Genetic characterization of Toxoplasma gondii from human and chicken isolates from Argentina.

This study aimed to determine the nPCR-RFLP genotypes of newly obtained T. gondii isolates from human congenital toxoplasmosis cases in Argentina and to determine their allelic profiles for virulence genes ROP18/ROP5. In addition, the ROP18/ROP5 profiles were also determined for previously characterized T. gondii samples. Isolation from congenital toxoplasmosis cases was carried out in mouse bioassay from two placentas (P1 and P2). Genotyping for the new human isolates was performed by nPCR-RFLP using 10 markers. The samples analyzed for ROP18/ROP5 included the two newly obtained isolates (from the congenital toxoplasmosis cases) and nine previously genotyped T. gondii DNA samples from humans and chickens. The results for P1 and P2 named as TgHm18-02Arg and TgHm19-01Arg showed ToxoDB genotypes #14 (non-archetypal) and #2 (clonal type III), respectively. Non-archetypal #14 has been isolated from human cases before in Argentina. However, this is the first report of T. gondii clonal type III in a human case in the country. The ROP18/ROP5 combination was detected in nine samples: 3/3 (n = 1), 4/3 (n = 4), 4/4 (n = 3), and 3-4/4 (n = 1). Notably, the 4/4 profile was identified for the first time and exclusively in T. gondii samples from Misiones province (which borders southern Brazil). Further studies are required to corroborate the regionalization of the ROP18/ROP5 profiles in Argentina.

Consenso Argentino de toxoplasmosis congénita 2020 / Argentine Consensus of congenital toxoplasmosis

Resumen La transmisión vertical de la infección por Toxoplasma gondii ocurre cuando la madre se infecta por primera vez en el transcurso del embarazo. El diagnóstico de la infección materna y la del re cién nacido se logra con el conjunto de pruebas serológicas, hallazgos clínicos y ecográficos. El reconocimiento temprano de la infección materna permite un tratamiento que reduce la tasa de transmisión y el riesgo de daño en el producto de la concepción. El objetivo de este consenso de expertos fue revisar la literatura científica para actualizar las recomendaciones de práctica clínica respecto de la prevención, el diagnóstico y el tratamiento de la toxoplasmosis congénita en nuestro país.

Abstract Mother-to-child transmission in Toxoplasma gondii infection occurs only when the infection is acquired for the first time during pregnancy. Diag nosis of maternal infection and the newborn is achieved by a combination of serological tests, clinical features and ultrasound images. An early diagnosis of maternal infection allows treatment that offers a reduction both in transmission rate and risk of congenital damage. The aim of this expert consensus was to review the scientific literature which would enable an update of the clinical practice guideline of prevention, diagnosis and treatment of congenital toxoplasmosis in our country.

[Argentine consensus of congenital toxoplasmosis]. / Consenso argentino de toxoplasmosis congénita 2020.

Mother-to-child transmission in Toxoplasma gondii infection occurs only when the infection is acquired for the first time during pregnancy. Diagnosis of maternal infection and the newborn is achieved by a combination of serological tests, clinical features and ultrasound images. An early diagnosis of maternal infection allows treatment that offers a reduction both in transmission rate and risk of congenital damage. The aim of this expert consensus was to review the scientific literature which would enable an update of the clinical practice guideline of prevention, diagnosis and treatment of congenital toxoplasmosis in our country.

La transmisión vertical de la infección por Toxoplasma gondii ocurre cuando la madre se infecta por primera vez en el transcurso del embarazo. El diagnóstico de la infección materna y la del recién nacido se logra con el conjunto de pruebas serológicas, hallazgos clínicos y ecográficos. El reconocimiento temprano de la infección materna permite un tratamiento que reduce la tasa de transmisión y el riesgo de daño en el producto de la concepción. El objetivo de este consenso de expertos fue revisar la literatura científica para actualizar las recomendaciones de práctica clínica respecto de la prevención, el diagnóstico y el tratamiento de la toxoplasmosis congénita en nuestro país.

Interferon-γ and IL-10 Release Assay for Patients with Ocular Toxoplasmosis.

Peripheral blood mononuclear cells (PBMC) from patients with ocular toxoplasmosis were challenged with total antigens from Toxoplasma gondii lysate (TATL) in a cytokine release assay (CRA), run during the inactive period of the disease. Increased interferon gamma (IFN-γ) levels were detected after PBMC stimulation with either ME49 reference strain (P = 0.0015) or local TgCkAr-11-9 isolate (P = 0.0012), as compared with those recorded under basal conditions. TATL from TgCkAr11-9 isolate induced a higher release of IFN-γ than ME49 strain in CRA from all tested patients (P = 0.02). The median value of IFN-γ release on TgCkAr-11-9 stimulation (26.03 pg/mL) allowed the classification of patients into high- or low-/non-IFN-γ releasers. Clinical correlations were established with both groups. The results obtained in this study suggest the need to include local strains when performing CRA with TATL.

Evaluation of biological behavior of Toxoplasma gondii atypical isolates # 14 and # 163.

Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals, including humans. A highly diverse genetic population has been reported in Central and South America, predominating mainly atypical genotypes. Different genotypes showed different biological behavior in mice. The aim of this study was to evaluate the biological behavior of T. gondii isolates obtained from Macropus rufogriseus (TgMr) and Saimiri boliviensis (TgSb) identified as atypical genotypes # 14 and # 163, respectively. Strains RH, ME49 and VEG were used as reference for clonal types I, II and III, respectively. In vitro invasion and replication capacity assays were analyzed at 6 and 18 hpi, respectively. In vivo assay was performed in Swiss mice (n = 30) using 1 × 102 and 1 × 103 parasites/mouse as infective doses (ME49, VEG, TgMr, TgSb and negative control). Morbi-mortality and tissues PCR were assessed. Lymphoproliferation assays were performed and gamma interferon was measured by ELISA. The ME49 strain showed the highest invasion, followed by TgSb and VEG, while RH and TgMr presented the lowest invasions. The RH strain and the TgSb isolate showed more endodyogeny events (fastest doubling times) than VEG and ME49 strains and the TgMr isolate. Both atypical isolates showed high virulence (100% morbi-mortality, at 8-10 dpi) and parasite DNA was detected in all tissue samples. Splenocytes from mice inoculated with TgMr and TgSb registered the highest values of gamma interferon. An in vitro invasion-replication index was established which correlates inversely with virulence in mice. In conclusion, T. gondii atypical isolates # 14 and # 163 showed a different in vitro behavior than clonal strains, with low invasion-replication indexes but being highly virulent in mouse model.

Ovine Abortion by Neospora caninum: First Case Reported in Argentina.

PURPOSE: The aim of this study was to describe for the first time a natural case of ovine abortion associated with Neospora caninum in a flock with reproductive losses in Argentina. MATERIALS AND METHODS: The analyzed flock consisted of 256 Texel sheep, of which 134 had been mated. A single blood sample was obtained by jugular vein puncture from 220 ewes (116 adult ewes, 104 yearling ewes) and 93 lambs. Serum samples and fetal fluid were tested using the indirect fluorescence antibody test to detect antibodies against N. caninum and T. gondii. Fetal and placental tissues from aborted fetus were examined by standard gross pathology procedures and were tested using histopathology and immunohistochemistry. Moreover, DNA from fetal and placental tissues was isolated and a PCR assay to detect N. caninum, T. gondii and Chlamydia spp. was used. RESULTS AND DISCUSSION: The pregnancy rate was 89% (119/134), the abortion rate was 8.4% (10/119) whereas the perinatal mortality rate was 15% (16/109). Out of 116 adult ewes sampled, 34.48% presented anti-N. caninum antibodies. Ten ewes had aborted, and one fetus was directly submitted to the diagnostic laboratory for further processing. Antibodies against N. caninum were detected in fetal fluid and in the aborted dam. Histopathological analysis in fetal tissues showed multifocal lymphohistiocytic glossitis, diffuse mild lymphohistiocytic endocarditis, pericarditis and focally extensive myocarditis. Severe multifocal necrotizing placentitis and diffuse mild lymphohistiocytic placentitis with the presence of lymphohistiocytic vasculitis were observed in placenta. N. caninum was immunolabeled in the placenta and fetal tongue. In addition, N. caninum DNA was detected in placenta, central neural system, lung and heart of the aborted fetus. There was no evidence of other infectious abortifacients in the aborted fetus. CONCLUSION: The present study described for the first time an ovine abortion caused by N. caninum in Argentina. Further investigations at a larger scale are required to establish the role of N. caninum as an important cause of reproductive losses in sheep flocks from the region.

Toxoplasma gondii and Trichinella infections in wild boars (Sus scrofa) from Northeastern Patagonia, Argentina.

Wild boar (Sus scrofa) was introduced in many countries of the world and is recognized as carrier of many infectious diseases. Wild game meat consumption is recognized as a source of transmission of Toxoplasma gondii and Trichinella spp. The aim of the present study was to evaluate the prevalence of antibodies to T. gondii and Trichinella spp. in free-range wild boars in Northeastern Argentine Patagonia. Between 2014 and 2018, 144 blood samples and 423 muscle samples from 423 carcasses were collected. To detect T. gondii IgG, 144 sera were processed by an immunofluorescent antibody test, and to detect anti-Trichinella IgG, 125 sera and 304 muscle juice samples were processed by ELISA. Detection of first stage larvae in muscle was performed by artificial digestion. A total of 423 wild boars muscle samples were negative to Trichinella spp. by artificial digestion. Antibodies to Trichinella spp. were detected in 2.4% (3/125) of serum samples and in 1.64% (5/304) of meat juice samples. Antibodies to T. gondii infection were detected in 12.5% (18/144) of the serum samples. This is the first study to reveal the presence of antibodies to T. gondii in wild boars from Argentina. The present results suggest that consumption of raw or undercooked wild boar meat could represent a potential source risk for toxoplasmosis in humans and that Trichinella spp. is infrequent and/or that it circulates in low burdens among wild boars in Northeastern Patagonia.

Toxoplasma gondii and Neospora caninum infections in synanthropic rodents from Argentina.

Toxoplasma gondii and Neospora caninum are closely related coccidian parasites (phylum Apicomplexa). This is the first study from urban synanthropic rodent species that involved serological and molecular diagnosis of T. gondii and N. caninum infection, and genotyping of T. gondii in Argentina. A total of 127 rodent samples were trap captured: Mus musculus (n = 78), Rattus norvegicus (n = 26) and Rattus rattus (n = 23). Antibodies against T. gondii and N. caninum were detected by IFAT in 32.8% (40/122) and 0.8% (1/122) of rodent samples, respectively, demonstrating contact with these protozoans. Additionally, T. gondii DNA was detected in 3.3% (4/123) of rodent central nervous system samples and 2 samples were genotyped by multilocus nPCR-RFLP. Neospora caninum DNA was not detected by PCR. The 2 genotyped samples were type III allele for all markers except for SAG-1 (type I for Rat1Arg and type II/III for Rat2Arg) and were identified as #48 and #2 (likely) according to the allele combinations reported on Toxo DB (Toxo-DB). The results of the present study revealed a wide distribution of T. gondii and less for N. caninum, in synanthropic rats and mice in the studied area.

Toxoplasma gondii and Neospora caninum infections in synanthropic rodents from Argentina / Infecções por Toxoplasma gondii e Neospora caninum em roedores sinantrópicos da Argentina

Abstract Toxoplasma gondii and Neospora caninum are closely related coccidian parasites (phylum Apicomplexa). This is the first study from urban synanthropic rodent species that involved serological and molecular diagnosis of T. gondii and N. caninum infection, and genotyping of T. gondii in Argentina. A total of 127 rodent samples were trap captured: Mus musculus (n = 78), Rattus norvegicus (n = 26) and Rattus rattus (n = 23). Antibodies against T. gondii and N. caninum were detected by IFAT in 32.8% (40/122) and 0.8% (1/122) of rodent samples, respectively, demonstrating contact with these protozoans. Additionally, T. gondii DNA was detected in 3.3% (4/123) of rodent central nervous system samples and 2 samples were genotyped by multilocus nPCR-RFLP. Neospora caninum DNA was not detected by PCR. The 2 genotyped samples were type III allele for all markers except for SAG-1 (type I for Rat1Arg and type II/III for Rat2Arg) and were identified as #48 and #2 (likely) according to the allele combinations reported on Toxo DB (Toxo-DB). The results of the present study revealed a wide distribution of T. gondii and less for N. caninum, in synanthropic rats and mice in the studied area.

Resumo Toxoplasma gondii e Neospora caninum são parasitas coccídeos intimamente relacionados (filo Apicomplexa). Este é o primeiro estudo de espécies de roedores sinantrópicos urbanos, o qual envolveu diagnósticos sorológicos e moleculares da infecção por T. gondii e N. caninum e genotipagem de T. gondii na Argentina. Um total de 127 amostras de roedores foram obtidas: Mus musculus (n = 78), Rattus norvegicus (n = 26) e Rattus rattus (n = 23). Anticorpos contra T. gondii e N. caninum foram detectados pela IFAT em 32,8% (40/122) e 0,8% (1/122) das amostras de roedores, respectivamente, demonstrando contato com esses protozoários. Adicionalmente, o DNA de T. gondii foi detectado em 3,3% (4/123) das amostras do sistema nervoso central de roedores e duas amostras foram genotipadas por nPCR-RFLP multilocus. O DNA de N. caninum não foi detectado por PCR. As 2 amostras genotipadas eram do tipo III para todos os marcadores, exceto para SAG-1 (tipo I para Rat1Arg e tipo II / III para Rat2Arg) e foram identificadas como # 48 e # 2 (provavelmente) de acordo com as combinações de alelos relatadas no Toxo DB (Toxo-DB). Os resultados do presente estudo indicam uma ampla distribuição de T. gondii e menor para N. caninum , em ratos e camundongos sinantrópicos na área estudada.

Congenital human toxoplasmosis caused by non-clonal Toxoplasma gondii genotypes in Argentina.

Toxoplasmosis, a worldwide distributed zoonosis, can be transmitted congenitally affecting fetuses and developing variable clinical signs. Different Toxoplasma gondii genotypes and infective dose are related factors with different clinical manifestations. Several studies indicate that atypical strains could produce more severe clinical manifestations compared to typical strains. Umbilical cord blood (n = 37) and placenta (n = 19) were collected at birth from women with acute T. gondii infection and processed for isolation by mice bioassay. Six isolates were obtained and identified as TgHm14-4Arg, TgHm15-02Arg, TgHm16-01Arg, TgHm16-02Arg, TgHm17-01Arg and TgHm17-02Arg. Three genotypes described previously on Toxo-DB were identified: #138 identified in chickens from Brazil, #182 isolated from eared doves from Brazil, #14 from wallaby kangaroos and chickens from Argentina, chickens from Brazil, Colombia, Chile and Venezuela, cats and dogs from Brazil and Colombia and also coyotes from USA indicating worldwide distribution of these genotypes. Two new allele combinations were obtained showing high genotypes diversity in Argentina. Four of the isolates (TgHm14-4Arg, TgHm15-02Arg, TgHm16-01Arg, TgHm16-02Arg) and two of them (TgHm17-01Arg, TgHm17-02Arg) produced chronic and acute infections in mice, respectively. Until now, seven T. gondii isolates have been obtained from humans in Argentina, and all were atypical or non-clonal genotypes. The identification of atypical strains causing congenital toxoplasmosis and circulating in our region, make important to perform the serological screenings according Argentine Consensus of Toxoplasmosis and to apply and monitoring treatments earlier in pregnancy. To achieve this aim, it is necessary to inform general population about T. gondii infection, diagnostics and control measures. These results should serve to generate awareness about congenital toxoplasmosis in South America.

Population structure of Toxoplasma gondii in Argentina.

The protozoan Toxoplasma gondii is worldwide distributed showing a particular population structure that may differ among continents and countries. The aim of this study was to analyze the T. gondii population structure in Argentina and compare it with genotyping information from other South American countries. For the analysis, 39 samples from Argentina (isolates from the provinces of Buenos Aires, Misiones, Entre Ríos and San Luis) were genotyped using 10 multilocus PCR-RFLP markers including SAG1, SAG2 (5'-3'SAG2, alt. SAG2), SAG3, BTUB, GRA6, C22-8, C29-2, L358, PK1, and Apico. The T. gondii DNA samples were obtained from domestics animals (chickens n = 20; cats n = 3; pigs n = 2; goat n = 1; rabbit n = 1), humans (n = 6), zoo animals (n = 5) and a rat (n = 1). Phylogenetic relationship of these Argentinean isolates together with representative reference genotypes was determined by phylogenetic network analysis. Thirty-seven Argentinean samples belonged to 21 genotypes and two samples were genotyped at 8 of the 10 loci and considered incomplete characterized. Among these 37 typed samples, five genotypes were not previously reported. The majority of the samples grouped with the Type III (ToxoDB PCR-RFLP genotype #2) lineage. The clonal Type II (ToxoDB genotypes #1 and #3) was also identified. Our results suggest a unique population structure with combination of unique genotypes and the common Type II and Type III lineages in Argentina. Nevertheless, different regions showed distinctive pattern of genotypes, revealing a higher variability in Northern provinces.

Prevalência de anticorpos e fatores de risco associados à infecção por Toxoplasma gondii em galinhas domésticas da zona rural de Santa Maria, Rio Grande do Sul / Prevalence of antibodies and risk factors associated with infection by Toxoplasma gondii in free range chickens of rural area of Santa Maria, Rio Grande do Sul, Brazil

RESUMO: Toxoplasma gondii é um protozoário apicomplexa que infecta animais de sangue quente, podendo ser considerado um dos principais parasitas capazes de infectar os seres humanos. Galinhas domésticas podem ser facilmente infectadas por protozoários, uma vez que estas podem ingerir oocistos encontrados no solo, sendo consideradas boas indicadoras de contaminação ambiental por T. gondii. O objetivo deste estudo foi determinar a presença de anticorpos anti-T. gondii em galinhas domésticas criadas extensivamente e avaliar os fatores de risco associados ao protozoário, na zona rural de Santa Maria, RS, Brasil. No período de março de 2013 a fevereiro 2014 foram coletadas 597 amostras de sangue de galinhas domésticas em 74 propriedades, oriundas de nove estratos que representam cada distrito da zona rural. Para avaliar os fatores de risco, nessas propriedades foi aplicado um questionário epidemiológico aos moradores. As amostras de soro foram testadas por imunofluorescência indireta, e 49,2% (294/597) foram positivas para anticorpos anti-T. gondii, com títulos variando de 16 a 4096. Das 74 propriedades analisadas, em 63 (85,1%) houve relatos que os gatos têm acesso ao deposito de alimentos, com associação significativa quando associado à presença de galinhas positivas (p=0,04) e o OR de 4,07. A variável "abate de animais" (aves e bovinos), em 51 (68,9%) das propriedades foi relatado o abate de bovinos e aves na propriedade, com valor de p significativo (p=0,05). A maioria das propriedades 59 (79,7%) foi relatada a presença de gatos domésticos, o que poderia estar associada com a alta soroprevalência encontrada em galinhas e a taxa de contaminação ambiental. A elevada prevalência de anticorpos encontrada neste estudo, além da alta frequência de propriedades com casos positivos, sugere uma grande contaminação ambiental nos distritos pesquisados, sendo assim um risco potencial para a saúde humana e animal.

ABSTRACT: Toxoplasma gondii is an apicomplex protozoan that infects warm-blooded animals and can be considered a major parasite capable of infecting humans. Domestic chickens can be easily infected by protozoa, since they can ingest oocysts found in the soil and are considered good indicators of environmental contamination by T. gondii. The aim of this study was to determine the presence of anti-T. gondii antibodies in free range chickens and to evaluate the risks factors associated with the protozoan in rural area of Santa Maria, RS, Brazil. From March 2013 to February 2014, 597 blood samples from domestic chickens were collected from 74 farms, from nine layers representing each district in the rural area. To evaluate the risk factors, in these farms an epidemiological questionnaire was applied to the residents. Serum samples were tested by indirect immunofluorescence and 49.2% (294/597) were positive for anti-T.gondii antibodies, with titres varying from 16 to 4096. Of the 74 analyzed farms, 63 (85.1%) reported that cats had access to food deposits, with a significant association when positive chickens were present (p = 0.04) and the OR of 4.07. The variable "slaughter of animals" (poultry and cattle) in 51 (68.9%) of the farms was reported the slaughter of cattle and birds in the farm, with significant p value (p = 0.05). Most farms 59 (79.7%) reported the presence of domestic cats, which could be associated with the high seroprevalence found in chickens and the rate of environmental contamination. The high prevalence of antibodies found in this study, in addition to the high frequency of farms with positive cases, suggests a great environmental contamination in the studied districts, thus being a potential risk to human and animal health.

[Congenital toxoplasmosis: Serology, PCR, parasite isolation and molecular characterization of Toxoplasma gondii]. / Toxoplasmosis congénita: Diagnóstico serológico, RPC, aislamiento y caracterización molecular de Toxoplasma gondii.

BACKGROUND: Congenital toxoplasmosis diagnosis in the newborn is a very important issue due to the need for early treatment to prevent future sequels. Aim To compare available methods at the institution for the diagnosis of congenital toxoplasmosis. Material and Methods In this study we have evaluated the different diagnostic tests used in 67 congenital exposed newborns, including serological tests, PCR, parasite isolation and molecular characterization. Results The ISAGA IgM and IgA tests showed sensitivity (Se) of 87 and 91%, respectively, and specificity (Sp) of 100%. When ISAGA IgM and IgA were performed simultaneously, the Se increased to 98% and the Sp was 100%. The presence of IgE contributed to the diagnosis when it was detected in the child's serum but not in maternal blood. In four congenital infected children the parasite was isolated and genotyped: one was genotype II and the other three were "atypical" genotypes. No parasite was isolated in children without congenital toxoplasmosis. Discussion Overall, serological tests showed a good diagnostic performance although in one case they were all negative and isolation was the only tool to identify the infection. We conclude that it is essential to use all diagnostic tests in every single exposed child, including if possible, molecular characterization due to its epidemiological implication.

Evaluation of frequency of antibodies against Toxoplasma gondii,Neospora caninum and Sarcocystis spp. and transmission routes in sheep from Humid Pampa, Argentina.

The aim of this study was to describe the frequency of ovine specific antibodies to Toxoplasma gondii, Neospora caninum and Sarcocystis spp. and to estimate different transmission routes of these infections. One hundred and thirty Texel sheep and their 117 Texel lambs were included in the study. Serum samples were tested for antibodies to T. gondii, N. caninum and Sarcocystis spp. using IFAT. Toxoplasma gondii seroprevalence was 10.00% in sheep (IC95%: 4.80-15.20%), being higher in adult sheep (≥12 year) than in younger sheep (OR 1.30; 95% CI, 1.10-1.50). N. caninum and Sarcocystis spp. seroprevalences were 1.54% (IC95%: 0.00-5.70) and 72.09% (IC95%: 67.70-82.70), respectively, with no association between age and seropositivity in sheep (P>0.05). T. gondii seroprevalence in lambs was 4.27% (IC95%: 0.61-7.94). No association between T. gondii serological status in sheep and their lambs was detected (P = 0.07). Two T. gondii and Sarcocystis spp. seropositive lambs were euthanized and T. gondii and Sarcocystis spp. DNA was detected by PCR in their tissues. In conclusion, the increase of T. gondii seropositivity in relationship with sheep age and the lack of association between sheep-lamb serological status, suggest that horizontal infection is the main transmission route in this flock as reported before. Due to the low number of N. caninum-seropositive ewes no assumptions can be done about the impact of this parasite in this flock. According with previous reports, the main transmission route for Sarcocystis spp. in this species in the present study was horizontal.

Fatal canine encephalitozoonosis in Latin America, first report.

Encephalitozoon cuniculi is an obligate, intracellular microsporidian organism capable of establish infection in a wide variety of animals. In carnivores it may cause a sporadic, severe disease in the first few months of life, which usually culminates with the death of the animal. The objective of this study was to report a natural fatal case of encephalitozoonosis in a puppy from Argentina. Clinical signs included reduced appetite, depression, vocalizing, weight loss, weakness, convulsions and recumbency. No significant gross lesions were noticed at necropsy. Microscopically, severe, diffuse, lymphocytic encephalitis was seen. Large cytoplasmic vacuoles containing spores, morphologically compatible with E. cuniculi, were present within endothelial cells of brain and kidney, in renal tubular epithelium and hepatocytes. Encephalitozoon cuniculi DNA was detected by PCR in the kidney. Antibody titers to E. cuniculi in serum from the surviving puppies and the dam were ≥1:200. This report contributes to our understanding of neurologic disease in puppies. Encephalitozoonosis should be considered as a differential diagnosis in cases of fatal encephalitis in puppies.

Toxoplasmosis congénita: Diagnóstico serológico, RPC, aislamiento y caracterización molecular de Toxoplasma gondii / Congenital toxoplasmosis: Serology, PCR, parasite isolation and molecular characterization of Toxoplasma gondii

Resumen Introducción El diagnóstico de toxoplasmosis congénita (TC) en el recién nacido es muy importante porque debe recibir tratamiento siempre, sintomático o no, para evitar o aminorar las secuelas de la enfermedad. Objetivo Evaluación comparativa de los métodos disponibles en la institución para el diagnóstico de TC. Materiales y Métodos Se evaluaron métodos diagnósticos en 67 niños cuyas madres cursaron toxoplasmosis aguda durante el embarazo. Se utilizó la técnica de Sabin Feldman para IgG al nacimiento y durante el seguimiento serológico hasta el año de vida. Para determinar IgM, IgA e IgE se utilizó la técnica immunosorbent agglutination assay (ISAGA). El diagnóstico directo se realizó por reacción de polimerasa en cadena (RPC), aislamiento y caracterización molecular del parásito. Resultados La sensibilidad (S) de ISAGA IgM fue 87%, ISAGA IgA 91% y la especificidad (E) fue 100% para ambas; cuando se realizaron en conjunto, la S aumentó a 98%. La detección de IgE contribuyó al diagnóstico cuando se la detectó sólo en la sangre del neonato y no en sangre materna. Se aisló el parásito en cuatro casos de TC, uno fue genotipo II y los otros tres, genotipos "atípicos". La S del aislamiento fue 80% y la E 100%. Conclusión Los métodos serológicos utilizados mostraron una buena eficacia diagnóstica. Un caso fue detectado sólo por el aislamiento y la caracterización molecular tiene gran valor epidemiológico.

Background. Congenital toxoplasmosis diagnosis in the newborn is a very important issue due to the need for early treatment to prevent future sequels. Aim To compare available methods at the institution for the diagnosis of congenital toxoplasmosis. Material and Methods In this study we have evaluated the different diagnostic tests used in 67 congenital exposed newborns, including serological tests, PCR, parasite isolation and molecular characterization. Results The ISAGA IgM and IgA tests showed sensitivity (Se) of 87 and 91%, respectively, and specificity (Sp) of 100%. When ISAGA IgM and IgA were performed simultaneously, the Se increased to 98% and the Sp was 100%. The presence of IgE contributed to the diagnosis when it was detected in the child's serum but not in maternal blood. In four congenital infected children the parasite was isolated and genotyped: one was genotype II and the other three were "atypical" genotypes. No parasite was isolated in children without congenital toxoplasmosis. Discussion Overall, serological tests showed a good diagnostic performance although in one case they were all negative and isolation was the only tool to identify the infection. We conclude that it is essential to use all diagnostic tests in every single exposed child, including if possible, molecular characterization due to its epidemiological implication.

Seroprevalence of Toxoplasma gondii and Neospora caninum infections in goats from two Argentinean provinces.

The aim of this study was to compare the seroprevalence of Toxoplasma gondii and Neospora caninum in goats from two Argentinean provinces raised under different management conditions. A total of 2922 serum samples from adult goats of Córdoba (n=2187) and Buenos Aires provinces (n= 735), Argentina, were assayed by indirect fluorescence antibody test (IFAT, cut-off 1:100) for antibodies to T. gondii and N. caninum. Seroprevalence was 40.8% (CI 39.0%-42.6%) and 5.5% (CI 4.7%-6.4%) for T. gondii and N. caninum, respectively. The seropositivity for both infections was higher in goats from dairy farms, resulting as follows: for T. gondii 32.7% (CI 30.8%-34.8%) in extensive farms and 59.3% (CI 56.1%-62.6%) in dairy farms and for N. caninum 4.1% (CI 3.2%-4.9%) in extensive farms and 8.8% (CI 6.9%-10.7%) in dairy farms. This is the first extensive seroepidemiology investigation for T. gondii and N. caninum in goats in Argentina.

Sarcocystis masoni, n. sp. (Apicomplexa: Sarcocystidae), and redescription of Sarcocystis aucheniae from llama (Lama glama), guanaco (Lama guanicoe) and alpaca (Vicugna pacos).

There is considerable confusion concerning the species of Sarcocystis in South American camelids (SAC). Several species names have been used; however, proper descriptions are lacking. In the present paper, we redescribe the macroscopic sarcocyst forming Sarcocystis aucheniae and describe and propose a new name, Sarcocystis masoni for the microscopic sarcocyst forming species. Muscles samples were obtained from llamas (Lama glama) and guanacos (Lama guanicoe) from Argentina and from alpacas (Vicugna pacos) and llamas from Peru. Individual sarcocysts were processed by optical and electron microscopy, and molecular studies. Microscopic sarcocysts of S. masoni were up to 800 µm long and 35-95 µm wide, the sarcocyst wall was 2·5-3·5 µm thick, and had conical to cylindrical villar protrusions (vp) with several microtubules. Each vp had 11 or more rows of knob-like projections. Seven 18S rRNA gene sequences obtained from sarcocysts revealed 95-96% identity with other Sarcocystis spp. sequences reported in the GenBank. Sarcocysts of S. aucheniae were macroscopic, up to 1·2 cm long and surrounded by a dense and laminar 50 µm thick secondary cyst wall. The sarcocyst wall was up to 10 µm thick, and had branched vp, appearing like cauliflower. Comparison of the 11 sequences obtained from individual macroscopic cysts evidenced a 98-99% of sequence homology with other S. aucheniae sequences. In conclusion, 2 morphologically and molecularly different Sarcocystis species, S. masoni (microscopic cysts) and S. aucheniae (macroscopic cysts), were identified affecting different SAC from Argentina and Peru.

Toxoplasma gondii isolates from chickens in an area with human toxoplasmic retinochoroiditis.

The aim of this study was to detect, isolate and genetically characterize Toxoplasma gondii from tissues obtained from free range chickens which were breed in farms from patients with toxoplasmic retinochoroiditis in Misiones, Argentina. Thirty three samples of head (refrigerated = 18 and frozen = 15) from free range chickens were processed. Refrigerated (n = 18) chicken central nervous systems (CNS) were bioassay in mice. DNA was obtained from all samples (n = 33) and PCR was performed using TOX5-TOX8 T. gondii specific primers. Positive PCR samples were characterized by nested-PCR and restriction fragment length polymorphism using the markers SAG2, BTUB, GRA6, SAG3, PK1, L358, C22-8, C29-2 and Apico. T. gondii DNA was amplified in 30.3% (10/33) of CNS samples. Isolates were obtained in 27.7% (5/18) of inoculated CNS samples (TgCk11-9Arg, TgCk13-5Arg, TgCk14-5Arg, TgCk14-6Arg and TgCk14-7Arg). Seven samples showed a restriction pattern to all markers and were identified as atypical with several alleles type III. Genotyping of T. gondii from samples of patients with retinochoroiditis in the same area could improve the understanding of the epidemiology of toxoplasmosis in the region.

First isolation and molecular characterization of Toxoplasma gondii from a human placenta in Argentina.

Blood sample and placenta were taken from a 37-week pregnant woman; serologic results indicated acute toxoplasmosis. Placenta was inoculated into mice. Seropositive mice were sacrificed and tissue cysts from brain were inoculated into new mice. Specific DNA was detected by PCR, and the isolate was characterized as Type II by nPCR-RFLP for nSAG2, SAG3, BTUB, GRA6, c29-2, c22-8, L358, PK1 and Apico markers. This is the first isolation and molecular characterization of Toxoplasma gondii from humans in Argentina.