Serological response to nifurtimox in adult patients with chronic Chagas disease: An observational comparative study in Argentina.

Nifurtimox is indicated in Chagas disease but determining its effectiveness in chronic disease is hindered by the length of time needed to demonstrate negative serological conversion. We manually reviewed long-term follow-up data from hospital records of patients with chronic Chagas disease (N = 1,497) in Argentina diagnosed during 1967-1980. All patients were aged ≥18 years at diagnosis and were either treated with nifurtimox (n = 968) or received no antitrypanosomal treatment (n = 529). The primary endpoint was negative seroconversion (the "event"), defined as a change from positive to negative in the serological or parasitological laboratory test used at diagnosis. Time to event was from baseline visit to date of endpoint event or censoring. The effectiveness of nifurtimox versus no treatment was estimated with Cox proportional hazard regression using propensity scores with overlap weights to calculate the hazard ratio and 95% confidence interval. The nifurtimox group was younger than the untreated group (mean, 32.4 vs. 40.3 years), with proportionally fewer females (47.9% vs. 60.1%), and proportionally more of the nifurtimox group than the untreated group had clinical signs and symptoms of Chagas disease at diagnosis (28.9% vs. 14.0%). Median maximum daily dose of nifurtimox was 8.0 mg/kg/day (interquartile range [IQR]: 8.0-9.0) and median treatment duration was 44 days (IQR: 1-90). Median time to event was 2.1 years (IQR: 1.0-4.5) for nifurtimox-treated and 2.4 years (IQR: 1.0-4.2) for untreated patients. Accounting for potential confounders, the estimated hazard ratio (95% confidence interval) for negative seroconversion was 2.22 (1.61-3.07) favoring nifurtimox. Variable treatment regimens and follow-up duration, and an uncommonly high rate of spontaneous negative seroconversion, complicate interpretation of this epidemiological study, but with the longest follow-up and largest cohort analyzed to date it lends weight to the benefit of nifurtimox in adults with chronic Chagas disease. Trial registration: The study protocol was registered at ClinicalTrials.gov: NCT03784391.

Assessing antibody decline after chemotherapy of early chronic Chagas disease patients.

BACKGROUND: Chagas disease remains a significant public health problem in Latin America. There are only two chemotherapy drugs, nifurtimox and benznidazole, and both may have severe side effects. After complete chemotherapy of acute cases, seropositive diagnosis may revert to negative. However, there are no definitive parasitological or serological biomarkers of cure. METHODS: Following a pilot study with seven Bolivian migrants to Spain, we tested 71 serum samples from chronic patients (mean age 12.6 years) inhabiting the Argentine Chaco region. Benznidazole chemotherapy (5-8 mg/kg day, twice daily for 60 days) was administered during 2011-2016. Subsequently, pre-and post-chemotherapy serum samples were analysed in pairs by IgG1 and IgG ELISA using two different antigens and Chagas Sero K-SeT rapid diagnostic tests (RDT). Molecular diagnosis by kDNA-PCR was applied to post-treatment samples. RESULTS: Pilot data demonstrated IgG1 antibody decline in three of seven patients from Bolivia 1 year post-treatment. All Argentine patients in 2017 (averaging 5 years post-treatment), except one, were positive by conventional serology. All were kDNA-PCR-negative. Most (91.5%) pre-treatment samples were positive by the Chagas Sero K-SeT RDT, confirming the predominance of TcII/V/VI. IgG1 and IgG of Argentine patients showed significant decline in antibody titres post-chemotherapy, with either lysate (IgG, P = 0.0001, IgG1, P = 0.0001) or TcII/V/VI peptide antigen (IgG, P = 0.0001, IgG1, P = 0.0001). IgG1 decline was more discriminative than IgG. Antibody decline after treatment was also detected by the RDT. Incomplete treatment was associated with high IgG1 post-treatment titres against lysate (P = 0.013), as were IgG post-treatment titres to TcII/V/VI peptide (P = 0.0001). High pre-treatment IgG1 with lysate was associated with Qom ethnicity (P = 0.045). No associations were found between gender, age, body mass index and pre- or post-treatment antibody titres. CONCLUSIONS: We show that following chemotherapy of early chronic Chagas disease, significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure. We show that following chemotherapy of early chronic Chagas disease, a significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure.

IgG Subclasses and Congenital Transmission of Chagas Disease.

The mechanism of vertical transmission of Trypanosoma cruzi is poorly understood. In this study, we evaluated the role of IgG subclasses in the congenital transmission of Chagas disease. We conducted a case-control study in a public maternity hospital in Santa Cruz, Bolivia, enrolling women at delivery. Thirty women who transmitted T. cruzi to their newborns (cases), and 51 women who did not (controls) were randomly selected from 676 total seropositive women. Trypanosoma cruzi-specific IgG1, IgG2, and IgG3 levels were measured by in-house ELISA. The IgG4 levels were unmeasurable as a result of low levels in all participants. Quantitative polymerase chain reaction results and demographic factors were also analyzed. One-unit increases in normalized absorbance ratio of IgG1 or IgG2 levels increased the odds of congenital T. cruzi transmission in Chagas-seropositive women by 2.0 (95% CI: 1.1-3.6) and 2.27 (95% CI: 0.9-5.7), adjusted for age and previous blood transfusion. Odds of congenital transmission were 7.0 times higher in parasitemic mothers (95% CI: 2.3-21.3, P < 0.01) compared with nonparasitemic mothers. We observed that all mothers with IgG1 ≥ 4 were transmitters (sensitivity = 20%, specificity = 100%). Additionally, no mothers with IgG2 < 1.13 were transmitters (sensitivity = 100%, specificity = 21.6%). We demonstrated that IgG subclasses and parasite presence in blood are associated with vertical transmission of T. cruzi and could identify women at increased risk for congenital transmission by measuring IgG subclasses. These measures have potential as objective screening tests to predict the congenital transmission of Chagas.

HIV and Chagas Disease: An Evaluation of the Use of Real-Time Quantitative Polymerase Chain Reaction to Measure Levels of Trypanosoma cruzi Parasitemia in HIV Patients in Cochabamba, Bolivia.

This cross-sectional study evaluated epidemiologic characteristics of persons living with HIV (PWH) coinfected with Trypanosoma cruzi in Cochabamba, Bolivia, and estimated T. cruzi parasitemia by real-time quantitative polymerase chain reaction (qPCR) in patients with and without evidence of reactivation by direct microscopy. Thirty-two of the 116 HIV patients evaluated had positive serology for T. cruzi indicative of chronic Chagas disease (27.6%). Sixteen of the 32 (50%) patients with positive serology were positive by quantitative polymerase chain reaction (qPCR), and four of the 32 (12.5%) were positive by direct microscopy. The median parasite load by qPCR in those with CD4+ < 200 was 168 parasites/mL (73-9951) compared with 28.5 parasites/mL (15-1,528) in those with CD4+ ≥ 200 (P = 0.89). There was a significant inverse relationship between the degree of parasitemia estimated by qPCR from blood clot and CD4+ count on the logarithmic scale (rsBC= -0.70, P = 0.007). The correlation between T. cruzi estimated by qPCR+ blood clot and HIV viral load was statistically significant with rsBC = 0.61, P = 0.047. Given the significant mortality of PWH and Chagas reactivation and that 57% of our patients with CD4+ counts < 200 cells/mm3 showed evidence of reactivation, we propose that screening for chronic Chagas disease be considered in PWH in regions endemic for Chagas disease and in the immigrant populations in nonendemic regions. Additionally, our study showed that PWH with advancing immunosuppression have higher levels of estimated parasitemia measured by qPCR and suggests a role for active surveillance for Chagas reactivation with consideration of treatment with antitrypanosomal therapy until immune reconstitution can be achieved.

Assessment of Liaison XL Murex Chagas diagnostic performance in blood screening for Chagas disease using a reference array of chimeric antigens.

BACKGROUND: Chagas disease (CD) serological screening at blood banks is usually performed by a single highly sensitive serological assay, with chemiluminescent immunoassays (CLIAs) being the method of choice. CLIAs employ recombinant, fusion peptides and/or chimeric antigens that selectively capture anti-Trypanosoma cruzi antibodies. However, despite high sensitivity, the ability of these tests to identify CD-positive cases should be evaluated against T. cruzi strains circulating in specific locales. Herein, we used a latent class analysis (LCA) approach employing an array of four chimeric antigens to assess the diagnostic performance of the Liaison XL Murex Chagas CLIA for the detection of anti-T. cruzi IgG in serum samples. STUDY DESIGN AND METHODS: The study included a panel of 5014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia, submitted to anti-T. cruzi antibody detection using Liaison Chagas CLIA and LCA as a reference test in the absence of a gold standard. RESULTS: LCA classified 4993 samples as negative, while positivity for T. cruzi antibodies was predicted in 21 samples. Compared with LCA, CLIA demonstrated sensitivity and specificity of 76.2% and 99.5%, respectively, providing an overall accuracy of 99.4%. DISCUSSION: In blood banks lacking a de facto highly sensitive screening immunoassay, the low sensitivity offered by Liaison Chagas CLIA renders it unsuitable for standalone use in serological screening procedures for CD. Moreover, blood banks are encouraged to carefully assess the ability of diagnostic methods to identify local T. cruzi strains in circulation.

Risk of Trypanosoma cruzi infection among travellers visiting friends and relatives to continental Latin America.

BACKGROUND: Chagas disease (CD) is regarded as a possible risk for travellers to endemic areas of continental Latin America (LA). The aim of the study is to determine the risk of Trypanosoma cruzi (TC) infection among travellers to CD endemic areas and to identify risk factors for acquiring TC infection. METHODS/PRINCIPAL FINDING: We designed a multicenter cross-sectional study among travellers in Spain (Badalona, Barcelona and Madrid). All available adults with laboratory confirmed proof of absence of TC infection from January 2012 to December 2015 were contacted. Participants referring a trip to LA after the negative TC screening were offered to participate. We performed a standardized questionnaire of travel related factors and measurement of TC antibodies in serum. A total of 971 participants with baseline negative TC serology were selected from the microbiology records. After excluding participants not meeting inclusion criteria, eighty participants were selected. Sixty three (78.8%) were female, and the median age was 38 (IQR 34-47) years. The reason to travel was visiting friends and relatives in 98.8% of the participants. The median duration of travel was 40 (IQR 30-60) days, with 4911 participants-day of exposure. Seventy seven cases (96.25%) participants had two negative TC serology tests after the travel, two cases (2.5%) had discordant serology results (considered false positive results) and one case was infected before travelling to LA. According to our data, the upper limit of the 95% confidence interval of the incidence rate of TC acquisition in travellers is 0.8 per 1000 participant-days. CONCLUSIONS/SIGNIFICANCE: Among 79 non-CD travellers to TC endemic areas, we found no cases of newly acquired TC infection. The incidence rate of TC acquisition in travellers to endemic countries is less than or equal to 0.8 per 1000 traveller-days.

Sensitivity and Specificity of two rapid tests for the diagnosis of infection by Trypanosoma cruzi in a Colombian population.

OBJECTIVE: To evaluate diagnostic precision of two rapid diagnostic tests (RDT's) on patients with chronic Chagas disease. METHODOLOGY: Prospective study with the following inclusion criteria: subjects older than 3 years, signed informed consent. Exclusion criterion: subjects could not have previously received treatment for infection with T. cruzi. The study population were participants in a screening process undertaken in rural and urban zones of the department Boyacá, Colombia. Two RDT's were performed to all participants: the Chagas Detect Plus InBios (CDP) and the Chagas Stat-Pak (CSP) and as a reference standard the ELISA Chagas III GrupoBios and the Chagas ELISA IgG+IgM I Vircell tests were used. In the case of discordant results between the two ELISA tests, an indirect immunofluorescence was done. RESULTS: Three hundred-five (305) subjects were included in the study (38 patients with leishmaniasis), of which 215 tested negative for T cruzi and 90 tested positive according to the reference standard. The sensitivity of the RDT's were 100% (CI 95% 95.9-100), and the specificity of the CDP was 99.1% (CI 95% 96.6-99.8) and for CSP was 100% (CI 95% 98.3-100). The agreement of CDP was 99.5% and for CSP was 100% with Kappa values of (k = 99.1; CI 95% 92.6-99.8%) and (k = 100; CI 95% 94.3-100), respectively. RDT's did not present cross-reactions with samples from patients who were positive for leishmaniasis. CONCLUSIONS: The findings demonstrate excellent results from the RDT's in terms of validity, safety, and reproducibility. The results obtained provide evidence for the recommendation for using these tests in a Colombian epidemiological context principally in endemic areas in which laboratory installations necessary to perform conventional tests are not available, or they are scarce and to help in diagnosing chronic Chagas disease in order to provide access to treatment as soon as possible.

In vitro and in vivo trypanocidal activity of a benzofuroxan derivative against Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, is a major public health problem and is described as one of the most neglected diseases worldwide. It affects about 6-7 million people. Currently, only two drugs are available for the treatment of this disease: nifurtimox and benznidazole. However, both drugs are highly toxic and have several side effects, which lead many patients to discontinue treatment. Moreover, these compounds show a significant curative efficacy only in the acute phase of the disease. Therefore, searching for new drugs is necessary. The objective of this study was to evaluate the in vitro and in vivo activity of a benzofuroxan derivative (EA2) against T. cruzi, and to evaluate the hematological and biochemical changes induced by its treatment in animals infected with T. cruzi. The results were then compared with those of healthy controls. In vitro testing was first performed with T. cruzi epimastigote forms. In this experiment, EA2 was diluted at three different concentrations (0.25, 0.50, and 1%). In vitro evaluation of the trypanocidal activity was performed 24, 48, and 72 h after incubation. In vivo assays were performed using three different doses (10, 5, and 2,5 mg/kg). Mice were divided into 10 groups (five animals/group), wherein four groups comprised non-infected animals (A, G, H, I) and six groups comprised infected animals (B, C, D E, F, J). Groups B and J represented the negative and positive controls, respectively. Groups G, H, and I were used to confirm that EA2 was not toxic to non-infected animals. Parasitemia was measured in infected animals and the hematological and biochemical profiles (urea, creatinine, albumin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) were evaluated in all animals. EA2 demonstrated in vitro trypanocidal activity at all concentrations tested. Although it did not demonstrate a curative effect in vivo, EA2 was able to retard the onset of parasitemia, and significantly reduced the parasite count in groups D and E (treated with 5 and 2.5 mg/kg, respectively). EA2 did not induce changes in hematological and biochemical parameters in non-infected animals, demonstrating that it is not toxic. However, further assessments should aim to confirm the safety of EA2 since this was the first in vitro and in vivo study conducted with this molecule.

Measurement of multiple cytokines for discrimination and risk stratification in patients with Chagas' disease and idiopathic dilated cardiomyopathy.

Chagas' disease (CD), caused by the hemoflagellate protozoan, Trypanosoma cruzi, is endemic in most countries of Latin America. Heart failure (HF) is often a late manifestation of chronic CD, and is associated with high morbidity and mortality. Inflammatory processes mediated by cytokines play a key role in the pathogenesis and progression of CD. Keeping in view the inflammatory nature of CD, this study investigated the possible role of 21 different inflammatory cytokines as biomarkers for prediction and prognosis of CD. The plasma concentration of these cytokines was measured in a group of patients with CD (n = 94), and then compared with those measured in patients with dilated cardiomyopathy (DCM) from idiopathic causes (n = 48), and with control subjects (n = 25). Monovariately, plasma levels of cytokines such as stem cell growth factor beta (SCGF beta), hepatocyte growth factor (HGF), monokine induced by interferon gamma (CXCL9), and macrophage inhibitory factor (MIF) were significantly increased in CD patients with advanced HF compared to control group. None of the cytokines could demonstrate any prognostic potency in CD patients, and only MIF and stromal derived factor-1 alpha (CXCL12) showed significance in predicting mortality and necessity for heart transplant in DCM patients. However, multivariate analysis prognosticated a large proportion of CD and DCM patients. In CD patients, HGF and Interleukin-12p40 (IL-12p40) together separated 81.9% of 3-year survivors from the deceased, while in DCM patients, CXCL12, stem cell factor (SCF), and CXCL9 together discriminated 77.1% of survivors from the deceased. The significant increase in plasma concentrations of cytokines such as HGF and CXCL9 in CD patients, and the ability of these cytokines to prognosticate a large proportion of CD and DCM patients multivariately, encourages further studies to clarify the diagnostic and prognostic potential of cytokines in such patients.

Prevalence of Cardiac Arrhythmias and Distal Conduction Disorders in Patients With Chronic Chagas' Disease and Elevated Autoantibodies Against M2 Muscarinic Acetylcholine Receptors.

Chagas' disease (ChD) is a parasitic disease endemic to regions of Latin America and with an increasingly global reach. Up to 30% of patients with ChD develop severe dilated cardiomyopathy, ventricular arrhythmias, conduction disorders and/or sudden cardiac death. Autoantibodies against M2 muscarinic acetylcholine receptors (M2 mAChR) have been implicated in the pathogenesis of ChD. We sought to understand whether there was an association between anti-M2 mAChR autoantibody titers in patients with chronic ChD and the presence of distal cardiac conduction disorders or cardiac arrhythmias. We conducted a cross-sectional study in 79 patients from Argentina and Bolivia with chronic ChD without evident structural heart disease. Autoantibody titers were measured using indirect enzyme-linked immunosorbent assay. Elevated anti-M2 mAChR autoantibody titers were associated with the presence of distal conduction disease but not with cardiac arrhythmias. High anti-M2 mAChR autoantibody levels could assist with identifying early structural heart disease in patients with chronic ChD.

New Biomarker in Chagas Disease: Extracellular Vesicles Isolated from Peripheral Blood in Chronic Chagas Disease Patients Modulate the Human Immune Response.

Chagas disease, a neglected tropical disease (NTD) caused by the flagellated protozoan Trypanosoma cruzi (T. cruzi), is a major public health problem. It was initially restricted to Latin America, but it is now expanding globally. Host and pathogen interactions are crucial in the establishment of disease, and since 1970, it has been known that eukaryotic cells release extracellular vesicles (EVs), which in turn have an important role in intercellular communication in physiological and pathological conditions. Our study proposed to characterize and compare circulating EVs isolated from the plasma of chronic Chagas disease (CCD) patients and controls. For this, peripheral blood was collected from patients and controls, and mononuclear cells (PBMCs) were isolated and stimulated with parasite EVs, showing that patient cells released fewer EVs than control cells. Then, after plasma separation followed by EV total shedding enrichment, the samples were subjected to ultracentrifugation to isolate the circulating EVs, which then had their size and concentration characterized by nanoparticle tracking analysis (NTA). This showed that patients had a lower concentration of circulating EVs while there were no differences in size, corroborating the in vitro data. Additionally, circulating EVs were incubated with THP-1 cells (macrophages) that, after the interaction, had their supernatant analyzed by ELISA for cytokine detection. In relation to their ability to induce cytokine production, the CCD patient EVs were able to induce a differential production of IFN-γ and IL-17 in relation to controls, with differences being more evident in earlier/less severe stages of the disease. In summary, a decreased concentration of circulating EVs associated with differential activation of the immunological system in patients with CCD is related to parasite persistence and the establishment of chronic disease. It is also a potential biomarker for monitoring disease progression.

A novel Trypanosoma cruzi secreted antigen as a potential biomarker of Chagas disease.

Chagas drug discovery has been hampered by a lack of validated assays to establish treatment efficacy in pre-clinical animal models and in patients infected with T. cruzi. Reduced levels of parasite secreted antigens in the blood of infected hosts could be used to demonstrate treatment efficacy. A published proteomic study of parasite secreted antigens identified the hypothetical protein Tc_5171 as a secreted antigen. In this report, we developed Tc_5171 specific antibodies and showed that the native protein was expressed by the three life cycle stages of the parasite. Anti-peptide antibodies were able to detect the parasite antigen in blood of infected mice during the acute and the chronic phase of infection. Benznidazole treatment of infected mice significantly reduced their blood antigen levels. Of clinical significance, patients diagnosed with Chagas disease, either asymptomatic or with cardiac clinical symptoms had significantly higher Tc_5171 antigen levels compared to endemic controls. Pair-wise analysis, before and after Benznidazole treatment, of patients with asymptomatic Chagas disease showed a significant reduction in antigen levels post treatment. Taken together, our results indicate that Tc_5171 could be used as a novel biomarker of Chagas disease for diagnosis and to assess treatment efficacy.

Declining antibody levels to Trypanosoma cruzi correlate with polymerase chain reaction positivity and electrocardiographic changes in a retrospective cohort of untreated Brazilian blood donors.

BACKGROUND: Although infection with Trypanosoma cruzi is thought to be lifelong, less than half of those infected develop cardiomyopathy, suggesting greater parasite control or even clearance. Antibody levels appear to correlate with T. cruzi (antigen) load. We test the association between a downwards antibody trajectory, PCR positivity and ECG alterations in untreated individuals with Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: This is a retrospective cohort of T. cruzi seropositive blood donors. Paired blood samples (index donation and follow-up) were tested using the VITROS Immunodiagnostic Products Anti-T.cruzi (Chagas) assay (Ortho Clinical Diagnostics, Raritan NJ) and PCR performed on the follow-up sample. A 12-lead resting ECG was performed. Significant antibody decline was defined as a reduction of > 1 signal-to-cutoff (S/CO) unit on the VITROS assay. Follow-up S/CO of < 4 was defined as borderline/low. 276 untreated seropositive blood donors were included. The median (IQR) follow-up was 12.7 years (8.5-16.9). 56 (22.1%) subjects had a significant antibody decline and 35 (12.7%) had a low/borderline follow-up result. PCR positivity was lower in the falling (26.8% vs 52.8%, p = 0.001) and low/borderline (17.1% vs 51.9%, p < 0.001) antibody groups, as was the rate of ECG abnormalities. Falling and low/borderline antibody groups were predominantly composed of individuals with negative PCR and normal ECG findings: 64% and 71%, respectively. CONCLUSIONS/SIGNIFICANCE: Low and falling antibody levels define a phenotype of possible spontaneous parasite clearance.

Aportaciones de las técnicas moleculares en la fase crónica de la Enfermedad de Chagas en ausencia de tratamiento / Contributions of molecular techniques in the chronic phase of Chagas disease in the absence of treatment

INTRODUCCIÓN: La fase crónica de la enfermedad de Chagas se caracteriza por una parasitemia baja e intermitente. En esta fase la sensibilidad de la reacción en cadena de la polimerasa (PCR) es muy variable, limitando su utilización como técnica diagnóstica. A pesar de ello, la realización de la PCR en pacientes no tratados puede aportar datos sobre el comportamiento del parásito y su presencia en sangre periférica. MÉTODOS: Se realizó PCR a tiempo real de forma puntual en una cohorte de 495 pacientes con enfermedad de Chagas crónica en ausencia de tratamiento. También se realizó seguimiento de una subcohorte de 29 pacientes mediante PCR a tiempo real seriadas, entre 8 y 12 meses en los que no tuvieron acceso al tratamiento por falta de suministro. RESULTADOS: El porcentaje de positividad de PCR a tiempo real fue de 42%. Este porcentaje fue significativamente mayor en pacientes con 5 años o menos de residencia en España (p = 0,041). La detección de ADN no se relacionó con la existencia de alteraciones cardíacas y/o digestivas. En el subgrupo de pacientes a los que se realizaron determinaciones seriadas, el resultado de PCR fue sostenidamente positivo en el 13,8% de los pacientes, negativo en el 31% e intermitente en el 55,2%. CONCLUSIONES: Las diferencias de resultados de PCR a tiempo real en función del tiempo de residencia apuntan que existen factores externos que pueden influir en la presencia del parásito en sangre periférica. Así mismo, factores propios del hospedador parecen influir en la dinámica parasitaria a lo largo del tiempo

INTRODUCTION: The chronic phase of Chagas disease (CD) is characterised by a low and intermittent parasitaemia. The Polymerase Chain Reaction (PCR) presents a variable sensitivity in this stage limiting its use as a diagnostic tool. Despite this, the use of PCR in untreated patients can provide information on the parasite behaviour and its presence in peripheral blood. METHODS: A timely real-time PCR determination was performed on a cohort of 495 untreated chronic CD patients. Also, a subcohort of 29 patients was followed-up by serial real-time PCR during a period from 8 to 12 months in which they could not have access to the treatment due to lack of supply. RESULTS: The positive percentage of real-time PCR in our series was 42%. Nevertheless, real-time PCR positive results were significantly higher in patients with five years or less of residence in Spain (P = .041). The detection of DNA was not related to the existence of cardiac and/or gastrointestinal abnormalities. In the follow-up subgroup, real-time PCR was consistently positive in 13.8% of patients, consistently negative in 31%, and intermittent in 55.2%. CONCLUSIONS: The different real-time PCR results regarding the time of residence suggests the possible relationship of external factors in the parasite presence in peripheral blood. On the other hand, specific host factors may be involved in the behaviour of parasitaemia over time

Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.

Human Chagas-Flow ATE-IgG1 for advanced universal and Trypanosoma cruzi Discrete Typing Units-specific serodiagnosis of Chagas disease.

The molecular and serological methods available for Discrete Typing Units (DTU)-specific diagnosis of Trypanosoma cruzi in chronic Chagas disease present limitations. The study evaluated the performance of Human Chagas-Flow ATE-IgG1 for universal and DTU-specific diagnosis of Chagas disease. A total of 102 sera from Chagas disease patients (CH) chronically infected with TcI, TcVI or TcII DTUs were tested for IgG1 reactivity to amastigote/(A), trypomastigote/(T) and epimastigote/(E) antigens along the titration curve (1:250-1:32,000). The results demonstrated that "AI 250/40%", "EVI 250/30%", "AII 250/40%", "TII 250/40%" and "EII 250/30%" have outstanding accuracy (100%) to segregate CH from non-infected controls. The attributes "TI 4,000/50%", "EI 2,000/50%", "AVI 8,000/60%" and "TVI 4,000/50%" were selected for DTU-specific serotyping of Chagas disease. The isolated use of "EI 2,000/50%" provided the highest co-positivity for TcI patients (91%). The combined decision tree algorithms using the pre-defined sets of attributes showed outstanding full accuracy (92% and 97%) to discriminate "TcI vs TcVI vs TcII" and "TcI vs TcII" prototypes, respectively. The elevated performance of Human Chagas-Flow ATE-IgG1 qualifies its use for universal and TcI/TcVI/TcII-specific diagnosis of Chagas disease. These findings further support the application of this method in epidemiological surveys, post-therapeutic monitoring and clinical outcome follow-ups for Chagas disease.

Presence of Acute Chagas Disease Among Febrile Patients in the Western Coast of Mexico.

Chagas disease (ChD) is a parasitosis caused by the protozoan Trypanosoma cruzi (Tc). It is endemic to almost all Latin American countries, including the southern United States. The acute form of ChD and its actual incidence have rarely been described in Mexico, despite the extensive presence of favorable niches for its transmission. The objective of this study was to estimate the frequency of acute ChD in febrile patients at the central Pacific coast of Mexico. For this, we surveyed patients with persistent fever (5 to 10 days) in five hospitals at the Mexican states of Jalisco, Colima, and Nayarit in 2012. Samples were taken from a total of 485 patients to detect Tc in blood using the polymerase chain reaction (PCR) test and direct microscopic examination. Of these subjects, 10 were positive for PCR and none for microscopic examination (2% in 12 months). We adjusted this rate by the total people at risk in the area and obtained an incidence of 7.4/100,000 habs./year. The positive cases showed no association with sex, rural settlement, or pet ownership, only with the contact with Triatominae insects (odds ratio = 9.22 and confidence interval: 1.93-44.06). The clinical picture of positive patients showed an association with the diagnosis of lower respiratory tract infections. Meanwhile, only one fatal case showed the typical picture of acute fatal cardiomyopathy. The pulmonary manifestations of our patients suggest possible lung pathogenicity of Tc, which merits further investigation. Our findings differ markedly from the official reports for ChD. This difference suggests an underestimation of the disease. These findings urge the Mexican health authorities to implement more vigorous actions aimed at improving medical skills in the timely diagnosis of ChD, as well as to apply efficient preventive programs.

Kinins and nitric oxide in patients with chronic chagas disease and systemic arterial hypertension.

BACKGROUND: Chronic Chagas disease (CCHD) associated with Systemic Arterial Hypertension (SAH) is frequently found in areas where the disease is endemic. The pathogenesis of patients with both pathologies (CCHD-SAH) is unsettled. Nitric Oxide (NO) and Kinins are important players in the myocardial inflammation process in experimental CCHD. No previous study has addressed this question in patients with CCHD, particularly in those with CCHD-SAH. Accordingly, this study was undertaken in an attempt to contribute to the understanding of the pathogenesis of patients with CCHD-SAH. METHODS: Thirty-seven patients with a positive serology for Chagas disease were enrolled; 15 had CCHD alone, 22 had CCHD-SAH (abnormal ECG/Doppler echocardiogram plus a systolic blood pressure > 140 mmHg or diastolic blood pressure > 90 mmHg on admission), and 11 had SAH alone. Thirty healthy individuals matched by age and sex served as controls. Plasma High-molecular (Hkg) and low-molecular weight (LKg) kininogens, plasma kallikrein levels (Pkal and Tcal), Kininase II, and plasma NO were measured. RESULTS: HKg and LKg were lower in CCHD-SAH patients in comparison with other groups (P < .0001). Pkal and Tcal were higher in CCHD-SAH patients in comparison with the other groups (P< .0001). Kininase II levels were similar in SAH, CCHD, and CCHD-SAH patients, but lower in comparison with controls (P< .0001). NO levels were similar in CCHD and CCHD-SAH patients, but higher in comparison with SAH patients and controls (P > .0001). CONCLUSION: Such findings suggest increased Kinin and NO activity in patients with CCHD-SAH, thus contributing to the understanding of the pathogenesis of this condition.

Distal contractile integral measurement and vascular compression in the esophagus: a problem unsolved?

BACKGROUND: Distal contractile integral (DCI) is influenced by factors other than esophageal smooth muscle contractility, such as intrabolus pressure and vascular and respiratory movements' artifacts. We aimed to determine the size of the contribution of pressures generated by vascular compression on the esophagus to the DCI measured in HRM recordings in symptomatic patients. METHODS: HRM manometry recordings obtained from 383 subjects referred to the GI motility laboratory at a tertiary center (2012-2016) were evaluated by visual inspection for evidence of strong vascular compression (SVC) of the esophagus. Clinical, demographic, manometric, and serologic data for Chagas disease were obtained. Subjects were classified, respectively, as asymptomatics (ASYM) or symptomatics (SYMP). DCI and SVC-DCI were measured, and the SVC-DCI/DCI ratio was expressed as a percentage and the difference between DCI and SVC-DCI (neat-DCI) was calculated. DCI, SVC-DCI, SVC-DCI/DCI % and neat-DCI from SYMP and ASYM were compared. RESULTS: SVC was conspicuous in 42 of 383 subjects (11%). In 33 subjects, SVC was detected only in supine position. SVC was localized in middle esophagus in 21 subjects (50%), in distal esophagus in 12 subjects (29%) and in both regions in 9 subjects (21%). In 9 subjects, SVC vanished from the swallowing window analysis (21%). CONCLUSIONS: SVC is a common finding in esophageal HRM study, particularly in the supine position. Occasionally, its contribution to DCI value is sufficiently great to masquerade esophageal hypocontractility. Different manometric protocols may be required in patients with SVC.

Thioridazine aggravates skeletal myositis, systemic and liver inflammation in Trypanosoma cruzi-infected and benznidazole-treated mice.

While thioridazine (Tio) inhibits the antioxidant defenses of Trypanosoma cruzi, the gold standard antitrypanosomal drug benznidazole (Bz) has potent anti-inflammatory and pro-oxidant properties. The combination of these drugs has never been tested to determine the effect on T. cruzi infection. Thus, we compared the impact of Tio and Bz, administered alone and in combination, on the development of skeletal myositis and liver inflammation in T. cruzi-infected mice. Swiss mice were randomized into six groups: uninfected untreated, infected untreated, treated with Tio (80 mg/kg) alone, Bz (50 or 100 mg/kg) alone, or a combination of Tio and Bz. Infected animals were inoculated with a virulent T. cruzi strain (Y) and treated by gavage for 20 days. Mice untreated or treated with Tio alone developed the most intense parasitemia, highest parasitic load, elevated IL-10, IL-17, IFN-γ, and TNF-α plasma levels, increased N-acetylglucosaminidase and myeloperoxidase activity in the liver and skeletal muscle, as well as severe myositis and liver inflammation (P < 0.05). All parameters were markedly attenuated in animals receiving Bz alone (P < 0.05). However, the co-administration of Tio impaired the response to Bz chemotherapy, causing a decrease in parasitological control (parasitemia and parasite load), skeletal muscle and liver inflammation, and increased microstructural damage, when compared to the group receiving Bz alone (P < 0.05). Altogether, our findings indicated that Tio aggravates systemic inflammation, skeletal myositis and hepatic inflammatory damage in T. cruzi-infected mice. By antagonizing the antiparasitic potential of Bz, Tio limits the anti-inflammatory, myoprotectant and hepatoprotective effects of the reference chemotherapy, aggravating the pathological remodeling of both organs. As the interaction of T. cruzi infection, Bz and Tio is potentially toxic to the liver, inducing inflammation and microvesicular steatosis; this drug combination represents a worrying pharmacological risk factor in Chagas disease.