Usefulness of real time PCR to quantify parasite load in serum samples from chronic Chagas disease patients

Abstract

BackgroundInconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi load in peripheral blood samples. A promising perspective refers to the possibility of parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening.MethodsIn order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction.ResultsThe comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 105 to 0.5 par. eq./mL) between serum, blood or culture samples (T. cruzi epimastigotes – Cl Brener strain) was found. When patient samples were evaluated, no significant differences in parasite load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples.ConclusionsTaken together, our data indicate the potential of using serum samples for molecular diagnosis and parasite load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients.

A therapeutic nanoparticle vaccine againstTrypanosoma cruziin a BALB/c mouse model of Chagas disease

“…60 Briefly, 5£10 6 splenocytes were stained with CFSE (Life Technologies, C34554) and then incubated with 50 mg/mL Tc24, 5 mg/mL ConA, or media only, 72 hrs, 37 C, 5% CO 2 . Cells were stained with LIVE/DEAD TM Fixable Blue Stain (Life Technologies, L-34961), and then incubated with Purified Rat Anti-Mouse CD16/CD32 (BD Biosciences, 553142).…”

Sequential measurement of Trypanosoma cruzi parasitic load in endomyocardial biopsies for early detection and follow‐up of Chagas disease reactivation after heart transplantation

“…Besides the careful search for parasites in EMB, monitoring for CDR is based on microscopic examination of blood smears and fresh buffy coat preparations, and more recently blood PCR for T cruzi DNA. 5,9,16,17 As non-immunosupressed, chronic chagasic patients can show positive PCR in the blood, 18 probably due to parasite persistence and fortuitous blood circulation, one proposed criterion for the diagnoses of CDR is sequential positive blood PCR results (at least two) of increasing parasitic load. 17 In this study, we showed sequential measurement of T cruzi parasitic load in EMB is useful for the early detection and treatment follow-up of CDR.…”

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“…Ascaris ascaris worm long, cylindrical and thin ends of the milky white color and flower color. ascaris adult females 20 to 45 cm in length and diameter of 3 to 6 mm and length of 15 to 30 cm shorter female Ascaris male and 2 to 4 mm diameter [6]. Worm egg.…”

“…Although malaria is suspected as required at any time to collect samples, respecting the time of blood sampling, species and life stages of the parasite makes more possible [8]. The best time for blood in between attacks of chills and fever before, and usually due to the impossibility of finding the parasite in blood samples once collected blood smear should continue until three consecutive days every 6-8 hours [6]. It is clear that the use of anti-malaria drug samples has diagnostic value.…”