Genetic Diversity and Natural Selection of Plasmodium vivax Merozoite Surface Protein 8 in Global Populations.

Plasmodium vivax Merozoite Surface Protein 8 (PvMSP8) is a promising candidate target for the development of multi-component vaccines. Therefore, determining the genetic variation pattern of Pvmsp8 is essential in providing a reference for the rational design of the P. vivax malaria vaccines. This study delves into the genetic characteristics of the Pvmsp8 gene, specifically focusing on samples from the China-Myanmar border (CMB) region, and contrasts these findings with broader global patterns. The study uncovers that Pvmsp8 exhibits a notable level of conservation across different populations, with limited polymorphisms and relatively low nucleotide diversity (0.00023-0.00120). This conservation contrasts starkly with the high polymorphisms found in other P. vivax antigens such as Pvmsp1. A total of 25 haplotypes and 14 amino acid mutation sites were identified in the global populations, and all mutation sites were confined to non-functional regions. The study also notes that most CMB Pvmsp8 haplotypes are shared among Burmese, Cambodian, Thai, and Vietnamese populations, indicating less geographical variance, but differ notably from those found in Pacific island regions or the Panama. The findings underscore the importance of considering regional genetic diversity in P. vivax when developing targeted malaria vaccines. Non departure from neutral evolution were found by Tajima's D test, however, statistically significant differences were observed between the kn/ks rates. The study's findings are crucial in understanding the evolution and population structure of the Pvmsp8 gene, particularly during regional malaria elimination efforts. The highly conserved nature of Pvmsp8, combined with the lack of mutations in its functional domain, presents it as a promising candidate for developing a broad and effective P. vivax vaccine. This research thus lays a foundation for the rational development of multivalent malaria vaccines targeting this genetically stable antigen.

Polymorphism of Drug Resistance Genes dhfr and dhps in Plasmodium falciparum Isolates among Chinese Migrant Workers Who Returned from Ghana in 2013.

In 2013, an epidemic of falciparum malaria involving over 820 persons unexpectedly broke out in Shanglin County, Guangxi Zhuang Autonomous Region, China, after a large number of migrant workers returned from Ghana, where they worked as gold miners. Herein, we selected 146 isolates randomly collected from these patients to investigate the resistance characteristics of the parasite to sulfadoxine-pyrimethamine (SP) by screening mutations in the dhfr and dhps genes. All 146 isolates were successfully genotyped for dhps, and only 137 samples were successfully genotyped for dhfr. In the dhfr gene, point mutations occurred at three codons: 51 (83.2%, 114/137), 59 (94.9%, 130/137), and 108 (96.4%, 132/137). In the dhps gene, mutations occurred at four codons: 436 (36.3%, 53/146 for S436A, 0.7%, 1/146 for S436Y), 437 (95.2%, 139/146), 540 (3.4%, 5/146), and 613 (2.7%, 4/146). All 146 isolates had mutations in at least one codon, either within dhfr or dhps. Quadruple mutation I51R59N108/G437 (41.1%, 60/146) of partial or low resistance level was the most prevalent haplotype combination. Quintuple I51R59N108/G437E540 accounted for 2.1% (3/146). Sextuple I51R59N108/A436G437S613 was also found and accounted for 1.4% (2/146). A chronological assay incorporating two sets of resistance data from the studies of Duah and Amenga-Etego provided an overview of the resistance trend from 2003 to 2018. During this period, the results we obtained generally coincided with the total development tendency of SP resistance. It can be concluded that Plasmodium falciparum samples collected from Chinese migrant workers from Ghana presented prevalent but relatively partial or low resistance to SP. A chronological assay incorporating two sets of data around 2013 indicates that our results possibly reflect the SP resistance level of Ghana in 2013 and that the possibility of increased resistance exists. Therefore, reasonable drug use and management should be strengthened while also maintaining a continuous screening of resistance to SP. These findings also underscore the need to strengthen the prevention of malaria importation from overseas and focus on preventing its reintroduction and transmission in China.

Genetic diversity and natural selection of rif gene (PF3D7_1254800) in the Plasmodium falciparum global populations.

To reveal the genetic characteristics of one member of the Plasmodium falciparum repetitive interspersed family (rif), we sequenced the rif gene (PF3D7_1254800) in 53 field isolates collected from Ghana-imported cases into China and compared them with 350 publicly available P. falciparum rif sequences from global populations. In the Ghana-imported population, the nucleotide diversities were 0.05714 and 0.06616 for the full length and variable region of rif gene, respectively. Meanwhile, 22 and 20 haplotypes were identified for the full length and variable region of rif gene (Hd = 0.843 and 0.838, respectively). Diversity of rif gene in Ghana-imported population was higher than that observed in Cambodia, Thailand, Vietnam, Myanmar, Mali, Ghana, and Senegal populations. In this analysis, we found high genetic diversity of rif gene in global P. falciparum populations and identified 158 haplotypes. Tajima's D-test shows that there are large differences in the direction of selection between the conserved and variable region of rif gene. Tajima's D value for the variable region was 0.20074, indicating that balancing selection existed in this region. We found that the variable region was the main target of selection for positive diversification, and most mutation sites were located in this region. The population structure suggested optimized cluster values of K = 6. The five groups in Ghana-imported population included a unique subpopulation. Our results reveal the dynamics of the rif gene (PF3D7_1254800) in P. falciparum populations, which can aid in the rational design of P. falciparum rif-based vaccines.

Retrospective analysis of Plasmodium vivax genomes from a pre-elimination China inland population in the 2010s.

Introduction: In malaria-free countries, imported cases are challenging because interconnections with neighboring countries with higher transmission rates increase the risk of parasite reintroduction. Establishing a genetic database for rapidly identifying malaria importation or reintroduction is crucial in addressing these challenges. This study aimed to examine genomic epidemiology during the pre-elimination stage by retrospectively reporting whole-genome sequence variation of 10 Plasmodium vivax isolates from inland China. Methods: The samples were collected during the last few inland outbreaks from 2011 to 2012 when China implemented a malaria control plan. After next-generation sequencing, we completed a genetic analysis of the population, explored the geographic specificity of the samples, and examined clustering of selection pressures. We also scanned genes for signals of positive selection. Results: China's inland populations were highly structured compared to the surrounding area, with a single potential ancestor. Additionally, we identified genes under selection and evaluated the selection pressure on drug-resistance genes. In the inland population, positive selection was detected in some critical gene families, including sera, msp3, and vir. Meanwhile, we identified selection signatures in drug resistance, such as ugt, krs1, and crt, and noticed that the ratio of wild-type dhps and dhfr-ts increased after China banned sulfadoxine-pyrimethamine (SP) for decades. Discussion: Our data provides an opportunity to investigate the molecular epidemiology of pre-elimination inland malaria populations, which exhibited lower selection pressure on invasion and immune evasion genes than neighbouring areas, but increased drug resistance in low transmission settings. Our results revealed that the inland population was severely fragmented with low relatedness among infections, despite a higher incidence of multiclonal infections, suggesting that superinfection or co-transmission events are rare in low-endemic circumstances. We identified selective signatures of resistance and found that the proportion of susceptible isolates fluctuated in response to the prohibition of specific drugs. This finding is consistent with the alterations in medication strategies during the malaria elimination campaign in inland China. Such findings could provide a genetic basis for future population studies, assessing changes in other pre-elimination countries.

Genetic Diversity and Natural Selection of Plasmodium vivax Duffy Binding Protein-II From China-Myanmar Border of Yunnan Province, China.

Malaria incidence has declined dramatically over the past decade and China was certified malaria-free in 2021. However, the presence of malaria in border areas and the importation of cases of malaria parasites are major challenges for the consolidation of the achievements made by China. Plasmodium vivax Duffy binding protein (PvDBP) performs a significant role in erythrocyte invasion, and is considered a promising P. vivax vaccine. However, the highly polymorphic region of PvDBP (PvDBP-II) impedes the development of blood-stage vaccine against P. vivax. In this study, we investigated the genetic diversity and natural selection of PvDBP-II among 124 P. vivax isolates collected from the China-Myanmar border (CMB) in Yunnan Province, China, during 2009-2011. To compare genetic diversity, natural selection, and population structure with CMB isolates, 85 pvdbp-II sequences of eastern Myanmar isolates were obtained from GenBank. In addition, global sequences of pvdbp-II were retrieved from GenBank to establish genetic differentiation relationships and networks with the CMB isolates. In total, 22 single nucleotide polymorphisms reflected in 20 non-synonymous and two synonymous mutations were identified. The overall nucleotide diversity of PvDBP-II from the 124 CMB isolates was 0.0059 with 21 haplotypes identified (Hd = 0.91). The high ratio of non-synonymous to synonymous mutations suggests that PvDBP-II had evolved under positive selection. Population structure analysis of the CMB and eastern Myanmar isolates were optimally grouped into five sub-populations (K = 5). Polymorphisms of PvDBP-II display that CMB isolates were genetically diverse. Mutation, recombination, and positive selection promote polymorphism of PvDBP-II of P. vivax population. Although low-level genetic differentiation in eastern Myanmar was identified along with the more effective malaria control measures, the complexity of population structure in malaria parasites has maintained. In conclusion, findings from this study advance knowledge of the understanding of the dynamic of P. vivax population, which will contribute to guiding the rational design of a PvDBP-II based vaccine.

Intensive surveillance, rapid response and border collaboration for malaria elimination: China Yunnan's ''3 + 1''strategy.

BACKGROUND: Eliminating malaria and preventing re-establishment of malaria transmission in border areas requires universal coverage of malaria surveillance and a rapid response to any threats (i.e. malaria cues) of re-establishing transmission. MAIN TEXT: Strategy 1: Intensive interventions within 2.5 km-wide perimeter along the border to prevent border-spill malaria. The area within 2.5 km along the international border is the travel radius of anopheline mosquitoes. Comprehensive interventions should include: (1) proactive and passive case detection, (2) intensive vector surveillance, (3) evidence-based vector control, and (4) evidence-based preventative treatment with anti-malarial drugs. Strategy 2: Community-based malaria detection and screening of migrants and travellers in frontier townships. Un-permitted travellers cross borders frequently and present in frontier townships. Maintenance of intensified malaria surveillance should include: (1) passive malaria detection in the township hospitals, (2) seek assistance from villager leaders and health workers to monitor cross border travellers, and refer febrile patients to the township hospitals and (3) the county's Centre for Disease Control and Prevention maintain regular proactive case detection. Strategy 3: Universal coverage of malaria surveillance to detect malaria cues. Passive detection should be consolidated into the normal health service. Health services personnel should remain vigilant to ensure universal coverage of malaria detection and react promptly to any malaria cues. Strategy + 1: Strong collaborative support with neighbouring countries. Based on the agreement between the two countries, integrated control strategies should be carried out to reduce malaria burden for both countries. There should be a clear focus on the border areas between neighbouring countries. CONCLUSION: The 3 + 1 strategy is an experience summary of border malaria control and elimination, and then contributed to malaria elimination in Yunnan's border areas, China. Nevertheless, Yunnan still has remaining challenges of re-establishment of malaria transmission in the border areas, and the 3 + 1 strategy should still be carried out.

Genome-Wide Scans for Ghanaian Plasmodium falciparum Genes Under Selection From Local and Chinese Host Populations.

Initial malarial infection mostly causes symptomatic illness in humans. Infection that is not fatal induces complete protection from severe illness and death, and thus complete protection from severe illness or death is granted with sufficient exposure. However, malaria parasite immunity necessitates constant exposure. Therefore, it is important to evaluate lowered immunity and recurrent susceptibility to symptomatic disease in lower transmission areas. We aimed to investigate selection pressure based on transmission levels, antimalarial drug use, and environmental factors. We whole genome sequenced (WGS) P. falciparum clinical samples from Chinese hosts working in Ghana and compared the results with the WGS data of isolates from native Ghanaians downloaded from pf3k. The P. falciparum samples were generally clustered according to their geographic origin, and Chinese imported samples showed a clear African origin with a slightly different distribution from the native Ghanaian samples. Moreover, samples collected from two host populations showed evidence of differences in the intensity of selection. Compared with native Ghanaian samples, the China-imported isolates exhibited a higher proportion of monoclonal infections, and many genes associated with RBC invasion and immune evasion were found to be under less selection pressure. There was no significant difference in the selection of drug-resistance genes due to a similar artemisinin-based combination therapy medication profile. Local selection of malarial parasites is considered to be a result of differences in the host immunity or disparity in the transmission opportunities of the host. In China, most P. falciparum infections were imported from Africa, and under these circumstances, distinct local selective pressures may be caused by varying acquired immunity and transmission intensity. This study revealed the impact of host switching on the immune system, and it may provide a better understanding of the mechanisms that enable clinical immunity to malaria.

Genetic Diversity and Selection of Plasmodium vivax Apical Membrane Antigen-1 in China-Myanmar Border of Yunnan Province, China, 2009-2016.

Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is an important vaccine candidate for vivax malaria. However, antigenic variation within PvAMA-1 is a major obstacle to the design of a global protective malaria vaccine. In this study, we analyzed the genetic polymorphism and selection of the PvAMA-1 gene from 152 P. vivax isolates from imported cases to China, collected in the China-Myanmar border (CMB) area in Yunnan Province (YP) during 2009-2011 (n = 71) and 2014-2016 (n = 81), in comparison with PvAMA-1 gene information from Myanmar (n = 73), collected from public data. The overall nucleotide diversity of the PvAMA-1 gene from the 152 YP isolates was 0.007 with 76 haplotypes identified (Hd = 0.958). Results from the population structure suggested three groups among the YP and Myanmar isolates with optimized clusters value of K = 7. In addition, YP (2014-2016) isolates generally lacked some K components that were commonly found in YP (2009-2011) and Myanmar. Meanwhile, PvAMA-1 domain I is found to be the dominant target of positive diversifying selection and most mutation loci were found in this domain. The mutation frequencies of D107N/A, R112K/T, K120R, E145A, E277K, and R438H in PvAMA-1 were more than 70% in the YP isolates. In conclusion, high genetic diversity and positive selection were found in the PvAMA-1 gene from YP isolates, which are significant findings for the design and development of PvAMA-1-based malaria vaccine.

Genome-Wide Analysis of the Malaria Parasite Plasmodium falciparum Isolates From Togo Reveals Selective Signals in Immune Selection-Related Antigen Genes.

Malaria is a public health concern worldwide, and Togo has proven to be no exception. Effective approaches to provide information on biological insights for disease elimination are therefore a research priority. Local selection on malaria pathogens is due to multiple factors including host immunity. We undertook genome-wide analysis of sequence variation on a sample of 10 Plasmodium falciparum (Pf) clinical isolates from Togo to identify local-specific signals of selection. Paired-end short-read sequences were mapped and aligned onto > 95% of the 3D7 Pf reference genome sequence in high fold coverage. Data on 266 963 single nucleotide polymorphisms were obtained, with average nucleotide diversity π = 1.79 × 10-3. Both principal component and neighbor-joining tree analyses showed that the Togo parasites clustered according to their geographic (Africa) origin. In addition, the average genome-wide diversity of Pf from Togo was much higher than that from other African samples. Tajima's D value of the Togo isolates was -0.56, suggesting evidence of directional selection and/or recent population expansion. Against this background, within-population analyses identifying loci of balancing and recent positive selections evidenced that host immunity has been the major selective agent. Importantly, 87 and 296 parasite antigen genes with Tajima's D values > 1 and in the top 1% haplotype scores, respectively, include a significant representation of membrane proteins at the merozoite stage that invaded red blood cells (RBCs) and parasitized RBCs surface proteins that play roles in immunoevasion, adhesion, or rosetting. This is consistent with expectations that elevated signals of selection due to allele-specific acquired immunity are likely to operate on antigenic targets. Collectively, our data suggest a recent expansion of Pf population in Togo and evidence strong host immune selection on membrane/surface antigens reflected in signals of balancing/positive selection of important gene loci. Findings from this study provide a fundamental basis to engage studies for effective malaria control in Togo.

Genetic diversity of Merozoite surface protein 1-42 (MSP1-42) fragment of Plasmodium vivax from Indonesian isolates: Rationale implementation of candidate MSP1 vaccine.

Morbidity and mortality related to malaria in Indonesia are attributed to both Plasmodium falciparum and P. vivax parasites. In addition to vaccines for P. falciparum, vaccines against P. vivax are urgently needed for the prevention of the disease. An extensively studied antigen is the carboxyl-terminus of the 42 kDa region of P. vivax merozoite surface protein-1 (PvMSP1-42). The design of a vaccine based on this antigen requires an understanding of the extent of polymorphism. However, there is no information on the genetic diversity of the antigen in Indonesia. This study aimed to profile the diversity of PvMSP1-42 and its two subdomains (PvMSP1-33 and PvMSP1-19) among Indonesian P. vivax isolates. A total of 52 P. vivax-infected blood samples were collected from patients in two different endemic areas in Indonesia: Banjarmasin (Kalimantan) and Sumba Timur (Nusa Tenggara Timur). The polymorphic characteristics and natural selection of PvMSP1-42 were analyzed using the DnaSP, MEGA, and Structure software. Thirty distinct haplotypes of PvMSP1-42 were identified. They displayed amino acid changes compared to the reference PVP01 sequence. Most of the mutations were concentrated in the 33 kDa fragment. PvMSP1-42 of the Indonesian isolates appeared to be under positive selection. Recombination may also play a role in the resulting genetic diversity of PvMSP1. In conclusion, PvMSP1-42 of Indonesian isolates displayed allelic polymorphisms caused by mutation, recombination, and positive selection. These results will aid the understanding of the P. vivax population in Indonesia and to develop a PvMSP1 based vaccine against P. vivax.

High multiple mutations of Plasmodium falciparum-resistant genotypes to sulphadoxine-pyrimethamine in Lagos, Nigeria.

BACKGROUND: Plasmodium falciparum-resistance to sulphadoxine-pyrimethamine (SP) has been largely reported among pregnant women. However, the profile of resistance markers to SP dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) in the general population are varied and not frequently monitored. Currently, SP is used as partner drug for artemisinin combination therapy (SP-artesunate) in some sub-Saharan African countries or as a prophylactic drug in intermittent preventive treatment of malaria during pregnancy and infants and in seasonal malaria chemoprevention (SMC). Profiling of P. falciparum-resistant genotypes to SP is dynamic and critical in providing data that would be useful for malaria control programmes. This study assessed the profile of dhfr and dhps genes genotypes among individuals with malaria in Lagos, Nigeria. METHODS: Molecular markers of SP resistance were identified by nested PCR and sequenced among malaria positive dried blood spots (DBS) that were collected from individuals attending health facilities from January 2013 to February 2014 and during community surveys from October 2010 to September 2011 across different Local Government Areas of Lagos State, Nigeria. RESULTS: A total of 242 and 167 samples were sequenced for dhfr and dhps, respectively. Sequence analysis of dhfr showed that 95.5% (231/242), 96.3% (233/242) and 96.7% (234/242) of the samples had N51I, C59R and S108N mutant alleles, respectively. The prevalence of dhps mutation at codons A437G, A613S, S436A, A581G, I431V and K540E were 95.8% (160/167), 41.9% (70/167), 41.3% (69/167), 31.1% (52/167), 25.1% (42/167), and 1.2% (2/167) respectively. The prevalence of triple mutations (CIRNI) in dhfr was 93.8% and 44.3% for the single dhps haplotype mutation (SGKAA). Partial SP-resistance due to quadruple dhfr-dhps haplotype mutations (CIRNI-SGKAA) and octuple haplotype mutations (CIRNI-VAGKGS) with rate of 42.6% and 22.0%, respectively has been reported. CONCLUSIONS: There was increased prevalence in dhfr triple haplotype mutations when compared with previous reports in the same environment but aligned with high prevalence in other locations in Nigeria and other countries in Africa. Also, high prevalence of dhfr and dhps mutant alleles occurred in the study areas in Lagos, Nigeria five to eight years after the introduction of artemisinin combination therapy underscores the need for continuous monitoring.

Establishment and application of the National Parasitic Resource Center (NPRC) in China.

The National Parasitic Resource Center (NPRC) was created in 2004. It is a first-level platform under the Basic Condition Platform Center of the Ministry of Science and Technology of China. The resource centre involves 21 depository institutions in 15 regions of the country, including human parasite and vector depository, animal parasite depository, plant nematode characteristic specimen library, medical insect characteristic specimen library, trematode model specimen library, parasite-vector/snail model specimen library, etc. After nearly 15 years of operation, the resource centre has been built into a physical library with a database of 11 phyla, 23 classes, 1115 species and 117,814 pieces of parasitic germplasm resources, and three live collection bases of parasitic germplasm resources. A variety of new parasite-related immunological and molecular biological detection and identification technologies produced by the resource centre are widely used in the fields of public health responses, risk assessments on food safety, and animal or plant quarantine. The NPRC is the largest and top level resource centre on parasitology in China, and it is a leading technology platform for collecting and identifying parasitic resources.

Kelch 13 propeller gene polymorphism among Plasmodium falciparum isolates in Lagos, Nigeria: Molecular Epidemiologic Study.

OBJECTIVE: To assess polymorphism in Kelch 13 gene of Plasmodium falciparum isolates in Lagos, Nigeria. METHODS: 195 Plasmodium falciparum-positive dried blood spots collected from individuals that accessed diagnostic care at some health facilities and during community surveys across several Local Government Areas of Lagos State, Nigeria, were investigated for the presence of mutations in the K13 gene by nested polymerase chain reaction (PCR) using haplotype-specific probes and sequencing. RESULTS: Three mutant genotypes of K13 gene were observed: A578S in 0.5%, D464N in 0.5% and Q613H in 1.5%. The frequency of K13 polymorphism was 3.1%, while the remaining parasite population had the wild K13 propeller genes. CONCLUSION: No validated Kelch 13 polymorphism associated with artemisinin resistance was seen among P. falciparum isolates from Lagos, Nigeria. As no clinical study was done, this could not be correlated with artemisinin sensitivity.

OBJECTIF: Evaluer le polymorphisme du gène Kelch 13 dans les isolats de Plasmodium falciparum à Lagos, au Nigéria. MÉTHODES: 195 gouttes de sang séchées positives pour Plasmodium falciparum recueillies auprès d'individus ayant accédé à des soins de diagnostic dans certains centres de santé et lors d'enquêtes communautaires menées dans plusieurs zones du gouvernement local de l'Etat de Lagos, au Nigéria, ont été examinées pour rechercher la présence de mutations du gène K13 par la réaction en chaîne imbriquée de la polymérase (PCR) en utilisant des sondes spécifiques à l'haplotype et par le séquençage. RÉSULTATS: Trois génotypes mutants du gène K13 ont été observés: A578S dans 0,5%, D464N dans 0,5% et Q613H dans 1,5%. La fréquence du polymorphisme K13 était de 3,1%, alors que la population parasitaire restante avait les gènes sauvages de l'hélice K13. CONCLUSION: Aucun polymorphisme validé de Kelch 13 associé à une résistance à l'artémisinine n'a été observé parmi les isolats de P. falciparum de Lagos, au Nigéria. Aucune étude clinique n'ayant été réalisée, il n'a pas été possible d'établir une corrélation entre cette observation et la sensibilité à l'artémisinine.

Contribution of Plasmodium immunomics: potential impact for serological testing and surveillance of malaria.

Introduction: Plasmodium vivax (Pv) and P. knowlesi account together for a considerable share of the global burden of malaria, along with P. falciparum (Pf). However, inaccurate diagnosis and undetectable asymptomatic/submicroscopic malaria infections remain very challenging. Blood-stage antigens involved in either invasion of red blood cells or sequestration/cytoadherence of parasitized erythrocytes have been immunomics-characterized, and are vital for the detection of malaria incidence. Areas covered: We review the recent advances in Plasmodium immunomics to discuss serological markers with potential for specific and sensitive diagnosis of malaria. Insights on alternative use of immunomics to assess malaria prevalence are also highlighted. Finally, we provide practical applications of serological markers as diagnostics, with an emphasis on dot immunogold filtration assay which holds promise for malaria diagnosis and epidemiological surveys. Expert commentary: The approach largely contributes to Pf and Pv research in identifying promising non-orthologous antigens able to detect malaria incidence and to differentiate between past and recent infections. However, further studies to profiling naturally acquired immune responses are expected in order to help discover/validate serological markers of no cross-seroreactivity and guide control interventions. More so, the application of immunomics to knowlesi infections would help validate the recently identified antigens and contribute to the discovery of additional biomarkers of exposure, immunity, or both.

Whole-genome sequencing and analysis of Plasmodium falciparum isolates from China-Myanmar border area.

BACKGROUND: China has made progress in malaria control and aims to eliminate malaria nationwide, but implementing effective interventions along the border regions remain a huge task. The Plasmodium falciparum cases imported from Southeast Asia has frequently reported especially in the China-Myanmar border (CMB) area. Though, information is scant on P. falciparum genetic variability in this area. METHODS: This study reported P. falciparum isolates genome sequence of six clinical isolates in the CMB area. Furthermore, we estimated the nucleotide diversity, Watterson's estimator and Tajima's D value for the whole genome mutation rate in slide window. RESULTS: Our data were aligned onto 96.05-98.61% of the reference 3D7 genome in high fold coverages. Principal component analysis result showed that P. falciparum clustered generally according to their geographic origin. A total of 91 genes were identified as positive selection with Ka/Ks ratio significantly higher than 1, and most of them were multigene families encoding variant surface antigens (VSAs) such as var, rif and stevor. The enrichment of the positive selection on VSA genes implied that the environment complexity subjected CMB's P. falciparum to more pressure for survival. CONCLUSIONS: Our research suggests that greater genetic diversity in CMB area and the positive selection signals in VSA genes, which allow P. falciparum to fit the host immune system well and aggravate the difficulty of treatment. Meanwhile, results obtained from this study will provide the fundamental basis for P. falciparum population genomic research in CMB area.

Immunomics analysis of Babesia microti protein markers by high-throughput screening assay.

Babesia microti is a protozoan considered to be a major etiological agent of emerging human babesiosis. It imposes an increasing public-health threat and can be overlooked because of low parasitemia or mixed infection with other pathogens. More sensitive and specific antigens are needed to improve the diagnosis of babesiosis. To screen the immune diagnostic antigens of B. microti, 204 sequences from homologue proteins between B. microti and B. bovis genome sequences in PiroplasmaDB were selected. The high throughput cloned and expressed B. microti proteins were screened with the sera from the BALB/c mice infected by B. microti using protein arrays. Ten (5.9%, 10/169) highly immunoreactive proteins were identified, and most (80%, 8/10) of these highly immunoreactive proteins had not been characterized before, making them potentially useful as candidate antigens for the development of diagnostic tools for babesiosis.

Study roadmap for high-throughput development of easy to use and affordable biomarkers as diagnostics for tropical diseases: a focus on malaria and schistosomiasis.

BACKGROUND: Interventions are currently being used against 'infectious diseases of poverty', which remain highly debilitating and deadly in most endemic countries, especially malaria, schistosomiasis, echinococcosis and African sleeping sickness. However, major limitations of current 'traditional' methods for diagnosis are neither simple nor convenient for population surveillance, and showed low sensitivity and specificity. Access to novel technologies for the development of adequate and reliable tools are expressly needed. A collaborative project between African Network for Drugs and Diagnostics Innovation and partner institutions in Africa and China aims to screen suitable serological biomarkers for diagnostic pipelines against these 'diseases of the poor'. METHODS: Parasite-specific exposed versus unexposed individuals were screened and sera or urine/stools were collected through case-control studies in China and African countries. Target genes/open reading frames were selected, then will be cloned and cell-free expressed, quantified and immuno-detected. Target antigens/epitopes will be probed and screened with sera from exposed or unexposed individuals using a high-throughput antigen screening platform as the study progresses. The specificity and sensitivity of highly immunoreactive biomarkers will be evaluated as well, using enzyme-linked immunosorbent assays or dipsticks. DISCUSSION: This roadmap explicitly unfolds the integrated operating procedures with focus on malaria and schistosomiasis, for the identification of suitable biomarkers that will aid the prioritization of diagnostics for population use. However, there is need to further validate any new diagnostic through comparison with standard methods in field deployable tests for each region. Our expectations for the future are to seek regulatory approval and promote the use of diagnostics in endemic areas.

Genome-wide scans for the identification of Plasmodium vivax genes under positive selection.

BACKGROUND: The current trend of Plasmodium vivax cases imported from Southeast Asia into China has sharply increased recently, especially from the China-Myanmar border (CMB) area. High recombination rates of P. vivax populations associated with varied transmission intensity might cause distinct local selective pressures. The information on the genetic variability of P. vivax in this area is scant. Hence, this study assessed the genetic diversity of P. vivax genome sequence in CMB area and aimed to provide information on the positive selection of new gene loci. RESULTS: This study reports a genome-wide survey of P. vivax in CMB area, using blood samples from local patients to identify population-specific selective processes. The result showed that considerable genetic diversity and mean pair-wise divergence among the sequenced P. vivax isolates were higher in some important gene families. Using the standardized integrated haplotype score (|iHS|) for all SNPs in chromosomal regions with SNPs above the top 1% distribution, it was observed that the top score locus involved 356 genes and most of them are associated with red blood cell invasion and immune evasion. The XP-EHH test was also applied and some important genes associated with anti-malarial drug resistance were observed in high positive scores list. This result suggests that P. vivax in CMB area is facing more pressure to survive than any other region and this has led to the strong positive selection of genes that are associated with host-parasite interactions. CONCLUSIONS: This study suggests that greater genetic diversity in P. vivax from CMB area and positive selection signals in invasion and drug resistance genes are consistent with the history of drug use during malaria elimination programme in CMB area. Furthermore, this result also demonstrates that haplotype-based detecting selection can assist the genome-wide methods to identify the determinants of P. vivax diversity.

Whole-genome sequencing of a Plasmodium vivax clinical isolate exhibits geographical characteristics and high genetic variation in China-Myanmar border area.

BACKGROUND: Currently in China, the trend of Plasmodium vivax cases imported from Southeast Asia was increased especially in the China-Myanmar border area. Driven by the increase in P. vivax cases and stronger need for vaccine and drug development, several P. vivax isolates genome sequencing projects are underway. However, little is known about the genetic variability in this area until now. RESULTS: The sequencing of the first P. vivax isolate from China-Myanmar border area (CMB-1) generated 120 million paired-end reads. A percentage of 10.6 of the quality-evaluated reads were aligned onto 99.9% of the reference strain Sal I genome in 62-fold coverage with an average of 4.8 SNPs per kb. We present a 539-SNP marker data set for P. vivax that can identify different parasites from different geographic origins with high sensitivity. We also identified exceptionally high levels of genetic variability in members of multigene families such as RBP, SERA, vir, MSP3 and AP2. The de-novo assembly yielded a database composed of 8,409 contigs with N50 lengths of 6.6 kb and revealed 661 novel predicted genes including 78 vir genes, suggesting a greater functional variation in P. vivax from this area. CONCLUSION: Our result contributes to a better understanding of P. vivax genetic variation, and provides a fundamental basis for the geographic differentiation of vivax malaria from China-Myanmar border area using a direct sequencing approach without leukocyte depletion. This novel sequencing method can be used as an essential tool for the genomic research of P. vivax in the near future.

[Digital Description and Identification of 11 Kinds of Principal Parasite Eggs].

Objective: To facilitate the identification of parasite eggs using computer technology, establish the automation-based applications, and propose an algorithm for egg classification. Methods: Eggs of 11 parasites, Clonorchis sinensis, Taenia solium, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Spirometra mansoni, Diphyllobothrium latum, Ancylostoma duodenale, Schistosoma japonicum, Paragonimus westermani and Fasciolopsis buski, were selected and divided into two groups, the training group and the testing group, and were microphotographed. The eigenvalue was extracted using the VC++-based method. The eigenvalue database was constructed, and the training data set was tested with a variety of classification algorithms. The classifier was constructed using algorithm with the highest efficiency and an identification method was established by multi-feature fusion. Results: After removal of images with invalid values, the training group received 19 844 egg images, and the testing group, 3 721 images. Based on the 14 eigenvalues, there were significant differences in the size and color among the eggs of 11 parasite species. For example, the length, width, area and brightness of the smallest parasite egg of Clonorchis sinensis were 292.24 µm, 192.64 µm, 43 416.61 µm2 and 53.84, respectively, while those of the largest parasite egg of Fasciolopsis buski were 945.31 µm, 610.88 µm, 536 002.60 µm2 and 100.54, respectively. When using dynamic weights to construct the classifier, the discrimination rate on the training data set was 88.89%（17 641/19 844）, and that on the verification data set was 91.83%（3 004/3 271）, with an average modeling time of 0.01 s. Conclusion: The algorithm for egg classification has been established, which pravides a basis for further study on its feasibility.