Demographic characteristics of imported malaria cases in Qatar
Out of 583 patients (all expatriates) examined for malaria, between January 2013 and October 2016 in Hamad General Hospital and Al-Khor Hospital, 448 (76.8%) tested positive: 318 P. vivax (70.9%), 118 P. falciparum (26.3%) and 12 (2.7%) mixed P. vivax / P. falciparum infection (Table 1). The vast majority of the malaria-positive patients were males, P. vivax (94.3%); P. falciparum (84%), with a mean age of 32 yrs and 33 yrs, respectively, reflecting the fact that the predominate attending patients were young male.
The major origin of those presenting with P. vivax was the Indian Subcontinent (83%%, n =264): India (46%, n = 146), Pakistan (32.1%, n = 102) and Nepal (3.8%, n = 12). A smaller proportion of the P. vivax cases were from Africa (16%, n = 53) (Table 1).
Unlike P. vivax, the main origin of P. falciparum infection was Africa (East African [76.1%, n = 67] West & Central African countries [23.9%, n = 21], followed by the Indian Subcontinent (20.3%, n = 24) and other countries (5.1%, n = 6) (Table 1).
Parasitaemia and gametocytaemia among imported malaria cases
Ninety of the 118 P. falciparum infections were further examined for total parasite and gametocyte density, using qPCR and qRT-PCR, respectively, and diversity of 10 microsatellites and alleles of four genes linked to drug resistance.
The total P. falciparum density among imported cases ranged widely between 32 and 9,218,498 parasites/ml blood with a median 82783 parasites/ml. The median parasite density among imported cases from the Indian Subcontinent (99,572 parasites/ml) was not significantly higher than that from Africa (88,504 parasites/ml) (p=0.394).
Seventy-three P. falciparum isolates were examined by qRT-PCR to detect and quantify transcripts of genes expressed in early (Pfpeg4) and late gametocytes (Pfs25). The prevalence of all gametocytes was 74% (n=54); with 9.6% (n=7), 37% (n=27) and 27.4% (n=20) of patients carrying only early stages, only late stages or a mixture of both stages, respectively.
Early gametocytes were found at a relatively lower density, ranging between 14 and 3781/ml blood, with a median of 1011 early gametocytes/ml blood, compared to late gametocytes, which presented at a higher density overall (Mann Whitney U test, p = 0.003, range 16 - 15289 gametocytes/ml blood, median 136 gametocytes/ml blood). No correlation was found between total parasitaemia (18S rRNA copy numbers) and either late gametocytes (Pfs25 copy number) (rs = 0.008, p = 0.946) or early gametocytes (Pfpeg4 copy number) (rS = 0.031, p = 0.835) (Figure1) and early gametocyte density (p = 0.857).
Genetic diversity and structure of imported P. falciparum parasites
Microsatellite polymorphism
All the examined microsatellites were highly polymorphic among P. falciparum isolates originating from both Africa and the Indian Subcontinent (Table 2). The number of alleles per locus was higher among the African isolates, ranging from 5 for pfg377 to 18 for polyα, compared to the Indian Subcontinent isolates, ranging from 3 for 2490 to 7 for both TA1 and PfPK2 (Table 2; Supplementary Table 1). However, allelic diversity, summarized as mean expected heterozygosity (He) across the 10 microsatellite loci, was not significantly different among parasites in the Indian Subcontinent (mean He = 0.78) compared to that in Africa (mean He = 0.76).
Multi-locus haplotypes were constructed using predominant alleles at all of the examined loci. All 90 isolates differ from each other in at least one of the examined loci, with exception of two isolates from Africa, sharing an identical haplotype, both from Sudan. Thus, almost every isolate in each of the examined sites carried a unique genotype.
Multiplicity of infection (MOI)
Seventy-six (84.4%) out of the 90 imported P. falciparum isolates with complete set of data harbored multiple genotypes. A similar mean of multiple genotype infections was seen among parasites in the Indian Subcontinent (84.6%) and Africa (84.4%). The minimum number of genotypes per infected person (the mean maximum number of alleles observed at all loci) was slightly lower in Africa (2.16 genotypes) that in the Indian Subcontinent (2.38 genotypes), but this was not statistically significant (P > 0.05).
Genetic differentiation
Alleles of most microsatellites were distributed widely across P. falciparum among imported malaria cases from Africa (n =77) and the Indian Subcontinent (n = 13). However, a large number of private alleles (detected only in one region) were seen in Africa (n = 50) compared to the Indian Subcontinent (n = 5), which may reflect the smaller sample size. Nonetheless, no evidence of genetic differentiation was observed between imported P. falciparum from Africa and the Indian Subcontinent, (FST =0.055). The genetic relatedness between the P. falciparum populations was further illustrated by PCoA analysis (Figure 2). Analysis of molecular variance (AMOVA) revealed that the majority of the differences were due to variation between individuals within the same group (95%), and only 5% could be attributed to differences between populations.
Distribution of drug resistance genes among imported cases
Seventy imported P. falciparum isolates were examined using amplicon sequencing for four putative drug resistance genes, PfK13, Pfmdr1, Pfcrt and Pfmrp1 (Table 3). With exception of K13, there was no differences in the prevalence of wild type of the examined genes among parasites originated from Africa or Asia. There was a significantly higher prevalence of mutant PfK13 haplotypes among parasites from Africa than Asia. One nonsynonymous mutation in PfK13 (K189T) was observed at a high prevalence (36%) among parasites originating from Sudan, similar to another African countries [28]. Moreover, ten additional nonsynonymous SNPs K108E, L119L, H136N, T149S, K189T, K189N, N217H, R255K, I354V, E433D, G453A, all existed at very low prevalence ranging from 1-3% (Table 3; Supplementary Table 2).
However, the PfK13 mutations bringing about the amino acid substitutions C580Y, Y493H, and R539T, associated with slow artemisinin clearance of P. falciparum [29], were not detected among the P. falciparum isolates imported into Qatar. However,
Pfmdr1 alleles encoding the polymorphisms N86Y (33%) and Y184F (77%) were prevalent among imported P. falciparum isolates. In addition, six rare nonsynonymous SNPs were detected (Table 3). The N86F184D1246 and Y86F184D1246 haplotype associated with Artemether-Lumefantrine (AL) and Chloroquine/Amodiaquine (CQ/AQ) treatment failure existed were highly prevalent among imported P. falciparum cases, at 43% and 33%, respectively.
Strikingly, the Pfcrt K76T substitution associated with CQR was found at low frequency [4/70 (6%)], however, other SNPs were seen at high prevalence, e.g. A220S (53%), Q271E (49%), N326D/S (36%), I356L (6%) and R371I (47%). The CQ sensitive haplotype C72V73M74N75K76 was common (94%), while the CQ resistant haplotypes, S72V73M74N75T76 and C72V73M74N75T76 were detected in one and three isolates, respectively.
Regarding Pfmrp1 eight mutations were seen among imported P. falciparum in Qatar, ranging from high I876V (46%) to low D1533V (3%) (Table 3). The most prevalent 6 mutations (0.2 to 0.46) detected among imported cases in Qatar, have been previously reported in southeast Asian and Africa [30, 31]. Pfmrp1 polymorphisms that have previously been associated with decreased in vitro susceptibility to SP, artemisinin, mefloquine, and lumefantrine were common among imported malaria in Qatar. For example, the most prevalent SNP I876V (46%), was found to be under significant selection pressure after AL treatment [30].