Bin Zheng

The online version contains supplementary material available at 10.1038/s41598-026-42288-3.

Background: Anopheles sinensis , the primary malaria vector in China, is a potential threat to the prevention of re-establishment of malaria transmission following imported cases. The extensive use of insecticides has led to widespread resistance. Conventional bioassays and molecular detection are limited in sensitivity, throughput, and efficiency, underscoring the need for a rapid and cost-effective genotyping tool for large-scale multiple insecticide-resistance markers surveillance. Methods: A multiplex SNaPshot assay was developed to simultaneously detect six SNPs ( kdr1014-F , kdr1014-R , Ace-1 , RDL296 , RDL327 , and RDL345 ) in An. sinensis . Positive plasmids carrying known resistance-associated genotypes and laboratory-reared An. sinensis samples were used to optimize and validate the reactions. Wild An. sinensis collected from Henan Puyang (HN-PY) and Anhui Shucheng (AH-SC) were used for bioassays with 0.05% deltamethrin, 0.15% beta-cyfluthrin, 5% malathion, 0.1% propoxur, and 0.06% flufenoxuron. Then samples randomly selected on the basis of 24 h survival status were genotyped via PCR–Sanger sequencing and the SNaPshot assay. Consistency was assessed using the Kappa testing, and genotype–phenotype correlations were evaluated by chi-squared tests. Results: Bioassays showed that the HN-PY An. sinensis population was resistant to deltamethrin, beta-cyfluthrin, malathion, propoxur, and flufenoxuron, with adjusted mortality rates of 5.2%, 24.4%, 9.1%, 24.3%, and 5.8%, respectively. In contrast, the AH-SC population remained susceptible to malathion (100% mortality) but exhibited resistance to deltamethrin, beta-cyfluthrin, propoxur, and flufenoxuron, with adjusted mortality rates of 71.6%, 78.6%, 44.9%, and 33.4%, respectively. Multiplex SNaPshot genotyping of 272 field-collected mosquitoes revealed high frequencies of resistance-associated mutations at the kdr 1014-F, kdr 1014-R, Ace-1 , RDL 296, RDL 327, and RDL 345 loci, with frequencies of 4.7%, 99.3%, 53.3%, 89.7%, 96.0%, and 53.3% in the HN-PY population and 25.5%, 78.0%, 55.3%, 89.8%, 89.8%, and 46.3% in the AH-SC population, respectively. Additionally, the SNaPshot assay showed strong agreement with PCR–Sanger sequencing (Kappa > 0.90 at all loci). Conclusions: The multiplex SNaPshot assay provides a reliable, rapid, and economical tool for detecting multiple resistance mutations in An. sinensis . Its application in routine entomological surveillance will provide an alternative tool for early warning of insecticide resistance. Graphical

Background The frequent occurrences of Babesia microti ( B. microti ) infection have emerged as a significant global public health safety concern. The diagnosis of patients constitutes a crucial component in the prevention and control of babesiosis, necessitating the urgent establishment of efficient and accurate molecular biology diagnostic methods for B. microti detection. Purpose This study aimed to develop a sensitive, specific, and rapid fluorescent recombinase polymerase amplification (RPA) technique for the detection of B. microti ( B. microti ) and assess its suitability for field use. Methods This study developed and optimized a fluorescent RPA assay targeting the B. microti 18S rRNA gene fragment. The method's sensitivity (detection limit down to 10 fg/µL genomic DNA), specificity (distinguishing Plasmodium samples), and detection limit for spiked mouse blood samples (across varying parasitemia levels) were evaluated. The detection capability was validated using samples from an infected mouse model (tail-tip blood collected 3–21 days post-infection) and two confirmed human cases. Furthermore, 119 blood samples from patients presenting with fever accompanied by thrombocytopenia were tested using both nested PCR and the newly developed fluorescent RPA assay. The agreement between the two methods was analyzed using the Kappa value, and the significance of their difference was assessed using McNemar's test. Results A fluorescent RPA assay was developed targeting two B. microti 18S rRNA fragments (243 bp/191 bp). The 191 bp fragment demonstrated superior sensitivity (detection limit: 1 fg/µL genomic DNA) and was selected for assay establishment. Optimized at 39°C for 20 min, its detection threshold was defined by the lowest detectable concentration (1 fg/µL). Specificity testing with 8 P. falciparum and 8 P. ovale samples showed fluorescence below this threshold. The assay achieved a detection limit of 0.046 parasites/µL blood, demonstrating a 600-fold increase in sensitivity compared to nested PCR, which detects 28.49 parasites/µL. In infected mice, both RPA and nested PCR detected parasites by day 9 post-infection (microscopy: day 15), with RPA showing higher positivity. The method confirmed infection in 2 patients. Among 119 febrile patients with thrombocytopenia from Xinyang City, Henan Province, nested PCR detected 20 B. microti- positive cases while fluorescent RPA detected 21. The Kappa value for agreement between the methods was 97.05% (91.31% − 100%), and McNemar's test indicated no statistically significant difference in their detection rates (S = 1, P > 0.1). Based on the combined results of both tests, the molecular positivity rate for B. microti in this sample set was 17.65%, with co-infections involving Bunya virus present, accounting for 10.08% of the tested samples. Conclusion The fluorescence RPA method provides a rapid, sensitive, and specific tool for detecting B. microti and is effective for field screening.

Background: Necator americanus is the predominant species causing hookworm infections in humans. Despite advancements in prevention strategies, mild cases of infection still occur, highlighting the need for improved detection technology. Recombinase Polymerase Amplification (RPA) is an isothermal molecular diagnostic known for its sensitivity, speed, portability, and widespread application in detecting various pathogens. Although several molecular assays are available for N. americanus, they have limitations in detecting mild N. americanus infections. Methods: Fluorescent RPA primers and probes targeting the N. americanus internal transcribed spacer 2 (ITS2) gene were developed. The method’s detection limit was assessed via serial dilution of genomic DNA. Specificity was confirmed against Clonorchis sinensis, Schistosoma japonicum, Fasciola hepatica, Ascaris lumbricoides, Enterobius vermicularis and Ancylostoma duodenale. Thirty samples identified as positive by Kato-Katz, along with 11 samples identified as negative by the method, were tested to evaluate the sensitivity and specificity of fluorescent RPA. Additionally, 287 field samples were tested for validation with these methods. All positive samples were identified as either N. americanus or A. duodenale. Results: This study successfully developed a fluorescent RPA assay targeting the ITS2 gene of N. americanus. The length of the amplified fragment was 237 bp. Optimized conditions were achieved, resulting in a minimum detection limit of 1fg/µL, with no cross-reactivity with other pathogens. In laboratory validation, the fluorescent RPA assay demonstrated 100% sensitivity (30/30) and 100% specificity (11/11) compared to the Kato-Katz, and 100% sensitivity (29/29) and 91.7% specificity (11/12) when compared to the semi-nested PCR. In field validation using human fecal samples, the fluorescent RPA assay showed a sensitivity of 90.0% (36/40) and a specificity of 91.1% (225/247) compared to the Kato-Katz. And the sensitivity of the fluorescent RPA method compared to the semi-nested PCR method was 100% (34/34), while the specificity was 90.5% (229/252). Conclusions: The fluorescent RPA assay presents a rapid and dependable method for detecting N. americanus in fecal samples. Its high sensitivity and specificity provide significant utility for field surveillance and early identification of N. americanus infections. This advancement could facilitate the rapid molecular diagnosis of N. americanus disease in hookworm-endemic regions.

Rhipicephalus sanguineus, a repulsive obligate blood feeder, is a three-host tick inflicting tremendous damage. Blood-sucking initiates tick-pathogen-host interactions along with alterations in the expression levels of numerous bioactive ingredients. Key molecules regulating blood meals were identified using the transcriptomic approach. A total number of 744 transcripts showed statistically significantly differential expression including 309 significantly upregulated transcripts and 435 significantly downregulated transcripts in semiengorged female ticks compared to unfed ticks, all collected in 2021. The top 10 differentially upregulated transcripts with explicit functional annotations included turripeptide OL55-like protein, valine tRNA ligase-like protein and ice-structuring glycoprotein-like protein. The top 10 differentially down-regulated transcripts were uncharacterized proteins. Gene Ontology (GO) enrichment analysis revealed four associated terms in the cellular component category and 16 in the molecular function category among the top 20 terms. Differentially expressed genes (DEGs) were enriched in GO terms ID 0000323 (lytic vacuole) and ID 0005773 (vacuole). The top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included metabolism, cellular processes, organismal systems and human diseases. The DEGs were enriched in the KEGG term ID: ko-04142 (lysosome pathway) associated with intracellular digestion in the tick midgut epithelium. Molecular markers annotated via comparative transcriptomic profiling were expected to be candidate markers for the purpose of tick control.

Background: Following China’s official designation as malaria-free country by WHO, the imported malaria has emerged as a significant determinant impacting the malaria reestablishment within China. The objective of this study is to explore the application prospects of machine learning algorithms in imported malaria risk assessment of China. Methods: The data of imported malaria cases in China from 2011 to 2019 was provided by China CDC; historical epidemic data of malaria endemic country was obtained from World Malaria Report, and the other data used in this study are open access data. All the data processing and model construction based on R, and map visualization used ArcGIS software. Results: A total of 27,088 malaria cases imported into China from 85 countries between 2011 and 2019. After data preprocessing and classification, clean dataset has 765 rows (85 * 9) and 11 cols. Six machine learning models was constructed based on the training set, and Random Forest model demonstrated the best performance in model evaluation. According to RF, the highest feature importance were the number of malaria deaths and Indigenous malaria cases. The RF model demonstrated high accuracy in forecasting risk for the year 2019, achieving commendable accuracy rate of 95.3%. This result aligns well with the observed outcomes, indicating the model’s reliability in predicting risk levels. Conclusions: Machine learning algorithms have reliable application prospects in risk assessment of imported malaria in China. This study provides a new methodological reference for the risk assessment and control strategies adjusting of imported malaria in China.

Background: Babesia is a unique apicomplexan parasite that specifically invades and proliferates in red blood cells and can be transmitted via blood transfusion, resulting in transfusion-transmitted babesiosis. However, detecting Babesia in blood before transfusion has not received enough attention, and the risk of transfusing blood containing a low density of Babesia microti (B. microti) is unclear, possibly threatening public health and wellness.Purpose: This study aimed to determine the lower detection limit of B. microti in blood and to evaluate the transmission risk of blood transfusion containing low-density B. microti.Methods: Infected BALB/c mouse models were established by transfusing infected whole blood with different infection rates and densities of B. microti. Microscopic examination, nested Polymerase Chain Reaction (nested PCR), and an enzyme-linked immunosorbent assay (ELISA) were used to evaluate the infection status of the mouse models. Meanwhile, the nested PCR detection limit of B. microti was obtained using pure B. microti DNA samples with serial concentrations and whole blood samples with different densities of B. microti-infected red blood cells. Thereafter, whole mouse blood with a B. microti density lower than that of the nested PCR detection limit and human blood samples infected with B. microti were transfused into healthy mice to assess the transmission risk in mouse models. The infection status of these mice was evaluated through microscopic examination, nested PCR tests, and ELISA.Results: The mice inoculated with different densities of B. microti reached the peak infection rate on different days. Overall, the higher the blood B. microti density was, the earlier the peak infection rate was reached. The levels of specific antibodies against B. microti in the blood of the infected mice increased sharply during the first 30 days of infection, reaching a peak level at 60 days post-infection, and maintaining a high level thereafter. The nested PCR detection limits of B. microti DNA and parasite density were 3 fg and 5.48 parasites/μL, respectively. The whole blood containing an extremely low density of B. microti and human blood samples infected with B. microti could infect mice, confirming the transmission risk of transfusing blood with low-density B. microti.Conclusion: Whole blood containing extremely low density of B. microti poses a high transmission risk when transfused between mice and mice or human and mice, suggesting that Babesia detection should be considered by governments, hospitals, and disease prevention and control centers as a mandatory test before blood donation or transfusion.

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.

Purpose. The current study quantitatively assessed research trends in tropical medicine in China via a bibliometric method, which used the Web of Science database to analyse the research-based literature related to tropical medicine published from 2010 to 2019. Methods. Articles were analysed according to the output and research performance of institutes and countries that collaborated with China. Distribution of keywords was applied to evaluate research trends. Results. Our findings showed that 3372 articles in the field of tropical medicine have been indexed under the Web of Science database during the past 10 years, indicating that studies pertaining to tropical medicine have been growing from 2010 to 2019. The Chinese Center for Disease Control and Prevention (China CDC) system, which published 549 articles on tropical medicine, may be considered as the flagship of tropical medicine in China. The United States ranked first in internationally collaborative articles with China. Furthermore, Parasite & Vectors, which published 707 papers, emerged as the top journal according to the number of publications. Conclusions. This study highlights the key institutes and topics pertaining to tropical medicine research in China. Although there has been rapid progress in research on tropical medicine in China, some gaps still remain.

Background There are three epidemiological types of visceral leishmaniasis in China, which are caused by Leishmania strains belonging to the L. donovani complex. The mechanisms underlying their differences in the population affected, disease latency, and animal host, etc., remain unclear. We investigated the protein abundance differences among Leishmania strains isolated from three types of visceral leishmaniasis endemic areas in China. Methods Promastigotes of the three Leishmania strains were cultured to the log phase and harvested. The protein tryptic digests were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive mass spectrometer. Raw spectra were quantitatively analyzed with the MaxQuant software (ver 1.3.0.5) and matched with the reference database. Differentially expressed proteins were analyzed using the bioinformatics method. The MS analysis was repeated three times for each sample. Results A total of 5012 proteins were identified across the KS-2, JIASHI-5 and SC6 strains in at least 2 of the three samples replicate. Of them, 1758 were identified to be differentially expressed at least between 2 strains, including 349 with known names. These differentially expressed proteins with known names are involved in biological functions such as energy and lipid metabolic process, nucleotide acid metabolic process, amino acid metabolic process, response to stress, cell membrane/cytoskeleton, cell cycle and proliferation, biological adhesion and proteolysis, localization and transport, regulation of the biological process, and signal transduction. Conclusion The differentially expressed proteins and their related biological functions may shed light on the pathogenicity of Leishmania and targets for the development of vaccines and medicines.