Background
Lack of sensitive screening tools for detecting asymptomatic malaria in blood donors is believed to have contributed significantly to the underestimation of the global burden of transfusion-transmitted malaria (TTM) [
1]. In Ghana, the prevalence of malaria among blood donors have been reported to be between 8 and 13.7% by rapid diagnostic testing (RDT) and 3–4.7% by microscopy [
2‐
4]. In 2016, cases of TTM were reported from different provinces in Iran [
5] and in Ghana [
2]. In blood recipients with no or reduced immunity to malaria, TTM can be fatal, if infections are not detected and treated quickly [
6]. The parasite species most frequently associated with transfusion malaria are
Plasmodium falciparum, P. malariae and
P. vivax [
7,
8]. Blood donors with semi-immunity could harbor low levels of the
Plasmodium parasites, which are mostly below the detection threshold of currently available assays. This will cause malaria parasites to persist for several years in infected blood recipients [
7,
9]. During storage, malaria parasites have been found to survive in donated blood at temperatures between 2 °C and 6 °C for 3 weeks, with the estimated inoculum in transfusions from one to 10 parasites per donation [
10,
11]. Transfusing one malaria parasite per microliter of infected blood, converts to about 500,000 infected red cells in one unit of blood [
2]. Most reported cases of TTM were either through whole blood or red blood cell concentrates, with a few cases of TTM occurring after platelets and leukocytes transfusions [
12].
Genetic diversity of
P. falciparum has been attributed partly to its ability to evade host immune system [
13] and adapt to anti-malarials [
14]
. Alleles of
P. falciparum chloroquine resistance transporter gene (
Pfcrt) have been associated with chloroquine resistance in
P. falciparum [
15].
P. falciparum multi-drug resistance (
Pfmdr) gene is also associated with both chloroquine and amodiaquine resistance [
16]. Mutations in
P. falciparum dihydropteroate-synthetase (
Pfdhps) gene confer resistance to sulfadoxine [
17] while
P. falciparum dihydrofolate-reductase (
Pfdhfr) gene alleles are also associated with resistance to pyrimethamine [
18]. High prevalence of mutations in
Pfdhfr and
Pfdhps will undermine the prophylactic effect of sulphadoxine–pyrimethamine in pregnant women and immigrants visiting malaria endemic regions from non-endemic countries [
19]. Finally, non-synonymous single nucleotide polymorphisms in the
Kelch 13 propeller domain on chromosome 13 (
Kelch 13) were found to be strongly associated with resistance to artemisinin derivatives [
20].
Artemisinin-based combination therapies (ACTs) have contributed to the substantial decline in malaria burden. However, the efficacy of ACTs is threatened by the emergence of artemisinin resistance in
P. falciparum that led to treatment failure of ACTs in South-East Asian countries namely, Thailand, Cambodia, Vietnam and Myanmar [
21].
In previous studies in Ghana, high frequencies of mutation in
Pfcrt,
Pfmdr1,
Pfdhfr and
Pfdhps were identified in
P. falciparum infected children aged 4–15 years [
22,
23]. However, such study is yet to done in Ghanaian adults and for that matter, blood donors. Hence, this study was designed to determine the magnitude of the risk associated with the transmission of these putative anti-malaria drug resistant biomarkers through blood transfusion.
Discussion
In malaria-endemic countries, asymptomatic carriers of malaria parasites have been shown to exist and blood donors are likely to harbour the malaria parasites, though asymptomatically, which could later be transfused to patients who may require transfusion. Many of blood transfusion users are pregnant women, immunocompromised patients such as children under 5 years, HIV, TB, cancer and malnourished patients. These groups of people are particularly at higher risk of transfusion-transmissible malaria (TTM) which may be compounded by possible anti-malaria drug resistance strains.
This study therefore describes putative anti-malaria drug resistance biomarkers in blood donors that were recruited in the Greater Accra region of Ghana. The genes of interest were
Pfcrt,
Pfmdr1, Pfdhps,
Pfdhfr and
Kelch 13 propeller genes. Allelic forms of these genes have been linked to anti-malaria drug resistance in several studies [
15‐
18,
20].
In this study, the
Pfcrt gene were found to contain three different amino acids at positions 74, 75 and 76 resulting in M74I, N75E and K76T. Importantly, K76T mutation has been implicated in chloroquine resistance [
29,
30]. In Ghana, incidence of K76T has been reported in several studies [
22,
31,
32], however, K76T mutation may not complicate TTM, since chloroquine is no longer the drug of choice for the treatment of malaria. In Ghana, the drug of choice for management of uncomplicated malaria is artemisinin-based combination therapy (ACT). Non-synonymous mutations in
Kelch 13 propeller gene is associated with artemisinin resistance especially in parasites causing infection in South-East Asia (SEA) [
21].
In this study, five non-synonymous mutations P615L, A578S, I543V, A676S and C580Y were identified in the
Kelch 13 gene. The P615L mutation was detected in Pakistan [
33]. Also, A676S has been identified in Africa and South-East Asia [
34]. Again, S466N was identified in Peru [
35] and subsequently in Colombia [
36]. On the other hand, I543V allele has not been previously identified in any parasite strain. However, I543T, a very close allele to I543V, has been previously identified in SEA [
34]. Outside SEA [
34], C580Y has previously been identified in Africa specifically Cameroon [
34] and Guyana in South America [
37]. C580Y has been validated as artemisinin anti-malaria drug resistance allele [
20,
34,
38‐
40], however, association of C580Y with anti-malaria drug resistance outside SEA has not been reported.
Kelch 13 A578S allele, as was observed in this study, has also been previously detected in Ghanaian parasite isolates. In Ghana, A578S has been previously identified in three regions, Eastern, Bono (formerly Brong Ahafo) and in the Upper West Regions [
23]. The A578S allele is two codons away from C580Y allele. Malaria endemic regions that have previously identified A578S, must enhance surveillance for C580Y allele. Matrevi et al. [
23] have also detected C580V (very close allele of C580Y) in Central region of Ghana. Furthermore, Feng et al. [
41] in 2013 also detected three isolates of C580Y SNP in Chinese migrant workers from Ghana. Two years later (in 2015), Huang et al. [
42] also detected some cases of C580Y from Ghanaian immigrants to China. These reports highlight the fact that C580Y allele is present in Africa and in Ghana in particular. It could, therefore, be concluded that C580Y allele could be an emerging
Kelch 13 SNP in Ghana. It is therefore suggested that enhanced surveillance for C580Y in Ghana be implemented to unearth the actual burden of the SNP.
Mutations in the
Pfmdr1 gene, have also been associated with chloroquine, amodiaquine, lumefantrine and mefloquine resistance [
43,
44]. Amodiaquine, lumefantrine and mefloquine are artemisinin partner drugs. The most reported mutations in
Pfmdr1 gene globally is N86Y, Y184F and N1246Y [
16,
40,
42,
43]. In Ghana, these alleles have also been identified to be associated with reduced sensitivity of ACTs and other anti-malaria mono-therapies [
45]. This study identified the double mutant, Y184F/D1246Y represented as NFY haplotype in 10.8% of the isolates while the triple mutant, YFN (N86Y/Y184F/D1246N) was observed in 8.1% of the isolates. Two single mutants, NFD (Y184F) and NYY (D1246Y) were identified in 5.4% of the parasite isolates. Transmission of these haplotypes through blood transfusion, possibly will reduce the sensitivity of artemisinin partner drugs if infection is established. In Nigeria, N86Y and Y184F alleles have been associated with recurrent parasitaemia following Artemether-Lumefantrine therapy [
46]. If even blood recipients are treated for malaria as suggested in other studies [
47], they risk experiencing symptomatic infections due to the presence of N86Y and Y184F alleles.
In a study in the Volta region of Ghana, Kweku et al estimated malaria in pregnancy (MIP) to be 20.3% [
48]. MIP is associated with severe maternal anaemia, low birth weight, increased perinatal mortality, pre-term birth, still birth and infant deaths [
49,
50]. World Health Organization recommends intermittent preventive treatment of malaria in pregnancy (IPTp-MIP) with sulphadoxine-pyrimethamine (SP) to reduce the risk of poor MIP outcomes. With or without malaria parasitological testing, sulphadoxine-pyrimethamine for IPTp (IPTp-SP) is given to pregnant women. IPTp-SP is administered to either treat patient with parasites or provide a prophylactic effect to non-infected patients [
51]. Mutations in two genes of
P. falciparum, dihydropteroate synthase (
Pfdhps) and dihydrofolate reductase (
Pfdhfr) have been associated with resistance against sulfadoxine and pyrimethamine respectively. Accumulation of single point mutations in these genes have synergistic effect on reducing the efficacy of SP. The most important mutant biomarkers that increase the resistance of SP are combined triple
Pfdhfr (N51I, C59R, S108N) and double
Pfdhps (A437G, K540E) mutations resulting in quintuple mutation [
52]. Analysis of genomic data obtained in this study indicated 24.0% triple mutations in the
Pfdhfr gene (N51I, C59R, S108N resulting in IRNI haplotype) and 17.7% double mutations in
Pfdhps gene (A437G, K540E resulting in AGESS haplotype). Further analysis revealed that 12 isolates representing 15.1% contained the quintuple mutations in the
Pfdhfr and
Pfdhps genes. This study reveal that quintuple mutations in the
Pfdhfr and
Pfdhps genes associated with infected donor blood could reduce the prophylactic efficacy of SP in pregnant women transfused with infected donor blood.
There is paucity of data with regards to previous findings on the prevalence and frequencies of anti-malaria drug resistant genes in blood donors in both endemic and non-endemic countries. However, several cases of anti-malaria drug resistant genes have been imported, by asymptomatic carriers, into non-endemic countries from endemic countries [
41,
52‐
54]. Imported anti-drug resistant malaria is of public health concern since it has the potential to subvert global efforts to eradicate malaria. And also in regions with females
Anopheles mosquitoes during transmission seasons, residents risk being transmitted with
P. falciparum and more importantly,
P. falciparum carrying drug resistant genes.
In a previous study [
41], authors reported that imported
P. falciparum malaria sharply increased in China, due to migrant workers who had returned from Ghana, a sub-Saharan malaria-endemic country. Of the 118 imported
P. falciparum parasites that were evaluated, high prevalence of anti-malaria drug resistant gene polymorphisms, notably, N86Y, N86Y and D1246Y in
Pfmdr1 genes and K76T in
Pfcrt were reported. Again, four non-synonymous mutations in
Kelch 13 gene were also identified (R539T, C580Y, C580F, D584V), one of which was identified in this study (C580Y). Findings from Feng et al. [
41] and results obtained in this study confirm high prevalence of putative anti-malaria drug resistant biomarkers in asymptomatic infections in both Ghanaians and migrants from Ghana.
These findings raise concerns about the possible emergence of artemisinin resistance in Ghana. The impact of Pfmdr1 and Kelch 13 gene polymorphisms on Ghanaian P. falciparum parasite clearance times following ACT treatment need to be determined in further studies.
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