Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

Development and validation a prognostic model based on natural killer T cells marker genes for predicting prognosis and characterizing immune status in glioblastoma through integrated analysis of single-cell and bulk RNA sequencing.

BACKGROUND: Glioblastoma (GBM) is an aggressive and unstoppable malignancy. Natural killer T (NKT) cells, characterized by specific markers, play pivotal roles in many tumor-associated pathophysiological processes. Therefore, investigating the functions and complex interactions of NKT cells is great interest for exploring GBM. METHODS: We acquired a single-cell RNA-sequencing (scRNA-seq) dataset of GBM from Gene Expression Omnibus (GEO) database. The weighted correlation network analysis (WGCNA) was employed to further screen genes subpopulations. Subsequently, we integrated the GBM cohorts from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases to describe different subtypes by consensus clustering and developed a prognostic model by least absolute selection and shrinkage operator (LASSO) and multivariate Cox regression analysis. We further investigated differences in survival rates and clinical characteristics among different risk groups. Furthermore, a nomogram was developed by combining riskscore with the clinical characteristics. We investigated the abundance of immune cells in the tumor microenvironment (TME) by CIBERSORT and single sample gene set enrichment analysis (ssGSEA) algorithms. Immunotherapy efficacy assessment was done with the assistance of Tumor Immune Dysfunction and Exclusion (TIDE) and The Cancer Immunome Atlas (TCIA) databases. Real-time quantitative polymerase chain reaction (RT-qPCR) experiments and immunohistochemical profiles of tissues were utilized to validate model genes. RESULTS: We identified 945 NKT cells marker genes from scRNA-seq data. Through further screening, 107 genes were accurately identified, of which 15 were significantly correlated with prognosis. We distinguished GBM samples into two distinct subtypes and successfully developed a robust prognostic prediction model. Survival analysis indicated that high expression of NKT cell marker genes was significantly associated with poor prognosis in GBM patients. Riskscore can be used as an independent prognostic factor. The nomogram was demonstrated remarkable utility in aiding clinical decision making. Tumor immune microenvironment analysis revealed significant differences of immune infiltration characteristics between different risk groups. In addition, the expression levels of immune checkpoint-associated genes were consistently elevated in the high-risk group, suggesting more prominent immune escape but also a stronger response to immune checkpoint inhibitors. CONCLUSIONS: By integrating scRNA-seq and bulk RNA-seq data analysis, we successfully developed a prognostic prediction model that incorporates two pivotal NKT cells marker genes, namely, CD44 and TNFSF14. This model has exhibited outstanding performance in assessing the prognosis of GBM patients. Furthermore, we conducted a preliminary investigation into the immune microenvironment across various risk groups that contributes to uncover promising immunotherapeutic targets specific to GBM.

SARS-CoV-2 Omicron (B.1.1.529) infection in rhesus macaques, hamsters, and BALB/c mice with severe lung histopathological damage.

Since the first SARS-CoV-2 outbreak in late 2019, the SARS-CoV-2 genome has harbored multiple mutations, especially spike protein mutations. The currently fast-spreading Omicron variant that manifests without symptoms or with upper respiratory diseases has been recognized as a serious global public health problem. However, its pathological mechanism is largely unknown. In this work, rhesus macaques, hamsters, and BALB/C mice were employed as animal models to explore the pathogenesis of Omicron (B.1.1.529). Notably, Omicron (B.1.1.529) infected the nasal turbinates, tracheae, bronchi, and lungs of hamsters and BALB/C mice with higher viral loads than in those of rhesus macaques. Severe histopathological damage and inflammatory responses were observed in the lungs of Omicron (B.1.1.529)-infected animals. In addition, viral replication was found in multiple extrapulmonary organs. Results indicated that hamsters and BALB/c mice are potential animal models for studies on the development of drugs/vaccines and therapies for Omicron (B.1.1.529).

Utilizing biochar to decorate nanoscale FeS for the highly effective decontamination of Se(IV) from simulated wastewater.

Selenium (Se) as an essential nutrient for human beings at trace concentrations, the allowable concentration for the human is only 40 µg/L. Iron sulfide (FeS) nanoparticles have been applied for excessive of selenium (Se) remediation in surface water and groundwater. In this study, FeS nanoparticles were anchored onto biochar (BC) to reduce agglomeration of FeS and prepared into the composite of FeS-BC by pyrolysis to economically and efficiently remove Se(IV) from simulated wastewater based on the excellent performance of FeS and the low cost of BC. Characterizations presented the uniform anchorage of FeS on the BC surface to prevent agglomeration. The results of batch experiments revealed that the removal of Se(IV) by FeS-BC nanomaterials significantly depended on the pH value, with the maximum removal of ∼174.96 mg/g at pH 3.0. A pseudo-second-order kinetic model well reflected the kinetic removal of Se(IV) in pure Se(IV) solution with different concentration, as well as the coexistence of K+, Ca2+, Cl-, and SO42- ions. The presence of K+ ions significantly inhibited the removal of Se(IV) with the increase of K+ ion concentration compared with the effect of the other three ions. SEM-EDS and XPS analyses indicated that the removal process was achieved through adsorption by surface complexation, and reductive precipitation of Se(IV) into Se0 with the electron donor of Fe(II) and S(-II) ions. The FeS-BC nanomaterial exhibited an excellent application prospect in the remediation of Se(IV).

Genetic polymorphism of circumsporozoite protein of Plasmodium falciparum among Chinese migrant workers returning from Africa to Henan Province.

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

Molecular Surveillance of Drug Resistance of Plasmodium falciparum Isolates Imported from Angola in Henan Province, China.

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

Molecular investigation of the Pfmdr1 gene of Plasmodium falciparum isolates in Henan Province imported from Africa.

Efficacious antimalarial drugs are important for malaria control and elimination, and continuous monitoring of their efficacy is essential. The prevalence and distribution of Pfmdr1 were evaluated in African migrant workers in Henan Province. Among 632 isolates, 13 haplotypes were identified, NYSND (39.87%, 252/632), YYSND (2.85%, 18/632), NFSND (31.01%, 196/632), NYSNY (0.47%, 3/632), YFSND (13.77%, 87/632), NFSNY (0.32%, 2/632), YYSNY (2.06%, 13/632), YFSNY (0.16%, 1/632), N/Y YSND (1.90%, 12/632), N Y/F SND (6.17%, 39/632), N/Y Y/F SND (0.47%, 3/632), YYSN D/Y (0.16%, 1/632) and N/Y FSND (0.79%, 5/632). The highest frequency of NYSND was observed in individuals from North Africa (63.64%, 7/11), followed by South Africa (61.33%, 111/181), Central Africa (33.33%, 56/168), West Africa (28.94%, 68/235) and East Africa (27.03%, 10/37) (χ2 = 54.605, P < 0.05). The highest frequency of NFSND was observed in East Africa (48.65%, 18/37), followed by West Africa (39.14%, 92/235), Central Africa (26.79%, 45/168), South Africa (22.65%, 41/181) and North Africa (9.09%, 1/11) (χ2 = 22.368 P < 0.05). The mutant prevalence of codons 86 and 184 decreased. These data may provide complementary information on antimalarial resistance that may be utilized in the development of a treatment regimen for Henan Province.

Polymorphisms analysis of the Plasmodium ovale tryptophan-rich antigen gene (potra) from imported malaria cases in Henan Province.

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

Analysis of polymorphisms in the circumsporozoite protein gene of Plasmodium vivax isolates from Henan Province, China.

BACKGROUND: Plasmodium vivax malaria has historically been a major source of disease in Henan, China. In the 1970s, the morbidity of malaria was highest in the country. With support from the government and the efforts of healthcare personnel, the reported malaria cases have declined dramatically and a national elimination programme was launched in 2010. To achieve the goal, it is essential to study the diversity of autochthonous malaria and transmission of Plasmodium parasites, which will provide baseline data for disease control and management. METHODS: Thirty-two P. vivax isolates from Henan province were collected from 2008 to 2011, and circumsporozoite protein (csp) genes were analysed to estimate the genetic diversity of this parasite. RESULTS: The assessment of csp sequences indicated that all the isolates were the VK210 type, however, none of them was identical to the VK210 strain. The sequences displayed variations in the central region, and eight sub-types were observed. Among the sub-types, HN7 was the most prevalent (37.5%), followed by HN3 (34.4%). A total of 653 repeat units were discovered in 32 Henan isolates. Nucleotide sequences were grouped in 13 unique repeat nucleotide sequence allotypes that coded for 7 different repeated amino acid allotypes. B (GNGAGGQAA) and D (GDRAAGQPA) were more frequent based on the results; they represented 53.9% (352/653) of the total. In comparison to the basic repeat units of VK210, more than 75% of the central repeat units had at least one non-synonymous nucleotide change. CONCLUSIONS: Recent P. vivax populations in Henan province showed some degree of genetic diversity in csp, with 8 sub-types among 32 samples. Meantime, the results also suggested its relative conserved parasite populations. This could provide interesting baseline data that allow identifying whether potential new cases differ from the parasites already circulating in the area.

Polymorphisms of Plasmodium falciparum k13-propeller gene among migrant workers returning to Henan Province, China from Africa.

BACKGROUND: Henan Province has been in the malaria elimination stage, with all reports of the disease being imported since 2012 and over 90% coming from Africa. Surveillance and population studies are essential for the early detection and subsequent prevention of the spread of drug resistance. The K13-propeller gene was recently identified as a proposed molecular marker of artemisinin (ART) resistance. In this study, we detected mutations of the K13-propeller gene in samples taken from imported malaria cases in Henan Province from 2012 to 2015. METHODS: There were 483 samples that were obtained from Plasmodium falciparum-infected malaria migrant workers who returned to Henan Province from Africa between 2012 and 2015. The single nucleotide polymorphisms in the K13-propeller gene were assessed by nested PCR with DNA sequencing. Frequency and geographic difference of K13-propeller gene mutant types were analyzed. RESULTS: Of 483 patients, 476 were cured and 7 died. There were no K13-propeller mutations in the blood samples from the 7 patients who died, but there were 23 different genotypes of the K13-propeller that were observed in 24 (4.97%) of the samples. C580Y, which was the predominant one in the resistance of ART, was not detected in the samples, but R539T and P574L which have also been associated with ART resistance, were observed in two samples from Angola and Equatorial Guinea. No mutations were detected in 11 samples from North Africa. The frequency of the K13-propeller was 6.50% (8/123) in Central Africa, followed by East Africa (1/19, 5.26%), West Africa (9/198, 4.55%) and South Africa (6/132, 4.55%). There was no significant difference among these four areas (P = 0.795). CONCLUSION: R539T and P574L were found in migrant workers who traveled from Africa to Henan Province, although the frequency of the K13-propeller mutants was low. These data may enrich the molecular surveillance of antimalarial resistance and will be helpful for developing and updating the antimalarial policy in Henan Province.

Ecological control line: A decade of exploration and an innovative path of ecological land management for megacities in China.

Ecological control line is a system innovation in the field of ecological environment protection in China and it has become as an important strategy of national ecological protection. Ten years have passed since the first ecological control line in Shenzhen was delimited in 2005. This study examines the connotations of ecological control line and the current study status in China and abroad, and then takes a brief description about the delimitation background and existing problems of the ecological control line in Shenzhen. The problem-solving strategy is gradually transforming from extensive management to refined management. This study proposes a differential ecological space management model that merges the space system, management system, and support system. The implementation paths include the following five aspects: delimiting ecological bottom lines to protect core ecological resources; formulating access systems for new construction projects to strictly control new construction; implementing construction land inventory reclamation assisted by market means; regulating boundary adjusting procedures and processes; and constructing ecological equity products by using multiple means to implement rights relief. Finally, this study illustrates the progress of the implementation and discusses the rigorousness and flexibility problems of ecological control line and calls for the promotion of the legislation. The management model and implementation paths proposed in this study have referential significance for developing countries and megacities to achieve ecological protection and sustainable development.

[Polymorphisms of tryptophan-rich antigen gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province].

Objective: To analyze the genetic sequence of tryptophan-rich antigen (PoTRA) gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province in 2015. Methods: Blood samples were collected from 22 imported ovale malaria cases in Henan Province in 2015. After DNA extraction, PoTRA was amplified by nested PCR, and was inserted into the pMD18-T vector. The plasmid was extracted and sequenced, and the results were blasted in GenBank to determine the subspecies of P. ovale. The sizes and species of the PoTRA gene were analyzed. The amino-acid sequence of PoTRA was also aligned to analyze the difference in amino acid sequence. The phylogenetic tree was constructed to analyze the genetic relationship among the samples by neighbor-joining. Results: Of the 22 imported cases, eight (36.4%) were infected with P. ovale wallikeri, which had two sizes, the predominant 245 and 299 bp. The other 14 cases (63.6%) were infected with P. ovale curtisi, which had three sizes, the predominant 299, 317 and 335 bp. Amino-acid sequence alignment revealed that the two types of P. ovale wallikeri differed in two amino-acid units, MANPINMANPIN and AITPIN, while the three types of P. ovale curtisi differed in amino-acid units TITPIS and TINPIN. The phylogenetic tree showed that the 22 samples belonged to two subpopulations of P. ovale curtisi and P. ovale wallikeri, wherein the P. ovale curtisi was further divided into two sub-branches, and samples with sizes of 317 and 335 bp were in the same sub-branch with a closer genetic relationship. Conclusion: Two subspecies, P. ovale curtisi and P. ovale wallikeri, are identified from the imported ovale malaria cases in Henan Province in 2015. The P. ovale curtisi has three genetic types and P. ovale wallikeri has two genetic types of PoTRA gene, revealing genetic polymorphisms of PoTRA.

Molecular mutation profile of pfcrt in Plasmodium falciparum isolates imported from Africa in Henan province.

BACKGROUND: Anti-malarial drug resistance is a primary public health problem. Haplotypes of pfcrt gene have been implicated to be molecular markers of chloroquine (CQ) resistance. This study aims to explore the prevalence of polymorphisms in pfcrt in Plasmodium falciparum-infected patients imported from Africa in Henan province. METHODS: Blood samples were collected from 502 patients who were infected with P. falciparum returning from Africa in Henan province during 2012-2015. The single nucleotide polymorphisms in pfcrt (codons 72-76) were assessed by nested PCR with DNA sequencing and restriction digestion, the haplotype prevalences were also determined. RESULTS: Four haplotypes coding 72-76 of pfcrt were found including CVMNK (wild type), CVIET (mutation type), CVIEK (mutation type), and CV M/I N/E/D/K K/T (mixed type), with 61.95 % (311/502), 33.07 % (166/502), 0.20 % (1/502), and 4.78 % (24/502) prevalence, respectively. Except mixed type, CVIET and CVIEK were the largest proportion of the mutant type in West Africa, accounting for 44.83 % (91/203), followed by East Africa (8/21, 38.10 %), North Africa (4/11, 36.36 %), Central Africa (36/135, 26.67 %), and South Africa (28/132, 21.21 %). There was significant difference among the groups (χ(2) = 23.78, P < 0.05). Mixed type was the largest proportion in North Africa (9.09 %), followed by Central Africa (6.67 %), East Africa (4.76 %), South Africa (4.55 %), and West Africa (3.45 %). There was no significant difference among the groups (χ(2) = 2.31, P > 0.05). The position 72 and 73 of pfcrt showed predominance for the wild type with rates of 100 % (502/502). CONCLUSIONS: This study identified four haplotypes of pfcrt in P. falciparum-infected patients imported from Africa in Henan province. The prevalence of mutations in the pfcrt was dropped comparing with other people's researches. It establishes fundamental data for detection of P. falciparum CQR with molecular markers for the imported P. falciparum in China, and it also provides complementary information of CQR for the malaria endemic countries and assesses the evolution of anti-malarial drug resistance.

[Medical Costs on Malaria in Henan Province and Analysis of the Influential Factors].

Objective: To analyze the costs for the diagnosis and treatment of malaria in Henan Province and the influential factors. Methods: Malaria cases diagnosed in and reported by medical institutions in Henan Province from November 2013 to October 2014 were selected. General information and clinical information of those with further microscopic confirmation were also collected. The rank sum test for two or more independent samples and stepwise regression analysis were used to investigate the influential factors for medical costs. Results: A total of 218 malaria cases were finally included, of whom 73.4% were from rural areas. On average, the medical costs for patients from rural areas and cities/towns were 1 503 yuan and 4 833 yuan respectively. The average medical cost per patient with first-visit in rural hospitals was 2 600 yuan, and that with first-visit in provincial hospitals was 7 800 yuan. The average medical costs for patients diagnosed in county/city-level hospitals and provincial hospitals were 1 022.5 yuan and 6 170 yuan, respectively. There was null cost for patients diagnosed at the first-visit, while for those diagnosed after 3 or more visits the average cost per patient was 5 621 yuan. Factors significantly associated with medical costs were the current living locality of patients, the hospital level of first-visit, the hospital level of diagnosis and the number of visits before diagnosis（P＜0.05）. Stepwise regression analysis showed that the hospital level of first-visit was the most important influential factor for medical cost, followed by the hospital level of diagnosis and the number of visits before diagnosis. The higher hospital levels of first-visit and diagnosis, the higher cost. The same applied to the number of visits before diagnosis. Conclusion: There is an considerable correlation between medical cost and health seeking behavior in malaria patients.

[ Effect of Malaria Prevention and Control in Henan Province from 2010 to 2014].

During 2010-2014 a total of 1 779 malaria cases were reported in Henan Province, including 958 indigenous cases, and 821 imported cases, with 12 deaths. No indigenous malaria cases were reported during 2012-2014, but the number of imported cases showed a trend of increase, rising from 106 cases in 2010 to 216 in 2014. Blood examination was conducted in 5 210 428 patients with fever during 2010-2014. A total of 4 829 anophelines were captured, of which 4 741 were Anopheles sinensis and 88 were Anopheles anthropophagus. A total of 28 067 patients were given pre-transmission medications. In addition, 3 112 staff for Plasmodium microscopical examinations and 783 epidemiological researchers received training in the five years. Seventy-two counties (cities, districts) passed the evaluation for malaria elimination. Future work should focus on the enhancement of inspection and management of imported malaria to prevent its further dissemination and to achieve the final goal of malaria elimination in the province.

[The Role of Medical Institutions in the Control of Imported Malaria Since the Initiation of Malaria Elimination Program in Henan Province].

Objective: To retrospectively overview the diagnosis and treatment of malaria during 2010-2014 in Henan Province and understand the role of medical institutions in imported malaria control. Methods: Information on malaria epidemic situation as well as its diagnosis and treatment during 2010-2014 in Henan Province was collected from the infectious disease surveillance system and information management system for the prevention and treatment of parasitic disease. Descriptive analysis was performed to determine the role of medical institutions in the reporting, diagnosis and treatment of malaria. Results: A total of 821 imported malaria cases were reported during 2010-2014 in Henan Province, among whom 12 died, with a case fatality rate of 1.7%. The 12 deaths were all due to falciparum malaria and from Africa. The number of cases reported by medical institutions and disease control agencies were 432（52.6%） and 389（47.4%）, respectively. Among the 569 imported malaria cases with diagnosis records, 380 were determined to be malaria at first-diagnosis, with a diagnostic accuracy of 66.8%（380/569）. The accuracy of first diagnosis by medical institutions（49.2%, 178/362） was significantly lower than that by disease control agencies（97.6%, 202/207）（χ2=139.147, P<0.01）. The accuracy of first diagnosis by medical institutions at the town-ship level or below, the county level, the city/prefecture level and the provincial level was 14.2%（18/127）, 43.4% （23/53）, 73.6%（67/97） and 76.9%（70/91）, respectively（χ2=112.764, P<0.01）. However, there was no significant difference in the accuracy among disease control agencies of the above levels（χ2=0.380, P＞0.05）. The cases diagnosed by medical institutions and disease control agencies constituted 48.9%（278/569） and 51.1%（291/569）, respectively, with no significant difference（χ2=0.594, P＞0.05）. In addition, the cases diagnosed by medical institutions at the town-ship level or below, the county level, the city/prefecture level and the provincial level constituted 1.2%（7/569）, 3.7%（21/569）, 12.5%（71/569） and 31.5%（179/569）, respectively（χ2=299.143, P<0.01）. Similar results were also obtained for disease control agencies of the above levels（χ2=91.569, P<0.01）. Conclusion: There are considerable differneces of the first diagnosis accuracy and the diagnosis rate among medical institutions of different levels. Medical institutions of lower levels, which establish a diagnosis mainly based on microscopic examination, have lower diagnosis rate than the disease control agencies at same levels.

[Analysis of Pfcrt Gene Polymorphism in Imported Falciparum Malaria Cases in Henan Province in 2015].

Objective: To analyze Plasmodium falciparum chloroquine resistant transporter (Pfcrt) gene polymorphism in imported falciparum malaria cases in Henan Province in 2015. Methods: Blood samples were collected from 132 cases of imported falciparum malaria in Henan Province in 2015. DNA was extracted from the samples, and the Pfcrt was amplified by nested PCR using specific primers. The PCR products were digested by restriction endonuclease enzyme Apol I and sequenced. Pfcrt gene polymorphism and distribution were analyzed. Results: Most of the 132 cases of imported malaria were young male adults returning from the Africa, with the highest percentage in those from West Africa（38.6%, 51/132）, then Central Africa（26.5%, 35/132）, South Africa（25.0%, 33/132）, East Africa（8.3%, 11/132）, and North Africa（1.5%, 2/132）. The nested PCR yielded a 145-bp product for each sample, and 66.7%（88/132） of the products were completely digested by Apol I, resulting in two fragments of 114 bp and 31 bp; 32.6%（43/132） could not been digested and only a single fragment of 145 bp was shown; and 0.8%（1/132） were incompletely digested, yielding three fragments of 145 bp, 114 bp and 31 bp. By blasting against chloroquine sensitive strain 3D7, we found mutations of Pfcrt at sites correspondig to residues 74, 75 and 76 from ATG, AAT and AAA to ATT, GAA and ACA （i.e. M74I, N75E and K76T） in 43 of the 132 blood samples, and mixed type mutations into ATG/T, A/GAA/T and AA/CA at sites correspondig to residues 74, 75 and 76（CVM/I, N/E/D/K, T/K） in one blood sample. The other 88 blood samples showed a wild type with no mutation (CVMNK). Mutations occurred mainly in cases from West Africa（41.2%, 21/51）, then East Africa（36.4%, 4/11）, South Africa（30.3%, 10/33）, and Central Africa（22.9%, 8/27）（χ2=4.07, P＞0.05）. The 2 cases from the North Africa both had wild type Pfcrt; the one with mixed type mutation was from West Africa. Conclusion: Three haplotypes of Pfcrt have been found, including wild type (CVMNK), mutation type (CVIET) and mixed type (CVM/I, N/E/D/K, K/T) in the imported malaria cases. The wild type occupies the highest proportion （66.7%）, while the mutation type possesses a high proportion of 41.2% in cases from West Africa.

Knockdown resistance of Anopheles sinensis in Henan province, China.

BACKGROUND: Vivax malaria was historically epidemic in Henan Province of China and Anopheles sinensis was the main vectors and poor farming communities bare the greatest burden of disease. Knockdown resistance in An. sinensis is one of the mechanisms of resistance against pyrethroids. In the present study, the frequency of mutations from An. sinensis was examined in Henan province, China. METHODS: Anopheles was collected from Kaifeng, Tongbai, Tanghe, Pingqiao, Shihe, and Yongcheng counties of Henan province in 2013. Molecular identification of Anopheles species was conducted by polymerase chain reaction (PCR) amplifying the internal transcribed spacer 2 (ITS2). Part of the IIS6 domain of the para-type sodium channel protein gene was polymerase chain reaction-amplified and directly sequenced. Frequency and geographic difference of kdr gene mutant types were analysed. RESULTS: 208 Anopheles were received molecular identification, of which 169 (81.25%) were An. sinensis, 25 (12.02%) were Anopheles yatsushiroensis, and 12 (5.77%) were Anopheles lesteri. A 325 bp fragment of the para-type sodium channel gene including position 1014 was successfully sequenced from 139 Anopheles, of which 125 (89.93%) were An. sinensis, 12 (8.63%) were An. yatsushiroensis, 2 (1.44%) were An. lesteri. The molecular analyses revealed that mutations existed at codon 1014 in An. sinensis but not in An. yatsushiroensis and An. lesteri. Frequency of kdr mutation was 73.60% (92/125) from population of An. sinensis in Henan province, of which L1014F (TTT + TTC) allele frequencies accounted for 46.40% (58/125), and was higher than that of L1014C(TGT) which accounted for 27.20% (34/125) ( χ2 = 55.423, P < 0.001). The frequency of kdr mutation in Kaifeng county was 100% (49/49), and was higher than that of 37.93% (11/29) in Tongbai, 54.55% (6/11) in Pingqiao, 50.00% (3/3) in Shihe, and 62.50% (10/16) in Yongcheng county, respectively (χ2 = 39.538, P < 0.001; χ2 = 24.298, P < 0.001; χ2 = 25.913, P < 0.001; χ2 = 20.244, P < 0.001). While 92.86% (13/14) frequency of kdr mutation was found in Tanghe county, which was higher than that in Tongbai county (χ2 = 11.550, P = 0.0018). CONCLUSIONS: A high frequency of kdr gene mutations from population of An. sinensis in Henan province was found.

[Tracing Investigation of One Vivax Malaria Case by Detecting the Gene Encoding Circumsporozoite Protein in Henan].

A vivax malaria case in Henan Province was diagnosed as an indigenous case firstly in June 2013, and replased in April 2014. The clinical data of this case were collected and the epidemiological investigation was conducted. The blood samples were examined by Giemsa-stained blood smear, rapid diagnostic test strip (RDT) and nested PCR. This patient stayed at Myanmar for about one week in May 2013, had the symptoms of chills, fever and sweating in June, and was diagnosed as vivax malaria. After treated with artesunate, the symptoms disappeared. The CSP sequence was amplified from the blood samples of the first and second attack, and there was no difference in the central repeat domain of CSP gene. The identity of our two CSP gene sequences to that of Myanmar isolates (GenBank accessssion No. ABS95455, ABS95456) was 95.1% and 100%, while their nucleotide sequence was with 88.8% and 67.1% identity with that of Henan isolates (accessssion No. KP888996, KP889000), respectively. This patient is therefore confirmed as an imported relapse case of Plasmodium vivax infection.

First imported relapse case of Plasmodium vivax malaria and analysis of its origin by CSP sequencing in Henan Province, China.

In recent years, there has been a substantial increase of imported Plasmodium vivax incidence in Henan Province. As China is in a pre-elimination phase, the surveillance of imported malaria is essential, but there is no good way to distinguish imported cases from indigenous cases. This paper reports a case of a 39-year-old man who acquired P. vivax while staying in Indonesia for one month in 2013, and relapsed in Henan, China in 2014. This was diagnosed as vivax malaria based on rapid diagnostic test, Giemsa-stained peripheral blood smear and Plasmodium species-specific nested PCR. The genetic sequence for the circumsporozoite protein genes was analysed and the genetic variations were compared with a previously constructed database of Chinese isolates. The results from the circumsporozoite protein (CSP) gene sequence analysis centered on the repeat patterns showed that the imported cases had completely different sequences from any subtypes from Chinese isolates, but well matched with the countries travelled by the patient. The imported vivax cases were able to clearly distinguish from the indigenous vivax cases by detecting the CSP gene and were able to confim its origin by genotyping.