Search

Abstract A novel combined measurements techniques has been designed in this work, enabling the acquisition of laser-induced breakdown spectroscopy (LIBS) and Raman spectral signals at the same point on a sample. The application of this combined technique to the analysis of multi-layered mock-up blocks painted with orpiment and realgar pigments has yielded significant insights. By correlating variations in the emission line intensity of characteristic elements within the LIBS spectra with depth-specific Raman spectra, the number of laser pulses that penetrated the pigment layers has been accurately determined, thereby establishing a method to measure layer thickness. Finally, the technique wasto analysis the actual mural fragment from Mogao Cave 196, determining the types of pigment and the thickness of the pigment layers. Graphical Abstract

Abstract Background Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease has been underestimated. Plasmodium ovale curtisi Duffy binding protein domain region II (PocDBP-RII) is an essential ligand for reticulocyte recognition and host cell invasion by P. ovale curtisi. However, the genomic variation, antigenicity and immunogenicity of PocDBP-RII remain major knowledge gaps. Methods A total of 93 P. ovale curtisi samples were collected from migrant workers who returned to China from 17 countries in Africa between 2012 and 2016. The genetic polymorphism, natural selection and copy number variation (CNV) were investigated by sequencing and real-time PCR. The antigenicity and immunogenicity of the recombinant PocDBP-RII (rPocDBP-RII) protein were further examined, and the humoral and cellular responses of immunized mice were assessed using protein microarrays and flow cytometry. Results Efficiently expressed and purified rPocDBP-RII (39 kDa) was successfully used as an antigen for immunization in mice. The haplotype diversity (Hd) of PocDBP-RII gene was 0.105, and the nucleotide diversity index (π) was 0.00011. No increased copy number was found among the collected isolates of P. ovale curtisi. Furthermore, rPocDBP-RII induced persistent antigen-specific antibody production with a serum IgG antibody titer of 1: 16,000. IFN-γ-producing T cells, rather than IL-10-producing cells, were activated in response to the stimulation of rPocDBP-RII. Compared to PBS-immunized mice (negative control), there was a higher percentage of CD4+CD44highCD62L− T cells (effector memory T cells) and CD8+CD44highCD62L+ T cells (central memory T cells) in rPocDBP-RII‑immunized mice. Conclusions PocDBP-RII sequences were highly conserved in clinical isolates of P. ovale curtisi. rPocDBP-RII protein could mediate protective blood-stage immunity through IFN-γ-producing CD4+ and CD8+ T cells and memory T cells, in addition to inducing specific antibodies. Our results suggested that rPocDBP-RII protein has potential as a vaccine candidate to provide assessment and guidance for malaria control and elimination activities. Graphical Abstract

Abstract Background Predicting the risk of malaria in countries certified malaria-free is crucial for the prevention of re-introduction. This review aimed to identify and describe existing prediction models for malaria re-introduction risk in eliminated settings. Methods A systematic literature search following the PRISMA guidelines was carried out. Studies that developed or validated a malaria risk prediction model in eliminated settings were included. At least two authors independently extracted data using a pre-defined checklist developed by experts in the field. The risk of bias was assessed using both the prediction model risk of bias assessment tool (PROBAST) and the adapted Newcastle–Ottawa Scale (aNOS). Results A total 10,075 references were screened and 10 articles describing 11 malaria re-introduction risk prediction models in 6 countries certified malaria free. Three-fifths of the included prediction models were developed for the European region. Identified parameters predicting malaria re-introduction risk included environmental and meteorological, vectorial, population migration, and surveillance and response related factors. Substantial heterogeneity in predictors was observed among the models. All studies were rated at a high risk of bias by PROBAST, mostly because of a lack of internal and external validation of the models. Some studies were rated at a low risk of bias by the aNOS scale. Conclusions Malaria re-introduction risk remains substantial in many countries that have eliminated malaria. Multiple factors were identified which could predict malaria risk in eliminated settings. Although the population movement is well acknowledged as a risk factor associated with the malaria re-introduction risk in eliminated settings, it is not frequently incorporated in the risk prediction models. This review indicated that the proposed models were generally poorly validated. Therefore, future emphasis should be first placed on the validation of existing models.

Abstract Background Malaria is a worldwide infectious disease. For countries that have achieved malaria elimination, the prevention of re-establishment due to infections in returned travellers has become important. The accurate and timely diagnosis of malaria is the key in preventing re-establishment, and malaria rapid diagnostic tests (RDTs) are frequently used due to their convenience. However, the RDT performance in Plasmodium malariae (P. malariae) infection diagnosis remains unknown. Methods This study analysed epidemiological features and diagnosis patterns of imported P. malariae cases from 2013 to 2020 in Jiangsu Province and evaluated the sensitivity of four parasite enzyme lactate dehydrogenase (pLDH)-targeting RDTs (Wondfo, SD BIONLINE, CareStart and BioPerfectus) and one aldolase-targeting RDT(BinaxNOW) for P. malariae detection. Furthermore, influential factors were investigated, including parasitaemia load, pLDH concentration and target gene polymorphisms. Results The median duration from symptom onset to diagnosis among patients with P. malariae infection was 3 days, which was longer than that with Plasmodium falciparum (P. falciparum) infection. The RDTs had a low detection rate (39/69, 56.5%) among P. malariae cases. All tested RDT brands had poor performance in P. malariae detection. All the brands except the worst-performing SD BIOLINE, achieved 75% sensitivity only when the parasite density was higher than 5000 parasites/μL. Both pLDH and aldolase showed relatively conserved and low gene polymorphism rates. Conclusions The diagnosis of imported P. malariae cases was delayed. The RDTs had poor performance in P. malariae diagnosis and may threaten the prevention of malaria re-establishment from returned travellers. The improved RDTs or nucleic acid tests for P. malariae cases are urgently needed for the detection of imported cases in the future.

Abstract Background Malaria is still one of the major infectious diseases affecting human health, and the World Health Organization (WHO) has attached special importance to malaria-related technical training for its global elimination efforts. The Jiangsu Institute of Parasitic Diseases (JIPD), designated as a WHO Collaborating Centre for Research and Training on Malaria Elimination, has conducted numerous international malaria training programmes during the last 2 decades. Methods A retrospective analysis of international training programmes organized and facilitated by JIPD in China since 2002 was conducted. A web-based questionnaire was designed to gather respondents’ basic information, evaluation of course topics, methodology, trainers, and facilitators, course impact, and suggestions for future trainings. Individuals who participated in the training courses from 2017 to 2019 were invited to participate in this assessment. Results Since 2002, JIPD has conducted 62 malaria-related international trainings attended by 1935 participants from 85 countries, covering 73% of malaria endemic countries. Of 752 participants enrolled, 170 responded to the online survey. A majority of respondents (160/170, 94.12%) gave a high evaluation of the training, with an average score of 4.52 (5 maximum score). Also, survey respondents gave a 4.28 score on “knowledge and skills gained in the training useful for the national malaria programme”, 4.52 on “topics appropriate to their professional needs”, and 4.52 on “knowledge and skills gained in the training useful to their career”. Surveillance and response was the most important topic discussed and field visit was the most effective method of training. For future training programmes, with increasing length of training, more field visits and demonstration, improving language barrier, and sharing experience were what the respondents requested most. Conclusion JIPD, as a professional institute for malaria control, has conducted a great quantity of training in the past 20 years, providing training opportunities to both malaria and non-malaria endemic countries globally. For future training, survey respondents’ suggestions will be considered to provide a more effective capacity building activity to better contribute to global malaria elimination.

Abstract Background Imported malaria cases remains a key health concern, especially during the COVID-19 pandemic. Providing accurate health information is important to improving people’s awareness of malaria. WeChat is an excellent social media tool for health information dissemination, especially during the pandemic. This study explored the effect of malaria knowledge dissemination via a WeChat public account. Methods A questionnaire for data collection was constructed using the online survey tool Sojump. Questionnaires were sent to users who followed the Jiangsu institute of Parasitic Disease WeChat public account during the National Malaria Day 2021 period. A small incentive (WeChat Red Packet) was distributed to everyone who answered the questionnaire correctly on time. Results A total of 13,169 valid questionnaires were collected during the China National Malaria Day period. Questions in which participants focused mainly on information pertaining to themselves, such as infection, symptoms, and epidemic areas, reached highest accuracy (above 90%). Questionnaires were submitted through smartphones and most of them were completed during the period of 4 days from April 23 to April 26, 2021 when a WeChat Red Packet was offered. The accuracy of responses was related to bolded words and location and number of knowledge points that were shown at the beginning of the questionnaire. The number of users of the WeChat public account in question increased from 5961 to 12,339 in just 4 days of the activity. Conclusion A WeChat public account is a convenient and accessible tool for spreading malaria-related health information to the public. Distribution of incentives (Red Packets) can effectively increase public attention to popular science and health information and activities.

Abstract Background The rapid emergence and global spread of COVID-19 have caused substantial global disruptions that have impacted malaria programs worldwide. Innovative strategies to enable countries aiming to eliminate malaria as well as those that are already certified as malaria-free, are needed to address malaria importation in the context of the COVID-19 pandemic. China was certified as malaria-free in 2021 and now aims to prevent the malaria re-establishment. Nonpharmaceutical interventions such as entry screening, quarantining, and health education for individuals returning from international travel during the COVID-19 pandemic present both opportunities and challenges to the management of imported malaria. This study aimed to describe and analyze the operational challenges associated with an integrated surveillance and case management program in which malaria re-establishment prevention measures were incorporated into the COVID-19 program in China. Methods After the integration of malaria re-establishment prevention activities into the COVID-19 program for 10 months in Jiangsu Province, China, a focus-group discussion of public health workers working on preventing malaria re-establishment and controlling COVID-19 was held in June 2021, aiming to explore the operational challenges and lessons learned from the integrated approach. Results From 01 August 2020 to 31 May 2021, 8,947 overseas travelers with Yangzhou as the final destination underwent 14-day managed quarantine and 14-day home isolation. Of these travelers, 5,562 were from malaria-endemic regions. A total of 26,026 education booklets and materials were distributed to expand malaria-related knowledge. Twenty-two patients with unknown fever were screened for malaria with rapid diagnostic tests, and one patient was confirmed to have imported malaria. The challenges associated with the implementation of the integrated malaria surveillance and case management program include neglect of malaria due to COVID-19, lack of a standard operating procedure for malaria screening, mobility of public health providers, and difficulties in respecting the timeline of the “1–3-7” surveillance strategy. Conclusions China’s experience highlights the feasibility of integrated case surveillance and management of existing infectious diseases and new emerging infections. It also demonstrates the importance of a sound public health infrastructure with adequate, trained field staff for screening, testing, contact tracing, and providing health education, all of which are crucial for the success of both malaria re-establishment prevention program and the effective control of COVID-19.

Abstract Background Following initiation of China’s National Malaria Elimination Action Plan (NMEAP) in 2010, the ‘1-3-7’ approach was developed and rolled out in China to facilitate the malaria elimination programme and accelerate malaria elimination. This study aims to summarize and condense these experiences through a retrospective analysis in Jiangsu Province, which could be adapted and applied in other malaria elimination settings worldwide. Methods A retrospective analysis of imported malaria cases into China identified through an improved surveillance and response system in Jiangsu Province was carried out for the period of 2001–2020. To improve the malaria surveillance and response system, Centers for Diseases Control and Prevention from the prefectures and counties in Jiangsu province conducted population-level health education to improve healthcare seeking behavior, strengthened capacity of health facilities to improve performance of malaria diagnosis and treatment, and raised the capacity of public health providers to improve implementation of the ‘1-3-7’ approach. Categorical variables were carried out by Chi square tests with Fisher’s exact correction. Results From 2001 to 2020, a total of 9,879 malaria cases were reported in Jiangsu Province. Since 2012, no indigenous malaria cases have been reported in Jiangsu Province. However, in recent years, there has been a substantial increase of imported falciparum malaria cases. Between 2012 and 2020, an estimated 61.57 million individuals have benefited from population-level health education in Jiangsu Province. For healthcare-seeking services among the 2,423 imported malaria cases, 687 (28.4%) and 1,104 (45.6%) cases visited hospitals on the first day and the second day from symptom onset, respectively. A total of 1,502 (61.9%) cases were diagnosed on the first day at medical facilities. Jiangsu Province achieved 100%, 99.4% and 98.3% completion rate in terms of case detection and notification (within one day), case investigation (within three days) and foci response and disposition (within seven days), respectively. The improved surveillance and response system in Jiangsu Province plays an important role in preventing the re-introduction of malaria and maintaining the malaria-free status. Conclusions Jiangsu Province has maintained its malaria-free status since 2012. The continuous improvement of a surveillance and response system plays an important role in the early detection and rapid response of potential malaria-related outbreaks in Jiangsu, China, and has important lessons for other malaria eliminating settings. Remaining vigilant in the detection of imported malaria cases and maintaining an active surveillance and response system is critical to sustain the success of malaria elimination. Graphic Abstract

Summary: The emergence and spread of artemisinin-tolerant malaria parasites threatens malaria control programmes worldwide. Mutations in the propeller domain of the Kelch13 protein confer Plasmodium falciparum artemisinin resistance (ART-R). ART-R is linked to the reduced susceptibility of temporary growth-arrested ring-stage parasites, but the metabolic mechanisms remain elusive. We generated two PfKelch13 mutant lines via CRISPR-Cas9 gene editing which displayed a reduced susceptibility accompanied by an extended ring stage. The metabolome of ART-induced ring-stage growth arrest parasites carrying PfKelch13 mutations showed significant alterations in the tricarboxylic acid (TCA) cycle, glycolysis, and amino acids metabolism, pointing to altered energy and porphyrin metabolism with metabolic plasticity. The critical role of these pathways was further confirmed by altering metabolic flow or through chemical inhibition. Our findings uncover that the growth arrestment associated with ART-R is potentially attributed to the adaptative metabolic plasticity, indicating that the defined metabolic remodeling turns out to be the trigger for ART-R.

Plasmodium ovale curtisi and P. ovale wallikeri are both endemic in sub-Saharan Africa, the Middle East and Southeast Asia. Molecular surveillance data for drug resistance in P. ovale spp. is limited at present. We analysed polymorphisms in the podhfr, pocrt and pocytb genes of P. ovale spp. in 147 samples collected from travelers returning to China from Africa. Two podhfr mutations, S58R and S113N/T were detected in P. ovale curtisi with high/moderate frequencies of 52.17% and 17.39%, respectively. Evidence of positive selection (dN/dS = 2.41) was found for podhfr in P. ovale curtisi and decreased diversity (He) of microsatellite markers flanking the mutant alleles suggests that selective sweeps have occurred for both. Mutations E34G (1.50%) and L43V (1.50%) in pocrt of P. ovale curtisi, and E34G (3.70%), I102M (1.80%) and V111F (1.80%) of P. ovale wallikeri were found at low frequencies. Mutations R66K (6.20%), R75K (11.63%) and R95K (3.88%) of pocytb were found in both P. ovale curtisi and P. ovale wallikeri. These results suggest that the podhfr gene of P. ovale curtisi may be subject to drug selection in Africa, warranting further attention. We observed significant differences in the prevalence and distribution of podhfr mutations between the two P. ovale species, suggestive of fundamental biological differences between them.

Abstract Background Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa region of MSP1 has long been considered one of the major candidate antigens for a malaria blood-stage vaccine against Plasmodium falciparum. However, there is limited information on the C-terminal 19-kDa region of Plasmodium ovale MSP1 (PoMSP119). This study aims to analyze the genetic diversity and immunogenicity of PoMSP119. Methods A total of 37 clinical Plasmodium ovale isolates including Plasmodium ovale curtisi and Plasmodium ovale wallikeri imported from Africa into China and collected during the period 2012–2016 were used. Genomic DNA was used to amplify P. ovale curtisi (poc) msp1 19 (pocmsp1 19 ) and P. ovale wallikeri (pow) msp1 19 (powmsp1 19 ) genes by polymerase chain reaction. The genetic diversity of pomsp1 19 was analyzed using the GeneDoc version 6 programs. Recombinant PoMSP119 (rPoMSP119)-glutathione S-transferase (GST) proteins were expressed in an Escherichia coli expression system and analyzed by western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assay. In addition, antigen-specific T cell responses were assessed by lymphocyte proliferation assays. A total of 49 serum samples from healthy individuals and individuals infected with P. ovale were used for the evaluation of natural immune responses by using protein microarrays. Results Sequences of pomsp1 19 were found to be thoroughly conserved in all the clinical isolates. rPoMSP119 proteins were efficiently expressed and purified as ~ 37-kDa proteins. High antibody responses in mice immunized with rPoMSP119-GST were observed. rPoMSP119-GST induced high avidity indexes, with an average of 92.57% and 85.32% for rPocMSP119 and rPowMSP119, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found in the splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for the detection of natural immune responses in patients infected with P. ovale were 89.96% and 75%, respectively. Conclusions This study revealed highly conserved gene sequences of pomsp1 19 . In addition, naturally acquired humoral immune responses against rPoMSP1 were observed in P. ovale infections, and high immunogenicity of rPoMSP119 in mice was also identified. These instructive findings should encourage further testing of PoMSP119 for rational vaccine design. Graphical abstract

ABSTRACT Arboviruses and insect-specific viruses (ISVs) are two major types of viruses harbored by mosquitoes that are distinguished by the involvement of vertebrate hosts in their transmission cycles. While intensive studies have focused on the transmission, tissue tropism, and evolution of arboviruses, these characteristics are poorly investigated in ISVs, which dominate the mosquito virome. Therefore, in this study, we collected two mosquito species, Anopheles sinensis and Culex quinquefasciatus, in the field and used a metatranscriptomics approach to characterize their RNA viromes in different tissues, such as the midgut, legs, salivary gland, eggs, and the remainder of the carcass. Blood-engorged individuals of these species were captured in 3 locations, and 60 mosquitoes were pooled from each species and location. A total of 40 viral species from diverse viral taxa associated with all viral RNA genome types were identified, among which 19 were newly identified in this study. According to the current viral taxonomy, some of these viruses, such as Yancheng Anopheles associated virus 2 (Narnaviridae) and Jiangsu Anopheles-related virus (Ghabrivirales), were novel. The two investigated mosquito species generally harbored distinct viromes. Nevertheless, the viruses were generally shared among different tissue types to various degrees. Specifically, the eggs possessed a viral community with significantly lower diversity and abundance than those in other tissues, whereas the legs and salivary glands exhibited higher viral abundance. The compositions and distributions of the viromes of different mosquito tissues were demonstrated for the first time in our study, providing important insight into the virome dynamics within individual mosquitoes. IMPORTANCE ISVs are considered to be ancestral to arboviruses. Because of their medical importance, arboviruses have been well studied from the aspects of their transmission mode, evolution of dual-host tropism, and genetic dynamics within mosquito vectors. However, the mode of ISV maintenance is poorly understood, even though many novel ISVs have been identified with the emergence of sequencing technology. In our study, in addition to the identification of a diverse virus community, the tissue tropism of RNA viromes harbored by two field-collected mosquito species was demonstrated for the first time. According to the results, the virus communities of different tissues, such as the salivary glands, midguts, legs, and eggs, can help us understand the evolution, transmission routes, and maintenance modes of mosquito-specific viruses in nature.

Insecticide-based vector control measures play an important role in the prevention and control of insect-borne infectious diseases such as malaria; however, insecticide resistance has become a severe global problem for vector control. To date, the metabolic mechanism by which Anopheles sinensis, the most widely distributed malaria vector in China and Asia, detoxifies insecticides is not clear. In this study, the molecular metabolite changes in both the larval and adult stages of deltamethrin susceptible (DS) and deltamethrin-resistant (DR) An. sinensis mosquitoes were analysed by using liquid chromatography tandem mass spectrometry (LC-MS/MS) after exposure to deltamethrin. There were 127 differential metabolites in larval DR An. sinensis and 168 in adults. Five metabolites (glycerophosphocholine, deoxyguanosine, DL-methionine sulfoxide, D-myo-inositol-3-phosphate and N-acetyl-alpha-D-glucosamine1-phosphate) were downregulated in both DR larvae and adults, and one metabolite (aspartyl-glutamine) was upregulated, and the ratio of down- and up-regulation of these metabolites was 5:1. The differential metabolites between the DS and DR mosquitos were mainly classified into organic oxygen compounds, carboxylic acids and their derivatives, glycerophospholipids and purine nucleotides, and the common pathway enriched in both the larval and adult DR An. sinensis was glycerophospholipid metabolism. The findings of this study provide further mechanistic understanding of insecticide resistance in An. sinensis.

Abstract Background Current methods to classify local and imported malaria infections depend primarily on patient travel history, which can have limited accuracy. Genotyping has been investigated as a complementary approach to track the spread of malaria and identify the origin of imported infections. Methods An extended panel of 26 microsatellites (16 new microsatellites) for Plasmodium falciparum was evaluated in 602 imported infections from 26 sub-Saharan African countries to the Jiangsu Province of People’s Republic of China. The potential of the 26 microsatellite markers to assign imported parasites to their geographic origin was assessed using a Bayesian method with Markov Chain Monte Carlo (MCMC) as implemented in the program Smoothed and Continuous Assignments (SCAT) with a modification to incorporate haploid genotype data. Results The newly designed microsatellites were polymorphic and are not in linkage disequilibrium with the existing microsatellites, supporting previous findings of high rate of recombination in sub-Saharan Africa. Consistent with epidemiology inferred from patients’ travel history, no evidence for local transmission was found; nearly all genetically related infections were identified in people who travelled to the same country near the same time. The smoothing assignment method assigned imported cases to their likely geographic origin with an accuracy (Angola: 59%; Nigeria: 51%; Equatorial Guinea: 40%) higher than would be achieved at random, reaching statistical significance for Angola and Equatorial Guinea. Conclusions Genotyping using an extended microsatellite panel is valuable for malaria case classification and programme evaluation in an elimination setting. A Bayesian method for assigning geographic origin of mammals based on genetic data was adapted for malaria and showed potential for identification of the origin of imported infections.

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China. Author summary The mosquitoes Aedes albopictus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens and Cx. tritaeniorhynchus are native to China and the major vectors in the transmission of arboviruses, protozoans and nematodes. Recently, an innovative biocontrol strategy has been developed and evaluated based on the ability of Wolbachia to induce cytoplasmic incompatibility (CI), as well as interfere with the infection and replication of pathogens. Since the ability to induce CI largely depends on the density and diversity of Wolbachia, we investigated and characterized the natural infection of Wolbachia in above-mentioned five species of field-collected mosquitoes in 25 provinces of China. The results showed that the positive rates of Wolbachia infection were high in mosquitoes of Ae. albopictus, Ar. subalbatus and Cx. pipiens in large-scale regions of China and low in Cx. tritaeniorhynchus in Guizhou province. Phylogenetic analysis based on Wolbachia surface protein (wsp) gene and five multilocus sequence typing (MLST) loci indicated the high polymorphism of Wolbachia in Ae. albopictus, and low polymorphisms in Ar. subalbatus and Cx. pipiens. This finding contributes to the understanding of the nationwide distribution of Wolbachia and the potential application of this biocontrol strategy in China.

Abstract Background Plasmodium ovale is widely distributed across tropical regions and has two closely related but distinct species, namely P. ovale curtisi and P. ovale wallikeri. Combining genetic characterization with the immunogenicity of merozoite surface protein-8 (MSP-8) supports considering MSP-8 as a candidate target for blood-stage vaccines against malaria. However, no previous studies have focused on characterizing the genetic diversity and immunogenicity of PoMSP-8. Methods Blood samples were collected from 42 patients infected with P. ovale. The patients were migrant workers returning to the Jiangsu Province from Africa; genomic DNA was extracted from their blood samples for sequencing and protein expression. The recombinant PoMSP-8 (rPoMSP-8) proteins were expressed and purified to assess their immune responses in BALB/c mice. Results The sequences of the P. ovale curtisi and P. ovale wallikeri msp8 genes were completely conserved in each isolate. The rPoMSP-8 proteins were successfully expressed and purified as ~70 kDa proteins. Antibodies raised against rPoMSP-8 in mice showed appropriate immunoreactivity, as evidenced by immunoblotting. These specific antibodies were detected at day 7 post-immunization, and their levels increased throughout the whole immunization period. rPoMSP-8-raised antibodies had high endpoint titers (1:5,120,000) and high avidity (PocMSP-8: 94.84%, PowMSP-8: 92.69%). Cross-reactivity between rPocMSP-8 and rPowMSP-8 was observed with each anti-PoMSP8-specific antibody. Conclusions Remarkable conservation and high immunogenicity was observed in both rPoMSP-8s. Intriguingly, cross-reaction between rPocMSP-8 and rPowMSP-8 was detected, suggesting that a single PoMSP8-based construction might be applicable for both species.

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.

Increased population movement has increased the risk of reintroducing parasites to elimination areas and also dispersing drug-resistant parasites to new regions. Therefore, reliable and repeatable methods to trace back to the source of imported infections are essential. The recently developed 23-single-nucleotide polymorphism (SNP) barcode from organellar genomes of mitochondrion (mt) and apicoplast (apico) provides a valuable tool to locate the geographic origin of Plasmodium falciparum. This study aims to explore the feasibility of using the 23-SNP barcode for tracking P. falciparum by polymerase chain reaction and sequencing, while providing geographical haplotypes of isolates that originated from Central Africa. Based on 23-SNP barcode analysis, SNPs were found at seven loci; 27 isolates were confirmed to have originated in West Africa, and this study also showed four isolates from Central Africa (Equatorial Guinea, 3; Republic of Congo, 1) that originated in East Africa. This study provides the sequence data from Central Africa and fills 23-SNP barcode data gaps of sample origins.

Abstract Background Plasmodium merozoite surface protein-1 (MSP-1) is released into the bloodstream during merozoite invasion, and thus represents a crucial malarial vaccine target. Although substantial research effort has been devoted to uncovering the genetic diversity of MSP-1 for P. falciparum and P. vivax, there is minimal information available regarding the genetic profiles and structure of P. ovale. Therefore, the aim of the present study was to determine the extent of genetic variation among two subspecies of P. ovale by characterizing the MSP-1 N-terminal sequence at the nucleotide and protein levels. Methods N-terminal of MSP-1 gene were amplified from 126 clinical samples collected from imported cases of malaria in migrant workers returning to Jiangsu Province from Africa using a conventional polymerase chain reaction (PCR) assay. The PCR products were then sequenced and analyzed using the GeneDoc, MegAlign, MEGA7 and DnaSP v.6 programs. Results The average pairwise nucleotide diversities (π) of P. ovale curtisi and P. ovale wallikeri MSP-1 genes (pomsp1) were 0.01043 and 0.01974, respectively, and the haplotype diversity (Hd) were 0.746 and 0.598, respectively. Most of the nucleotide substitutions detected were non-synonymous, indicating that the genetic variations of pomsp1 were maintained by positive diversifying selection, thereby suggesting their role as a potential target of a protective immune response. Amino acid substitutions of P. ovale curtisi and P. ovale wallikeri MSP-1 could be categorized into five and three unique amino acid variants, respectively. Conclusions Low mutational diversity was observed in pomsp1 from the Jiangsu Province imported malaria cases; further studies will be developed such as immunogenicity and functional analysis.

Abstract Background Local malaria transmission has decreased rapidly since the National Malaria Elimination Action Plan was launched in China in 2010. However, imported malaria cases from Africa and Southeast Asia still occur in China due to overseas laborers. Diagnosis by microscopy is the gold standard for malaria and is used in most hospitals in China. However, the current capacity of microscopists to manage malaria cases in hospitals and public health facilities to meet the surveillance needs to eliminate and prevent the reintroduction of malaria is unknown. Methods Malaria diagnoses were assessed by comparing the percentage of first visit and confirmed malaria diagnoses at Centers for Disease Control and Prevention (CDCs) and hospitals. The basic personnel information for public health departments and hospitals at different levels was investigated. The skills of microscopists for blood smear preparation and slide interpretation were also examined at the county and township levels. Results Inaccurate rate with 13.49% and 7.32%, respectively, in 2013 and 2014, from 341 and 355 reported cases from sub-provincial levels in Jiangsu province. Most of the 523 malaria cases reported in Nantong Prefecture from 2000 to 2014 involved patients who first visited county CDCs seeking treatment, however, none of these cases received confirmed diagnosis of malaria in townships or villages.The staff at county CDCs and hospitals with a higher education background performed better at making and interpreting blood smears than staff from townships. Conclusions The network for malaria elimination in an entire province has been well established. However, an insufficient capacity for malaria diagnosis was observed, especially the preparing and reading the blood smears at the township and village levels, which is a challenge to achieving and maintaining malaria elimination.

Abstract Background 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. Methods 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. Results 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. Conclusions 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.

Abstract Background Anopheles sinensis is a dominant natural vector of Plasmodium vivax in China, Taiwan, Japan, and Korea. Recent genome sequencing of An. sinensis provides important insights into the genomic basis of vectorial capacity. However, the lack of a physical genome map with chromosome assignment and orientation of sequencing scaffolds hinders comparative analyses with other genomes to infer evolutionary changes relevant to the vector capacity. Results Here, a physical genome map for An. sinensis was constructed by assigning 52 scaffolds onto the chromosomes using fluorescence in situ hybridization (FISH). This chromosome-based genome assembly composes approximately 36% of the total An. sinensis genome. Comparisons of 3955 orthologous genes between An. sinensis and Anopheles gambiae identified 361 conserved synteny blocks and 267 inversions fixed between these two lineages. The rate of gene order reshuffling on the X chromosome is approximately 3.2 times higher than that on the autosomes. Conclusions The physical map will facilitate detailed genomic analysis of An. sinensis and contribute to understanding of the patterns and mechanisms of large-scale genome rearrangements in anopheline mosquitoes.

Currently, malaria rapid diagnostic tests (RDTs) are widely used for malaria diagnosis, but test performance and the factors that lead to failure of Plasmodium ovale detection are not well understood. In this study, three pLDH-based RDTs were evaluated using cases in China that originated in Africa. The sensitivity of Wondfo Pf/Pan, CareStart pLDH PAN and SD BIOLINE Pf/Pan in P. ovale detection was 70, 55 and 18%, respectively. CareStart was worse at detecting P. o. curtisi (36.5%) than at detecting P. o. wallikeri (75.0%), and SD could not detect P. o. curtisi. The overall detection ratio of all three RDTs decreased with parasite density and pLDH concentration. Wondfo, CareStart and SD detected only 75.0, 78.1 and 46.9% of the P. ovale cases, respectively, even when the parasitemia were higher than 5000 parasites/μL. Subspecies of P. ovale should be considered while to improve RDT quality for P. ovale diagnosis to achieve the goal of malaria elimination.

Abstract Background Anopheles lesteri and Anopheles sinensis are two major malaria vectors in China and Southeast Asia. They are dramatically different in terms of geographical distribution, host preference, resting habitats, and other traits associated with ecological adaptation and malaria transmission. Both species belong to the Anopheles hyrcanus group, but the extent of genetic differences between them is not well understood. To provide an effective way to differentiate between species and to find useful markers for population genetics studies, we performed a comparative cytogenetic analysis of these two malaria vectors. Results Presented here is a standard cytogenetic map for An. lesteri, and a comparative analysis of chromosome structure and gene order between An. lesteri and An. sinensis. Our results demonstrate that much of the gene order on chromosomes X and 2 was reshuffled between the two species. However, the banding pattern and the gene order on chromosome 3 appeared to be conserved. We also found two new polymorphic inversions, 2Lc and 3Rb, in An. lesteri, and we mapped the breakpoints of these two inversions on polytene chromosomes. Conclusions Our results demonstrate the extent of structural divergence of chromosomes between An. lesteri and An. sinensis, and provide a new taxonomic cytogenetic tool to distinguish between these two species. Polymorphic inversions of An. lesteri could serve as markers for studies of the population structure and ecological adaptations of this major malaria vector.

Abstract Background Anopheles sinensis is one of the most important malaria vectors in Asian countries. The rapid spread of insecticide resistance has become a major obstacle for insecticide-based strategies for vector control. Therefore, it is necessary to prepare an insecticide-resistant strain of An. sinensis to further understand the insecticide resistance mechanisms in this species to facilitate genetic approaches to targeting the insecticide-resistant population of this important malaria vector. Methods An. sinensis mosquitoes were collected from regions where pyrethroid resistance had been reported. The mosquitoes were subjected to continuous pyrethroid selection after species confirmation, and the forced copulation method was used to increase the mating rate. In addition, the knockdown-resistance (kdr) mutation frequencies of each generation of An. sinensis were measured; and the metabolic enzyme activities of cytochrome P450 monoxygenases (P450s) and glutathione S-transferases (GSTs) were detected. Results The identification of field-captured An. sinensis was confirmed by both morphological and molecular methods. The population of An. sinensis exhibited stable resistance to pyrethroid after continuous generations of pyrethroid selection in the laboratory with high kdr mutation frequencies; and elevated levels of both P450s and GSTs were significantly found in field selected populations comparing with the laboratory susceptible strain. So far, the colonised strain has reached its eleventh generation and culturing well in the laboratory. Conclusions We colonised a pyrethroid-resistant population of An. sinensis in the laboratory, which provides a fundamental model for genetic studies of this important malaria vector.

Anopheles sinensis is the most important vector of malaria in Southeast Asia, including China. Currently, the most effective measure to prevent malaria transmission relies on vector control through the use of insecticides, primarily pyrethroids. Extensive use of insecticides poses strong selection pressure on mosquito populations for resistance. Resistance to insecticides can arise due to mutations in the insecticide target site (target site resistance), which in the case of pyrethroids is the para-type sodium channel gene, and/or the catabolism of the insecticide by detoxification enzymes before it reaches its target (metabolic detoxification resistance). In this study, we examined deltamethrin resistance in An. sinensis from China and investigated the relative importance of target site versus metabolic detoxification mechanisms in resistance. A high frequency (>85%) of nonsynonymous mutations in the para gene was found in populations from central China, but not in populations from southern China. Metabolic detoxification as measured by the activity of monooxygenases and glutathione S-transferases (GSTs) was detected in populations from both central and southern China. Monooxygenase activity levels were significantly higher in the resistant than the susceptible mosquitoes, independently of their geographic origin. Stepwise multiple regression analyses in mosquito populations from central China found that both knockdown resistance (kdr) mutations and monooxygenase activity were significantly associated with deltamethrin resistance, with monooxygenase activity playing a stronger role. These results demonstrate the importance of metabolic detoxification in pyrethroid resistance in An. sinensis, and suggest that different mechanisms of resistance could evolve in geographically different populations.