Objective To establish a dual TaqMan probe-based qPCR method for determining the copy number of the geneti-cally modified pertussis toxin(PT) gene in detoxified pertussis vaccine strains, and to identify optimal reference genes forevaluating the genetic stability of modified strains during serial passage.MethodsSpecific primers and probes of modifiedgene PT and multiple housekeeping genes were designed. The optimal reference genes were screened by GeNorm, Norm-Finder, BestKeeper and Delta CT based on dual TaqMan probe qPCR. The copy number of PT gene of genetically detoxifiedpertussis vaccine strain was calculated by relative quantitative analysis. The specificity, linearity and repeatability of theestablished method were verified. The genomic DNA of P1, P3, P5, P10 and P15 strains used in the production of geneticallydetoxified pertussis vaccine was used as templates, and the passage stability of the strains was evaluated by using the estab-lished dual TaqMan probe qPCR method.ResultsThe stability of candidate reference genes was analyzed using GeNorm,NormFinder, BestKeeper, and Delta CT software, with Rpob identified as the most stable reference gene. Both the target PTgene and the reference Rpob gene were amplified in standard strain 58003. A good linear correlation(R~2≥ 0. 990) wasobserved between Ct values and template quantities within the concentration range of 0. 001 to 10 ng/μL. For P1 generationstrains, the 2~(-△△Ct)values from six replicate assays consistently ranged between 0. 87 and 1. 25, with an RSD of less than 20%.All tested generations exhibited single-copy PT target genes, demonstrating good genetic stability during serial passages.ConclusionThe established dual TaqMan probe qPCR assay demonstrates good primer specificity and reproducibility,making it suitable for evaluating the genetic stability of bacterial strains during serial passages in the production of geneti-cally detoxified pertussis vaccines, thereby further evaluating the quality of strains.

Objective:To understand the status of intestinal helminth infections in Lunan area of Shandong Province (Jining City, Rizhao City, Linyi City, Heze City, and Zaozhuang City), and provide scientific basis for formulating parasitic disease prevention and control strategies.Methods:From 2016 to 2023, a stratified sampling method was used to conduct surveillance in 33 counties (cities, districts, hereinafter referred to as counties) in Lunan area of Shandong Province. Each county was divided into five areas (east, south, west, north, and center), with one administrative village (community) selected from each area. And ≥200 permanent residents (aged ≥3 years old, having lived locally for ≥6 months) were sampled from each village (community), and one stool sample was collected from each participant. The Kato-Katz method (two slides per sample) was used for parasite detection, and the results were analyzed.Results:From 2016 to 2023, a total of 49 436 people were surveyed, including 23 861 males and 25 575 females, with an age range of 3 - 105 years old. The testing identified 687 intestinal helminth infections, with an overall infection rate of 1.39%. The predominant infection was whipworm (582 cases), with an infection rate of 1.18%; followed by roundworm and hookworm, with infection rates of 0.13% (62 cases) and 0.05% (23 cases) respectively; other types of helminths ( Clonorchis sinensis and pinworm) totaled 20 cases, with an infection rate of 0.04%. From 2016 to 2023, the annual intestinal helminth infection rates were 2.76% (201/7 292), 0.90% (56/6 327), 0.84% (52/6 200), 1.70% (124/7 282), 1.23% (88/7 133), 1.45% (104/7 150), 0.34% (17/5 058), and 1.46% (45/3 084), showing an overall downward trend (χ 2trend = 42.40, P < 0.001). The ≥60 age group had the highest intestinal helminth infection rate (2.39%, 323/13 489), while the 30 - 39 age group had the lowest rate (0.68%, 48/7 016). There was statistically significant difference in infection rates among different age groups (χ 2 = 172.25, P < 0.001). There was no statistically significant difference in infection rates between genders (χ 2 = 0.03, P = 0.862). Farmers had the highest infection rate (1.69%, 560/33 118), with statistically significant differences among different occupational groups (χ 2 = 64.88, P < 0.001). Those with primary school education or below had the highest infection rate (1.82%, 445/24 469), with statistically significant differences among different education levels (χ 2 = 64.93, P < 0.001). Conclusions:In Lunan area of Shandong Province, whipworm is the predominant intestinal helminth infection, with elderly people being the high-risk group. Although the intestinal helminth infection rate in this region remains at a low level, there is still a risk of transmission. Continuous surveillance is needed, along with strengthened prevention and control measures for key populations.

Autophagy is a conserved physiological phenomenon commonly found in all eukaryotic cells,which promotes cell survival by degrading intracellular proteins and organelles during starvation or nutrient deficiency.Dysfunc-tion autophagy is closely related to various diseases.Soluble N-elthylmaleimide-sensitive factor attachment protein recep-tor(SNARE)complex is a protein complex that mediates membrane fusion and plays a key role in the fusion process of au-tophagosomes and lysosomes.The aberrant assembly of the SNARE complex results in the arrest of autophagic flow,there-by leading to cellular dysfunction and the occurrence of diseases.In this review,we summarized the roles of SNARE pro-teins in the autophagosome-lysosome fusion and the latest research in related diseases,providing a reference for studying the mechanism of autophagy and diseases targeting the SNARE complex.

Objective:To understand the status of intestinal helminth infections in Lunan area of Shandong Province (Jining City, Rizhao City, Linyi City, Heze City, and Zaozhuang City), and provide scientific basis for formulating parasitic disease prevention and control strategies.Methods:From 2016 to 2023, a stratified sampling method was used to conduct surveillance in 33 counties (cities, districts, hereinafter referred to as counties) in Lunan area of Shandong Province. Each county was divided into five areas (east, south, west, north, and center), with one administrative village (community) selected from each area. And ≥200 permanent residents (aged ≥3 years old, having lived locally for ≥6 months) were sampled from each village (community), and one stool sample was collected from each participant. The Kato-Katz method (two slides per sample) was used for parasite detection, and the results were analyzed.Results:From 2016 to 2023, a total of 49 436 people were surveyed, including 23 861 males and 25 575 females, with an age range of 3 - 105 years old. The testing identified 687 intestinal helminth infections, with an overall infection rate of 1.39%. The predominant infection was whipworm (582 cases), with an infection rate of 1.18%; followed by roundworm and hookworm, with infection rates of 0.13% (62 cases) and 0.05% (23 cases) respectively; other types of helminths ( Clonorchis sinensis and pinworm) totaled 20 cases, with an infection rate of 0.04%. From 2016 to 2023, the annual intestinal helminth infection rates were 2.76% (201/7 292), 0.90% (56/6 327), 0.84% (52/6 200), 1.70% (124/7 282), 1.23% (88/7 133), 1.45% (104/7 150), 0.34% (17/5 058), and 1.46% (45/3 084), showing an overall downward trend (χ 2trend = 42.40, P < 0.001). The ≥60 age group had the highest intestinal helminth infection rate (2.39%, 323/13 489), while the 30 - 39 age group had the lowest rate (0.68%, 48/7 016). There was statistically significant difference in infection rates among different age groups (χ 2 = 172.25, P < 0.001). There was no statistically significant difference in infection rates between genders (χ 2 = 0.03, P = 0.862). Farmers had the highest infection rate (1.69%, 560/33 118), with statistically significant differences among different occupational groups (χ 2 = 64.88, P < 0.001). Those with primary school education or below had the highest infection rate (1.82%, 445/24 469), with statistically significant differences among different education levels (χ 2 = 64.93, P < 0.001). Conclusions:In Lunan area of Shandong Province, whipworm is the predominant intestinal helminth infection, with elderly people being the high-risk group. Although the intestinal helminth infection rate in this region remains at a low level, there is still a risk of transmission. Continuous surveillance is needed, along with strengthened prevention and control measures for key populations.

Autophagy is a conserved physiological phenomenon commonly found in all eukaryotic cells,which promotes cell survival by degrading intracellular proteins and organelles during starvation or nutrient deficiency.Dysfunc-tion autophagy is closely related to various diseases.Soluble N-elthylmaleimide-sensitive factor attachment protein recep-tor(SNARE)complex is a protein complex that mediates membrane fusion and plays a key role in the fusion process of au-tophagosomes and lysosomes.The aberrant assembly of the SNARE complex results in the arrest of autophagic flow,there-by leading to cellular dysfunction and the occurrence of diseases.In this review,we summarized the roles of SNARE pro-teins in the autophagosome-lysosome fusion and the latest research in related diseases,providing a reference for studying the mechanism of autophagy and diseases targeting the SNARE complex.

Objective:To understand the knowledge and skills of professional technicians in prevention and control of parasitic diseases in Shandong Province, and to provide a basis for further capacity building.Methods:On October 12 and 13, 2017, five professional technicians from each of 17 prefectures and cities in Shandong Province participated in the "Shandong Province Parasitic Diseases Prevention and Control Job Skills Competition in 2017". This competition included theoretical knowledge examination (written test) and operation skill examination (operation test). The written test included basic knowledge of parasites life history, pathogen, diagnosis, epidemic and prevention; the operation test included blood smear making, Kato-Katz thick smear making, microscopic examination of Plasmodium and Helminth eggs. The competition results of the competitors were statistically analyzed, and the pass rate, correct answer rate and accuracy of microscopic examination were calculated. According to the report of malaria cases from 2010 to 2017, 17 prefectures and cities in Shandong Province were divided into malaria classⅠreport area (≥100 cases) and malaria classⅡreport area (< 100 cases), and the competition results of the two types of areas were compared. Results:A total of 85 competitors in Shandong Province participated in the competition, including 19 males (22.35%) and 66 females (77.65%); the age was (34.67±6.04) years old, the youngest was 25 years old, and the oldest was 51 years old. The scores of written test and operation test were (67.06±12.73) and (59.31±14.23) points, respectively, and the difference between them was statistically significant ( t=4.949, P < 0.01). The pass rate of written test was 74.12% (63/85); the correct answer rates of morphology, life history, pathogenicity, diagnosis, epidemic and prevention were 71.76% (366/510), 71.61% (913/1 275), 67.94% (462/680), 71.18% (847/1 190), 66.91% (455/680) and 65.76% (1 062/1 615), respectively, there was statistically significant difference in the correct answer rates of different knowledge points (χ 2=18.185, P < 0.01). The pass rate of operation test was 55.29% (47/85); among them, the pass rates of blood smear making, Kato-Katz thick smear making, microscopic examination of Plasmodium and Helminth eggs were 98.82% (84/85), 98.82% (84/85), 70.59% (60/85) and 31.76% (27/85), respectively. Four Plasmodium species were examined, and the overall accuracy of microscopic examination was 61.41% (261/425), there was no statistically significant difference in the accuracy of microscopic examination between different Plasmodium species (χ 2=4.791, P > 0.05). Nine common Helminth eggs were examined, and the overall accuracy of microscopic examination was 47.29% (402/850), there was statistically significant difference in the accuracy of microscopic examination between different Helminth eggs (χ 2=180.064, P < 0.01). The scores [(28.27±3.74) and (23.20±3.39) points, n=30] of microscopic examination of Plasmodium and Helminth eggs in the malaria classⅠ report area were higher than those in the malaria classⅡreport area [(22.40±5.81) and (18.25±3.41) points, n=55], and the differences were statistically significant ( t=2.217, 2.860, P < 0.05). Conclusions:For professional technicians, the mastery of theoretical knowledge in prevention and control of parasitic diseases is better than operation skills in Shandong Province. So the training and assessment of prevention and control skills of parasitic diseases should be strengthened in areas with weak abilities.

Objective:To understand epidemiological characteristics of imported cases of malaria in Shandong province and provide scientific basis for timely adjustment of prevention and control measures.Methods:The incidence data of malaria, case investigation data and case review data by Shandong Provincial Reference Laboratory for Malaria Diagnosis from 2017 to 2018 were collected. Software SPSS 18.0 was used for statistical analysis and software QGIS 2.18 was used for mapping.Results:A total of 442 imported cases of malaria were reported in Shandong from 2017 to 2018, and the main infection source was in Africa (97.96%, 433/442). All the 17 prefectures in Shandong reported imported malaria cases, mainly in Jining (88 cases), Yantai (65 cases), Weihai (46 cases), Qingdao (44 cases) and Dezhou (42 cases), accounting for 64.48% (285/442). The cases were distributed in 77.37%(106/137) of counties of the province. The cases were reported in every month without seasonal characteristics. The median ( M) of time interval between onset and the first medical care seeking was 2 days, and the interquartile range ( IQR) was 3 days. The M of time interval between the first medical care seeking and final diagnosis was 0 day, and the IQR was 3 days. The proportion of medical care seeking on onset day was only 27.83% (123/442). Only 69.68% (308/442) of cases were diagnosed with malaria in the first medical care seeking, and the diagnostic accuracy of medical institutions below the county level was lower than other medical institutions (all P<0.01). Only 51.13% (226/442) of cases were diagnosed with malaria in the first medical care seeking, the differences in the rates among medical institutions at different levels were not significant ( P>0.05). Conclusions:The imported malaria in Shandong was characterized by a large number of cases, multiple infection sources and wide area distribution during 2017-2018. The awareness of timely medical care seeking in the cases was low, meanwhile the awareness and ability of malaria diagnosis and treatment in primary medical institutions were still inadequate. It is necessary to adjust the prevention and control measures accordingly.

Objective To understand the malaria epidemic situation and characteristics in Shandong Province in 2014,so as to provide evidences for taking targeted strategies and measures for malaria elimination. Methods The data of malaria epi?demic and malaria cases of Shandong Province in 2014 were collected from Information Management System for Infectious Dis?eases Report and Information Management System for Parasitic Diseases Control and Prevention. The epidemiological character?istics of malaria situation and the diagnosis and treatment of malaria cases were analyzed by Microsoft Excel 2007. Results A total of 150 malaria cases were reported in Shandong Province in 2014,which increased by 14.50% compared with that in 2013 (131 cases). All the cases were imported from other countries except one imported from Yunnan Province in China,and most of the imported cases were from Africa(142 cases,95.30%). All the cases were confirmed in Shandong Provincial Reference Labo?ratory and there were 121 cases(80.67%)infected with Plasmodium falciparum,16 cases(10.67%)infected with P. vivax,9 cases(6.00%)infected with P. ovale and 4 cases(2.67%)infected with P. malariae. The cases were mainly distributed in Tai’ an(40 cases,26.67%),Yantai(17 cases,11.33%),Weihai(13 cases,8.67%),Jining(11 cases,7.33%)and Heze(11 cases,7.33%)cities. Conclusions There have been no local malaria cases reported in Shandong Province in the continuous 3 years. However,the reported number of imported malaria cases shows an increasing trend in the province in recent years,and the species of infected Plasmodium are diverse. Imported malaria from other countries is the key of malaria control in Shandong Province at present.

Objective To explore the association between malaria epidemic situation and some natural and social factors in high?incidence areas of Shandong Province,so as to provide evidences for malaria elimination in these areas. Methods Twenty towns of 10 counties(cities,districts,)in the high incidence areas of malaria in Shandong Province were selected as the study sites,and the residents in the study sites were investigated by questionnaires with one household as a unit,so as to understand the related natural and social factors. In addition,the malaria epidemic data in the study sites from 2006 to 2010 were collected, and the correlation between these factors and the epidemic situation of malaria was analyzed by Spearman rank correlation and multiple stepwise regression. Results The square root of malaria incidence rate(Y)was negatively related to the rate of house?holds using insecticide(X3),and the rate of households using screen doors and windows(X4)(both P<0.05),but was positive?ly related to the rate of housing surrounding water environment and exposure ratio(X6)(both P<0.05). The regression equa?tion established was Y=0.032X5+0.048X6-0.495,R2=0.973. Conclusions Malaria incidence is obviously associated with some natural and social factors. The measures such as clearing the breeding place of mosquito,protecting the exposure popula?tion at nightfall,as well as using door?window screen and repellents correctly,can effectively control malaria.

Objective To understand the status of intestinal parasitic infections and the related knowledge and behavior in residents of Jiaodong area of Shandong Province,so as to provide the evidence for making an appropriate preventive and control strategy. Methods A total of 18 villages from 6 counties in Jiaodong area were selected as investigation sites according to the stratified sampling method. The feces samples of the permanent residents aged above 3 years were collected and examined by Kato-Katz technique to find the intestinal parasite eggs,and the children under 12 years old were examined by the method of cellophane anal swab to detect the Enterobius vermicularis eggs. In addition,50 households in each survey sites were randomly selected to in-vestigate the basic family situation and the condition of awareness on prevention knowledge and formation of correct behavior of res-idents by using a structured questionnaire. Results Totally 6 163 residents involved in the feces examinations,and the total in-fection rate of intestinal parasites was 6.91%. The infection rates of Trichuris trichiura,Ascaris lumbricoides and hookworm were 6.56%,0.62%and 0.21%,respectively. The infection rate of E. vermicularis in children under 12 years old was 0.51%. The eggs of Clonorchis sinensis and Taenia solium were not found in this survey. The awareness rate of knowledge about preventing parasitic diseases was 49.54%. The formation rates of washing hands before eating,washing hands after using the toilet,never eating raw fruit and vegetable without washing clean,never working in the field with bare feet,and never drinking unboiled water were 97.78%,91.95%,88.81%,92.42%and 86.48%respectively. Conclusions The infection rate of intestinal parasites is low in Ji-aodong area,but there is a significant difference among different counties. The awareness rate of knowledge about preventing para-sitic diseases is low,but the formation rate of healthy behavior is high. In the future,the health education and the strategy of tak- ing medicine among the key population should be enhanced,and the project of reconstructing safe water supply and lavatory should be advanced.