Among the 535 samples confirmed by PCR for P. falciparum, 446 samples were included for the analysis from eight different locations in Yambio County, in order to ensure enough DNA quality and quantity. The mean age of patients was 4.41, as the majority of the samples were collected from children (Table 3).
Allelic genotyping of pfmsp1 and pfmsp2 genes
The frequency of pfmsp1 and pfmsp2 allelic families was 91.9% (410/446) and 83.6% (373/446), respectively. 61.2% (251/410) of the samples positive for pfmsp1 were single - allelic infections (Table 1). From the total sample, monomorphic K1 allele infection was predominant (37.0%, 152/410) in every location (Figure 2a). Among the polyclonal infections (47.6%, 195/410) the most frequent was RO33+K1 identified in 20.9% (86/410) of the samples, followed by trimorphic infections (RO33+K1+MAD20). However, these polymorphic genotypes were detected in different frequencies by location.
Among isolates positive for pfmsp2 locus, 60.9% (227/373) were monomorphic, being single allele 3D7 predominant (44.8%, 167/373). 39.1% of isolates presented double-allelic genotype 3D7+FC27, with a wide range of number of fragments per locus from 2 to 11 (Table 1). The frequency of different allele combinations varies among different collection sites (Figure 2b).
Table 1. Frequency of genotypes of allelic families of pfmsp1 and pfmsp2 genes.
Family alleles
|
Genotype frequency
% among pfmsp family
(Npfmsp1 = 410; Npfmsp2 = 373)
|
Number of alleles per genotype
|
Pfmsp1
|
RO33
|
13.7% (56)
|
1
|
K1
|
37.1% (152)
|
1 – 3
|
MAD20
|
5.1% (21)
|
1 - 3
|
RO33+K1
|
21.0% (86)
|
2 – 6
|
RO33+MAD20
|
4.6% (19)
|
2 – 6
|
K1+MAD20
|
7.3% (30)
|
2 – 8
|
RO33+K1+MAD20
|
10.2% (42)
|
3 – 9
|
Pfmsp2
|
3D7
|
44.8% (167)
|
1 – 5
|
FC27
|
16.1% (60)
|
1 – 6
|
3D7/FC27
|
39.1% (146)
|
2 – 11
|
pfmsp1 and pfmsp2 genes allelic polymorphism, diversity and multiplicity
Combining results of pfmsp1 and pfmsp2, 71.9% of samples (321/446) presented polyclonal infections, while 28.1% (126/446) had monoclonal infections. The overall MOI was 1.96; the MOI of pfmsp1 and pfmsp2 was 1.53 and 1.85 respectively (Table 2).
In the pfmsp1 locus, K1 allelic family was the most frequent (75.6%) and the most diverse, with 189 different allele fragments ranging from 70 bp to 629 bp (Figure 3a). RO33 family showed less diversity, ranging from 100 to 284, the 150 bp fragment was the most predominant. The expected heterozygosity (HE) of pfmsp1 was 0.63.
In the pfmsp2 locus, 3D7 was the predominant allelic family (83.9%, 313/373), the predominant fragment size was between 100 and 119 (Figure 3b). FC27 allelic family, identified in less frequency (55.2%, 206/373), presented more diversity with 242 different fragments. The expected heterozygosity was 0.48.
Table 2. Genotyping of allelic variants of pfmsp1 and pfmsp2. HE : expected heterozygosity; MOI : Multiplicity of infection
Allelic family
|
Allelic family frequency
|
Fragment size
|
Number of fragments per locus
|
Number of alleles per family
|
HE
|
MOI
|
pfmsp1 gene (N = 410)
|
RO33
|
49.5% (203)
|
100 - 284
|
1 - 3
|
56
|
|
|
K1
|
75.6% (310)
|
70 – 629
|
1 - 5
|
189
|
MAD20
|
27.3% (112)
|
100 - 464
|
1 - 6
|
122
|
Total pfmsp1
|
0.63
|
1.53
|
pfmsp2 gene (N = 373)
|
3D7
|
83.9 % (313)
|
100 - 848
|
1 – 6
|
178
|
|
|
FC27
|
55.2 % (206)
|
101 - 948
|
1 - 7
|
242
|
Total pfmsp2
|
0.48
|
1.85
|
Total pfmsp1 & pfmsp2
|
|
1.96
|
Variability of multiplicity of infection by age and village
No statistical significant difference was observed in multiplicity of infection (MOI) by location, although pfmsp2 showed a wide range of MOI (1.48 – 2.25) (Table 3).
Contrastingly, the MOI significantly varied according to age, increasing from the under-five age group to reach the highest MOI in the age group between 5 and 14 years (Figure 4).
Table 3. Multiplicity of infection by location and age group
|
pfmsp1 MOI
|
pfmsp2 MOI
|
pfmsp1 & pfmsp2 MOI
|
Location
|
Nambia
|
1.57
|
1.95
|
2.14
|
Gitikiri
|
1.37
|
1.84
|
2.01
|
Bakiwiri
|
1.55
|
2.25
|
1.95
|
Mamboi
|
1.55
|
1.99
|
2.07
|
Masumbu
|
1.67
|
1.78
|
1.96
|
Birisi
|
1.69
|
1.79
|
2.01
|
Yambio State Hospital
|
1.27
|
1.85
|
1.42
|
Kasia
|
1.52
|
1.48
|
1.91
|
p-value
|
0.111
|
0.217
|
0.163
|
Age group
|
< 5 (N=119)
|
1.49
|
1.86
|
1.91
|
5 – 14 (N=170)
|
1.67
|
1.93
|
2.30
|
>14 (N=138)
|
1.41
|
1.73
|
1.81
|
p-value
|
0.007
|
0.107
|
0.022
|
Distribution of the allelic variants and multiplicity of infection by malaria infection severity
The overall mean MOI of uncomplicated malaria infections (2.01) was significantly higher than the overall mean MOI of severe malaria infections (1.40) (Figure 5). However, neither pfmsp1 MOI nor pfmsp2 MOI was independently associated with severe malaria (Table 4).
The frequency of pfmsp1 and pfmsp2 alleles according to severity of infection is presented in Table 5. No significant differences in allelic distribution between both groups were detected.
Table 4. Relation between multiplicity of infection (MOI) and severity of malaria infection.
|
pfmsp1 MOI
|
pfmsp2 MOI
|
pfmsp1 & pfmsp2 MOI
|
N
|
MOI
|
N
|
MOI
|
N
|
MOI
|
Severity of infection
|
Severe malaria
|
17
|
1.40
|
8
|
1.93
|
18
|
1.40
|
Uncomplicated malaria
|
429
|
1.54
|
360
|
1.85
|
400
|
2.01
|
P - value
|
0.251
|
0.289
|
0.011
|
Table 5. Distribution of pfmsp1 and pfmsp2 allelic families by infection severity.
|
Severe malaria
|
Uncomplicated malaria
|
p - value
|
Fragment size (bp)
|
F% (n)
|
Fragment size (bp)
|
F% (n)
|
pfmsp1 allele family
|
|
N=17
|
N = 378
|
|
RO33
|
102 - 151
|
41.2% (7)
|
100 - 284
|
51.9% (196)
|
0.620
|
K1
|
133 - 383
|
70.6% (12)
|
70 - 842
|
78.8% (298)
|
0.779
|
MAD20
|
234 - 320
|
23.5% (4)
|
100 - 461
|
28.6% (108)
|
0.916
|
pfmsp2 allele family
|
|
|
N = 7
|
|
N = 359
|
|
3D7
|
106 - 513
|
85.7% (6)
|
100 - 848
|
84.1% (302)
|
0.883
|
FC27
|
286 - 560
|
57.1% (4)
|
101 - 948
|
54.9% (197)
|
0.912
|
Allelic frequencies and multiplicity of infections relation with antimalarial drug resistance haplotypes
All 446 samples included in the study were analysed for molecular haplotypes associated with resistance to sulfadoxine – pyrimethamine, which included SNPs of pfdhfr and pfdhps genes, and to amodiaquine and chloroquine, which included SNPs of pfmdr1 and pfcrt genes.
The overall mean MOI showed a gradual increase following the level of resistance, from 2.36 for the partially resistant haplotype 51I/59R/108N + pfdhps 437G), to 3.03 for the fully resistant haplotype (3.03) (pfdhfr 51I/59R/108N + pfdhps 437G/540E), and 3.88 for the super resistant haplotype (pfdhfr 51I/59R/108N + pfdhps 437G/540E/581G), (Figure 6). Nevertheless, the difference between the mean MOI of the haplotypes was not significant (Table 6).
Concerning the frequency of alleles of the pfmsp1 family, no significant differences were observed between the resistance haplotypes (Table 7). For pfmsp2 family alleles, although the differences between the four groups were not statistically significant, the three haplotypes associated with resistance showed high frequency of the allelic family 3D7 gene (> 80%) than isolates without resistance haplotypes (10.6%). Parasite infections that did not have any of the 3 resistance haplotypes presented lower pfmsp1 MOI (1.71 and 1.79, respectively) but higher pfmsp2 MOI (2.94 and 3.15, respectively) (Table 6).
Table 6. Relation between multiplicity of infection (MOI) and antimalarial drugs resistance haplotypes.
|
Pfmsp1 MOI
|
Pfmsp2 MOI
|
pfmsp1 & pfmsp2 MOI
|
pfdhfr & pfdhps mutated haplotypes
|
Partially resistant
|
1.65
|
2.44
|
2.36
|
Fully resistant
|
2.20
|
2.82
|
3.03
|
Super resistant
|
2.50
|
3.13
|
3.88
|
Non - associated with resistance haplotype
|
2.06
|
2.87
|
2.90
|
p-value
|
0.383
|
0.8
|
0.061
|
pfmdr1 haplotype
|
Pfmdr1 86Y+1246Y
|
1.71
|
2.94
|
2.95
|
Non - associated with resistance haplotype
|
2.16
|
2.83
|
2.99
|
p-value
|
0.185
|
0.785
|
0.665
|
Pfmdr1 + pfcrt haplotype
|
Pfmdr1 86Y + pfcrt 76T
|
1.79
|
3.14
|
3.21
|
Non - associated with resistance haplotype
|
2.17
|
2.81
|
2.97
|
p-value
|
0.157
|
0.790
|
0.647
|
Table 7. Distribution of pfmsp-1 and pfmsp-2 allelic families according to sulfadoxine – pyrimethamine resistance haplotypes.
|
Non - associated with resistance haplotype
|
Partially resistant
|
Fully resistant
|
Super resistant
|
P - value
|
|
F% (n)
|
F% (n)
|
F% (n)
|
F% (n)
|
|
pfmsp1 allele family
|
|
N = 123
|
N=20
|
N = 247
|
N = 16
|
|
RO33
|
45.5% (56)
|
50% (10)
|
51% (126)
|
68.8% (11)
|
0.342
|
K1
|
77.2% (95)
|
65% (13)
|
77.3% (191)
|
68.8% (11)
|
0.547
|
MAD20
|
23.6% (29)
|
20% (4)
|
30.4% (75)
|
25% (4)
|
0.462
|
pfmsp2 allele family
|
|
N = 104
|
N = 16
|
N = 237
|
N = 16
|
|
3D7
|
10.6% (11)
|
81.3% (13)
|
81% (192)
|
81.3% (13)
|
0.284
|
FC27
|
45.2% (47)
|
50% (8)
|
54.9% (130)
|
62.5% (10)
|
0.913
|