Molecular monitoring of the diversity of human pathogenic malaria species in blood donations on Bioko Island, Equatorial Guinea

Abstract

Background: Malaria can be transmitted by blood transfusion from human to human and it is responsible for the majority of transfusion-transmitted infectious diseases worldwide. In sub-Saharan Africa, it had been estimated that almost a quarter of blood donations contain malaria parasites. Since rapid diagnostic tests and thick blood smear microscopy lack sensitivity for low density parasitaemia, particularly in asymptomatic adults, the most reliable method to assess the problem of transfusion-transmitted malaria are nucleic acid-based molecular approaches such as quantitative polymerase chain reaction. The study was undertaken to determine the prevalence of sub-microscopic malaria parasite infection among blood donors in Malabo, Equatorial Guinea.

Methods: Between July and August 2017, a total of 200 individual blood samples from blood donors at the Malabo Blood Bank were collected and screened by rapid diagnostic tests and thick blood smear microscopy. Retrospectively, the same samples were analysed for the presence of undetected, low-density malaria parasites using quantitative polymerase chain reaction.

Results: In comparison to 6.5% (13/200) by rapid diagnostic test and 2.0% (4/200) by microscopy, the proportion of Plasmodium falciparum positive blood donations analysed by quantitative polymerase chain reaction was significantly higher (26%, 52/200). Densities of P. falciparum positive blood donations were ranging from 0.06 to 3707.0 parasites/µL with 79.6% below 100 parasites/µL and therefore not detectable by non-molecular malaria diagnostic tests. qPCR based species identification revealed that P. falciparum was the dominating species responsible for 88.1% (52/59) of positive blood donations, followed by Plasmodium malariae (15.3%, 9/59) and Plasmodium ovale (3.4%, 2/59).

Conclusions: This study confirms that in malaria endemic settings, sub-patent malaria infections among blood donors are prevalent. In blood collected from healthy donors living in Malabo, P. falciparum, P. malariae and P. ovale parasites were identified. Currently widely used malaria diagnostic tools have missed more than 75% of P. falciparum containing blood donations, demonstrating the value of quantitative polymerase chain reaction to reliably detect low density P. falciparum infections. Since the availability of molecular diagnostic methods in malaria endemic countries is still limited, the blood recipients living in malaria endemic countries should be treated following WHO recommendations.

Keywords: P. falciparum; P. malariae; P. ovale; Transfusion-transmitted malaria; qPCR.

Fig. 1

Analytical performance of PlasQ (a) and PlasID (b) assay. a Correlation of P. falciparum standards and the Cq values for both targets, Pspp18S (black circles) and PfvarATS (white circles) of the PlasQ assay. Based on these quadruple replicates of the WHO standards, LODs and qPCR efficiencies were calculated. b The ability of the PlasID assay identifying five different malaria species is shown by a representative, composite amplification plot. DNA from the six Plasmodium species were analysed in separate tubes during the same qPCR experiment

Fig. 2

Overview of blood sample analysis. Blood donations were systematically screened for the presence of malaria parasites by microscopy, RDT and qPCR assays

Fig. 3

Parasite densities of positive blood donations obtained from qPCR assays. Strong correlation between the two targets is observed (Spearman r 0.894). The green dots highlight samples containing non-falciparum malaria species. An offset of 0.05 parasites/µL was used

Fig. 4

Association between parasitaemia and sensitivity of diagnostic methods applied in this study. Median and interquartile ranges of non-cumulative P. falciparum parasitaemia are grouped according to different diagnostic tests used in this study (left y-axis, scatter plot). The dashed line at 100 parasites/µL represents the widely accepted LOD for RDTs. Proportional rates of positive blood donations are represented as bars (right y-axis)