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New Multiplex qPCR Assay May Enable Better Chagas Disease Monitoring


NEW YORK (GenomeWeb) – About eight million people worldwide are infected with Trypanosoma cruzi, the parasite that causes Chagas disease, but research into epidemiology and treatment of this neglected tropical disease has been hampered by the lack of ideal molecular tests.

Now, scientists in Argentina have developed a multiplex PCR assay that may simplify the process, and have demonstrated its potential by typing a large set of international samples.

The protozoa T. cruzi can be classed into six "discrete typing units," or DTUs, based on three nuclear loci, and DTUs are differentially distributed in endemic areas, Alejandro Schijman, a researcher at El Instituto de Investigaciones en Ingeniería Genética y Biología Molecular inBuenos Aires, told GenomeWeb in an email.

In the study published last week in PLoS Neglected Tropical Diseases, Shijman and his collaborators described a multiplex, TaqMan probe-based, real-time PCR assay for the six DTUs.

They then used the new assay to type 307 different biological samples and 39 cultured stocks of T. cruzi. Samples from Chagas patients, animal reservoirs, and insect vectors came from about a dozen of the 21 endemic countries in the Americas as well as a number of European research labs.

The standardized DTU nomenclature, developed about five years ago by experts in the field, was meant to enable things like new drug discovery and outbreak monitoring. However, using conventional PCR for this typing can be cumbersome and time consuming and often relies on initial culturing of the parasites, the study noted.

Parasite culture "may under-represent the complexity of the natural infection because of the coexistence of polyclonal populations that may belong to different DTUs, and because in vitro culturing favors detection of the most competitive parasite clones," Shijman explained.

In the study, the new typing protocol characterized all DTUs from patients with acute Chagas infection in total agreement with conventional PCR techniques.

The method also gave positive results in a high percentage of direct samples and culture isolates from the most common Chagas insect vector, triatomine bugs, as well as samples from mammalian reservoirs like raccoons, opossums, and dogs.

The assay detected a few mixed infections, but the incidence of mixed infection in patients is actually unknown, since previous typing was usually done from culture, allowing a single DTU to dominate.

The results of the current study suggest that there is "a higher frequency of mixed infections than previously observed," Shijman noted.

The molecular targets used for typing are not as repetitive as the satellite DNA or minicircle DNA sequences used for typical molecular diagnosis, Schijman said, so known cases with low parasite load were more difficult to type.

The test was less successful on samples from patients with chronic disease, for example.

In that stage, parasites hide in the heart and digestive tract, and overall parasitic load is low. "This is the main reason that PCR is unsuitable for diagnosis of chronic Chagas disease, and consequently its laboratory diagnosis depends on serological methods," Shijman said.

Chronic disease may ultimately be amenable to approaches that characterize the human immune response to Chagas disease, as recently reported by GenomeWeb.

The researchers will now use this assay for molecular epidemiology, and have published the primer and probe sequences "so everybody can reproduce the test for their own interests," Shijman said.

The study also points out that the assay would be appropriate for creation of a Chagas disease typing kit.

"At present we are working on a kit prototype for molecular diagnosis of congenital Chagas disease using a similar PCR approach in the context of a private-public consortium financed by the [Argentina] National Ministry of Science, Technology and Innovation, but have not initiated actions towards the development of a kit for DTU typing," said Shijman. 

In the US, there are an estimated 300,000 cases of Chagas disease but access to care is limited, according to one recent study. The US blood supply has also been routinely screened for serological markers of T. cruzi infection since 2007. One examination of the blood supply in New York State found a total of 204 donors were positive for T. cruzi antibodies between 2007 and 2011.