Effects of specific treatment on parasitological and histopathological parameters in mice infected with different Trypanosoma cruzi clonal genotypes

The goal of this study was to verify the effect of specific treatment on parasitological and histopathological parameters in mice experimentally infected with different Trypanosoma cruzi clonal genotypes. Twenty cloned stocks were selected, representative of the whole phylogenetic diversity of the protozoan and belonging to the clonal genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II). The stocks were inoculated in 40 BALB/c mice divided into four groups: (i) treated with benznidazole, (ii) treated with itraconazole and (iii and iv) untreated control groups (NT) for each drug, respectively. Seven parameters related to parasitaemia curves and histopathological lesions were analysed. Four during the acute phase (AP) and three during both the AP and chronic phase (CP) of infection. Statistical comparison between benznidazole-treated and NT groups for the biological parameters showed significant differences for all genotypes. Benznidazole treatment led to lower patent period, maximum of parasitaemia, day of maximum parasitaemia and area under the parasitaemia curve for all genotypes analysed. Percentage of positive haemoculture during AP and CP was lower for genotypes 19 and 32. Tissue parasitism (TP) and inflammatory process (IP) during AP were lower for genotypes 19 and 32, respectively. In general, itraconazole treatment induced a smaller reduction in these same parameters between treated and NT animals in relation to benznidazole treatment. Our results indicate that phylogenetic divergence among T. cruzi clonal genotypes must be taken in account in chemotherapy and studies dealing with all aspects of the parasite and the disease.     

Impact of dual infections on chemotherapeutic efficacy in BALB/c mice infected with major genotypes of Trypanosoma cruzi

The aim of this work was to investigate the impact of dual infections with stocks of Trypanosoma cruzi major genotypes on benznidazole (BZ) treatment efficacy. For this purpose, T. cruzi stocks representative of the genetic T. cruzi lineages, displaying different susceptibilities to BZ, belonging to the major T. cruzi genotypes broadly dispersed in North and South America and important in Chagas' disease epidemiology were used. Therapeutic efficacy was observed in 27.8% of the animals treated. Following BZ susceptibility classification, significant differences were observed in dual infections on the major genotype level, demonstrating that combinations of genotypes 19+39 and genotypes 19+32 led to a shift in the expected BZ susceptibility profile toward the resistance pattern. Analysis on the T. cruzi stock level demonstrated that 9 out of 24 dual infections shifted the expected BZ susceptibility profile compared with the respective single infections, including shifts toward lower and higher BZ susceptibilities. Microsatellite identification was able to identify a mixture of T. cruzi stocks in 7.7% of the T. cruzi isolates from infected and untreated mice (6.9%) and infected and treated but not cured mice (9.0%), revealing in some mixtures of BZ-susceptible and -resistant stocks that the T. cruzi stock identified after BZ treatment was previously susceptible in single infections. Considering the clonal structure and evolution of T. cruzi, an unexpected result was the identification of parasite subpopulations with distinct microsatellite alleles in relation to the original stocks observed in 12.2% of the isolates. Taken together, the data suggest that mixed infections, already verified in nature, may have an important impact on the efficacy of chemotherapy.     

Chemotherapy with benznidazole and itraconazole for mice infected with different Trypanosoma cruzi clonal genotypes

The benznidazole (BZ) and itraconazole (ITC) susceptibilities of a standard set of Trypanosoma cruzi natural stocks were evaluated during the acute phase and the chronic phase of experimental chagasic infection in BALB/c mice. Twenty laboratory-cloned stocks representative of the total phylogenetic diversity of T. cruzi, including genotypes 20 and 19 (T. cruzi I) and genotypes 39 and 32 (T. cruzi II), were analyzed. Our results demonstrate important differences among stocks that could be pointed out as markers of biological behavior. Members of the T. cruzi I group were highly resistant to both BZ and ITC, whereas members of the T. cruzi II group were partially resistant to both drugs, despite their susceptibilities to ITC during the chronic phase of infection. The resistance to BZ observed for T. cruzi I was mainly triggered by genotype 20 isolates, whereas resistance to ITC was due to both genotype 20 and 19 isolates. Two polar patterns of response to BZ observed for genotype 39 isolates had a major impact on the partial resistance pattern observed for members of the T. cruzi II group. Genotype 32 isolates showed a typical profile of susceptibility. The correlation between the response to treatment and phylogenetic classification of T. cruzi stocks was clearer for ITC than for BZ. In conclusion, the data presented show a correlation between phylogenetic divergence among T. cruzi stocks and their susceptibilities to chemotherapeutic agents in vivo. Our results warn of the necessity to take into account the lesser genetic subdivisions of T. cruzi stocks since the upper subdivisions (T. cruzi I and II) show a great deal of heterogeneity for in vivo drug susceptibility.     

Killing in vitro of Trypanosoma cruzi by macrophages from mice immunized with T. cruzi or BCG, and absence of cross-immunity on challege in vivo

Peritoneal macrophages from T. cruzi-immune mice were resistant to infection in vitro with culture forms of the parasite. Macrophage resistance appeared in infected mice about 21 days postinfection when parasitemia was still rising. Resistance in vitro was nonspecific since macrophages from BCG-immune mice were resistant to T. cruzi, and since macrophages from T. cruzi-immune mice were resistant to infection in vitro with Listeria were not resistant to challenge with T. cruzi even when the parasites were opsonized.     

Evaluating pathogen reduction of Trypanosoma cruzi with riboflavin and ultraviolet light for whole blood

BACKGROUND: Trypanosoma cruzi, the protozoan parasitic agent of Chagas disease, can be transmitted by blood transfusion. In 2007, most US blood banks started screening blood donations for T. cruzi, but the cost and perceived need of the test have been the subject of ongoing discussion. In this study, we evaluated the ability of the Mirasol System (CaridianBCT), which uses riboflavin (RB) and ultraviolet light to inactivate pathogens, to reduce the levels of infectious T. cruzi in whole blood (WB). STUDY DESIGN AND METHODS: WB units were inoculated with 4, 40, 400, and 4000 trypomastigotes/mL. After addition of RB and illumination at various energy levels, the samples were tested for the presence of live parasites by hemoculture. RESULTS: All preillumination samples exhibited T. cruzi growth in hemoculture, while postillumination samples from units containing 4 and 40 trypomastigotes/mL showed no signs of viable parasites after 16 weeks of culture. In contrast, at both 400 and 4000 parasites/mL, two of the three units were positive for viable parasites. CONCLUSIONS: The total log reduction observed for T. cruzi was 3.5 log or greater, but less than 4.5 log. This level of reduction is likely to be orders of magnitude higher than what would be expected in a tainted blood donation, indicating that the Mirasol System could be effective at preventing transfusion of the causative agent of Chagas disease.     

Epidemiologic and laboratory findings from 3 years of testing United States blood donors for Trypanosoma cruzi

BACKGROUND: At most blood centers in the United States routine testing of donations for Trypanosoma cruzi using an enzyme‐linked immunosorbent assay (ELISA) is followed by supplemental testing by radioimmunoprecipitation assay (RIPA). The objective of this study was to report the results of routine testing and risk factor data from allogeneic blood donors. STUDY DESIGN AND METHODS: T. cruzi testing data from January 2007 through December 2009 were analyzed, and risk factor interviews and follow‐up studies were conducted on seroreactive donors. Prevalences of confirmed infection and risk factors associated with infection were assessed using logistic and multivariable logistic regression. RESULTS: Of 2,940,491 allogeneic donations from 1,183,076 donors, 305 (0.01% per donation tested and 0.026% per blood donor) were repeat reactive (RR) and 89 of those were confirmed positive by RIPA, yielding an overall seroprevalence of 1 per 33,039 donations and 1 per 13,292 donors. Country of birth and US blood center location differences in the seroprevalence of T. cruzi were evident. The odds of confirmed infection were highest if the donor reported having been bitten by the reduviid (kissing) bug (odds ratio [OR], 76.1; 95% confidence interval [CI], 11.1‐3173) followed by having lived in a rural area of Latin America (OR, 38.6; 95% CI, 15.1‐102.5). In multivariable analyses, having spent 3 months or more in Mexico or Central and/or South America was associated with the highest odds of RIPA‐confirmed infection (OR, 8.5; 95% CI, 2.7‐26.5). Polymerase chain reaction (PCR) testing of ELISA RR donors exhibited low sensitivity (1/22 [4%] RIPA‐confirmed donors was PCR positive). CONCLUSION: Risk factors for confirmed infection in US blood donors are consistent with the known epidemiology of Chagas disease. Blood donors or transfusions do not substantially contribute to the burden of T. cruzi infection in the United States.     

Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of S-adenosylmethionine decarboxylase.

A structural analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxy adenosine (MDL 73811), of decarboxy S-adenosyl-L-methionine, the product of the reaction catalyzed by S-adenosyl-L-methionine (AdoMet) decarboxylase (DC), was found to inhibit Trypanosoma brucei brucei AdoMet DC. The inhibition was time dependent (tau 50, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 microM), and was apparently irreversible. The natural substrate of the reaction, AdoMet, protected the enzyme from inactivation, suggesting that MDL 73811 was directed at the enzyme active site and was probably catalytically activated. Administration of MDL 73811 to T. b. brucei-infected rats resulted in rapid inhibition of AdoMet DC activity, a decrease in spermidine, and an increase in putrescine in the trypanosomes isolated from treated rats. Treatment of T. b. brucei-infected mice with MDL 73811 (20 mg/kg of body weight intraperitoneally twice daily for 4 days) resulted in cures of the trypanosome infections. Additionally, drug-resistant T. brucei rhodesiense infections in mice were cured by either a combination of MDL 73811 (50 mg/kg intraperitoneally three times per day for 5 days) and relatively low oral doses of alpha-difluoromethylornithine or MDL 73811 (50 mg/kg per day for 7 days) administered alone in implanted miniosmotic pumps. These data suggest that MDL 73811 and, perhaps, other inhibitors of AdoMet DC have potential for therapeutic use in various forms of African trypanosomiasis.     

Prevention of transfusion-associated Chagas' disease: efficacy of white cell-reduction filters in removing Trypanosoma cruzi from infected blood

BACKGROUND : Transfusion-associated Chagas' disease (TA-CD) is a worldwide problem. Measures adopted to prevent TA-CD include the clinical and serologic screening of blood donors and/or the inactivation of Trypanosoma cruzi present in collected blood, using gentian violet as the trypanocidal agent. This study investigated the efficacy of white cell-reduction filters in removing T. cruzi from infected blood. STUDY DESIGN AND METHODS : Human blood was contaminated with 2 or 150 T. cruzi parasites per mL and then left unfiltered or filtered with white cell-reduction filters that provided either 2, 3, or 6 log10 white cell removal. The efficacy of the parasite removal of these filters was evaluated by microscopic enumeration of active forms of T. cruzi both in vivo and in vitro. The in vivo experiments were done in Swiss mice that had been intraperitoneally inoculated with T. cruzi-infected human blood. The in vitro experiments were performed with fresh human blood that had been deliberately contaminated with T. cruzi. RESULTS : The number of parasites seen in mice inoculated with unfiltered blood containing 2 or 150 parasites per mL was significantly higher than the number of parasites seen in mice inoculated with blood from the same sample, but filtered with white cell-reduction filters providing 3 or 6 log10 white cell removal. Fifty to 70 percent of the mice given T. cruzi-infected (2 parasites/mL) filtered blood did not develop T. cruzi infection. In vitro, the use of white cell-reduction filters, providing 2, 3, or 6 log10 white cell removal, significantly reduced the number of parasites seen in culture. CONCLUSION : The present experimental data provide evidence that white cell-reduction filters are effective in reducing the number of parasites in T. cruzi- infected blood and that this efficacy depends, in part, on the concentration of parasites in the artificially infected blood. Properly designed clinical studies of known carriers of T. cruzi must be conducted to determine whether the use of white cell-reduction filters may be an alternative method of reducing the incidence of TA-CD.     

Estimation of sensitivity and specificity of several Trypanosoma cruzi antibody assays in blood donors in Argentina

BACKGROUND: The absence of a gold standard test for Trypanosoma cruzi antibodies represents a problem not only for the evaluation of screening tests, but also for appropriate blood donor counseling. The aim of this study was to estimate the sensitivity and specificity of multiple blood donor screening tests for T. cruzi antibodies in Argentina.
STUDY DESIGN AND METHODS: From June 2006 to March 2007 a sample of 1455 blood donors was recruited from two blood banks in Chaco province, an area of Argentina with highly endemic T. cruzi infection. Samples were tested by three epimastigote lysate enzyme immunoassays (EIAs), one recombinant antigen EIA, two indirect hemagglutination assay (IHA) tests, a particle agglutination assay (PA), and a research trans -sialidase inhibition assay (TIA). Sensitivity and specificity were estimated using latent class analysis (LCA).
RESULTS: LCA estimated the consensus prevalence of T. cruzi infection at 24.5%. Interassay correlation was higher among the four EIA tests and TIA compared to IHA tests. Assay sensitivities varied from 96 to 99.7 for different EIAs, 91% for TIA, 84% for PA, and 66 to 74% for IHA tests. Relative to the LCA, assay specificities were from 96% to almost 100%.
CONCLUSION: Based on the comparison of several tests in a large population from an endemic area for T. cruzi infection, our data showed an adequate sensitivity for EIA tests in contrast to PA and IHA assays. The latter tests should no longer be used for blood donor screening.     

Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta)

The effects of transforming growth factor beta (TGF-beta) on interferon gamma-mediated killing of the intracellular protozoan parasite Trypanosoma cruzi and on the course of T. cruzi infection in mice were investigated. Spleen cells from mice with acute T. cruzi infections were found to produce elevated levels of biologically active TGF-beta in vitro, and the possibility that TGF-beta may mediate certain aspects of T. cruzi infection was then addressed. When mouse peritoneal macrophages were treated with TGF-beta in vitro, the ability of IFN-gamma to activate intracellular inhibition of the parasite was blocked. This occurred whether cells were treated with TGF-beta either before or after IFN-gamma treatment. TGF-beta treatment also blocked the T. cruzi-inhibiting effects of IGN-gamma on human macrophages. Additionally, treatment of human macrophages with TGF-beta alone led to increased parasite replication in these cells. The effects of TGF-beta on T. cruzi infection in vivo were then investigated. Susceptible C57BL/6 mice developed higher parasitemias and died earlier when treated with TGF-beta during the course of infection. Resistant C57BL/6 x DBA/2 F1 mice treated with TGF-beta also had increased parasitemias, and 50% mortality, compared with no mortality in infected, saline-treated controls. A single dose of TGF-beta, given at the time of infection, was sufficient to significantly decrease resistance to infection in F1 mice and to exacerbate infection in susceptible C57BL/6 mice. Furthermore, a single injection of TGF-beta was sufficient to counter the in vivo protective effects of IFN-gamma. We conclude that TGF-beta, produced during acute T. cruzi infection in mice, is a potent inhibitor of the effects of macrophage activating cytokines in vivo and in vitro and may play a role in regulating infection.     

Reactivity of chagasic sera with crude and highly purified glycosphingolipid fractions from Trypanosoma cruzi epimastigotes

The reactivities of sera from patients with Chagas disease or from T. cruzi-immunized rabbits with two different lipid preparations of T. cruzi were assessed using epimastigote antigens. Serum reactivities were determined using a quantitative enzyme-linked immunosorbent assay (ELISA). Antigen 1 represents the lower phase obtained from crude lipid extract after Folch partition (LCL). Antigen 2 is a highly purified glycosphingolipid fraction (GSL). The LCL antigen discriminated quite well the reactivities of Chagasic patients' sera and sera from healthy individuals, as well as between the serum from a T. cruzi-immunized rabbit (TIRS) and normal rabbit serum (NRS). A strong reactivity with GSL was obtained with TIRS. Reactivity with GSL was also obtained with human Chagasic sera. Compared to a group of normal individuals, the reactions of antibodies directed against lipid antigens were considerably increased in sera of patients with Chagas disease. Chagasic sera did not differentiate between glycolipids with terminal β-glucosyl or β-galactosyl nonreducing units. They discriminated, however, glucosylceramides with differences in the ceramide structure. To determine the specificity of Chagasic sera, antibodies isolated on LCL-immunosorbent (LCL-Ch Abs) as well as on laminin-immunosorbent (Lam-Ch Abs) were tested against laminin and LCL antigens. We found that Lam-Ch Abs reacted with murine laminin, whereas the reaction was negative with LCL. In contrast, the LCL-Ch Abs reacted either with LCL antigens or with laminin. The reactivity with laminin was strong in comparison with LCL. Results suggest that although the glycolipids are recognized by Chagasic patients' sera, the reactive antibodies are not specific since they reacted quite well with murine laminin. Polyspecific antibodies from Chagasic sera have already been described using other unrelated antigens. © 1994 Wiley-Liss, Inc.     

南充市Rh阴性献血者血清学表型和不规则抗体调查

目的调查南充市Rh阴性无偿献血者的血清学表型和不规则抗体情况。方法应用血型血清学方法对初检Rh阴性的献血者进行Rh阴性确认、血清学表型鉴定及不规则抗体筛选鉴定。结果在433份送检标本中共确认Rh阴性402份(92.8%),检出D变异体23份(5.3%),其余8份(1.9%)为检验科RhD检测错误;共检出不规则抗体12份,在Rh确认阴性献血者中检出7份抗-D(1.7%);Rh阴性献血者的血清学表型分布结果主要以dccee和dCcee为主,其余为dCCee、dccEe和dCcEe。结论通过本次调查,掌握了南充市Rh阴性无偿献血者的血清学表型和不规则抗体的分布情况,保障了临床Rh阴性患者输血安全性、有效性和及时性。     

The validity of serologic tests for Trypanosoma cruzi and the effectiveness of transfusional screening strategies in a hyperendemic region

BACKGROUND: This study aims at obtaining unbiased estimates of the sensitivity and specificity of existing screening tests for Trypanosoma cruzi and at simulating the effectiveness of alternative screening strategies at different prevalence rates. STUDY DESIGN AND METHODS: A systematic random sample of 400 was taken from 1200 banked serum samples of donors screened between August 1998 and January 1999 in Santa Cruz, Bolivia. Samples were tested with indirect hemagglutination test (IHA), indirect immunofluorescence assay (IFA), and four enzyme-linked immunosorbent assays (ELISAs). Sensitivity and specificity of tests were estimated through latent class analysis. RESULTS: The sensitivity of individual tests ranged from 96.5 to 100 percent, and their specificity from 87.0 to 98.9 percent. Combinations of two tests used in parallel would, even at 40 percent prevalence, only miss approximately 1 infected unit per 10,000 screened. At 5 percent prevalence, however, they would yield 75 to 120 false-positive units per 1000 units screened. Parallel testing with IHA plus ELISA or with IHA plus IFA is marginally more cost-effective, compared to single IHA testing, than single ELISA or single IFA testing, regardless of the T. cruzi prevalence. CONCLUSIONS: Routine blood donor screening for T. cruzi with a single test results in unacceptable numbers of false-negative samples in highly endemic areas or in at risk population groups. Adding a second test seems mandatory, but which one to choose depends on local cost components and feasibility.     

HBV基因型在不同临床感染者中的分析和干扰素治疗应答

目的:探讨山东地区HBV基因型在不同病情感染者之间的分布和基因型与干扰素治疗应答的关系.方法:随机选择249例慢性HBV感染者,用巢式PCR进行A-F基因型别分析,对其中126例慢性乙型肝炎患者采用α干扰素治疗,300万u/次,肌肉注射,3次/周,疗程6个月,所有患者均在治疗前、治疗期间及治疗后检测肝功能、HBV血清学指标及HBV DNA.结果:249例中无症状携带者53例,慢性肝炎126例,肝硬化38例,重型肝炎32例.在A-F基因分型中,无症状携带者组以B基因型为主28/53(52.8%),而C基因型则在活动性肝病中占优势:慢性肝炎69/126(54.8%);肝硬化20/38(52.6%);重型肝炎17/32(53.1%),差异有统计学意义(P<0.05).在慢性肝炎组中,B基因型组HBV DNA定量及ALT水平均较C基因型组和B C基因型组为低,差异有统计学意义(P<0.01).不同基因型组间,年龄、性别差异无统计学意义(P>0.05).126例慢性乙型肝炎患者用α干扰素治疗6个月,完全应答率:B型19/38(50%),C型9/69(13%),B C型5/19(26.3%),与B基因型和B C基因型相比,C基因型对干扰素抗病毒治疗应答率显著低(P<0.05).结论:山东地区HBV C基因型在活动性肝病中占优势;HBV C基因型对α干扰素抗病毒治疗的完全应答率较低. 炎组中,B基因型组HBV DNA定量及ALT水平均较C基因型组和B C基因型组为低,差异有统计学意义(P<0.01).不同基因型组问,年龄、性别差异无统计学意义(P>0.05).126例慢性乙型肝炎患者用α干扰素治疗6个月,完全应答率:B型19/38(50%),C型9/69(13%),B C型5/19(26.3%),与B基因型和B C基因型相比 C基因型对干扰素抗病毒治疗应答率显著低(P<0.05).结论:山东地区HBV C基因型在活动性肝病中占优势;HBV C基因型对α干扰素抗病毒治疗的完全应答率较低. 炎组中,B基因型组HBV DNA定量及ALT水平均较C基因型组和B C基因型组为低,差异有统计学意义(P<0.01).不同基因型组问,年龄、性别差异无统计学意义(P>0.05).126例慢性乙