Still Searching for a Suitable Molecular Test to Detect Hidden Plasmodium Infection: A Proposal for Blood Donor Screening in Brazil

Abstract

BackgroundEfforts have been made to establish sensitive diagnostic tools for malaria screening in blood banks in order to detect malaria asymptomatic carriers. Microscopy, the malaria reference test in Brazil, is time consuming and its sensitivity depends on microscopist experience. Although molecular tools are available, some aspects need to be considered for large-scale screening: accuracy and robustness for detecting low parasitemia, affordability for application to large number of samples and flexibility to perform on individual or pooled samples.MethodologyIn this retrospective study, we evaluated four molecular assays for detection of malaria parasites in a set of 56 samples previously evaluated by expert microscopy. In addition, we evaluated the effect of pooling samples on the sensitivity and specificity of the molecular assays. A well-characterized cultured sample with 1 parasite/μL was included in all the tests evaluated. DNA was extracted with QIAamp DNA Blood Mini Kit and eluted in 50 μL to concentrate the DNA. Pools were assembled with 10 samples each. Molecular protocols targeting 18S rRNA, included one qPCR genus specific (Lima-genus), one duplex qPCR genus/Pf (PET-genus, PET-Pf) and one duplex qPCR specie-specific (Rougemont: Roug-Pf/Pv and Roug-Pm/Po). Additionally a nested PCR protocol specie-specific was used (Snou-Pf, Snou-Pv, Snou-Pm and Snou-Po).ResultsThe limit of detection was 3.5 p/μL and 0.35p/μl for the PET-genus and Lima-genus assays, respectively. Considering the positive (n = 13) and negative (n = 39) unpooled individual samples according to microscopy, the sensitivity of the two genus qPCR assays was 76.9% (Lima-genus) and 72.7% (PET-genus). The Lima-genus and PET-genus showed both sensitivity of 86.7% in the pooled samples. The genus protocols yielded similar results (Kappa value of 1.000) in both individual and pooled samples.ConclusionsEfforts should be made to improve performance of molecular tests to enable the detection of low-density parasitemia if these tests are to be utilized for blood transfusion screening.

Field evaluation of malachite green loop-mediated isothermal amplification as a malaria parasite detection tool in a health post in Roraima state, Brazil

“…The reproducibility of PCR assays in the detection of samples with very low parasitemia was shown to alternate between positive and negative in about 38% of PCR replicates tested 25 . Similar inconsistencies, with low parasitemia samples, were observed in other studies 15, 26 . While PET-PCR remains a more sensitive assay overall when diagnosing low-density infections, it is a far more complicated procedure compared to the MG-LAMP, which requires costly equipment and resources.…”

Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates

“…Regarding the disagreement between molecular techniques and microscopy results, this finding has also been reported by other authors who analyzed blood samples from New World primates (DUARTE et al, 2008;YAMASAKI et al, 2011;FIGUEIREDO et al, 2015) and humans (COLEMAN et al, 2006;BOONMA et al, 2007). In general, PCR has been found to be more sensitive and specific than thick and thin-drop blood smears and RDT for both human (LIMA et al, 2016) and New World primates blood samples, particularly in cases of low levels of parasitemia or mixed infections (BROWN et al, 1992;MOODY, 2002;RUBIO et al, 1999).…”

“…In all the reactions, ultra-pure water (Invitrogen  , Carlsbad, CA, USA) was used as a negative control. Plasmodium vivax and P. malariae DNA samples from human infected patients (HRISTOV et al, 2014;LIMA et al, 2016) and P. falciparum DNA (strains K1 and Palo Alto obtained through in vitro culturing) were used as positive controls. The products were then subjected to a second reaction, using 2 μL of the amplified product from the first reaction, in a final reaction volume of 25 μL, containing 1X Taq buffer, 2.5 mM of MgCl 2 , 0.1 mM of deoxynucleotide triphosphate mixture, 0.5 μM of PLF, 0.064 μM of MAR, 0.3 μM of FAR, 0.05 μM of VIR and 1.25 U of Taq DNA polymerase.…”

“…Construction of plasmid standards containing Plasmodium sp. 18S rRNA gene fragments Plasmodium falciparum and P. malariae (HRISTOV et al, 2014;LIMA et al, 2016) 18S rRNA gene fragments were amplified in conventional PCR assays using the same set of primers that are used in qPCR assays (GAMA et al, 2007). The amplification reactions were performed in a T100 thermal cycler (Bio Rad), using 4 μL of DNA in a 46 μL mixture, containing 1X PCR buffer, 2 mM of MgCl 2 , 1.25 U of Taq Platinum DNA Polymerase (Life Technologies  , Carlsbad, CA, USA), 200 μM of each dNTP and 200 nM of each M60 and M61 primer.…”

Molecular Epidemiology of Plasmodium Falciparum by Multiplexed Amplicon Deep Sequencing in Senegal

“…Each experimental run included both a negative (no template) and a positive (3D7 P. falciparum strain) control. Samples with a cycle threshold (CT) of 40 or less were scored as positive [33][34]. The two Pfmsp1 and Pfmsp2 polymorphic genes were ampli ed by multiplex PCR.…”