Modifications to the parasitological technique of Rugai increase the diagnostic sensitivity for strongyloidiasis.

Strongyloidiasis is a neglected tropical disease caused mainly by Strongyloides stercoralis, a nematode that can persist for decades in the human host with a very low parasitic burden and without specific symptoms. Hence, it is difficult to diagnose and control. Larval concentration and culture methods with fecal samples show higher sensitivity for the diagnosis of Strongyloides-infected individuals; however, these techniques are not routinely used, primarily due to the challenges associated with processing a substantial volume of fecal samples. In the current study, we comparatively evaluated the sensitivity and applicability of modifications made to the Rugai parasitological method for the diagnosis of strongyloidiasis in fecal samples of experimentally infected rats and in 68 individuals from an urban community close to Maceió, Brazil. The presence and quantity of parasite larvae in the feces were comparatively evaluated using different parasitological techniques. In the experimental model, we demonstrated that the modified Rugai technique (RMOD) allowed for significantly higher recovery of larvae than the original Rugai technique (RO). Moreover, the sediment was cleaner and easier to evaluate using optical microscopy. Compared to other parasitological techniques, such as agar-plate culture (A-PC) and spontaneous sedimentation (SS), the RMOD technique showed higher sensitivity in the detection of larvae in all infected groups and presented comparatively better performance, especially in rats with a low parasite burden. In the human population, among the 68 stool samples evaluated, Strongyloides larvae were detected in the feces of six individuals with an estimated prevalence of 8.82%. However, the performance of each parasitological method was remarkably different. SS identified Strongyloides larvae in only two individuals and A-PC in three, whereas RMOD was able to identify six infected individuals, resulting in sensitivities of 33.3%, 50%, and 100%, respectively. In conclusion, the modifications introduced to the Rugai technique resulted in improved sensitivity for the detection of Strongyloides spp. infections, especially in stool samples with a low parasite burden, in comparison with other routinely used parasitological techniques.

Evaluation of isotype-based serology for diagnosis of Schistosoma mansoni infection in individuals living in endemic areas with low parasite burden.

Intestinal schistosomiasis is a chronic and debilitating disease that affects public health systems worldwide. Control interventions to reduce morbidity primarily involve the diagnosis and treatment of infected individuals. However, the recommended Kato-Katz (KK) parasitological method shows low sensitivity in individuals with low parasite loads and is not useful for monitoring elimination of parasite transmission at later stages. In the current study, we evaluated the accuracy of serum reactivity levels of different immunoglobulin isotypes in an enzyme-linked immunosorbent assay (ELISA), utilizing Schistosoma mansoni crude extracts, with the aim to improve the diagnosis of infected individuals with low parasite loads. The serum reactivity of IgM and IgG subclass antibodies (IgG1, IgG3, and IgG4) against soluble adult worm and egg antigen preparations was evaluated in residents from a schistosomiasis-endemic area in northern Minas Gerais, Brazil. The parasitological status of the study population was determined through fecal examination with multiple parasitological tests to create a consolidated reference standard (CRS) plus a fecal DNA detection test (q-PCR). Twelve months after praziquantel treatment, a second serum sample was obtained from the population for reexamination. A two-graph receiver operating characteristic curve (TG-ROC) analysis was performed using the serum reactivity of non-infected endemic controls and egg-positive individuals, and the cut-off value was established based on the intersection point of the sensibility and specificity curves in TG-ROC analyses. The diagnostic accuracy of each serological test was evaluated in relation to the parasitological CRS and to the combination of CRS plus qPCR results. The data revealed that serum reactivity of IgM and IgG3 against S. mansoni antigens did not allow identification of infected individuals from the endemic area. In contrast, serum IgG1 and IgG4-reactivity against schistosome antigens could distinguish between infected and non-infected individuals, with AUC values ranging between 0.728-0.925. The reactivity of IgG4 anti-soluble egg antigen - SEA (sensitivity 79 %, specificity 69 %, kappa = 0.49) had the best diagnostic accuracy, showing positive reactivity in more than 75 % of the infected individuals who eliminated less than 12 eggs per gram of feces. Moreover, serum IgG4 reactivity against SEA and against soluble worm antigen preparation (SWAP) was significantly reduced in the serum of infected individuals after 12 months of confirmed parasitological cure and in the absence of re-infection. These results reinforce that the described IgG4 anti-SEA ELISA assay is a sensitive alternative for the diagnosis of active intestinal schistosomiasis in individuals from endemic areas, including in those with a very low parasite load.

Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome

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.

Acute Disseminated Toxoplasmosis in Two Specimens of Macropus rufogriseus Caused by a Genotype so far Exclusive to South America.

Macropods are included among the species considered highly susceptible to Toxoplasma gondii infection. Clinically, it is difficult to distinguish between acute toxoplasmosis due to primary infection and reactivation of chronic latent infection in susceptible species until pathologic studies are performed. Here, we described the clinical cases and lesions found in two deceased Bennett's wallabies (Macropus rufogriseus) with a presumptive diagnosis of toxoplasmosis, as well as the genetic characterization of the T. gondii isolates obtained from these specimens. Both animals presented acute infection lesions in the lungs, liver, spleen and lymph nodes associated to T. gondii infection. Histopathology and immunohistochemistry also demonstrated tissue cysts of different sizes, indicating that the wallabies were previously infected with this parasite. Two isolates were obtained, one from each specimen and the molecular characterization was done; both isolates were the ToxoDB #116 genotype. This is the first study that reports the isolation of this particular genotype outside South America, and given the histopathological findings, it could be considered virulent for this species. The dynamics of infection that T. gondii is causing in definitive and intermediate hosts in a region allows us to know the risks to which the animals and humans that live in the area are exposed, and in the future to implement a preventive medicine plan against this parasite.

Experimental infection with Schistosoma mansoni isolated from the wild rodent Holochilus sciureus shows a low parasite burden but induces high schistosomiasis severity in BALB/c mice.

Wild mammals, especially rodents, can participate in the life cycle of Schistosoma mansoni; however, the impact of these parasite strains on the severity of schistosomiasis remains unclear. The aim of this study was to comparatively evaluate the parasitological and immunopathological alterations induced by an S. mansoni strain isolated from the wild rodent Holochilus sciureus (HS strain) and a parasite strain isolated from a human (LE strain) in experimentally infected mice. Male BALB/c mice were subcutaneously infected with 50 cercariae/mouse of either the HS or the LE strain and were evaluated for 12 weeks. In the experimental groups, the parasite burden was estimated by worm and egg (feces and tissues) count, and immunopathological alterations were evaluated in the liver and intestines. Compared to experimental infection with the LE parasite strain, HS-infected mice showed reduced number of parasite worms but higher fecundity rate, significant reduction in IL-5, IL-10 and IL-13 concentrations, lower EPO-activity in liver homogenate and higher concentrations of TNF-α, IFN-γ, IL-12 and IL-17 in the small intestine homogenate. Moreover, HS infection resulted in higher concentrations of NO end-products in both the liver and intestine, suggesting a predominance of the Th1/Th17 immune response. HS-infected mice also showed higher plasma transaminase levels, formed larger granulomas, and had a higher mortality rate in comparison with LE-infected mice. Data indicate that BALB/c mice infected with the HS strain of S. mansoni showed reduced susceptibility to the parasite but stronger tissue inflammation and high disease severity.

Status of gastrointestinal nematode infections and associated epidemiological factors in sheep from Córdoba, Colombia.

Gastrointestinal nematodes cause serious economic losses in sheep production systems. To determine the prevalence and risk factors associated with these parasites, a study was conducted on 595 sheep in Córdoba, Colombia. Prevalence and parasite burden were determined using the McMaster technique. Larvae were cultured from feces to identify the nematode genera. For the analysis of associated factors and parasite burden, data means, medians, and confidence intervals were compared. A georeferenced data analysis was performed and an epidemiological map was constructed. An overall prevalence of 88.2% was found, with the highest prevalence and parasite burden for Strongylida (83.2%) and Strongyloides (41%) nematodes. The mean parasite burden was 1255 eggs per gram. The gastrointestinal nematode genera identified were Trichostrongylus, Strongyloides, Haemonchus, Oesophagostomum, Bunostomum, and Cooperia. In general, a significant association was found between parasite burden and dewormed animals, anthelmintic used, date of last deworming, and breeds. The FAMACHA® method and body condition showed a significant association with the Strongylida parasite burden. Clusters with higher intensity of gastrointestinal nematode burden and high prevalence were observed in the regions of Bajo Sinú, Sinú Medio, and San Jorge.

Parasitic Load Correlates With Left Ventricular Dysfunction in Patients With Chronic Chagas Cardiomyopathy.

Background: Chronic Chagas disease (CChD), one of the infectious parasitic diseases with the greatest social and economic impact upon a large part of the American continent, has distinct clinical manifestations in humans (cardiac, digestive, or mixed clinical forms). The mechanisms underlying the development of the most common and ominous clinical form, the chronic Chagas cardiomyopathy (CCC) have not been completely elucidated, despite the fact that a high intensity of parasite persistence in the myocardium is deemed responsible for an untoward evolution of the disease. The present study aimed to assess the parasite load CCC and its relation to left ventricular ejection fraction (LVEF), a definite prognostic marker in patients with CCC. Methods: Patients with CCC were clinically evaluated using 12-lead-electrocardiogram, echocardiogram, chest X-ray. Peripheral blood sampling (5 ml of venous blood in guanidine/EDTA) was collected from each patient for subsequent DNA extraction and the quantification of the parasite load using real-time PCR. Results: One-hundred and eighty-one patients with CCC were evaluated. A total of 140 (77.3%) had preserved left ventricular ejection fraction (of ≥40%), and 41 individuals had LV dysfunction (LVEF of <40%). A wide variation in parasite load was observed with a, mean of 1.3460 ± 2.0593 (0.01 to 12.3830) par. Eq./mL. The mean ± SD of the parasite load was 0.6768 ± 0.9874 par. Eq./mL and 3.6312 ± 2.9414 par. Eq./mL in the patients with LVEF ≥ 40% and <40%, respectively. Conclusion: The blood parasite load is highly variable and seems to be directly related to the reduction of LVEF, an important prognostic factor in CCC patients.

Nitric-oxide releasing chitosan nanoparticles towards effective treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a major public health problem caused by Leishmania parasites that produce destructive and disfiguring skin conditions. There is an urgent need for alternative topical therapies due to the limitations of current systemic treatments. Recently, we have synthesized nitric oxide-releasing chitosan nanoparticles (NONPs) and shown their potential in vitro against Leishmania amazonensis. Herein we evaluated the application of NONPs for the treatment of CL on infected BALB/c mice. Mice were treated with topical administration of increasing concentrations of NONPs and disease progression was investigated regarding parasite load, lesion thickness, and pain score. As a result, we observed a dose-dependent NONPs effect. Parasite burden and lesion thickness were substantially lower on animals receiving NONPs at a 2 mM concentration compared to untreated control. Moreover, the clinical presentation of the lesions did not show any visible signs of ulcer, suggesting clinical healing in these animals. This successful outcome was sustained for at least 21 days after therapy even in one single dose. Thus, we demonstrate that NONPs are suitable for topical administration, and represent an attractive approach to treat CL.

Chronic Chagas disease: Quantification of Trypanosoma cruzi in peripheral blood and dejections of Triatoma infestans fed by xenodiagnosis in patients with and without cardiopathy.

It is not currently known which individuals with chronic Chagas disease (ChD) will develop cardiopathy in a determined period and which will be maintained asymptomatic with normal routine laboratory tests all their lives. The parasite burden is a factor that could explain this different evolution. The objective of this study was to quantify Trypanosoma cruzi burden by real-time PCR in blood (qPCR-B) and dejections of triatomines fed by xenodiagnosis (qPCR-XD) in 90 individuals with chronic ChD untreated, classified according to XD results and the presence or absence of cardiopathy. All individuals came from hyperendemic areas of Chile and participated in the study under Informed Consent. The standard qPCR curves for qPCR-B and qPCR-XD were elaborated with a mixture of known concentrations of T. cruzi strains, performing DNA serial dilutions (1/10) with a dynamic range between 105 and 10-1 parasite equivalents/mL. The TaqManⓇ detection system was applied in a Stratagene Mx3000P thermocycler (Agilent Technologies, USA) with cruzi 1 and cruzi 2 satellite primers. 22.2% and 15.6% of cases with cardiopathy or without cardiopathy were XD positive. There was no significant difference between the groups. The positivity of qPCR-B and qPCR-XD in the positive XD group was 82.35% and 100%, respectively, while in the negative XD group was 55.26% and 42.10%, respectively. A superior qPCR value in chronic ChD patients with and without cardiopathy was determined for qPCR in cases with positive XD and positive qPCR-XD. The receiver operating characteristic (ROC) curve analyses show better accuracy for detecting parasite burden (area under the curve, AUC) for qPCR-XD in comparison to qPCR-B. That is to say, major performance in DNA samples obtained of positive XD (gold standard for viable T. cruzi) detected and quantified by qPCR-XD. A high percentage of cases with XD and qPCR-XD positive (80-100%) have result concordant with qPCR-B. In absence of XD, future challenges are especially related to the low parasitic load of chronic ChD patients treated with trypanocidal drugs and post-therapy parasitological evaluations by qPCR-B. Finally, no statistically significant differences were found between presence or absence of cardiopathy and XD, qPCR-B or qPCR-XD.

Correlation of Parasite Burden, kDNA Integration, Autoreactive Antibodies, and Cytokine Pattern in the Pathophysiology of Chagas Disease.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), is the main parasitic disease in the Western Hemisphere. Unfortunately, its physiopathology is not completely understood, and cardiomegaly development is hard to predict. Trying to explain tissue lesion and the fact that only a percentage of the infected individuals develops clinical manifestations, a variety of mechanisms have been suggested as the provokers of CD, such as parasite persistence and autoimmune responses. However, holistic analysis of how parasite and host-related elements may connect to each other and influence clinical outcome is still scarce in the literature. Here, we investigated murine models of CD caused by three different pathogen strains: Colombian, CL Brener and Y strains, and employed parasitological and immunological tests to determine parasite load, antibody reactivity, and cytokine production during the acute and chronic phases of the disease. Also, we developed a quantitative PCR (qPCR) protocol to quantify T. cruzi kDNA minicircle integration into the mammalian host genome. Finally, we used a correlation analysis to interconnect parasite- and host-related factors over time. Higher parasite load in the heart and in the intestine was significantly associated with IgG raised against host cardiac proteins. Also, increased heart and bone marrow parasitism was associated with a more intense leukocyte infiltration. kDNA integration rates correlated to the levels of IgG antibodies reactive to host cardiac proteins and interferon production, both influencing tissue inflammation. In conclusion, our results shed light into how inflammatory process associates with parasite load, kDNA transfer to the host, autoreactive autoantibody production and cytokine profile. Altogether, our data support the proposal of an updated integrative theory regarding CD pathophysiology.

C57BL/6 mice immunized with synthetic peptides from Toxoplasma gondii surface and microneme immunodominant antigens are able to decrease parasite burden in the brain tissues.

Toxoplasmosis is a disease caused by Toxoplasma gondii, an intracellular protozoan able to infect a wide range of hosts. The infection is particularly severe in immunocompromised patients or during pregnancy, circumstances in which the parasite could find a more favorable microenvironment to replicate and invade host tissues. The current treatment consists in toxic drugs for the patients, being not appropriate for the fetuses and immunodeficient patients. So far, there is a lack of available vaccine to prevent the disease. The present study aimed to evaluate the immune response induced by peptides derived from parasite immunodominant proteins from key components, as surface, rhoptry, microneme and dense granule antigens. A panel of eleven peptides was selected considering the highest scores for B cell epitope prediction by in silico analyses. The peptides were divided in groups, according to the parasite organelle locations, and used to immunize C57BL/6 mice. The animals were submitted to three doses of immunization and infected by 10 cysts of T. gondii ME49 strain. Blood samples were collected and used to measure the production of antibodies and cytokines, while the brains were collected to determine the parasite burden by quantitative polymerase chain reaction (qPCR). It was found that synthetic peptides from all targets were able to induce IgG synthesis in immunized mice, as well as to modulate the Th1/Th2 cytokine production, particularly the MIC and SRS groups, which presented the IFN-γ/IL-10 and TNF-α/IL-10 ratios 30 and 10 times higher, respectively, when compared with non-immunized group. Interestingly, the animals from MIC and SRS groups had significantly lower levels of T. gondii DNA in their brains. In summary, it can be concluded that peptides mainly from SRS and MIC parasite components constitute relevant targets to design vaccine candidates against parasite burden observed during chronic toxoplasmosis.

Modulation of Allergic Reactivity in Humans Is Dependent on Schistosoma mansoni Parasite Burden, Low Levels of IL-33 or TNF-α and High Levels of IL-10 in Serum.

Helminth infections and allergies are characterized by a predominant type-2 immune response. In schistosomiasis, the Th-2 response is usually accompanied by induction of immunoregulatory mechanisms that contribute to worm survival and less severe schistosomiasis. Although helminth-induced immunomodulatory mechanisms seem to affect atopy, epidemiological studies on the relationship between helminths and allergy have been inconsistent, and data suggest that the modulatory effects may be influenced by helminth species, chronicity of infection, and parasite burden. Here we performed a cross-sectional study to investigate the effects of Schistosoma mansoni parasite burden and immune response on allergic reactivity of individuals living in a schistosomiasis endemic area in Brazil. Fecal samples from the participants were collected for extensive parasitological examinations by spontaneous sedimentation, Kato-Katz, Helmintex and Saline Gradient tests and molecular detection of S. mansoni by qPCR. Additionally, the concentrations of cytokines and chemokines, total IgE and IgE-reactivity to common house dust allergens were quantified from serum samples. IgE reactivity to dust allergens was detected in 47 individuals (23.8%), and 140 individuals (54.4%) were diagnosed with S. mansoni infection. Most of the infected population (108 individuals) presented very low parasite burden (≤12 eggs/g of feces). The frequency and intensity (p ≤ 0.03) of allergic reactivity were lower in S. mansoni-infected compared with non-infected individuals. Multivariable logistic regression models adjusted by age revealed that allergic reactivity was positively associated with low IL-10 response (OR, 4.55, 95% CI, 0.56-7.36) and high concentration of the inflammatory mediators IL-33 (OR, 2.70, 95% CI, 1.02-7.15) or TNF-α (OR, 6.88, 95% CI, 0.32-143.39) in serum, and inversely associated with S. mansoni infection (OR, 0.38, 95% CI, 0.16-0.87). Most importantly, the logistic regression demonstrated that the modulatory effects of Schistosoma infection depend on parasite burden, with individuals infected with ≤12 eggs/g of feces showing allergic IgE-reactivity similar to non-infected individuals Altogether, our data show that immunomodulation of allergic reactivity depends on S. mansoni burden, low type-2 inflammatory response, and high level of IL-10.

Causes and consequences of higher Leishmania infantum burden in patients with kala-azar: a study of 625 patients.

BACKGROUND: An infected host's Leishmania infantum load in blood is considered to be an estimate of his or her total parasite burden. Therefore, the measurement of blood parasite burden is important in the identification of factors involved in parasite control. METHODS: Quantitative polymerase chain reaction was performed on blood samples from 625 patients with kala-azar consecutively admitted to a reference hospital in Teresina, Brazil. Primers were used to amplify a segment of kDNA using the TaqMan system. Non-parametric statistical tests were applied. RESULTS: The median blood parasite burden was 499.2 amastigote equivalents (AE)/ml. Children <1 year old (yo) had a high parasite burden, which dropped sharply after the first year of life (192.8, AE/ml at 1 < 2 yo) and remained lower until adolescence. Following adolescence, the parasite burden increased with age, peaking among elderly individuals. Men had a higher parasite burden than women. HIV-infected patients had a much higher parasite burden than non-infected patients. The parasite burden of children under 5 years with acute moderate to severe malnourishment (weight-for-age and body mass index z-scores <-2) was almost three times greater than that of better-nourished children. The parasite burden identified in deceased patients was more than twice that of surviving patients; those with a higher risk of death, sepsis, pneumonia and jaundice also had increased parasite burdens. All of these differences were statistically significant at P-values <0.05. CONCLUSIONS: These data indicate that the parasite burden in patients with kala-azar was associated with age- and gender-associated factors and with HIV infection status. Acute malnutrition could be either a cause or a consequence of a higher parasite burden. An individual's parasite burden influences his or her clinical profile, disease severity and mortality risk. The best explanation for the presence of a higher parasite burden in individuals with these immunoregulatory conditions and severe disease is the occurrence of acquired immunosuppression followed by heightened innate immunity.

Quantification by real-time PCR of Trypanosoma cruzi DNA in samples of Triatoma infestans used in xenodiagnosis of chronic Chagas disease patients.

BACKGROUND: Trypanosoma cruzi multiplies and differentiates in the digestive tract of triatomine insects. Xenodiagnosis (XD) is a parasitological tool in which the insect vectors acts as a biological culture medium to amplify and detect T. cruzi infection in mammals. The sensitivity of XD has been overcome by the application of PCR in fecal samples (FS) of XD (PCR-XD). In this study, T. cruzi amplified in Triatoma infestans fed by XD on individuals with chronic Chagas disease (CChD) is quantified by real-time PCR (qPCR-XD). FINDINGS: Under informed consent, 100 individuals were evaluated. In 21 of them XD, PCR-XD and qPCR-XD were positive. For the contrary, 79 were negative XD. In 58 (73.4 %) and 66 cases (83.5 %) of them, PCR-XD (Fisher's exact test P = 0.005) and qPCR-XD (Fisher's exact test: P = 0.037) respectively, were positive. In cases with positive XD, qPCR-XD allowed to establish that in 9/21 cases (42.9 %) the parasite burden fluctuated between 100 and 1,000 par. eq./ml. Otherwise, in 32/79 (40.5 %) cases with negative XD, a parasite burden between 1 and 10 par. eq./ml was determined. All samples showed amplification of exogenous internal control (X12, Ct average: 31.8), so problems in the DNA extraction (excess or loss of genetic material), unspecific amplification and/or inhibition in qPCR-XD reactions were ruled out. Additionally, in all the patients qPCR in blood (qPCR-B) was performed. In the cases with positive XD, the concordance between the positivity of qPCR-XD and qPCR-B was 100 %, nevertheless, the parasite burden in blood was lower and different than XD (Chi-square test: χ (2) = 91.82, df = 5, P = 0.0001). In the cases with negative XD the ranges of qPCR-XD and qPCR-B were similar (Chi-square test: χ (2) = 6.71, df = 5, P = 0.1520). CONCLUSIONS: This study allowed the detection and quantification of T. cruzi by qPCR-XD in FS of Tr. infestans fed on patients with CChD. The highest parasite burden was observed in positive XD cases. qPCR-XD could be used in different studies related with the complex T. cruzi-vector-host interactions.

Chronic Chagas cardiopathy in Chile. Importance of Trypanosoma cruzi burden and clinical evaluation.

Currently there are no biological markers to indicate which individuals with chronic indeterminate period of Chagas disease develop heart disease and who will remain all his life in this phase. The aim of this survey was to determine if Trypanosoma cruzi burden is related to the presence of heart disease in patients with chronic Chagas disease. 200 patients who had not been treated, 100 with cardiopathy and 100 without, groups A and B respectively, were submitted to clinical study and electrocardiogram, Echo-Doppler was performed for group A in which all important known causes of cardiopathy were discarded. In both groups xenodiagnosis, conventional PCR and quantitative PCR were undertaken. The 100 cardiopaths had 133 electrocardiographic alterations most of them in grade II of the New York Heart Association classification. 98 cardiopaths were classified in grade I by Echo-Doppler and only 2 cases were in grade III due to low ejection fraction. The difference in average parasitemia in patients of group A and B was not significant and no statistically differences were observed between average parasitemia of cardiopaths grade II versus grade I of NYHA. This results allow to characterize same clinical, electrocardiographical and parasitological features in chagasic cardiopaths of Chile.

Performance of POC-CCA® in diagnosis of schistosomiasis mansoni in individuals with low parasite burden

Abstract: INTRODUCTION: Schistosomiasis, caused by Schistosoma mansoni, is a public health concern in Brazil. However, the most popular diagnostic method, the Kato-Katz technique, exhibits low sensitivity in low-endemicity areas. We aimed to compare the performance of an immunological assay, the point-of-care circulating cathodic antigen (POC-CCA®) test, with that of two parasitological techniques in a low-endemicity population. METHODS: Our study included 141 individuals living in Estreito de Miralta, Minas Gerais, Brazil. Fecal samples were obtained from all participants and analyzed for schistosomiasis using two parasitological techniques: the Kato-Katz technique and the saline gradient technique. Additionally, POC-CCA® strips were utilized for testing urine samples. The results obtained by the different techniques were compared. RESULTS: Analysis of two or 24 slides using the Kato-Katz technique resulted in a positivity rate of 10.6% (15/141) or 19.1% (27/141), respectively. The saline gradient technique yielded a positivity rate of 17.0% (24/141). The prevalence according to both parasitological techniques was 24.1% (34/141). The POC-CCA® test yielded a positivity rate of 22.7% (32/141); however, the positivity rate was merely 2.1% if trace results were considered negative. The agreements observed between POC-CCA® and the parasitological techniques were good (Kappa indexes > 0.64). The POC-CCA® test was more sensitive than the two-slide Kato-Katz technique (p < 0.05) in detecting cases of S. mansoni infection when trace results were considered positive. CONCLUSIONS: These findings reinforce the importance of using multiple diagnostic techniques in low-endemicity areas for effective control of disease.

Distinct courses of infection with Leishmania (L.) amazonensis are observed in BALB/c, BALB/c nude and C57BL/6 mice.

Leishmania (L.) amazonensis [L. (L.) amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects of L. (L.) amazonensis infection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden by in vivo imaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophages in vitro, indicating that in vivo milieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course of L. (L.) amazonensis infection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.

Strength and Aerobic Physical Exercises Are Able to Increase Survival of Toxoplasma gondii-Infected C57BL/6 Mice by Interfering in the IFN-γ Expression.

Physical exercise has been implicated in several immunophysiological improvements, particularly during the aging process, when an immunocompromised status could be established. Toxoplasma gondii is a protozoan parasite that causes a widespread opportunistic infection, which may present severe consequences, mainly to the fetus and immunocompromised patients. It is estimated that one-third of the human population worldwide has been infected by this parasite, being the reactivation during immunesenescence an unexplored public health issue. The major purpose of the present study was to observe the immunophysiological differences between exercised vs. sedentary C57BL/6 male mice that have been experimentally infected by T. gondii. In the first set of experiments, the animals were infected after exercising and three groups were set up: experimental groups-infected sedentary (IS, n = 6); infected exercised (IEx, n = 6) and control group-non-infected sedentary (NIS, n = 6). When stimulated in vitro by T. gondii-soluble tachyzoite antigen, it was found that splenocytes from exercised group produced higher levels of IFN-γ, as well as of IFN-γ/IL-10 ratios in comparison with splenocytes from sedentary animals (P < 0.001). However, it was not found significant differences concerning quantification of T. gondii genomic DNA by qRT-PCR and immunohistochemistry analysis in brain cysts from both group of animals (P > 0.05). In order to further investigate the consequences of these data for the host, a second set of experiments was performed, when the animals were infected before exercising and four groups of animals were established for comparison purpose, as follows: experimental groups-infected sedentary (IS, n = 7); infected exercised (IEx, n = 6) and control groups-non-infected sedentary (NIS, n = 6) and non-infected exercised (NIEx, n = 6). It was found significant differences in the survival rates of the exercised group the animals, as they survived longer than sedentary groups (P = 0.0005). In both sets of experiments, mice have been submitted to moderate exercises: aerobic (14 m/min; 3 x/week) and strength (60-80% of one maximum repetition; 2 x/week). Overall, our findings are showing that the aerobic and strength exercises are able to modulate immune response against T. gondii infection, being these immunological features beneficial to the host.

Schistosoma mansoni in a low-prevalence area in Brazil: the importance of additional methods for the diagnosis of hard-to-detect individual carriers by low-cost immunological assays

Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.

Experimental infection with Leishmania chagasi in immunosuppressed Balb/c mice: cytokines and parasite burdens

The immune response in leishmaniasis may result in a polarization of the T lymphocyte subpopulation, altering cell phenotype and resulting in immune protection or disease exacerbation. Leishmania may persist in the body either during asymptomatic infections or after treatment, which represents high risk under immunosuppression. The objective of this study was to evaluate the effect of infection with immunosuppression by dexamethasone associated with pentoxifylline on animal weight, spleen weight, spleen and hepatic parasitic load and immunopathology, as well as the IFN-ã and IL-10 production in spleen cell culture of Balb/c mice infected with Leishmania chagasi. The infection did not cause body weight gain in animals, but both the weight and size of the spleen were increased. The immunosuppression using dexamethasone associated with pentoxifylline affected body weight gain and spleen weight and size in both infected and non-infected animals. The immunosuppression did not significantly alter the course of the splenic or hepatic parasite burden. Dexamethasone and pentoxifylline significantly affected cytokine production, but did not influence the Th1/Th2 ratio in infected animals.(AU)