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

Abstract

Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10^-1 to 5 × 10⁵ versus >1 × 10^-1 to 6 × 10³ parasite equivalents/mL, respectively [P < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia (P = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.

Keywords: Chagas disease; LAMP; Trypanosoma cruzi; acute reactivation; chronic infection; congenital infection; molecular diagnosis; parasite load; parasitemia quantification; qPCR.

FIG 1

Flow of sample analysis. (A) Samples from congenital infections. (B) Samples from imported chronic infections. (C) Samples from individual cases in follow-up to monitor changes in parasitemia levels before treatment. T₁, time of first sample collection; T_n, time of consecutive sample collection, in one case T₂; in two cases T₂ and T₃; in two other cases T₂, T₃, and T₄; and in the last case T₂ up to T₇.

FIG 2

Distribution of cases according to the phase of Chagas disease (CD) and other characteristics.

FIG 3

Comparison of the absolute and relative rates of positivity between kDNA and Sat-DNA tests. The rates of positivity in congenital (Cong) infection were similar (Q = 3 [P = 0.392]), whereas the differences in imported (Imp) chronic infection were significant (Q = 57.5 [P < 0.001]); there were no significant differences between Sat-DNA tests (Q = −0.43 [P = 0.321]) and kDNA tests (Q = −0.78 [P = 0.74]). CD, Chagas disease. The number of cases is indicated in white in each column, and the percentages of positivity for each test are indicated in black.

FIG 4

Relationship between C_T and time-to-positivity (T_p) values. (A) Sat-TcLAMP versus Sat-qPCR. (B) Sat-TcLAMP versus kDNA-qPCR. (C) kDNA-qPCR versus Sat-qPCR. The T_p of Sat-TcLAMP is expressed as the inverse to improve the linear shape of the association. Red dots, congenital infection cases diagnosed before 9 months of age; black dots, children with congenital infection diagnosed at between 1 and 5 years of age; gray dots, imported chronic infection cases diagnosed at between 10 and 65 years of age. Dashed lines highlight cases with C_T values outside the quantification range. R, coefficient of regression.

FIG 5

Standard curve for Sat-TcLAMP. Shown are polynomial (A) and linear (B) regressions between the time to positivity (T_p) and parasite equivalents (par eq) (P < 0.0001 for both models).

FIG 6

Bland-Altman plots for agreement analysis of parasitemia level estimations by Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR. (A to C) All cases with results by both comparison tests. (D to F) Congenital infection group. (G to I) Imported chronic infection group. The black dashed lines represent the limits of agreement. The red dashed lines represent the linear regression of data points. Χ, mean difference (bias regarding difference values of 1); SD, standard deviation; R, coefficient of regression; par eq, parasite equivalents. Axes are on logarithmic scales.

FIG 7

Estimation of parasite loads by Sat-qPCR, kDNA-qPCR, and Sat-TcLAMP. (A) Cases of congenital infection diagnosed before 9 months of age. (B) Cases of congenital infection diagnosed after 9 months and before 5 years of age. (C) Cases of imported chronic infection. The black lines in the dots represent the median values of parasitemia levels. The color background is defined considering the limit of detection of microhematocrit, 0 to 40 parasite equivalents/mL (white), >40 to 500 parasite equivalents/mL (very light blue), >500 to 10,000 parasite equivalents/mL (light blue), and >10,000 parasite equivalents/mL (blue) (31–33). F, Friedman test value. The vertical axis is on a semilogarithmic scale.

FIG 8

Parasite loads according to age at diagnosis. (A) Data interpretation from this study. (B) Parasitemia levels from other studies. Values are in parasite equivalents (par eq) per milliliter. According to our data, the strength of the association between parasitemia levels and age was moderate from 0 to 38 years of age (Sat-TcLAMP versus age, r_s = −0.456 [P < 0.001]; Sat-qPCR versus age, r_s = −0.647 [P < 0.001]; kDNA-qPCR versus age, r_s = −0.583 [P < 0.001]). ^a, reference ; ^b, reference ; ^c, reference ; ^d, reference ; ^e, reference ; ^f, reference ; ^g, reference ; ^h, reference ; ⁱ, reference ; ^j, reference ; ^k, reference . Bol, Bolivia; SC, Santa Cruz; Cba, Cochabamba; Arg, Argentina; BA, Buenos Aires; Col, Colombia; Flo, Florinda; Bo, Bogota. Dashed lines are trend lines. The color background is defined considering the limit of detection of microhematocrit, 0 to 40 parasite equivalents/mL (white), >40 to 500 parasite equivalents/mL (very light blue), >500 to 10,000 parasite equivalents/mL (light blue), and >10,000 parasite equivalents/mL (blue) (31–33).

FIG 9

Parasitemia monitoring in terms of C_T and T_p values. Low C_T values represent high levels of parasitemia. (A) Congenital infection. The horizontal axis represents days of life, with zero being the birth date. (B) Imported chronic infection. The horizontal axis represents days before treatment, with zero being the day of the beginning of treatment.