Genotyping of Toxoplasma gondii: DNA extraction from formalin-fixed paraffin-embedded autopsy tissues from AIDS patients who died by severe disseminated toxoplasmosis.

This study investigated the genetic features of Toxoplasma gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes identified in 14 patients were not described. TgHuDis1 was the most frequent and was determined in 8 patients. TgHuDis3 and TgHuDis5 were identified in two patients each. TgHuDis2 and TgHuDis4 have been identified in one patient each. These suggestive genotypes could be considered as virulent, since they caused severe tissue damage and had similar characteristics as Toxo # DB 11.

Toxoplasma gondii isolates: multilocus RFLP-PCR genotyping from human patients in Sao Paulo State, Brazil identified distinct genotypes.

This study investigated the genetic characteristics of Toxoplasma gondii samples collected from 62 patients with toxoplasmosis in Sao Paulo State, Brazil. DNA samples were isolated from blood, cerebrospinal fluid and amniotic fluids of 25 patients with cerebral toxoplasmosis and AIDS, two patients with acute toxoplasmosis, 12 patients with ocular toxoplasmosis, six newborns with congenital toxoplasmosis and 17 pregnant women with acute infection. Diagnosis of toxoplasmosis was based in clinical, radiological and laboratory features. Genotyping was performed using multilocus PCR-RFLP genetic markers including SAG1, SAG2, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico. Among the 62 clinical samples, 20 (32%) were successfully genotyped at eight or more genetic loci and were grouped to three distinct genotypes. Eighteen samples belonged to ToxoDB Genotype #65 and the other two samples were identified as ToxoDB Genotypes #6 and #71, respectively (http://toxodb.org/toxo/). Patients presenting Genotypes #6 and #71 had severe and atypical cerebral toxoplasmosis, characterized by diffuse encephalitis without extensive brain lesions. These results indicate that T. gondii Genotype #65 may have a high frequency in causing human toxoplasmosis in Sao Paulo State, Brazil. This unusual finding highlights the need to investigate the possible association of parasite genotypes with human toxoplasmosis.

Toxoplasmic retinochoroiditis caused by Toxoplasma gondii strain ToxoDB#65.

Ocular toxoplasmosis, caused by Toxoplasma gondii, is the most common cause of inflammation in the back of the eye. Analysis of the infecting strain may provide information regarding disease behavior and recurrence. Here, we describe clinical and epidemiological data for toxoplasmic retinochoroiditis in two Brazilian women infected by T. gondii strain ToxoDB#65, living in an urban region of São Paulo State, Brazil.

Genotyping of Toxoplasma gondii: DNA extraction from formalin-fixed paraffin-embedded autopsy tissues from AIDS patients who died by severe disseminated toxoplasmosis

This study investigated the genetic features of Toxoplasma gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes identified in 14 patients were not described. TgHuDis1 was the most frequent and was determined in 8 patients. TgHuDis3 and TgHuDis5 were identified in two patients each. TgHuDis2 and TgHuDis4 have been identified in one patient each. These suggestive genotypes could be considered as virulent, since they caused severe tissue damage and had similar characteristics as Toxo # DB 11

Genotyping of toxoplasma gondii: DNA extraction from formalin-fixed paraffin-embedded autopsy tissues from AIDS patients who died by severe disseminated toxoplasmosis

This study investigated the genetic features of T. gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes...(AU)

Genotipagem de cepas polimórficas de Toxoplasma gondii provenientes de pacientes com toxoplasmose / Genotyping of polymorphic strains of Toxoplasma gondii from patients with toxoplasmosis

Este estudo investigou as características genéticas de amostras de Toxoplasma gondii provenientes de 62 pacientes com toxoplasmose no estado de São Paulo, Brasil. Amostras de DNA foram isoladas de sangue, líquido cefalorraquidiano e líquido amniótico de 25 pacientes com toxoplasmose cerebral e AIDS, 2 pacientes com toxoplasmose aguda, 12 pacientes com toxoplasmose ocular, 6 recém-nascidos com toxoplasmose congênita e 17 mulheres grávidas com infecção aguda. O diagnóstico da toxoplasmose foi baseado em características clínicas, radiológicas e laboratoriais. A genotipagem foi realizada por multilocus PCR-RFLP incluindo os marcadores SAG1, SAG2 (5'- SAG2 e 3'-SAG2, alt.SAG2) SAG3, BTUB, GRA6, C22-8, C29-2, L358, PK1 e APICO. Entre as 62 amostras clínicas, 20 (32%) foram genotipadas com sucesso em 8 ou mais loci gênicos, e somente estas comparadas com exames clínicos e laboratoriais. As outras 42 amostras (68%) obtiveram resultado em até 7 loci gênicos. Dentre as 20 amostras, três genótipos foram identificados e comparados aos listados no ToxoDB. Dezoito (90%) amostras pertencem ao genótipo ToxoDB # 65 e as outras duas amostras foram identificadas com os genótipos ToxoDB # 6 e # 71, respectivamente (http://toxodb.org/toxo/). Os pacientes portadores de genótipos # 6 e # 71 tiveram toxoplasmose cerebral atípica e severa caracterizada por encefalite difusa sem lesões cerebrais expansivas. Estes resultados indicam que o genótipo # 65 de T. gondii podem ter uma alta frequência na toxoplasmose humana no Estado de São Paulo, Brasil. Este achado incomum destaca a necessidade de investigar a possível associação dos genótipos do parasita com toxoplasmose humana.

Toxoplasma gondii isolates: multilocus RFLP–PCR genotyping from human patients in Sao Paulo State, Brazil identified distinct genotypes

This study investigated the genetic characteristics of Toxoplasma gondii samples collected from 62 patients with toxoplasmosis in Sao Paulo State, Brazil. DNA samples were isolated from blood, cerebrospinal fluid and amniotic fluids of 25 patients with cerebral toxoplasmosis and AIDS, two patients with acute toxoplasmosis, 12 patients with ocular toxoplasmosis, six newborns with congenital toxoplasmosis and 17 pregnant women with acute infection. Diagnosis of toxoplasmosis was based in clinical, radiological and laboratory features. Genotyping was performed using multilocus PCR–RFLP genetic markersincluding SAG1, SAG2, 50- and 30-SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico. Among the 62 clinical samples, 20 (32%) were successfully genotyped at eight or more genetic loci and were grouped to three distinct genotypes. Eighteen samples belonged to ToxoDB Genotype #65 andthe other two samples were identified as ToxoDB Genotypes #6 and #71, respectively (http://toxodb. org/toxo/). Patients presenting Genotypes #6 and #71 had severe and atypical cerebral toxoplasmosis, characterized by diffuse encephalitis without extensive brain lesions. These results indicate that T.gondii Genotype #65 may have a high frequency in causing human toxoplasmosis in Sao Paulo State, Brazil.This unusual finding highlights the need to investigate the possible association of parasite genotypes with human toxoplasmosis

Genotipagem de isolados de Toxoplasma gondii em pacientes com Aids e toxoplasmose cerebral / Genotyping of Toxoplasma gondii isolates form patients with Aids and cerebral toxoplasmosis

Este estudo investigou as características genotípicas de isolados de 87 pacientes com toxoplasmose cerebral e AIDS tratados no Instituto de Infectologia Emílio Ribas -São Paulo. O diagnóstico laboratorial da toxoplasmose cerebral foi baseado na sorologia e PCR positivas realizadas em amostras de sangue e/ou líquido cefalorraquidiano. As genotipagens foram realizadas utilizando-se quatro marcadores (5’- SAG2, 3’- SAG2,SAG3 e GRA6). Cada um dos marcadores distingue as três linhagens clonais de Toxoplasma gondii designadas como tipos I, II e III, tratando-se os produtos de PCR amplificados com enzimas de restrição (PCR-RFLP). Foram obtidos os seguintes resultados: 40 pacientes (46%) foram infectados com cepas do tipo I; 4 (4%) com as do tipo III; 13 (15%), com cepas polimórficas (genótipos incomuns); 6 pacientes com cepas do tipo I ou II; e 15 (17%) dos pacientes foram infectados com cepas incapazes de serem genotipadas pelos marcadores utilizados. A PCR-RFLP também identificou 9 (11%) isolados clínicos como sendo do tipo II, genótipo ainda não descrito na América do Sul. Contudo, o sequênciamento dos produtos gerados na “nested”-PCR de uma amostra classificada como tipo II (marcador SAG3) e de isolados polimórficos (marcadores 5’-SAG2, SAG3 e GRA6) mostrou diferenças nucleotídicas quando comparadas com as cepas clonais dos tipos I, II e III. Estes dados confirmam estudos prévios realizados na América do Sul demonstrando que cepas de T.gondii prevalentes no Brasil são altamente polimórficas. (AU)

Genotipagem de isolados de Toxoplasma gondii em pacientes com Aids e toxoplasmose cerebral

Este estudo investigou as características genotípicas de isolados de 87 pacientes com toxoplasmose cerebral e AIDS tratados no Instituto de Infectologia Emílio Ribas -São Paulo. O diagnóstico laboratorial da toxoplasmose cerebral foi baseado na sorologia e PCR positivas realizadas em amostras de sangue e/ou líquido cefalorraquidiano. As genotipagens foram realizadas utilizando-se quatro marcadores (5’- SAG2, 3’- SAG2,SAG3 e GRA6). Cada um dos marcadores distingue as três linhagens clonais de Toxoplasma gondii designadas como tipos I, II e III, tratando-se os produtos de PCR amplificados com enzimas de restrição (PCR-RFLP). Foram obtidos os seguintes resultados: 40 pacientes (46%) foram infectados com cepas do tipo I; 4 (4%) com as do tipo III; 13 (15%), com cepas polimórficas (genótipos incomuns); 6 pacientes com cepas do tipo I ou II; e 15 (17%) dos pacientes foram infectados com cepas incapazes de serem genotipadas pelos marcadores utilizados. A PCR-RFLP também identificou 9 (11%) isolados clínicos como sendo do tipo II, genótipo ainda não descrito na América do Sul. Contudo, o sequênciamento dos produtos gerados na “nested”-PCR de uma amostra classificada como tipo II (marcador SAG3) e de isolados polimórficos (marcadores 5’-SAG2, SAG3 e GRA6) mostrou diferenças nucleotídicas quando comparadas com as cepas clonais dos tipos I, II e III. Estes dados confirmam estudos prévios realizados na América do Sul demonstrando que cepas de T.gondii prevalentes no Brasil são altamente polimórficas.

Genotipagem de isolados de Toxoplasma gondii em pacientes com Aids e toxoplasmose cerebral / Genotyping of Toxoplasma gondii isolates form patients with Aids and cerebral toxoplasmosis

Este estudo investigou as características genotípicas de isolados de 87 pacientes com toxoplasmose cerebral e AIDS tratados no Instituto de Infectologia Emílio Ribas -São Paulo. O diagnóstico laboratorial da toxoplasmose cerebral foi baseado na sorologia e PCR positivas realizadas em amostras de sangue e/ou líquido cefalorraquidiano. As genotipagens foram realizadas utilizando-se quatro marcadores (5’- SAG2, 3’- SAG2,SAG3 e GRA6). Cada um dos marcadores distingue as três linhagens clonais de Toxoplasma gondii designadas como tipos I, II e III, tratando-se os produtos de PCR amplificados com enzimas de restrição (PCR-RFLP). Foram obtidos os seguintes resultados: 40 pacientes (46%) foram infectados com cepas do tipo I; 4 (4%) com as do tipo III; 13 (15%), com cepas polimórficas (genótipos incomuns); 6 pacientes com cepas do tipo I ou II; e 15 (17%) dos pacientes foram infectados com cepas incapazes de serem genotipadas pelos marcadores utilizados. A PCR-RFLP também identificou 9 (11%) isolados clínicos como sendo do tipo II, genótipo ainda não descrito na América do Sul. Contudo, o sequênciamento dos produtos gerados na “nested”-PCR de uma amostra classificada como tipo II (marcador SAG3) e de isolados polimórficos (marcadores 5’-SAG2, SAG3 e GRA6) mostrou diferenças nucleotídicas quando comparadas com as cepas clonais dos tipos I, II e III. Estes dados confirmam estudos prévios realizados na América do Sul demonstrando que cepas de T.gondii prevalentes no Brasil são altamente polimórficas.