Antibody drop in newborns congenitally infected by Trypanosoma cruzi treated with benznidazole

Objective  To compare the drop of Chagas antibody titres between non-infected and congenitally infected newborns treated by two doses of benznidazole, aiming at evaluating the recovery time and giving recommendations regarding serological criteria of recovery.
Methods  During a clinical trial, the drop of Trypanosoma cruzi antibody titres measured by ELISA tests was followed during the first year of life in congenitally infected newborns treated with different doses of benznidazole and compared to T. cruzi antibody titres in non-parasitaemic newborns. Confirmation of recovery was given by two negative serological tests: Chagas Stat-Pak^® (CSP) (immunochromatography) and Chagatest^® v3.0 (ELISA).
Results  In non-parasitaemic infants of infected mothers, antibodies of maternal origin disappeared in <8 months while in infected infants, T. cruzi antibodies decreased more slowly and disappeared in 9–16 months allowing to confirm the recovery. All CSP tests were negative before the ninth month while about 10% of ELISA tests remained positive at the 12th month.
Conclusions  Recovery may be confirmed in most cases at 10 months. The CSP test was compared to Chagatest^® v3.0 ELISA and appeared to give a reliable response. The decrease rate of antibodies does not depend on treatment modes.     

Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples

The aim of this study was to develop a real-time PCR technique to detect Trypanosoma cruzi DNA in blood of chagasic patients. Analytical sensitivity of the real-time PCR was assessed by two-fold serial dilutions of T. cruzi epimastigotes in seronegative blood (7.8 down to 0.06 epimastigotes/mL). Clinical sensitivity was tested in 38 blood samples from adult chronic chagasic patients and 1 blood sample from a child with an acute congenital infection. Specificity was assessed with 100 seronegative subjects from endemic areas, 24 seronegative subjects from non-endemic area and 20 patients with Leishmania infantum-visceral leishmaniosis. Real-time PCR was designed to amplify a fragment of 166 bp in the satellite DNA of T. cruzi. As internal control of amplification human RNase P gene was coamplified, and uracil-N-glycosylase (UNG) was added to the reaction to avoid false positives due to PCR contamination. Samples were also analysed by a previously described nested PCR (N-PCR) that amplifies the same DNA region as the real-time PCR. Sensitivity of the real-time PCR was 0.8 parasites/mL (50% positive hit rate) and 2 parasites/mL (95% positive hit rate). None of the seronegative samples was positive by real-time PCR, resulting in 100% specificity. Sixteen out of 39 patients were positive by real-time PCR (41%). Concordance of results with the N-PCR was 90%. In conclusion, real-time PCR provides an optimal alternative to N-PCR, with similar sensitivity and higher throughput, and could help determine ongoing parasitaemia in chagasic patients.     

Congenital transmission of Trypanosoma cruzi: an operational outline for detecting and treating infected infants in north-western Argentina

We designed a set of procedures for first-line local health services to detect and treat the congenital transmission of Trypanosoma cruzi at a province-wide scale, and field-tested the programme in the province of Tucumán, northwestern Argentina, from 1992 to 1994. The programme consists of routine screening of pregnant women for seroreactivity to T. cruzi, serological and parasitological follow-up of the newborn at least twice during the first year of age, treatment of the infected infants, and evaluation of the outcome. 927 (5.5%) of 16 842 pregnant women were seroreactive to T. cruzi by indirect haemagglutination assay and ELISA. Twenty-one (6.7%) of 315 newborns to seroreactive mothers were diagnosed as infected with T. cruzi parasites microhaematocrit concentration before 30 days of age. Five newborns who initially tested negative had a T. cruzi infection detected by microhaematocrit and/or serological techniques at 3 or 6 months of age. Thus, congenital infection was diagnosed in 26 (7.1%) infants born to seroreactive women and residing in houses free of triatomine bugs. Four of 6 infants born to seroreactive mothers died during the first year of age and had some evidence of T. cruzi infection; one of the deaths was attributed to T. cruzi based on clinical evidence. After specific treatment with nifurtimox or benznidazole, 30 of 32 infants remained parasitologically and serologically negative. This study shows the feasibility of controlling the incidence of congenitally acquired T. cruzi infections at a province-wide scale by means of a specific screening programme at first-line health services level.     

Trypanosoma cruzi TcIII / Z3 genotype as agent of an outbreak of Chagas disease in the Brazilian Western Amazonia

Chagas’ disease is an emerging and neglected disease in the Brazilian Amazon region, where T. cruzi I predominates among the acute cases of the disease; and T. cruzi III/Z3, a population cluster from sylvatic areas of the Amazon basin, is rarely associated with human infections. On 23rd April 2007, the Foundation for Health Surveillance of the State of Amazonas, Brazil reported an outbreak of acute Chagas disease in the municipality of Coari on the Solimões River banks. Fresh blood examination confirmed the infection in 25 patients. Parasite culture in LIT medium was successful for 18 isolates. Molecular characterization was performed by PCR of the non‐transcribed spacer of the mini‐exon and by sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene. The T. cruzi isolates were all from genotype Z3, and sequencing revealed that all isolates had equal COII sequences compatible with TcIII type, suggesting a single source of infection. To our knowledge, this is the first outbreak of acute cases caused uniquely by the genotype TcIII/Z3. Wild vectors harbouring TcIII stocks contribute to transmission when the triatomine species reaches human food chain or when humans invade the forest environment, where sylvatic cycle constitutes a reservoir of parasites that might be associated with specific epidemiological and clinical traits of the emergent Chagas disease in the Amazon.     

Infestation of peridomestic Attalea phalerata palms by Rhodnius stali,a vector of Trypanosoma cruzi in the Alto Beni,Bolivia

Objectives To determine (i) whether peridomestic Attalea phalerata palms in fragmented human‐occupied areas of the Alto Beni, Bolivia, are infested by triatomines; (ii) the specific status of triatomines captured in the area; and (iii) the rate of natural Trypanosoma cruzi infection among those triatomines. Methods One hundred and twenty‐five live‐bait traps were used to sample 47 A. phalerata palms in three Alto Beni localities. Active search for vectors was also performed in 10 chicken coops and three rice storage units. Only Rhodnius specimens were found. As nymphs of closely related Rhodnius species are morphologically undistinguishable, and because of controversy in the literature regarding which Rhodnius species occur in Bolivia, collected insects were identified through molecular taxonomy. Phylogenetic analyses of DNA sequences obtained for a fragment of the mitochondrial cytochrome b gene and for the nuclear ITS‐2 ribosomal region were used as molecular markers. Natural infection rates were determined using a pair of primers that PCR‐amplify a 330‐bp fragment of the parasite’s kDNA. Results Twelve nymphs were captured in five A. phalerata palms (from two of the three localities studied), and an adult was collected from a chicken coop in Iniqua (and morphologically identified as Rhodnius stali). All nymphs (as well as the adult) were molecularly identified as R. stali based on the two molecular markers used. A single nymph was found to be infected with T. cruzi. Conclusions Attalea phalerata palms represent an important sylvatic ecotope occupied by R. stali in the Alto Beni region of Bolivia, where there are signs of T. cruzi transmission to humans, despite the preliminary indication of low level of natural infection of the vectors.     

Comparison of Trypanosoma cruzi detection by PCR in blood and dejections of Triatoma infestans fed on patients with chronic Chagas disease

In this study, we compare the sensitivity of detecting Trypanosoma cruzi in dejections of Triatoma infestans nymphs that had fed on the blood of chronic chagasic patients, with detection of T. cruzi in peripheral blood, using a polymerase chain reaction assay (PCR-D and PCR-B, respectively). Fifty-seven chronic patients were evaluated who were positive (group I) or negative (group II) by xenodiagnosis (XD). Patients showed 84.8 and 75% positive PCR results in both kinds of samples in groups I and II, respectively. Six cases (10.5%) showed positive PCR-D and negative PCR-B, five of them belonged to group I. In contrast, five cases of group II showed negative PCR-D and positive PCR-B. Overall, the PCR-D assay gave positive results in 52 out of 57 samples (91.2%), while 51 out of 57 (89.5%) were positive by PCR-B. In comparison, only 57.9% were positive by XD (p = 0.0001). In conclusion, PCR performed in dejection or blood was more sensitive for the parasite detection than xenodiagnosis. All patients (100%) were detected positive when both, PCR-D and PCR-B, were applied.     

Myocardial scars correlate with eletrocardiographic changes in chronic Trypanosoma cruzi infection for dogs treated with Benznidazole

Objectives The cardiac form of Chagas disease is evidenced by a progressive cardiac inflammation that leads to myocarditis, fibrosis and electrocardiographic (ECG) conduction abnormalities. Considering these characteristics, the aim of this study was to prospectively evaluate the early ECG changes in dogs that were experimentally inoculated with Benznidazole (Bz)‐susceptibly (Berenice‐78) and Bz‐resistant (VL‐10, and AAS) Trypanosoma cruzi strains and, later, evaluate the efficacy of Bz treatment for preventing these ECG alterations. Methods Electrocardiographic changes of treated and untreated animals were prospectively evaluated for up to 270 days after infection, at which point collagen (right atrium) quantification was performed. Results All infected dogs had a high intensity of heart fibrosis (4616.00 ± 1715.82 collagen/74931 μm² in dogs infected with Berenice‐78 strain, 5839.2 ± 1423.49 collagen/74931 μm² in infected by AAS and 6294.40 ± 896.04 collagen/74931 μm² in animals infected with VL‐10 strain), while 78.57% of all infected dogs showed ECG alterations. Bz Therapy reduced or prevented fibrosis in Bz‐susceptible Berenice‐78 (2813.00 ± 607.13 collagen/74931 μm²) and Bz‐resistant AAS strains (4024 ± 1272.44 collagen/74931 μm²), coincident with only 10% de ECG alterations at 270 days. However, in those animals infected with a Bz‐resistant VL‐10 strain, specific treatment did not alter collagen deposition (6749.5 ± 1596.35 collagen/74931 μm²) and there was first atrioventricular block and chamber overload at 120 and 270 days after infection, with 75% abnormal ECG exams. Conclusions These findings indicate that an effective antiparasitic treatment in the early stage of Chagas disease can lead to a significant reduction in the frequency and severity of the parasite‐induced cardiac disease, even if parasites are not completely eliminated.     

Inconclusive results in conventional serological screening for Chagas' disease in blood banks: evaluation of cellular and humoral response

Objective To find the most reliable screening method for Trypanosoma cruzi infection in blood banks. Material and methods Epidemiological data, lymphoproliferation assay, parasitological, conventional serological tests: immunofluorescence, haemagglutination, ELISA with epimastigote and trypomastigote antigens and reference serological tests: trypomastigote excreted‐secreted antigens (TESA) blot and chemiluminescent ELISA assay with mucine from trypomastigote forms were applied to individuals with inconclusive serology, non‐chagasic individuals and chronic chagasic patients. Results TESA blot had the best performance when used as a single test in all the groups. In the inconclusive group 20.5% of individuals were positive for TESA blot, 23.3% for either lymphoproliferation or TESA blot, and 17.8% for lymphoproliferation only. Positive lymphoproliferation without detectable antibodies was observed in 5.47% of all inconclusive serology cases. Analysis of six parameters (three serological assays, at least one parasitological test, one lymphoproliferation assay and epidemiological data) in the inconclusive group showed that diagnosis of Chagas’ disease was probable in 15 patients who were positive by two or more serological tests or for whom three of those six parameters were positive. Conclusion TESA blot is a good confirmatory test for Chagas’ disease in the inconclusive group. Although lymphoproliferation suggests the diagnosis of Chagas’ disease in the absence of antibodies when associated with a high epidemiological risk of acquiring Chagas’ disease, the data from this study and the characteristics of the lymphoproliferation assay (which is both laborious and time‐consuming) do not support its use as a confirmatory test in blood‐bank screening. However, our findings underscore the need to develop alternative methods that are not based on antibody detection to improve the diagnosis when serological tests are inconclusive.     

Metabolic,mental health,behavioural and socioeconomic characteristics of migrants with Chagas disease in a non‐endemic country

Objectives Chronic Chagas disease causes cardiopathy in 20–40% of the 8–10 million people affected. The prevalence of atherogenic factors increases rapidly in Latin America. Somatic, mental, behavioural and social characteristics of the 80 000 Latino migrants with Chagas disease in Europe are not known. We postulate that they may accumulate these factors for poor health – notably cardiovascular‐outcomes. Methods This study took place at the Geneva University Hospitals in 2011. Latin American migrants with Chagas disease diagnosed in Geneva since 2008 were contacted. Interviews and blood tests assessed behavioural, socioeconomic, metabolic and cardiovascular factors. Results One hundred and thirty‐seven patients (women: 84.7%; median age: 43 years) with chronic Chagas disease were included in the study. The majority were Bolivians (94.2%), undocumented (83.3%), uninsured (72.3%) and living below the Swiss poverty line (89.1%). Prevalence of obesity was 25.5%, of hypertension 17.5%, of hypercholesterolemia 16.1%, of impaired fasting glucose 23.4%, of diabetes 2.9%, of metabolic syndrome 16.8%, of anxiety 58.4%, of depression 28.5%, of current smoking 15.4% and of sedentary lifestyle 62.8%. High (>10%) 10‐year cardiovascular risk affected 12.4%. Conclusions Latin American migrants with Chagas disease accumulate pathogenic chronic conditions of infectious, non‐transmissible, socioeconomic and behavioural origin, putting them at high risk of poor health, notably cardiovascular, outcomes. This highlights the importance of screening for these factors and providing interventions to tackle reversible disorders; facilitating access to care for this hard‐to‐reach population to prevent delays in medical interventions and poorer health outcomes; and launching prospective studies to evaluate the long‐term impact of these combined factors on the natural course of Chagas disease.     

Detection of Trypanosoma cruzi infection in naturally infected dogs and cats using serological,parasitological and molecular methods

Domestic dogs and cats are major domestic reservoir hosts of Trypanosoma cruzi and a risk factor for parasite transmission. In this study we assessed the relative performance of a polymerase chain reaction assay targeted to minicircle DNA (kDNA-PCR) in reference to conventional serological tests, a rapid dipstick test and xenodiagnosis to detect T. cruzi infection in dogs and cats from an endemic rural area in northeastern Argentina. A total of 43 dogs and 13 cats seropositive for T. cruzi by an immunosorbent assay (ELISA) and an indirect hemagglutination assay (IHA), which had been examined by xenodiagnosis, were also tested by kDNA-PCR. kDNA-PCR was nearly as sensitive as xenodiagnosis for detecting T. cruzi-infectious dogs and cats. kDNA-PCR was slightly more sensitive than xenodiagnosis in seropositive dogs (91% versus 86%, respectively) and cats (77% against 54%, respectively), but failed to detect all of the seropositive individuals. ELISA and IHA detected all xenodiagnosis-positive dogs and both outcomes largely agreed (kappa coefficient, κ = 0.92), whereas both assays failed to detect all of the xenodiagnosis-positive cats and their agreement was moderate (κ = 0.68). In dogs, the sensitivity of the dipstick test was 95% and agreed closely with the outcome of conventional serological tests (κ = 0.82). The high sensitivity of kDNA-PCR to detect T. cruzi infections in naturally infected dogs and cats supports its application as a diagnostic tool complementary to serology and may replace the use of xenodiagnosis or hemoculture.     

Benznidazole treatment in chronic children infected with Trypanosoma cruzi: Serological and molecular follow-up of patients and identification of Discrete Typing Units

A total of 221 children from two rural settlements in Northeast Argentina were examined for T. cruzi infection. Blood samples were taken for serology tests and PCR assays. In addition, T. cruzi Discrete Typing Units (DTUs) were determined by hybridization with specific DNA probes of the minicircle hypervariable regions (mHVR). Serological results indicated that 26% (57/215) were reactive against T. cruzi antigens. PCR analyses were performed on seropositive samples showing presence of parasite DNA in 31 out of 53 samples (58.5%). All seropositive children underwent specific chemotherapy with Benznidazole (5 mg/kg/day) for a period of two months and were monitored two and five years after treatment. Overall the treatment was well tolerated and low side effects were observed. Serological conversion was observed at two years post -treatment in one child form Pampa Ávila and at five years in two children from Tres Estacas. However, at the end of the follow-up period, T. cruzi DNA could not be detected by PCR in samples from treated children, except in two cases. In addition, the results of hybridizations with specific DNA probes showed that DTU TcV was detected in 68% (21/31), TcVI in 7% (2/31) and TcV/VI in 3% (1/31) of the samples. Altogether, results of the follow-up of treated children showed a low rate of seroconversion; however trend toward seroconversion was evident at five years post-treatment. On the other hand, detection of T. cruzi DNA by PCR significantly decreased after Benznidazole treatment. The existence of data regarding serological and molecular follow-ups from controlled studies in the Chaco Region will be important for future treatment efforts against T. cruzi infection in this region. The results obtained in the present study represent a contribution in this regard.     

The efficacy of photochemical treatment with methylene blue and light for the reduction of Trypanosoma cruzi in infected plasma

Background and Objectives  Chagas disease is a transfusion-transmitted infection. This study evaluates the efficacy of a methylene blue (MB) and light system for reducing the viability of Trypanosoma cruzi in plasma.
Materials and Methods  Trypanosoma cruzi strains were spiked in plasma pools. Treatment arms included combined filtration, MB, light and freezing. Post-treatment parasite viability was assayed through in vitro cultures and in vivo inoculation in inducible nitric oxide synthase- and interferon-γ-receptor-deficient mice.
Results  The filtration, MB and light combined treatment showed a log reduction of > 3·4 in in vitro cultures, and log reductions that ranged from > 4·9 to > 5·8 in deficient mice inoculated with different T. cruzi strains.
Conclusion  The treatment of plasma units with the MB and light system reduces the T. cruzi burden and could be useful in preventing transfusion-transmitted Chagas disease.     

Door-to-door screening as a strategy for the detection of congenital Chagas disease in rural Bolivia

Objective To demonstrate the feasibility of a house‐to‐house screening system used for congenital Chagas disease in rural areas based on an active search for pregnant women and newborns in their homes in addition to passive case detection in health facilities. Methods Exploratory phase conducted by the research team followed by an operational period coordinated by municipal health service. A blood sample was taken for serological and parasitological tests of Trypanosoma cruzi from pregnant women who were searching antenatal care or visited at home by field investigators. Infants born to T. cruzi‐infected women were examined for infection at birth and again at 1 and 7 months of age. Results 64.5% of the pregnant women were infected. Congenital infection was diagnosed at birth in 4.0% (12/299) of the children born to seroreactive mothers. Twelve additional cases of infection (4%) were diagnosed in children between 1 and 7 months of age. Finally, 37% of the children were lost to follow‐up in the exploratory phase and 53% during the operational phase (P = 0.002), significantly fewer than in most passive case detection studies. Conclusion Despite poorer outcomes after door‐to‐door screening activities have been transferred to the health system, a combined strategy based on active and passive case detection appeared to be efficient for identifying rural cases of congenital Chagas disease.