Diagnosis of Chagas Disease by Detecting Species-Specific Repetitive Genomic DNA from Filtered Human Urine Samples.

INTRODUCTION: Chagas disease caused by Trypanosoma cruzi (T cruzi) found in the Americas is often missed during the early stage of infection due to lack of sensitive diagnostic tests. The classic immunological and parasitological tests often fail in the acute phase due to the nonspecific and low antibody level nature of the infection and in the chronic phase due to low levels of trypanosomes in the blood. For successful control strategies, there must be a sensitive and specific diagnostic test. OBJECTIVE/METHODS: We have demonstrated the possibility (proof of concept) of detecting T cruzi-specific repeat DNA via polymerase chain reaction (PCR) by (1) spiking 15 urine samples collected from volunteers free of prior infection with 3 different concentrations of T cruzi (3 strains), Trypanosoma brucei, and Trypanosoma rhodesiense (African strain) genomic DNA and (2) from filtered collected clinical samples from Argentina. Three sets of primers were used. RESULTS: Our approach detected repeat DNA specific for T cruzi strains from 1 clinical sample by 2 sets of primer and from spiked urine by all 3 sets of primer but not the African species. A serial dilution (spiking) also was performed on T cruzi strains to detect sensitivities of the assay. One set of primers constantly detected satellite DNA for all T cruzi strains from 70 pg/µl to 175 fg/µl. CONCLUSIONS: We were able to demonstrate the feasibility of detecting T cruzi-specific DNA from filtered urine samples by sensitive and specific PCR assay. Besides the evident increased sensitivity and specificity of primers, our approach can be used to explore Chagas prevalence in endemic areas - especially in congenital Chagas newborn screening - and in the acute phase.

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients.

BACKGROUND: Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts. CONCLUSION: Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients.

Development of a LC-MS/MS methodology for the monitoring of the antichagasic drug benznidazole in human urine.

Monitoring the drug benznidazole in biological fluids is a powerful tool for clinical diagnostic and pharmacological studies in chagasic patients. However, research in this concern needs to be done. The accurate quantitation of this drug in complex matrices represents a highly challenging task complicated by the absence of sensitive analytical methods. It follows that sample processing strategies, preparation/cleanup procedures, and chromatographic/ionization/detection parameters, were evaluated for method optimization. The summation of this work generated a rapid, selective, sensitive methodology based on reversed-phase chromatography-tandem mass spectrometry for the analysis of benznidazole in urine samples. To the best of our knowledge, this is a first report of a LC-MS/MS platform employed for this application. Matrix effect was determined; a 90% of signal suppression was observed. The limits of detection and quantification were 0.75 and 4.85 µg L(-1); respectively. The latter allowed the method's application to the detection of benznidazole in clinical studies and pharmacological monitoring analysis.

Detection of soluble antigen and DNA of Trypanosoma cruzi in urine is independent of renal injury in the guinea pig model.

The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA). T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa) were detected in the acute phase (67.5%) and the chronic phase (45%). Parasite DNA in urine was detected only in the acute phase (45%). Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1) and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients.

Validation of Mycobacterium tuberculosis Rv1681 protein as a diagnostic marker of active pulmonary tuberculosis.

The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis.

Avaliaçäo urodinâmica de pacientes chagásicos / Urodynamic assessement in patients with Chagas disease

No presente trabalho estudou-se a fisiologia urinária de pacientes com Doença de Chagas nas suas diversas formas. Grupo A (controle) com 21 indivíduos normais; Gupo B: 16 pacientes com sorologia positiva; Grupo C: com 16 pacientes com cardiopatia chagásica e Grupo D: 21 pacientes com esofagopatia e/ou megacolon chagásicos. Os resultados mostraram que näo houve diferenças significantes (p > 0,05) entre os 4 grupos estudados para os parâmetros: capacidade cistométrica, pressäo de micçäo e fluxo urinário. Entretanto, houve diferenças significantes (p < 0,05) para os parâmetros: pressäo abdominal, pressäo do detrusor, pressäo uretral e tempo de micçäo na análise comparativa entre o grupo D e o controle A. Esses achados sugerem que a Doença de Chagas, em sua forma digestiva, também pode comprometer o trato urinário baixo, alterando sua fisiologia.

Trypanosoma cruzi tubulin eliminated in the urine of the infected host.

In previous studies we have identified and characterized an 80-kDa Trypanosoma cruzi urinary antigen (UAg) eliminated during acute infection. Polyclonal antibodies raised against this antigen revealed by western blotting and immunoprecipitation analyses showed the existence of another antigenic component of 50-55 kDa in the UAg preparation. The antiserum was also used for screening of a T. cruzi expression library. Sequencing of inserts from selected cDNA clones showed high homology with the 3' end of the T.cruzi beta-tubulin gene sequence encoding for the C-terminus of the protein. The presence of T. cruzi tubulin in the UAg was confirmed by immunoprecipitation of a 50-55-kDa protein from 125I-labeled UAg with monoclonal antibodies (MAbs) to human alpha/beta-tubulin. Interestingly, MAbs recognized radiolabeled T. cruzi tubulin eliminated in the urine of infected mice 24 hr postinoculation of [35S]methionine-labeled viable trypomastigotes. Tubulin found in the urine proved to be of T. cruzi origin because this protein could not be identified in urinary specimens from uninfected animals or mice acutely infected with Leishmania infantum or Toxoplasma gondii. We conclude that tubulin is one of the parasite antigens eliminated in the urine of T. cruzi-infected hosts. This finding may be used to develop a noninvasive procedure for early diagnosis of Chagas' disease.

Presence of IgM antibodies to Trypanosoma cruzi urinary antigen in sera from patients with acute Chagas' disease.

An 80-kilodalton Trypanosoma cruzi antigen is eliminated in the urine of infected hosts during the acute stage of Chagas' disease. We show that affinity-purified urinary antigen is recognised by IgM antibodies in the sera from acute chagasic patients. Comparing our urinary antigen assay with that using a whole T. cruzi lysate antigen for IgM antibody detection, we demonstrated that ELISA with urinary antigen increases the diagnostic sensitivity and specificity of IgM serology in recent chagasic infection. Twenty-six of 30 patients with acute T. cruzi infection had serum IgM antibodies that reacted with urinary antigen by ELISA, while lysate antigen IgM was detected in 24 sera. When sera from patients suffering other parasitoses were tested, strong cross-reactions occurred in ELISA with T. cruzi lysate antigen, whereas ELISA with urinary antigen proved to better discriminate acute chagasic patients. Human antibodies to urinary antigen immunoprecipitated this T. cruzi urinary antigen and also inhibited the binding of monoclonal antibody to urinary antigen in an inhibition assay. These findings suggest that urinary antigen may be useful for the development of serodiagnostic procedures for acute T. cruzi infection.

An iron-binding Trypanosoma cruzi urinary antigen.

An 80-kDa Trypanosoma cruzi urinary antigen (UAg) was affinity-purified from the urine of infected dogs. We demonstrated that UAg is structurally and functionally related to proteins belonging to the transferrin family, as shown by amino acid sequence and iron binding experiments. Nevertheless, monoclonal antibodies raised against UAg specifically and selectively recognized this parasite's circulating antigen. The existence of an 80-kDa T. cruzi antigen co-migrating with UAg could be confirmed when epimastigotes were metabolically labelled with [35S] methionine and then immunoprecipitated with the above mentioned antibodies. We conclude that UAg is an iron-binding T. cruzi component eliminated in the urine of the infected host.

Trypanosoma cruzi: detection of a circulating antigen in urine of chagasic patients sharing common epitopes with an immunodominant repetitive antigen.

A monospecific antiserum raised to a Trypanosoma cruzi recombinant antigen, with tandem repeat of 68 amino acids, was used to screen urine samples of chagasic and nonchagasic patients. The antiserum detected a specific 150-160 kDa antigen in urine of 60% of chronic chagasic patients, but not in urine samples from nonchagasic patients and healthy control individuals. The reactivity to 150-160 kDa urinary antigen could be abolished by adsorption with the recombinant repetitive antigen. These results suggest that 150-160 kDa urinary antigen is a T. cruzi-derived antigen and specific for Chagas' disease.

Hemoglobinopatias e hepatoglobinas em portadores de doença de Chagas / Hemoglobinopathies and haptoglobin in patients with Chagas disease

A endemia chagásica é uma das doenças tropicais mais difundidas e graves, tornando-se um problema de saúde pública de primeira ordem nas Américas do Sul e Central. As condiçöes básicas para a transmissåo da doença indicam que esta endemia é de característica de áreas rurais de baixo nível sócio-econômico. A evoluçåo da doença manifesta-se diferentemente entre os indivíduos e, também, regionalmente. Diferenças de ordem individual e regional såo igualmente observadas em alguns tipos de hemoglobinas anormais e haptoglobinas. Com o objetivo de verificar a prevalência de hemoglobinas anormais e a freqüência de fenótipos de haptoglobinas em chagásicos, analisamos 2.739 amostras de sangue, dos quais 337 pertenciam a pacientes com diagnóstico clínico e laboratorial da doença de Chagas, 1402 tinham sorologia negativa para doença de Chagas (Grupo A) e 1.000 estudantes de 2§ gru de Såo José do Rio Preto (Grupo B). Na análise geral de hemoglobinas anormais, observou-se que a hemoglobina A2 (HbA2) aumentada foi mais prevalente entre chagásicos (8,3 por cento) em comparaçåo com os grupos A (1,21 por cento) e B (0,70 por cento). Análises posteriores comprovaram que este fato nåo se devia à associaçåo da doença de Chagas com talassemia beta heterozigota, principal causa de aumento desta fraçåo hemoglobínica, fato que pode ser indicativo de condiçöes adquiridas. A presença de agregados intraeritrocitários de hemoglobina H (HbH), que é comum na talassemia alfa, também foi mais prevalente entre os portadores da doença de Chagas, notadamente na forma indeterminada da doença (FIDC). Os estudos evidenciaram que esta prevalência também se deve à forma adquirida. A presença de outras hemoglobinas anormais nåo motrou diferenças signficativas entre os portadores da doença de Chagas e os grupos controle. Com relaçåo à estimativa dos valores quantitativos de hemoglobina Fetal (HbF), a média foi menor em portadores de Chagas quando comparados com os grupos sem esta doença, enquanto que para HbA2, sua concentraçåo média foi maior em chagásicos que em nåo chagásicos. Na análise de haptoglobinas obteve-se a qualificaçåo dos três fenótipos mais freqüentes (Hp 1-1, Hp 2-1, Hp 2-2). Os resultados mostraram que tanto em chagásico quanto nos grupos controle o fenótipo Hp 2-1 foi o mais prevalente, seguido de Hp 2-2 e Hp 1-1

Antigenuria in chronic chagasic patients detected by a monoclonal antibody raised against Trypanosoma cruzi.

A monoclonal antibody (B2/5) raised against Trypanosoma cruzi was able to immunoprecipitate a major 100 kDa polypeptide in 84% of the urines collected from chronic chagasic patients. Other polypeptides were also detected. The antibody recognized polypeptides on the surface of epimastigotes (150-25 kDa) and metacyclic trypomastigotes (150-50 kDa), suggesting that the antigens share a common epitope.

Antigenuria in infants with acute and congenital Chagas' disease.

Detection and partial characterization of Trypanosoma cruzi soluble antigens (SAg) in urine, as well as demonstration of parasite circulating antigens (CAg) in serum from pediatric patients with acute (10 patients) and congenital (10 patients) Chagas' disease, are reported. Classical techniques for parasite detection and antibody serology were also conducted in both groups. Samples collected before the onset of parasiticidal drug treatment were tested by an enzyme-linked immunosorbent assay for SAg and CAg demonstration. The control population consisted of 6 children with acute toxoplasmosis, 6 with cutaneous leishmaniasis, and 20 healthy individuals. Patients with acute cases were 100% positive for both SAg and CAg, whereas patients with congenital disease were 80% CAg positive and 100% SAg positive. Controls yielded negative results in all cases. Partial characterization of SAg from two patients with acute disease was performed by iodination, affinity chromatography, immunoprecipitation, and two-dimensional gel electrophoresis. Two different antigenic glycoproteins (80 kilodaltons, pI 6 to 6.5 and 55 kilodaltons, pI 6.5 to 7) were identified by these methods. Traditional serology and classical parasitologic tests failed, each in a different way, to provide an accurate diagnosis in the total of our patients. The enzyme-linked immunosorbent assay for SAg detection proved to be the most effective procedure for achieving early and precise proof of infection in acute and congenital cases of Chagas' disease.