Polymerase chain reaction detection of Trypanosoma cruzi in Macaca fascicularis using archived tissues

Abstract

This study describes conventional and real-time polymerase chain reaction (PCR) methods developed to detect and quantify Trypanosoma cruzi DNA in cynomolgus monkeys (Macaca fascicularis) using formalin-fixed paraffin-embedded blocks archived for periods of up to 6 years. The highest concentration of T. cruzi DNA was found in the myocardium, urinary bladder, stomach, lymph node, adrenal gland, and colon. The concentration of T. cruzi DNA detected in cardiac tissues was 10-100-fold greater than found elsewhere; the mean concentrations of T. cruzi DNA in non-cardiac tissues were otherwise comparable. Trypanosoma cruzi DNA was amplified from cerebrum but not cerebellum or kidney. Successful use of DNA from formalin-fixed, paraffin-embedded blocks is important because most pathology laboratories routinely archive wax blocks. This archived resource can be used for further studies on the prevalence of this disease.

Figure 1

A, Gross pathology in a 3-year-old Macaca fascicularis that died of naturally acquired Chagas disease, illustrating pulmonary edema, hydrothorax, hydropericardium, hepatomegaly, and rounded heart (arrow). Ascites have been drained. B, Histopathologic appearance of cardiac tissue in an infected M. fascicularis, illustrating the presence of focal to diffuse, primarily lymphocytic, myocardial infiltrates. H&E stain; bar = 200 μm. C, High power magnification of Panel B showing the presence within the cardiac muscle of a nest of organisms morphologically consistent with the amastigote form of T. cruzi (arrows). H&E stain; bar = 50 μm.

Figure 2

A, Polymerase chain reaction (PCR) detection of Trypanosoma cruzi DNA in cardiac tissue using primers TCZ1 and TCZ2. Lane 1: control animal; Lanes 2–5: infected animals; Lanes 6–7: positive controls (T. cruzi DNA, Tulahuen strain); Lane 8: distilled water; lane MK: molecular weight markers. B, PCR detection of T. cruzi DNA in spleen using primers TCZ1 and TCZ2. Lanes 1–5: uninfected control animal; Lanes 6–8: infected animal; Lane 9: distilled water; lane MK: molecular weight markers.

Figure 3

Concentration of Trypanosoma cruzi DNA (in pg) by real-time polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded (FFPE) tissue samples from six Macaca fascicularis positive for lymphocytic myocarditis and/or presence of T. cruzi organisms. Only non-zero concentrations are shown.