Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients.

BACKGROUND: 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. METHODS: CSF samples from AIDS and HIV-negative patients were analyzed. Patients were divided into two groups according to the Centre for Disease Control and Prevention (CDC) criteria for AIDS-related TE: AIDS patients with suspected neurotoxoplasmosis and AIDS and HIV-negative patients with other confirmed neurological diseases but no suspicions of TE. Predictive values, diagnostic accuracy, sensitivity and specificity of the PCR B1 method were calculated. RESULTS: The results obtained from 190 patients showed that this assay has a good sensitivity and specificity (83.3% and 95.7%, respectively) for the diagnosis of TE in AIDS patients. CONCLUSION: PCR using the B1 gene and B22/B23 set of primers is a single, rapid and reliable method that may be valuable for discrimination between toxoplasmosis and other central nervous system (CNS) diseases.

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.

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.

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.

Control de la calidad del diagnóstico coproparasitológico en la provincia de Ciudad de la Habana, Cuba / External quality assessment in coproparasitology in Havana City Province, Cuba

Se realizó un estudio sobre la calidad del diagnóstico coproparasitológico en 77 laboratorios de la red de salud pública de la província Ciudad de La Habana, Cuba. El procedimiento se basó en la entrega a cada jefe de laboratorio de un modelo de encuesta, y una bolsa de nylon conteniendo 10 viales plásticos con distintos especímenes parasitarios, preservados en formaldehído al 7 por ciento. Recogidos los resultados en las primeras 72 horas después de su entrega, se realizó la evaluación mediante una escala de puntuación establecida. La mayoría de los laboratorios aprobaron (70 por ciento); sin embargo aún existen centros, sobre todo policlínicas, con calificaciones deficientes. Los municipios con resultados más desfavorables fueron, Lisa, Marianao y Habana del Este, alcanzándose mejores resultados en los hospitales que en las policlínicas. En el análisis de Protozooarios, el mejor diagnosticado fué Giardia lamblia, con solo un centro que erró al identificarlo. Las mayores dificultades se presentaron en Blastocystis hominis con 61 por ciento de fallas, Endolimax nana, con 24,6 por ciento, y Entamoeba histolytica, con 22 por ciento. Entre los helmintos, la mayor aprobación fué en Trichuris trichiura y los errores diagnósticos predominaron con Fasciola hepatica y Taenia sp., ambos con 66,2 por ciento de fallas. Dados los resultados obtenidos, hemos organizado una intervención educativa en la red de laboratorios de la provincia.

Intervención educativa para mejorar la calidad del diagnóstico coproparasitológico en la red de salud de Ciudad Habana, Cuba / An educational intervention to improve the quality of coproparasitological diagnosis in laboratories of Havana City, Cuba

Evalua la efectividad de una intervención educativa con participación voluntaria realizada en 1993, a partir de los resultados obtenidos en un primer control de la calidad del diagnóstico coproparasitológico, en 77 laboratorios de la red de salud de Ciudad Habana. Se comparan los resultados obtenidos por los centros que adiestraron con los que no participaron en la actividad; se obtuvieron resultados superiores en los municipios 10 de Octubre, Plaza y Cerro y en la totalidad de los laboratorios que enviaron personal para el adiestramiento. Se encontró un mejor diagnóstico de los helmintos Trichuris trichiura, Taenia sp. y Fasciola hepatica, y de los protozoos Blastocystis hominis y Enndolimax nana, en los laboratorios que recibieron el curso. Además se observó que la mayoría de los laboratorios que adiestraron un técnico subieron significativamente sus notas, mientras que los que no lo efectuaron las bajaron, lo que demuestra la efectividad de la intervención. Proponemos que estas actividades de Control de la Calidad y Educación Continuada se mantengan de forma periódica y con un carácter obligatorio, para garantizar una mejoría creciente del diagnóstico coproparasitológico en la red de salud.

Anticuerpos IgG anti-Toxoplasma gondii en pacientes con síntomas atribuibles a toxoplasmosis / IgG anti-Toxoplasma gondii antibodies in patients with possible toxoplasmosis symptomatology

Se evaluaron 6.520 pacientes con síntomas atribuíbles a toxoplasmosis por la presencia de anticuerpos IgG anti- Toxoplasma gondii mediante la técnica de inmunofluorescencia indirecta. El 51,27 por ciento de los evaluados resultaron positivos. El 60,85 por ciento de los pacientes con trastornos oculares son seropositivos a IgG anti- T. gondii; seguidos del 56,25 y 48,10 por ciento para aquellos con trastornos generales y gineco-obstétricos. Los síntomas o manifestaciones que evidenciaron mayor porcentaje de positividad fueron: astenia, coriorretinitis, trastornos menstruales, cefaleas y uveítis