Human extracellular vesicles and correlation with two clinical forms of toxoplasmosis.

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.

Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS.

Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation.

[Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii]. / Polimorfismos en los genes de dihidrofolato-reductasa ( dhfr ) y dihidropteroato-sintasa ( dhps ) y modelado estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii.

INTRODUCTION: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. OBJECTIVE: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . MATERIALS AND METHODS: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. RESULTS: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. CONCLUSIONS: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Polimorfismos en los genes de dihidrofolato-reductasa ( dhfr ) y dihidropteroato-sintasa ( dhps ) y modelado estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii / Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii

Introducción. No existen reportes sobre las variaciones en la secuencia de los genes blanco de los medicamentos anti- Toxoplasma en aislamientos provenientes de Suramérica. Objetivo. Clonar y secuenciar los genes de la dihidrofolato-reductasa ( dhfr ) y la dihidropteroato-sintetasa ( dhps ) de la cepa de referencia RH y de dos aislamientos colombianos de Toxoplasma gondii. Materiales y métodos. Se obtuvieron dos aislamientos de T. gondii en líquido céfalorraquídeo de pacientes colombianos positivos para HIV con toxoplasmosis cerebral. Se extrajo el ADN de los genes dhfr y dhps y se amplificaron mediante reacción en cadena de la polimerasa (PCR). Los productos fueron clonados en el vector pGEM-T y secuenciados. Resultados. Se encontró un cambio de adenina por guanina (A « G) en la posición 235 del exón 2 del gen dhps , dos cambios de guanina por citocina (G « C) en las posiciones 259 y 260 y un cambio de timina por guanina (T « G) en la posición 371 del exón 4 del gen dhps. Por análisis bioinformático, en este último exón se identificó un polimorfismo no sinónimo en la región codificante, que podría llevar al cambio de una Glu (CAA o CAG) por una His (codificada por los codones AAU o AAC). Se calculó el modelo estructural de la enzima dihidropteroato-sintetasa (DHPS) de T. gondii y se identificaron las modificaciones en la estructura secundaria ocasionadas por las mutaciones. Conclusiones. La metodología estandarizada puede servir como base para la búsqueda de polimorfismos en muestras de pacientes con diferentes manifestaciones clínicas de toxoplasmosis y para establecer su posible relación con los cambios en la sensibilidad a los antifolatos y la reacción al tratamiento.

Introduction: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Materials and methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. Results: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Features to validate cerebral toxoplasmosis.

INTRODUCTION: Neurotoxoplasmosis (NT) sometimes manifests unusual characteristics. METHODS: We analyzed 85 patients with NT and AIDS according to clinical, cerebrospinal fluid, cranial magnetic resonance, and polymerase chain reaction (PCR) characteristics. RESULTS: In 8.5%, focal neurological deficits were absent and 16.4% had single cerebral lesions. Increased sensitivity of PCR for Toxoplasma gondii DNA in the central nervous system was associated with pleocytosis and presence of >4 encephalic lesions. CONCLUSIONS: Patients with NT may present without focal neurological deficit and NT may occur with presence of a single cerebral lesion. Greater numbers of lesions and greater cellularity in cerebrospinal fluid improve the sensitivity of PCR to T gondii.

Features to validate cerebral toxoplasmosis

Introduction Neurotoxoplasmosis (NT) sometimes manifests unusual characteristics. Methods We analyzed 85 patients with NT and AIDS according to clinical, cerebrospinal fluid, cranial magnetic resonance, and polymerase chain reaction (PCR) characteristics. Results In 8.5%, focal neurological deficits were absent and 16.4% had single cerebral lesions. Increased sensitivity of PCR for Toxoplasma gondii DNA in the central nervous system was associated with pleocytosis and presence of >4 encephalic lesions. Conclusions Patients with NT may present without focal neurological deficit and NT may occur with presence of a single cerebral lesion. Greater numbers of lesions and greater cellularity in cerebrospinal fluid improve the sensitivity of PCR to T gondii. .

Conventional polymerase chain reaction for the diagnosis of neurotoxoplasmosis: comparison of three sets of primers for the B1 gene using CSF samples.

Polymerase chain reaction (PCR) has made a significant improvement in the diagnosis of toxoplasmic encephalitis (TE). Nevertheless, a wide variety of targets and primers has been used in different assays, and few comparative studies had been carried out. The aim of the present study was to compare the efficiency of 3 conventional PCR methods by using 3 sets of primers targeting the repetitive B1 gene in the diagnosis of TE. Diagnostic sensitivity and specificity of PCR and nested-PCR protocols were assessed for 207 (nested-PCR/T1-T4), 200 (nested-PCR/S1-AS1), and 206 (PCR/B22-B23) cerebrospinal fluid (CSF) samples, including AIDS and HIV-negative patients. The diagnostic sensitivity of PCR and nested-PCR assays was 50.85%, 68.97%, and 72.41% for T1-T4, S1-AS1, and B22-B23, respectively. The diagnostic specificity was high for all the assays showing values between 95% and 97%. In general, the best results were obtained for the B22-B23 set of primers, suggesting their usefulness compared with 2 nested-PCR protocols and showing that this simple and rapid strategy may be the preferred one for the diagnosis of TE in AIDS patients.

Influence of neurotoxoplasmosis characteristics on real-time PCR sensitivity among AIDS patients in Brazil.

Cerebral toxoplasmosis among individuals with AIDS may be difficult to diagnose and needs to be differentiated from other neurological diseases. A validation study was performed on real-time PCR for detecting the B1 gene of Toxoplasma gondii in the blood and cerebrospinal fluid (CSF) of AIDS patients with cerebral toxoplasmosis. The study included 135 AIDS patients divided into two groups: Group I comprised 85 patients with neurotoxoplasmosis; and Group II comprised 50 patients with non-toxoplasmic neurological diseases. Real-time PCR on blood showed a sensitivity of 1.5%, specificity of 100.0%, positive predictive value (PPV) of 100.0% and negative predictive value (NPV) of 36.5%. CSF testing produced better results, with a sensitivity of 35.3%, specificity of 100.0%, PPV of 100.0% and NPV of 44.7%. The group presenting with pleocytosis and four or more encephalic lesions was associated with greater CSF positivity on PCR. In conclusion, real-time PCR on blood was not useful for diagnosis. CSF testing showed low sensitivity but high specificity. Greater numbers of lesions and greater CSF cellularity may improve the sensitivity of the method.

Cerebrospinal fluid levels of chemokines in HIV infected patients with and without opportunistic infection of the central nervous system.

Chemokines are chemoattractant cytokines involved in the immune response of a wide variety of diseases. There are few studies assessing their role in opportunistic infections in HIV-infected patients. In this study, we measured CC and CXC chemokines in cerebrospinal fluid (CSF) samples obtained from 40 HIV-infected patients with or without opportunistic infections of the central nervous system (CNS). CSF samples were also analyzed for quantification of total protein, cell count and HIV-1 RNA. HIV+ patients with cryptococcal meningitis had higher levels of CCL2, CCL3, CCL5, CXCL9 and CXCL10 when compared to patients without opportunistic neurological infections. Furthermore, HIV+ patients with associated cryptococcal meningitis had higher levels of CCL3, CXCL9 and CXCL10 when compared to HIV+ patients with associated toxoplasmic encephalitis. CCL3 and CXCL9 levels were positively correlated with CSF HIV-1 RNA levels, CSF protein concentration, and CSF cell count. CXCL10 level was correlated with the CSF viral load and the CSF cell count and CCL5 level was correlated with the CSF cell count. In conclusion, the profile of chemokines in CSF of HIV patients may differ according to the modality of the presented opportunistic infection and according to other biological markers, such as viral load in CSF. These differences are probably related to different patterns of neuroinflammatory responses displayed by patients with different opportunistic neurological infections.

Neurotoxoplasmosis diagnosis for HIV-1 patients by real-time PCR of cerebrospinal fluid.

Encephalitis caused by Toxoplasma gondii is the most common cause of central nervous system damage in patients with acquired immunodeficiency syndrome (AIDS). Toxoplasma may infect any of the brain cells, thus leading to non-specific neurotoxoplasmosis clinical manifestations including focused or non-focused signs and symptoms of central nervous system malfunction. Clinical development ranges from insidious display during weeks to experiencing acute general confusion or ultimately fatal onset. Cerebral toxoplasmosis occurs in advanced stages of immunodeficiency, and the absence of anti-toxoplasmosis antibodies by the immunofluorescence method does not allow us to rule out its diagnosis. As specific therapy begins, diagnosis confirmation is sought through clinical and radiological response. There are few accurate diagnosis methods to confirm such cases. We present a method for T. gondii DNA detection by real time PCR-Multiplex. Fifty-one patients were evaluated; 16 patients had AIDS and a presumptive diagnosis for toxoplasmosis, 23 patients were HIV-positive with further morbidities except neurotoxoplasmosis, and 12 subjects were HIV-negative control patients. Real time PCR-Multiplex was applied to these patients' cephalorachidian liquid with a specific T. gondii genome sequence from the 529bp fragment. This test is usually carried out within four hours. Test sensitivity, specificity, positive predictive value, and negative predictive value were calculated according to applicable tables. Toxoplasma gondii assay by real time Multiplex of cephalorachidian fluid was positive for 11 out of 16 patients with AIDS and a presumptive diagnosis for cerebral toxoplasmosis, while none of the 35 control patients displayed such a result. Therefore, this method allowed us to achieve 68.8% sensitivity, 100% specificity, 100% positive predictive value, and 87.8% negative predictive value. Real time PCR on CSF allowed high specificity and good sensitivity among patients who presumably had cerebral toxoplasmosis. Since this is a low invasive method, it could be included in the diagnosis algorithm of patients with AIDS and central nervous system damage.

Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP analysis.

Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR-B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba.

Neurotoxoplasmosis diagnosis for HIV-1 patients by real-time PCR of cerebrospinal fluid

Encephalitis caused by Toxoplasma gondii is the most common cause of central nervous system damage in patients with acquired immunodeficiency syndrome (AIDS). Toxoplasma may infect any of the brain cells, thus leading to non-specific neurotoxoplasmosis clinical manifestations including focused or non-focused signs and symptoms of central nervous system malfunction. Clinical development ranges from insidious display during weeks to experiencing acute general confusion or ultimately fatal onset. Cerebral toxoplasmosis occurs in advanced stages of immunodeficiency, and the absence of anti-toxoplasmosis antibodies by the immunofluorescence method does not allow us to rule out its diagnosis. As specific therapy begins, diagnosis confirmation is sought through clinical and radiological response. There are few accurate diagnosis methods to confirm such cases. We present a method for T. gondii DNA detection by real time PCR-Multiplex. Fifty-one patients were evaluated; 16 patients had AIDS and a presumptive diagnosis for toxoplasmosis, 23 patients were HIV-positive with further morbidities except neurotoxoplasmosis, and 12 subjects were HIV-negative control patients. Real time PCR-Multiplex was applied to these patients' cephalorachidian liquid with a specific T. gondii genome sequence from the 529bp fragment. This test is usually carried out within four hours. Test sensitivity, specificity, positive predictive value, and negative predictive value were calculated according to applicable tables. Toxoplasma gondii assay by real time Multiplex of cephalorachidian fluid was positive for 11 out of 16 patients with AIDS and a presumptive diagnosis for cerebral toxoplasmosis, while none of the 35 control patients displayed such a result. Therefore, this method allowed us to achieve 68.8 percent sensitivity, 100 percent specificity, 100 percent positive predictive value, and 87.8 percent negative predictive value. Real time PCR on CSF allowed high specificity...

Comparison of four DNA extraction methods from cerebrospinal fluid for the detection of Toxoplasma gondii by polymerase chain reaction in AIDS patients.

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% and the diagnostic specificity was 100%. 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.

Factors influencing cerebrospinal fluid and plasma HIV-1 RNA detection rate in patients with and without opportunistic neurological disease during the HAART era.

BACKGROUND: In the central nervous system, HIV replication can occur relatively independent of systemic infection, and intrathecal replication of HIV-1 has been observed in patients with HIV-related and opportunistic neurological diseases. The clinical usefulness of HIV-1 RNA detection in the cerebrospinal fluid (CSF) of patients with opportunistic neurological diseases, or the effect of opportunistic diseases on CSF HIV levels in patients under HAART has not been well defined. We quantified CSF and plasma viral load in HIV-infected patients with and without different active opportunistic neurological diseases, determined the characteristics that led to a higher detection rate of HIV RNA in CSF, and compared these two compartments. METHODS: A prospective study was conducted on 90 HIV-infected patients submitted to lumbar puncture as part of a work-up for suspected neurological disease. Seventy-one patients had active neurological diseases while the remaining 19 did not. RESULTS: HIV-1 RNA was quantified in 90 CSF and 70 plasma samples. The HIV-1 RNA detection rate in CSF was higher in patients with neurological diseases, in those with a CD4 count lower than 200 cells/mm3, and in those not receiving antiretroviral therapy, as well as in patients with detectable plasma HIV-1 RNA. Median viral load was lower in CSF than in plasma in the total population, in patients without neurological diseases, and in patients with toxoplasmic encephalitis, while no significant difference between the two compartments was observed for patients with cryptococcal meningitis and HIV-associated dementia. CSF viral load was lower in patients with cryptococcal meningitis and neurotoxoplasmosis under HAART than in those not receiving HAART. CONCLUSION: Detection of HIV-1 RNA in CSF was more frequent in patients with neurological disease, a CD4 count lower than 200 cells/mm3 and detectable plasma HIV-1. Median HIV-1 RNA levels were generally lower in CSF than in plasma but some patients showed higher CSF levels, and no difference between these two compartments was observed in patients with cryptococcal meningitis and HIV-associated dementia, suggesting the presence of intrathecal viral replication in these patients. HAART played a role in the control of CSF HIV levels even in patients with cryptococcal meningitis and neurotoxoplasmosis in whom viral replication is potentially higher.

Estudo retrospectivo dos exames de líquido cefalorraquidiano (LCR) de / Retrospective study on cerebrospinal fluid (CSF) evaluation in HIV positive-patients with clinically suspected neurotoxoplasmosis

A toxoplasmose é uma infecção comum, observada tanto em indivíduos sadios como em pacientes imunocomprometidos. Este trabalho tem como objetivo apresentar um estudo retrospectivo dos resultados obtidos de exames realizados no Instituto Adolfo Lutz, laboratório Regional de Sorocaba, no período de 01/01/2003 a 29/11/2004 em amostras de líquido cefalorraquidiano (LCR) de pacientes HIV positivos com sintomatologia clínica e suspeita de neurotoxoplasmose, atendidos no Conjunto Hospitalar de Sorocaba (CHS). Selecionamos 109 pacientes HIV positivos, onde foi realizada a técnica de Imunofluorescência Indireta (IFI) para pesquisa de anticorpos IgG contra Toxoplasma gondii no LCR. Dos 109 pacientes HIV positivos selecionados, 46 foram do sexo feminino e 63 do sexo masculino, cuja faixa etária de maior freqüência foi de 21 a 40 anos. Nenhuma paciente era gestante. Dos 46 pacientes femininos, 48% apresentaram anticorpos anti-Toxoplasma gondii e dos 63 pacientes masculinos, 36,5% apresentaram positividade. O número de exames realizados que apresentaram resultados positivos no ano de 2003 foi de 61,5%, e em 2004 até o período estudado, foi de 38,5%. Os resultados mostram que é importante o acompanhamento de todos os pacientes portadores de HIV, mesmo os que apresentam ausência de anticorpos no LCR, pois podem ter a atividade baixa do toxoplasma no SNC, sem manifestação clínica aparente. Assim, seria oportuno realizar o segmento destes pacientes a fim de determinar sinais de reativação da doença no futuro

Evaluación de la técnica de PCR en la detección de toxoplasmosis encefálica en pacientes con inmunodeficiencia

La Toxoplasmosis encefálica (TE) es una de las infecciones oportunistas más comunes en personas con el síndrome de inmunodeficiencia adquirida, se ha estimado que la tercera parte de estos pacientes desarrollan TE, siendo la mayoría de los casos producto de una reactivación endógena de una infección latente por Toxoplasma gondii, sin embargo la serología no es un marcador lo suficiente sensible para el diagnóstico de TE. Los métodos diagnósticados utilizados son coloraciones con Giemsa de fluídos corporales, inoculación en ratones, cultivos celulares pero tienen baja sensibilidad, también se puede realizar por biopsia de cerebro, pero implica peligro al paciente. A fin de superar estos problemas en el presente trabajo se ha evaluado la utilización de la Reacción en cadena de la Polimerasa (PCR) para el diagnóstico de TE en diferentes muestras clínicas, usando "Primers" derivados del Gen B1 del parásito. Se usó como referencia o "Gold standard" la inoculación en ratones y/o el diagnóstico clínico. Se trabajó con una población de 68 pacientes divididos en tres grupos, uno de pacientes sospechosos (24 pacientes) y los otros dos de controles negativos (44 pacientes) las muestras que se evaluaron fueron líquido cefalorraquídeo (LCR) y sangre. Utilizando ya sea LCR o sangre la sensibilidad fue de 67 por ciento (8/12, 6/9) y cuando fue posible utilizar ambas muestras del mismo paciente, la sensibilidad se incrementó al 89 por ciento (8/9). La especificidad fue del 100 por ciento. El 77.7 por ciento (7/9) de los pacientes positivos a PCR presentaron anticuerpos contra t.gondii. Los niveles de CD4 por debajo de 200 células por µL de sangre predisponen al paciente a la adquisición de TE y otras enfermedades. El número de muestras trabajadas fue muy pequeño, no obstante esta limitación, consideramos como un importante aporte la iniciación del uso del PCR para el diagnóstico de Toxoplasma.

Toxoplasmosis encefálica en Sida / Encephalic toxoplasmosis in AIDS

La encefalitis toxoplásmica es la complicación oportunista del SNC que con mayor frecuencia ocasiona manifestaciones neurológicas focales en personas infectadas por el virus de la inmunodeficiencia humana (VIH). Desde 1986, cuando se diagnosticó el primer caso de toxoplasmosis encefálica en el sindrome de inmunodeficiencia adquirida (Sida) en nuestro país, hasta enero de 1997, en la Clínica y Servicio de Enfermedades Infecciosas (CEI-SEIC) se han observado 40 casos de esta enfermedad. La inaccesibilidad a ciertas técnicas diagnosticas y la imposibilidad de realizar autopsias en nuestro medio, hacen que esta complicación esté seguramente subdiagnosticada. La encefalitis toxoplásmica es sospechada por el cuadro clinico-tomográfico y reafirmada por la respuesta favorable al tratamiento. En 1 caso se tuvo la confirmación del diagnóstico por cultivo del germen que fue aislado del LCR. La mayoría de los enfermos tuvieron fiebre y manifestaciones neurológicas focales. Ante la sospecha clínica se realizó tomografía axial computada (TAC) de cráneo, la que se hizo en forma urgente, con la finalidad de iniciar precozmente el tratamiento específico y mejorar el pronóstico. Todos los casos presentaron lesiones características. En 1 sin lesiones tomográficas focales se hizo diagnóstico de encefalitis toxoplásmica difusa. En 27, a los que se les practicó estudio serológico de anticuerpos lgG específicos antitoxoplasma los resultados fueron positivos con títulos variables. En 20 enfermos en quienes se realizó estudio de subpoblaciones linfocitarias todos tenían un estado severo de inmunodepresión. En un alto porcentaje de casos se comprobaron otras infecciones oportunistas concomitantes. Con la sospecha diagnóstica se inició el tratamiento específico. Se observaron reacciones adversas a las drogas en 6 casos. Finalizado el período de tratamiento de la enfermedad activa, se continuó con terapia supresiva, observándose recaídas en 6 enfermos que lo habían discontinuado. Para prevenir esta grave complicación que compromete la vida del enfermo está indicado realizar quimioprofilaxis especialmente en aquellos con serología positiva para toxoplasmosis cuando su estado de inmunodepresión es grave, mientras que no es considerada prioritaria en los seronegativos para toxoplasma, en quienes la frecuencia de neurotoxoplasmosis es escasa

Toxoplasmosis encefálica en Sida / Encephalic toxoplasmosis in AIDS

La encefalitis toxoplásmica es la complicación oportunista del SNC que con mayor frecuencia ocasiona manifestaciones neurológicas focales en personas infectadas por el virus de la inmunodeficiencia humana (VIH). Desde 1986, cuando se diagnosticó el primer caso de toxoplasmosis encefálica en el sindrome de inmunodeficiencia adquirida (Sida) en nuestro país, hasta enero de 1997, en la Clínica y Servicio de Enfermedades Infecciosas (CEI-SEIC) se han observado 40 casos de esta enfermedad. La inaccesibilidad a ciertas técnicas diagnosticas y la imposibilidad de realizar autopsias en nuestro medio, hacen que esta complicación esté seguramente subdiagnosticada. La encefalitis toxoplásmica es sospechada por el cuadro clinico-tomográfico y reafirmada por la respuesta favorable al tratamiento. En 1 caso se tuvo la confirmación del diagnóstico por cultivo del germen que fue aislado del LCR. La mayoría de los enfermos tuvieron fiebre y manifestaciones neurológicas focales. Ante la sospecha clínica se realizó tomografía axial computada (TAC) de cráneo, la que se hizo en forma urgente, con la finalidad de iniciar precozmente el tratamiento específico y mejorar el pronóstico. Todos los casos presentaron lesiones características. En 1 sin lesiones tomográficas focales se hizo diagnóstico de encefalitis toxoplásmica difusa. En 27, a los que se les practicó estudio serológico de anticuerpos lgG específicos antitoxoplasma los resultados fueron positivos con títulos variables. En 20 enfermos en quienes se realizó estudio de subpoblaciones linfocitarias todos tenían un estado severo de inmunodepresión. En un alto porcentaje de casos se comprobaron otras infecciones oportunistas concomitantes. Con la sospecha diagnóstica se inició el tratamiento específico. Se observaron reacciones adversas a las drogas en 6 casos. Finalizado el período de tratamiento de la enfermedad activa, se continuó con terapia supresiva, observándose recaídas en 6 enfermos que lo habían discontinuado. Para prevenir esta grave complicación que compromete la vida del enfermo está indicado realizar quimioprofilaxis especialmente en aquellos con serología positiva para toxoplasmosis cuando su estado de inmunodepresión es grave, mientras que no es considerada prioritaria en los seronegativos para toxoplasma, en quienes la frecuencia de neurotoxoplasmosis es escasa(AU)

Detection of Toxoplasma gondii antigen in cerebrospinal fluid samples using a dot-enzyme-linked immunosorbent assay

Toxoplasma encephalitis (TE) is the most common manifestations of a recurrent Toxoplasma gondii infection in immunocompromised individuals, especially in AIDS patients. The infection is associated with considerable morbidity and mortality, even with improved diagnostic procedures and adequate treatment. The diagnosis of TE is difficult to establish by the demonstration of the parasite in the central nervous system or in cerebrospinal fluid (CSF). In order to diagnose TE we have developed a TGA-Dot-ELISA for antigen detection using rabbit anti-toxoplasma antiserum. We have applied this test to specimens of cerobrospinal fluid from patients with toxoplasma encephalitis (180 patients), neurocysticercosis (15), or bacterial meningitis (15). The relative sensitivity and specificity obtained for the TGA-Dot-ELISA were 0.930 and 0.730, respectively. TGA-Dot-ELISA is a promising method for the rapid diagnosis and effective prophylaxis of toxoplasma encephalitis.