Evaluation of an EDTA version of CATT/Trypanosoma brucei gambiense for serological screening of human blood samples.

CATT/Trypanosoma brucei gambiense, a direct card agglutination test designed for field surveys on human African trypanosomosis, is currently used with freshly collected heparinized blood samples. When testing serum samples, it has been observed earlier that, at lower sample dilutions, a complement-mediated inhibition phenomenon may cause false negative test results. This can be avoided by adding an anticomplementary agent such as di-sodium ethylenediaminetetraacetate dihydrate (EDTA) to the reaction. As the sensitivity of the blood assay might be improved in the same way, this possibility has been examined under both laboratory and field conditions, by adding EDTA to the test buffer or, as an anticoagulant, to the blood samples. The CATT-EDTA versions proved up to 7% more sensitive but also 1-2% less specific than the current test. CATT buffer supplemented with EDTA remained stable for at least 2 years at +45 degrees C.     

Cryopreservation of Trypanosoma brucei gambiense in a commercial cryomedium developed for bull semen

There have been major advances in the formulation of cryomedia for spermatozoa owing to their economic importance. In this study, the suitability of the commercial cryomedium Triladyl developed for bull semen was evaluated for the cryopreservation of Trypanosoma brucei gambiense. Cryopreservation efficacy was determined by direct counting of motile trypanosomes and by viability assessment using in vitro and in vivo methods. Culture medium containing 10% glycerol was used as the control. Trypanosomes cryopreserved in Triladyl demonstrated a higher in vitro viability than those in culture medium with 10% glycerol. Similar results were obtained in vivo in immunosuppressed Mastomys natalensis. Trypanosomes cryopreserved in Triladyl showed better growth characteristics than those in culture medium with glycerol. It can be concluded that the use of Triladyl in the cryopreservation of T. b. gambiense leads to a better survival of the trypanosomes which could lead to an improved isolation of T. b. gambiense from sleeping sickness patients.     

Evaluation of LATEX/T.b.gambiense for mass screening of Trypanosoma brucei gambiense sleeping sickness in Central Africa

We compared the Card Agglutination Test for Trypanosomiasis (CATT), which consists of lyophilized bloodstream form trypomastigotes of Trypanosoma brucei gambiense (T.b.g.) variable antigen type LiTat 1.3, with LATEX/T.b.g., which consists of a lyophilized suspension of latex particles coated with variable surface glycoproteins of T.b.g. variable antigen types LiTat 1.3, 1.5 and 1.6. This study was carried out during two mass screening surveys in 1998 in Campo, a sleeping sickness focus in Cameroon, with a low prevalence (0.3%) and in 1999 in Batangafo which belongs to the Central African focus of Ouham which has a higher prevalence (3%). In Campo, we compared the CATT performed on whole blood with the LATEX/T.b.g. on diluted blood. In Batangafo, both tests were performed on diluted blood. In all circumstances, the specificity of the LATEX/T.b.g. was higher than of CATT. The use of LATEX/T.b.g. on diluted blood instead of CATT results in an important decrease of workload and as a consequence, of costs related to parasitological examinations. In the case of Campo the workload was up to 12 times less than when using CATT 1.3 on whole blood and the cost divided by 3. In Batangafo the workload was decreased by nearly 20% with the LATEX/T.b.g. Finally, it should be noted that in Batangafo, one of the parasitologically confirmed sleeping sickness patients was negative in CATT and positive in LATEX/T.b.g. and that the reading of the test result in LATEX/T.b.g. is easier than in CATT.     

Monitoring the developmental status of Trypanosoma brucei gambiense in the tsetse fly by means of PCR analysis of anal and saliva drops

Teneral Glossina palpalis gambiensis (Diptera: Glossinidae) were infected with a culture of procyclic forms of Trypanosoma brucei gambiense using a single-bloodmeal membrane feeding technique. The infection was monitored by analysing the saliva (mature infection) and anal drop (midgut infection) of each fly at different post-infection times both by microscopic observation and polymerase chain reaction (PCR). Amplification revealed many more positive anal drops than microscopy. The monitoring showed that the installation of T. b. gambiense in Glossina took place at least 11 days after the infection and that maturation occurred after 29 days. It also reflected precisely the parasitic status of each tsetse fly as determined by the dissection, microscopic examination and PCR amplification of the midguts and salivary glands 47 days post-infection. Twice as many tsetse flies with mature salivary glands infection were revealed by PCR than by microscopic examination, but the two techniques gave exactly the same results regarding the proportion of flies with midgut infection. This study also demonstrated the ability of natural non-infective procyclic forms of T. b. gambiense, to colonise the midgut and subsequently establish in the salivary glands of G. p. gambiensis.     

Detection of Trypanosoma brucei gambiense in sleeping sickness suspects by PCR amplification of expression-site-associated genes 6 and 7

We have developed a sensitive and specific method to identify Trypanosoma brucei ssp. using PCR to amplify conserved expression-site-associated gene 6 and 7 DNA target sequences. Amplification of 10% of the DNA in a single trypanosome produced sufficient PCR product to be visible as a band in an agarose gel stained with ethidium bromide. We analysed 59 blood samples of serologically positive cases of sleeping sickness by PCR, and directed parasitological examination of tissue fluids. The PCR test detected 87% of the parasitologically positive cases, with a specificity of 97%. In 5 cases, the parasite was demonstrated by the PCR test 4-6 months prior to parasitological detection. This result shows the potential of the assay in early diagnosis of actual T. b. gambiense infections in apparently aparasitaemic sleeping sickness patients.     

Detection of Trypanosoma brucei rhodesiense in animals from sleeping sickness foci in East Africa using the serum resistance associated (SRA) gene

The human serum resistance associated (SRA) gene has been found exclusively in Trypanosoma brucei rhodesiense, allowing the unequivocal detection of this pathogen in reservoir hosts and the tsetse vector without recourse to laborious strain characterisation procedures. We investigated the presence of the SRA gene in 264 T. brucei ssp. isolates from humans, domestic animals and Glossina pallidipes from foci of human trypanosomiasis in Kenya and Uganda. The SRA gene was present in all isolates that were resistant to human serum, and absent from all serum sensitive isolates tested. Further, the gene was present in all isolates that had previously been shown to be identical to human infective trypanosomes by isoenzyme characterisation. The SRA gene was detected in isolates from cattle, sheep, pigs, dog, reedbuck, hyena and G. pallidipes from sleeping sickness foci, but was not found in Trypanosoma evansi or in Trypanosoma brucei gambiense isolates. The present study indicates that the SRA gene may be invaluable in detecting and differentiating T. brucei rhodesiense from other T. brucei ssp. in reservoir hosts and tsetse.     

Multiple-strain infections of Trypanosoma brucei across Africa

It is becoming increasingly clear that parasitic infections frequently contain multiple strains of the same parasite species. This may have important consequences for the parasite dynamics in the host and thus alter disease and transmission dynamics. In Trypanosoma brucei, the causal agent of human African trypanosomiasis (sleeping sickness), multiple-strain infections have previously been demonstrated to occur. Here, we analyzed field isolates of T. b. gambiense, T. b. rhodesiense, and T. b. brucei, isolated throughout Africa to assess the commonness of multiple-strain infections across the natural range of this parasite. Using eight highly variable microsatellite loci, we found multiple strains in 8.8% of our isolates. Due to the technical challenges of detecting multiple infections this number represents a minimum estimate and the true frequency of multiple-strain infections is likely to be higher. Multiple-strain infections occurred across the entire East-West range of the parasite. Together with previous results, these findings strongly suggest that multiple-strain infections are common for this parasite and that their consequences for epidemiology and parasite evolution should be investigated in detail.     

Trypanosoma brucei: the mechanism of remission in murine infections. A calculator simulation

A simulation (using an electronic calculator program) of the growth of Trypanosoma brucei in the mouse is presented. This suggests that remission of the infection is effected by the removal of ever increasing numbers of the organisms as the immune (antibody) response develops, rather than that a gradual build-up of antibody on each trypanosome occurs until a fatal concentration is reached, simultaneously, on all of them. The program has enabled a theoretical examination to be made of the effect, on the trypanosome growth curve, of altering the doubling time of the organism, the rate of development of the immune response, and the efficiency of the antibodies. Suggestions are made for further extensions of the program to cover other parameters that may vary during the infections. It should also be possible to use similar simple programs, that do not employ advanced mathematics, to problems of the growth of many organisms.     

Sleeping sickness in Zaire: a nested polymerase chain reaction improves the identification of Trypanosoma (Trypanozoon) brucei gambiense by specific kinetoplast DNA probes

Blood samples collected in the sleeping sickness focus of Boma, Zaire, from human patients and domestic animals were analysed by polymerase chain reaction (PCR) for the presence of trypanosome DNA. The comparison of PCR and miniature anion exchange centrifugation technique (m‐AECT) results clearly shows that in domestic animals mixed infections ( Trypanozoon/Trypanosoma [ Nannomonas ] congolense ) are more frequently diagnosed by PCR than by m‐AECT. Trypanozoon positive blood samples were further analysed for Trypanosoma ( Trypanozoon ) brucei gambiense . For that purpose amplified minicircle kinetoplast DNA (minicircle kDNA) was differentiated in gambiense and non‐ gambiense by hybridization with DNA probes. To analyse blood samples, especially those with low parasite numbers, the amplification step had to be improved by a nested PCR. Subsequent hybridiz‐ation was done with kDNA probes generated by PCR from blood samples which had been obtained from a human patient infected with T. ( T. ) b. gambiense and a pig infected with Trypanozoon . The hybridization results clearly show that at least two genotypes of Trypanozoon parasites occur in the sleeping sickness focus of Boma, Bas‐Zaire. One obviously corresponds to T. ( T. ) b. gambiense and was present in humans and two domestic animals (pig, dog). The other genotype seems to be associated with T. ( T. ) b. brucei and could be detected only in the blood of domestic animals. This is the first time that field samples could be analysed by a technique which facilitates the molecular identification of T. ( T. ) b. gambiense without prior cloning, propagation, and/or isolation of the parasites. Therefore, this technique seems to be a promising tool to elucidate the significance of the animal reservoir for the epidemiology of the gambiense sleeping sickness in Africa.     

Studies of quinapyramine-resistance of Trypanosoma brucei evansi in China

In the present article, we summarize our studies of antrycide-resistance of Trypanosoma brucei evansi in four aspects in the last recent several years, the analysis of quinapyramine-sensitive situation of T. b. evansi in China, biological characteristics of T. b. evansi population in quinapyramine-resistance and biological materials of quinapyramine-resistance in T. b. evansi population. Firstly, the correlative assays of effective dosage of quinapyramine on T. b. evansi disease between in vivo and in vitro methods showed that their relationship was parabolic with positive correlation. On the other hand, the IC₅₀ and CD₁₀₀ values of 12 T. b. evansi isolates, AHB, GDB1, GDB2, HNB, JSB1, JSB2, YNB, ZJB, GDH, GXM, HBM and XJCA, collected from buffaloes, horses, mules and camels across nine provinces of China were examined using the two methods, respectively. Among them, the nine isolates, AHB, GDB1, GDB2, HNB, JSB1, JSB2, YNB, ZJB and GDH, became quinapyramine-sensitive T. b. evansi. Secondly, T. evansi populations could rapidly obtain antrycide-resistance when they were passed through immunosuppressed mice treated with low doses of the drug. But, the replication rate of trypanosomes with antrycide-resistance decreases as the level of drug-resistance increases. Thirdly, the analysis of the HK, G6PDH, ALAT and ASAT isoenzymes showed that they were not involved in the quinapyramine-resistance of T. b. evansi. But the protein bands of 15.79 kDa and 19.76 kDa might be involved in the antrycide-resistance of T. b. evansi population. At genetic level, the gene, TbTA1, could be amplified from the T. b. evansi isolate sensitive to quinapyramine-sensitivity but the T. b. evansi isolate with quinapyramine-resistance using not only the RT-PCR technique, but also PCR technique. We used the SSH (Suppression Subtractive Hybridization) to clone highly or low expressed cDNA fragments caused by production of antrycide-resistance in T. b. evansi. The 5 low and 9 high expressed new cDNA fragments were amplified. Among them, the 3 low expressed cDNA fragments had the same sequence of 65 amino acids and the 3 high expressed cDNA fragments were located in chromosome VI, like T. brucei. Lastly, more work needs to be done in order to elucidate the mechanism of quinapyramine-resistance of T. b. evansi.     

Influence of habitat and seasonal variation on wild mammal diversity and distribution with special reference to the Trypanosoma brucei gambiense host-reservoir in Bipindi (Cameroon)

To evaluate the role of wildlife in the resurgence and perenisation of human African trypanosomiasis (HAT), we investigated the influence of habitat and seasonal variations on the diversity and spatial distribution of wild mammals, with special reference to those recognised as potential host-reservoirs of Trypanosoma brucei gambiense in Bipindi (southwestern Cameroon). To achieve this, we carried out transect surveys in four habitat types over two years. A total of 31 mammal species were recorded, of which 14 occurred in the undisturbed forest, 9 in cocoa plantations, 11 in farmlands and 11 in village-adjacent gallery forests. Among them, six species (Cephalophus monticola, Cephalophus dorsalis, Atherurus africanus, Cricetomys emini, Nandinia binotata and Cercopithecus nictitans), known as reservoir hosts of T. b. gambiense, occurred in all kinds of habitats suitable or unsuited to Glossina palpalis palpalis and in all seasons. These species are the most involved in the transmission cycle (human being/tsetse flies/wild animals). Cercopithecus cephus, Miopithecus talapoin and Heliosciurus rufobrachium host Trypanosoma brucei spp.; however, only C. cephus does not occur permanently in the suitable habitat of G. palpalis palpalis. In general, some species (C. monticola, Tragelaphus spekei and C. emini) showed a slight density increase from the long dry to the heavy rainy season within the undisturbed and farmland habitats, and a slight decrease within cocoa plantations and village-adjacent forests in the same period. The density of A. africanus increased greatly from the long dry season to the heavy rainy season in the undisturbed forest while, the density of primates in this habitat decreased slightly from the long dry season to the heavy rainy season. These variations indicate a permanent movement of wild mammal reservoir or feeding hosts from one biotope to another over the seasons. Thryonomys swinderianus needs to be investigated because it occurs permanently in the suitable habitat of G. palpalis palpalis and Potamochoerus porcus for its genetic similarities to domestic pigs, favourable feeding hosts of G. palpalis palpalis.     

Metabolism and distribution of phenanthridine trypanocides in Trypanosoma brucei

Phenanthridine trypanocides (isometamidium chloride hydrochloride, ISM, and Ethidium bromide, EBr) have been widely used to treat African trypanosomiasis in livestock for more than 40 years. Their main action is to inhibit nucleic acid synthesis in trypanosome parasites, by intercalation between the DNA base pairs. They can also linearise selectively kinetoplast DNA minicircles; a form of mitochondrial DNA unique to this group of parasites. However, the metabolism of these compounds by trypanosomes has not been reported. Indeed, it is not known whether or not their metabolism by the parasite contributes to their activity, selective toxicity for these parasites or to the development of chemoresistance. Therefore, we studied the metabolism of EBr and ISM, and their distribution in Trypanosoma brucei (TREU 927) using high performance liquid chromatography (HPLC), liquid chromatography combined with mass spectrometry (LC-MS) and confocal laser scanning microscopy (CLSM). Incubation of EBr with trypanosomes led to the formation of a small amount (0.606+/-0.191%) of one metabolite (MI). Ion chromatograms extracted from an LC-MS analysis using electrospray ionisation (ESI), showed that the difference in mass between the parent compound and its metabolite was 30. This may correspond to the addition of a hydroxyl and a methyl group. No metabolites could be detected for ISM. The distribution of the two drugs in trypanosomes was investigated by CLSM, using their intrinsic fluorescence. ISM and EBr showed differences in their distribution in trypanosomes. ISM had a greater affinity for the kinetoplast than EBr and it stained other organelles like the flagellum; in contrast the distribution of EBr was more diffuse.     

The elimination of Trypanosoma brucei gambiense sleeping sickness in the focus of Luba, Bioko Island, Equatorial Guinea

After the resurgence of sleeping sickness in Luba, Equatorial Guinea, a major campaign to control the disease was established in 1985. The campaign comprised no vector control, but intensive active and passive surveillance using serology for screening, and treatment of all parasitological and suspected serological cases. Total prevalence was used to classify villages as endemic, at risk, anecdotal and non-endemic which also allowed defining the geographic extent of the focus. Active case-finding was implemented from 1985 to 2004. The frequency of surveys was based on parasitological prevalence: twice a year during intensified control, once a year during ordinary control and once every 2 years during the control consolidation phase, when the parasitological prevalence in the whole focus fell to 0.1%. From 1985 to 1999, the indirect immunofluorescent antibody test (IFAT) was used as an initial screening tool, followed by parasitological confirmation of IFAT positive cases, and the Card Agglutination Trypanosomiasis Test (CATT) if necessary. In 2000, the IFAT was replaced by the CATT. Serum-positive individuals without parasitological confirmation were subsequently tested on serial dilution. All cases underwent lumbar puncture to determine the stage of the disease. First-stage cases were treated with pentamidine and second-stage cases with melarsoprol. A few relapses and very advanced cases were treated with eflornithine. The last sleeping sickness case was identified and treated in 1995.     

Variables that modulate the spatial distribution of Trypanosoma cruzi and Trypanosoma evansi in the Brazilian Pantanal

An evaluation was made on how the landscape and cattle ranching affect the transmission cycles and the patterns of tripanosomatid infection (Trypanosoma cruzi and Trypanosoma evansi) of small wild mammals in the Pantanal. This region comprises a large natural environment with a multiplicity of habitats, wide variety of biodiversity besides the presence of livestock. T. cruzi and T. evansi infections were evaluated by parasitological and serological methods in one preserved and one cattle ranching area. The diversity of the small mammal fauna showed to be the same in the two studied areas, however, their relative abundance was different. Distinct enzootiological scenarios of both trypanosomatids could be observed. Transmission of T. cruzi occurred mainly in forested areas, in the two study areas, while T. evansi occurred dispersed among all habitats studied in the unpreserved area. The arboreal rodent Oecomys mamorae, the most abundant species in both areas, displayed high T. cruzi and T. evansi serum prevalence and parasitemias. Also, the caviomorph rodent Thrichomys pachyurus was shown to be an important host due to its expressive relative abundance, prevalence of infection by both trypanosomatid species and a broad range use of habitats. The role of the small mammal fauna in the transmission cycle of both trypanosomes species seems to be distinct according to land use since we found a broad range of T. evansi infected hosts in the preserved area in contrast to cattle ranching area and a half number of the rodents species infected with T. cruzi in unpreserved in comparison to protect area. The present study showed that cattle ranching in this study area did not enhance overall prevalence of T. cruzi infection among small wild mammals. Together with the observation that small mammals diversity in FA is similar to RN area suggest that ranching activity may also not necessarily conduct to biodiversity loss or risk of Chagas disease.     

Uptake of pentamidine in Trypanosoma brucei brucei is mediated by the P2 adenosine transporter and at least one novel, unrelated transporter.

Diamidine drugs such as pentamidine and berenil (diminazene aceturate) are vital drugs for the treatment of early stage human African trypanosomiasis and the corresponding veterinary condition, respectively. The action of diamidines on trypanosomes is critically dependent on their efficient uptake by the parasite. We have therefore investigated the mode of uptake of pentamidine by Trypanosoma brucei brucei, using [¹²⁵I]iodopentamidine as a permeant. [¹²⁵I]Iodopentamidine uptake was linear for up to 15 min and inhibited by adenosine with a K_i value of 0.64±0.03 μM, to a maximum of 50–70%. The adenosine-sensitive flux was also inhibited by adenine with a K_i value of 0.44±0.04 μM. Iodopentamidine uptake was saturable, with the adenosine-insensitive flux displaying a K_m of 22±2 μM and a V_max of 2.2±0.9 pmol(10⁷ cells)⁻¹ s⁻¹, whereas the adenosine-sensitive flux was inhibited by much lower iodopentamidine concentrations. These results clearly demonstrate that iodopentamidine is taken up by at least two different T. b. brucei transporters, an adenosine-sensitive pentamidine transporter (ASPT1) and a low-affinity pentamidine transporter (LAPT1). The identity of these transporters was investigated, and their significance for drug uptake and resistance in African trypanosomes is discussed.     

IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test

An increased IgM concentration in cerebrospinal fluid (CSF), occurring as a consequence of massive intrathecal IgM synthesis, is a marker of interest for diagnosis of the meningo-encephalitic stage in human African trypanosomiasis. However, in current practice, IgM in CSF is not determined because of the lack of a simple and robust test that is applicable in African rural regions where the disease prevails. We describe the development of a sensitive semiquantitative card agglutination test, LATEX/IgM, for IgM quantification in CSF. The test is simple and fast and the lyophilized reagent remains stable even at 45 degrees C. CSF end-titres obtained with LATEX/IgM parallel the IgM concentrations determined by nephelometry and enzyme-linked immunosorbent assay. Detection of intrathecal IgM synthesis is the most sensitive marker for CNS involvement in sleeping sickness. At a cut-off value of >or= 8, the sensitivity and specificity of LATEX/IgM for intrathecal IgM synthesis are 89.4 and 92.7%. As a consequence, patients with LATEX/IgM end-titres >or= 8 are likely to have intrathecal IgM synthesis, thus central nervous system involvement and therefore should be treated accordingly. Further studies should concentrate on the relationship between the LATEX/IgM end-titres, presence of intrathecal IgM synthesis and occurrence of treatment failures in patients treated with pentamidine.     

Diversity and spatial distribution of vectors and hosts of T. brucei gambiense in forest zones of, Southern Cameroon: Epidemiological implications

Host and vector distribution of Trypanosoma brucei gambiense was studied in relation to habitat types and seasons. Six (19.35%) of the 31 mammal species recorded in Bipindi were reservoir hosts. Cercopithecus nictitans was confined to the undisturbed forest and the low intensive shifting cultivation zones, while Cephalophus monticola, Cephalophus dorsalis, Cricetomys gambianus, Atherurus africanus and Nandinia binotata occurred in all the habitat types. As for vectors of human African trypanosomiasis (HAT), Glossina palpalis palpalis, was the most abundant (99.13%) among tsetse fly species. It occurs in all biotopes with its highest density recorded in the village-adjacent forest. The village-adjacent forest is therefore the most risky transmission zone for HAT mainly during the short rainy season when G. palpalis palpalis’ density is highest (2.91); while, the high and low intensive shifting cultivation zones are the most important contact zones between humans, G. palpalis palpalis and wild mammals in all seasons.     

Biological characterization of Trypanosoma cruzi stocks from domestic and sylvatic vectors in Sierra Nevada of Santa Marta, Colombia

Sierra Nevada of Santa Marta is one of the most endemic regions of Chagas disease in Colombia. In this study, we compared the biological behavior and genetic features of Trypanosoma cruzi stocks that were isolated from domestic and sylvatic insects in this area. Rhodnius prolixus (from domestic environments) and Triatoma dimidiata (from sylvatic, peridomestic and domestic environments) are the most important vectors in this region. Genetic characterization showed that all stocks corresponded to T. cruzi I, but LSSP-PCR analyses indicated that some genotypes were present in both environments. Biological characterization in vitro showed a low growth rate in sylvatic T. cruzi stocks and in some domestic T. cruzi stocks, possibly indicating the presence of stocks with similar behavior in both transmission cycles. In parallel, in vivo behavioral analysis also indicated that T. cruzi stocks are variable and this species did not show a correlation between the environments where they were isolated. In addition, all stocks demonstrated a low mortality rate and histopathological lesions in heart, skeletal muscle and colon tissue. Moreover, our data indicated that experimentally infected chagasic mice displayed a relation between their myocardial inflammation intensity, parasitism tissue and parasite load using the qPCR.In conclusion, our results indicate that the T. cruzi stocks present in SNSM have similar biological behavior and do not show a correlation with the different transmission cycles. This could be explained by the complex transmission dynamics of T. cruzi in Sierra Nevada of Santa Marta, where hosts, vectors (e.g., T. dimidiata) and reservoirs circulate in both environments due to the close contact between the two transmission cycles, favoring environment overlapping. This knowledge is an important key to understanding the epidemiology and pathology of Chagas disease in this Colombian region. Furthermore, our findings could be of significant use in the design of control strategies restricted to a specified endemic region.     

On the occurrence of thioredoxin in Trypanosoma cruzi

The full coding sequence for thioredoxin from Trypanosoma cruzi (TcTRX) strain Tulahuen O has been cloned into the pRSETA vector. The protein was expressed in Escherichia coli with an N-terminal extension of six histidine residues for purification through metal ion chromatography. The biological activity of recombinant TcTRX was proved utilizing the insulin reduction assay. Amino acid sequence alignment indicates a high identity of TcTRX with thioredoxins from different sources. Immunocytochemistry assays showed that TcTRX is present in epimastigote forms of T. cruzi, thus, indicating that the gene is expressed in vivo, rather than being a pseudogene. The in vivo occurrence of TcTRX points out the necessity of considering this protein as a molecular component of the redox metabolism in trypanosomatids.     

TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi

We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T. cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, São Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T. cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi.