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

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

Detection of apoptotic cells in cardiac tissue of guinea pigs infected with <i>T. cruzi</i>.

<p>(A). Apoptosis in inflammatory infiltrate (arrows) at 165 dpi, 400×. (B) Apoptosis of cardiomyocytes (asterisks) surrounded by inflammatory infiltrate (arrows) at 365 dpi, 1000×. (C). Apoptosis of endothelial cells (arrowheads) and inflammatory infiltrate (arrows) at 25 dpi, 400×. (D). Apoptosis of epicardial adipose tissue (arrowheads) and inflammatory cells (asterisks) at 25 dpi, 400×. (E). Apoptosis-like death (green nucleus) in amastigote nests at 25 dpi, 400×. (F). Apoptosis-like death in amastigote nest (arrowheads) detected in cardiac tissue and surrounded by adipocyte cells and inflammatory infiltrate at 25 dpi, 400×. Apoptotic cells: green or yellow nucleus). Non-apoptotic cells: red nucleus.</p

Levels of serum creatinine, serum urea, urine protein and urine KIM-1 in guinea pigs.

<p>Bars represent mean values per group; lines on the bars represent the standard deviation * Statistically significant (p<0.05). Number of animals in infected group: 5 days = 8, 15 days = 8, 20 days = 8, 25 days = 8, 40–55 days = 16, 115–365 days = 19. Number of animals in non-infected group: 5 days = 2, 15 days = 2, 20 days = 2, 25 days = 2, 40–55 days = 4, 115–365 days = 6.</p

Serum levels of PICP and PIIINP in guinea pig infected with <i>T. cruzi</i>.

<p>A. Serum levels of procollagen type I carboxy-terminal propeptide (PICP) were significantly higher in guinea pigs with cardiac fibrosis than those without fibrosis: * p = 0.019. ** p = 0.022. Fibrotic cases were considered those with more than 15% of microscopic fields with collagen I deposition. B. Serum levels of procollagen type III amino-terminal propeptide (PIIINP) significantly higher in guinea pigs with cardiac fibrosis than those without fibrosis: * p = 0.028. Fibrotic cases were considered those with more than 15% of microscopic fields with collagen III deposition. Acute phase: n = 12, late acute phase: n = 11, early chronic phase: n = 11 and chronic phase: n = 6. The bars and error bars represent mean ± standard deviation. T-test with unequal variances.</p

Histopathological changes in kidney tissue of guinea pigs infected with <i>T. cruzi</i>.

<p>Hematoxylin-eosin stain: A). Amastigote nests (arrows) and tubular necrosis, 25 dpi (500x). B). Focal and mild periglomerular and interstitial inflammation (arrows), 25 dpi (100x). C). Glomerulus of non-infected guinea pig. Note the number of nucleus (1 to 3) in the mesangium (1000x). D). Mesangial hypercellularity and congestion (arrows), 365 dpi (400x). E). Dilatation of proximal tubules (arrows) and periglomerular inflammation, 40 dpi, (200x). <b>Masson’s Trichromic stain:</b> F). Kidney tissue of non-infected guinea pig (400x). G). Mild increase in interstitial collagen, 365 dpi (400x). H). Moderate increase in interstitial collagen and tubular atrophy, 25 dpi (200x).</p

Kinetics of antigen detection in urine samples of guinea pig infected with <i>Trypanosoma cruzi</i>.

<p>Bands were detected by Western Blot using a polyclonal antibody against excretory-secretory trypomastigote <i>T. cruzi</i> antigen.</p><p>N: Number of animals per group.</p><p>n: Number of animals with antigenuria test positive.</p><p>%: Percentage of positive animals.</p><p>The antigenuria test was considered positive when any of the bands of 75, 80, 120 or 150 kDa were detected.</p

Distribution of collagen isotypes in cardiac tissue of guinea pig infected with <i>T. cruzi</i>.

<p>Deposition of collagen I (A), collagen III (B) and collagen IV (C) during the course of infection. Moderate increase of interstitial collagen I, at 365 dpi, 400× (D). Increase of perivascular collagen I, at 365 dpi, 400× (E). Severe increase of interstitial collagen III and moderate inflammation, at 365 dpi, 400× (F). Increase of collagen III near epicardial adipose tissue at 165 dpi, 400× (G). Detection of collagen IV in non-infected guinea pig (H). Increase of collagen IV in the basement of cardiac fibers, at 365 dpi, 400× (I). Acute phase (20–25 dpi): n = 12, late acute phase (40–55 dpi): n = 11, early chronic phase (115–165 dpi): n = 11 and chronic phase (365 dpi): n = 6.</p

Histopathological and biochemical changes during kidney injury in <i>C. porcellus</i> infected with <i>T. cruzi.</i>

a<p>Parasites in kidney were detected by H&E and PCR. In order to be considered positive one or more parasites needed to be observed in two entire tissue sections.</p>b<p>Corresponds to the increase of level of two or more biochemical parameters: serum urea, urine proteins and urine KIM-1.</p><p>n = Number of animals analyzed.</p