Chagas' disease diagnosis: comparative analysis of parasitologic, molecular, and serologic methods.

Abstract

Abstract. During the course of chronic chagasic infection, low parasitemia levels prevent parasite detection by current techniques such as hemoculture and xenodiagnosis. Since serologic tests have sensitivity but lack specificity, molecular assays based on the polymerase chain reaction (PCR) have been proposed as alternative tools for parasite detection in individuals with chronic Chagas' disease. A variable degree of PCR efficiency has been reported in the literature and illustrates the need for further evaluation of large numbers of chagasic patients. In this study, we compared an optimized PCR technique with hemoculture and complement-mediated lysis (CoML) in 113 individuals from or living in endemic areas of Brazil who had conventional serologic results that were either positive, negative, or inconclusive. The PCR amplification yielded positive results in 83.5% (66 of 79) of individuals with positive serology, 47.6% (10 of 21) with negative serology, and 46.2% (6 of 13) with inconclusive serology. Of 10 patients with negative serology and positive PCR result, eight (80%) had positive CoML, indicating that they could have been chagasic but were not mounting immune responses. The PCR results were also positive for all individuals who had positive hemoculture, for 37 individuals with negative hemoculture and positive serology, and for two of six individuals with inconclusive serology and negative hemoculture. Thirteen individuals living in nonendemic areas who had negative serology were used as a negative control group: 100% had negative PCR results. Our results show that the optimized PCR protocol used here was very sensitive in detecting the presence of Trypanosoma cruzi in chronic chagasic patients. The PCR and CoML results were well correlated in all of the groups studied, which suggests that our PCR protocol may be effective in the evaluation of cure in patients who receive anti-parasite treatment.Different approaches have been used in the diagnosis of chronic Chagas' disease. Serologic tests are used to detect antibodies against Trypanosoma cruzi and not the presence of the parasite itself. These tests have high sensitivity but lack specificity because of antigenic cross-reactivity with parasites such as Leishmania sp. and T. rangeli.1,2 Parasitologic tests such as hemoculture or xenodiagnosis have proven to be highly specific, but the sensitivity of these techniques is low. Recently, molecular assays such as the polymerase chain reaction (PCR), which amplify certain repetitive sequences of trypanosome kinetoplast DNA (kDNA) have been proposed as a good alternative tools for detection of T. cruzi in human blood.3-5 The ഠ330-basepair (bp) fragment of the kinetoplast minicircles is normally used as a target for amplification.The PCR assay has shown a variable degree of efficiency. Initial sensitivity reports ranged from 96% to 100% compared with serologic diagnosis.3,4 A lower sensitivity level was observed by Britto and others 6 and Junqueira and others. 7 These inconsistencies illustrate the need for addi...

Diagnosis of Chagas disease: what has been achieved? What remains to be done with regard to diagnosis and follow up studies?

“…Both donor and recipient tested serologically negative for T. cruzi prior to the procedure. This finding shows the possibility of false-negative results and highlights the need for more accurate diagnostic criteria, which should include not only serological tests, but also careful consideration of the epidemiological background of Chagas disease and an active search for the parasite using PCR methods as suggested by Gomes et al (1999).…”

Transmission of Tropical and Geographically Restricted Infections during Solid-Organ Transplantation

“…Parasitological methods are the methods of choice for the diagnosis of the infection in the acute phase, when there is a high circulating parasitemia (137). These methods include the identification of T. cruzi in Giemsa-stained thick and thin blood films or by means of concentration techniques.…”

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

“…Both methods have a tendency to increase their positivity with the number of tests performed, amount of blood employed, cultivation medium, interval of time between blood collection and cultivation and other factors emphasized by Chiari et al (1989). Moreover, these tests are time-consuming and laborious, requiring special laboratory biosecurity conditions (Brener 1962, Gomes et al 1999). In addition, post-therapeutic monitoring in the chronic phase of Chagas disease still constitutes a major challenge due to the long-term persistence of specific antibodies that are detected by conventional serology for several years, despite repeated negative direct parasite detection tests (Andrade et al 1988(Andrade et al , 1991; nonconventional serology is not frequently used .…”

“…However, in the 1990s, assays based on PCR were used to detect T. cruzi DNA in the blood of chronic chagasic patients with an adequate sensitivity (a single parasite cell in 10-20 mL of whole blood) and proven to be a promising tool for evaluating parasitological failure after specific etiologic treatment in chronic infections (Avila et al 1991, Britto et al 1993, 1995, Wincker et al 1994a, b, Junqueira et al 1996, Gomes et al 1999. Britto et al (1995) proposed for the first time the use of PCR tests to monitor chagasic patients submitted to specific treatment as a complement to serological investigation of cure.…”