Characterization and profile of samples
A total of 147 PCR confirmed parasite genomic DNA samples were available for molecular characterization in this study. Seven-microsatellite-marker typing was performed for 85 samples, however only 49 samples produced positive allele peaks at >5 microsatellite markers and were considered for analysis. Thirty-six samples (n=36) that produced allele peaks at 1 – 4 microsatellite markers only were excluded from further analyses on MOIs and parasite genetic relatedness. Clean DNA sequences of the pfhrp2 exon 2 was obtained from 62 samples. The entire sample of 147 genomic DNA and their demographics came from a study that previously characterized the pfhpr2 and pfhrp3 genes [31].
Baseline characteristics samples
The majority of these samples was from males (54.4%), aged ≥ 5 years (65.9%) and from the eastern region of Uganda (53.7%). Most had a parasite density ≥1,000 /μl (65.3%). All the genomic DNA were from PCR confirmed P. falciparum infected samples (Table 1)
Table 1: Baseline characteristics of samples
Variable
|
Frequency
|
Proportion (%)
|
Sequenced (%)
|
Microsatellite typing (%)
|
Total (n=147)
|
(n=62)
|
(n=85)
|
Age
|
|
|
|
|
<5
|
50
|
34.01
|
40.3
|
29.4
|
≥5
|
97
|
65.99
|
59.6
|
70.6
|
Gender
|
|
|
|
|
Male
|
80
|
54.42
|
54.8
|
54.1
|
Female
|
67
|
45.58
|
45.2
|
35.9
|
Region
|
|
|
|
|
Western
|
68
|
46.3
|
37.1
|
51.7
|
Eastern
|
79
|
53.7
|
62.9
|
48.2
|
Density
|
|
|
|
|
<1,000
|
51
|
34.69
|
38.7
|
45.8
|
≥1,000
|
96
|
65.31
|
61.3
|
54.1
|
Endemicity
|
|
|
|
|
low transmission
|
87
|
59.18
|
45.2
|
69.4
|
Moderate
|
60
|
40.82
|
54.8
|
30.9
|
Legend for table 1: <5 and ≥5 means less than 5 and equal or above years of age respectively. < 1,000 and ≥1,000 means parasite densities of less than 1,000 and equal or greater than 1,000 parasites/µL respectively.
Polyclonality of infections
Microsatellite typing revealed a high proportion samples examined had more than one allele on at least one of the seven markers indicative of multi-clone infections. Overall, 75.51 (95%, CI:61.1 - 85.8) of samples examined were classified as multiple-clone (mixed genotype) infections, significantly higher compared to single clone 24.5% (95% CI: 14.2 - 38.9), P<0.05. This high level of polyclonality in samples indicates frequent infections consistent with high malaria transmission intensity in the study areas. Although not statistically significant, proportions of multiclonal infections were 71.8% (91% CI: 53.4 - 85.1) and 82.4% (95% CI: 55.5 - 94.6), P>0.05 in >5 and ≥5 years respectively. Multiclonal infections significantly occurred in isolates collected from Eastern 73.7% (95% CI: 48.8 - 89.1) compared to Western region 26.3% (95% CI: 10.9 - 51.2), P<0.05.
Multiplicity of Infections (MOI)
The number of alleles at any of the 7 markers varied between markers. The highest number of alleles at any marker was 4 (Table 2). MOI at individual marker varied between 1 and 1.51. The overall MOI in this set of samples was 1.92 (95% CI:1.72 - 2.12), consistent with high transmission intensity. Though not statistically significant the MOI was higher in children <5 than ≥5 years old (1.9 and 1.8) respectively. We did not see geographical differences in MOIs in parasites collected from Eastern and Western Uganda (1.9 vs 1.8), P>0.05 respectively (Table 2). Importantly, 5/7 samples with gene deletions were multi-clone infections (pfhrp2-/pfhrp3+ =2, pfhrp2-/pfhrp3- n=3, pfhrp2+/pfhrp3- n=1).
Table 2: Microsatellite markers, Typing and overall MOI
Microsatellite marker
|
No. of samples typed
|
No. of alleles revealed
|
Range in No. of alleles
|
MOI
|
95%CI
|
2409
|
49
|
65
|
1 – 3
|
1.33
|
NA
|
TA1
|
48
|
56
|
1 – 2
|
1.17
|
NA
|
383
|
30
|
38
|
1 – 2
|
1.27
|
NA
|
TA109
|
47
|
69
|
1 – 4
|
1.47
|
NA
|
313
|
19
|
19
|
1 – 1
|
1
|
NA
|
PolyA
|
49
|
74
|
1 – 3
|
1.51
|
NA
|
PK2
|
42
|
50
|
1 – 4
|
1.19
|
NA
|
Overall MOI*
|
49
|
94
|
1 – 4
|
1.92
|
1.72 - 2.12
|
Overall MOI* (RDT+/PCR+)
|
33
|
65
|
1 – 3
|
1.97
|
NA
|
Overall MOI* (RDT-/PCR+)
|
16
|
29
|
1 – 4
|
1.81
|
NA
|
Overall MOI* Age
<5 years
≥5 years
|
26
23
|
48
27
|
1-4
1-2
|
1.88
1.94
|
1.52 - 2.24
1.68 - 2.20
|
Overall MOI* Region
Western
Eastern
|
19
31
|
36
51
|
1-2
1-4
|
1.89
1.93
|
1.54 - 2.25
1.68 - 2.19
|
Table 2 Legend: *calculated by selecting the highest number of alleles at any of the markers typed. NA= not applicable
Number of 5 to 7-microsatellite marker haplotypes
Five to 7 marker haplotypes were obtained from 29 samples: 12 single clone infected samples and 17 multi-clone infected samples where multiple alleles were observed in only one marker. This resulted in a total of 42 unique haplotypes from 29 samples. The remaining samples had multiple alleles at more than one marker resulting in haplotypes unassigned due to the complexity in number of possible haplotype combinations. The high level of diversity and multiplicity in haplotypes detected in this set of samples indicates large parasite population size consistent with high transmission intensity in the areas.
Only two of the 42 haplotypes (H29 and H31) were shared between 2 (D196 and D204) and between 3 (D130, D176 and D204) different samples. Interestingly, both these haplotypes H29 and H31 were identified from parasite samples that had both pfhrp2 and pfhrp3 deletions. Haplotypes H25, H37 and H38 were identified in samples determined to have deleted pfhrp2 only, while H26 and H27 were identified in a sample determined to have deleted pfhrp3 only (Table 3). This observation suggests that parasites having pfhrp2 or pfhrp3 gene deletions had a lower genetic diversity compared to parasites without gene deletions indicating clonal expansion of gene deleted parasites. Furthermore, parasites with 5 different haplotypes (H29, H31, H25, H37 and H38) and two different haplotypes (H26 and H27) were observed to have deleted the pfhrp2 gene and pfhrp3 gene respectively, suggesting that pfhrp2 and pfhrp3 gene deletions have occurred in parasites with different genetic backgrounds.
Table 3: Haplotype determination in pfhrp2/3 deleted samples
Haplotype
|
Identified in Sample
|
pfhrp2/3 status
|
H29
|
D196, D204
|
pfhrp2-/pfhrp3-
|
H31
|
D130, D176, D196
|
pfhrp2-/pfhrp3-
|
H25
|
D154
|
pfhrp2-/pfhrp3+
|
H37
|
D169
|
pfhrp2-/pfhrp3+
|
H38
|
D169
|
pfhrp2-/pfhrp3+
|
H26
|
D263
|
pfhrp2+/pfhrp3-
|
H27
|
D263
|
pfhrp2+/pfhrp3-
|
Table 3 legend: pfhrp2/3 detected:+; undetected:-
Genetic relatedness of parasites
In order to investigate the possible origin of pfhrp2/3 gene deleted parasites, we conducted genetic relatedness analysis among 42 Ugandan parasite haplotypes as well as between Ugandan and Eritrean parasites. Among parasites from Uganda, there was a major cluster of parasites having deleted both pfhrp2 and pfhrp3 (H29, H30 and H31), and small clusters of pfhrp2-/pfhrp3+ (H37 and H38), pfhrp2+/pfhrp3- (H26 and H27) suggesting that these parasites are genetically closely related (Figure 2A). There were several small clusters of closely related pfhrp2+/hrp3+ parasites. The biggest of clustering consisted of parasites with dual pfhrp2/3 deletions indicating possible clonal expansion of these RDT undetectable parasites possibly selected for by RDT usage.
Haplotypes of Ugandan parasites (gene deleted and not deleted) did not cluster with parasite haplotypes from Eritrea (E1-E14), suggesting parasites with gene deletions in Uganda did not share common genetic origins with Eritrean parasites (different genetic lineages) and hence, were not likely to have resulted from spread from Eritrea. There is a trend indicating that parasites have formed small clusters within the Western and Eastern regions. However, there were two clusters that included parasites from both regions (Figure 2B).
Genetic diversity in the pfhrp2 gene
To investigate genetic diversity of the pfhrp2 positive parasite population, Exon 2 of the pfhrp2 gene was sequenced from 62 pfhrp2 positive samples to reveal genetic diversity in the gene. Sequencing analysis revealed 60 unique sequence types in 62 samples sequenced, i.e. almost every sample had a different pfhrp2 sequence, demonstrating a high level of genetic diversity in pfhrp2 of the sequenced parasite population. Only two sequence types were shared between two different samples: the unique sequence types 39 (shared by D102 and D103) and 49 (shared by C041 and D165). Details are indicated in Supplementary Information Table 5.
Sequence length of pfhrp2 exon2 which encodes the HRP2 protein
Exon2 length of the 62 sequences ranged between 537-801 bp. Generally, sequence length in the two sample sub-sets was comparable between the RDT-/LM+ and the RDT+/LM+ samples (p=0.28) Figure 3. Detailed data on pfhrp2 exon length is included in Supplementary Information Table 5.
Phylogeny and sequence clustering
A phylogeny bootstrap consensus neighbor-join tree based on 62 pfhrp2 sequences was constructed. No major clustering of parasites was observed in relation to RDT positivity, sequence length or sequence types. Interestingly, the three pfhrp2+/hrp3- parasites (D126, D146 and C139) did not appear to be clustered as they are on 3 separate branches (Fig 4). This suggests that parasites with pfhrp3 deletions have emerged independently from different genetic backgrounds.
Putative amino acid repeat types in HRP2
Among 62 sequences, 15 types of amino acid repeats were identified. Their numbers and ranges are shown in Table 3. There was no significant correlation between the number of any repeats and geographical origin or RDT positivity.
Table 4: pfhrp2 Amino Acid Repeat types, translated sequences and frequencies (n=62)
PfHRP2
|
Total Region
|
RDT and LM
|
|
East West
|
RDT-/LM+
|
RDT+/LM+
|
p-value
|
Code
|
Repeat sequences
|
(N=62) (N=40) (N=22)
|
(N=24)
|
(N=38)
|
1
|
AHHAHHVAD
|
2.7 ± 1.4 1-7 0-6
|
2.9 ± 1.5
|
2.6 ± 1.3
|
0.504
|
2
|
AHHAHHAAD
|
11.8 ± 2.2 10-17 8-13
|
12.1 ± 1.9
|
11.7 ± 2.4
|
0.427
|
3
|
AHHAHHAAY
|
1.2 ± 0.7 0-2 0-3
|
1.1 ± 0.7
|
1.3 ± 0.7
|
0.457
|
4
|
AHH
|
0.5 ± 0.8 0-3 0-1
|
0.3 ± 0.6
|
0.6 ± 0.9
|
0.146
|
5
|
AHHAHHASD
|
0.7 ± 0.5 0-2 0-1
|
0.8 ± 0.5
|
0.6 ± 0.5
|
0.221
|
6
|
AHHATD
|
3.0 ± 1.4 1-6 2-5
|
3.0 ± 1.3
|
3.0 ± 1.4
|
1
|
7
|
AHHAAD
|
6.5 ± 2.4 4-11 4-8
|
6.2 ± 2.1
|
6.8 ± 2.5
|
0.317
|
8
|
AHHAAY
|
1.1 ± 0.6 0-2 1-2
|
1.2 ± 0.8
|
1.1 ± 0.5
|
0.85
|
9
|
AAY
|
0.0 ± 0.0 0.0 0.0
|
0.0 ± 0.0
|
0.0 ± 0.0
|
NA
|
10
|
AHHAAAHHATD
|
1.6 ± 0.7 0-3 0-2
|
1.9 ± 0.7
|
1.5 ± 0.6
|
0.018
|
11
|
AHN
|
0.0 ± 0.0 0.0 0.0
|
0.0 ± 0.0
|
0.0 ± 0.0
|
NA
|
12
|
AHHAAAHHEAATH
|
0.0 ± 0.0 0 0
|
0.0 ± 0.0
|
0.0 ± 0.0
|
NA
|
13
|
AHHASD
|
0.0 ± 0.0 0 0
|
0.0 ± 0.0
|
0.0 ± 0.0
|
NA
|
14
|
AHHAHHATD
|
0.3 ± 0.4 0-1 0-1
|
0.4 ± 0.5
|
0.2 ± 0.4
|
0.097
|
*New
|
|
0.0 ± 0.3 0-2 0
|
0.0 ± 0.0
|
0.1 ± 0.3
|
0.324
|
Table legend: *New= are sequences observed in Ugandan isolates that had not been reported before. East and West are the Eastern and Western regions of Uganda. Data was given by mean +-SD. P-value was calculated by Student’s t-test or Wilcoxon’s Rank Sum test as appropriate. LM= microscopy
The geographical distribution of the different HRP2 sequences is shown in Fig 3.
HRP2 Amino acid sequence grouping
The amino acid sequences were grouped following the previously published formula (Type 2 x type 7) [16]. Proportions of samples with Type A sequence (≥100), Type B (44-99) and Type C (≤43) were: 12.9% (8/62), 77.4% (48/62) and 9.7% (6/62), respectively. Similar to sequences reported from other countries [16] the majority of parasites in the Ugandan samples had a Type B pfhrp2 sequence. Interesting to note is that 10% of HRP2 sequences are of type C that was initially reported to be associated with reduced performance of some HRP2-based RDTs [17] but later found to have no association when a larger sample size was analyzed [16] . The proportion of Type C sequence accounted for 13% in the RDT-/LM+ compared to 8% in RDT+/LM+ samples (Table 5).
Table 5. Unique pfhrp2 sequences, sequence types and their proportions (n=62).
Genomic DNA sub-sets
|
No. sequenced
|
Unique Sequence types identified
|
Sequence type A
|
Sequence type B
|
Sequence type C
|
|
Total
|
N
|
%
|
N
|
%
|
N
|
%
|
N
|
%
|
RDT+/LM+
|
38
|
38
|
100%
|
5
|
13%
|
30
|
79%
|
3
|
8%
|
RDT-/LM+
|
24
|
23
|
96%
|
3
|
13%
|
18
|
75%
|
3
|
13%
|
Overall
|
62
|
61
|
98%
|
8
|
13%
|
48
|
77%
|
6
|
10%
|
Table legend: RDT+/LM+ and RDT-/LM+ are genomic DNA samples that were initially (HRP2 RDT negative and microscopy positive) and those that were both HRP2 RDT and microscopy positive respectively