Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by SFTS virus (SFTSV), is a widely distributed infection with significant mortality. Diagnosis in resource-limited settings remains challenging. For rapid and convenient diagnosis, we developed a portable rapid diagnosis method that combines Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a technology with immunochromatographic test strips. The SFTSV RNA was amplified by reverse transcription loop-mediated isothermal amplification (RT-LAMP). The homologous target sequence and single stranded DNA (ssDNA) reporter gene were cleaved by CRISPR/Cas12a in parallel, and ssDNA probes labeled with FAM fluorescein and biotin were captured by an immunochromatographic strip. Finally, the signal on the immunochromatographic strips became visible to the naked eye. Based on CRISPR/Cas12a, a rapid SFTSV detection method was developed, featuring simplicity, rapidity, low cost, and ease of use. The method was applied for the nucleic acid detection of SFTSV in 40 clinical serum samples and compared with RT-polymerase chain reaction (PCR). The new method showed 100% sensitivity and 100% specificity with a detection agreement rate of 100%. The minimum detection limit of the method was 2.5 copies/μL, and no cross-reactivity with nucleic acids from other common pathogens was observed. Detection can be completed within 80 min, and results are observable with the naked eye. For the analysis of clinical samples, the method exhibits good detection performance and thus provides an attractive option for the nucleic acid detection of SFTSV in point-of-care and resource-limited medical settings.

In this study, we selected Acinetobacter baumannii Ab2897, a strain from our laboratory, as the host, while the lytic bacteriophage vB_AbaP_IME546 was isolated from untreated hospital sewage. We examined the phage's biological characteristics and found that its latent period was under 5 min, with complete lysis of the bacteria occurring within 60 min. The plaques formed by the bacteriophage showed a halo, indicating the presence of an antibacterial protein. Consequently, this phage underwent next-generation sequencing. A BLAST analysis of the assembled genome sequence indicated that vB_AbaP_IME546 is a novel bacteriophage. After carrying out RAST annotation, it was proposed that the tail fiber protein of the bacteriophage might possess depolymerase activity. To further explore this, the ORF49 gene encoding the tail fiber protein was cloned and expressed. The resulting protein, Dp49, was successfully generated. When the protein was applied to a double-layer plate assay, a transparent halo formed in the area surrounding the bacterial growth. This finding confirms that the IME546 phage exhibits depolymerase activity during the lysis of Ab2897.

Chlamydia trachomatis, a pathogen of global public health importance, is prioritized by the World Health Organization as a major sexually transmitted infection. Its continued worldwide spread poses persistent challenges to reproductive health and disease control systems, underscoring the urgent need for rapid and accurate diagnostic methods. While current clinical diagnosis primarily relies on nucleic acid amplification tests (NAATs), their dependence on expensive thermal cyclers, complex laboratory setups, and sophisticated procedures limits widespread use in low-resource settings. Recent advances in recombinase-aided amplification (RAA) combined with CRISPR-Cas12a have revolutionized molecular diagnostics. This study established an integrated one-tube CRISPR fluorescence detection system by combining rapid nucleic acid lysis, isothermal amplification, and CRISPR-Cas12a cleavage. The system is coupled with a portable palm-sized 4-channel fluorescence detector and a terminal app for visual readout. Experimental results demonstrated a detection limit of 10¹ copies/mL with no cross-reactivity to common reproductive tract pathogens including Neisseria gonorrhoeae, Ureaplasma urealyticum, Candida albicans, Trichomonas vaginalis, and Gardnerella vaginalis. Clinical validation against qPCR showed 100.00

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-β-naphthylamide dihydrochloride (PaβN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 10 2 copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.

The global proliferation of carbapenemase-producing bacteria (CPB) has garnered significant attention worldwide. Early diagnosis of CPB and accurate identification of carbapenemases are crucial for preventing the spread of CPB and ensuring targeted antibiotic therapy. Therefore, efficient and accurate identification of carbapenemases is paramount in clinically treating diseases associated with CPB. In this study, 58 CPB strains were collected and detected using the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method, a rapid detection platform based on CRISPR-Cas12a gene editing and isothermal amplification. Additionally, four conventional methods (the APB/EDTA method, PCR, NG-test Carba 5, and GeneXpert Carba-R) were employed and compared against whole genome sequencing (WGS) results, considered the gold standard, to evaluate their efficacy in detecting carbapenemases. Detection by the APB/EDTA method revealed that 29 strains were positive for Class A serine endopeptidases, while 29 strains were positive for Class B metalloenzymes. The classification of these zymotypes was consistent with the sequencing result. All target carbapenemases for KPC were identified with 100% sensitivity using NG-test Carba 5, PCR, DETECTR, and GeneXpert Carba-R. In the case of NDM, both Xpert Carba-R and DETECTR showed a sensitivity of 100%. In contrast, NG-test Carba 5 and PCR had a slightly lower sensitivity of 96.7%, each missing one target carbapenemase. n this study, the APB/EDTA method is capable of identifying the zymotype classification but not the specific resistant genes, while Xpert Carba-R and DETECTR are able to detect all target carbapenemases.

Objective To observe the in vitro susceptibility test result of ceftazidime/avibactam（CZA） combined with aztreonam（ATM）, as well as imipenem（IPM） and meropenem（MEM） respectively combined with ceftazidime（CAZ）, cefoperazone/sulbactam（SCF）, amikacin（AK）, levofloxacin（LEV） and fosfomycin（FOS） to KPC-, NDM-and IMP-producing carbapenem-resistant Enterobacterales（CRE）. Methods 38 non-repetitive CRE strains were isolated from clinical specimens. The minimal inhibitory concentration（MIC） of antimicrobial agents against CRE strains was determined by micro-broth dilution method. Combined antimicrobial susceptibility test was performed by the chessboard dilution method. Fractional inhibitory concentration（FIC） index was calculated to determine the combined effect. Results The MICs of CZA to 22 KPC-producing strains were ≤4 μg/mL, to 16 class B metallo-enzyme（NDM and IMP）-producing strains were ＞128 μg/mL. The synergy rate of CZA was 100% after combined with ATM. The synergy rate of IPM or MEM combined with SCF was the highest, which were 63.2% and 68.4% respectively, and the sum of synergy rate and addition rate was 100%, while the sum of synergy rate and addition rate of IPM combined with FOS was the lowest（40.5%）. Among Klebsiella pneumoniae（KP） strains, the synergy rates of IPM or MEM combined with SCF were both 68%, the sum of synergy rate and addition rate was 100%, the sum of synergy rate and addition rate of IPM combined with FOS was the lowest（24%）. Among Escherichia coli（ECO）, the synergy rates of IPM or MEM combined with SCF were 44.4% and 77.8% respectively, the sum of synergy rate and addition rate was 100%, the sum of synergy rate and addition rate of IPM combined with FOS was the lowest（66.7%）. Among genotype KPC strains, the synergy rates of IPM or MEM combined with SCF were 72.7% and 63.6% respectively, the sum of synergy rate and addition rate was 100%, the sum of synergy rate and addition rate of IPM combined with FOS was the lowest（22.7%）. Among genotype NDM strains, the synergy rates of IPM or MEM combined with SCF were 46.7% and 80.0% respectively, the sum of synergy rate and addition rate was 100%, the sum of synergy rate and addition rate of IPM combined with FOS was the lowest（64.3%）. All combined schemes had no antagonistic effect. Conclusion CZA alone or in combination with ATM is effective for CRE strains. Among all the combinations of IPM and MEM respectively with other antimicrobial agents, the sum of synergy rate and addition rate of IPM and MEM combined with SCF is the highest, and the sum of synergy rate and addition rate of IPM combined with FOS is the lowest. Not all combination schemes are effective. The effect of different combination schemes on the same strain is different, and the effect of the same combination scheme on different strains is also different. The dose ratio between two agents is also important.

Abstract Background Tigecycline (TGC) and polymyxin B (PMB)are important antibiotics for the treatment of multidrug-resistant bacterial infections. However, with the widespread applications, the resistant strains have been isolated. To optimize the use of TGC and PMB, there is an urgent need to develop a rapid detection method for the antibiotics. In recent years, a novel Direct-on-Target Micro-droplet Growth Assay (DOT-MGA) has been developed that can detect antibiotic resistance within hours. The study aims to verify the feasibility and accuracy of DOT-MGA in rapidly identification of TGC and PMB-susceptible and non-susceptible bacteria. Methods A total of 67 strains of Klebsiella pneumoniae were collected for DOT-MGA. According to FDA/EUCAST breakpoint concentrations, 6 µL droplets with or without TGC or PMB were added in triplicate into the wells of the MALDI plate with four incubation time points (3 h, 4 h, 6 h, and 8 h). The results were compared with standard databases using Bruker Biotyper software and classified as susceptible and non-susceptible based on the scores. Results Compared with the micro-broth dilution method, the DOT-MGA achieved the best detection performance after 4 h of incubation. The results showed that the growth efficiency of TGC and PMB were both 100%, and the classification agreement rates were 98.15% and 96.15%, respectively. Conclusion With the 4h incubation time, DOT-MGA can accurately identify the resistance of TGC and PMB, which has good consistency compared with the micro-dilution method. DOT-MGA can provide rapid and reliable drug susceptibility diagnosis, which is of great significance for the clinical anti-infective treatment.

目的 探讨基于锥形束计算机断层扫描(cone beam computed tomography,CBCT)图像引导评估热塑膜体位固定对不同体重指数(body mass index,BMI)胸部肿瘤放疗患者摆位精度和受照剂量的影响.方法 收集2018年9月至2021年12月安徽医科大学附属阜阳医院收治的胸部肿瘤患者70例.按照随机数字表法将患者分为A组和B组,每组各35例.A组患者采用双手上举交叉置于颅顶热塑膜体位固定方式,B组采用双手置于体侧热塑膜体位固定方式.根据BMI将患者分为体瘦组(11例)、正常组(28例)、超重组(19例)和肥胖组(12例).所有患者在放疗前均采用CBCT进行图像扫描和数据采集.比较患者不同BMI指数水平下的摆位误差;比较不同热塑膜体位固定方式下的摆位误差;比较不同BMI水平指数下患者脊髓、心脏和肺部受照射剂量;比较不同热塑膜体位固定方式下患者脊髓、心脏和肺部受照射剂量.结果 体瘦组和正常组患者不同方向摆位误差及不同组织受照射剂量相比,差异无显著性(P>0.05).与正常组相比,超重组和肥胖组患者不同方向摆位误差增大(P<0.05).与正常组相比,超重组和肥胖组不同组织受照射剂量增加(P<0.05).B组患者在不同方向的摆位误差小于A组患者(P<0.05).B组患者心脏和肺受照射剂量明显小于A组(P<0.05).两组患者脊髓受照射剂量比较,差异无显著性(P>0.05).结论 胸部肿瘤患者不同热塑膜体位固定方式和不同BMI对放疗时摆位精度和受照射剂量有不同程度的影响.

Introduction: Carbapenemase-mediated antimicrobial resistance is currently a hot spot of global concern.Carbapenem-resistant organisms are highly prevalent in hospitals associated with difficult-to-treat infections, resulting in poor clinical outcome due to limited treatment options.It is urgently needed to have a rapid, efficient, and convenient molecular assay for identifying such resistant strains.Methods: For this end, we developed a new laboratory assay targeting Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) based on loop-mediated isothermal amplification, CRISPR-Cas12a, and lateral flow immunochromatographic strip (CRISPR-Cas-LAMP-lateral flow strip).The method was designed to use a guide RNA (gRNA) to recognize the target DNA and guide Cas12a to cleave the target DNA, and simultaneously cleave any single-stranded DNA within the cleavage reaction system. Results:The cleavage products are visible to the naked eye on the lateral flow strip.This method is highly sensitive in direct detection of bacteria in samples containing at least 3×10 5 CFU/mL without the need for bacterial culture.Discussion: It provides shorter turnaround time and higher specificity than the conventional bacterial culture and susceptibility testing method.This new assay is applicable for extensive use in hospital infection control, as well as identification and treatment of resistant strains due to simple operation and inexpensive apparatuses.

Introduction:To examine the in vitro and in vivo antimicrobial activities of ceftazidime/avibactam (CZA) alone or in combination with aztreonam (ATM) against KPC-, NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains.Methods:A total of 67 clinical non-repetitive carbapenem-resistant Enterobacterales (CRE) strains were selected for the microdilution broth method that was performed to analyze the minimal inhibitory concentration (MIC) and the combination antimicrobial susceptibility test using checkerboard titration method. The fractional inhibitory concentration (FIC) was calculated to determine the antimicrobial effect. The time-kill assays and the mouse infection model were used to study the bactericidal effect and therapeutic effect of CZA alone or in combination with ATM.Results:The CZA minimal inhibitory concentration (MIC) values of CZA revealed that 29 KPC-producing strains and 1 OXA-producing strain were ≤4µg/mL. The CZA MIC values of 37 metal-β-lactamase (MBLs)-producing strains such as NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains were ≥128µg/mL, after combining with ATM, the FIC values were all below 0.51. The time-kill assays revealed that CZA at various concentrations of 2, 4 and 8 MIC showed significant bactericidal efficiency to the KPC-producing strains. For NDM-, IMP-producing strains, no colony growth was detected after 8 hours of incubation with CZA in combination with ATM. Six percent of the mice in the treatment group and 58% of the mice in the infection group died within 3 days.Conclusion:Our in vitro results showed that CZA had a good antimicrobial effect on the KPC-producing and OXA-producing strains. CZA combined with ATM showed synergistic bacteriostatic or bactericidal activity against NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains. The combination of CZA and ATM reduced mortality and prolonged lifespan of mice infected with NDM-, IMP-, KPC+IMP-, and KPC+NDM-producing strains, which provides fundamental knowledge for improving treatment strategies and initializing clinical trials.

Aim To reduce the inspection time for urinary tract pathogens and provide a rapid and effective therapeutic plan for clinical anti-infection treatment, this study developed a rapid identification (ID) and antimicrobial sensitivity test (AST) method by DOT-MGA. Methods We grouped midstream urine samples with single bacteria according to the number of bacteria (≤5/5–15/≥ 15) under per oil microscope after Gram staining. Then we adopted differential centrifugation to process the grouped samples to collect precipitate. MALDI-TOF MS was performed using precipitate directly or after short-term culture. If succeed, we resuspended the precipitate into droplets with or without antibiotics at a MALDI target. Four hours later, mass spectrometer (MS) was used to identify the culture on the target and to analyse AST. Results Samples (count ≥ 15), which precipitate can be directly identified by MS; otherwise, the precipitate need a short-term cultured for 3–6 h before ID. The consistency of the ID results between conventional culture and the precipitate is 100%. Compared with broth microdilution method, DOT-MGA for predicting AST had a high consistency. EA and CA for IPM, LEV, CAZ, NIT, and FOT were 100%/100%, 98%/90%, 98%/92%, 100%/90%, 98%/94%, respectively. No VME was observed in all tests. Besides, MIC50 for the five antibiotics by DOT-MGA and broth microdilution method were ≤1/≤0.5,>2/2,≤4/≤2,≤32/≤16,≤64/≤32 and MIC90 were ≤1/≤0.5, >2/>4, 16/16, 128/128, 128/64. Conclusion This study can shorten the ID time (minimum 0.5h) and AST (minimum 4h) of the main pathogens of urinary tract infection to 5–10 hours, which greatly reduce the inspection time and provide substantial help for the rapid diagnosis and treatment of patients with urinary tract infection.

INTRODUCTION:The recently developed DOT-MGA (direct-on-target microdroplet growth assay) has shown the desirability of direct application of this approach in positive blood cultures and its good performance in detection. This study selected 44 Enterobacteriaceae strains and implemented a DOT-MGA assay on blood cultures to detect their resistance to seven antibiotics. The results of DOT-MGA were compared with the other two antimicrobial susceptibility testing (AST) methods to analyze the detection performance of DOT-MGA.METHODS:We adopted the differential centrifugation to process positive blood-culture (BC). Processed BC broth was directly used for rapid AST using DOT-MGA. Droplets of 6 µL with and without antibiotics at the EUCAST breakpoint concentration were spotted in triplicates onto the surface of a MALDI target. The plates were incubated in a wet box for 4 h before the broth was removed with filter paper. Bruker Biotyper software was used to analyze the test results compared with standard database, and the scores were used to quantify and determine the results.RESULTS:DOT-MGA results were compared with the direct-from-BC disk-diffusion method and results were reported by broth microdilution method, respectively. The comparison demonstrated a 100% growth efficiency in DOT-MGA, a 100% classification consistency for ampicillin, ceftriaxone, and gentamicin, and >93% classification consistency for tobramycin, aztreonam, trimethoprim-sulfamethoxazole (TMP-SMX), and ceftazidime.DISCUSSION:These study results have shown that DOT-MGA is suitable for directly identifying bacterial resistance to positive blood cultures in clinical microbiology laboratories. Furthermore, it is conducive for early diagnosis and treatment of patients with bloodstream infection due to its convenience, time efficiency, and good performance in identifying multiple antibiotic-insensitive bacteria.

目的 研究红细胞分布宽度(red blood cell distribution width,RDW)、血小板分布宽度(platelet distribution width,PDW)联合QT校正离散度(QT corrected dispersion,QTcd)在急性心肌梗死(acute myocardial infarction,AMI)临床诊断中的应用价值.方法 选取2017年7月至2020年12月安徽医科大学附属阜阳医院收治的147例胸痛或胸闷患者为研究对象,均详细采集病史,查体,进行心电图检查并测量QTcd,同时检测外周血RDW和PDW.分析各指标对AMI和冠状动脉完全闭塞的诊断价值.结果 AMI组RDW、PDW和QTcd高于非AMI组,且冠状动脉闭塞组RDW、PDW和QTcd均高于非闭塞组,差异有统计学意义(P＜0.05).多因素Logistic回归分析结果显示RDW、PDW和QTcd与AMI及冠状动脉闭塞程度均密切相关(P＜0.05);RDW、PDW、QTcd及三者联合检测诊断AMI的曲线下面积(area under the curve,AUC)分别为0.817、0.775、0.861和0.938,敏感度分别为60.42％、80.21％、77.08％和87.50％,特异度分别为92.16％、66.67％、84.31％和88.24％;RDW、PDW、QTcd及三者联合检测诊断AMI患者冠状动脉闭塞的AUC分别为0.671、0.824、0.680和0.865,敏感度分别为64.86％、91.89％、51.35％和81.08％,特异度分别为69.49％、61.02％、84.75％和83.05％.结论 RDW、PDW和QTcd用于AMI诊断具有良好价值,同时可为评估AMI患者冠状动脉狭窄程度提供参考信息,对临床诊断和治疗均具有重要意义.

目的:了解我院肺炎克雷伯菌的临床分布及耐药性,为临床合理用药和预防感控提供依据.方法:采用自动化仪器法或纸片扩散法进行抗菌药物敏感性测试,按照临床和实验室标准化协会(C L S I)2017版标准判读结果.结果:我院自开诊两年来共分离肺炎克雷伯菌361株,其中2017年7月—2018年6月,分离160株,2018年7月—2019年6月共分离201株;标本来源主要是痰、尿、血标本,分别占68.42％、14.40％、6.65％;科室分布以I C U、神经外科、呼吸内科为主,分别占16.34％、14.68、8.86％;耐药现象严重,对头孢曲松、氨苄西林/舒巴坦耐药性最高,分别占42.74％、42.66％.结论:我院肺炎克雷伯菌耐药现象不容乐观,相关部门应做好预防感控措施,及时监测细菌耐药现象,合理使用抗生素.

目的:分析本院2017年～2019年常见病原菌分布和细菌耐药性情况,以指导临床用药.方法:将送检标本中的病原菌进行鉴定,参照CLSI 2017版判读药敏结果.结果:收集2017年7月～2019年6月临床分离菌共2292株,其中革兰阴性菌1862株,占81.2％,革兰阳性菌430株,占18.8％.大肠埃希菌占比最高,占25.7％.主要分离于尿液标本(55.9％)、血液标本(12.2％)和痰液标本(10.8％).金黄色葡葡球菌中MRSA(耐甲氧西林金黄色葡萄球菌)和凝固酶阴性葡萄球菌中耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)的检出率分别为61.4％和74.4％.MRSA主要分布于普外科、神经外科和骨科.未检出替考拉宁、利奈唑胺或万古霉素耐药的葡萄球菌.肠球菌属中屎肠球菌对测试药物的耐药率均较高.16株肺炎链球菌,其中3株对青霉素耐药.肠杆菌科细菌中,肺炎克雷伯菌对碳青霉烯耐药率为14.5％.鲍曼不动杆菌对碳青霉烯的耐药率为76.9％.铜绿假单胞菌为19.3％.14株流感嗜血杆菌中,β内酰胺酶检出率为35.7％(5/14).结论:2017年～2019年我院常见病原菌以革兰阴性杆菌为主,病原菌的耐药性较高,临床应加强科室管理,合理应用抗生素,防耐药菌株的流行,降低医院感染的发生风险.