Abstract
BackgroundImprovements on malarial diagnostic methods are currently needed for the correct detection in low-density Plasmodium falciparum infections. Microfluorimetric DNA-based assays have been previously used for evaluation of anti-malarial drug efficacy on Plasmodium infected erythrocytes. Several factors affecting the sensitivity of these methods have been evaluated, and tested for the detection and quantification of the parasite in low parasitaemia conditions.MethodsParasitaemia was assessed by measuring SYBRGreen I® (SGI) and PicoGreen® (PG) fluorescence of P. falciparum Dd2 cultures on human red blood cells. Different modifications of standard methods were tested to improve the detection sensitivity. Calculation of IC50 for chloroquine was used to validate the method.ResultsRemoval of haemoglobin from infected red-blood cells culture (IRBC) increased considerably the fluorescent signal obtained from both SGI and PG. Detergents used for cell lysis also showed to have an effect on the fluorescent signal. Upon depletion of haemoglobin and detergents the fluorescence emission of SGI and PG increased, respectively, 10- and 60-fold, extending notably the dynamic range of the assay. Under these conditions, a 20-fold higher PG vs. SGI fluorescent signal was observed. The estimated limits of detection and quantification for the PG haemoglobin/detergent-depleted method were 0.2% and 0.7% parasitaemia, respectively, which allow the detection of ~10 parasites per microliter. The method was validated on whole blood-infected samples, displaying similar results as those obtained using IRBC. Removal of white-blood cells prior to the assay allowed to increase the accuracy of the measurement, by reducing the relative uncertainty at the limit of detection from 0.5 to 0.1.ConclusionThe use of PG microassays on detergent-free, haemoglobin-depleted samples appears as the best choice both for the detection of Plasmodium in low-density infections and anti-malarial drugs tests.
Highlights
Improvements on malarial diagnostic methods are currently needed for the correct detection in low-density Plasmodium falciparum infections
DNA standard curves in the presence of detergents SYBRGreen I® (S33102) and PicoGreen® (P7589) were purchased to Invitrogen and diluted as indicated by the manufacturer in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) or TE plus different combinations of saponin and Triton X-100 detergents to obtain a range of concentrations from 1000 ng/mL to 5 ng/mL
Higher signal was detected for PG (706 ± 33 above. Fluorescence Unit (AU) per 1 μg/mL) as compared to SYBRGreen I® (SGI) (141 ± 40 AU per 1 μg/mL) in TE
Summary
Improvements on malarial diagnostic methods are currently needed for the correct detection in low-density Plasmodium falciparum infections. Several factors affecting the sensitivity of these methods have been evaluated, and tested for the detection and quantification of the parasite in low parasitaemia conditions. The early detection and monitoring of malarial disease has become a key requirement for the efficient control of Plasmodium infections. Detection of malaria in pregnancy poses a challenge due to the low parasitaemia in peripheral blood [3]. While microscopic methods are simple, they require trained microscopist and do not allow detection at densities below 50 parasites/μL. Real-time PCR showed to be highly sensitive and allows the specific detection of species, but it includes labour-consuming methods such as DNA extraction, and requires expensive equipment and temperature-labile consumables [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
