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The reintroduction of captive animals to the wild helps restore endangered species, but it risks pathogen transmission, harming wild populations. Such transmission can impact the genetic diversity and long-term viability of these populations. This study assessed parasite diversity and load in captive Pecari tajacu, a species native to the Americas and culturally significant to Brazilian indigenous culture, prior to reintroduction. Samples from 24 peccaries were analyzed for ectoparasites, hemopathogens, and stool parasites with direct and molecular analysis. Findings showed that various parasites were present. Two peccaries (8.3%) were infested by the adult tick Amblyomma sculptum. Six (25.0%) tested positive for Trypanosoma evansi, four (16.7%) for hemobacteria of the family Anaplasmataceae, twelve (50.0%) for hemotropic Mycoplasma, and seven (29.2%) for Leishmania braziliensis. Stool samples indicated multiple parasites, with sixteen (66.7%) peccaries infected by Strongylida order parasites, Spiruridae in three (12.5%), and Ascaris suum in one (4.2%) animal. Cysts of Balantidium sp. were found in twenty (83.3%), Entamoeba polecki in five (20.8%), and Iodamoeba bütschlii in two (8.3%) peccaries. To our current knowledge, this is the first global report of Leishmania braziliensis, Iodamoeba bütschlii, and Entamoeba polecki in P. tajacu, irrespective of the environment, including both captivity and wild conditions. Some of these parasites are common in domestic animals, and others are zoonotic, indicating potential interspecies pathogen transmission.
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Background: Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania. There are still no vaccines, and therapeutic options are limited, indicating the constant need to understand the fine mechanisms of its pathophysiology. An approach that has been explored in leishmaniasis is the participation of microRNAs (miRNAs), a class of small non-coding RNAs that act, in most cases, to repress gene expression. miRNAs play a role in the complex and plastic interaction between the host and pathogens, either as part of the host's immune response to neutralize infection or as a molecular strategy employed by the pathogen to modulate host pathways to its own benefit. Methods: Monocyte-derived macrophages from healthy subjects were infected with isolates of three clinical forms of L. braziliensis: cutaneous (CL), mucosal (ML), and disseminated (DL) leishmaniasis. We compared the expression of miRNAs that take part in the TLR/NFkB pathways. Correlations with parasite load as well as immune parameters were analyzed. Results: miRNAs -103a-3p, -21-3p, 125a-3p -155-5p, -146a-5p, -132- 5p, and -147a were differentially expressed in the metastatic ML and DL forms, and there was a direct correlation between miRNAs -103a-3p, -21-3p, -155-5p, -146a-5p, -132-5p, and -9-3p and parasite load with ML and DL isolates. We also found a correlation between the expression of miR-21-3p and miR-146a-5p with the antiapoptotic gene BCL2 and the increase of viable cells, whereas miR-147a was indirectly correlated with CXCL-9 levels. Conclusion: The expression of miRNAs is strongly correlated with the parasite load and the inflammatory response, suggesting the participation of these molecules in the pathogenesis of the different clinical forms of L. braziliensis.
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Leishmania , Leishmaniose Cutânea , MicroRNAs , Humanos , Estados Unidos , MicroRNAs/genética , MicroRNAs/metabolismo , Macrófagos/metabolismo , PeleRESUMO
In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 104 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs-TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials.
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Toxoplasma gondii is the causative agent of toxoplasmosis, a disease that affects warm-blooded animals and one third of the human population worldwide. Pregnant women who have never been exposed to the parasite constitute an important risk group, as infection during pregnancy often leads to congenital toxoplasmosis, the most severe form of the disease. Current therapy for toxoplasmosis is the same as it was 50 years ago and has little or no effect when vertical transmission occurs. Therefore, it is urgent to develop new strategies to prevent mother-to-fetus transmission. The implementation of experimental animal models of congenital toxoplasmosis that reproduces the transmission rates and clinical signs in humans opens an avenue of possibilities to interfere in the progression of the disease. In addition, knowing the parasite load in maternal and fetal tissues after infection, which may be related to organ abnormalities and disease outcome, is another important step in designing a promising intervention strategy. Therefore, we implemented here a murine model of congenital toxoplasmosis with outbred Swiss Webster mice infected intravenously with tachyzoites of the ME49 strain of T. gondii that mimics the frequency of transmission of the parasite, as well as important clinical signs of human congenital toxoplasmosis, such as macrocephaly, in addition to providing a highly sensitive quantitative real-time PCR assay to assess parasite load in mouse tissues. As the disease is not restricted to humans, also affecting several domestic animals, including companion animals and livestock, they can also benefit from the model presented in this study.
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BACKGROUND: The adjuvants' optimal dose and the administration route can directly influence the epitope recognition patterns and profiles of innate response. We aimed to establish the effect and the optimal dose of adjuvant systems for proposing a vaccine candidate to be employed with Leishmania (Viannia) braziliensis. METHODS: We evaluated the adjuvants saponin (SAP), monophosphoryl lipid A (MPL) and resiquimod (R-848) isolated and combined as adjuvant systems in a lower dose corresponding to 25%, 33%, and 50% of each adjuvant total dose. Male outbred BALB/c mice were divided into 13 groups, SAP, MPL, and R-848 isolated, and the adjuvant systems SAP plus MPL (SM), SAP plus R-848 (SR), and MPL plus R-848 (MR). RESULTS: SM50 increased levels of all chemokines analyzed and TNF production, while it presented an increased inflammatory cell infiltrate in the skin with macrophage recruitment. Thus, we proposed a vaccine candidate employing L. (V.) braziliensis antigen associated with the SM adjuvant system against experimental L. (Leishmania) infantum challenge. We observed a significant increase in the frequency of cells expressing the central and effector memory CD4+ T cells phenotype in immunized mice with the LBSM50. In the liver, there was a decreased parasite load when mice received LBSM50. CONCLUSIONS: When combined with L. (V.) braziliensis antigen, SM50 increases TNF and IFN-γ, which generates central and effector memory CD4+ T cells. Therefore, using an adjuvant system can promote an effective innate immune response with the potential to compose future vaccines.
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Leishmaniasis is a zoonotic disease with worldwide distribution. In the Americas, the causative agent of the visceral form is the protozoa Leishmania (Leishmania) infantum. Transmission to the host or vertebrate reservoir occurs through the bite of infected arthropod females like Lutzomyia longipalpis. The epidemiological connection between the infection in dogs and humans generate constant studies about the relationship between the parasite and the canine host, including the development of methods and tests for the detection and quantification ofLeishmania (L.) infantum. Both conventional PCR (cPCR) and quantitative PCR (qPCR) can be used in the diagnosis of the parasite. Dropet Digital PCR (ddPCR) is another useful tool. Knowing the parasite load and its relationship with the clinical signs of naturally infected dogs is useful in research development and for establishing treatments that reduce the transmission of the disease. In this study, thirty-nine clinical samples of spleen from dogs naturaly infected by L. infantum were collected after necropsy. Two molecular tools were used to quantify the parasite load (qPCR and ddPCR) and there was 100% agreement in the results of the them. The tools developed in this work are important for the detection of L. infantum in dogs and humans. Droplet Digital PCR does not require a standard curve and is easy to standardize. In such manner, this new tool can generate more in-depth information in the broad debate about parasitic loads and the pathogenesis of leishmaniasis.
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The transmission cycles of Trypanosoma cruzi, the causative agent of Chagas disease, include a wide variety of mammals and hematophagous triatomine insects. Infection with this blood parasite has been confirmed in many armadillo species; however, information on infection in Zaedyus pichiy, a small armadillo that inhabits areas endemic to Chagas disease, is scarce. Our objective was to determine the infection frequency and parasite load of T. cruzi in 49 wild Z. pichiy confiscated dead from poachers in Mendoza, Argentina, 2010-2017. We detected T. cruzi DNA in 32 of 49 armadillos (65%) using real-time PCR, confirming infection with T. cruzi in a high proportion of confiscated pichis. No differences were found related to sex, age, or ecoregion origin of the assessed pichis. Parasite loads ranged between <0.1 and 8.88 parasite equivalents/microgram cardiac tissue. Additional studies on the infection status of Z. pichiy are needed to determine their role in the maintenance of the sylvatic transmission cycle and the potential zoonotic risk from hunted pichis.
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Doença de Chagas , Trypanosoma cruzi , Xenarthra , Animais , Trypanosoma cruzi/genética , Tatus , Argentina , Doença de Chagas/epidemiologia , Doença de Chagas/veterinária , MamíferosRESUMO
ABSTRACT Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5'and 3' SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 102 parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 102 and ≤ 103 (one mild, 13 moderate and three severe), and three had load > 103 parasites/mL (three severe); G2 with 22 samples (5'and 3' SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 102 parasites/mL (18 asymptomatic and four mild); G3 with five samples (5' and 3' SAG2 allele I + SAG3 allele II), parasite load ≤ 102 parasites/mL (three asymptomatic and two mild); G4 with one sample (5' and 3' SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 102 parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5'-SAG2 allele I; 98.7% of 3'-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.
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Trypanosoma cruzi, the causal agent of Chagas disease, is mainly transmitted by insects of the Triatominae subfamily. In Colombia, there are 26 triatomine species, and 16 of them are naturally infected with the parasite. The parasite loads of naturally infected vectors can be significant in targeting specific species that can affect the epidemiology of the disease. Studying their ecology and behavior is vital to understand their role in T. cruzi transmission dynamics. We evaluated the parasite loads of 182 field-collected triatomines corresponding to 10 species in 13 departments across Colombia. We standardized a methodology to quantify T. cruzi DNA in these insects. We obtained a LOD (limit of detection) of 3.05 p-eq/mL. The 82% of triatomines we evaluated were positive for T. cruzi infection, with loads ranging from hundreds to millions of equivalent parasites per milliliter. Panstrongylus geniculatus, Rhodnius prolixus, and Triatoma dimidiata were the species with the highest loads of T. cruzi; however, other species whose role as vectors is still unknown were also found with high loads of parasites. Our results suggest the relevance of secondary species for T. cruzi transmission in Colombia. We hope our data can help improve entomological surveillance and vector control programs in the country and the region.
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A total of 123 DNA samples from Panamanian patients with cutaneous leishmaniasis (CL) lesions were evaluated. These samples were previously confirmed with CL by a specific KDNA-Viannia PCR but had a negative parasitological diagnosis (Group A). Epidemiological variables, such as age, sex, geographic origin, evolution time, and the number and location of the lesions, were analyzed. No significant differences (p < 0.05) were found when these variables were evaluated against a control panel of 123 CL lesion samples from CL patients with positive parasitological diagnoses (Group B). Of the 123 samples (Group A), 67% (82/123) gave positive results when re-analyzed by PCR-hsp70. An analysis of 69 of these samples via PCR-hsp70-RFLP showed that 59.4% (41/69) of the found restriction patterns corresponded to Leishmania (Viannia) panamensis and 40.6% (28/69) to Leishmania (Viannia) guyanensis. Finally, the sequence and phylogenetic analysis of 32 of the samples confirmed the species in 21 (65.6%, 21/32) samples, originally characterized as L. (V.) panamensis. However, 11 samples (34.4%, 11/32), initially identified via RFLP-Hsp70 as L. (V.) guyanensis, matched the sequence of a genetic variant known as Leishmania sp.1. These results point out the species/genetic variants of Leishmania in the case of CL lesions with an apparently low parasite load.
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Tilapia has a high socioeconomic value in many countries worldwide. However, it has been identified as a zoonotic parasite reservoir. A systematic literature search and meta-analysis were carried out in order to estimate the global prevalence of zoonotic parasites that affect tilapia. The search was performed by three field experts to avoid reviewer bias. Polled prevalence was estimated using a logistic-normal random-effect regression model in the R software. We dealt with the heterogeneity among studies through subgroup analysis, taking into account the continent, country, genus of the host, parasite taxonomic group, sample origin, and type of diagnostic test as moderator variables. Fifty-two eligible articles were identified covering five tilapia genera with a pooled prevalence of 0.14 (95% CI: 0.10−0.20) showed significant heterogeneity (I2 = 98.4; p < 0.001). The subgroup analysis revealed that the most affected host was Sarotherodon, with a prevalence of 0.42 (95% CI: 0.22−0.65). Cestode was the taxonomic group with the largest prevalence (0.40; 95% CI:0.32−0.48), followed by amoeba (0.24; 95% CI: 0.16−0.35) and nematode (0.22; 95% CI: 0.11−0.38), among which, Schyzocotyle spp., Opistorchis spp., Gnathostoma spp. and Vermamoeba spp. have an impact on public health. Significant differences (p < 0.004) were found among continents and countries, with the highest value of prevalence detected in the African continent (0.28; 95% CI: 0.20−0.37), specifically in Tanzania (0.56; 95% CI: 0.22−0.87) and Egypt (0.43; 95% CI: 0.20−0.55). The origin of samples had a significant effect (p < 0.0001) on the detected prevalence, especially from those that showed the highest prevalence (0.24; 95% CI: 0.17−0.33). Finally, there were no differences in prevalence according to the diagnostic test (p = 0.97). Our results provide useful information on the development of epidemiological programs for the control of zoonoses associated with parasites in tilapia and in the design, planning, and implementation of future research.
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We present the first study that compares phenological variation in parasite load and inflammatory response in a lizard with asynchronous male and female gonadal cycles. Other studies have used many species with seasonal and synchronous reproductive cycles, in which it is difficult to decouple the effects of internal and external factors that can affect parasite abundance in each sex. Species with asynchronous reproductive cycles provide the opportunity to study the effects of seasonality and reproductive condition separately, but few studies have documented variation in parasite abundance in these species. We made an extensive comparison of parasite load and inflammatory response of the lizard Sceloporus torquatus, a species with asynchronous reproductive cycles, throughout its active period. We hypothesized that the parasite load would be higher in the period of maximum gonadal activity for each sex, negatively related to body condition and inflammatory response. Our results partially support the hypothesis; males had more parasites in summer than in spring and autumn, while females had more parasites in spring and summer than in autumn; however, we do not find a relationship between parasite load, body condition and inflammatory response. Our results indicated that host-parasite interactions are complex and depend upon both environmental and internal factors. Therefore, longer-term studies may provide a more comprehensive picture of host-parasite dynamics in populations of wild lizards.
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Lagartos , Parasitos , Animais , Feminino , Interações Hospedeiro-Parasita , Lagartos/fisiologia , Masculino , Carga Parasitária , Reprodução , Estações do AnoRESUMO
Trypanosoma cruzi is the causal agent of Chagas disease, a parasitic zoonosis transmitted mainly through the feces of triatomine insects. Triatoma infestans is the main triatomine vector of this disease in South America. Previous research has shown that T. cruzi infection modifies the behavior of triatomines. We evaluated, for the first time, the effect of parasite load on feeding and defecation behavior, which we quantified by using real-time PCR. The detection time of the host was shorter in infected individuals, and the number of bites increased, while the dejection time was reduced when compared with the non-infected group. A significant correlation between the parasite load and the behavioral changes registered in the infected triatomines was found. These results would indicate that the intensity of T. cruzi infection modulates the feeding and defecation behavior of T. infestans, increasing the vector competence of this triatomine vector.
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American trypanosomiasis is a disease caused by the flagellate protozoan Trypanosoma cruzi, which is transmitted mainly in endemic areas by blood-sucking triatomine vectors. Triatoma infestans is the most important vector in the southern cone of South America, exhibiting a nocturnal host-seeking behavior. It has been previously documented that the parasite produces changes in some triatomine species, but this is the first time that the behavior of a vector has been evaluated in relation to its parasite load. After comparing the movement events and distance traveled of infected and non-infected T. infestans, we evaluated the change produced by different T. cruzi parasite loads on its circadian locomotor activity. We observed differences between infected and non-infected triatomines, and a significant relation between the parasite load and the increase in locomotor activity of T. infestans, which was accentuated during the photophase. This could have direct implications on the transmission of T. cruzi, as the increased movement and distance traveled could enhance the contact of the vector with the host, while increasing the predation risk for the vector, which could both constitute a risk for vectorial and oral transmission to mammals.
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BACKGROUND: The distribution of parasite load across hosts may modify the transmission dynamics of infectious diseases. Chagas disease is caused by a multi-host protozoan, Trypanosoma cruzi, but the association between host parasitemia and infectiousness to the vector has not been studied in sylvatic mammalian hosts. We quantified T. cruzi parasite load in sylvatic mammals, modeled the association of the parasite load with infectiousness to the vector and compared these results with previous ones for local domestic hosts. METHODS: The bloodstream parasite load in each of 28 naturally infected sylvatic mammals from six species captured in northern Argentina was assessed by quantitative PCR, and its association with infectiousness to the triatomine Triatoma infestans was evaluated, as determined by natural or artificial xenodiagnosis. These results were compared with our previous results for 88 humans, 70 dogs and 13 cats, and the degree of parasite over-dispersion was quantified and non-linear models fitted to data on host infectiousness and bloodstream parasite load. RESULTS: The parasite loads of Didelphis albiventris (white-eared opossum) and Dasypus novemcinctus (nine-banded armadillo) were directly and significantly associated with infectiousness of the host and were up to 190-fold higher than those in domestic hosts. Parasite load was aggregated across host species, as measured by the negative binomial parameter, k, and found to be substantially higher in white-eared opossums, cats, dogs and nine-banded armadillos (range: k = 0.3-0.5) than in humans (k = 5.1). The distribution of bloodstream parasite load closely followed the "80-20 rule" in every host species examined. However, the 20% of human hosts, domestic mammals or sylvatic mammals exhibiting the highest parasite load accounted for 49, 25 and 33% of the infected triatomines, respectively. CONCLUSIONS: Our results support the use of bloodstream parasite load as a proxy of reservoir host competence and individual transmissibility. The over-dispersed distribution of T. cruzi bloodstream load implies the existence of a fraction of highly infectious hosts that could be targeted to improve vector-borne transmission control efforts toward interruption transmission. Combined strategies that decrease the parasitemia and/or host-vector contact with these hosts would disproportionally contribute to T. cruzi transmission control.
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Doença de Chagas/transmissão , Mamíferos/parasitologia , Triatoma/parasitologia , Trypanosoma cruzi , Animais , Animais Selvagens/parasitologia , Argentina/epidemiologia , Tatus/parasitologia , Gatos , Doença de Chagas/diagnóstico , Doença de Chagas/prevenção & controle , Didelphis/parasitologia , Reservatórios de Doenças/parasitologia , Vetores de Doenças , Cães , Florestas , Genes de Protozoários , Humanos , Insetos Vetores/parasitologia , Carga Parasitária/estatística & dados numéricos , Parasitemia/parasitologia , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificação , Doenças Transmitidas por Vetores/diagnóstico , Doenças Transmitidas por Vetores/prevenção & controle , Doenças Transmitidas por Vetores/transmissão , XenodiagnósticoRESUMO
In this study, the prevalence of T. cruzi infection was estimated in dogs and their owners from a rural community in Mexico using serological techniques for chronic infection cases, qPCR for acute phase cases, and a combination of both techniques to detect chronic and acute infections. Eighty-nine blood samples were collected from owners and their dogs for obtaining serum and parasite DNA. Prevalence was calculated using (i) positive cases detected in a serological test (ELISA and Western blot), (ii) positive cases detected in a qPCR test, and (iii) positive cases detected by both techniques. Sensitivity, specificity, and predictive values were determined individually for serology, qPCR and for both techniques used simultaneously. The prevalence observed varied: for serology, 25.8% of the dogs and 7.9% of the owners were seropositive, while for qPCR 29.2% of the dogs and 10.1% of the owners were identified as positive. Combination of serological and molecular techniques resulted in a prevalence of 38.2% for dogs and 12.4% for their owners. The sensitivity, specificity and predictive values calculated for both techniques improved when both techniques were used simultaneously (sensitivity of 92.4% and specificity of 100% for infected dogs and sensitivity of 93.4% and specificity of 100% for infected owners). Combined use of serological tests and qPCR allowed identifying a greater number of positive cases in dogs and their owners. This strategy may help implement adequate and timely epidemiological surveillance of American trypanosomiasis in order to prevent the appearance of new cases of Trypanosoma cruzi infections in endemic zones.
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The golden hamster is a suitable model for studying cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis. Immunopathological mechanisms are well established in the L. (L.) major-mouse model, in which IL-4 instructs a Th2 response towards progressive infection. In the present study, we evaluated the natural history of L. braziliensis infection from its first stages up to lesion establishment, with the aim of identifying immunological parameters associated with the disease outcome and parasitism fate. To this end, hamsters infected with 104, 105, or 106 promastigotes were monitored during the first hours (4h, 24h), early (15 days, 30 days) and late (50 days) post-infection (pi) phases. Cytokines, iNOS and arginase gene expression were quantified in the established lesions by reverse transcription-quantitative PCR. Compared to the 105 or 106 groups, 104 animals presented lower lesions sizes, less tissue damage, and lower IgG levels. Basal gene expression in normal skin was high for TGF-ß, and intermediary for TNF, IL-6, and IL-4. At 4hpi, no cytokine induction was observed in the 104 group, while an upregulation of IL-6, IL-10, and IL-4 was observed in the 106 group. At 15dpi, lesion appearance was accompanied by an increased expression of all assessed cytokines, markedly in the 105 and 106 groups. Upregulation of all investigated cytokines was observed in the late phase, although less expressive in the 104 group. IFN-γ was the depending variable influencing tissue damage, while IL-6 was associated to parasite load. The network correlating gene expression and clinical and laboratorial parameters indicated inoculum-independent associations at 15 and 30dpi. A strong positive network correlation was observed in the 104 group, but not in the 105 or 106 groups. In conclusion, IL-4, IL-6, IL-10, and TGF-ß are linked o L. braziliensis progression. However, a balanced cytokine network is the key for an immune response able to reduce the ongoing infection and reduce pathological damage.
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Citocinas/metabolismo , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Transdução de Sinais , Animais , Biomarcadores , Biologia Computacional/métodos , Cricetinae , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/imunologia , Imunomodulação , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Carga ParasitáriaRESUMO
Strains of Trypanosoma cruzi, etiological agent of Chagas disease, are classified into different discrete typing units that may present distinct dynamics of infection and susceptibility to benznidazole (BZ) treatment. Mice that were orally inoculated with T. cruzi IV strains exhibited a more intense course of infection compared with intraperitoneally inoculated mice, reflected by higher parasite loads. We evaluated the efficacy of BZ treatment in Swiss mice that were inoculated with T. cruzi IV strains from the Western Brazilian Amazon. The mice were orally (OR) or intraperitoneally (IP) inoculated with 2 × 106 culture-derived metacyclic trypomastigotes of the AM14, AM16, AM64, and AM69 strains of T. cruzi that were obtained from two outbreaks of orally acquired acute Chagas disease in the state of Amazonas, Brazil. The animals were treated with BZ (100 mg/kg/day for 20 days). Fresh blood examination, hemoculture, conventional and quantitative real-time polymerase chain reaction were performed to monitor the therapeutic effects of BZ. Significant reductions in five of 24 parameters of parasitemia and parasite load were found in different tissues in the OR group, indicating worse response to BZ treatment compared with the IP group, in which significant reductions in nine of those 24 parameters were observed. The cure rates in the OR groups ranged from 18.2% (1/11) to 75.0% (9/12) and in the IP groups from 58.3% (7/12) to 91.7% (11/12), for the AM14 and AM69 strains, respectively. These findings indicate that treatment with BZ had fewer beneficial effects with regard to reducing parasitemia and parasite load in different tissues of mice that were OR inoculated with four TcIV strains compared with IP inoculation. Therefore, the route of infection with T. cruzi should be considered when evaluating the therapeutic efficacy of BZ in patients with Chagas disease.
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Doença de Chagas/parasitologia , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/classificação , Parede Abdominal/parasitologia , Animais , Brasil/epidemiologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/epidemiologia , Esôfago/parasitologia , Coração/parasitologia , Camundongos , Nitroimidazóis/farmacologia , Carga Parasitária , Parasitemia/tratamento farmacológico , Parasitemia/epidemiologia , Parasitemia/parasitologia , Estômago/parasitologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
In order to improve the diagnosis of giardiasis, fecal samples (high/medium/low concentration of cysts) were processed by the parasitological methods used in the routine: Faust, Lutz e Ritchie modified (replacement of formaldehyde by distilled water). The cysts were quantified; the DNA was extracted and amplified by semi-nested PCR (GDH gene). Fifteen clinical samples were analyzed to validate the study by PCR-RFLP. The results showed that the parasite was only detected and genotyped correctly when samples from children with high, medium, and low parasitic load, belonging to genotype AII, were processed by the modified Ritchie method, different from what was observed for the other methods used in laboratory routine (Faust and Lutz). The modified Ritchie method proved to be more suitable, recovering a greater number of cysts from samples, regardless of parasitic load, which reduces the chance of false negative results and has epidemiological repercussions since individuals with low parasite load are usually asymptomatic and the main disseminators of this infection.
Assuntos
Técnicas de Genotipagem/métodos , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/isolamento & purificação , Giardíase/parasitologia , Reação em Cadeia da Polimerase/métodos , Pré-Escolar , Fezes/parasitologia , Feminino , Genótipo , Giardia lamblia/genética , Giardíase/diagnóstico , Humanos , Lactente , Estágios do Ciclo de Vida , Masculino , Técnicas de Diagnóstico Molecular/métodos , Carga Parasitária , Proteínas de Protozoários/genéticaRESUMO
We aimed to characterize the genetic constitution of natural T. cruzi populations involved in an Oral Chagas Disease (OCD) outbreak at a rural school of the community of Chichiriviche de la Costa, Venezuela, which affected patients did not respond to the etiological treatment. Peripheral blood samples and/or hemocultures were obtained from twenty-nine OCD patients at time of diagnosis or along nine years of Post-treatment (Tx) follow-up. The IgG serology, T. cruzi discrete typing units (DTU), satellite DNA-qPCR parasitic loads, and minicircle signatures were determined at Pre-Tx and after Tx. The serological titles and parasitic loads changed after treatment, with a significant decrease of IgG titers (Spearman's r value= -0.961) and median parasite loads from 2.869 [IQR = 2.113 to 3.720] to 0.105 [IQR = -1.147 to 1.761] log10 par eq. /mL at Pre-Tx and Post-Tx, respectively, suggesting infection evolution from acute to chronic phase, without seroconversion or parasitological eradication, which was indicative of treatment failure. All patients were infected with T. cruzi DTU I populations. At Pre-Tx their median Jaccard genetic distances were 0.775 [IQR = 0.708 to 0.882], decreasing in genetic variability towards the end of follow-up (Mann-Whitney U test p= 0.0031). Interestingly, no Post-Tx minicircle signature was identical to its Pre-Tx counterpart population in a same patient, revealing selection of parasite subpopulations between the primary infection and Post-Tx. The parasitic populations isolated from hemocultures showed a lower number of bands in the minicircle signatures with respect to the signatures obtained directly from the patients' blood samples, demonstrating a process of parasitic selection and reduction of the population variability that initially infected the patients. Decrease of parasitic loads after treatment as well as Pre- and Post-Tx intra-TcI diversity might be a consequence of both, natural evolution of the acute infection to the chronic phase and persistence of refractory populations due to Tx selection.