Demographic characteristics of imported malaria cases in Qatar
Among the 583 patients (all expatriates) tested for malaria between January, 2013 and October, 2016 in Hamad General Hospital and Al-Khor Hospital, Qatar, 448 (76.8%) tested positive: 318 for P. vivax (70.9%), 118 for P. falciparum (26.3%) and 12 (2.7%) for P. vivax / P. falciparum coinfection (Table 1). The majority of patients that tested positive for malaria were men, P. vivax (94.3%), and P. falciparum (84.0%), with a mean age of 32 yrs and 33 yrs, respectively, reflecting the fact that the predominate attending patients were young men (Supplementary Table 1).
The primary origin of those presenting with P. vivax was the Indian Subcontinent: India (46.0%, n = 146), Pakistan (32.1%, n = 102) and Nepal (3.8%, n = 12). A smaller proportion of P. vivax cases were from Africa (16%, n = 53) as a whole (Table 1). Unlike P. vivax, the primary origin of P. falciparum infection was Africa: East Africa (76.1%, n = 67) West and Central Africa (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 discovered P. falciparum infections were further examined for total parasite and gametocyte density (using qPCR and qRT-PCR, respectively) as well as the diversity of 10 microsatellites and the alleles of four genes linked to drug resistance. The total P. falciparum density among imported cases varied widely, ranging between 32 and 9,218,498 parasites/ml blood with a median of 82,783 parasites/ml. The median parasite density among imported cases from the Indian Subcontinent (99,572 parasites/ml) was not statistically greater than that of Africa (88,504 parasites/ml) (P = 0.394).
Seventy-three P. falciparum isolates were successfully 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) of patients carrying only early stages, 37% (n =27) only late stages, and 27.4% (n =20) a mixture of both stages. Early gametocytes were found at a relatively lower density, ranging between 14 and 3,781/ml blood, with a median of 1,011 early gametocytes/ml blood, compared to late gametocytes, which possessed a higher density overall (range 16 – 15,289 gametocytes/ml blood, median 136 gametocytes/ml blood) (Mann-Whitney U test, P = 0.003). 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) (Figure 1) and early gametocyte density (P = 0.857).
Genetic diversity and structure of imported P. falciparum parasites
Microsatellite polymorphisms
All of the examined microsatellites were highly polymorphic among P. falciparum isolates originating from Africa and the Indian Subcontinent (Table 2). The number of alleles per locus was higher among African isolates, ranging from 5 for pfg377, to 18 for polyα, compared to Indian Subcontinent isolates, which ranged from 3 for 2490 to 7 for TA1 and PfPK2 (Table 2; Supplementary Table 2). Nevertheless, allelic diversity (summarized as mean expected heterozygosity (He) across 10 microsatellite loci) was not significantly different among parasites in the Indian Subcontinent (mean He = 0.78) compared to those in Africa (mean He = 0.76).
Multi-locus haplotypes were constructed using predominant alleles at all examined loci. All 90 isolates differed from each other in at least one of the examined loci, with the exception of two isolates from Sudan, Africa, that shared an identical haplotype. 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 data) harbored multiple genotypes. A similar mean of multiple genotype infections was observed among parasites of the Indian Subcontinent (84.6%). 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) than in the Indian Subcontinent (2.38 genotypes), but this trend was not calculated to be statistically significant (a < 0.05).
Genetic differentiation
Alleles of most microsatellites were distributed widely across P. falciparum among imported malaria cases from both Africa (n = 77) and the Indian Subcontinent (n = 13). 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 those from the Indian Subcontinent (FST = 0.055). The genetic relatedness between P. falciparum populations was further illustrated using PCoA analysis (Figure 2). Analysis of molecular variance (AMOVA), comparing between P. falciparum isolates imported from the Indian Subcontinent, revealed that the majority of differences were due to variation between individuals within the same group (95%), while only 5% could be attributed to differences between populations (P < 0.001).
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 the exception of PfK13, there was no difference in the prevalence of wild type genes among parasites originating from Africa versus the Indian subcontinent. There was, however, a significantly higher prevalence of mutant PfK13 haplotypes among parasites from Africa than the Indian subcontinent. One nonsynonymous mutation in PfK13 (K189T) was observed at a high prevalence (36%) among parasites originating from Sudan, analogous to reported findings from other African countries [28]. Furthermore, ten additional nonsynonymous SNPs: K108E (2%), L119L (1%), H136N (1%), T149S (2%), K189N (2%), N217H (1%), R255K (3%), I354V (1%), E433D (1%), G453A (1%), all existed at very low prevalence ranging from 1-3% (Table 3; Supplementary Table 3).
PfK13 mutations including the amino acid substitutions of C580Y, Y493H, R539T and M579I associated with slow artemisinin clearance of P. falciparum [29, 30] 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 were prevalent among imported P. falciparum cases, at 43% and 33%, respectively.
Notably, while the Pfcrt K76T substitution associated with CQR was found at low frequency (n = 70, 6%), other SNPs implicated in CQR were observed at high prevalence, e.g. A220S (53%), Q271E (49%), N326D/S (36%), I356L (6%) and R371I (47%). Overall, the CQ sensitive haplotype C72V73M74N75K76 was common (94%), while the CQ resistant haplotypes, S72V73M74N75T76 and C72V73M74N75T76, were detected in only one and three isolates, respectively.
Regarding Pfmrp1, eight mutations were observed among imported P. falciparum in Qatar, ranging from high I876V (46%) to low D1533V (3%) (Table 3). The most prevalent six mutations (20% to 46%) detected among imported cases in Qatar have been previously reported in the Indian subcontinent and Africa [31, 32]. 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 [31].