Surveillance of Antimalarial Resistance Pfcrt, Pfmdr1, and Pfkelch13 Polymorphisms in African Plasmodium falciparum imported to Shandong Province, China

Introduction

Malaria is one of the most important parasitic diseases presented in many countries that causing a serious global public health problem. There were approximately 214 million malaria cases and 438,000 deaths worldwide in 2015, of which approximately 88% occurred in Africa¹. P. falciparum, the most threatening among five species of malaria parasites associated with human infection, has been mainly responsible for the majority of the morbidity and mortality around the world². During the past decades, drug resistance of P. falciparum has become a critical obstacle to control and eliminate malaria. Long-term anti-malarial monotherapy is capable of causing the emergence of drug resistant parasite strains and may provide a chance to spread the resistance to sensitive parasite population in other epidemic areas under conditions suitable Anopheles mosquitoes for transmission³. Since the early 1940s, chloroquine (CQ) was continuously used for treatment of malaria in many countries, which has been confirmed one of the most important antimalarial drugs with quick metabolism, good curative effects, and low prices⁴. However, due to excessive drug usage over the years, the CQ resistance (CQR) of P. falciparum isolates was found initial emerging from Thailand-Cambodia border in Southeast Asia in 1957 and Venezuela-Colombia border in Northern South America in 1959^5,6, and eventually spread to other countries around the world. In 2006, the World Health Organization (WHO) recommended artemisinin-based combination therapies (ACTs) as the first-line treatment for uncomplicated P. falciparum⁷. ACTs serve as an important therapeutic method to avoid or defer the development of drug resistance for P. falciparum infection. Unfortunately, P. falciparum resistance to artemisinin (ART), the cornerstone of ACTs, was emergence in western Cambodia and subsequently spreading across several neighboring countries in Greater Mekong Subregion (GMS) of Southeast Asia in recent years^8–13. Moreover, the emergence of ART resistant indigenous isolates in Africa has been reported more recently, which would be anxious in the future since the suitable replacement drugs are limited¹⁴. Thus, it is urgent to monitor the drug resistant trend of P. falciparum, so as to assess the possibility of reintroducing conventional drugs and also attempt to block the emergence of potential large-scale ART resistant transmission.

Molecular marker detection for parasite’s drug resistance is one of several methods for the surveillance of resistant prevalence and antimalarial efficacy¹⁵. The single nucleotide polymorphisms (SNPs) at codons 72, 74, 75 and 76 of P. falciparum chloroquine resistance transporter gene (Pfcrt), and 86, 184, 1034, 1042, and 1246 of P. falciparum multidrug resistance 1 gene (Pfmdr1) have been shown to be associated with parasite’s CQR^16,17. Moreover, Pfkelch13, a gene locating on chromosome 13 of P. falciparum and encoding K13-propeller protein, was identified playing a vital role in conferring ART resistance through whole genome sequencing of ART resistance isolate and vitro ring-stage survival assays (RSA_0–3h)¹⁸. To date, 13 nonsynonymous mutations (P441L, F446I, G449A, N458Y, Y493H, R539T, I543T, P553L, R561H, V568G, P574L, C580Y, A675V) on Pfkelch13 gene of P. falciparum have been reported to be associated with clinical ART resistance that occurred in Southeast Asia¹⁹. Among them, Y493H, R539T, I543T, and C580Y were validated ART-resistant mutations, whereas the rest were candidate resistance mutations²⁰.

In China, owing to substantial efforts for strategies and intervention over the past decades, the country has achieved a great success in controlling malaria transmission, with morbidity and mortality dramatically reduced to low levels²¹. In 2010, the Chinese Government initiated the National Malaria Elimination Program (NMEP), in order to eliminate malaria nationwide by the year 2020²². Currently, indigenous malaria parasite was almost absented in majority of regions in China other than some local transmission still occurred in Yunnan Province and Tibet Autonomous Region²³. However, due to the intensive commercial intercourse, travelling and migrant laborers, a markedly rise of imported cases in recent years has posed a severe threat to eradicate malaria²⁴. There has been no indigenous malaria patient reported in Shandong Province of China since 2012, whereas the imported cases have increased gradually, especially P. falciparum coming back from Africa was predominant²⁵. Since little is known about the molecular basis of drug resistance of imported P. falciparum in Shandong Province, we investigated polymorphisms and haplotypes distribution of Pfcrt and Pfmdr1, and mutations in Pfkelch13 of P. falciparum isolates returned from Africa in Shandong between 2012 and 2015, in order to accumulate and update baseline data for molecular surveillance linked to antimalarial drug resistance in China.

Results

Discussion

In response to uncomplicated P. falciparum infections, ACTs including oral compound tablets of dihydroartemisinin plus piperaquine (DHA + PQ), artesunate plus amodiaquine (AS + AQ), and ART + PQ were routine regimen for the current treatment in China²⁷. The widespread resistance to CQ, AQ, and the emergence of ART resistant isolates in Africa could attract China’s attention since the resistant parasites would enter the country with increasing migrants. Therefore, understanding the molecular mutation profiles and geographical distribution of drug resistance of P. falciparum is urgent and important for the effective treatment of malaria in China.

Shandong Province is an important coastal economic region located in Eastern China. Historically, Shandong was one of the most severe malaria transmission areas in China, with millions of annual malaria case numbers outbreak in the province during the 1960s and 1970s²⁸. Relying on substantial efforts for anti-malarial campaigns over the decades, the malaria cases in the province decreased sharply and epidemics was well controlled. Nevertheless, there was an increasing trend of the malaria cases presented in Shandong from 2012 to 2015 due to the proportion of imported malaria (100%, 586/586) and the majority of them were P. falciparum cases returning from African countries (79.9%, 468/586)^29,30. Since 2009, DHA + PQ and AS + AQ have been applied to against uncomplicated P. falciparum in Shandong as recommended by WHO in China^7,26. Nevertheless, little is known about current drug resistance of imported malaria cases. Thus, we assessed polymorphisms of the Pfcrt, Pfmdr1, and Pfkelch13 genes in order to provide genetic data for antimalarial drug resistance.

Polymorphisms in the amino acid positions 72–76 of Pfcrt gene are reliable markers for CQR of P. falciparum parasites, of which K76T mutation is the predominant³¹. In our study, Pfcrt mutant allele was found together with K76T mutation, which was consistent with above conclusion. For CQR parasites, CVIET and SVMNT are the two main mutant haplotypes prevalent worldwide^32,33. The CVIET haplotype has been shown to be predominant in many African countries and is almost the unique haplotype with high frequency in some areas of Africa³⁴. In this study, CVIET (26.9%) was more frequently than other mutant haplotypes and had no significant difference among four regions of Africa, was consistent with above conclusions. Interestingly, CVMNT haplotype was detected in an isolate returned from Equatorial Guinea in this study, which was also observed in 7% of Nigerian isolates and 70.6% of Ghananian isolates previously^35,36, suggesting a distinct difference was present in epidemic distribution of CVMNT in Africa. Except mutant types, mixed genotypes of Pfcrt had been detected in 6.6% isolates from Equatorial Guinea³⁷, and then 4.8% mixed types were found in parasites returned from ten countries in Africa³⁸. In present study, 9.3% (26/279) isolates with Pfcrt mixed genotypes were found coming back from 11 source countries in Africa. It further enriched the geographical range of Pfcrt mixed types and also suggested Pfcrt mixed alleles were widely prevalent in African countries. It was known that the isolates with SVMNT haplotype were found most prevalent in South America and Southeast Asia but considered rare in Africa³⁹. This was consistent with our results showing that no isolate carried SVMNT from Africa. However, several recent studies indicated that SVMNT had been detected in parasite strains from Tanzania and Angola. This could be associated with relatively low efficacy of AQ monotherapy in the countries^40,41. Therefore, continuous surveillance of SVMNT haplotype is still required for African imported malaria in China. The cessation of CQ for a period of time may lead to the restoration of CQ sensitive parasites. In Malawi, the prevalence of CQR Pfcrt genotype decreased from 85% to 13% during 10 years after withdrawal of CQ⁴². The same situation also happened to patients from Southern Ethiopia and travelers returned from West and Central African countries^43,44. In our samples, the parasites returned from Western Africa and Central Africa carried 70.3% and 67.0% of wild CVMNK haplotype in contrast to 21.6% and 24.7% of mutant CVIET haplotype respectively, which was consistent with above conclusion.

SNPs of Pfmdr1 gene was selected for CQR, and they also had been reported to be associated with regulating drug susceptibility or tolerance to several antimalarials, for example, quinine (QN), mefloquine (MQ), lumefantrine (LU), and even ART⁴⁵. In this study, Pfmdr1 allelic variants was only observed in codons 86, 184 and 1246. Previous studies suggested that Pfmdr1 N86Y mutation was a potential marker for CQR while Y184F may also play a role in mediating resistance to several antimalarial drugs^3,46. Among the isolates, a high frequency of N86Y (30.2%) and Y184F (60.7%) were observed in our study, of which Y184F was more prevalent. It was similar to the previous results in Senegal (14.9% and 71.8%) and Equatorial Guinea (50.3% and 87.3%)^37,47. In addition, linkage disequilibrium between K76T and N86Y has been observed in Africa previously⁴⁸. In this study, both Pfcrt K76T and Pfmdr1 N86Y were detected in 8.9% (25/282) parasite isolates. The correlation of two mutations associated with drug resistance will be of concern in further survey. Considering Pfmdr1 codons 1034, 1042, and 1246, the mutational haplotypes has occurred frequently among CQR parasites in South America, whereas wild haplotype is common in CQR isolates from Africa and Asia³⁵. Interestingly, D1246Y mutation were found in our samples, one reason for this might be ascribed to population flows between Africa and America. For Pfmdr1 wild haplotypes and mutant haplotypes, frequency diversity was observed among four regions of Africa (P < 0.05) respectively. It might be related to diversity of drug pressure and transmission intensity among the countries in Africa.

ACTs are the first-line treatment for P. falciparum in the majority of endemic countries and has been identified as the most successful antimalarial drug over the past 10 years⁴⁹. Pfkelch13 gene is essential for molecular surveillance of malaria parasites with ART resistance. So far, more than 150 nonsynonymous mutations contracted in Pfkelch13 gene have been reported¹⁹. In this study, 9 nonsynonymous Pfkelch13 mutations were observed in the isolates and most of which were returned from Equatorial Guinea and Angola. One possible reason was that samples from two countries were significantly more than others. Previous study reported that R539T mutation was detected in isolates returned from Angola and Equatorial Guinea respectively⁵⁰. In addition, R539T and C580Y was found in migrant workers returning from Ghana to China³⁶. In our study, no validated ART-resistant Pfkelch13 mutations was observed, the difference could be probably explained by sample sizes. Especially, a candidate ART-resistant mutation P553L was found in an isolate returned from Angola in this study, which also had been detected in Cambodia, Vietnam and West Africa¹⁹. These available data suggested that the extensive distribution of low frequent Pfkelch13 nonsynonymous mutations in African P. falciparum population and the emergence of Pfkelch13 mutations in Africa might lead the risk of global resistance transmission. The A578S mutation, which was commonly observed in Africa and several Southeast Asian countries, has been proven to be not related to clinical ART resistance¹⁹. In our study, A578S was the most prevalent nonsynonymous mutations among the samples, which was consistent with above results. Notably, one recent report showed a Chinese patient in Jiangsu Province carried ART resistant P. falciparum from Equatorial Guinea, indicating the emergence of indigenous ART resistant isolate in Africa¹⁴. Although there is no evidence that ART resistant P. falciparum parasite has emerged in Shandong, the attention should be paid to the increased imported malaria in the province. Therefore, routine molecular surveillance, clinical investigation and field research should be continuously strengthened for awareness of potential emergence of resistance to ACTs from Africa.

In conclusion, our study evaluated polymorphisms and geographic distribution of haplotypes of Pfcrt gene and Pfmdr1 gene in uncomplicated P. falciparum cases imported from Africa to Shandong Province of China. The prevalence of Pfcrt K76T and Pfmdr1 N86Y were still modestly present, indicating the presence of CQR in imported P. falciparum cases. We also detected one synonymous and 9 nonsynonymous mutations in propeller domain of Pfkelch13 gene, among which a candidate ART resistance mutation P553L was observed and 3 mutations were unreported before. Nevertheless, no validated ART resistance mutation of Pfkelch13 gene was found in this study, suggesting no immediate risk to the effect of ART. The study establishes fundamental data for the detection of CQR and ART resistance with molecular markers of the imported P. falciparum in China, and it also enriches the genetic data of drug resistance for the malaria endemic countries in Africa.

Methods

Electronic supplementary material

Author Contributions

C.X. and Q.W. made major contributions to writing the manuscript; B.H. conceived and initiated the study; K.Y., H.S., J.L., T.X. and G.Z. participated in sample collection and molecular markers detection; X.K., Y.W., S.Z. and G.Y. participated in the data collection and analyzed the data; K.Y., H.S. and J.K. provided some suggestions for the revision; All authors read and approved the final manuscript.

Data availability

All data generated or analyzed during this study are included in this published article.

Electronic supplementary material

Supplementary information accompanies this paper at 10.1038/s41598-018-31207-w.