)
Asunto(s)
Humanos , Toxoplasma/inmunología , Toxoplasmosis/líquido cefalorraquídeo , Toxoplasmosis/diagnóstico , Síndrome de Inmunodeficiencia AdquiridaRESUMEN
BACKGROUND: Toxoplasmosis is a serious and often life-threatening disease in immunodeficient patients. Polymerase chain reaction (PCR) assays allow a rapid diagnosis of Toxoplasma infection by direct detection of the parasite's DNA. To perform a sensitive, specific, and reliable PCR-based diagnostic test, the availability of pure DNA lacking PCR inhibitors as well as a rapid and easy-to-perform DNA extraction protocol are essential. The aim of the present study was to compare four DNA extraction methods for the detection of T. gondii on cerebrospinal fluid (CSF) using the PCR technology. MATERIAL/METHODS: Four DNA extraction methods (boiling, lysis + centrifugation, the miniMAG commercial system, and phenol-chloroform) were compared with respect to the time of completion, the manual labor involved, and PCR analytical sensitivity for the detection of T. gondii in CSF. The optimal DNA extraction method for the detection of the parasite was evaluated in CSF from 43 AIDS patients using the nest-PCR B1 assay. RESULTS: According to the time required for completion, labor, and PCR analytical sensitivity, the lysis + centrifugation protocol proved to be a simple, efficient, and economical in-house procedure to recover the T. gondii DNA present in the CSF. The diagnostic sensitivity of nest-PCR, according to Centers for Disease Control and Prevention (CDC) criteria, was 86.3 percent and the diagnostic specificity was 100 percent. CONCLUSIONS: We report a simple, rapid, reproducible, and economical in-house method for T. gondii DNA extraction from CSF. This method is recommended for diagnostic PCR of Toxoplasmic encephalitis (TE) in places with economical shortage(AU)
ANTECEDENTES: La toxoplasmosis es una grave y, a menudo, las enfermedades que amenazan la vida en pacientes inmunodeficientes. Reacción en cadena de polimerasa (PCR) los ensayos de permitir un diagnóstico rápido de infección por Toxoplasma detección directa del ADN del parásito. Para realizar una sensible, específico y fiable basada en PCR-prueba de diagnóstico, la disponibilidad de ADN puro falta inhibidores de la PCR, así como una rápida y fácil de realizar el protocolo de extracción de ADN son esenciales. El objetivo del presente estudio fue comparar cuatro métodos de extracción de ADN para la detección de T. gondii en líquido cefalorraquídeo (LCR), utilizando la tecnología PCR. MATERIAL Y MÉTODOS: Cuatro métodos de extracción de ADN (punto de ebullición, + lisis centrifugación, el sistema comercial miniMAG, y fenol-cloroformo) con respecto a la hora de finalización, la mano de obra en cuestión, y PCR de análisis de sensibilidad para la detección de T. gondii en el LCR. El método óptimo de extracción de ADN para la detección del parásito se evaluó en el LCR de 43 pacientes con SIDA mediante el nido-PCR B1 ensayo. RESULTADOS: Según el tiempo necesario para la realización, la mano de obra, análisis de sensibilidad y la PCR, el protocolo de lisis centrifugación + demostrado ser un simple, eficiente y económico en el seno del procedimiento de recuperación de T. gondii de ADN presentes en el MCA. La sensibilidad diagnóstica de PCR-nido, de acuerdo con los Centros para Control y Prevención de Enfermedades (CDC) de los criterios, fue 86,3 por ciento y la especificidad diagnóstica fue del 100 por ciento. CONCLUSIONES: Se presenta un sencillo, rápido, reproducible y económica en el seno de un método de extracción de ADN T. gondii de PPC. Se recomienda este método de PCR para el diagnóstico de la encefalitis toxoplásmica (TE) en lugares con escasez económica(AU)
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/parasitología , ADN Protozoario/líquido cefalorraquídeo , ADN Protozoario/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Toxoplasmosis/líquido cefalorraquídeo , Toxoplasmosis/parasitologíaAsunto(s)
Humanos , Masculino , Femenino , Recién Nacido , Lactante , Preescolar , Niño , Adolescente , Adulto , Persona de Mediana Edad , Toxoplasmosis/líquido cefalorraquídeo , Enfermedades del Sistema Nervioso/líquido cefalorraquídeo , Toxoplasmosis/diagnóstico , Reacción en Cadena de la Polimerasa , Enfermedades del Sistema Nervioso/diagnósticoRESUMEN
The behavior of CSF inflammatory pattern in patients with AIDS and/or toxoplasmosis of the CNS is studied in 176 patients, divided in three groups. In the first group, 96 patients with toxoplasmosis and AIDS are considered; in the second group, 50 patients with toxoplasmosis without AIDS; in the third group, 30 AIDS patients without toxoplasmosis nor any other opportunistic infection. It is possible to conclude that patients with toxoplasmosis associated to AIDS exhibit CSF inflammatory pattern similar to patients with neurotoxoplasmosis without AIDS, except in respect to gamma globulin rates for which a cumulative effect can be detected.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/líquido cefalorraquídeo , Encefalopatías/líquido cefalorraquídeo , Toxoplasmosis/líquido cefalorraquídeo , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Adolescente , Adulto , Encefalopatías/complicaciones , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Toxoplasmosis/complicacionesRESUMEN
Foram estudados 176 pacientes com o objetivo de avaliar o comportamento da reaçäo inflamatória no líquido cefalorraqueano (LCR) de pacientes com toxoplasmose do sistema nervoso central (SNC). Foram considerados três grupos de pacientes: o primeiro com toxoplasmose do SNC associada à síndrome de imunodeficiência adquirida (AIDS), com 96 pacientes; o segundo, com toxoplasmose, sem AIDS, com 50 pacientes; o terceiro, apenas com AIDS, sem qualquer infecçäo oportunista, com 20 pacientes. Verificou-se que o comportamento da reaçäo inflamatória da toxoplasmose do SNC estudada, pelo exame de LCR predomina sobre as características de base da AIDS, excepto no que diz respeito aos teores de globulinas gama; para estas, parece haver efeito comulativo das duas patologias