INTRODUCTION
Malaria continues to be a major health problem in Southeast Asia and Africa.1 Globally, approximately 8% of estimated cases are due to Plasmodium vivax in Africa, but it increases to 47% in Southeast Asia. In the recent years, a trend of increasing malaria prevalence and malaria morbidity in South and Southeast Asian region has also been witnessed. However, it is not entirely clear at this point if this trend is the result of failure of interventions or if other causes, such as changing environments and population movements are important cofactors.1,2 Furthermore, until recently, P. vivax malaria has received less attention than Plasmodium falciparum malaria. However, in recent years, several studies have documented the contribution of P. vivax to severe malaria including multiple organ dysfunctions in the Indian subcontinent.3,4
Qatar is situated in the Arabian Peninsula, with a total population of 2.6 million (2016 census according to the Ministry of Development Planning and Statistics, Qatar) and more than 88% of these are expatriates from the Indian subcontinent, North and East Africa, Southeast Asia and Middle East. Indigenous malaria transmission has been eliminated from Qatar but the risk of imported malaria still exists because of the large number of immigrant workers originating from the Indian subcontinent and Africa.5,6 The incidence of malaria, mostly due to P. vivax, has increased over the past decade in Qatar and the majority of cases seen in the period between August and October, which confirms the importation of infection by expatriates from their respective countries on their return from summer vacation.6,7
Antimalarial drug resistance is the most persistent problem confronting malaria control programs in many endemic countries.1,8 Moreover, the spread of chloroquine (CQ) and sulphadoxine–pyrimethamine (SP) drug resistance in P. vivax is becoming more widespread and has a significant public health implication in the control program.9–11 Currently, CQ followed by primaquine is the first-line regimen for the treatment of P. vivax malaria in Qatar.
Fansidar (SP), an antifolate drug combination, is one of the extensively used antimalarial drugs, after CQ resistance rise in P. falciparum, throughout the world because of its low cost and relative safety.12 SP has never been recommended for treatment of P. vivax malaria in Qatar; however, the coexistence of P. falciparum and P. vivax and misdiagnosis of Plasmodium species suggest that unnecessary treatment of P. vivax malaria with SP may be inadvertently led to the simultaneous selection of SP resistant P. vivax by inducing drug pressure on P. vivax isolates. Furthermore, in countries where the SP has been used intensively for P. falciparum malaria, resistance has also been appearing in P. vivax populations.13–18
Molecular epidemiology studies have suggested that P. vivax resistance to SP is associated with point mutations in Pvdhfr and Pvdhps genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), major proteins involved in the folate biosynthesis pathways.13,14 Four nonsynonymous mutations of Pvdhps at positions S382F/A, A437G, K540E, and A581G have been implicated, these are equivalent to known nonsynonymous mutations in P. falciparum dhps at positions S436F/A, A437G, K540E, and A581G, respectively.19–21 Among these mutations, the A383G single mutation, A383G A553G double mutation, and S383A A553G triple mutation were reported in many endemic countries.18 With regard to Pvdhfr, twenty nonsynonymous mutations have been described.14,17 Of these, mutations at codons 57, 58, 61, 117, and 173 described to be involved in antifolate resistance,13,22 which corresponded to 51, 59, 108, and 164 in P. falciparum.22 Similarly, five mutations in the Pvdhps at codon 382, 383, 512, 553, and 585 have also been suspected to be involved in SP resistance.19–22 However, it has been speculated that there may be a known innate resistance of P. vivax to sulphadoxine because of the steric hindrance caused by the V585 in Pvdhps to the binding site of sulphadoxine.23
The drug resistance to CQ in P. vivax has been reported from different malaria endemic regions.9 But, unlike in P. falciparum, the molecular mechanisms behind CQ resistance in P. vivax remain unclear. Nevertheless, mutations in K10 insertion in the Pvcrt-o gene have been identified as a possible molecular marker of CQ resistance in P. vivax.24 In addition, recently, a CQ mutant allele of P. vivax (Pvcrt-o) has shown an association with in vivo and in vitro drug susceptibility in malaria-endemic countries.25
With the increasing reports of severe manifestations because of P. vivax in South Asia region,2 and the rampant use of CQ and SP for the treatment of malaria in these countries, there is a need for monitoring antimalarial drug efficacy and drug resistance in Qatar for prompt management of imported P. vivax malaria as most of the immigrant workers come from the Indian subcontinent.5–7,26 Therefore, this study aimed to determine the distribution of mutant Pvdhfr, Pvdhps, and Pvcrt-o genes involved in resistance to different antimalarial drugs in P. vivax. The pattern of mutations present in this study will provide valuable molecular information on antifolate and CQ drug resistance, and that may be useful for epidemiological mapping of drug-resistant P. vivax malaria in Qatar.
MATERIALS AND METHODS
Study site, ethics, and sample collection.
The study was conducted in Doha, Qatar (25.3548° N and 51.1839° E). This study received ethical approval from the Institutional Review Board of WCM-Q and HMC (Protocol no. 14-00097). During January 2013–September 2016, on a written informed consent from adult and parents of minors, a total of 314 P. vivax malaria cases from 12 countries were enrolled in this study (Figure 1). Blood samples were collected from the patients with uncomplicated malaria (N = 314) attending the Hamad General Hospital, a premier nonprofit health care provider in Doha, Qatar.
A map showing the countries of origin of the patients from whom Plasmodium vivax samples were collected in Doha, Qatar.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 6; 10.4269/ajtmh.17-0436
A map showing the countries of origin of the patients from whom Plasmodium vivax samples were collected in Doha, Qatar.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 6; 10.4269/ajtmh.17-0436
A map showing the countries of origin of the patients from whom Plasmodium vivax samples were collected in Doha, Qatar.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 6; 10.4269/ajtmh.17-0436
All of the samples were screened for the Plasmodium infection by light microscopic examination of Giemsa-stained blood smears and subsequently confirmed by nested polymerase chain reaction (PCR).27 The patient’s sociodemographics and clinical parameters such as age, nationality, clinical history, parasitaemia, and hemoglobin were collated from a patient’s medical records. In addition, information on risk factors such as travel history and blood transfusion was also recorded on structured questionnaire.
DNA extraction and amplification of Pvdhfr, Pvdhps, and Pvcrt-o.
Genomic DNA was isolated using the QIAamp DNA blood mini kit following the manufacturer’s instructions (Qiagen, Valencia, CA). Target sequences of the Pvdhfr, Pvdhps, and Pvcrt-o genes harboring putative mutations associated with SP and CQ resistance were amplified and sequenced. The outer and nested primers and thermal cycling conditions adapted as previously described16,24,28 and are summarized in Table 1. The expected band sizes for Pvdhfr, Pvdhps, and Pvcrt were 785, 703, and 1194 bp, respectively.
Primer sequence and PCR conditions used for amplification of Plasmodium vivax drug resistance genes
| Genes | Primers | Sequence | PCR conditions | PCR amplified product (bp) | References | |
|---|---|---|---|---|---|---|
| Pvdhfr | PF | Outer | 5′-accgcaccagttgattcctac-3′ | 94°C × 10 minutes, 35 cycles of [94°C × 30 seconds, 58°C × 60 seconds, 72°C × 60 seconds], 72°C × 10 minutes | 1,200 | 16 |
| PR | 5′-tgttaaagctgaagtacacgag-3′ | |||||
| NF | Nested | 5′-atggaggacctttcagatgt-3′ | 94°C × 10 minutes, 30 cycles of [94°C × 30 seconds, 54°C × 60 seconds, 72°C × 60 seconds], 72°C × 10 minutes | 785 | ||
| NR | 5′-aacgcattgcagttctccga-3′ | |||||
| NF | Sequencing | 5′-atggaggacctttcagatgt-3′ | Purified and sequenced using Sanger method | – | ||
| NR | 5′-aacgcattgcagttctccga-3′ | |||||
| Pvdhps | PF | Outer | 5′-attccagagtataagcacagcacatttgag-3′ | 94°C × 10 minutes, 35 cycles of [94°C × 30 seconds, 58°C × 60 seconds, 72°C × 60 seconds], 72°C × 10 minutes | 1,463 | 28 |
| PR | 5′-ctaaggttgatgtatccttgtgagcacatc-3′ | |||||
| NF | Nested | 5′-aatggcaagtgatggggcgagcgtgattga-3′ | 94°C × 10 minutes, 30 cycles of [94°C × 30 seconds, 58°C × 60 seconds, 72°C × 60 seconds], 72°C × 10 minutes | 703 | ||
| NR | 5′-cagtctgcactccccgatggccgcgccacc-3′ | |||||
| NF | Sequencing | 5′-aatggcaagtgatggggcgagcgtgattga-3′ | Purified and sequenced using Sanger method | – | ||
| NR | 5′-cagtctgcactccccgatggccgcgccacc-3′ | |||||
| Pvcrt | PF | Outer | 5′-cgctgtcgaagagcc-3′ | 94°C × 10 minutes, 35 cycles of [94°C × 60 seconds, 54°C × 60 seconds, 72°C × 60 seconds], 72°C × 10 minutes | 1,194 | 24 |
| PR | 5′-agtttccctctacacccg-3′ | |||||
| NF | Sequencing | 5′-cgctgtcgaagagcc-3′ | Purified and sequenced using Sanger method | – | ||
| NR | 5′-agtttccctctacacccg-3′ | |||||
NF = nested forward; NR = nested reverse; PCR = polymerase chain reaction; PF = primary forward; PR = primary reverse.
Sequencing and data analysis.
Amplified secondary PCR products of Pvdhfr, Pvdhps, and Pvcrt genes were purified using the QIAquick PCR Purification Kit according to manufacturer’s instructions (Qiagen). The sequencing reaction for all three genes was performed in the forward and reverse directions using respected nested PCR primers. Sanger sequencing was performed (Genewiz Inc., South Plainfield, NJ) and sequence analysis was performed using Leaser gene (SeqMan™ Version 7.0; DNASTAR, Madison, WI) and Geneious version R10 (www.geneious.com). Sequences of a P. vivax crt-o isolate (GenBank: EU333972.1), PPPK-dhps isolate (GenBank: EU478871.1), and dhfr-TS isolate (GenBank: EU478858.1) were used as references for studying polymorphisms in Pvcrt, Pvdhps, and Pvdhfr, respectively.
Statistical analysis.
A descriptive analysis was performed and estimates of prevalence along with their 95% confidence intervals (CI) were obtained (Table 2).
Mutations analysis of Pvcrt, Pvdhfr, and Pvdhps genes in study populations
| Gene | Mutation type | India | Nepal | Pakistan | Sudan | Rest of Africa | Total | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N (%) | 95% CI | N (%) | 95% CI | N (%) | 95% CI | N (%) | 95% CI | N (%) | 95% CI | N (%) | 95% CI | ||
| Pvdhfr | 117N | 3 (2.52) | (0.52–7.13) | – | – | 22 (27.5) | (18.10–38.62) | 1 (3.33) | (0.08–17.21) | 1 (9.09) | (0.23–41.28) | 27 (10.93) | (7.33–15.50) |
| 58R | 1 (0.84) | (0.02–4.56) | – | – | 2 (2.5) | (0.30–8.74) | 3 (10) | (2.11–26.53) | – | – | 6 (2.43) | (0.90–5.21) | |
| 58R, 117N | 59 (49.58) | (39.93–58.45) | 4 (57.14) | (18.41–90.10) | 21 (26.25) | (17.04–37.29) | 9 (30) | (14.73–49.40) | 7 (63.64) | (30.79–89.07) | 100 (40.49) | (34.31–46.89) | |
| Wild | 56 (47.06) | (37.51–56.00) | 3 (42.86) | (9.90–81.59) | 35 (43.75) | (32.67–55.30) | 17 (56.67) | (37.43–74.54) | 3 (27.27) | (6.02–60.97) | 114 (46.15) | (39.81–52.59) | |
| Pvdhps | 383G | 10 (7.41) | (3.61–13.20) | 2 (18.18) | (2.28–51.78) | 9 (9.28) | (4.33–16.88) | 7 (22.58) | (9.59–41.10) | 2 (10.00) | (1.23–31.70 | 30 (10.20) | (6.99–14.25) |
| 383G, 553G | 28 (20.74) | (14.25–28.56) | 3 (27.27) | (6.02–60.97) | 2 (2.06) | (0.25–7.25) | 2 (6.45) | (0.79–21.42) | 2 (10.00) | (1.23–31.70 | 37 (12.59) | (9.02–16.93) | |
| Wild | 97 (71.85) | (63.47–79.25) | 6 (54.55) | (23.38–83.25) | 86 (88.66) | (80.61–94.20) | 22 (70.97) | (51.96–85.78) | 16 (80.00) | (56.34–94.27) | 227 (77.21) | (71.98–81.88) | |
| Pvcrt | Mutant | 11 (8.33) | (4.23–14.42) | 1 (10.00) | (0.25–44.50) | 13 (16.46) | (9.06–26.50) | 1 (3.45) | (0.09–17.76) | – | – | 26 (9.96) | (6.61–14.25) |
| Wild | 121 (91.67) | (85.58–95.77) | 9 (90.00) | (55.50–99.74) | 66 (83.54) | (73.51–90.94) | 28 (96.55) | (82.24–99.91) | 11 (100.00) | – | 235 (90.04) | (85.75–93.39) | |
CI = confidence interval; N = number.
RESULTS
Demographic characteristics of the study population.
A total of 314 blood samples were collected from patients with microscopically confirmed P. vivax in HMC hospital, Doha. The age range was 3–73 years with a mean age of 32.1 years (SD = 11.9). The majority of patients were from India (46.5%, N = 146) with the rest belonging to Pakistan (32.5%, N = 102), Sudan (10.8%, N = 34), Africa (6%, N = 19), Nepal (3.8%, N = 12) and Canada (N = 1, 0.3%) who had a travel history of India (Figure 1). All patients had a history of travel to their country of origin ranged between 1 day and 13 months, but none of them had blood transfusion history.
Mutations and haplotypes in P. vivax dhfr, dhps, and crt-o gene.
For Pvdhfr, Pvdhps, and Pvcrt-o, 247 (78.7%), 294 (93.6%), and 261 (83.1%) P. vivax isolates, respectively were successfully amplified and sequenced.
With regard to Pvdhfr, 46.2% (N = 114) contained the wild-type Pvdhfr genotype ANSSI (codons 15, 50, 58, 117, and 173) and 53.8% (N = 133) of samples possessed mutation at codons (58R and 117N) (Table 2). Of 53.8%, Pvdhfr double mutation, 58R/117N, was observed in 40.5% (N = 100) cases of P. vivax infection (95% CI: 34.31–46.89%) with 10.9% (N = 27) single mutant at codon 117N (95% CI: 7.33–15.50%) followed by 2.4% (N = 6) at codon 58R (95% CI: 0.90–5.21%) but no case exhibited mutation at codon 15, 50, and 173 of Pvdhfr (Table 2). The haplotype analysis of Pvdhfr revealed four distinct allelic forms from all samples except for samples originating from Nepal, where only two haplotype (wild type and double mutant) was recorded (Table 2).
With respect to the Pvdhps gene, 294 (92.4%) samples were successfully sequenced and the majority (77.1%, N = 227) of the isolates had a wild type Pvdhps allele SAKAV (codons 382, 383, 512, 553, and 585). Of the remaining isolates, 10.2% (N = 30) harbored single mutation at codon 383G (95% CI: 6.99–14.25%) and the frequency distribution of double mutant alleles (383G/553G) was 12.6% (N = 37) (95% CI: 9.02–16.93%) (Table 2). Furthermore, we detected three haplotypes in our populations and single mutant haplotype (383G) as well as double mutant haplotype (383G/553G) found from all over the country (Table 2).
In addition, 11 different haplotypes were found, when we grouped Pvdhfr and Pvdhps genes. The majority of the isolates showed the wild type (40.0%) followed by double mutant alleles (Figure 2). Triple (8.0%) and quadruple (10.0%) mutations were also detected in this study, especially among samples collected from patients of Indian origin (Table 3).
Prevalence of grouped Pvdhfr/Pvdhps mutant alleles in Plasmodium vivax linked to sulphadoxine-pyrimethamine treatment failure.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 6; 10.4269/ajtmh.17-0436
Prevalence of grouped Pvdhfr/Pvdhps mutant alleles in Plasmodium vivax linked to sulphadoxine-pyrimethamine treatment failure.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 6; 10.4269/ajtmh.17-0436
Prevalence of grouped Pvdhfr/Pvdhps mutant alleles in Plasmodium vivax linked to sulphadoxine-pyrimethamine treatment failure.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 6; 10.4269/ajtmh.17-0436
Different haplotypes of Pvdhfr and Pvdhps genes from the different countries
| Gene | Mutation type | India (N = 120) | Nepal (N = 9) | Pakistan (N = 80) | Sudan (N = 31) | Rest of Africa (N = 11) |
|---|---|---|---|---|---|---|
| Pvdhfr | 117N | 1 (0.83) | 0.00 | 18 (22.50) | 1 (3.23) | 0 |
| 58R | 1 (0.83) | 0.00 | 1 (1.25) | 3 (9.68) | 0 | |
| 58R, 117N | 32 (26.67) | 1 (11.11) | 19 (23.75) | 2 (6.45) | 4 (36.36) | |
| Pvdhps | 383G | 4 (3.33) | 0.00 | 2 (2.50) | 0.00 | 0.00 |
| 383G, 553G | 5 (4.17) | 2 (22.22) | 2 (2.50) | 2 (6.45) | 0.00 | |
| Pvdhfr + Pvdhps | 117N + 383G | 1 (0.83) | 0.00 | 4 (5.00) | 0.00 | 0.00 |
| 58R + 383G | 0.00 | 0.00 | 1 (1.25) | 0.00 | 0.00 | |
| 117N + 383G, 553G | 1 0.83 | 0.00 | 0.00 | 0.00 | 1 (9.09) | |
| 58R, 117N + 383G | 5 (4.17) | 2 (22.22) | 2 (2.50) | 7 (22.58) | 2 (18.18) | |
| 58R, 117N + 383G, 553G | 22 (18.33) | 1 (11.11) | 0.00 | 0.00 | 1 (9.09) | |
| Wild | 48 (40.00) | 3 (33.33) | 31 (38.75) | 16 (51.61) | 3 (27.27) | |
| Total | 120 (100.00) | 9 (100.00) | 80 (100.00) | 31 (100.00) | 11 (100) |
N = number.
The Pvcrt-o gene was successfully sequenced in 251 (78.9%) isolates. The majority of isolates (90.0%, N = 235) carried wild type, without K10 insertion (95% CI: 86.22–95.52%) (Table 2). Of note, synonymous mutations or K10 insertions (AAG) were found in 11.3% (N = 24/212) of isolates from India and Pakistan (95% CI: 6.48–13.78%) and was absent in P. vivax parasites from Africa, except one (3.45%) patient from Sudan.
DISCUSSION
The molecular markers associated to antimalarial drug resistance are valuable tools for the surveillance of resistant malarial parasites. They are useful to anticipate risks associated with the emergence, the spread of antimalarial parasite resistance, and to alert policy makers. The associations of point mutations in the Pvdhfr and Pvdhps genes have been well established with antifolate drug resistance.29,30 A number of in vitro and in vivo studies have also suggested that these molecular markers provide valuable information about the trends of SP resistance in P. vivax.15,31,32 Furthermore, in Pvdhfr gene, it has been well established that mutations at S58R and S117N arise first and increases resistance more than wild type under drug pressure.13–15,33
In the present study, high prevalence (54%) of both Pvdhfr 58R and 117N mutation was observed. However, mutations at codons I13L, P33L, F57L, T61M, I172V, and I173L, which have been reported earlier from different regions,16,34–38 were not found in our studied population. Furthermore, delayed parasite clearance and treatment failure of SP has been associated with double (N117/R58), triple (N117/R58/L57), and quadruple mutations (L57/R58/M61/T11).13,15,22,34–38 In this study, among the mutants identified, the Pvdhfr double mutant at positions 58R and 117N were predominant (40%), suggesting the drug pressure on these parasites. Our observation corroborates the previous reports from Pakistan, Nepal, and Sudan.39–41 However, no triple/quadruple mutations were detected, which is in contrast with previous findings from India16,36 as India has different degrees of malaria endemicity, and the levels of mutations from the different geographical state is also varied.42
With regard to the Pvdhps gene, majority of our isolates (77.2%) carried wild type alleles and frequency of single mutant (383G) and double mutant genotypes (383G/553G) was 10.2% and 12.6%, respectively, which have been found to be directly related to sulphadoxine resistance43 and associated with reduced sensitivity to both sulfa drugs and sulfones.23,28 In addition, the association of SP treatment failure has also been reported with Pvdhps 553G/383G in combination with Pvdhfr double mutation 58R/117N and increasing prevalence of these mutations may further reduce the sensitivity to antifolate drugs in P. vivax population.44,45 The multiple mutation pattern (combination of 58, 117 + 383 triple mutant) was found from all over the country, although it was in low level. However, the other triple (117 + 383, 553) and quadruple (58, 117 + 383, 553) combinations were observed mainly in the patients originating from India. Such multiple mutations pattern was also reported previously from India.34
The association of molecular markers i.e., Pfcrt and Pfmdr1 with the CQ drug resistance in P. falciparum malaria has been well documented; however, little is known about the possible mechanisms of CQ resistance (Pvmdr1 and Pvcrt-o genes) in P. vivax malaria.46,47 In the present study, wild type allele was found to be predominant and K10 insertions (AAG) was found in 11.2% of the Indian subcontinent samples, suggesting commencement of a trend toward decreased CQ sensitivity in these countries.
Overall, in the present study, a large proportion of the isolates harbored wild-type genotype of Pvdhps and Pvcrt-o genes. However, P. vivax parasites carried the same frequency of wild-type Pvdhfr allele (46.5%) and double mutations at 58R/117N (53.8%), suggesting that both the alleles are still common in the Indian subcontinent and Africa and could cause structural changes in Pvdhfr and lead to decreased binding to pyrimethamine.48,49 Nevertheless, it has been shown that P. vivax isolates with multiple mutations in Pvdhfr and Pvdhps can respond adequately to SP.28 Thus, in the present study, the absence of triple/quadruple mutations strongly suggests that imported P. vivax infection in Qatar can respond adequately to antifolate drugs. However, the presence of double mutations in Pvdhfr and Pvdhps genes indicates the regular monitoring is essential for malaria treatment point of view. The limitation of the present study was that we were unable to perform the therapeutic efficacy study; hence, couldn’t correlate the molecular findings with treatment outcome and in vitro drug sensitivity. Therefore, in future, a therapeutic efficacy study should be performed to correlate genotypic observations with phenotypic data.
The current guidelines for treatment of malaria at HMC include administration of chloroquine, followed by primaquine for patients with P. vivax malaria and coartem or quinine plus doxycycline (adults)/or clindamycine (children) for patients with P. falciparum malaria. In addition, those with mixed infections were usually treated as P. falciparum malaria.50 In the present study, all the patients were from the Indian subcontinent and Africa, where SP alone has never been recommended for the treatment of P. vivax malaria but artesunate + SP combination is used for treatment of P. falciparum malaria in some African countries.1 The incidence of P. falciparum/P. vivax co-infection is most common in malaria endemic countries but because of a wrong diagnosis and extensive use of SP to treat P. falciparum malaria patients,16 P. vivax population often exposed accidentally to SP, resulting in a selection pressure which might be reflected in the observed mutation pattern in the Pvdhfr and Pvdhps genes in our parasite populations.
Plasmodium vivax may cause relapse within a few weeks to a few months after initial infection.51 As reported indigenous malaria transmission has been eliminated from Qatar, the patients who were positive for P. vivax and did not have a recent travel history may likely be the cases of relapses.
In conclusion, this was the first comprehensive molecular study carried out in Qatar focusing on mutation in Pvdhfr, Pvdhps, and Pvcrt-o genes that were strongly associated with SP and CQ resistance. This study shows relatively low distribution of these mutations associated with antifolate and chloroquine resistance, which suggest that the P. vivax parasites may still be susceptible to SP and CQ and any combination of antimalarial drug with antifolates that have been recommended for P. falciparum might be effective in P. vivax malaria also. However, the presence of high prevalence of double mutation in the Pvdhfr gene demonstrates a need of continuous surveillance studies. The development and spread of drug-resistant parasite strains is a major obstacle to the malaria control and elimination program. Therefore, the results of the present study provide baseline data on the extent of SP drug resistance in P. vivax, which might be helpful for the enrichment of molecular surveillance of antimalarial resistance and will be useful for developing and updating antimalarial guidance for imported cases in Qatar.
Acknowledgments:
We would like to thank Musaed Saad Al Samawi, Peter Cameroon, and Paramedical staff of the Emergency Department, HMC, Doha, Qatar, for helping in sample collection.
REFERENCES
- 1.↑
World Health Organization, 2016. World Malaria Report. Available at: http://www.who.int/malaria/publications/world-malaria-report-2016/report/en/. Accessed February 2, 2017.
- 2.↑
Behrens RH et al. 2010. The incidence of malaria in travellers to south-east Asia: is local malaria transmission a useful risk indicator? Malar J 9: 266.
- 3.↑
Naha K, Dasari S, Prabhu M, 2012. Spectrum of complications associated with Plasmodium vivax infection in a tertiary hospital in south-western India. Asian Pac J Trop Med 5: 79–82.
- 4.↑
Zubairi AB, Nizami S, Raza A, Mehraj V, Rasheed AF, Ghanchi NK, Khaled ZN, Beg MA, 2013. Severe Plasmodium vivax malaria in Pakistan. Emerg Infect Dis 19: 1851–1854.
- 5.↑
Beljaev AE, 2000. The malaria situation in the WHO eastern Mediterranean region. Med Parazitol (Mosk) 2: 12–15.
- 7.↑
Khan FY, Lutof AK, Yassin MA, Khattab MA, Saleh M, Rezeq HY, Almaslamani M, 2009. Imported malaria in Qatar: a one year hospital-based study in 2005. Travel Med Infect Dis 7: 111–117.
- 8.↑
Verlinden BK, Louw A, Birkholtz LM, 2016. Resisting resistance: is there a solution for malaria? Expert Opin Drug Discov 11: 395–406.
- 9.↑
Price RN, von Seidlein L, Valecha N, Nosten F, Baird JK, White NJ, 2014. Global extent of chloroquine-resistant Plasmodium vivax: a systematic review and meta-analysis. Lancet Infect Dis 14: 982–991.
- 10.
Ferreira MU, Castro MC, 2016. Challenges for malaria elimination in Brazil. Malar J 15: 284.
- 11.↑
Ghanchi NK, Shakoor S, Thaver AM, Khan MS, Janjua A, Beg MA, 2016. Current situation and challenges in implementing Malaria control strategies in Pakistan. Crit Rev Microbiol 42: 588–593.
- 12.↑
WHO, 2004. Drug Resistance in Malaria. Available at: http://www.who.int/csr/resources/publications/drugresist/malaria.pdf. Accessed March 7, 2017.
- 13.↑
Imwong M, Pukrittakayamee S, Looareesuwan S, Pasvol G, Poirreiz J, White NJ, Snounou G, 2001. Association of genetic mutations in Plasmodium vivax dhfr with resistance to sulfadoxine-pyrimethamine: geographical and clinical correlates. Antimicrob Agents Chemother 45: 3122–3127.
- 14.↑
Imwong M, Pukrittayakamee S, Rénia L, Letourneur F, Charlieu JP, Leartsakulpanich U, Looareesuwan S, White NJ, Snounou G, 2003. Novel point mutations in the dihydrofolate reductase gene of Plasmodium vivax: evidence for sequential selection by drug pressure. Antimicrob Agents Chemother 47: 1514–1521.
- 15.↑
Hastings MD, Porter KM, Maguire JD, Susanti I, Kania W, Bangs MJ, Sibley CH, Baird JK, 2004. Dihydrofolate reductase mutations in Plasmodium vivax from Indonesia and therapeutic response to sulfadoxine plus pyrimethamine. J Infect Dis 189: 744–750.
- 16.↑
Alam MT et al. 2007. Similar trends of pyrimethamine resistance-associated mutations in Plasmodium vivax and P. falciparum. Antimicrob Agents Chemother 51: 857–863.
- 17.↑
Hawkins VN et al. 2008. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase. Malar J 7: 72.
- 18.↑
Saralamba N, Nakeesathit S, Mayxay M, Newton PN, Osorio L, Kim JR, White NJ, Day NP, Dondorp AM, Imwong M, 2016. Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia. Malar J 15: 484.
- 19.↑
Imwong M, Sudimack D, Pukrittayakamee S, Osorio L, Carlton JM, Day NP, White NJ, Anderson TJ, 2006. Microsatellite variation, repeat array length, and population history of Plasmodium vivax. Mol Biol Evol 23: 1016–1018.
- 20.
Triglia T, Cowman AF, 1994. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc Natl Acad Sci USA 91: 7149–7153.
- 21.↑
Hawkins VN, Suzuki SM, Rungsihirunrat K, Hapuarachchi HC, Maestre A, Na-Bangchang K, Sibley CH, 2009. Assessment of the origins and spread of putative resistance-conferring mutations in Plasmodium vivax dihydropteroate synthase. Am J Trop Med Hyg 81: 348–355.
- 22.↑
Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH, 2007. Antifolates can have a role in the treatment of Plasmodium vivax. Trends Parasitol 23: 213–222.
- 23.↑
Korsinczky M, Fischer K, Chen N, Baker J, Rieckmann K, Cheng Q, 2004. Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site. Antimicrob Agents Chemother 48: 2214–2222.
- 24.↑
Suwanarusk R et al. 2007. Chloroquine resistant Plasmodium vivax: in vitro characterisation and association with molecular polymorphisms. PLoS One 2: e1089.
- 25.↑
Nyunt MH, Han JH, Wang B, Aye KM, Aye KH, Lee SK, Htut Y, Kyaw MP, Han KT, Han ET, 2017. Clinical and molecular surveillance of drug resistant vivax malaria in Myanmar (2009–2016). Malar J 16: 117.
- 27.↑
Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA, 1999. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg 60: 687–692.
- 28.↑
Imwong M, Pukrittayakamee S, Cheng Q, Moore C, Looareesuwan S, Snounou G, White NJ, Day NP, 2005. Limited polymorphism in the dihydropteroate synthetase gene (dhps) of Plasmodium vivax isolates from Thailand. Antimicrob Agents Chemother 49: 4393–4395.
- 29.↑
Marfurt J et al. 2008. Molecular markers of in vivo Plasmodium vivax resistance to amodiaquine plus sulfadoxine-pyrimethamine: mutations in pvdhfr and pvmdr1. J Infect Dis 198: 409–417.
- 30.↑
Sá JM, Nomura T, Neves Jd, Baird JK, Wellems TE, del Portillo HA, 2005. Plasmodium vivax: allele variants of the mdr1 gene do not associate with chloroquine resistance among isolates from Brazil, Papua, and monkey-adapted strains. Exp Parasitol 109: 256–259.
- 31.↑
Hastings MD, Maguire JD, Bangs MJ, Zimmerman PA, Reeder JC, Baird JK, Sibley CH, 2005. Novel Plasmodium vivax dhfr alleles from the Indonesian archipelago and Papua New Guinea: association with pyrimethamine resistance determined by a Saccharomyces cerevisiae expression system. Antimicrob Agents Chemother 49: 733–740.
- 32.↑
Rungsihirunrat K, Na-Bangchang K, Hawkins VN, Mungthin M, Sibley CH, 2007. Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from Thailand. Am J Trop Med Hyg 76: 1057–1065.
- 33.↑
Zakeri S, Motmaen SR, Afsharpad M, Djadid ND, 2009. Molecular characterization of antifolates resistance-associated genes, (dhfr and dhps) in Plasmodium vivax isolates from the Middle East. Malar J 8: 20.
- 34.↑
Kaur S, Prajapati SK, Kalyanaraman K, Mohmmed A, Joshi H, Chauhan VS, 2006. Plasmodium vivax dihydrofolate reductase point mutations from the Indian subcontinent. Acta Trop 97: 174–180.
- 35.
Valecha N, Joshi H, Eapen A, Ravinderan J, Kumar A, Prajapati SK, Ringwald P, 2006. Therapeutic efficacy of chloroquine in Plasmodium vivax from areas with different epidemiological patterns in India and their Pvdhfr gene mutation pattern. Trans R Soc Trop Med Hyg 100: 831–837.
- 36.↑
Prajapati SK, Joshi H, Dev V, Dua VK, 2011. Molecular epidemiology of Plasmodium vivax anti-folate resistance in India. Malar J 10: 102.
- 37.
Khatoon L, Baliraine FN, Bonizzoni M, Malik SA, Yan G, 2009. Prevalence of antimalarial drug resistance mutations in Plasmodium vivax and P. falciparum from a malaria-endemic area of Pakistan. Am J Trop Med Hyg 81: 525–528.
- 38.↑
Schmider N, Peyerl-Hoffmann G, Restrepo M, Jelinek T, 2003. Short communication: point mutations in the dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Colombia. Trop Med Int Health 8: 129–132.
- 39.↑
Khattak AA, Venkatesan M, Khatoon L, Ouattara A, Kenefic LJ, Nadeem MF, Nighat F, Malik SA, Plowe CV, 2013. Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan. Malar J 12: 310.
- 40.
Ranjitkar S, Schousboe ML, Thomsen TT, Adhikari M, Kapel CM, Bygbjerg IC, Alifrangis M, 2011. Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal. Malar J 10: 75.
- 41.↑
Pirahmadi S, Talha BA, Nour BY, Zakeri S, 2014. Prevalence of mutations in the antifolates resistance-associated genes (dhfr and dhps) in Plasmodium vivax parasites from Eastern and Central Sudan. Infect Genet Evol 26: 153–159.
- 42.↑
Mishra N et al. 2016. Monitoring the efficacy of antimalarial medicines in India via sentinel sites: outcomes and risk factors for treatment failure. J Vector Borne Dis 53: 168–178.
- 43.↑
Lu F et al. 2010. Mutations in the antifolate-resistance-associated genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium vivax isolates from malaria-endemic countries. Am J Trop Med Hyg 83: 474–479.
- 44.↑
Tjitra E, Baker J, Suprianto S, Cheng Q, Anstey NM, 2002. Therapeutic efficacies of artesunate-sulfadoxine-pyrimethamine and chloroquine-sulfadoxine-pyrimethamine in vivax malaria pilot studies: relationship to Plasmodium vivax dhfr mutations. Antimicrob Agents Chemother 46: 3947–3953.
- 45.↑
Zakeri S, Afsharpad M, Ghasemi F, Raeisi A, Safi N, Butt W, Atta H, Djadid ND, 2010. Molecular surveillance of Plasmodium vivax dhfr and dhps mutations in isolates from Afghanistan. Malar J 9: 75.
- 46.↑
Srivastava HC, Yadav RS, Joshi H, Valecha N, Mallick PK, Prajapati SK, Dash AP, 2008. Therapeutic responses of Plasmodium vivax and P. falciparum to chloroquine, in an area of western India where P. vivax predominates. Ann Trop Med Parasitol 102: 471–480.
- 47.↑
Singh RK, 2000. Emergence of chloroquine-resistant vivax malaria in south Bihar (India). Trans R Soc Trop Med Hyg 94: 327.
- 48.↑
Kongsaeree P, Khongsuk P, Leartsakulpanich U, Chitnumsub P, Tarnchompoo B, Walkinshaw MD, Yuthavong Y, 2005. Crystal structure of dihydrofolate reductase from Plasmodium vivax: pyrimethamine displacement linked with mutation-induced resistance. Proc Natl Acad Sci USA 102: 13046–13051.
- 49.↑
Mokmak W, Chunsrivirot S, Hannongbua S, Yuthavong Y, Tongsima S, Kamchonwongpaisan S, 2014. Molecular dynamics of interactions between rigid and flexible antifolates and dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium falciparum. Chem Biol Drug Des 84: 450–461.
- 50.↑
Khan FY, Elshafie SS, Almaslamani M, Abu-Khattab M, El Hiday AH, Errayes M, Almaslamani E, 2010. Epidemiology of bacteraemia in Hamad general hospital, Qatar: a one year hospital-based study. Travel Med Infect Dis 8: 377–387.