Sample collection and analysis.

Blood was drawn from each patient and divided into samples of whole blood for direct microscopy and qPCR, blood clot for qPCR, and serum for serological testing.

Immediately after collection, guanidine was added to the sample of whole blood with ethylenediaminetetraacetic acid in a 1:1 ratio. Samples were brought to the CEADES laboratory in Cochabamba for direct observation microscopy and serology tests (recombinant ELISA, conventional ELISA, and hemagglutination). The microhematocrit concentration method was performed before microscopic examination. Six heparinized capillary tubes were filled with peripheral venous blood sample and centrifuged in a microhematocrit centrifuge at 12,000 rpm for 5 minutes. After centrifugation, the buffy coat within the capillary tubes was observed under the light microscope. The samples reported as positive had one to three parasites per high power field observed at magnification 400×. The samples for qPCR were stored at –20°C at a CEADES laboratory in Cochabamba and transported 1 to 12 months after collection to a laboratory at Universidad Peruana Cayetano Heredia in Lima, Peru, for qPCR.

Diagnosis of chronic Chagas disease was made if samples were positive by two of the three serology tests in accordance with WHO criteria for chronic Chagas disease.¹⁸ The diagnosis of Chagas reactivation was made if trypomastigotes of T. cruzi were visualized by direct microscopy by the microhematocrit method described earlier.

DNA was extracted following a standard phenol-chloroform protocol.¹⁹ Real-time quantitative (q)PCR was performed on the basis of previously published methods,²⁰ with a few modifications (Supplemental File 1). All laboratory workers were blinded from results of clinical evaluations, and technicians who performed the qPCR assays were blinded to the serologic results.

ECG abnormalities included as potentially caused by Chagas disease were bradycardia (< 60 beats/min), first- or second-degree heart block, right bundle branch block, left anterior fascicular block, atrial fibrillation, and atrial flutter. Patients were evaluated for cardiomegaly on chest radiograph defined by cardiothoracic ratio > 50% on PA film. Score of < 25 on the Folstein’s Mini-Mental Status Exam met criteria for mild cognitive deficit, ≤ 20 or less for moderate cognitive deficit, and ≤ 10 or less for severe cognitive deficit.²¹

Data were entered into Excel, and the analysis was carried out in STATA/IC version 10.1 (Stata Corp LP, College Station, TX). Continuous variables were described as median and 25 to 75 percentiles because they were not normally distributed. Categorical variables were described as absolute or relative frequencies. For continuous variables, medians were compared using Mann-Whitney test. For categorical variables, χ² test or Z-test was used, and Fisher’s exact test was used for the comparison of two proportions. The correlation between estimated parasitemia by qPCR (number of parasites per milliliter) and CD4+ count (in the logarithm scale), as well as correlation between parasitemia by qPCR and HIV viral load were estimated through the Spearman’s rank correlation coefficient.