Resumen Introducción. Toxoplasma gondii es un parásito con gran potencial zoonótico que puede infectar un amplio rango de huéspedes de sangre caliente, incluidos los animales del sector pecuario, lo que causa pérdidas a la industria. En el humano, es patógeno en personas inmunosuprimidas y afecta el desarrollo del feto en infecciones congénitas. Además, se asocia con diversos trastornos del comportamiento en personas sanas. El humano puede adquirir T. gondii al consumir carnes contaminadas mal cocidas. Objetivo. Determinar la positividad de T. gondii en carnes de consumo humano (res, pollo y cerdo) en Ibagué, Colombia. Materiales y métodos. Se utilizó la PCR convencional anidada y la secuencia del gen B1 de T. gondii como blanco de amplificación. Se tomaron 186 muestras de carne comercializada en la zona urbana de Ibagué (62 de res, 62 de pollo y 62 de cerdo) y se obtuvo el porcentaje de positividad en cada tipo de carne evaluada. Resultados. Se encontró un porcentaje de positividad de 18,8 % en las muestras, siendo la carne de cerdo la del mayor porcentaje (22,5 %; 14/62), seguida por las muestras de carne de res (19,3 %; 12/62) y de pollo (14,5 %; 9/62). Los mejores productos amplificados fueron secuenciados en Macrogen, y alineados con las secuencias del gen B1 depositadas en el GenBank, con lo que se confirmó su identidad. Conclusiones. Este es el primer estudio sobre prevalencia de T. gondii en carnes para consumo humano en Ibagué y el departamento del Tolima. Se demostró que los tres tipos de carne representan un riesgo para la infección en humanos a nivel local.

Abstract Introduction: Toxoplasma gondii is a parasite with great zoonotic potential. It can infect a broad range of warm-blooded hosts (including livestock) and causes significant losses in the industry. In humans, it has been described as a pathogen in immunosuppressed people, it affects the fetus development in congenital infections, and is associated with various behavioral disorders in healthy people. Humans can acquire T. gondii by consuming undercooked, contaminated meat. Objective: To determine T. gondii positivity (currently unknown) in meat for human consumption (i.e., beef, chicken, and pork) in the city of Ibague, Colombia. Materials and methods: We used conventional nested PCR and the T. gondii B1 gene sequence as amplification target. We collected samples of meat (N=186) sold in the urban area of Ibagué (62 beef, 62 chicken, and 62 pork samples) and determined the T. gondii positivity percentage for each type of meat. Results: The study found an average of 18.8% positivity for all meat samples, pork having the highest percentage (22.5%; 14/62), followed by beef (19.3%; 12/62) and chicken (14.5%; 9/62). The best-amplified products were sequenced by macrogen and aligned with the B1 gene sequences in GenBank, thereby confirming their identity. Conclusions: This is the first study of T. gondii prevalence in meat for human consumption carried out in the city of Ibagué and the department of Tolima. All three types of meat sampled represent a risk for local human infection.

Resumo O objetivo deste estudo foi identificar membros da família Sarcocystidae em aves silvestres de vida livre naturalmente infectadas e resgatadas no estado de Minas Gerais, Brasil. Coração e cérebro de 44 aves silvestres foram avaliados por bioensaio em camundongos para detecção de T. gondii e extração de DNA para Nested-PCR do gene 18S do DNA ribossomal de membros da família Sarcocystidae. As amostras positivas foram sequenciadas, analisadas, editadas e comparadas com sequências depositadas no GenBank. Toxoplasma gondii foi isolado de seis (13,6%) das 44 aves. DNA de T. gondii foi identificado em 10/44 (22,7%) das 44 aves. As sequências amplificadas exibiram 100% de similaridade com o DNA da cepa ME49 de T. gondii. DNA de Sarcocystis (99% de similaridade) foi identificado em 5/44 (11,4%) das 44 aves. T. gondii e Sarcocystis spp. são encontrados, comumente, em aves silvestres no estado de Minas Gerais, Brasil.

Abstract This study aimed to identify members of the Sarcocystidae family in naturally infected wild birds at a rescue center in the state of Minas Gerais, southeastern Brazil. The heart and brain of 44 wild birds were evaluated by bioassay in mice to detect T. gondii, and extracted DNA was used for nested PCR of the 18S ribosomal DNA gene to detect members of the Sarcocystidae family. The positive samples were sequenced, assembled, edited and compared with sequences deposited in GenBank. Toxoplasma gondii was isolated from six (13.6%) out of 44 birds. Toxoplasma gondii DNA was identified in 10/44 (22.7%) of the birds. The amplified sequences exhibited 100% similarity with the DNA of the ME49 strain of T. gondii. Sarcocystis DNA (99% similarity) was identified in 5/44 (11.4%) of the birds. T. gondii and Sarcocystis spp. are common in wild birds in Minas Gerais, Brazil.

Resumo Toxoplasma gondii apresenta alta prevalência mundial, capaz de infectar diversos animais. Felinos são considerados os hospedeiros definitivos e entre os hospedeiros intermediários destacamos os mamíferos e as aves. O homem pode se infectar ingerindo cistos teciduais presentes na carne das aves e mamíferos. O conhecimento dos aspectos biológicos e moleculares do parasito possibilitam melhor entendimento da epidemiologia da toxoplasmose. Neste trabalho foi realizada triagem sorológica por hemaglutinação indireta (HI) em 58 galinhas caipiras (Gallus gallus domesticus) utilizadas para consumo humano, provenientes do estado do Espírito Santo, Brasil. Treze galinhas apresentaram sorologia positiva para T. gondii. O coração e o cérebro de cinco galinhas positivas foram colhidos, tratados com pepsina e inoculados separadamente, em dois camundongos Swiss, por via intraperitoneal. Observou-se taquizoítos no peritônio de todos os camundongos, entre sete e 10 dias após o inóculo. Foram obtidos 10 novos isolados de T. gondii os quais foram estudados em camundongos BALB/C inoculados com 101 a 104 taquizoítos por animal. Todos os isolados foram considerados virulentos ou de virulência intermediária. A caracterização molecular dos isolados, realizada por PCR-RFLP, demonstrou a ocorrência de três genótipos distintos. Nenhum isolado apresentou genótipo clonal ou linhagem clonal do Brasil. Não foi observada diferença molecular (PCR-RFLP) entre os isolados obtidos a partir do cérebro ou do coração da mesma ave. Dois isolados já haviam sido relatados na literatura como causadores de doenças em humanos.

Abstract Toxoplasma gondii presents a high prevalence worldwide, infecting several animals. Felines are considered the definitive hosts and among the intermediate hosts we highlight mammals and birds. The man can become infected by ingesting tissue cysts present in birds and mammals. Biological and molecular aspects of T. gondii allows a better understanding of the epidemiology of toxoplasmosis. This work is a serologic screening of 58 chickens grown (Gallus gallus domesticus) for human consumption in Espírito Santo State, by means of indirect haemagglutination assay (IHA). Thirteen chickens tested positive for anti-T. gondii antibodies. The heart and brain of five positive chickens were harvested, treated with pepsin and inoculated separately, in two Swiss mice, intraperitoneally. Tachyzoites were observed in the peritoneum of all the animals, between seven and 10 days after the inoculum. Ten isolates were obtained and biologically characterised in BALB/c mice inoculated with 101 to 104 tachyzoites. All isolates were classified as virulent or intermediately virulent. Isolates were genotyped by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, revealing three different genotypes. None of the isolates exhibited the clonal type I, II or III genotype. No genotypic differences were observed between the isolates from the brain or heart from the same bird.

Resumo A filogenia da subfamília Toxoplasmatinae tem sido amplamente investigada com diversos marcadores moleculares. Neste estudo, a filogenia molecular da subfamília Toxoplasmatinae foi analisada através de genes de apicoplasto e mitocondriais. Foram analisadas sequências parciais de genes de apicoplasto codificadores da protease caseinolítica (clpC), e da subunidade beta da RNA polimerase (rpoB) e de gene mitocondrial codificador de citocromo B (cytB). Foram investigadas cepas adaptadas em laboratório de Sarcocystis neurona eSarcocystis falcatula, parasitos estreitamente relacionados, além de Neospora caninum, Neospora hughesi, Toxoplasma gondii (cepas RH, CTG e PTG),Besnoitia akodoni, Hammondia hammondi e duas linhagens geneticamente divergentes de Hammondia heydorni. A análise molecular, baseada em genes de organelas, não diferenciou claramenteN. caninum de N. hughesi, porém foi possível confirmar as duas linhagens de H. heydorni. Foram encontradas pequenas diferenças entre as cepas adaptadas em laboratório deS. falcatula e S. neurona em todos os marcadores moleculares avaliados. Concluindo, filogenias congruentes foram reconstruídas com os três diferentes genes que podem ser úteis em triagem de parasitos sarcocistídeos não identificados, para identificar sua relação com organismos da família Sarcocystidae. Os estudos evolutivos com genes organelares confirmam que o gênero Hammondia é parafilético. Osprimers utilizados para amplificação declpC e rpoB foram capazes de amplificar sequências genéticas de organismos do gênero Sarcocystis e da subfamília Toxoplasmatinae.

Abstract Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG), Besnoitia akodoni, Hammondia hammondiand two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genusHammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystisand organisms of the subfamily Toxoplasmatinae as well.

The aim of this work was to evaluate the utility of ELISA-based testing of total IgG (IgGt) antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii for diagnosing congenital toxoplasmosis. Sera from 217 newborns initially testing positive for specific IgM in filter paper dried blood spots were tested for specific IgM and IgG by ELFA-VIDAS®. Congenital toxoplasmosis was confirmed in 175 and ruled out in 42 infants. The validity of the ELISA tests was determined using the persistence of IgG antibodies (ELFA-VIDAS® kit) at the end of 12 months, which is considered the reference test for the diagnosis of congenital toxoplasmosis. The frequency of positivity with IgGt against STAg, rSAG1 and rMIC3 was found in 97.2%, 96.3% and 80.2%, respectively, of the newborns with confirmed congenital toxoplasmosis. IgG1 reacted with all three antigens, while IgG3 and IgG4 reacted preferentially with rMIC3. Higher mean values of reactivity (sample optical density/cut-off) were found for all subclasses when using rMIC3. All of the antigens showed high sensitivity and low specificity in detecting anti-T. gondii IgGt and IgG1 and low sensitivity and high specificity in detecting IgG3 and IgG4. In conclusion, the combined detection of IgG antibody subclasses against recombinant toxoplasmic antigens may be useful for the early diagnosis of congenital toxoplasmosis.

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

A toxoplasmose congênita pode causar déficit neurossensorial em até 20% dos casos e o tratamento no primeiro ano de vida melhora o prognóstico. No Brasil, desconhece-se o impacto da infecção na hipoacusia. OBJETIVO: Avaliar a audição de crianças com toxoplasmose congênita identificadas pela triagem neonatal. MATERIAL E MÉTODO: Estudo prospectivo de crianças com toxoplasmose congênita identificadas pela triagem neonatal (IgM anti-T. gondii) em Belo Horizonte, durante 2003/2004. Realizada sorologia confirmatória (mãe/filho) e consideradas positivas as crianças apresentando IgM e/ou IgA nos primeiros seis meses ou IgG aos 12 meses de vida. Avaliações auditivas ao diagnóstico e após 12 meses incluíram Audiometria Comportamental, Emissões Otoacústicas, Imitanciometria, Audiometria de Tronco Encefálico. RESULTADOS: Dentre 30.808 crianças triadas (97% dos nascidos vivos), 20 apresentavam toxoplasmose congênita, 15 (75%) com infecção subclínica. Dezenove crianças realizaram avaliação auditiva. Quatro apresentaram déficit neurossensorial (21,1%). Uma criança apresentou outros fatores de risco para hipoacusia; nas outras três, a toxoplasmose foi o único fator observado. Duas crianças, tratadas adequadamente com antiparasitários, apresentaram déficit auditivo, em desacordo com a literatura. CONCLUSÃO: Os achados sugerem que a toxoplasmose congênita, prevalente no Brasil, é um fator de risco para hipoacusia e o impacto dessa infecção nas perdas auditivas deve ser estudado.

Congenital toxoplasmosis may cause sensorineural deficit in up to 20% of the patients and proper treatment in the first year improves prognosis. In Brazil, this infection’s impact on hearing impairment is unknown. AIM: To evaluate hearing of newborns with congenital toxoplasmosis identified by the newborn screening service. METHOD: This prospective study analyzed children with congenital toxoplasmosis identified by newborn screening (IgM anti-T.gondii) in Belo Horizonte during 2003/2004. The presence of IgM and/or IgA in the first 6 months or IgG at 12 months-of age in serology was used as case definition. Hearing tests were carried out at the time of diagnosis and 12 months later, including behavioral audiometry, evoked otoacoustic emission and brainstem evoked responses audiometry. RESULTS: Among 30.808 screened children (97% of live births), 20 had congenital toxoplasmosis, 15 (75%) were asymptomatic at birth. Nineteen children were evaluated by hearing tests. Four had sensorineural impairment (21.1%). One child had other risk factors for hearing impairment; the other three had no other risk factors but toxoplasmosis. Two properly children treated still had hearing loss, in disagreement with current literature. CONCLUSION: Results suggest that congenital toxoplasmosis, common in Brazil, is a risk factor for hearing impairment and its impact on hearing loss deserves further studies.

A toxoplasmose é infecção freqüente em todo o mundo, mas na maioria dos casos não traz repercussões importantes para o paciente, exceto indivíduos imunodeprimidos e fetos, os quais podem apresentar graves seqüelas. O diagnóstico precoce durante a gravidez é altamente desejável, já que o tratamento da gestante reduz a freqüência e gravidade da infecção fetal. Neste estudo aplicou-se a técnica de PCR em 86 amostras de líquido amniótico de gestantes que apresentaram soroconversão durante a gravidez. O DNA foi amplificado usando-se iniciadores externos e internos, num sistema de nested PCR. As amostras foram também inoculadas em camundongos e os recém-nascidos acompanhados clinicamente através de sorologia, RX de crânio, ultrassom transfontanela, exame de fundo de olho e punção lombar. Pela PCR, sete casos foram positivos e 79 negativos. Entre os positivos, dois não se confirmaram pela inoculação em camundongo nem pela avaliação clínica da criança; dos negativos, três apresentaram clínica de toxoplasmose congênita, e em um deles o exame de inoculação em camundongo foi positivo. A sensibilidade e especificidade da PCR foram 62,5% e 97,4%, respectivamente; a inoculação em camundongos mostrou 42,9% de sensibilidade e 100% de especificidade. Embora a PCR não deva ser usada como único teste diagnóstico da toxoplasmose congênita, trata-se de método promissor e merece mais estudos para aumentar sua eficácia.

Toxoplasmosis is one of the most common infections all over the world. Most cases are asymptomatic, except in immunosuppressed individuals and fetuses, which can be seriously damaged. Prenatal diagnosis should be made as soon as possible since treatment of the mother can minimize fetal sequelae. Our aim in this study was to test the polymerase chain reaction technique (PCR) in 86 samples of amniotic fluid from women who seroconverted during pregnancy. DNA was amplified using external primers and, in a second step, internal primers, in a nested PCR system. Samples were also inoculated into mice and the newborn were evaluated by T. gondii serology, skull x-ray, transfontanel ultrasound, fundoscopic examination, lumbar puncture and clinical examination. PCR was positive in seven cases and negative in 79. Among PCR-positive cases, two were negative by inoculation into mice and by clinical evaluation; among PCR-negative ones, three had clinical evidence of toxoplasmosis and one was positive after inoculation into mice. PCR showed values of sensitivity = 62.5% and specificity = 97.4%; the values of inoculation into mice where 42.9% and 100%, respectively. Although PCR should not be used alone for prenatal diagnosis of congenital toxoplasmosis, it is a promising method and deserves more studies to improve its efficacy.

Objetivo: avaliar a eficácia da reação em cadeia da polimerase (PCR) no líquido amniótico para a detecção da contaminação fetal pelo Toxoplasma gondii em gestantes com infecção aguda e correlacionar com a técnica de inoculação e a histologia da placenta. Métodos: trinta e sete pacientes foram estudadas prospectivamente e o diagnóstico foi baseado na identificação da infecção aguda materna, seguido pela amniocentese guiada pela ultra-sonografia para obtenção de líquido amniótico para a realização de PCR e inoculação em camundongo. As pacientes foram tratadas com espiramicina durante a gestação; se a infecção fetal era demonstrada, pirimetamina e sulfadiazina eram acrescentadas ao esquema. As placentas foram encaminhadas para exame histológico. As crianças foram acompanhadas durante um período que variou de três a 23 meses para a confirmação ou exclusão da toxoplasmose congênita. Resultados: medidas de associação como sensibilidade, especificidade e valores preditivos foram calculadas para a PCR no líquido amniótico, a inoculação em camundongo e a histologia da placenta, mostrando os seguintes resultados: para a PCR, uma sensibilidade de 66,7% e especificidade de 87,1%; os respectivos valores para a inoculação em camundongo foram 50 e 100% e para a histologia da placenta foram 80 e 66,7%. Conclusões: embora a PCR não deva ser utilizada isoladamente para diagnóstico de toxoplasmose congênita, é um método promissor e necessita de maiores estudos para melhorar sua eficácia.

Objective: to test the effectiveness of the polymerase chain reaction (PCR) in the amniotic fluid for the detection of fetal contamination due to Toxoplasma gondii in pregnant women with acute infection and to correlate it with the inoculation technique and the histology of the placenta. Methods: thirty-seven patients were prospectively studied and the diagnosis was based on the identification of maternal acute infection followed by amniocentesis guided by ultrasound to obtain amniotic fluid for PCR and mice inoculation. The mothers were treated with spiramycin throughout pregnancy; when fetal infection was demonstrated, pyrimethamine and sulfadiazine were added to the regimen. The placentas were processed for histologic examination. The infants were followed for a period that varied from three to 23 months for the confirmation or exclusion of congenital toxoplasmosis. Results: association measures such as sensitivity, specificity and predictive values were calculated for PCR in the amniotic fluid, detection of the parasite through mice inoculation and placental histology and showed the following results: PCR values of sensitivity = 66.7% and specificity = 87.1%; the respective values for mice inoculation were 50 and 100% and for the placental histology were 80 and 66.7%. Conclusion: although PCR should not be used alone for the prenatal diagnosis of congenital toxoplasmosis, it is a promising method and deserves more studies to improve its efficacy.

O Dot-ELISA foi padronizado e avaliado para o imunodiagnóstico da toxoplasmose humana. Entre 538 amostras testadas pela reação de Imunofluorescência Indireta (teste de referência) 183 foram positivas considerando-se os soros reagentes nas diluições > 1:16 (34%). Utilizando a diluição 1:256 como título discriminante para o Dot-ELISA, foram observados 192 soros positivos (36%). A comparação entre as duas técnicas mostrou os seguintes resultados: co-positividade = 0,94, co-negatividade = 0,94 e youden = 0,88. Estes resultados mostram que o Dot-ELISA pode ser aplicado no imunodiagnóstico da toxoplasmose humana utilizando o título discriminante de 1:256. Entre as principais vantagens desta técnica podemos citar sua simplicidade, leitura visual sem uso de aparelho e utilização como teste qualitativo em levantamentos epidemiológicos ou como teste quantitativo para titulação de soros.

A Dot enzyme-linked immunosorbent assay (Dot-ELISA) was standardized and evaluated for the serodiagnosis of human toxoplasmosis. Out of 538 serum samples tested by the immunofluorescence test for toxoplasmosis (IFAT-IgG) as reference test, 183 (34%) were positive at cut off 1:16 and 192 (36%) were positive for Dot-ELISA-IgG at cut-off 1:256. For Dot-ELISA, co-positivity was 0.94, co-negativity 0.94 and concordance 0.88 in relation to IFAT-IgG. These results suggest the usefulness of Dot-ELISA (cut-off titer of 1:256) for the serodiagnosis of human toxoplasmosis. The main advantage of this technique is simplicity, positive test can be visually identified (colored precipitate). It does not require a special equipment and it can be used as a qualitative test to screen large numbers of samples or as a quantitative assay to determine end-point titration of individual sera.

This study evaluates whether blood collected on filter paper kept at 4 degrees C and tested at different intervals of time (1, 7, 15, 30 and 60 days after collection) would present similar results when compared to the serum samples and whether the type of filter paper influences the results. Eluates from filter paper samples were tested for Trypanosoma cruzi antibodies using indirect immunofluorescence antibody test (IFAT), indirect haemagglutination (IHA) and enzyme-linked immunosorbent assay (ELISA) as reference, the antibody titer in sera. Analysis of data showed that results obtained with IFAT, IHA (cut off point = 1:40) and ELISA in sera had similar sensitivity and good concordance among reactions. The use of a multiple linear regression model indicated that titer fall in eluates occurs up to the 7th day after the collection, and it is more marked for samples with lower antibodies titers. However, no significant differences were observed by IFAT, IHA (cut off point = 1:20) and ELISA in the proportion of positive reactions between sera and eluates. The results also showed that Melitta, Klabin or Whatman (reference) filter papers could be indicated for surveys, since they have shown similar capacity of maintenance of anti-T. cruzi immunoglobulins.