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

BackgroundEarly 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 FindingsT. 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 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.ConclusionChunap 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.Author SummaryReactivation of Chagas disease in people living with HIV is a serious clinical condition that is associated with high mortality. Hence, early diagnosis and treatment can be lifesaving. Although there are not well accepted criteria to identify patients at risk of reactivation, parasitemia levels are usually considered as the best predictor. Microscopy is used in Latin America for detection of parasitemia levels. However, this has low sensitivity, which usually leads to a delay in diagnosis and treatment. Quantitative PCR is used only for research proposes in endemic areas. Antigens in urine (antigenuria) are correlated with parasitemia levels in animal models, as well as in cases of congenital Chagas disease. We believe that antigenuria can also be used for prediction of parasitemia levels in T. cruzi/HIV co-infected patients. In this study, Chunap (Chagas urine nanoparticle test) was used for concentration and quantification of T. cruzi antigens in urine of T. cruzi/HIV co-infected patients. Values of more than 105 pg of T. cruzi antigens in urine were observed only in patients with reactivation of Chagas disease. This study shows that antigenuria levels are highly correlated to levels of parasitemia and can be used as a non-invasive technique for monitoring parasitemia levels in T. cruzi/HIV co-infected patients

Characteristics of HIV patients in the nested-case control study.

<p>Characteristics of HIV patients in the nested-case control study.</p

Levels of parasitemia and antigenuria in <i>T</i>. <i>cruzi</i>/HIV co-infected patients.

<p>A.) Levels of parasitemia determined by qPCR in <i>T</i>. <i>cruzi</i>/HIV co-infected patients. B.) Levels of antigenuria determined by Chunap in the three groups of <i>T</i>. <i>cruzi</i>/HIV co-infected patients. Levels of parasitemia: High parasitemia = positive by PCR and microscopy, Moderate parasitemia = positive by PCR, and negative by microscopy, Negative parasitemia = negative by PCR and microscopy but positive by serology, and No Chagas = negative by microscopy, PCR and serology. Bottom and top limits of the boxes correspond to first and third quartiles, and the line inside the boxes represents the second quartile (median). P-values were calculated using T-test with equal variances. * Statistically significant difference.</p

Antigenic bands in nanoparticles-concentrated urine samples of patients with HIV<i>/T</i>. <i>cruzi</i> co-infection.

<p>Bands were detected by Western Blot using a monoclonal antibody anti-lipophosphoglycan of <i>T</i>. <i>cruzi</i>. Bands of 20 kDa, 42 kDa, 58 kDa, 75 kDa, 82 kDa were considered specific for <i>T</i>. <i>cruzi</i>. Bands of other molecular weight were not considered for diagnosis criteria. Urine samples of <i>T</i>. <i>cruzi</i>+/HIV+ patients: Lanes 1–3, 5, 7, 9, 10–11, 13–14. Urine samples of <i>T</i>. <i>cruzi</i>-/HIV+ patients: Lanes 4, 6, 8, 12, and 15.</p