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Loop-Mediated Isothermal Amplification Kit Detects Chagas Disease

By LabMedica International staff writers
Posted on 07 Sep 2020
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Image: Visualization of Loopamp Trypanosoma cruzi results by the naked eye, Tubes 1 (Negative control) and 5 were negative, but #5 was positive on qPCR with a very low DNA load (Photo courtesy of Laboratorio de Biología Molecular de la Enfermedad de Chagas).
Image: Visualization of Loopamp Trypanosoma cruzi results by the naked eye, Tubes 1 (Negative control) and 5 were negative, but #5 was positive on qPCR with a very low DNA load (Photo courtesy of Laboratorio de Biología Molecular de la Enfermedad de Chagas).
Chagas disease (CD), also  known as American trypanosomiasis, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi that affects about six to seven million people worldwide, mainly in endemic areas of 21 Latin American countries.

The disease evolves from an acute phase when the infection is acquired, which is frequently asymptomatic, to a chronic phase that may develop, in up to 30% of cases, cardiac disease, and in 10% of cases digestive mega-syndromes, neurological and/or mixed complications.

Molecular Biologists at the Research Institute in Genetic Engineering and Molecular Biology (Buenos Aires, Argentina) and their colleagues developed as a ready-to-use diagnostic Loop-Mediated Isothermal Amplification method requiring minimal laboratory facilities to diagnose CD. They evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios.

In a retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 non-infected), four recipients of organs from CD donors, six orally–infected cases after consumption of contaminated guava juice and six CD patients co-infected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and one CSF sample). All were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). Duplex real-time qPCR using TaqMan probes targeted to T. cruzi satellite DNA, plus an internal amplification control (IAC), was carried out in an ABI7500 thermocycler (Applied Biosystems, Foster City, CA, USA).

The team reported that the sensitivity and specificity of Tc LAMP kit in blood samples from the pooled clinical groups was 93% (95% CI: 77–99) and 100% (95% CI: 80–100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62–1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations.

The authors concluded that Tc LAMP is a potentially useful rapid laboratory tool for the diagnosis and treatment monitoring of T. cruzi acute infections and cases of reactivation. Its advantages are that no complex laboratory infrastructure is needed, allowing its application in healthcare facilities with limited equipment, and giving results rapidly through detection by naked eye. The study was published on August 14, 2020 in the journal PLOS Neglected Tropical Diseases.

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Research Institute in Genetic Engineering and Molecular Biology
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