Chloroquine (CQ) was the cornerstone of anti-malarial treatment in Africa for almost 50 years, but has been widely withdrawn due to the emergence and spread of resistance. Recent reports have suggested that CQ-susceptibility may return following the cessation of CQ usage. Here, we monitor CQ sensitivity and determine the prevalence of genetic polymorphisms in the CQ resistance transporter gene (pfcrt) of Plasmodium falciparum isolates recently imported from Africa to China. Blood samples were collected from falciparum malaria patients returning to China from various countries in Africa. Isolates were tested for their sensitivity to CQ using the SYBR Green I test ex vivo, and for a subset of samples, in vitro following culture adaptation. Mutations at positions 72–76 and codon 220 of the pfcrt gene were analyzed by sequencing and confirmed by PCR-RFLP. Correlations between drug sensitivity and pfcrt polymorphisms were investigated. Of 32 culture adapted isolates assayed, 17 (53.1%), 6 (18.8%) and 9 (28.1%) were classified as sensitive, moderately resistant, and highly resistant, respectively. In vitro CQ susceptibility was related to point mutations in the pfcrt gene, the results indicating a strong association between pfcrt genotype and drug sensitivity. A total of 292 isolates were typed at the pfcrt locus, and the prevalence of the wild type (CQ sensitive) haplotype CVMNK in isolates from East, South, North, West and Central Africa were 91.4%, 80.0%, 73.3%, 53.3% and 51.7%, respectively. The only mutant haplotype observed was CVIET, and this was almost always linked to an additional mutation at A220S. Our results suggest that a reduction in drug pressure following withdrawal of CQ as a first-line drug may lead to a resurgence in CQ sensitive parasites. The prevalence of wild-type pfcrt CQ sensitive parasites from East, South and North Africa was higher than from the West and Central areas, but this varied greatly between countries. Further surveillance is required to assess whether the prevalence of CQ resistant parasites will continue to decrease in the absence of widespread CQ usage.

Anopheles sinensis is one of the most important malaria vectors in China and other Southeast Asian countries. High levels of resistance have been reported in this species due to the long-term use of insecticides, especially pyrethroids, for public health and agricultural purposes. Knockdown resistance (kdr) caused by a single base pair mutation in the gene encoding the sodium channel is strongly associated with pyrethroid insecticide resistance in many Anopheles mosquitoes. There are few methods currently available for detecting kdr mutations in An. sinensis. A novel AllGlo probe-based qPCR (AllGlo-qPCR) method was developed to screen for the predominant kdr mutations in An. sinensis mosquitoes from the Jiangsu Province. The results from AllGlo-qPCR, allele-specific PCR (AS-PCR), and TaqMan-MGB probe-based qPCR (TaqMan-qPCR) were compared. A comparative analysis of the equipment required, ease of use and cost of the available methods was also performed. Finally, the AllGlo-qPCR method was used to detect the frequencies of kdr mutations from the other four provinces in central China. Six kdr genotypes were detected in An. sinensis from the Jiangsu Province by DNA sequencing. The AllGlo-qPCR method detected all of the kdr genotypes with a high level of accuracy (97% sensitivity and 98% specificity). AllGlo-qPCR correctly determined the kdr genotypes of 98.73% of 158 An. sinensis samples, whereas TaqMan-qPCR and AS-PCR correctly identified 96.84% and 88.61% of mutations, respectively. Furthermore, the AllGlo-qPCR method is simpler to perform, requires less equipment, and exhibits a moderate expense cost comparing with the other tested methods of kdr mutation detection. Samples collected from four of the other provinces in central China showed a high frequency of kdr mutation in An. sinensis, as detected by the established AllGlo-qPCR method. The novel AllGlo-qPCR method developed for kdr mutation detection in An. sinensis exhibits greater specificity and sensitivity than currently available methods and is more cost-effective; therefore, it represents a useful tool for entomological surveillance.

Aedes albopictus is one of the most important vectors of Dengue, which poses a serious threat to public health. The bacterial microbiota has an effect on the parameters of mosquitos, such as larval development and fecundity, and it has emerged as a promising field to be explored for novel environmentally friendly control strategies. Rahnella sp. are present in many insects, including Ae. Albopictus, and play a role in bacterial–insect interactions; however, the role of the bacteria in mosquito biology has not yet been characterized. In this study, we characterized the Rahnella isolate RAeA1 obtained from Ae. albopcitus, and its colonization stability in Ae. albopictus was investigated by generating GFP-tagged bacteria. The influences of the bacteria on larval development and mosquito reproductive capacity were evaluated by inoculating RAeA1 in axenic larvae and antibiotic-treated adult mosquitoes, respectively. The results indicated that RAeA1, which is widespread in the field population of Ae. albopictus, can be transmitted directly from the parental strain to the progeny and can rescue axenic larvae developing into adults with a prolonged development time to pupation. RAeA1 inoculation can impair egg production and ovary maturation, as well as reducing the synthesis of ecdysteroids and vitellogenin in Ae. albopictus females. Overall, our results provide a thorough study of bacterium function characterization that will facilitate the development of potential strategies in relation to the design of microbiomes for vector control.

As more sporadic cases of chloroquine resistance occur (CQR) in Plasmodium vivax (P. vivax) malaria, molecular markers have become an important tool to monitor the introduction and spread of drug resistance. P. vivax multidrug resistance-associated protein 1 (PvMRP1), as one of the members of the ATP-binding cassette (ABC) transporters, may modulate this phenotype. In this study, we investigated the gene mutations and copy number variations (CNVs) in the pvmrp1 in 102 P. vivax isolates from China, the Republic of Korea (ROK), Myanmar, Papua New Guinea (PNG), Pakistan, the Democratic People’s Republic of Korea (PRK), and Cambodia. And we also obtained 72 available global pvmrp1 sequences deposited in the PlasmoDB database to investigate the genetic diversity, haplotype diversity, natural selection, and population structure of pvmrp1. In total, 29 single nucleotide polymorphisms reflected in 23 non-synonymous, five synonymous mutations and one gene deletion were identified, and CNVs were found in 2.9% of the isolates. Combined with the antimalarial drug susceptibility observed in the previous in vitro assays, except the prevalence of S354N between the two CQ sensitivity categories revealed a significant difference, no genetic mutations or CNVs associated with drug sensitivity were found. The genetic polymorphism analysis of 166 isolates worldwide found that the overall nucleotide diversity (π) of pvmrp1 was 0.0011, with 46 haplotypes identified (Hd = 0.9290). The ratio of non-synonymous to synonymous mutations (dn/ds = 0.5536) and the neutrality tests statistic Fu and Li’s D* test (Fu and Li’s D* = −3.9871, p < 0.02) suggests that pvmrp1 had evolved under a purifying selection. Due to geographical differences, genetic differentiation levels of pvmrp1 in different regions were different to some extent. Overall, this study provides a new idea for finding CQR molecular monitoring of P. vivax and provides more sequences of pvmrp1 in Asia for subsequent research. However, further validation is still needed through laboratory and epidemiological field studies of P. vivax samples from more regions.