How to improve the early diagnosis of Trypanosoma cruzi infection: relationship between validated conventional diagnosis and quantitative DNA amplification in congenitally infected children.

BACKGROUND: According to the Chagas congenital transmission guides, the diagnosis of infants, born to Trypanosoma cruzi infected mothers, relies on the detection of parasites by INP micromethod, and/or the persistence of T. cruzi specific antibody titers at 10-12 months of age. METHODOLOGY AND PRINCIPAL FINDINGS: Parasitemia levels were quantified by PCR in T. cruzi-infected children, grouped according to the results of one-year follow-up diagnosis: A) Neonates that were diagnosed in the first month after delivery by microscopic blood examination (INP micromethod) (n = 19) had a median parasitemia of 1,700 Pe/mL (equivalent amounts of parasite DNA per mL); B) Infants that required a second parasitological diagnosis at six months of age (n = 10) showed a median parasitemia of around 20 Pe/mL and 500 Pe/mL at 1 and 6 months old, respectively, and C) babies with undetectable parasitemia by three blood microscopic observations but diagnosed by specific anti - T. cruzi serology at around 1 year old, (n = 22), exhibited a parasitemia of around 5 Pe/mL, 800 Pe/mL and 20 Pe/mL 1, 6 and 12 month after delivery, respectively. T. cruzi parasites were isolated by hemoculture from 19 congenitally infected children, 18 of which were genotypified as DTU TcV, (former lineage TcIId) and only one as TcI. SIGNIFICANCE: This report is the first to quantify parasitemia levels in more than 50 children congenitally infected with T. cruzi, at three different diagnostic controls during one-year follow-up after delivery. Our results show that the parasite burden in some children (22 out of 51) is below the detection limit of the INP micromethod. As the current trypanocidal treatment proved to be very effective to cure T. cruzi - infected children, more sensitive parasitological methods should be developed to assure an early T. cruzi congenital diagnosis.

Evaluation of the ELISA-F29 test as an early marker of therapeutic efficacy in adults with chronic Chagas disease.

This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.

Evaluation of the ELISA-F29 test as an early marker of therapeutic efficacy in adults with chronic Chagas disease / Avaliação do tratamento tripanossomicida em coorte de adultos chagásicos crônicos através de técnicas sorológicas convencionais e ELISA-F29

This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.

Este trabalho comparou os tempos de soroconversão negativos obtidos pela sorologia convencional (CS) e teste ELISA-F29 em uma coorte de pacientes chagásicos crônicos tratados com nifurtimox ou benznidazol. Um estudo retrospectivo foi realizado com soro preservado de 66 adultos chagásicos assintomáticos com acompanhamento clínico e sorológico semestral ao longo de um seguimento médio de 23 anos. 29 pacientes receberam tratamento tripanossomicida e 37 outras permaneceram sem tratamento. O teste ELISA-F29 usou um antígeno recombinante obtido por expressão do gene de uma proteína flagelar de Trypanosoma cruzi de ligação de cálcio em Escherichia coli. Entre os pacientes não tratados, 36 mantiveram os títulos da CS. Um paciente apresentou sorologia duvidosa em alguns controles. ELISA-F29 apresentou reatividade constante em 35/37 e foi negativo no paciente com CS flutuante. Os pacientes tratados foram agrupados de acordo com os títulos da CS, em três grupos: 13 tornaram-se negativos, 12 diminuíram e quatro permaneceram inalterados. ELISA-F29 foi negativo nos dois primeiros grupos. O tempo de negativização foi significativamente menor para o teste ELISA-F29 do que para CS (14,5 ± 5,7 e 22 ± 4,9 anos, respectivamente). A soroconversão negativa foi observada somente nos pacientes tratados. Os resultados obtidos confirmam que o teste ELISA-F29 é útil como um indicador precoce de soronegativação em pacientes crônicos tratados.

Vertical transmission of Trypanosoma cruzi infection: quantification of parasite burden in mothers and their children by parasite DNA amplification.

The relationship between parasite burden and vertical transmission of Trypanosoma cruzi was studied in pairs of chronically infected women and their children in a non-endemic area. Parasitemia was quantified by quantitative polymerase chain reaction (qPCR) in the peripheral blood amplifying a nuclear T. cruzi DNA and expressed as equivalent amounts of CL Brener parasites DNA per ml (eP/ml). Similar levels of parasitemia were found in non-transmitting pregnant women and in non-pregnant women: 1.8 ± 0.5 and 1.5 ± 0.7 eP/ml, respectively. In women pregnant with infected children parasitemia was 11.0 ± 2.7 eP/ml (n=20). In 12 of their neonates the infection was detected by microscopic observation of the parasites in peripheral blood in the 1(st) month of age. These children had variable levels of parasitemia (13,000 ± 7000 eP/ml), that were about 600-fold higher than that found in their mothers. To our knowledge, this is the first quantitative evaluation of parasitemia in these three groups of women and in their congenitally infected children. These parasite quantifications could be a basis to plan the control of mother-to-child transmission of T. cruzi.

Leishmaniasis visceral: contribución a su control mediante el fortalecimiento del diagnóstico del reservorio canino en Posadas, Misiones, Argentina / Visceral leishmaniasis: control contribution by strengthening the diagnosis of dog reservoir in the city of Posadas, Misiones, Argentina

INTRODUCCION: La leishmaniasis visceral (LV) es una enfermedad emergente en Argentina. En 2006 se describió el primer caso autóctono en Posadas (Misiones), ciudad que se convirtió en epicentro de la LV.OBJETIVO: Determinar la prevalencia de la LV canica (LVC) y fortalecer su diagnóstico en Posadas.METODOS: Con un criterio entomológico, la ciudad de Posadas se dividió en islas de alta densidad (IAD) e islas de baja densidad (IBD) de flebótomos. Se seleccionó, de forma arbitraria y mediante imágenes satelitales, la manzana situada más al norte y más al sur de cada IAD e IBD. Se realizó un diseño observacional prospectivo, y se estudió a todos los perros de las manzanas seleccionadas. Se consideró un N inicial de 400 animales para detectar una diferencia de al menos 7% con una potencia de 0,90 y un nivel de 0,05 respecto de la prevalencia global supuesta del 20%. Se tomaron muestras de sangre, punción de ganglios linfáticos y raspado de piel. El estudio incluyó además un test serológico rápido (rK39 canino) y frotis de las muestras para búsqueda parasitológica, que se procesaron y evaluaron en el Instituto Municipal de Salud Animal de Posadas y en el Instituto Nacional de Parasitología Fatala Chabén.RESULTADOS: De los 182 animales evaluados, 33 fueron positivos para LVC, con una prevalencia global del 18,1%. En IAD la prevalencia fue 22% y en IBD, 13,4%. Los estudios parasitológicos fueron positivos en el 10% de los caninos en IAD y 1,2% en IBD. La LVC fue asintomática u oligosintomática, con una presentación más frecuente en IAD. La rK39 tuvo una sensibilidad del 90,3% y una especificidad del 98,9%.CONCLUSIONES: El análisis serológico con antígeno rK39 fue siempre más sensible que la búsqueda parasitológica para el diagnóstico. La LVC parece tener menor prevalencia en relación con estudios previos, quizás debido a diferentes medidas de control realizadas en el Municipio de Posadas.

INTRODUCCION: Visceral leishmanisis (VL) is an emerging disease in Argentina. The first autochthonous case was described in 2006 in a chld from Posadas (Misiones), the city which turned out to be the focus of VL.OBJECTIVE: To determine the prevalence of canine VL (CVL) and improve its diagnosis in Posadas.METHODS: According to entomological criteria, Posadas was divided in high density islands (HDI) and low density islands (LDI) of sandfly. Randomly and with satellite images, the northernmost and southernmost blocks of each HDI and LDI were selected. A prospective, experimental design was performed, studying all the dogs in the selected blocks. There was an initial sample of 400 animals to detect a difference of at least 7%, with a power of 0.90 and a level of 0.05, compared to the supposed prevalence of 20%. Blood samples were taken, with puncture of lymph nodes and skin scraping. The study also included a (canine rK39) rapid test for serological assessment adn sample smears for parasitological search. Samples were processed and evaluated in the Posadas institute of animal health (I.MU.S.A) and the Fatala Chabén National Institute of Parasitology.RESULTS: CVL was assessed in 33/182 animals with an overall prevalence of 18.1%. In HDI, the prevalence was 22%; in LDI, it was 13.4%. Parasitological studies were positive in 10% (HDI) and 1.2% (LDI). The CVL had a nonspecific clinical presentation; canines were asimptomatic or oligosymptomatic, with a higher trend in HDI. The rK39 had a sensitivity of 90.3% and a specificity of 98.9%.CONCLUSIONS: The antigen rK39 was always more sensitive than the search for parasites for the diagnosis of CVL. The disease appears to be less prevalent comapring to previous studies, perhaps due to different control measures performed in the Municipality of Posadas.

Leishmaniasis visceral: contribución a su control mediante el fortalecimiento del diagnóstico del reservorio canino en Posadas, Misiones, Argentina / Visceral leishmaniasis: control contribution by strengthening the diagnosis of dog reservoir in the city of Posadas, Misiones, Argentina

INTRODUCCION: La leishmaniasis visceral (LV) es una enfermedad emergente en Argentina. En 2006 se describió el primer caso autóctono en Posadas (Misiones), ciudad que se convirtió en epicentro de la LV.OBJETIVO: Determinar la prevalencia de la LV canica (LVC) y fortalecer su diagnóstico en Posadas.METODOS: Con un criterio entomológico, la ciudad de Posadas se dividió en islas de alta densidad (IAD) e islas de baja densidad (IBD) de flebótomos. Se seleccionó, de forma arbitraria y mediante imágenes satelitales, la manzana situada más al norte y más al sur de cada IAD e IBD. Se realizó un diseño observacional prospectivo, y se estudió a todos los perros de las manzanas seleccionadas. Se consideró un N inicial de 400 animales para detectar una diferencia de al menos 7% con una potencia de 0,90 y un nivel de 0,05 respecto de la prevalencia global supuesta del 20%. Se tomaron muestras de sangre, punción de ganglios linfáticos y raspado de piel. El estudio incluyó además un test serológico rápido (rK39 canino) y frotis de las muestras para búsqueda parasitológica, que se procesaron y evaluaron en el Instituto Municipal de Salud Animal de Posadas y en el Instituto Nacional de Parasitología Fatala Chabén.RESULTADOS: De los 182 animales evaluados, 33 fueron positivos para LVC, con una prevalencia global del 18,1%. En IAD la prevalencia fue 22% y en IBD, 13,4%. Los estudios parasitológicos fueron positivos en el 10% de los caninos en IAD y 1,2% en IBD. La LVC fue asintomática u oligosintomática, con una presentación más frecuente en IAD. La rK39 tuvo una sensibilidad del 90,3% y una especificidad del 98,9%.CONCLUSIONES: El análisis serológico con antígeno rK39 fue siempre más sensible que la búsqueda parasitológica para el diagnóstico. La LVC parece tener menor prevalencia en relación con estudios previos, quizás debido a diferentes medidas de control realizadas en el Municipio de Posadas.

INTRODUCCION: Visceral leishmanisis (VL) is an emerging disease in Argentina. The first autochthonous case was described in 2006 in a chld from Posadas (Misiones), the city which turned out to be the focus of VL.OBJECTIVE: To determine the prevalence of canine VL (CVL) and improve its diagnosis in Posadas.METHODS: According to entomological criteria, Posadas was divided in high density islands (HDI) and low density islands (LDI) of sandfly. Randomly and with satellite images, the northernmost and southernmost blocks of each HDI and LDI were selected. A prospective, experimental design was performed, studying all the dogs in the selected blocks. There was an initial sample of 400 animals to detect a difference of at least 7%, with a power of 0.90 and a level of 0.05, compared to the supposed prevalence of 20%. Blood samples were taken, with puncture of lymph nodes and skin scraping. The study also included a (canine rK39) rapid test for serological assessment adn sample smears for parasitological search. Samples were processed and evaluated in the Posadas institute of animal health (I.MU.S.A) and the Fatala Chabén National Institute of Parasitology.RESULTS: CVL was assessed in 33/182 animals with an overall prevalence of 18.1%. In HDI, the prevalence was 22%; in LDI, it was 13.4%. Parasitological studies were positive in 10% (HDI) and 1.2% (LDI). The CVL had a nonspecific clinical presentation; canines were asimptomatic or oligosymptomatic, with a higher trend in HDI. The rK39 had a sensitivity of 90.3% and a specificity of 98.9%.CONCLUSIONS: The antigen rK39 was always more sensitive than the search for parasites for the diagnosis of CVL. The disease appears to be less prevalent comapring to previous studies, perhaps due to different control measures performed in the Municipality of Posadas.

Identification, cloning and characterization of an aldo-keto reductase from Trypanosoma cruzi with quinone oxido-reductase activity.

Drugs currently used for treatment of Trypanosoma cruzi infection, the ethiological agent of Chagas' disease, have shown side effects and variable efficiency. With the aim to describe parasite enzymes involved in the mechanisms of action of trypanocidal drugs and since it has been reported that reductases are crucial in their metabolism, we attempted to identify novel NADPH-dependent oxido-reductases from T. cruzi. The percolation of a soluble fraction of epimastigote lysates through a Cibacron Blue-Sepharose column followed by elution by NADPH yielded a predominant protein with an apparent molecular weight of 32 kDa. This protein was identified by MALDI-TOF as an aldo-keto reductase (AKR) and hence denominated TcAKR. TcAKR was mainly localized in the cytosol and was also present in trypomastigote and amastigote lysates. The recombinant TcAKR (recTcAKR) showed NADPH-dependent reductase activity with the AKR substrates 4-nitrobenzaldehyde and 2-dihydroxyacetone. The saturation curves for both substrates were consistent with the Michaelis-Menten model. We also tested whether recTcAKR may reduce naphthoquinones (NQ), since many of these compounds have displayed important trypanocidal activity. recTcAKR reduced o-NQ (1,2-naphthoquinone, 9,10-phenanthrenquinone and beta-lapachone) with concomitant generation of free radicals but did not exhibit affinity for p-NQ (5-hydroxy-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, alpha-lapachone and menadione). The substrate saturation curve with o-NQ fitted to a sigmoidal curve, suggesting that recTcAKR presents a cooperative behavior. In addition, three peaks assigned to monomers, dimers and tetramers were obtained when recTcAKR was submitted to a Superose 12 gel chromatography column. TcAKR is the first member of the AKR family described in T. cruzi. Our results indicate that this enzyme may participate in the mechanisms of action of trypanocidal drugs.

Benzophenone-based farnesyltransferase inhibitors with high activity against Trypanosoma cruzi.

Less toxic drugs are needed to combat the human parasite Trypanosoma cruzi (Chagas's disease). One novel target for antitrypanosomal drug design is farnesyltransferase. Several farnesyltransferase inhibitors based on the benzophenone scaffold were assayed in vitro and in vivo with the parasite. The common structural feature of all inhibitors is an amino function which can be protonated. Best in vitro activity (LC50 values 1 and 10 nM, respectively) was recorded for the R-phenylalanine derivative 4a and for the N-propylpiperazinyl derivative 2f. These inhibitors showed no cytotoxicity to cells. When tested in vivo, the survival rates of infected animals receiving the inhibitors at 7 mg/kg body weight/day were 80 and 60% at day 115 postinfection, respectively.

In vitro anti-parasitic activity of Cyclosporin A analogs on Trypanosoma cruzi.

Cyclosporin A (CsA) nonimmunosuppressive analogs were evaluated against Trypanosoma cruzi and on TcCyP19, a cyclophilin of 19 kDa. Two out of eight CsA analogs, H-7-94 and F-7-62 showed the best anti-parasitic effects on all in vitro assays. Their IC(50) values were 0.82 and 3.41 microM, respectively, compared to CsA IC(50) value 5.39 microM on epimastigote proliferation; and on trypomastigote lysis their IC(50) values were 0.97 and 2.66 microM compared to CsA IC(50) value 7.19 microM. H-7-94 and F-7-62 were also more effective than CsA in inhibiting trypomastigote infection. The enzymatic activity of TcCyP19 was inhibited by all CsA derivatives, suggesting this target is involved in the trypanocidal effects observed.

Trypanosoma cruzi: molecular identification and characterization of new members of the Tc13 family. Description of the interaction between the Tc13 antigen from Tulahuén strain and the second extracellular loop of the beta(1)-adrenergic receptor.

Trypanosoma cruzi Tc13 antigens belong to the trans-sialidase superfamily. Their sequences have been described only partially and, up to now, their physiological activity has not been elucidated. Here we present two new members of this family from the Tulahuén strain (Tc13 Tul) and the CL Brener clone (Tc13 CL), being the latter the first Tc13 sequence fully described. Alignment of all Tc13 sequences allowed us to define two sub-families that differ in the number of repeats and the presence or absence of the GPI addition site. Chromoblots demonstrate that Tc13 antigens are mainly located in chromosome III and its homologous. Pull down assays suggest that recombinant MBP-Tc13 Tul interacts with the second extracellular loop of the beta(1)-adrenergic receptor. This is the first evidence that a Tc13 antigen acts as a ligand interacting with a neurotransmitter receptor. These observations might add some light to the development of chagasic pathology.

Genoma del Trypanosoma cruzi: correlacion entre el mapa transcripcional y el cariotipo caracterizacion molecular de un antigeno de superficie de la familia TC13 / Trypanosoma cruzi genome: transcriptional mapping and karyotype correlation: molecular characterization of a surface antigen from the Tc13 family

En este trabajo se describe la realización de un mapeo del genoma del parásito causal de la enfermedad de Chagas, el Trypanosoma cruzi, hibridando una genoteca construída en cósmidos y grillada en filtros de alta densidad, utilizando como sondas clones de AND copia provenientes de una genoteca de expresión en epimastigotes. Se muestra además la correlación de secuencias superpuestas de cósmidos (contigs) con bandas cromosomales del parásito. Utilizando estas mismas genotecas generadas por el Proyecto Genoma de T. cruzi, se caracterizó un nuevo miembro de la familia Tc 13 de la superfamilia de antígenos de superficie de tripomastigotes. A partir de un clon de la cepa Tulahuén stock. Tul2, con homología con estos antígenos, se secuenció y caracterizó el gen completo en la cepa de referencia CL clon CL Brener, encontrándose además homología con diferentes ESTs, lo cual posibilitaria conocer en su totalidad a esta familia de antígenos. (AU)

Genoma del Trypanosoma cruzi: correlacion entre el mapa transcripcional y el cariotipo caracterizacion molecular de un antigeno de superficie de la familia TC13 / Trypanosoma cruzi genome: transcriptional mapping and karyotype correlation: molecular characterization of a surface antigen from the Tc13 family

En este trabajo se describe la realización de un mapeo del genoma del parásito causal de la enfermedad de Chagas, el Trypanosoma cruzi, hibridando una genoteca construída en cósmidos y grillada en filtros de alta densidad, utilizando como sondas clones de AND copia provenientes de una genoteca de expresión en epimastigotes. Se muestra además la correlación de secuencias superpuestas de cósmidos (contigs) con bandas cromosomales del parásito. Utilizando estas mismas genotecas generadas por el Proyecto Genoma de T. cruzi, se caracterizó un nuevo miembro de la familia Tc 13 de la superfamilia de antígenos de superficie de tripomastigotes. A partir de un clon de la cepa Tulahuén stock. Tul2, con homología con estos antígenos, se secuenció y caracterizó el gen completo en la cepa de referencia CL clon CL Brener, encontrándose además homología con diferentes ESTs, lo cual posibilitaria conocer en su totalidad a esta familia de antígenos.

Trypanocidal effect of SKF525A, proadifen, on different developmental forms of Trypanosoma cruzi

SKF525A, an inhibitor and inducer of cytochrome P450, was tested on different developmental stages of Trypanosoma cruzi. Growth, motility and structure of epimastigotes, motility and infectivity of trypomastigotes, and infectivity of trypomastigotes to Vero cells in culture were abolished by the drug at 10-100 muM concentration. When blood from infected mice was treated with the drug, and used to infect 8 day-old-mice, no parasites were observed at 0.6-1 mM, and all animals survived. Blood cell morphology was well preserved, and the sleeping time of pentobarbital-treated mice inoculated with the same amount of drug was not increased. The present results suggest that SKF525A or other related inhibitors of cytochrome P450 coned be tested as an additive for blood sterilization in blood banks. (AU)

Trypanocidal effect of SKF525A, proadifen, on different developmental forms of Trypanosoma cruzi

SKF525A, an inhibitor and inducer of cytochrome P450, was tested on different developmental stages of Trypanosoma cruzi. Growth, motility and structure of epimastigotes, motility and infectivity of trypomastigotes, and infectivity of trypomastigotes to Vero cells in culture were abolished by the drug at 10-100 muM concentration. When blood from infected mice was treated with the drug, and used to infect 8 day-old-mice, no parasites were observed at 0.6-1 mM, and all animals survived. Blood cell morphology was well preserved, and the sleeping time of pentobarbital-treated mice inoculated with the same amount of drug was not increased. The present results suggest that SKF525A or other related inhibitors of cytochrome P450 coned be tested as an additive for blood sterilization in blood banks.