Prospective multicenter evaluation of real time PCR Kit prototype for early diagnosis of congenital Chagas disease

Abstract

Background: Current algorithm for Congenital Chagas Disease (cCD) diagnosis is unsatisfactory due to low sensitivity of the parasitological methods. Moreover, loss to follow-up precludes final serodiagnosis after nine months of life in many cases. A duplex TaqMan qPCR kit for Trypanosoma cruzi DNA amplification was prospectively evaluated in umbilical cord (UCB) and peripheral venous blood (PVB) of infants born to CD mothers at endemic and non-endemic sites of Argentina.

Methods: We enrolled and followed-up 370 infants; qPCR was compared to gold-standard cCD diagnosis following studies of diagnostic accuracy guidelines.

Findings: Fourteen infants (3·78%) had cCD. The qPCR sensitivity and specificity were higher in PVB (72·73%, 99·15% respectively) than in UCB (66·67%, 96·3%). Positive and negative predictive values were 80 and 98·73% and 50 and 98·11% for PVB and UCB, respectively. The Areas under the Curve (AUC) of ROC analysis for qPCR and micromethod (MM) were 0·81 and 0·67 in UCB and 0·86 and 0·68 in PVB, respectively. Parasitic loads ranged from 37·5 to 23,709 parasite equivalents/mL. Discrete typing Unit Tc V was identified in five cCD patients and in six other cCD cases no distinction among Tc II, Tc V or Tc VI was achieved.

Interpretation: This first prospective field study demonstrated that qPCR was more sensitive than MM for early cCD detection and more accurate in PVB than in UCB. Its use, as an auxiliary diagnostic tool to MM will provide more accurate records on cCD incidence.

Funding: FITS SALUD 001-CHAGAS (FONARSEC, MINCyT, Argentina) to the Public-Private Consortium (INGEBI-CONICET, INP-ANLIS MALBRAN and Wiener Laboratories); ERANET-LAC-HD 328 to AGS and PICT 2015-0074 (FONCYT, MinCyT) to AGS and FA.

Keywords: Congenital Chagas disease; Discrete typing unit; Early diagnosis; Parasitic load; Real Time PCR; Trypanosoma cruzi.

Fig. 1

Flowchart of Prospective Field study to evaluate qPCR kit prototype for diagnosis of cCD. PVB: Peripheral Venous Blood; UCB: Umbilical Cord Blood; MM: MicroMethod. *In four enrolled babies no sample could be withdrawn. No visit: the patient did not attend the appointment for collection of the follow-up sample.

Fig. 2

Analysis of ROC curves to compare the accuracy of qPCR (right panels) and MicroMethod (left panels) for diagnosis of cCD in UCB (a) and PVB (b) samples in infants born to seropositive women. AUC: Area Under the Curve. Std-Error: Standard-Error; CI: Confidence Interval.

Fig. 3

Genotyping of Discrete typing Units in qPCR positive samples from cCD patients. a. 1•5% Agarose gel electrophoresis showing identification of Tc II/V/VI samples using SL-IR II Heminested PCR. PCR positive samples 1001 and 2047 were obtained from the corresponding infants at ten months of age. b. 1•5% Agarose gel electrophoresis showing identification of Tc I samples using SL-IR I Heminested PCR. The arrows indicate the specific amplicon. SCN: Seronegative blood sample; NTC: Non Template control; Tc I control: Silvio X-10 stock; Tc V control: MNCl2 stock; Mk 1 kb+, 1 kilobase plus DNA ladder molecular weight marker. Numbers above wells in red font indicate UCB samples, numbers in green font indicate PVB withdrawn at first follow-up time point and numbers in blue font indicate PVB collected at ten months of age.

Fig. 4

Proposed algorithm for diagnosis of cCD. Detectable MM and/or qPCR findings of a neonate´s PVB-based DNA lysate are reported as cCD. P: Positive, N: Negative, *If one of the serological techniques is not reactive, a third technique should be performed.