In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

Hemolytic disease of the newborn due to anti-U

Anti-U is a rare red blood cell alloantibody that has been found exclusively in blacks. It can cause hemolytic disease of the newborn and hemolytic transfusion reactions. We describe the case of a female newborn presenting a strongly positive direct antiglobulin test due to an IgG antibody in cord blood. Anti-U was recovered from cord blood using acid eluate technique. Her mother presented positive screening of antibodies with anti-U identified at delivery. It was of IgG1 and IgG3 subclasses and showed a titer of 32. Monocyte monolayer assay showed moderate interaction of Fc receptors with maternal serum with a positive result (3.1%). The newborn was treated only with 48 hours of phototherapy for mild hemolytic disease. She recovered well and was discharged on the 4th day of life. We conclude that whenever an antibody against a high frequency erythrocyte antigen is identified in brown and black pregnant women, anti-U must be investigated.

Mitral valve disease with rheumatic appearance in the presence of left ventricular endomyocardial fibrosis

This is a report of a nine-year-old boy with both mitral stenosis and regurgitation and extensive endomyocardial fibrosis of the left ventricle. Focus is given to the singularity of the fibrotic process, with an emphasis on the etiopathogenic aspects.

Prevalence of Rheumatic Heart Disease in a Public School of Belo Horizonte

Background: Previous studies indicate that compared with physical examination, Doppler echocardiography identifies a larger number of cases of rheumatic heart disease in apparently healthy individuals. Objectives: To determine the prevalence of rheumatic heart disease among students in a public school of Belo Horizonte by clinical evaluation and Doppler echocardiography. Methods: This was a cross-sectional study conducted with 267 randomly selected school students aged between 6 and 16 years. students underwent anamnesis and physical examination with the purpose of establishing criteria for the diagnosis of rheumatic fever. They were all subjected to Doppler echocardiography using a portable machine. Those who exhibited nonphysiological mitral regurgitation (MR) and/or aortic regurgitation (AR) were referred to the Doppler echocardiography laboratory of the Hospital das Clínicas of the Universidade Federal of Minas Gerais (HC-UFMG) to undergo a second Doppler echocardiography examination. According to the findings, the cases of rheumatic heart disease were classified as definitive, probable, or possible. Results: Of the 267 students, 1 (0.37%) had a clinical history compatible with the diagnosis of acute rheumatic fever (ARF) and portable Doppler echocardiography indicated nonphysiological MR and/or AR in 25 (9.4%). Of these, 16 (6%) underwent Doppler echocardiography at HC-UFMG. The results showed definitive rheumatic heart disease in 1 student, probable rheumatic heart disease in 3 students, and possible rheumatic heart disease in 1 student. Conclusion: In the population under study, the prevalence of cases compatible with rheumatic involvement was 5 times higher on Doppler echocardiography (18.7/1000; 95% CI 6.9/1000-41.0/1000) than on clinical evaluation (3.7/1000-95% CI).

EVI antibodies in patients with Chagas' disease: relationship with anti-Trypanosoma cruzi immunoglobulins and effects of specific treatment

Antibodies against heart vascular structures and striated muscle cells interstitium (EVI antibodies) persist in Chagas' disease patients who had been cured by specific treatment as demonstrated by negative xenodiagnosis, conventional serology (CS) and complement mediated lysis (CoML). On the other hand, EVI antibodies are either present or absent in treated patients presenting positive CS but negative CoML. Since CoML detects antibodies associated to resistance, EVI antibodies are not likely to participate in the control of T. cruzi infections although they might be induced by cross-reacting antigens of heart cells and the parasite. They are neither necessarily related to antibodies responsible for CS. Absorption with T. cruzi and heart tissue confirms the suggestion that EVI antibodies are induced by a number of antigenic determinants, most from heart structures with a minor participation of T. cruzi antigens.

Propolis: anti-Staphylococcus aureus activity and synergism with antimicrobial drugs

Propolis is a natural resinous substance collected by bees from tree exudates and secretions. Its antimicrobial activity has been investigated and inhibitory action on Staphylococcus aureus growth was evaluated. The in vitro synergism between ethanolic extract of propolis (EEP) and antimicrobial drugs by two susceptibility tests (Kirby and Bauer and E-Test) on 25 S. aureus strains was evaluated. Petri dishes with sub-inhibitory concentrations of EEP were incubated with 13 drugs using Kirby and Bauer method and synergism between EEP and five drugs [choramphenicol (CLO), gentamicin (GEN), netilmicin (NET), tetracycline (TET), and vancomycin (VAN)] was observed. Nine drugs were assayed by the E-test method and five of them exhibited a synergism [CLO, GEN, NET, TET, and clindamycin (CLI)]. The results demonstrated the synergism between EEP and antimicrobial drugs, especially those agents that interfere on bacterial protein synthesis.

The utility of anti-trypomastigote lytic antibodies for determining cure of Trypanosoma cruzi infections in treated patients: an overview and perspectives

In previous work, we proposed alternative protocols for following patients with treated Chagas disease and these are reviewed herein. Evidence was provided to support the following: (i) functional anti-trypomastigote antibodies are indicative of ongoing chronic Trypanosoma cruzi infections; (ii) specific antibodies detected by conventional serology (CS) with epimastigote extracts, fixed trypomastigotes or other parasite antigens may circulate years after parasite elimination; (iii) functional antibodies are evidenced by complement-mediated lysis of freshly isolated trypomastigotes, a test which is 100% specific, highly sensitive, and the first to revert after T. cruzi elimination and (iv) the parasite target for the lytic antibodies is a glycoprotein of high molecular weight (gp160) anchored at the parasite surface. The complement regulatory protein has been cloned, sequenced and produced as a recombinant protein by other groups and is useful for identifying functional anti-T. cruzi antibodies in ELISA tests, thus dispensing with the need for live trypomastigotes to manage treated patients. If used instead of CS to define cures for Chagas patients, ELISA will avoid unnecessary delays in finding anti-T. cruzi drugs. Other highly sensitive techniques for parasite DNA detection, such as PCR, need to be standardized and included in future protocols for the management of patients with drug-treated Chagas disease.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Ergosterol biosynthesis and drug development for Chagas disease

This article presents an overview of the currently available drugs nifurtimox (NFX) and benznidazole (BZN) used against Trypanosoma cruzi, the aetiological agent of Chagas disease; herein we discuss their limitations along with potential alternatives with a focus on ergosterol biosynthesis inhibitors (EBI). These compounds are currently the most advanced candidates for new anti-T. cruzi agents given that they block de novo production of 24-alkyl-sterols, which are essential for parasite survival and cannot be replaced by a host's own cholesterol. Among these compounds, new triazole derivatives that inhibit the parasite's C14± sterol demethylase are the most promising, as they have been shown to have curative activity in murine models of acute and chronic Chagas disease and are active against NFX and BZN-resistant T. cruzi strains; among this class of compounds, posaconazole (Schering-Plough Research Institute) and ravuconazole (Eisai Company) are poised for clinical trials in Chagas disease patients in the short term. Other T. cruzi-specific EBI, with in vitro and in vivo potency, include squalene synthase, lanosterol synthase and squalene epoxidase-inhibitors as well as compounds with dual mechanisms of action (ergosterol biosynthesis inhibition and free radical generation), but they are less advanced in their development process. The main putative advantages of EBI over currently available therapies include their higher potency and selectivity in both acute and chronic infections, activity against NFX and BZN-resistant T. cruzi strains, and much better tolerability and safety profiles. Limitations may include complexity and cost of manufacture of the new compounds. As for any new drug, such compounds will require extensive clinical testing before being introduced for clinical use, and the complexity of such studies, particularly in chronic patients, will be compounded by the current limitations in the verification of true parasitological cures for T. cruzi infections.

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.

Adherence to anti-hypertensive treatment within a chronic disease management program: A longitudinal, retrospective study

Objective This study assessed pharmacological treatment adherence using the Morisky-Green Test and identified related variables. Method A longitudinal and retrospective study examined 283 patients with hypertension (62.5% women, 73.4 [10.9] years old) who were being monitored by a chronic disease management program for 17 months between 2011 and 2012. Nurses performed all the actions of the program, which consisted of advice via telephone and periodic home visits based on the risk stratification of the patients. Results A significant increase in treatment adherence (25.1% vs. 85.5%) and a decrease in blood pressure were observed (p<0.05). Patients with hypertension and chronic renal failure as well as those treated using angiotensin-converting enzyme inhibitors were the most adherent (p<0.05). Patients with hypertension who received angiotensin receptor blockers were less adherent (p<0.05). Conclusions Strategies such as nurse-performed chronic disease management can increase adherence to anti-hypertensive treatment and therefore contribute to the control of blood pressure, minimizing the morbidity profiles of patients with hypertension.

Anti-tumor necrosis factor-a for the treatment of steroid-refractory acute graft-versus-host disease

Allogeneic stem cell transplantation has been increasingly performed for a variety of hematologic diseases. Clinically significant acute graft-versus-host disease (GVHD) occurs in 9 to 50% of patients who receive allogeneic grafts, resulting in high morbidity and mortality. There is no standard therapy for patients with acute GVHD who do not respond to steroids. Studies have shown a possible benefit of anti-TNF-a (infliximab)for the treatment of acute GVHD. We report here on the outcomes of 10 recipients of related or unrelated stem cell transplants who received 10 mg/kg infliximab, iv, once weekly for a median of 3.5 doses (range: 1-6) for the treatment of severe acute GVHD and who were not responsive to standard therapy. All patients had acute GVHD grades II to IV (II = 2, III = 3, IV = 5). Overall, 9 patients responded and 1 patient had progressive disease. Among the responders, 3 had complete responses and 6 partial responses. All patients with cutaneous or gastrointestinal involvement responded, while only 2 of 6 patients with liver disease showed any response. None of the 10 patients had any kind of immediate toxicity. Four patients died, all of them with sepsis. Six patients are still alive after a median follow-up time of 544 days (92-600) after transplantation. Considering the severity of the cases and the bad prognosis associated with advanced acute GVHD, we find our results encouraging. Anti-TNF-a seems to be a useful agent for the treatment of acute GVHD.