Antimalarial activity of cecropin antimicrobial peptides derived from Anopheles mosquitoes.

The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles, and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro. We also demonstrate its anti-parasite activity in vivo, using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo. However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro. An-cecB-1 also showed better antimalarial activity in vivo. Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides.

Genome-wide landscape of ApiAP2 transcription factors reveals a heterochromatin-associated regulatory network during Plasmodium falciparum blood-stage development.

Heterochromatin-associated gene silencing controls multiple physiological processes in malaria parasites, however, little is known concerning the regulatory network and cis-acting sequences involved in the organization of heterochromatin and how they modulate heterochromatic gene expression. Based on systematic profiling of genome-wide occupancy of eighteen Apicomplexan AP2 transcription factors by ChIP-seq analysis, we identify and characterize eight heterochromatin-associated factors (PfAP2-HFs), which exhibit preferential enrichment within heterochromatic regions but with differential coverage profiles. Although these ApiAP2s target euchromatic gene loci via specific DNA motifs, they are likely integral components of heterochromatin independent of DNA motif recognition. Systematic knockout screenings of ApiAP2 factors coupled with RNA-seq transcriptomic profiling revealed three activators and three repressors of heterochromatic gene expression including four PfAP2-HFs. Notably, expression of virulence genes is either completely silenced or significantly reduced upon the depletion of PfAP2-HC. Integrated multi-omics analyses reveal autoregulation and feed-forward loops to be common features of the ApiAP2 regulatory network, in addition to the occurrence of dynamic interplay between local chromatin structure and ApiAP2s in transcriptional control. Collectively, this study provides a valuable resource describing the genome-wide landscape of the ApiAP2 family and insights into functional divergence and cooperation within this family during the blood-stage development of malaria parasites.

Poor performance of malaria rapid diagnostic tests for the detection of Plasmodium malariae among returned international travellers in China.

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.

Two decades of capacity building to support global malaria control and elimination: retrospective and prospective international trainings in Jiangsu Institute of Parasitic Diseases, China, 2002-2021.

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.

Predicting the risk of malaria re-introduction in countries certified malaria-free: a systematic review.

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.

Barriers to seeking health care among returning travellers with malaria: A systematic review.

OBJECTIVE: To identify barriers to seeking health care among returning travellers with malaria with the aim of developing targeted interventions that improve early health care-seeking behaviour, diagnosis and treatment. METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we conducted a systematic review of published medical literature, selecting studies that investigated and reported barriers to seeking health care among returning travellers and migrants with malaria. In total, 633 articles were screened, of which four studies met the inclusion criteria after a full-text review. RESULTS: The four studies reported barriers to seeking healthcare among returning travellers in China, the United States, Thailand and the Dominican Republic. Three studies had an observational design. The identified barriers were summarised based on the appraisal delay, illness delay and utilisation delay stages. During appraisal delays, low awareness of malaria was the most significant factor. Once the patient assessed that he or she was ill, belonging to a specific minority ethnicity, being infected with P. vivax and receiving a low level of social support were predictors of delayed health care-seeking. Finally, the most significant factor associated with utilisation delays was the monetary cost. CONCLUSION: The health care-seeking behaviour of returning travellers with malaria should be further investigated and improved. Addressing the identified barriers and gaps in health care-seeking behaviour among returning travellers with malaria, particularly among groups at high risk of travel-associated infections, is important to prevent severe disease and deaths as well as secondary transmission and epidemics.

Using social media for health education and promotion: a pilot of WeChat-based prize quizzes on China national malaria day.

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.

Implementation and challenges to preventing the re-establishment of malaria in China in the COVID-19 era.

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.

Differential metabolome responses to deltamethrin between resistant and susceptible Anopheles sinensis.

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.

Research supporting malaria control and elimination in China over four decades: a bibliometric analysis of academic articles published in chinese from 1980 to 2019.

BACKGROUND: China has accumulated considerable experience in malaria control and elimination over the past decades. Many research papers have been published in Chinese journals. This study intends to describe the development and experience of malaria control and elimination in China by quantitatively analysing relevant research using a bibliometric analysis. METHODS: A long-term, multistage bibliometric analysis was performed. Research articles published in Chinese journals from 1980 to 2019 were retrieved from the Wanfang and China National Knowledge Infrastructure (CNKI) databases. Year of publication, journal name and keywords were extracted by the Bibliographic Items Co-occurrence Matrix Builder (BICOMB). The K/A ratio (the frequency of a keyword among the total number of articles within a certain period) was considered an indicator of the popularity of a keyword in different decades. VOSviewer software was used to construct keyword co-occurrence network maps. RESULTS: A total of 16,290 articles were included. The overall number of articles continually increased. However, the number of articles published in the last three years decreased. There were two kinds of keyword frequency trends among the different decades. The K/A ratio of the keyword 'Plasmodium falciparum' decreased (17.05 in the 1980s, 13.04% in the 1990s, 9.86 in the 2000s, 5.28 in the 2010s), but those of 'imported case' and 'surveillance' increased. Drug resistance has been a continuous concern. The keyword co-occurrence network maps showed that the themes of malaria research diversified, and the degree of multidisciplinary cooperation gradually increased. CONCLUSIONS: This bibliometric analysis revealed the trends in malaria research in China over the past 40 years. The results suggest emphasis on investigation, multidisciplinary participation and drug resistance by researchers and policymakers in malaria epidemic areas. The results also provide domestic experts with qualitative evidence of China's experience in malaria control and elimination.

LncRNA ST8SIA6-AS1 promotes hepatocellular carcinoma progression by regulating MAGEA3 and DCAF4L2 expression.

Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer. In this study, we aimed to explore the role and mechanism of lncRNA ST8SIA6-AS1 in HCC. We found that ST8SIA6-AS1 was upregulated in HCC tissues and associated with poorer overall survival of HCC patients from TCGA. Moreover, ST8SIA6-AS1 was highly expressed in HCC in-house tissues and cells, and ST8SIA6-AS1 upregulation was related to aggressive tumor phenotypes and the poor overall survival of HCC patients. Downregulation of ST8SIA6-AS1 suppressed HCC cell proliferation, migration and invasion in vitro and restrained HCC tumorigenesis in vivo. In terms of mechanism, ST8SIA6-AS1 regulated melanoma-associated antigen (MAGE)-A3 (MAGEA3) and DDB1-and Cul4-associated factor 4-like 2 (DCAF4L2) expression, and rescue experiments verified that ST8SIA6-AS1 played a protumorigenic role in HCC via the regulation of MAGEA3 and DCAF4L2. ST8SIA6-AS1 partly directly bound to miR-129-5p and functioned as a competing endogenous RNA (ceRNA), subsequently facilitating the expression of the miR-129-5p target gene DCAF4L2 to play its role in HCC. In summary, our results identified ST8SIA6-AS1 as an oncogenic lncRNA predicting poor clinical outcomes of patients with HCC. These findings suggest that ST8SIA6-AS1 is a potential therapeutic target for HCC.

Confirmation of the absence of local transmission and geographic assignment of imported falciparum malaria cases to China using microsatellite panel.

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.

Classification of induced malaria case in an elimination setting: investigation of transfusion-transmitted malaria cases.

BACKGROUND: Since the National Malaria Elimination Action Plan was launched in China in 2010, local malaria transmission has decreased rapidly. Zero indigenous cases were reported since 2017. However, after 2010, the proportion of imported cases in China increased from 45.7% in 2010 to 99.9% in 2016, and almost all provinces of China have reported imported cases in recent years. Prevention of the reintroduction of malaria into China is crucial for the maintenance of its malaria-free status. Hence, it is of utmost importance to correctly identify the source of malaria infections within the country. CASE INTRODUCTION AND RESPONSE: In 2016 and 2017, three laboratory-confirmed cases of malaria caused by Plasmodium falciparum were identified in patients with no previous travel history to endemic areas were reported in Jiangsu Province, China, where malaria due to P. falciparum was eliminated about 30 years ago. These were diagnosed after 41, 31 and 39 days of seeking treatment, respectively, and all of them had received blood transfusions. Further investigations indicated that two of the cases had received blood from foreign students (from Indonesia and Ghana), and the other had received blood from an individual who had worked in Equatorial Guinea. All three blood donors were traced, and found to be carrying asymptomatic P. falciparum infections by microscopic examination and PCR. Furthermore, five polymorphic microsatellite markers (C1M4, C4M62, C13M13, C14M17, and C13M63) were typed and used to link parasites from the donors with those of the transfusion-receiving patients. CONCLUSIONS: Three transfusion-transmitted malaria cases were identified in China, all of which were due to the transfusion of blood donated by individuals who had contracted malaria outside the country. These cases can provide a reference for those faced with similar challenges in malaria case identification and classification in other regions. In addition, a stricter screening policy including the use of appropriate detection methods for malaria parasites should be developed and adopted for blood donation in regions undergoing malaria elimination.

The challenge of maintaining microscopist capacity at basic levels for malaria elimination in Jiangsu Province, China.

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.

Comparative physical genome mapping of malaria vectors Anopheles sinensis and Anopheles gambiae.

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.

The increasing importance of Plasmodium ovale and Plasmodium malariae in a malaria elimination setting: an observational study of imported cases in Jiangsu Province, China, 2011-2014.

BACKGROUND: Following initiation of China's National Malaria Elimination Action Plan in 2010, indigenous malaria infections in Jiangsu Province decreased significantly. Meanwhile imported Plasmodium infections have increased substantially, particularly Plasmodium ovale and Plasmodium malariae. Given the risk for malaria resurgence, there is an urgent need to understand the increase in imported P. ovale and P. malariae infections as China works to achieve national malaria elimination. METHODS: An observational study of imported malaria cases in Jiangsu Province, China was carried out for the period of 2011-2014. RESULTS: A total of 1268 malaria cases were reported in Jiangsu Province from 2011 to 2014. Although imported Plasmodium falciparum cases (n = 1058) accounted for 83.4 % of all reported cases in Jiangsu, P. ovale cases (14, 19, 30, and 46) and their proportion (3.7, 9.6, 8.8, and 13.0 %) of all malaria cases increased over the 4 years. Similarly, P. malariae cases (seven, two, nine, and 10) and proportion (1.9, 1.0, 2.6, and 2.8 %) of all malaria cases increased slightly during this time. A total of 98 cases of Plasmodium ovale curtisi (47/98, 48 %) and Plasmodium ovale wallikeri (51/98, 52 %) were identified as well. Latency periods were significant among these Plasmodium infections (p = 0.00). Also, this study found that the latency periods of P. ovale sp., P. malariae and Plasmodium vivax were significantly longer than P. falciparum. However, for both P. ovale curtisi and P. ovale wallikeri infections, the latency period analysis was not significant (p = 0.81). Misdiagnosis of both P. ovale and P. malariae was greater than 71.5 and 71.4 %, respectively. The P. ovale cases were misdiagnosed as P. falciparum (35 cases, 32.1 %), P. vivax (43 cases, 39.4 %) by lower levels of CDCs or hospitals. And, the P. malariae cases were misdiagnosed as P. falciparum (ten cases, 35.7 %), P. vivax (nine cases, 32.1 %) and P. ovale sp. (one case, 3.6 %). Geographic distribution of imported P. ovale sp. and P. malariae cases in Jiangsu Province mainly originated from sub-Saharan Africa such as Equatorial Guinea, Nigeria, and Angola. CONCLUSIONS: Although the vast majority of imported malaria cases were due to P. falciparum, the increase in other rare Plasmodium species originating from sub-Saharan Africa and Southeast Asia should be closely monitored at all levels of health providers focusing on diagnosis and treatment of malaria. In addition to a receptive vector environment, long latency periods and misdiagnosis of P. malariae and P. ovale sp. increase the risk of re-introduction of malaria in China.

Transcriptome profiling of pyrethroid resistant and susceptible mosquitoes in the malaria vector, Anopheles sinensis.

BACKGROUND: Anopheles sinensis is a major malaria vector in China and other Southeast Asian countries, and it is becoming increasingly resistant to the insecticides used for agriculture, net impregnation, and indoor residual spray. Very limited genomic information on this species is available, which has hindered the development of new tools for resistance surveillance and vector control. We used the 454 GS FLX system and generated expressed sequence tag (EST) databases of various life stages of An. sinensis, and we determined the transcriptional differences between deltamethrin resistant and susceptible mosquitoes. RESULTS: The 454 GS FLX transcriptome sequencing yielded a total of 624,559 reads (average length of 290 bp) with the pooled An. sinensis mosquitoes across various development stages. The de novo assembly generated 33,411 contigs with average length of 493 bp. A total of 8,057 ESTs were generated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. A total of 2,131 ESTs were differentially expressed between deltamethrin resistant and susceptible mosquitoes collected from the same field site in Jiangsu, China. Among these differentially expressed ESTs, a total of 294 pathways were mapped to the KEGG database, with the predominant ESTs belonging to metabolic pathways. Furthermore, a total of 2,408 microsatellites and 15,496 single nucleotide polymorphisms (SNPs) were identified. CONCLUSIONS: The annotated EST and transcriptome databases provide a valuable genomic resource for further genetic studies of this important malaria vector species. The differentially expressed ESTs associated with insecticide resistance identified in this study lay an important foundation for further functional analysis. The identified microsatellite and SNP markers will provide useful tools for future population genetic and comparative genomic analyses of malaria vectors.

Development and application of an AllGlo probe-based qPCR assay for detecting knockdown resistance (kdr) mutations in Anopheles sinensis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Assessing the risk of malaria local transmission and re-introduction in China from pre-elimination to elimination: A systematic review.

Assessing the risk of malaria local transmission and re-introduction is crucial for the preparation and implementation of an effective elimination campaign and the prevention of malaria re-introduction in China. Therefore, this review aims to evaluate the risk factors for malaria local transmission and re-introduction in China over the period of pre-elimination to elimination. Data were obtained from six databases searched for studies that assessed malaria local transmission risk before malaria elimination and re-introduction risk after the achievement of malaria elimination in China since the launch of the NMEP in 2010, employing the keywords "malaria" AND ("transmission" OR "re-introduction") and their synonyms. A total of 8,124 articles were screened and 53 articles describing 55 malaria risk assessment models in China from 2010 to 2023, including 40 models assessing malaria local transmission risk (72.7%) and 15 models assessing malaria re-introduction risk (27.3%). Factors incorporated in the 55 models were extracted and classified into six categories, including environmental and meteorological factors (39/55, 70.9%), historical epidemiology (35/55, 63.6%), vectorial factors (32/55, 58.2%), socio-demographic information (15/26, 53.8%), factors related to surveillance and response capacity (18/55, 32.7%), and population migration aspects (13/55, 23.6%). Environmental and meteorological factors as well as vectorial factors were most commonly incorporated in models assessing malaria local transmission risk (29/40, 72.5% and 21/40, 52.5%) and re-introduction risk (10/15, 66.7% and 11/15, 73.3%). Factors related to surveillance and response capacity and population migration were also important in malaria re-introduction risk models (9/15, 60%, and 6/15, 40.0%). A total of 18 models (18/55, 32.7%) reported the modeling performance. Only six models were validated internally and five models were validated externally. Of 53 incorporated studies, 45 studies had a quality assessment score of seven and above. Environmental and meteorological factors as well as vectorial factors play a significant role in malaria local transmission and re-introduction risk assessment. The factors related to surveillance and response capacity and population migration are more important in assessing malaria re-introduction risk. The internal and external validation of the existing models needs to be strengthened in future studies.