Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation

Abstract

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this 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 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp 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

Clinical profile and mortality in patients with T. cruzi/HIV co-infection from the multicenter data base of the “Network for healthcare and study of Trypanosoma cruzi/HIV co-infection and other immunosuppression conditions”

“…In this work, monitoring for CDR was made in 48.1% of patients with co-infection by parasitological enrichment methods, and more rarely, by PCR. Quantitative PCR represents a safety test to monitor parasitemia in immunocompromised hosts when able to differentiate between higher parasitemia in CDR and lower parasitemia in non-reactivation episodes of the co-infection [ 22 , 38 , 42 , 43 ]. Therefore, its implementation is essential for CDR detection as rapidly as possible in order to adapt immunosuppressive treatment and to start the anti-parasitic treatment.…”

Diagnostic Accuracy of Two Molecular Tools for Diagnosis of Congenital Chagas Disease

“…However, Besuschio et al tested a LAMP prototype kit, so far not commercially available, on a series of archival DNA samples from acutely infected patients and found sensitivity and specificity values of 93% and 100%, respectively [ 15 , 16 ]. All the samples they obtained from CCD-infected patients (average age 108 days old) yielded positive LAMP results and the parasitic loads found were between 5.27 and 3063.47 par.…”

“…All the samples they obtained from CCD-infected patients (average age 108 days old) yielded positive LAMP results and the parasitic loads found were between 5.27 and 3063.47 par. eq/mL [ 15 , 16 ]. Because the performance of the molecular tests will critically depend on the copy number of the target sequences, probably, increased sensitivity was possible because this prototype kit is targeted to satellite DNA sequences (a higher copy number than the 18S rRNA genes).…”

“…for T. cruzi detection [14][15][16]. Before implementation into routine clinical practice and incorporation into guidelines, every new diagnostic assay should go through a lengthy validation process.…”

“…Over 20 years, the PCR technique has been presented as a promising tool for sensitive and specific detection of T. cruzi parasites [ 11 – 13 ]. Furthermore, isothermal DNA amplification (LAMP), which requires less infrastructure, has been reported as a possible molecular diagnostic tool for T. cruzi detection [ 14 – 16 ]. Before implementation into routine clinical practice and incorporation into guidelines, every new diagnostic assay should go through a lengthy validation process.…”