Bin Xu

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.

The prevalence and infectious intensity of schistosomiasis japonica has decreased significantly in China in the past few decades. However, more accurate and sensitive diagnostic methods are urgently required for the further control, surveillance, and final elimination of the disease. In this study, we assessed the diagnostic efficacy of a real-time fluorescence quantitative PCR (qPCR) method and recombinase polymerase amplification (RPA) combined with a lateral-flow dipstick (LFD) assay for detecting early infections of Schistosoma japonicum and different infection intensities. The sensitivity of the qPCR at 40 days post-infection (dpi) was 100% (8/8) in mice infected with 40 cercariae, which was higher than in mice infected with 10 cercariae (90%, 9/10) or five cercariae (77.8%, 7/9). The results of the RPA–LFD assays were similar, with sensitivities of 55.6% (5/9), 80% (8/10), and 100% (8/8) in mice infected with 5, 10, and 40 cercariae, respectively. In goats, both the qPCR and RPA–LFD assays showed 100% (8/8) sensitivity at 56 dpi. In the early detection of S. japonicum infection in mice and goats with qPCR, the first peak in positivity appeared at 3–4 dpi, when the positivity rate exceeded 40%, even in the low infection, intensity mice. In the RPA–LFD assays, positive results first peaked at 4–5 dpi in the mice, and the positivity rate was 37.5% in the goats at 1 dpi. In conclusion, neither of the molecular methods produced exceptional results for the early diagnosis of S. japonicum infection. However, they were useful methods for the regular diagnosis of schistosomiasis in mice and goats.