Background
Plasmodium falciparum malaria remains a major public health problem in the sub-Saharan Africa (SSA). Increased global efforts in malaria control and elimination have resulted into significant reduction in the disease burden through scaling up of control interventions such as use of insecticide-treated nets (ITNs), indoor residual spraying (IRS) and early case diagnosis and prompt treatment using effective anti-malarial drugs [
1]. However, malaria control programmes are repeatedly challenged by rapid and widespread anti-malarial drug resistance [
2‐
4]. Following the widespread drug resistance to sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) [
5,
6], the World Health Organization (WHO) recommended a policy change from monotherapy to artemisinin-based combination therapy (ACT) [
7]. Despite policy changes as a result of widespread drug resistance against SP, the drug is still being recommended for intermittent preventive treatment during pregnancy (IPTp-SP) whereby in areas of moderate to high transmission SP dose is given at each scheduled antenatal care (ANC) visit at least monthly to prevent pregnancy associated malaria (PAM) and improve pregnancy outcomes [
8]. In addition, SP is still being used in the IPT in infants (IPTi-SP) to reduce malaria and anaemia among infants as well as seasonal malaria chemoprevention (SMC) programmes in some malaria endemic settings [
9]. Nonetheless, the chemoprophylactic effectiveness of IPTp-SP, IPTi-SP and SMC-SP strategies against malaria control amongst the most vulnerable is increasingly being compromised due to the rapid and widespread SP-resistance.
In Tanzania, SP was introduced as the first-line anti-malarial drug for treatment of uncomplicated falciparum malaria in 2001 as a result of high level chloroquine (CQ) resistance (CQR) and clinical treatment failures (TFs) [
10]. However, 5 years after its introduction, the policy was again revised in November 2006 due to widespread resistance against SP and remarkable TFs [
4,
11]. Thus, artemether-lumefantrine (AL), an ACT, was introduced as the first-line treatment for uncomplicated
falciparum malaria in the mainland Tanzania [
12]. The changes in malaria treatment policies were supported by data from molecular epidemiological resistance surveillance against CQ and SP. The in vivo molecular surveillance ascertained the treatment failures (TFs) [
4,
11] with
P. falciparum resistance against CQ and SP in clinical settings.
Sulfadoxine–pyrimethamine acts by inhibiting the
P. falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes [
13,
14]. Notably, several single nucleotide polymorphisms (SNPs) in the
Pfdhps gene at codons Serine 436 to Alanine (S436A), Alanine 437 to Glycine (A437G), Lysine 540 Glutamic acid (K540E), Alanine 581 to Glycine (A581G) and Alanine 613 to Serine (A613S) are associated with sulfadoxine resistance. Pyrimethamine resistance is conferred by mutations in the
Pfdhfr gene resulting from amino acid substitution at codons Cysteine 50 to Arginine (C50R), Asparagine 51 to Isoleucine (N51I), Cysteine 59 to Arginine (C59R), Serine 108 to Asparagine/Threonine (S108 N/T), and Isoleucine 164 to Leucine (I164L) [
15,
16]. Emergence and subsequent accumulation of the mutations in both the
Pfdhfr/Pfdhps genes is associated with SP clinical TF in several epidemiological settings [
11,
17‐
19]. The major
Pfdhfr haplotypes emerge as a result of combination of mutations of the wildtype cysteine-asparagine-cysteine-asparagine-isoleucine (CNCNI) followed by the gradual changes resulting to cysteine-isoleucine-cysteine-
asparagine-isoleucine (CIC
NI), cysteine-asparagine-
arginine-asparagine-isoleucine (CN
RNI), cysteine-isoleucine-
arginine-asparagine-isoleucine (C
IRNI) and cysteine-
isoleucine-arginine-asparagine-leucine (C
IRNL) as a single, double, triple and quadruple mutants, respectively (at amino acid positions C50
R, N51
I, C59
R, S108
N, and L164
I) [
20,
21]. For
Pfdhps, the wild type genotype serine-alanine-lysine-alanine-alanine (SAKAA) can change to a single
alanine–alanine-lysine-alanine-alanine or serine-
glycine-lysine- alanine–alanine (
AAKAA or S
GKAA), double (
AGKAA,
S
GK
GA or S
GEAA) or triple mutants (
AGEAA or S
GEGA, at amino acid positions S436
A, A437
G, K540
E, A581
G and A613
S/T) [
14,
22,
23]. Quintuple mutations as a result of the combination of
Pfdhfr triple (C
IRNI) and the
Pfdhps double (A437
G, K540
E) mutations were reported in East Africa [
24,
25] and have been shown to confer high level SP resistance rendering SP ineffective for treatment of
P. falciparum infections. In northeastern Tanzania, the rise in
Pfdhps mutations at codons A581
G has led to higher proportions of S
GEGA haplotypes [
26]. Combined
Pfdhfr-
Pfdhps S
GEGA-C
IRNI forming the sextuple haplotype has been associated with sub-optimal prophylactic effect and thus poor pregnancy outcomes particularly in areas where SP resistance is widespread in sub-Saharan Africa [
17,
18,
27].
In fact, WHO recommends stopping IPTp-SP in areas where the
Pfdhfr K540
E prevalence is >95 % and
Pfdhps A581
G is >10 % as SP is likely to be ineffective [
28]. The widely spread highly resistant haplotypes including quintuple (C
IRNI-S
GEAA) and sextuple (C
IRNI-S
GEGA) are likely to compromise effectiveness of IPTp-SP strategy. Continuous surveillance of SP effectiveness using molecular markers is therefore, critical and should be routinely implemented as recommended by WHO [
29].
Determination of molecular markers in the Pfdhfr and Pfdhps genes offers an invaluable tool for epidemiological surveillance of SP resistance in malaria endemic settings and will generate important data to assist and inform malaria control programmes on the status of resistance particularly due to emergence and rapid spread of highly resistant mutations and haplotypes that may highly compromise the usefulness of IPT strategies. This study aimed to assess the status of Pfdhfr-Pfdhps mutations and haplotypes in areas with different malaria transmission intensities in mainland Tanzania, 6 years after withdrawal of SP as first line drug for treatment of uncomplicated falciparum malaria.
Results
A total of 264 samples from the three sites (Muheza, Muleba and Nachingwea) were included in the analysis with each site contributing 88 samples. The demographic and parasitological characteristics of the study population between the study sites were comparable (Table
1). The study populations were similar with respect to age (χ
2 = 3.71, p = 0.16), gender (χ
2 = 1.92, p = 0.38) and the mean haemoglobin levels (g/dL) (F = 2.5, p = 0.08). However, the geometric mean parasite density was significantly lower at Muleba compared to Muheza and Nachingwea (F = 10.9, p < 0.001), (Table
1). Fever at health facility presentation (temperature ≥ 37.5 °C) was significantly higher at Muheza (χ
2 = 8.37, p = 0.02) compared to Muleba and Nachingwea. Only a few patients reported prior history of AL (n = 13) or SP (n = 2) usage 2 weeks prior to enrolment into the study.
Table 1
Demographic and parasitological characteristics of the study population in Tanzania
Median age (years) (25–75 % IQR) | 4.2 (2.1–6.3) | 4.4 (2.2–13.0) | 5.2 (2.9–12.6) | 0.16 |
Sex, female n (%) | 42 (47.7) | 48 (54.5) | 51 (58.0) | 0.38 |
Mean haemoglobin (g/dL), (SD)a
| 10.4 (1.8) | 9.9 (2.4) | 9.7 (2.4) | 0.08 |
GMPD (95 % CI)a
| 18,603 (13,280–26,060) | 3700 (1899–7211) | 12,968 (8066–20,848) | <0.001
|
Fever at presentation (≥37.5 °C), n (%) | 65 (73.9) | 59 (67.1) | 47 (53.4) |
0.02
|
Antimalarial treatment history, n (%) | NA | 10 (11.4) | 5 (6.2) | 0.23 |
Prevalence of SNPs associated with SP resistance in Plasmodium falciparumdhfr and dhps genes
Significantly higher prevalence of Pfdhfr mutations at codons C50/N51I was detected at Muheza (100 %) and Muleba (98.8 %) compared to Nachingwea (67.5 %), (p < 0.001). At Muheza, the Pfdhfr mutation at codon C59R had reached almost saturation level (96.6 %) while other sites, Muleba (73.1 %) and Nachingwea (81.8 %), had significantly lower prevalence (p < 0.001). For codon S108N, the prevalence was significantly higher at Muheza (98.7 %) and Nachingwea (95.5 %) compared to Muleba (83.3 %) (p = 0.003). The Pfdhfr mutation at codon I164L was not detected at any site.
For the Pfdhps, significantly high prevalence of parasite carrying mutant at codons S436A/A437G was detected (79.8 %) at Muheza as compared to Muleba (22.2 %) while Nachingwea had none (p < 0.001). However, Nachingwea had significantly higher prevalence (61.4 %) of double mutants S436A/A437G (p < 0.001). The prevalence of Pfdhps K540E at Muheza was significantly higher (95.4 %) compared to the other sites (p < 0.001). Another Pfdhps mutation also associated with high level resistance at codon A581G was significantly higher at Muheza (p < 0.001) reaching 48.9 % in comparison to Muleba (3.9 %) and Nachingwea at which the mutations was not detected. Low prevalence of Pfdhps A613S was detected at Muleba (2/77, 2.6 %) and Nachingwea (1/82, 1.2 %).
Prevalence of the major Plasmodium falciparumdhfr and dhps haplotypes
Similarly, the prevalence of double mutant haplotypes (CICNI, CNRNI and CIRSI) was low except for Muleba where a significantly higher prevalence (23.6 %) of double haplotype CICNI was reported compared to the other sites (p = 0.008). Overall, the triple Pfdhfr mutants (CIRNI) were predominant at all sites and almost near saturation at Muheza (93.3 %) but with significantly lower prevalence of and at Muleba (75 %) and Nachingwea (70.6 %), (p < 0.001).
The prevalence of wildtype
Pfdhps haplotype (SAKAA) was significantly low at Muheza (1.3 %) compared to other sites (p = 0.003). Similarly, the three major single mutant haplotypes were also low (Table
2). The double
Pfdhps haplotype S
GEAA was higher at Muheza (27.2 %) and Muleba (20.8 %) compared to Nachingwea (p < 0.001) while
AAKA
S was low at both site (p = 0.76). About 56 % of all isolates were triple mutants with Muheza having the highest prevalence of S
GEGA haplotype compared to
AGEAA which was more predominant at Muleba and Nachingwea (p < 0.001), (Table
2). The
Pfdhps quadruple haplotypes
AGEGA were generally low and varied significantly between the sites, whereby eight isolates (10.4 %) were detected at Muheza, two at Muleba (5.6 %) and none was detected at Nachingwea (p = 0.003) (Table
2).
Table 2
P. falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) haplotypes by study sites
Pfdhfr
| CNCSI | 0 (0) | 0 (0) | 3 (3.5) | 0.109 |
CICNI | 4 (5.2) | 17 (23.6) | 7 (8.4) |
0.002
|
CIRSI | 1 (1.3) | 0 (0) | 0 (0) | 0.642 |
CNRNI | 0 (0) | 1 (1.4) | 15 (17.7) | <0.001
|
CIRNI | 72 (93.3) | 54 (75.0) | 60 (70.6) | <0.001
|
Pfdhps
| SAKAA | 1 (1.3) | 11 (15.3) | 11 (16.5) |
0.003
|
AAKAA | 4 (5.2) | 4 (5.6) | 16 (18.8) |
0.008
|
SAKAS
| 0 (0) | 1 (1.4) | 0 (0) | 0.308 |
SAEAA | 0 (0) | 4 (5.6) | 0 (0) |
0.008
|
AAKAS
| 0 (0) | 1 (1.4) | 1 (1.2) | 0.76 |
SGEAA | 21 (27.2) | 15 (20.8) | 0 (0) | <0.001
|
SGEGA | 31 (40.3) | 2 (2.8) | 0 (0) | <0.001
|
AGEAA | 12 (15.6) | 30 (41.7) | 54 (63.5) | <0.001* |
AGEGA | 8 (10.4) | 4 (5.6) | 0 (0) |
0.003
|
Upon combination of the
Pfdhfr-
Pfdhps haplotypes (Table
3), quadruple mutant haplotypes with single
Pfdhps and triple
Pfdhfr mutation (
AAKAA-C
IRNI) was lowly distributed (n = 16) across all sites while the quintuple mutant haplotype, C
IRNI-S
GEAA was observed in only 31 isolates, at Muheza and Muleba. Sextuple C
IRNI-S
GEGA haplotypes (n = 32) were more predominant in Muheza whereas other sextuple haplotype combinations, (C
IRNI-
AGEAA (n = 69)) were mainly observed in Nachingwea. Interestingly, the emergence of the new septuple mutant haplotypes with three
Pfdhfr and four
Pfdhps mutant combination (C
IRNI
-AGEGA) was observed for the first time albeit in few samples (n = 11) and these were mainly from Muheza (n = 8) and (n = 3) were from Muleba whereas none was noted at Nachingwea. The occurrences of other
Pfdhps-
Pfdhfr haplotypes were generally low (Table
3).
Table 3
The pattern of combined P. falciparum
dhfr–dhps haplotypes
Wildtype | SAKAA | 0 | 4 | 0 | 2 | 20 | 26 |
Single |
AAKAA | 1 | 3 | 0 | 4 | 16 | 24 |
SAEAA | 0 | 0 | 0 | 0 | 4 | 4 |
SAKAS
| 0 | 0 | 0 | 0 | 1 | 1 |
Double | SGEAA | 0 | 5 | 0 | 0 | 31 | 36 |
AAKAS
| 0 | 0 | 0 | 0 | 2 | 2 |
Triple | SGEGA | 0 | 1 | 0 | 0 | 32 | 33 |
AGEAA | 2 | 14 | 1 | 10 | 69 | 96 |
Quadruple |
AGEGA | 0 | 1 | 0 | 0 | 11 | 12 |
Total | | 3 | 28 | 1 | 16 | 186 | 234 |
Discussion
The rapid and widespread anti-malarial drug resistance has necessitated frequent revisions of malaria treatment guidelines in P. falciparum malaria endemic regions. The emergence of super resistant mutations, such as the sextuple Pfdhfr/Pfdhps haplotypes has not only compromised malaria case management and treatment outcomes but also affected the effectiveness of the IPT strategies.
Few years after its adoption as the first-line treatment drug for uncomplicated malaria in Tanzania, several efficacy studies detected unacceptably high levels of molecular markers of parasite resistance to SP [
4,
35]. Despite its replacement by ACT, SP continued to be used in the IPT strategies which is likely to provide sub-optimal effect and, therefore, monitoring the spread of resistance using molecular markers (
Pfdhfr/Pfdhps) is still recommended [
36‐
38]. In this study, the prevalence of A581
G was almost 50 % in Muheza and this was significantly higher compared to Muleba (~4 %) and Nachingwea (0 %). This could confirm the earlier suggestions that this “super resistant” mutation may have originated in the north-eastern part of Tanzania and spread to other areas albeit at low prevalence [
37].
A remarkable difference was observed in the prevalence of
Pfdhps SNPs between the study sites with Muheza having the highest levels of
Pfdhps 436/437
SG, A581
G, A540
E SNPs and haplotypes compared to Muleba and Nachingwea. As expected areas with high malaria transmission intensities in Muleba and Nachingwea had higher prevalence of
Pfdhps wild types and single mutant 436/437A
G as compared to Muheza in north eastern where malaria endemicity has declined remarkably in recent years [
39,
40]. The current WHO recommendations suggest that SP-IPTp should be discontinued if the prevalence of this double
Pfdhps mutant, K540
E is more than 95 % and the A581
G is more than 10 % as it is considered to be ineffective [
29]. Obviously, these criteria are still met in Muheza confirming the findings of previous studies conducted in north Eastern Tanzania [
26]. In a cohort study conducted at Muheza, it was shown that IPTp–SP was associated with increased prevalence of parasites with mutations at codon A581
G and that use of IPTp-SP conferred no benefit in improvement of pregnancy outcomes [
17,
37]. Of note, this area is known to be the major focus of
Pfdhps A581
G mutations in East Africa which is believed to occur almost exclusively with
Pfdhfr K540
E leading to double mutant haplotypes [
41]. The high frequency of
Pfdhps A581
G at alarming level in this area clearly suggests for no beneficial protective effect from the IPTp-SP [
17,
18,
42]. This higher prevalence in north eastern Tanzania could be explained by the sustained drug pressure due to self-medication that could have elevated levels of SP resistance, Ringsted et al. [
43] reported 76 % volume sales of SP in private drug shops in this areas. Additionally, the high prevalence levels could be maintained due to selective pressure on
Pfdhfr and
Pfdhps as a result of co-trimoxazole (trimethoprim-sulfamethoxazole), another antifolate, used to prevent opportunistic infections in HIV infected individuals as cross-resistance might also occur [
44,
45].
The prevalence of the
Pfdhfr-
Pfdhps wild type haplotypes was low in all sites. The prevalence of mutant genotypes C51
I, was at saturation in Muheza (100 %) and equally at Muleba (98.8 %) with significantly lower prevalence (67.5 %) at Nachingwea (p < 0.001). Similarly, almost complete saturation (>96 %) of other mutations in
Pfdhfr (N51
I, C59
R, and S108
N) was observed at Muheza with marked differences between Muleba and Nachingwea (Table
2). In contrast, some previous studies have shown that other major resistance mutations in
Pfdhfr are well established throughout the country where the
Pfdhfr triple mutations (51
I, 59
R and 108
N) were above 84 % and close to saturation in six regions of Tanzania [
46]. Elsewhere, these data are in consistent with studies in West Africa where
Pfdhfr N51
I, N59
R, and S108
N have been shown to rise [
19]. The prevalence of I164L mutation conferring high pyrimethamine resistance was not detected at all the three sites which is in accordance to previous reports elsewhere [
47]. However, in other parts of Africa, only a few studies have reported occurrence of this high level mutation [
15,
16,
48]. Of note, the
Pfdhfr I164L mutation was first documented at low prevalence in Muheza in 1999 before the deployment of IPTp-SP [
49] and later one isolate (mixed allele) was reported in Rufiji [
50], but to date its presence has rarely been reported in the country.
The C
IRNI haplotype with triple mutations was present and highly prevalent at all study sites regardless of the transmission intensity. This is in line with several studies in Tanzania and elsewhere in the SSA [
51,
52]. The C
IRNI haplotype is associated with high level resistance to pyrimethamine and increases the risk of SP resistance if it occurs concurrently with
Pfdhps mutations [
21]. The increased double mutant
Pfdhps S
GEAA haplotype was observed in Muheza (28 %) and Muleba (22 %), but not in Nachingwea (0 %). In Muheza, the highly resistant triple S
GEGA
Pfdhps haplotypes was observed, (38.7 %).
The combinations of
Pfdhfr-
Pfdhps were detected at higher numbers including quintuple mutant C
IRNI-S
GEAA, sextuple haplotype which comprise triple mutations in both genes C
IRNI-S
GEGA and C
IRNI-
AGEGA have been highly associated with sub-optimal IPTp-SP effectiveness in previous studies [
17]. Interestingly, a septuple mutant haplotype C
IRNI-
AGEGA was observed which had not been previously reported in the study areas. From these observations, it is apparent that these mutant haplotypes associated with poor IPTp-SP are expanding in different epidemiological settings. Of worth noting, this study was not designed to correlate the clinical data with observed resistance pattern. Nonetheless previous literature showing the association of these haplotypes with clinical or treatment outcomes have been noted.
Ideally, in order to reduce the sustained drug pressure, efforts are required to limit the use of SP for IPTp purposes only through limiting over the counter SP prescriptions. Also the availability of ACT for the treatment of uncomplicated malaria and proper implementation of the national malaria treatment guidelines would also contribute in the reducing the selection pressure. However, alternative drugs for IPTp are urgently needed to replace the failing SP due to the saturation of the parasite population with Pfdhps-Pfdhfr mutations and haplotypes highly associated with SP resistance. Deployment of the sub-optimal IPTp-SP strategy is therefore unlikely to confer the anticipated effect on improving pregnancy outcomes.
Authors’ contributions
VB, RAM, DSI and JPG designed the study; VB, RAM, DN, DSI and JPG performed the research; VB, RAM, DTRM and FF analysed data; all authors contributed to the writing of the paper. All authors read and approved the final manuscript.