ABSTRACT
Malaria poses public health threats worldwide. Nigeria accounted for the highest numbers of cases (26.8%) and deaths (31.9%) among countries where malaria is endemic in 2020. Currently, monitoring molecular markers in imported malaria cases provides an efficient means to screen for emerging drug resistance in countries where malaria is endemic, particularly in those where field surveillance is challenging. Here, we investigated 165 Plasmodium falciparum infections imported from Nigeria to Zhejiang Province, China, between 2016 and 2020. Multiple molecular markers in k13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps were detected. The prevalences and patterns of mutations were analyzed. Polymorphism of k13 was limited to 5 of 156 (3.21%) isolates. The wild-type CVMNK allele of Pfcrt became predominant (65.36%) compared with the triple mutation CVIET. A low frequency (4.73%) of double mutations (N86Y and Y184F) in Pfmdr1 was observed. The dominant haplotypes of Pfdhfr and Pfdhps were IRNDI (92.41%) and ISGKAA (36.84%), respectively. The newly discovered mutant I431V was identified in 21.71% of isolates. A “fully resistant” combination of Pfdhfr-Pfdhps, IRN-GE, was found in eight (5.67%) samples, which was hardly seen in Nigeria. The current study demonstrated a high frequency of wild-type Pfcrt. Limited polymorphism of Pfmdr1 but a high prevalence of Pfdhfr and Pfdhps mutations was illustrated. Our data so far serve as comprehensive surveillance of molecular markers of the k13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes. Based on our findings, it has become crucial to evaluate the impact of the emerging fully resistant type of Pfdhfr-Pfdhps as well as its combination with I431V on the efficacy of sulfadoxine-pyrimethamine (SP) in Nigeria.
IMPORTANCE Monitoring the current resistance to antimalarial drugs is critical to enable timely action to prevent its spread and limit its impact. The high prevalence of wild-type Pfcrt found in our study is an optimistic signal to reevaluate chloroquine (CQ) sensitivity in Nigeria, which is cost-effective and once played a crucial role in the fight against malaria. Based on the continued emergence of fully resistant Pfdhfr-Pfdhps alleles illustrated in the current investigation, actions are needed in Nigeria, such as national systemic surveillance to monitor their updated epidemiology as well as assessments of their influence on SP efficacy to minimize any public health impact. These findings urge a response to the threat of drug resistance to facilitate appropriate drug policies in the study area.
KEYWORDS: molecular epidemiology, Nigeria, Plasmodium falciparum, drug resistance
INTRODUCTION
Malaria poses public health threats worldwide, especially in sub-Saharan Africa. There were an estimated 241 million cases of malaria in 2020 in 85 countries where malaria is endemic (1). The World Health Organization (WHO) Africa Region accounted for about 95% of cases, namely, 228 million cases (1). Among the countries where malaria is endemic, in 2020, Nigeria accounted for the highest percentages of cases (26.8%) and deaths (31.9%). Compared with 2018, in 2019 in Nigeria, there was an estimated increase of 2.4 million malaria patients, and >60 million cases were reported (2).
As an inexpensive and safe antimalarial drug, chloroquine (CQ) was once the drug most commonly prescribed in Nigeria (3). Sulfadoxine-pyrimethamine (SP), amodiaquine, quinine, halofantrine, and artemether were also used as monotherapies (3). However, the emergence of CQ-resistant parasites was reported in 1987 in the south of Nigeria and then spread throughout the country (4). Nigeria changed its national drug policy from CQ to artemisinin combination therapy (ACT) with artemether-lumefantrine (AL) in 2005 (4, 5). There has been a reduced response of Plasmodium falciparum infection to ACT in six geographical areas of Nigeria, 10 years after ACT was introduced as a first-line treatment (5). Although SP for intermittent preventive treatment has a positive impact on reducing peripheral parasitemia, placental parasitization, and low birth weight, there are also concerns about increasing resistance to SP (6, 7). These observations emphasize the importance of monitoring the current status and investigating the spread of antimalarial drug resistance in Nigeria.
An increased understanding of the molecular mechanisms underlying parasite resistance has led to a more efficient way to observe its trends by tracking the prevalence of molecular markers associated with drug resistance. The WHO updated a list of 10 validated P. falciparum k13 markers and 11 candidate markers of partial resistance to artemisinin (2). Since the emergence and clonal expansion of R561H mutant parasites were detected in Rwanda in 2014, which shared no genetic relatedness to those previously reported in Southeast Asia, concerns about their spread were raised in African countries (8). The CQ resistance transporter (Pfcrt) located on P. falciparum digestive vacuole membranes is associated with CQ treatment failure. Mutant Pfcrt (mainly the K76T mutation) is considered to be a highly sensitive and moderately specific marker for CQ treatment failure (9). The P. falciparum multidrug drug resistance gene Pfmdr1 encodes an ATP-binding cassette drug transporter and is believed to be the only gene associated with multidrug resistance. Mutant Pfmdr1, especially N86Y and an increased copy number, influences the sensitivity to a wide range of antimalarials, including CQ, artemisinin derivatives, and amodiaquine (10, 11). Mutations in the dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes of P. falciparum are associated with antifolate drug resistance.
In the current study, we collected P. falciparum isolates imported from Nigeria to Zhejiang Province, China, between 2016 and 2020 and analyzed the profiles of the k13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes, which are associated with drug resistance, to provide evidence for an update of antimalarial policies.
RESULTS
Overview of malaria infections imported from Nigeria to Zhejiang Province from 2016 to 2020.
A total of 839 malaria cases were imported to Zhejiang Province between 2016 and 2020. Nigeria was the main country of origin, accounting for 24.08% (202/839) of the total (Fig. 1; see also Table S2 in the supplemental material). Among the 202 cases imported from Nigeria, 81.68% (165/204) were caused by P. falciparum, followed by Plasmodium ovale (13.86%; 28/202). The proportions of cases infected by Plasmodium malariae and Plasmodium vivax were 2.97% (6/202) and 1.49% (3/202), respectively. All of the P. falciparum cases imported from Nigeria were included in the current survey. The median (range) age of the study population was 44 (29 to 69) years, and 152 were male. The numbers of P. falciparum samples from 2016 to 2020 were 53, 42, 29, 33, and 9, respectively.
FIG 1.
Proportion of imported malaria cases from Nigeria in Zhejiang Province (a) and proportion of P. falciparum cases from Nigeria (b).
Prevalence and patterns of k13 mutations.
A total of 156 sequences of k13 were successfully obtained from 165 samples. Sequence alignment revealed that 7 (4.49%; 7/156) isolates harbored a nucleotide mutation: C1321T in 2 isolates, A1372G in 1, C1767G in 2, G1948T in 1, and G2026T in 1. The prevalence of amino acid mutations was 3.21% (5/156). Specifically, nucleotide mutations C1321T, A1372G, G1948T, and G2026T resulted in amino acid mutations P441S, N458D, V650F, and A676S, respectively, while nucleotide mutation C1767G did not yield a nonsynonymous amino acid mutation (Table 1).
TABLE 1.
Prevalence of point mutations in k13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps in P. falciparum isolates imported to Zhejiang Province from Nigeria
| Gene | Codon mutation | No. of isolates from yr |
Total no. of isolates (%) | ||||
|---|---|---|---|---|---|---|---|
| 2016 | 2017 | 2018 | 2019 | 2020 | |||
| k13 (n = 156) | P441S | 0 | 1 | 1 | 0 | 0 | 2 (1.28) |
| N458D | 0 | 0 | 0 | 1 | 0 | 1 (0.64) | |
| V589V | 2 | 0 | 0 | 0 | 0 | 2 (1.28) | |
| V650F | 1 | 0 | 0 | 0 | 0 | 1 (0.64) | |
| A676S | 1 | 0 | 0 | 0 | 0 | 1 (0.64) | |
| Pfcrt (n = 153) | M74Ia | 18 | 19 | 6 | 10 | 0 | 53 (34.64) |
| N75Eb | 18 | 19 | 6 | 10 | 0 | 53 (34.64) | |
| K76Tc | 18 | 19 | 6 | 10 | 0 | 53 (34.64) | |
| Pfmdr1 (n = 158) | N86Y | 10 | 2 | 2 | 1 | 0 | 15 (9.49) |
| G102G | 0 | 0 | 0 | 1 | 0 | 1 (0.63) | |
| I107V | 0 | 1 | 0 | 0 | 0 | 1 (0.63) | |
| Y184F | 19 | 19 | 19 | 19 | 4 | 80 (50.63) | |
| Pfdhfr (n = 145) | N51I | 38 | 39 | 26 | 28 | 7 | 138 (95.17) |
| C59R | 38 | 38 | 25 | 29 | 7 | 137 (94.48) | |
| S108N | 39 | 39 | 28 | 31 | 7 | 144 (99.31) | |
| D139V | 0 | 0 | 0 | 0 | 0 | 0 (0) | |
| I164L | 0 | 0 | 0 | 0 | 0 | 0 (0) | |
| Pfdhps (n = 152) | I431V | 11 | 13 | 1 | 6 | 2 | 33 (21.71) |
| S436A | 19 | 18 | 9 | 16 | 4 | 66 (43.42) | |
| S436Y | 1 | 0 | 0 | 0 | 0 | 1 (0.66) | |
| A437G | 40 | 36 | 26 | 28 | 6 | 136 (89.47) | |
| K540E | 1 | 4 | 5 | 0 | 1 | 11 (7.24) | |
| A581G | 7 | 11 | 2 | 6 | 2 | 28 (18.42) | |
| A613S | 15 | 12 | 6 | 12 | 2 | 47 (30.92) | |
Mixed mutation M74M/I included.
Mixed mutation N75N/E/D/K included.
Mixed mutation K76K/T included.
Prevalence and patterns of Pfcrt mutations.
Of the 165 P. falciparum isolates from Nigeria described above, 153 were sequenced successfully. Wild-type (CVMNK), mutant-type (CVIET), and mixed mutant-type (CVM/IN/E/D/KK/T) Pfcrt alleles were detected (Tables 1 and 2). Among these alleles, the wild-type CVMNK allele was the most prevalent (65.36%; 100/153), followed by the triple mutant CVIET allele (29.41%; 45/153). Only eight (5.23%) isolates carried the mixed mutant type. By year of collection, the percentages of the triple mutation (CVIET) were 33.33% (17/51) in 2016, 39.02% (16/41) in 2017, 14.29% (4/28) in 2018, and 27.50% (19/29) in 2019. Regarding the mixed mutation (CVM/IN/E/D/KK/T), the proportions in 2017, 2018, and 2019 were similar, at about 7%, while the proportion was lower (1.96%; 1/51) in 2016. No mutant type was observed in 2020 due to the limited sample size.
TABLE 2.
Prevalence of haplotypes of Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps in isolates of P. falciparum imported to Zhejiang Province from Nigeria
| Gene | Haplotype | Codona | No. of isolates (%) |
|||||
|---|---|---|---|---|---|---|---|---|
| 2016 | 2017 | 2018 | 2019 | 2020 | Total | |||
| Pfcrt (n = 153) | Wild type | C72V73M74N75K76 | 33 (67.41) | 22 (53.66) | 22 (78.57) | 19 (65.52) | 4 (100.00) | 100 (65.36) |
| Triple mutant | CVIET | 17 (33.33) | 16 (39.02) | 4 (14.29) | 8 (27.50) | 0 | 45 (29.41) | |
| Mixed mutant | CVM/I N/E/D/K K/T | 1 (1.96) | 3 (7.32) | 2 (7.14) | 2 (6.90) | 0 | 8 (5.23) | |
| Pfmdr1 (n = 158) | Wild type | N86I107Y184 | 25 (50.00) | 19 (46.34) | 10 (34.48) | 12 (38.71) | 3 (42.86) | 69 (43.67) |
| Single mutant | YIY | 6 (12.00) | 2 (4.88) | 0 | 0 | 0 | 8 (5.06) | |
| NVY | 0 | 1 (2.44) | 0 | 0 | 0 | 1 (0.63) | ||
| NIF | 15 (30.00) | 19 (46.34) | 17 (58.62) | 18 (58.06) | 4 (57.14) | 73 (46.20) | ||
| Double mutant | YIF | 4 (8.00) | 0 | 2 (6.90) | 1 (3.23) | 7 (50.00) | 7 (4.43) | |
| Pfdhfr (n = 145) | Wild type | N51C59S108D139I164 | 1 (2.50) | 0 | 0 | 0 | 0 | 1 (0.69) |
| Single mutant | NCNDI | 0 | 0 | 1 (3.57) | 2 (6.45) | 0 | 3 (2.07) | |
| Double mutant | ICNDI | 1 (2.50) | 1 (2.56) | 2 (7.14) | 0 | 0 | 4 (2.76) | |
| NRNDI | 1 (2.50) | 0 | 1 (3.57) | 1 (3.23) | 0 | 3 (2.07) | ||
| Triple mutant | IRNDI | 37 (92.50) | 38 (97.44) | 24 (85.71) | 28 (90.32) | 7 (100.00) | 134 (92.41) | |
| Pfdhps (n = 152) | Wild type | I431S436A437K540A581A613 | 1 (2.22) | 1 (2.50) | 2 (6.90) | 2 (6.45) | 0 | 6 (3.95) |
| Single mutant | IAAKAA | 3 (6.67) | 2 (5.00) | 1 (3.45) | 1 (3.23) | 1 (14.29) | 8 (5.26) | |
| ISGKAA | 17 (37.78) | 14 (35.00) | 12 (41.38) | 11 (35.48) | 2 (28.57) | 56 (36.84) | ||
| ISAKAS | 0 | 1 (2.50) | 0 | 0 | 0 | 1 (0.66) | ||
| Double mutant | IAGKAA | 5 (11.11) | 3 (7.50) | 3 (10.34) | 4 (12.90) | 1 (14.29) | 16 (10.53) | |
| ISGEAA | 1 (2.22) | 4 (10.00) | 5 (17.24) | 0 | 1 (14.29) | 11 (7.24) | ||
| ISGKAS | 3 (6.67) | 0 | 0 | 1 (3.23) | 0 | 4 (2.63) | ||
| VSGKAA | 2 (4.44) | 0 | 0 | 0 | 0 | 2 (1.32) | ||
| IYAKAS | 1 (2.22) | 0 | 0 | 0 | 0 | 1 (0.66) | ||
| Triple mutant | IAGKAS | 3 (6.67) | 1 (2.50) | 4 (13.79) | 5 (16.13) | 0 | 13 (8.55) | |
| VAGKAA | 0 | 2 (5.00) | 0 | 0 | 0 | 2 (1.32) | ||
| VSGKAS | 1 (2.22) | 0 | 0 | 0 | 0 | 1 (0.66) | ||
| VSGKGA | 0 | 2 (5.00) | 0 | 0 | 0 | 2 (1.32) | ||
| ISGKGS | 0 | 0 | 1 (3.45) | 1 (3.23) | 0 | 2 (1.32) | ||
| Quadruple mutant | IAGKGS | 0 | 1 (2.50) | 0 | 0 | 0 | 1 (0.66) | |
| VAGKGA | 1 (2.22) | 0 | 0 | 0 | 0 | 1 (0.66) | ||
| VAGKAS | 1 (2.22) | 1 (2.50) | 0 | 0 | 0 | 2 (1.32) | ||
| Quintuple mutant | VAGKGS | 6 (13.33) | 8 (22.50) | 1 (3.45) | 5 (16.13) | 2 (28.57) | 22 (14.47) | |
The underlined amino acid of the haplotypes indicated that it was mutant.
Prevalence and patterns of Pfmdr1 mutations.
A total of 158 samples were assessed successfully for the Pfmdr1 codon polymorphisms N86F and Y184F (Tables 1 and 2). Point mutation N86Y was found in 9.49% (15/158) of the isolates, and Y184F was observed in half of the isolates (50.63%; 80/158). In addition, sequence alignments revealed that one sample from 2019 harbored a nucleotide mutation, T to C at position 306, and one sample from 2017 carried an amino acid mutation, isoleucine to valine at position 107. Analysis of the Pfmdr1 haplotype prevalence showed that a single mutation type (NIF) was observed the most frequently (46.20%; 73/158), followed by the wild type (NIY) (43.67%, 69/158). A double mutant (YIF) was also found in 4.43% (7/158) of the samples.
Prevalences and patterns of Pfdhfr mutations.
The haplotypes of Pfdhfr were determined for all 165 samples, but only 145 samples were detected successfully. Point mutations N51I, C59R, S108N, D139V, and I164L were recorded (Tables 1 and 2). Point mutation S108N predominated, at 99.31% (144/145). In addition, mutations N51I and C59R were frequently observed in 95.17% (138/145) and 94.48% (137/145) of the samples, respectively. However, there was no D139V or I164L mutation in these isolates. Regarding haplotypes of Pfdhfr, the prevalence of the triple mutation IRNDI was the highest (92.41%; 134/145). The proportions of the wild type, single mutations, and double mutations were as low as 0.69% to 2.76%.
Prevalence and patterns of Pfdhps mutations.
Of the 165 parasite-positive samples, we successfully amplified the Pfdhps gene from 152 isolates. Nucleotide polymorphisms at positions 431, 436, 437, 540, 581, and 613 were identified. The results for Pfdhps mutations are shown in Tables 1 and 2. Seven single nucleotide polymorphisms were observed. The highest-frequency point mutation was A437G (89.47%; 136/152), which was followed by S436A (43.42%; 66/152) and A613S (30.92%; 47/152). Notably, we also found I431V in 21.71% of the samples. For the haplotype analysis, the diversity of the mutant type was recorded. Specifically, haplotype ISGKAA predominated, at 36.84% (56/152), and double mutant type IAGKAA and triple mutant type IAGKAS were found in 10.53% (16/152) and 8.55% (13/152) of the samples, respectively. It is important to note that the combination of A437G and K540E was found in 7.24% (11/152) of the samples. There was no obvious pattern between years.
Patterns of Pfdhfr-Pfdhps mutations.
A total of 24 haplotypes were observed for Pfdhfr-Pfdhps mutations (Table 3). A quadruple mutant (IRNDI-ISGKAA) was the most frequent (48/142; 34.04%), followed by an octuple mutant (IRNDI-VAGKGS), at a frequency of 15.60% (22/141), and a quintuple mutant (IRNDI-IAGKAA), at a frequency of 11.35% (16/141). The quadruple combination of Pfdhfr-Pfdhps mutations (N51I, N59R, and S108N in Pfdhfr and A437G in Pfdhps) conferring partial resistance was found in 112 (79.43%) of the isolates. The quintuple combination (N51I, N59R, and S108N in Pfdhfr and A437G and K540E in Pfdhps) conferring full resistance was found in 8 (5.67%) isolates in 2016 (1 isolate), 2017 (2 isolates), 2018 (4 isolates), and 2020 (1 isolate). The sextuple combination (N51I, N59R, and S108N in Pfdhfr and A437G, K540E, and A581G in Pfdhps) conferring superresistance was not seen.
TABLE 3.
Prevalence of Pfdhps-Pfdhfr haplotypes in P. falciparum isolates imported to Zhejiang Province from Nigeria
| Haplotype | Codona | No. of isolates (%) |
|---|---|---|
| Single mutant | NCNDI-ISAKAA | 2 (1.42) |
| Double mutant | NRNDI-ISAKAA | 1 (0.71) |
| Triple mutant | ICNDI-ISGKAA | 3 (2.13) |
| IRNDI-ISAKAA | 3 (2.13) | |
| NRNDI-IAAKAA | 1 (0.71) | |
| Quadruple mutant | IRNDI-ISGKAA | 48 (34.04) |
| IRNDI-IAAKAA | 7 (4.96) | |
| IRNDI-ISAKAS | 1 (0.71) | |
| ICNDI-VSGKAA | 1 (0.71) | |
| NCSDI-VAGKAS | 1 (0.71) | |
| Quintuple mutant | IRNDI-IAGKAA | 16 (11.35) |
| IRNDI-ISGEAA | 8 (5.67) | |
| IRNDI-ISGKAS | 4 (2.84) | |
| IRNDI-VSGKAA | 1 (0.71) | |
| NRNDI-IAGKAS | 1 (0.71) | |
| Sextuple mutant | IRNDI-IAGKAS | 10 (7.09) |
| IRNDI-ISGKGS | 2 (1.42) | |
| IRNDI-VAGKAA | 3 (2.13) | |
| IRNDI-VSGKAS | 1 (0.71) | |
| IRNDI-VSGKGA | 2 (1.42) | |
| Septuple mutant | IRNDI-VAGKAS | 1 (0.71) |
| IRNDI-VAGKGA | 1 (0.71) | |
| IRNDI-IAGKGS | 1 (0.71) | |
| Octuple mutant | IRNDI-VAGKGS | 22 (15.60) |
| Total | 141 (100.00) | |
The underlined amino acid of the haplotypes indicated that it was mutant.
DISCUSSION
Due to the spread of drug-resistant parasites worldwide, especially the emergence of artemisinin-resistant P. falciparum k13 R561H mutant parasites in Rwanda (8), it is becoming increasingly important to monitor updated antimalarial resistance in Africa. Therefore, in the present study, we collected P. falciparum isolates imported from Nigeria to Zhejiang Province, China, between 2016 and 2020 to provide updated insight into the k13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genetic profiles. It is estimated that about one-quarter of malaria patients (22.78% to 26.42% between 2016 and 2020) in Zhejiang returned from Nigeria. The majority of these patients were infected by P. falciparum. The number of P. falciparum cases declined from 53 in 2016 to 9 in 2020, which could be explained by a sharp decrease in migrant workers because of the influence of coronavirus disease 2019 (COVID-19).
ACT was introduced in 2005 as the first-line treatment for malaria in Nigeria (4, 5), and AL is most commonly used in ACT (12). Ten years following ACT deployment as a first-line antimalarial treatment in Nigeria, a significant decline in the early responses of childhood P. falciparum infections to AL and artesunate-amodiaquine was reported and raised concern. However, we observed no validated molecular markers in the k13 propeller domain associated with artemisinin resistance, indicating an optimistic status concerning artemisinin resistance in Nigeria. Similarly, no validated k13 polymorphism associated with artemisinin resistance was seen in P. falciparum isolates in previous reports from the Lagos, Osun, and Anambra States of Nigeria (12–14). Additionally, our data showed 3.21% nonsynonymous mutations, P441S, N458D, V650F, and A676S, in five isolates. The A676S substitution was previously discovered in Ghana and Southeast Asia, and therapeutic efficacy data from Ghana still support its sensitivity to AL (15–17). As no clinical study or in vitro tests were performed here, the nonsynonymous mutations P441S, N458D, and V650F could not be correlated with artemisinin sensitivity. Therefore, this observation warrants a larger sample size to monitor these molecular markers as well as their response to antimalarials to evaluate their potential influence on and correlation with drug resistance.
Mutations at positions 72 to 76 of Pfcrt are thought to play a decisive role in conferring resistance to CQ (18). In Nigeria, CQ-resistant strains of P. falciparum were first observed in the southern region in 1987 and then spread across the country (4). ACT combined with artemether-lumefantrine was introduced as a first-line antimalarial treatment in 2005 (3, 4). Eleven years after the withdrawal of CQ, our study showed a low frequency of mutant type CVIET in Nigeria. Specifically, the overall prevalence of Pfcrt M74I, N75E, and K76T in the present study was found to be 34.64%, and 65.36% of the isolates carried the wild-type (CVMNK) Pfcrt gene, followed by triple mutants (29.41%), which is similar to the prevalences reported previously in isolates from Nigeria and other African countries such as Angola, the Republic of Guinea, Ghana, Equatorial Guinea, Cameroon, and Chad (19, 20). The high proportion of wild-type Pfcrt codons 72 to 76 indicates a return of CQ-sensitive P. falciparum isolates to Nigeria. There were also high levels of CQ-sensitive haplotypes of P. falciparum in China (21), Tanzania (22), Malawi (23), and Kenya (24) after CQ was no longer on the first-line list for the treatment of P. falciparum, which reflected decreasing drug pressure on the parasite population. However, our results are in contrast to those of several previous studies. For instance, the prevalence of haplotype CVIET was 76.37% in asymptomatic infections in Anambras, southeastern Nigeria (4); similarly, another two investigations carried out in Lagos and Edo in southwestern Nigeria showed that 38.7% and 4.2% of the isolates were of the wild type, respectively (25, 26). The high proportion of CVIET in regions of Nigeria might be explained by prescription practices for CQ in private facilities that do not follow the national drug policy (3). Conclusions cannot yet be drawn nationally in Nigeria on the dominant Pfcrt codons 72 to 76. It is necessary to perform systematic investigations in Nigeria to reevaluate the national Pfcrt situation and CQ efficacy.
Pfmdr1 is regarded as a pivotal factor in antimalarial drug resistance (10). We detected 9.49% of isolates with the N86F mutation in Pfmdr1. This finding was in line with the results of previous studies by Ikegbunam et al. in southeastern Nigeria (8.54%) (4) and Adam et al. in Kano State (11.3%) (27). Furthermore, our study revealed a higher proportion (50.63%) of isolates with the Y184F mutation, while a prevalence of only 29.27% was observed by Ikegbunam et al. The frequencies of Pfmdr1 gene mutations varied among the isolates in previous reports. Dokunmu et al. observed that 24% of isolates carried the N86F mutation among asymptomatic and symptomatic infections in Ota, southwestern Nigeria (28). In Lagos and Ede, Osun State, the N86F mutation was present in 44% of the isolates sequenced, while the Y184F mutation was found at a higher prevalence of 86.75% (13). The Pfmdr1 N86F mutation has been linked to lower parasite responses to a broad range of antimalarials, including CQ, artemisinin derivatives, mefloquine, and lumefantrine, while the Y184F variant has a limited effect. However, their combination (N86F and Y184F double mutant) is known to be associated with reduced piperaquine susceptibility (10). Fortunately, the frequency of the Pfmdr1 double mutant type (N86Y and Y184F) in the present study was as low as 4.73% (7/158). However, routine surveillance is still recommended to monitor the temporal and spatial distribution of molecular markers of Pfmdr1 given their varying regional prevalence in Nigeria (27).
Our study showed high frequencies of P. falciparum isolates with Pfdhfr and Pfdhps mutations in circulation in Nigeria. Point mutations N51I, C59R, and S108N in Pfdhfr were prevalent, which is comparable to the results of some other previously reported studies of asymptomatic pregnant women from Calabar as well as patients and asymptomatic persons from Lagos State, Nigeria (29, 30). We detected the newly discovered mutant I431V in 21.71% of isolates between 2016 and 2020, which was comparable to or higher than the rates reported in previous studies from Nigeria (7, 29, 31). Although no conclusive evidence was obtained about the effect of I431V on the drug response, molecular surveillance supports that I431V has been common in Nigeria and spread to neighboring countries (31, 32). In particular, we found an octuple mutant type, VAGKGS, in 22 isolates, for a prevalence of 15.60%, highlighting the spread of highly mutated Pfdhps in Nigeria. The significance of I431V remains unknown; however, based on our findings, it has become crucial to evaluate the impact of the combination of I431V with A437G, K540E, and A581G in Pfdhps. The high prevalence (92.41%) of triple mutant haplotype IRNDI of Pfdhfr found in this study confirms previously reported research showing that the efficacy of SP may be threatened in Nigeria (29, 30). Our analysis showed that the single mutant haplotype ISGKAA dominated in Nigeria. This was consistent with data from previous reports in Nigeria as well as other African countries (33). Alarmingly, 7.24% of the samples in our survey harbored a Pfdhps double mutation at codons 437 and 540, which is known for its contribution to clinical resistance to SP when acting in combination with triple mutant Pfdhfr (N51I, N51I, and S108N) (34). Considering Pfdhfr and Pfdhps together, a fully resistant set of quintuple Pfdhfr-Pfdhps mutations, A437G and K540E in Pfdhps and N51I, N59R, and S108N in Pfdhfr, has rarely been described in West Africa, whereas the combination of A437G and triple mutant Pfdhfr, conferring partial resistance, is common (35). To be specific to Nigeria, the quintuple Pfdhfr-Pfdhps mutation was not detected in recent studies (29, 30, 36). However, we found it in 8 (5.67%) isolates, along with the quadruple Pfdhfr-Pfdhps mutation in 112 (79.43%) isolates, indicating potentially increased resistance to SP in Nigeria. The current study clearly reveals the emergence and spread of the highly prevalent quintuple Pfdhfr-Pfdhps mutant in Nigeria, highlighting the importance of the continuous and strategic surveillance of molecular markers and the efficacy of SP.
The current study was limited by the region-specific distribution of molecular markers because data on the region of origin of imported cases were not collected when the epidemiological investigation was conducted, according to national guidelines. Second, the small sample size limited further interpretation by year. However, our analysis still provides data on molecular markers associated with resistance to artemisinin, CQ, and SP, from the view of P. falciparum isolates imported from Nigeria.
In conclusion, we demonstrated the prevalences and patterns of mutations in the k13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes in P. falciparum isolates imported from Nigeria to Zhejiang Province, China, between 2016 and 2020. Limited polymorphism in the k13 gene was detected. The wild-type CVMNK haplotype of Pfcrt became predominant. A low frequency of the double mutant type (N86Y and Y184F) in Pfmdr1 was observed. The prevalences of the triple mutation (IRNDI) in Pfdhfr and the single mutation (SGKAA) in Pfdhps were the highest. We found a quintuple combination of Pfdhfr-Pfdhps mutations in eight samples, which was rarely described in Nigeria, indicating the continued emergence and spread of SP resistance. The current study demonstrated a high frequency of wild-type Pfcrt, limited polymorphism of Pfmdr1, and a high prevalence of Pfdhfr and Pfdhps mutants. In this work, we present a comprehensive set of molecular markers for drug resistance from a sample of imported cases. Based on our findings, it has become crucial to evaluate the impact of the emerging “fully resistant” type of Pfdhfr-Pfdhps as well as its combination with I431V on the efficacy of SP in Nigeria.
MATERIALS AND METHODS
Study site.
Zhejiang Province is located in eastern China. The land area of Zhejiang is 105,500 km2, and the local population size was 50.38 million in 2019. It is located in the center of the subtropical zone, with an average annual temperature of 15°C to 18°C and annual rainfall of 1,200 to 2,000 mm. Anopheles sinensis is reported to be the only species of mosquito to transmit malaria. Labor export and trading are common between Zhejiang and Nigeria, and many migrant workers and business people travel between the two places each year.
Sample collection and DNA extraction.
We investigated 165 P. falciparum infections imported from Nigeria to Zhejiang Province between January 2016 and December 2020. Individuals with malaria-related symptoms and positive results by microscopy or rapid diagnostic tests (RDTs) are reported by hospitals or clinics according to national guidelines on malaria diagnosis. The local Center for Disease Control and Prevention (CDC) carried out an epidemiological investigation for each patient, including a detailed travel history, to track parasite origins. Thick and thin blood smears and PCR were used by the Zhejiang Provincial CDC to double-check and confirm the species. Whole blood was collected from each case before antimalarial treatment. All blood samples were stored at −80°C until use. Genomic DNA was extracted using the QIAamp DNA minikit (Qiagen Inc., Hilden, Germany).
DNA amplification and sequencing.
Multiple molecular markers were detected, including the k13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes. k13, Pfmdr1, Pfdhfr, and Pfdhps were detected by nested PCR, and Pfcrt was detected by regular PCR, as previously described (37–40). Sequences for positions 72 to 76 of Pfcrt and Pfmdr1 haplotypes (codons 86 and 184), Pfdhfr haplotypes (codons 51, 59, 108, 139, and 164), and Pfdhps haplotypes (codons 431, 436, 437, 540, 581, and 613) were amplified. The primers used for PCR are listed in Table S1 in the supplemental material (37, 38). Proofreading polymerase was used for each reaction to prevent amplification errors, which was contained in the high-fidelity PCR master mix (Sangon Biotech Co. Ltd., Shanghai, China). The cycling conditions for the k13, Pfcrt, Pfdhfr, and Pfdhps genes were described in previous studies (39, 40). Amplification conditions for both PCR rounds for Pfmdr1 were as follows: 1 cycle at 95°C for 5 min; 30 cycles at 95°C for 1 min, 54°C for 90 s, and 72°C for 1 min; and 1 extension cycle at 72°C for 10 min. PCR products were sequenced by Sangon Biotech.
Data availability.
Mega version 7.0.26 (https://www.megasoftware.net/) was used to align amplicon sequences to reference sequences retrieved from the NCBI database. The GenBank accession numbers for reference sequences are XM_001350122.1 for k13, NC_004328.3 for Pfcrt, X56851.1 for Pfmdr1, NC_004318.2 for Pfdhfr, and XM_001349382.1 for Pfdhps. A database was constructed using Microsoft Excel 2017.
ACKNOWLEDGMENTS
We thank the physicians and staff of the prefecture and county CDC for their invaluable assistance with sample collection and epidemiological investigation.
We have no competing interests.
This work was funded by grants from the Major Health Science and Technology Projects in Zhejiang Province (grant number WKJ-ZJ-2119) and the Medical Research Program of Zhejiang Province (grant numbers 2020PY038 and 2022KY723).
Footnotes
Supplemental material is available online only.
Contributor Information
Wei Ruan, Email: wruan@cdc.zj.cn.
Feng Ling, Email: fengl@cdc.zj.cn.
Laura A. Kirkman, Weill Cornell Medicine
REFERENCES
- 1.World Health Organization. 2021. World malaria report. World Health Organization, Geneva, Switzerland. [Google Scholar]
- 2.World Health Organization. 2020. World malaria report. World Health Organization, Geneva, Switzerland. [Google Scholar]
- 3.Gbotosho GO, Happi CT, Ganiyu A, Ogundahunsi O, Sowunmi A, Oduola AM. 2009. Potential contribution of prescription practices to the emergence and spread of chloroquine resistance in south-west Nigeria: caution in the use of artemisinin combination therapy. Malar J 8:313. doi: 10.1186/1475-2875-8-313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ikegbunam MN, Nkonganyi CN, Thomas BN, Esimone CO, Velavan TP, Ojurongbe O. 2019. Analysis of Plasmodium falciparum Pfcrt and Pfmdr1 genes in parasite isolates from asymptomatic individuals in southeast Nigeria 11 years after withdrawal of chloroquine. Malar J 18:343. doi: 10.1186/s12936-019-2977-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Sowunmi A, Ntadom G, Akano K, Ibironke FO, Ayede AI, Agomo C, Folarin OA, Gbotosho GO, Happi C, Oguche S, Okafor HU, Meremikwu M, Agomo P, Ogala W, Watila I, Mokuolu O, Finomo F, Ebenebe JC, Jiya N, Ambe J, Wammanda R, Emechebe G, Oyibo W, Useh F, Aderoyeje T, Dokunmu TM, Alebiosu OT, Amoo S, Basorun OK, Wewe OA, Okafor C, Akpoborie O, Fatunmbi B, Adewoye EO, Ezeigwe NM, Oduola A. 2019. Declining responsiveness of childhood Plasmodium falciparum infections to artemisinin-based combination treatments ten years following deployment as first-line antimalarials in Nigeria. Infect Dis Poverty 8:69. doi: 10.1186/s40249-019-0577-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Umemmuo MU, Agboghoroma CO, Iregbu KC. 2020. The efficacy of intermittent preventive therapy in the eradication of peripheral and placental parasitemia in a malaria-endemic environment, as seen in a tertiary hospital in Abuja, Nigeria. Int J Gynaecol Obstet 148:338–343. doi: 10.1002/ijgo.13085. [DOI] [PubMed] [Google Scholar]
- 7.Oguike MC, Falade CO, Shu E, Enato IG, Watila I, Baba ES, Bruce J, Webster J, Hamade P, Meek S, Chandramohan D, Sutherland CJ, Warhurst D, Roper C. 2016. Molecular determinants of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum in Nigeria and the regional emergence of dhps 431V. Int J Parasitol Drugs Drug Resist 6:220–229. doi: 10.1016/j.ijpddr.2016.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Uwimana A, Legrand E, Stokes BH, Ndikumana J-LM, Warsame M, Umulisa N, Ngamije D, Munyaneza T, Mazarati J-B, Munguti K, Campagne P, Criscuolo A, Ariey F, Murindahabi M, Ringwald P, Fidock DA, Mbituyumuremyi A, Menard D. 2020. Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda. Nat Med 26:1602–1608. doi: 10.1038/s41591-020-1005-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Andrea E, Lehane AM, Jérôme C, Fidock DA. 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28:504–514. doi: 10.1016/j.pt.2012.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Gil JP, Krishna S. 2017. pfmdr1 (Plasmodium falciparum multidrug drug resistance gene 1): a pivotal factor in malaria resistance to artemisinin combination therapies. Expert Rev Anti Infect Ther 15:527–543. doi: 10.1080/14787210.2017.1313703. [DOI] [PubMed] [Google Scholar]
- 11.Calçada C, Silva M, Baptista V, Thathy V, Silva-Pedrosa R, Granja D, Ferreira PE, Gil JP, Fidock DA, Veiga MI. 2020. Expansion of a specific Plasmodium falciparum PfMDR1 haplotype in Southeast Asia with increased substrate transport. mBio 11:e02093-20. doi: 10.1128/mBio.02093-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ikegbunam M, Ojo JA, Kokou K, Morikwe U, Nworu C, Uba C, Esimone C, Velavan TP, Ojurongbe O. 2021. Absence of Plasmodium falciparum artemisinin resistance gene mutations eleven years after the adoption of artemisinin-based combination therapy in Nigeria. Malar J 20:434. doi: 10.1186/s12936-021-03968-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Tola M, Ajibola O, Idowu ET, Omidiji O, Awolola ST, Amambua-Ngwa A. 2020. Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria. BMC Res Notes 13:497. doi: 10.1186/s13104-020-05334-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Igbasi U, Oyibo W, Omilabu S, Quan H, Chen SB, Shen HM, Chen JH, Zhou XN. 2019. Kelch 13 propeller gene polymorphism among Plasmodium falciparum isolates in Lagos, Nigeria: molecular epidemiologic study. Trop Med Int Health 24:1011–1017. doi: 10.1111/tmi.13273. [DOI] [PubMed] [Google Scholar]
- 15.Aninagyei E, Tetteh CD, Oppong M, Boye A, Acheampong DO. 2020. Efficacy of artemether-lumefantrine on various Plasmodium falciparum Kelch 13 and Pfmdr1 genes isolated in Ghana. Parasite Epidemiol Control 11:e00190. doi: 10.1016/j.parepi.2020.e00190. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Aninagyei E, Duedu KO, Rufai T, Tetteh CD, Chandi MG, Ampomah P, Acheampong DO. 2020. Characterization of putative drug resistant biomarkers in Plasmodium falciparum isolated from Ghanaian blood donors. BMC Infect Dis 20:533. doi: 10.1186/s12879-020-05266-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.MalariaGEN Plasmodium falciparum Community Project. 2016. Genomic epidemiology of artemisinin resistant malaria. Elife 5:e08714. doi: 10.7554/eLife.08714. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Bray PG, Martin RE, Tilley L, Ward SA, Kirk K, Fidock DA. 2005. Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance. Mol Microbiol 56:323–333. doi: 10.1111/j.1365-2958.2005.04556.x. [DOI] [PubMed] [Google Scholar]
- 19.Zhou RM, Zhang HW, Yang CY, Liu Y, Zhao YL, Li SH, Qian D, Xu BL. 2016. Molecular mutation profile of pfcrt in Plasmodium falciparum isolates imported from Africa in Henan province. Malar J 15:265. doi: 10.1186/s12936-016-1306-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Lu F, Zhang M, Culleton RL, Xu S, Tang J, Zhou H, Zhu G, Gu Y, Zhang C, Liu Y, Wang W, Cao Y, Li J, He X, Cao J, Gao Q. 2017. Return of chloroquine sensitivity to Africa? Surveillance of African Plasmodium falciparum chloroquine resistance through malaria imported to China. Parasit Vectors 10:355. doi: 10.1186/s13071-017-2298-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Wang X, Mu J, Li G, Chen P, Guo X, Fu L, Chen L, Su X, Wellems TE. 2005. Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People’s Republic of China. Am J Trop Med Hyg 72:410–414. doi: 10.4269/ajtmh.2005.72.410. [DOI] [PubMed] [Google Scholar]
- 22.Temu EA, Kimani I, Tuno N, Kawada H, Minjas JN, Takagi M. 2006. Monitoring chloroquine resistance using Plasmodium falciparum parasites isolated from wild mosquitoes in Tanzania. Am J Trop Med Hyg 75:1182–1187. doi: 10.4269/ajtmh.2006.75.1182. [DOI] [PubMed] [Google Scholar]
- 23.Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjalamala FK, Takala SL, Taylor TE, Plowe CV. 2006. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med 355:1959–1966. doi: 10.1056/NEJMoa062032. [DOI] [PubMed] [Google Scholar]
- 24.Mwai L, Ochong E, Abdirahman A, Kiara SM, Ward S, Kokwaro G, Sasi P, Marsh K, Borrmann S, Mackinnon M, Nzila A. 2009. Chloroquine resistance before and after its withdrawal in Kenya. Malar J 8:106. doi: 10.1186/1475-2875-8-106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Oboh MA, Singh US, Antony HA, Ndiaye D, Badiane AS, Ali NA, Bharti PK, Das A. 2018. Molecular epidemiology and evolution of drug-resistant genes in the malaria parasite Plasmodium falciparum in southwestern Nigeria. Infect Genet Evol 66:222–228. doi: 10.1016/j.meegid.2018.10.007. [DOI] [PubMed] [Google Scholar]
- 26.Oladipo OO, Wellington OA, Sutherland CJ. 2015. Persistence of chloroquine-resistant haplotypes of Plasmodium falciparum in children with uncomplicated malaria in Lagos, Nigeria, four years after change of chloroquine as first-line antimalarial medicine. Diagn Pathol 10:41. doi: 10.1186/s13000-015-0276-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Adam R, Mukhtar MM, Abubakar UF, Damudi HA, Muhammad A, Ibrahim SS. 2021. Polymorphism analysis of pfmdr1 and pfcrt from Plasmodium falciparum isolates in northwestern Nigeria revealed the major markers associated with antimalarial resistance. Diseases 9:6. doi: 10.3390/diseases9010006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Dokunmu TM, Adjekukor CU, Yakubu OF, Bello AO, Adekoya JO, Akinola O, Amoo EO, Adebayo AH. 2019. Asymptomatic malaria infections and Pfmdr1 mutations in an endemic area of Nigeria. Malar J 18:218. doi: 10.1186/s12936-019-2833-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Quan H, Igbasi U, Oyibo W, Omilabu S, Chen S-B, Shen H-M, Okolie C, Chen J-H, Zhou X-N. 2020. High multiple mutations of Plasmodium falciparum-resistant genotypes to sulphadoxine-pyrimethamine in Lagos, Nigeria. Infect Dis Poverty 9:91. doi: 10.1186/s40249-020-00712-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Esu E, Tacoli C, Gai P, Berens-Riha N, Pritsch M, Loescher T, Meremikwu M. 2018. Prevalence of the Pfdhfr and Pfdhps mutations among asymptomatic pregnant women in southeast Nigeria. Parasitol Res 117:801–807. doi: 10.1007/s00436-018-5754-5. [DOI] [PubMed] [Google Scholar]
- 31.Xu C, Sun H, Wei Q, Li J, Xiao T, Kong X, Wang Y, Zhao G, Wang L, Liu G, Yan G, Huang B, Yin K. 2019. Mutation profile of pfdhfr and pfdhps in Plasmodium falciparum among returned Chinese migrant workers from Africa. Antimicrob Agents Chemother 63:e01927-18. doi: 10.1128/AAC.01927-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Chauvin P, Menard S, Iriart X, Nsango SE, Tchioffo MT, Abate L, Awono-Ambéné PH, Morlais I, Berry A. 2015. Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in pregnant women in Yaoundé, Cameroon: emergence of highly resistant pfdhfr/pfdhps alleles. J Antimicrob Chemother 70:2566–2571. doi: 10.1093/jac/dkv160. [DOI] [PubMed] [Google Scholar]
- 33.Chaturvedi R, Chhibber-Goel J, Verma I, Gopinathan S, Parvez S, Sharma A. 2021. Geographical spread and structural basis of sulfadoxine-pyrimethamine drug-resistant malaria parasites. Int J Parasitol 51:505–525. doi: 10.1016/j.ijpara.2020.12.011. [DOI] [PubMed] [Google Scholar]
- 34.Ruizendaal E, Tahita MC, Traoré-Coulibaly M, Tinto H, Schallig H, Mens PF. 2017. Presence of quintuple dhfr N51, C59, S108-dhps A437, K540 mutations in Plasmodium falciparum isolates from pregnant women and the general population in Nanoro, Burkina Faso. Mol Biochem Parasitol 217:13–15. doi: 10.1016/j.molbiopara.2017.08.003. [DOI] [PubMed] [Google Scholar]
- 35.Naidoo I, Roper C. 2013. Mapping ‘partially resistant’, ‘fully resistant’, and ‘super resistant’ malaria. Trends Parasitol 29:505–515. doi: 10.1016/j.pt.2013.08.002. [DOI] [PubMed] [Google Scholar]
- 36.Kayode AT, Ajogbasile FV, Akano K, Uwanibe JN, Oluniyi PE, Eromon PJ, Folarin OA, Sowunmi A, Wirth DF, Happi CT. 2021. Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique. Sci Rep 11:471. doi: 10.1038/s41598-020-80017-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Zhang T, Xu X, Jiang J, Yu C, Tian C, Li W. 2018. Surveillance of antimalarial resistance molecular markers in imported Plasmodium falciparum malaria cases in Anhui, China, 2012-2016. Am J Trop Med Hyg 98:1132–1136. doi: 10.4269/ajtmh.17-0864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Voumbo-Matoumona DF, Kouna LC, Madamet M, Maghendji-Nzondo S, Pradines B, Lekana-Douki JB. 2018. Prevalence of Plasmodium falciparum antimalarial drug resistance genes in southeastern Gabon from 2011 to 2014. Infect Drug Resist 11:1329–1338. doi: 10.2147/IDR.S160164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Wang X, Wei R, Zhou S, Huang F, Lu Q, Feng X, Yan H. 2020. Molecular surveillance of Pfcrt and k13 propeller polymorphisms of imported Plasmodium falciparum cases to Zhejiang Province, China between 2016 and 2018. Malar J 19:59. doi: 10.1186/s12936-020-3140-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Pearce RJ, Drakeley C, Chandramohan D, Mosha F, Roper C. 2003. Molecular determination of point mutation haplotypes in the dihydrofolate reductase and dihydropteroate synthase of Plasmodium falciparum in three districts of northern Tanzania. Antimicrob Agents Chemother 47:1347–1354. doi: 10.1128/AAC.47.4.1347-1354.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1, Table S2. Download spectrum.00528-22-s0001.pdf, PDF file, 0.1 MB (124.9KB, pdf)
Data Availability Statement
Mega version 7.0.26 (https://www.megasoftware.net/) was used to align amplicon sequences to reference sequences retrieved from the NCBI database. The GenBank accession numbers for reference sequences are XM_001350122.1 for k13, NC_004328.3 for Pfcrt, X56851.1 for Pfmdr1, NC_004318.2 for Pfdhfr, and XM_001349382.1 for Pfdhps. A database was constructed using Microsoft Excel 2017.