Haemoglobin interference and increased sensitivity of fluorimetric assays for quantification of low-parasitaemia Plasmodium infected erythrocytes.
BACKGROUND
Improvements 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.


METHODS
Parasitaemia 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.


RESULTS
Removal 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.


CONCLUSIONS
The 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.
Moneriz
Carlos
C
Departamento de Bioquímica y Biología Molecular IV, Universidad Complutense de Madrid, Spain. cmoneriz@vet.ucm.es
Marín-García
Patricia
P
Bautista
José M
JM
Diez
Amalia
A
Puyet
Antonio
A
D000818:Animals; D052160:Benzothiazoles; D002469:Cell Separation; D002738:Chloroquine / Q000494:pharmacology*; D003592:Cytophotometry / Q000379:methods; D016054:DNA, Protozoan / Q000187:drug effects; D003959:Diamines; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000469:parasitology*; D005456:Fluorescent Dyes; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D057230:Limit of Detection; D016778:Malaria, Falciparum / Q000175:diagnosis; D009930:Organic Chemicals; D018512:Parasitemia / Q000235:genetics / Q000469:parasitology*; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D011804:Quinolines; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D052160:Benzothiazoles; D016054:DNA, Protozoan; D003959:Diamines; D005456:Fluorescent Dyes; D006454:Hemoglobins; D009930:Organic Chemicals; C099476:PicoGreen; D011804:Quinolines; C098022:SYBR Green I; D002738:Chloroquine