Introduction: Pregnant women have historically been underrepresented in clinical trials, resulting in limited evidence regarding malaria management in this population. This knowledge gap is particularly evident for imported malaria cases occurring in non-endemic regions. Case presentation: We describe two cases of imported Plasmodium falciparum malaria in pregnant women managed in a non-endemic setting. Both patients were successfully treated with intravenous artesunate, resulting in clinical recovery and parasite clearance. Both pregnancies resulted in favorable delivery outcomes, and no immediate neonatal adverse events were noted during the follow-up period. Conclusions: Timely diagnosis of imported malaria in pregnant women in non-endemic areas is essential, as it may be overlooked due to its low prevalence and unfamiliar clinical presentation. Clinicians should maintain a high index of suspicion and apply individualized treatment strategies according to clinical assessment and current guidelines to optimize maternal and fetal outcomes.

Background:The description of genetic diversity of malaria parasite populations in malaria-endemic areas may help determine the effectiveness of malaria interventions in the region. This study aimed to evaluate the genetic diversity and allele frequencies of the merozoite surface protein 1 gene (Msp1) and merozoite surface protein 2 gene (Msp2) in Plasmodium falciparum populations collected from Paletwa Township in western Myanmar and two regions along the China-Myanmar border (the Myitsone and Bhamo regions of Myanmar). Methods:Between 2013 and 2022, malaria cases in China were predominantly imported, with border counties in Yunnan reporting the highest number of cases nationwide. Furthermore, malaria transmission rates in adjacent townships in Myanmar remained relatively high. A total of 176 P. falciparum-infected blood samples were collected from local residents with infections in Bhamo and Paletwa Township in Myanmar, as well as from migrant malaria patients from Yunnan, China, who had worked in Myitsone, Myanmar. Among them, 96 samples were collected from Paletwa Township, Myanmar in 2015, and 28 from Bhamo, Myanmar in 2018-2019, both from local residents. In 2013, 55 samples were obtained from Chinese migrant workers returning from Myitsone, Myanmar, via the Tengchong border checkpoint in Yunnan, China. The genomic DNA was extracted, and the Msp1 and Msp2 genes were genotyped by nested PCR using allele-specific primers for P. falciparum. Results:A total of 176 and 162 samples were successfully genotyped at the Msp1 and Msp2 genes, respectively. Among them, 54 samples were successfully amplified for the Msp1 allelic families in Myitsone, 27 samples in Bhamo, and 95 samples in Paletwa Township. Additionally, 46 samples were successfully amplified for the Msp2 allelic families in Myitsone, 24 samples in Bhamo, and 92 samples in Paletwa Township. Among the three cities, MAD20 was the most frequent allele in the Msp1 gene, while 3D7 was the most frequent allele in the Msp2 gene. The MAD20 allele predominated with 89% (48/54), 85% (23/27) and 67% (64/95) in Myitsone, Bhamo and Paletwa Township respectively, followed by K1 with 65% (35/54) and 44% (12/27) in Myitsone and Bhamo, respectively. The second most frequent allele in Paletwa Township was RO33, representing 47% (45/95) of the population. In migrant workers, residents of Bhamo and residents of Paletwa Township, the multiplicity of infection (MOI) estimated from Msp1 was 1.87, 1.33 and 1.56, respectively And meanwhile, the MOI estimated from Msp2 was 1.17, 1.29 and 1.35 respectively. Migrant workers harbored MOI and rate of multi-infections more frequently (72%) than residents of Bhamo or Paletwa Township (43% and 45%, respectively) based on Msp1. Conclusions:The Msp1 revealed higher genetic diversity than Msp2. The prevalence of MOI and multi-infections among local residents was lower than that among migrant workers based on Msp1. The migrant workers in Myitsone exhibited higher MOI and the proportion of multi-infections compared to the locals, suggesting that low immunity among the migrant workers might be the primary reason for this observation, or that the transmission level in Myitsone is higher than in the local areas.

Plasmodium falciparum acetyl-CoA synthetase (PfAcAS) is an important source of acetyl-CoA. We detected mutations S868G and V950I in PfAcAS by whole-genome sequencing analysis in certain recrudescent parasites after treatment with artesunate and dihydroartemisinin-piperaquine. Using CRISPR/Cas9 technology, we engineered parasite lines to carry the PfAcAS S868G and V950I mutations in two genetic backgrounds and evaluated their susceptibilities to antimalarial drugs in vitro. The results demonstrated that PfAcAS S868G and V950I mutations alone or in combination affected the susceptibility of P. falciparum to several antimalarial drugs, including the artemisinin derivatives (dihydroartemisinin, artesunate, and artemether) and chloroquine, although absolute changes in susceptibilities were modest.IMPORTANCEMalaria, an infectious disease caused by Plasmodium parasites and transmitted by mosquitoes, continues to be one of the most pressing public health challenges worldwide. P. falciparum has demonstrated reduced sensitivity to artemisinin-based combination therapies (ACTs), thereby intensifying the difficulties associated with malaria management. Currently, only a limited number of molecular markers exist for identifying drug resistance in P. falciparum, and these markers do not fully elucidate the mechanisms behind this resistance. In this study, we performed whole-genome sequencing analysis on P. falciparum strains that reemerged following ACT treatment. We aim to identify molecules potentially associated with drug resistance, which may provide new molecular markers for monitoring drug resistance in P. falciparum.

Drug resistance in Plasmodium falciparum represents a significant challenge in malaria treatment. Identifying the molecular markers associated with P. falciparum resistance will effectively detect resistance and enhance treatment efficiency. In this study, we utilized the advanced CRISPR/Cas9 technology to precisely insert one, two, or three asparagine residues into the Kelch 13(K13) gene of the 3D7 strain, positioned after the 142nd amino acid residue, resulting in 1N-3D7, 2N-3D7, and 3N-3D7. Using ring-stage survival assays (RSA), drug sensitivity evaluations, and in vitro developmental assessments, our findings revealed a trend: 1) the insertion of asparagine residues into the parasite genome increased RSA, with more asparagine insertions leading to higher RSA. 2) According to the IC50 values, 1N-3D7 and 2N-3D7 exhibited similar sensitivity profiles across all ten tested drugs, with both demonstrating resistance to Naphthoquine, indicating that the insertions of one or two asparagines played an equivalent role in conferring resistance. However, the insertion of three asparagine residues resulted in significantly higher IC50 values compared to the first two forms when tested with Artesunate, Artemether, Dihydroartemisinin, Pyronaridine Phosphate, and Naphthoquine, showing resistance to all five drugs. Furthermore, 3N-3D7 exhibited a prolonged ring phase and a shortened trophozoite phase within red blood cells; the schizont phase appeared synchronous with the others, yet its mature schizonts contained fewer merozoites. Additionally, 3N-3D7 exhibited a fitness defect, with the proportion decreasing gradually during co-culture with 3D7, its fitness cost calculated as 14.88 ± 2.87. All these results support the opinion that the insertion of three asparagines was a molecular marker of resistance to artemisinin derivatives, Pyronaridine Phosphate, and Naphthoquine in P. falciparum.

Artemether-lumefantrine (AL) is the most widely used antimalarial drug for treating uncomplicated falciparum malaria. This study evaluated whether the K65Q mutation in the Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) gene was associated with alternated susceptibility to lumefantrine using clinical parasite samples from Ghana and the China-Myanmar border area. Parasite isolates from the China-Myanmar border had significantly higher IC50 values to lumefantrine than parasites from Ghana. In addition, the K65 allele was significantly more prevalent in the Ghanaian parasites (34.5%) than in the China-Myanmar border samples (6.8%). However, no difference was observed in the lumefantrine IC50 value between the Pfnfs1 reference K65 allele and the non reference 65Q allele in parasites from the two regions. These data suggest that the Pfnfs1 K65Q mutation may not be a reliable marker for reduced susceptibility to lumefantrine.

Plasmodium falciparum acetyl-CoA synthetase (PfACAS) protein is an important source of acetyl-CoA. We detected the mutations S868G and V949I in PfACAS by whole-genome sequencing analysis in some recrudescent parasites after antimalarial treatment with artesunate and dihydroartemisinin-piperaquine, suggesting that they may confer drug resistance. Using CRISPR/Cas9 technology, we engineered parasite lines carrying the PfACAS S868G and V949I mutations in two genetic backgrounds and evaluated their susceptibility to antimalarial drugs in vitro. The results demonstrated that PfACAS S868G and V949I mutations alone or in combination were not enough to provide resistance to antimalarial drugs.

Abstract Background Parasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China–Myanmar border. Methods We collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3β, by PCR-restriction fragment length polymorphism (PCR–RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations. Results PCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3β among the 370 and 378 samples, respectively. PCR–RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3β with Pst I. PCR–RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3β. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion. Conclusions Migrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR–RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

Purpose: Based on the analysis of factors that might affect the male fertility, and according to the practical experience and the current status of the male pregnancy preparation, the pre-pregnancy intervention measures were put forward in order to improve the quality for both male sperm and the embryo, to avoid the occurrence of birth defects, which would provide theoretical support for male preparation of pregnancy. Methods: Through the analysis of the current status and our practical experience on male pregnancy, to find out the factors that might affect the quality of male sperm, and put forward feasible measures. Results: There were many possible factors affecting the quality of male sperm, such as the rapid economic development, environmental pollution, the change of diet, the increase of unhealthy life style and the delay of childbearing age, with the increase of mental stress and iatrogenic injury, the quality of male sperm could be effectively improved by intervening the above factors, including nutrition supplement, change bad habits, antioxidant supplements, rational drug use, avoiding iatrogenic injury etc. Results: In this paper, we analyzed the factors that may influence the quality of male sperm, and put forward scientific suggestions and feasible practical strategies, it has theoretical and practical value for good pregnancy outcome and avoiding birth defects.

People are seeking fast, simple, easy and inexpensive method for DNA extraction form malaria parasites collected on filter paper. The quality and quantity of DNA is key for molecular diagnosis and analysis. Here, we developed a simple alkali lysis DNA extraction method from filter paper blood samples, and the PCR results gained from the method were compared with traditional heating method. We used different concentrations of NaOH to isolate DNA from filter paper blood, and then purging the sample filter with TE. After natural drying, the sample filters were used as template for nested-PCR. PCR results were examined by agarose gel electrophoresis or sequencing. The results showed that 0-50mM NaOH could isolate the DNA successfully, and 10mM NaOH was the best one.

青蒿琥酯延迟性溶血(post-artesunate delayed haemolysis，PADH)是指使用注射用青蒿琥酯抢救重症疟疾(尤其是高密度疟原虫血症)患者，其血内疟原虫消失后1~3周出现血红蛋白减少和乳酸脱氢酶升高的溶血性贫血表现。其机制是青蒿琥酯杀灭红细胞内的疟原虫后被排出红细胞外，而该红细胞仍继续存活，但其寿命缩短，导致被感染过的红细胞延迟破裂。PADH避开了重症疟疾患者抢救最危险的时期，减少了抢救过程中的风险。本研究对PADH的定义和机制、临床研究和治疗进行综述，提高临床医师对PADH的认识和处理水平。

成人学历教育作为继续教育的重要组成部分,存在工学矛盾突出、学生到课率低、学习动机不足、考试成绩不理想等亟待解决的问题.混合式教学利用技术超越时间和空间的限制,创建真实的学习环境,是解决这些问题的有效途径.以"医学细胞生物学"课程为例,从教学目标、教学内容、学习资源、教学模式与学习活动、学习支持、考核与评价六个方面,设计基于SPOC的成人医学学历教育混合式教学.经过实践,学生到课率、及格率及成绩都大幅度提升,取得了良好的教学效果,提高了教学质量,值得推广.

Background: The genetic diversity of malaria parasites traces the origin and spread of new variants and can be used to evaluate the effectiveness of malaria control measures. Therefore, this study aims to improve the understanding of the molecular epidemiology of Plasmodium vivax malaria at the China-Myanmar border by genotyping the PvMSP-3 alpha and PvMSP-3 ss genes. Methods: Blood samples were collected from P. vivax malaria patients along the China-Myanmar border. The PvMSP-3 alpha and PvMSP-3 ss genes were amplified by polymerase chain reaction (PCR) and the genetic polymorphism and haplotype of the two genes were analyzed. Results: A total of 422 blood samples were used for this study, of which 224 were analyzed at PvMSP-3 alpha and 126 at PvMSP-3 ss. Samples mainly were from young adults aged 18-45 years, although local patients were significantly younger than migrant laborers crossing the border at Tengchong (P < 0.0001). Molecular evolutionary analysis revealed that PvMSP-3 alpha and PvMSP-3 ss underwent diversifying natural selection, and intragenic recombination contributed to the diversity of the isolates. Based on the length of the genes, we identified three types of PvMSP-3 alpha [1.9-2.0 kb (Type-A), 1.4-1.5 kb (Type-B), and 1.1-1.3 kb (Type-C)] and two types of PvMSP3 ss [1.7-2.2 kb (Type-A) and 1.4-1.5 kb (Type-B)]. Migrant laborers returning to China through Tengchong bore P. vivax infections displaying significantly higher genetic diversity than local residents. Conclusions: Both PvMSP-3 paralogs were subjected to diversifying selection in each sample population. Clustering of alleles supports ephemeral endemic differentiation of alleles, but the broader phylogeny suggests that alleles transit the globe, perhaps accelerated by movements of migrants such as those transiting Tengchong.

Background: Chinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa.Methods: We developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection.Results: The new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5-100%] and 99.1% (95% CI 96.7-99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 102 copies/mu L, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/mu L of blood (equal to 0.04 parasites/mu L of DNA) using cultured P. falciparum and 1-7 parasites/mu L of blood (4-28 parasites/mu L of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays. Conclusion: The novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and costeffective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

目的 探讨PBL联合数字化微课在生殖医学本科生教学中的应用,可为提高生殖医学人才培养质量提供参考.方法 以389名昆明医科大学临床医学专业和生殖医学选修课班学生为研究对象,授课后收集学生在医学知识获取、医学思维培养、自主学习和实践能力等评价指标,可能影响授课效果的因素,并对结果进行评价分析.结果 该方法授课后学生在医学知识获取和医学思维培养,自主学习和实践能力方面有较明显优势,且分组结果存在显著差异(P<0.05).但有知识获取途径有限和课程准备时间长等问题.结论 PBL联合数字化微课是适合生殖医学教学的有效授课模式,有助于学生知识获取和能力提升,可推广应用.

在"新医科"背景下,云南地区医学发展迫切需要"五术"新型医学人才,医学细胞生物学作为医学专业基础核心课程,推进融入"五术"医学人才培养理念的"金课"建设势在必行.该文以"受体介导的胞吞"为例,以学生为中心进行融合整合式内容重构、课程思政、智慧教学、交互式模拟实验、项目式学习等创新性元素的混合式教学设计及实践.

课程思政就是要寓价值观引导于知识传授和能力培养之中,实现专业课与思政课同向同行,帮助学生塑造正确的世界观、人生观和价值观,这是人才培养的应有之义,更是必备内容.《医学细胞生物学》以细胞的生命现象和规律为研究对象,与人体各项生命活动密切相关,该课程是医学生专业基础必修课.该文以昆明医科大学为例,阐述《医学细胞生物学》课程思政建设路径探索,为医学类其他课程思政教育提供借鉴思路和经验.

目的 研究经氯喹合并伯氨喹啉根治后的间日疟患者复发的病因,探索对该类患者新的根治药物组合.方法 收集经氯喹合并伯氨喹啉8 d根治后复发的间日疟患者血样,检测患者CYP2D6酶基因状况,并对CYP2D6酶基因缺陷患者采用双氢青蒿素哌喹合并阿奇霉素进行根治.结果 102例非洲输入性间日疟患者经氯喹合并伯氨喹啉根治后,4例患者疟疾出现复发再就诊,PCR虫种检测均为间日疟,均为CYP2D6基因缺陷.其中3例经常规根治后因再次出国务工而终止观察,1例经青蒿素合并阿奇霉素进行一个疗程根治后,追踪观察4年余未见间日疟再复发.结论 经足量的氯喹合并伯氨喹啉根治后的疟疾复发患者,可能存在CYP2D6基因缺陷.双氢青蒿素哌喹合并阿奇霉素对CYP2D6基因缺陷患者的根治效果值得深入研究.

Drug-resistant Plasmodium vivax malaria impedes efforts to control, eliminate, and ultimately eradicate malaria in Southeast Asia. P. vivax resistance to antifolate drugs derives from point mutations in specific parasite genes, including the dihydropteroate synthase (pvdhps), dihydrofolate reductase (pvdhfr), and GTP cyclohydrolase I (pvgch1) genes. This study aims to investigate the prevalence and spread of drug resistance markers in P. vivax populating the China-Myanmar border. Blood samples were collected from symptomatic patients with acute P. vivax infection. Samples with single-clone P. vivax infections were sequenced for pvdhps and pvdhfr genes and genotyped for 6 flanking microsatellite markers. Copy number variation in the pvgch1 gene was also examined. Polymorphisms were observed in six different codons of the pvdhps gene (382, 383, 512, 549, 553, and 571) and six different codons of the pvdhfr gene (13, 57, 58, 61, 99, 117) in two study sites. The quadruple mutant haplotypes 57I/L/58R/61M/117T of pvdhfr gene were the most common (comprising 76% of cases in Myitsone and 43.7% of case in Laiza). The double mutant haplotype 383G/553G of pvdhps gene was also prevalent at each site (40.8% and 31%). Microsatellites flanking the pvdhfr gene differentiated clinical samples from wild type and quadruple mutant genotypes (F (ST)= 0.259-0.3036), as would be expected for a locus undergoing positive selection. The lack of copy number variation of pvgch1 suggests that SP-resistant P. vivax may harbor alternative mechanisms to secure sufficient folate.

Background: The spread of drug resistance has seriously impacted the effective treatment of infection with the malaria parasite, Plasmodium falciparum. Continuous monitoring of molecular marker polymorphisms associated with drug resistance in parasites is essential for malaria control and elimination efforts. Our study describes mutations observed in the resistance genes Pfkelch13, Pfcrt, and Pfmdr1 in imported malaria and identifies additional potential drug resistance-associated molecular markers.Methods: Chinese patients infected in Africa with P. falciparum were treated with intravenous (IV) injections of artesunate 240–360 mg for 3–5 days while hospitalized and treated with oral dihydroartemisinin-piperaquine (DHP) for 3 days after hospital discharge. Blood samples were collected and PCR sequencing performed on genes Pfkelch13, Pfcrt, and Pfmdr1 from all isolates.Results: We analyzed a total of 225 patients from Guangxi, China with P. falciparum malaria acquired in Africa between 2016 and 2018. All patients were cured completely after treatment. The F446I mutation of the Pfkelch13 gene was detected for the first time from samples of West African P. falciparum, with a frequency of 1.0%. Five haplotypes of Pfcrt that encode residues 72–76 were found, with the wild-type CVMNK sequence predominating (80.8% of samples), suggesting that the parasites might be chloroquine sensitive. For Pfmdr1, N86Y (13.1%) and Y184F (58.8%) were the most prevalent, suggesting that artemether-lumefantrine may not, in general, be a suitable treatment for the group.Conclusions: For the first time, this study detected the F446I mutation of the Pfkelch13 gene from Africa parasites that lacked clinical evidence of resistance. This study provides the latest data for molecular marker surveillance related to antimalarial drug resistance genes Pfkelch13, Pfcrt, and Pfmdr1 imported from Africa, in Guangxi, China from Chinese migrate workers.Clinical Trial Registration: ChiCTROPC17013106.

目的 探究融入思政教育的速课混合式教学在全科医学专业《医学细胞生物学》课程中的实践和效果评价。方法 以昆明医科大 学2020级全科医学专业学生为研究对象，随机分为实验班和对照班。实验班级开展速课混合式教学，对照班级采用传统多媒体教学。通过考 试成绩分析和问卷调查，对实践效果进行评价。结果 成绩分析结果提示，该混合式教学显著提升了学生及时理解和掌握知识点的能力，同时 对于终结性考核也有一定帮助。学生主观评价也对该教学方式给予高度认可。 结论 本研究将思政教育融入专业课程，并采用混合式教学方 法，在全科医学实践中取得了很好的课程育人实效。