Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease

Abstract

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected—namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen’s kappa index and Bland–Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = −0.58–0.62] and −0.04 [CI = −0.45–0.37] and a Cohen’s kappa coefficient of 0.78 [CI = 0.60–0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.

Parasitemia Levels in Trypanosoma cruzi Infection in Spain, an Area Where the Disease Is Not Endemic: Trends by Different Molecular Approaches

“…4 and 9 ; see also Table S1 in the supplemental material), Sat-TcLAMP allowed the monitoring of parasitemia levels like qPCR, and thus, they can be used interchangeably for the early detection of the risk of reactivation in chronic infection ( Fig. 9 ); similar findings were described previously by Muñoz-Calderón et al ( 40 ) but in qualitative terms.…”

Comparison of Simple RNA Extraction Methods for Molecular Diagnosis of Hepatitis C Virus in Plasma

“…Thus, it can be used as an alternative extraction method. A previous study also showed that magnetic-bead extracted DNA had high agreement with spin-column extracted DNA for downstream LAMP testing [ 12 ]. However, samples with low viral loads may cause a false negative result when used with RT-LAMP detection.…”

Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots

“…Similarly, it has been described it could be of major relevance for the detection of acute T . cruzi infections like those found upon congenital transmission of the parasite or during oral outbreaks [ 5 – 8 ].…”

“…Several LAMP procedures have already been proposed for the detection of T. cruzi infections [5][6][7][8][9][10]. The most studied of all has been the prototype kit developed by Eiken Chemical Company (Tokyo, Japan) based on T. cruzi nuclear satellite DNA (satDNA) sequences and containing dried reagents on the inside of the microtube caps, which has proven as sensitive as realtime quantitative PCR (qPCR) in blood samples collected from patients with acute, congenital and Chagas disease reactivations, as well as in patients under treatment monitoring [5][6][7][8].…”