As indigenous malaria has decreased over recent decades, the increasing number of imported malaria cases has provided a new challenge for China. The proportion of imported cases due to Plasmodium ovale has increased during this time, and the difference between P. ovale curtisi and P. ovale wallikeri is of importance. To better understand P. ovale epidemiology and the differences between the two subspecies, information on imported malaria in Henan Province was collected during 2010–2017. We carried out a descriptive study to analyze the prevalence, proportion, distribution, and origin of P. o. curtisi and P. o. wallikeri. It showed that imported P. ovale spp. accounts for a large proportion of total malaria cases in Henan Province, even more than that of P. vivax. This suggests that the proportion of P. ovale cases is underestimated in Africa. Among these cases, the latency period of P. o. curtisi was significantly longer than that of P. o. wallikeri. More attention should be paid to imported ovale malaria to avoid the reintroduction of these two subspecies into China.

Angola was the main origin country for the imported malaria in Henan Province, China. Antimalarial drug resistance has posed a threat to the control and elimination of malaria. Several molecular markers were confirmed to be associated with the antimalarial drug resistance, such as pfcrt, pfmdr1, pfdhfr, pfdhps, and K13. This study evaluated the drug resistance of the 180 imported Plasmodium falciparum isolates from Angola via nested PCR using Sanger sequencing. The prevalences of pfcrt C72V73M74N75K76, pfmdr1 N86Y184S1034N1042D1246, pfdhfr A16N51C59S108D139I164, and pfdhps S436A437A476K540A581 were 69.4%, 59.9%, 1.3% and 6.3%, respectively. Three nonsynonymous (A578S, M579I, and Q613E) and one synonymous (R471R) mutation of K13 were found, the prevalences of which were 2.5% and 1.3%, respectively. The single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, and pfdhps were generally shown as multiple mutations. The mutant prevalence of pfcrt reduced gradually, but pfdhfr and pfdhps still showed high mutant prevalence, while pfmdr1 was relatively low. The mutation of the K13 gene was rare. Molecular surveillance of artemisinin (ART) resistance will be used as a tool to evaluate the real-time efficacy of the artemisinin-based combination therapies (ACTs) and the ART resistance situation.

BackgroundPlasmodium ovale has two different subspecies: P. ovale curtisi and P. ovale wallikeri, which may be distinguished by the gene potra encoding P. ovale tryptophan-rich antigen. The sequence and size of potra gene was variable between the two P. ovale spp., and more fragment sizes were found compared to previous studies. Further information about the diversity of potra genes in these two P. ovale spp. will be needed.MethodsA total of 110 dried blood samples were collected from the clinical patients infected with P. ovale, who all returned from Africa in Henan Province in 2011–2016. The fragments of potra were amplified by nested PCR. The sizes and species of potra gene were analysed after sequencing, and the difference between the isolates were analysed with the alignment of the amino acid sequences. The phylogenetic tree was constructed by neighbour-joining to determine the genetic relationship among all the isolates. The distribution of the isolates was analysed based on the origin country.ResultsTotally 67 samples infected with P. o. wallikeri, which included 8 genotypes of potra, while 43 samples infected with P. o. curtisi including 3 genotypes of potra. Combination with the previous studies, P. o. wallikeri had six sizes, 227, 245, 263, 281, 299 and 335 bp, and P. o. curtisi had four sizes, 299, 317, 335 and 353 bp, the fragment sizes of 299 and 335 bp were the overlaps between the two species. Six amino acid as one unit was firstly used to analyse the amino acid sequence of potra. Amino acid sequence alignment revealed that potra of P. o. wallikeri differed in two amino acid units, MANPIN and AITPIN, while potra of P. o. curtisi differed in amino acid units TINPIN and TITPIS. Combination with the previous studies, there were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The phylogenetic tree showed that 11 isolates were divided into two clusters, P. o. wallikeri which was then divided into five sub-clusters, and P. o. curtisi which also formed two sub-clusters with their respective reference sequences. The genetic relationship of the P. ovale spp. mainly based on the number of the dominant amino acid repeats, the number of MANPIN, AITPIN, TINPIN or TITPIS. The genotype of the 245 bp size for P. o. wallikeri and that of the 299 and 317 bp size for P. o. curtisi were commonly exiting in Africa.ConclusionThis study further proved that more fragment sizes were found, P. o. wallikeri had six sizes, P. o. curtisi had four sizes. There were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The genetic polymorphisms of potra provided complementary information for the gene tracing of P. ovale spp. in the malaria elimination era.

Background Plasmodium falciparum malaria is recognized as a major global public health problem. The malaria vaccine was important because the case fatality rate of falciparum malaria was high. Plasmodium falciparum circumsporozoite protein (PfCSP) is one of the potential vaccine candidates, but the genetic polymorphism of PfCSP raises concerns regarding the efficacy of the vaccine. This study aimed to investigate the genetic polymorphism of PfCSP and provide data for the improvement of PfCSP-based vaccine (RTS,S malaria vaccine). Methods Blood samples were collected from 287 Chinese migrant workers who were infected with P. falciparum and returning from Africa to Henan Province during 2016–2018. The Pfcsp genes were analysed to estimate the genetic diversity of this parasite. Results The results showed that there were two mutations at the N-terminus of imported Pfcsp in Henan Province, including insertion amino acids (58.71%, 118/201) and A → G (38.81%, 78/201). The number of repeats of tetrapeptide motifs (NANP/NVDP/NPNP/NVDA) in the central repeat region ranged mainly from 39 to 42 (97.51%, 196/201). A total of 14 nonsynonymous amino acid changes were found at the C-terminus. The average nucleotide difference (K) of imported Pfcsp in Henan Province was 5.719, and the haplotype diversity (Hd) was 0.964 ± 0.004. The estimated value of dN-dS was 0.047, indicating that the region may be affected by positive natural selection. The minimum number of recombination events (Rm) of imported Pfcsp in Henan Province was close to that in Africa. The analysis of genetic differentiation showed that there may be moderate differentiation between East Africa and North Africa (Fst = 0.06484), and the levels of differentiation in the other regions were very small (Fst < 0.05). Conclusions The N-terminus of Pfcsp was relatively conserved, and the central repeat region and the Th2R and Th3R regions of the C-terminus were highly polymorphic. The gene polymorphism pattern among Chinese migrant workers returning from Africa to Henan Province was consistent with that in Africa. The geographical pattern of population differentiation and the evidence of natural selection and gene recombination suggested that the effect of polymorphism on the efficacy of PfCSP-based vaccines should be considered.

Malaria remains a major public health issue in Nigeria, and Nigeria is one of the main sources of imported malaria in China. Antimalarial drug resistance is a significant obstacle to the control and prevention of malaria globally. The molecular markers associated with antimalarial drug resistance can provide early warnings about the emergence of resistance. The prevalence of antimalarial drug resistant genes and mutants, including PfK13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, was evaluated among the imported Plasmodium falciparum isolates from Nigeria in Henan, China, from 2012 to 2019. Among the 167 imported P. falciparum isolates, the wild-type frequency of PfK13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps was 98.7, 63.9, 34.8, 3.1, and 3.1%, respectively. The mutation of PfK13 was rare, with just two nonsynonymous (S693F and Q613H) and two synonymous mutations (C469C and G496G) identified from four isolates. The prevalence of Pfcrt mutation at codon 74–76 decreased year-by-year, while the prevalence of pfmdr1 86Y also decreased significantly with time. The prevalence of Pfdhfr and Pfdhps mutants was high. Combined mutations of Pfdhfr and Pfdhps had a high prevalence of the quadruple mutant I51R59N108-G437 (39.0%), followed by the octal mutant I51R59N108-V431A436G437G581S613 (17.0%). These molecular findings update the known data on antimalarial drug-resistance genes and provide supplemental information for Nigeria.