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.

Molecular diagnosis of cerebral toxoplasmosis: comparing markers that determine Toxoplasma gondii by PCR in peripheral blood from HIV-infected patients

As cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS patients, this study evaluated three PCR markers for diagnosis, since some limitations remain present, such as low parasite levels in some clinical samples. The molecular markers were B22-B23 and Tg1-Tg2 (based on the B1 gene) and Tox4-Tox5 (non-coding fragment, repeated 200-300-fold). DNA samples from 102 AIDS patients with previously known diagnosis were analyzed. The cerebral toxoplasmosis group was constituted of DNA extracted from the blood of 66 AIDS patients, which was collected before or until the third day of the therapy for toxoplasmosis. DNA from the blood of 36 AIDS patients with other neurologic opportunistic infections was used as control group. Sensitivities of B22-B23, Tg1-Tg2, and Tox4-Tox5 markers were of 95.5 percent, 93.9 percent, and 89.3 percent, respectively. In the control group, the specificities were of 97.2 percent (B22-B23), 88.9 percent (Tg1-Tg2), and 91.7 percent (Tox4-Tox5). The association of at least two markers increased the PCR sensitivity and specificity. The concordance index between two markers varied from 83.3 percent to 93.1 percent. These data demonstrated that all markers evaluated here were highly sensitive for T. gondii determination, although B22-B23 has been shown to be the best. The association of two markers increases PCR sensitivity, but the procedure was more expensive and time-consuming.

Toxoplasma gondii infection and cerebral toxoplasmosis in HIV-infected patients

Cerebral toxoplasmosis is a major cause of morbidity and mortality among HIV-infected patients, particularly from developing countries. This article summarizes current literature on cerebral toxoplasmosis. It focuses on: Toxoplasma gondii genetic diversity and its possible relationship with disease presentation; host responses to the parasite antigens; host immunosupression in HIV and cerebral toxoplasmosis as well as different diagnostic methods; clinical and radiological features; treatment; and the direction that studies on cerebral toxoplasmosis will likely take in the future.

The role of MHC haplotypes H2d/H2b in mouse resistance/susceptibility to cyst formation is influenced by the lineage of infective Toxoplasma gondii strain

Toxoplasma gondii strains displaying the Type I/III genotype are associated with acquired ocular toxoplasmosis in humans. Here, we used a mice model to characterize some immunological mechanisms involved in host resistance to infection with such strains. We have chosen the Type I/III strains D8, G2 and P-Br, which cause a chronic infection in mice that resembles human toxoplamosis. Mice deficient of molecules MyD88, IFN-gamma, and IL-12 were susceptible to all three parasite strains. This finding indicates the importance of innate mechanisms in controlling infection. On the other hand, MHC haplotype did not influenced resistance/susceptibility; since mice lineages displaying a same genetic background but different MHC haplotypes (H2b or H2d) developed similar mortality and cyst numbers after infection with those strains. In contrast, the C57BL/6 genetic background, and not MHC haplotype, was critical for development of intestinal inflammation caused by any of the studied strains. Finally, regarding effector mechanisms, weobserved that B and CD8+ T lymphocytes controlled survival,whereas the inducible nitric oxide synthase influenced cyst numbers in brains of mice infected with Type I/III strains. These findings are relevant to further understanding of the immunologic mechanisms involved in host protection and pathogenesis during infection with T. gondii.

Cepas de Toxoplasma gondii que apresentam o genótipo I/III são associadas a toxoplasmose ocular adquirida em humanos. No presente trabalho, nós utilizamos um modelo da doença em camundongos para caracterizar mecanismos imunológicos envolvidos na resistência do hospedeiro à infecção por aquelas cepas. Escolhemos as cepas D8, G2 e P-Br, que causam infecção crônica em camundongos, semelhante à toxoplasmose humana. Camundongos deficientes em MyD88, IFN-G e IL-12 foram susceptíveis a infecções com todas as três linhagens do parasita. Esses dados indicam a importância de mecanismos inatos no controle da infecção. Por outro lado, o haplótipo do MHC não influenciou na resistência/susceptibilidade, na medida em que linhagens de camundongos com um mesmo "background'' genético, mas diferentes haplótipos de MHC (H2b e H2d) apresentam o índice de mortalidade e número de cistos semelhantes após a infecção com aquelas cepas do parasita. Em contraste, o "background'' genético de C57BL/6, mas não o haplótipo de MHC, foi crítico para o desenvolvimento de inflamação intestinal causada pelas cepas estudadas. Finalmente, com relação aos mecanismos efetores, observamos que linfócitos B e T CD8+ controlam a sobrevivência após infecção. Por outro lado, a ativação da enzima óxido nítrico sintase induzida foi um fator importante para controle do número de cistos cerebrais em camundongos infectados com cepas do Tipo I/III. Esses achados são relevantes para o melhor entendimento dos mecanismos imunológicos envolvidos na proteção e patogênese durante infecção com T. gondii.

Toxoplasmosis cerebral en pacientes con SIDA / Brain toxoplasmosis in AIDS patients

El presente trabajo abarcó el período comprendido entre el año 1987 y agosto de 1994. Se diagnosticaron 25 pacientes con toxoplasmosis cerebrales (23 hombres y 2 mujeres), con una media de 35 años. Doce fueron heterosexuales y 11 homosexuales, todos en etapa Sida con repercusión general. Las manifestaciones clínicas más frecuentes fueron síndrome neurológico en 19 pacientes (síndrome motor deficitario 12, síndrome arquineocerebeloso 6, síndrome frontal 3, estado confusional l); síndrome febril prolongado 11; síndrome de hipertensión endocraneana 14; síndrome poliadenomegálico 7. La TAC mostró en 24 pacientes imágenes compatibles con toxoplasmosis. Se observaron frecuentemente infecciones asociadas de las cuales 14 fueron marcadoras de etapa evolutiva SIDA; (Candidiasis orofaríngea P pacientes, neumocistosis 6 pacientes), otras infecciones asociadas (lúes, Bk, CMV, herpes). Siete pacientes cursaron con clínica de toxoplasmosis sin otras infecciones intercurrentes. El diagnóstico fue tomográfico, clínico y serológico. No hubo confirmación parasitológica, mediante biopsia cerebral. La determinación de las poblaciones linfocitarias mostró cifras de T4 menores a 200 en 15 pacientes. La serología para toxoplasmosis mostró títulos altos de anticuerpos en 13 pacientes, con IgM positiva en 2 de ellos. La mayor eficacia terapéutica se obtuvo con la combinación de pirimetamina, sulfadiazina y ácido folínico, obteniéndose en 10 pacientes buena respuesta clínica y paraclínica