Interferon-gamma release assays as a tool for differential diagnosis of gastrointestinal tuberculosis

In this editorial,we comment on an article published in a recent issue of the World Journal of Clinical Cases.There is a pressing need for reliable tools for diagnosing tuberculosis(TB)of the gastrointestinal tract.Despite advancements in the diagnosis and treatment,TB remains a global health challenge.Ali et al demon-strated that TB may mimic gastrointestinal conditions,such as gastric outlet obstruction,causing a delay in the diagnosis.Furthermore,the latter complication is frequently observed during infections,including Helicobacter pylori,and rarely is related to TB,as in the presented case.In line with this,we think that laboratory tests based on interferon-gamma release assays can be a helpful tool for diagnosing latent TB paced in the gastrointestinal tract.Innovative strategies and approaches for diagnosing latent/active extra pulmonary TB are crucial for establishing the diagnosis early and enhancing treatment strategies to mitigate the global burden of TB.

Reversed-Phase-HPLC Assay Method for Simultaneous Estimation of Sorbitol, Sodium Lactate, and Sodium Chlorides in Pharmaceutical Formulations and Drug Solution for Infusion

A rapid, straightforward, sensitive, efficient, and cost-effective reverse-phase high-performance liquid chromatographic method was employed for the simultaneous determination of Sorbitol, Sodium Lactate, and Chlorides in a drug solution for infusion. Sorbitol, Sodium lactate, and Chloride are all officially recognized in the USP monograph. Assay methods are provided through various techniques, with titrations being ineffective for trace-level quantification. Alternatively, IC, AAS, and ICP-MS, though highly accurate, are costly and often unavailable to most testing facilities. When considering methods, it’s important to prioritize both quality control requirements and user-friendly techniques. A simple HPLC simultaneous method was developed for the quantification of Chlorides, Sorbitol, and Sodium Lactate with a shorter run time. The separation utilized a Shimpack SCR-102(H) ion exclusion analytical column (7.9 mm × 300 mm, 7 μm), with a flow rate of 0.6 mL per min. The column compartment temperature was maintained at 40°C, and the injection volume was set at 10 μL, with detection at 200 nm. All measurements were conducted in a 0.1% solution of phosphoric acid. The analytical curves demonstrated linearity (r > 0.9999) in the concentration range of 0.79 to 3.8 mg per mL for Sodium Lactate (SL), 0.16 to 0.79 mg per mL for Sodium Chloride (SC), and 1.5 to 7.2 mg per mL for Sorbitol. Validation of the developed method followed the guidelines of the International Conference on Harmonization (ICH Q2B) and USP. The method exhibited precision, robustness, accuracy, and selectivity. In accelerated stability testing over 6 months, no significant variations were observed in organoleptic analysis and pH. Consequently, the developed method is deemed suitable for routine quality control analyses, enabling the simultaneous determination of Sodium Lactate, Sodium Chloride, and Sorbitol in pharmaceutical formulations and infusions.

Evaluation of Interferon-Gamma Release Assay Testing and Tuberculin Skin Test for Early Diagnosis of Tuberculosis in Children and Adolescents

Background: This study aimed to evaluate the diagnostic value of interferon-γ release assay (IGRA), a sensitive microbiological diagnostic method, in children and adolescents with suspected tuberculosis in a country with a high burden of tuberculosis. Method: This study included 581 children and adolescents aged 4 - 19 years who were suspected of having tuberculosis, were latently infected with Mycobacterium tuberculosis, and had received at least one dose of BCG vaccine between April 17, 2019, and February 24, 2021. The study evaluated the TST results of 106 patients who had a positive Quantiferon test and were suspected of having tuberculosis. Results: The study included 581 patients aged between 4 and 19 years. Of these, 106 patients tested positive for the Quantiferon test, while 19 were indeterminate and 456 were negative. The Quantiferon test positivity rate was 18.24%. Among the 106 QFT-Plus-positive cases, 23 patients also tested positive for TST. The difference in distribution was found to be statistically significant. Conclusion: The QFT-Plus test is considered an alternative to TST and other microbiological diagnostic methods for early tuberculosis diagnosis, particularly in children and adolescents.

Elispot assay检测牛奶中庆大霉素

为了建立快速检测牛奶中庆大霉素的Elispot assay,采用制备GM免疫抗原获得抗GM多克隆抗体。将检测抗原点阵在PVDF膜上,通过检测抗原和样品中GM竞争结合抗GM多克隆抗体,酶标结合物催化底物显色,根据颜色的有无及深浅判读结果,从而建立了检测牛奶中GM的Elispot assay。该方法的检测阈值为10 ng/mL,检测时间为40 min,可对样品实现半定量检测。该方法制备的试纸条于4℃密封保存90 d仍可用于检测;与多种结构类似物未见交叉反应;其结果与酶联免疫方法一致。

Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

Antioxidant and lipoxygenase inhibitory properties of a novel flavonoid from Pistacia chinensis Bunge and its molecular docking analysis

Background:Pistacia chinensis Bunge has been traditionally used to manage various conditions,including asthma,pain,inflammation,hepatoprotection,and diabetes.The study was conducted to investigate the antioxidant and anti-lipoxygenase(LOX)properties of the isolated compound 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one from Pistacia chinensis.Methods:LOX assay and antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl(DPPH)assay were performed.Molecular docking studies were conducted using a molecular operating environment.Results:The LOX assay revealed significant inhibitory effects at 0.2µM concentration,with an IC50 value of 37.80µM.The antioxidant effect demonstrated dose-dependency across 5 to 100µg/mL concentrations,reaching 93.09%at 100µg/mL,comparable to ascorbic acid’s 95.43%effect.Molecular docking studies highlighted strong interactions with the lipoxygenase enzyme,presenting an excellent docking score of-10.98 kcal/mol.Conclusion:These findings provide valuable insights into Pistacia chinensis’chemical components and biological effects,reinforcing its traditional medicinal applications.

RVP Fast与Seeplex PneumoBacter ACE Detection assay联合诊断呼吸道感染病原体

目的采用RVP Fast与Seeplex PneumoBacter ACE Detection assay两种方法,联合诊断呼吸道感染病原体。方法收集南京市呼吸道感染患者咽拭子标本67份,提取RNA及DNA,呼吸道病毒群快速检测(Respiratory Virus Panel Fast Array,RVP Fast)技术检测呼吸道病毒,Seeplex PneumoBacter ACE Detection assay检测呼吸道六重病原体。结果 67份标本中,呼吸道病毒阳性仅4例,主要为肠鼻病毒。呼吸道六重病原体检测中,阳性标本为26例,其中肺炎链球菌、流感嗜血杆菌、肺炎衣原体阳性较多。结论 RVP Fast与Seeplex PneumoBacter ACE Detection assay联合使用,有利于明确呼吸道感染病原体。

MassARRAY Assay高通量技术检测胃癌LTA单核苷酸多态性及其临床应用

目的探讨LTA基因单核苷酸多态性与胃癌易感性的相关性。方法选择60例胃癌患者和60例健康人群为研究对象。采用Mass ARRAY Assay高通量技术检测淋巴毒素-α（LTA）基因单核苷酸（SNP）位点rs909253基因多态性,分析其出现频率及与胃癌易感性的相关性。结果胃癌组与正常组在AA基因频率（20.00%vs.21.67%）差异无统计学意义;G等位基因、GA、GG＋GA基因型或变异基因型与胃癌易感风险相关[OR（95%CI）：1.48（1.15~1.99）、1.36（1.05~1.68、1.32（1.22~1.65）]。结论 LTA基因rs909253位点单核苷酸多态性与胃癌易感性相关,G等位基因、GG、GA、GG＋GA基因型或变异基因型可能是胃癌发病的危险因素。

Evaluation of the Ability to Apply Near-Infrared Spectroscopy on Direct Assay of Acyclovir in Tablets

This paper aimed at developing a simple and fast approach using chemometrics processing for direct assay of acyclovir in tablets by NIR （near infrared） spectroscopy in diffuse reflectance mode. In making trials with 5 different tablet matrices, the experimental results showed that regardless the matrix variation, it was always possible to construct a quantitative model with suitable linear range, accuracy and precision for direct assay of acyclovir in tablet from NIR spectra. Therefore, the approach used in this study was suitable for on-site fast assay of APIs in tablets during manufacturing process or in post-marketing surveillance of drug quality.

采用Allergy Lateral Flow Assay检测过敏原特异性IgE抗体的性能评价

目的Allergy Lateral Flow Assay(ALFA)是一种便捷、小巧的过敏原检测新技术,本研究以荧光酶联免疫全定量ImmunoCAP法为“金标准”,旨在探讨ALFA的检验效能及在中国南方地区的应用价值。方法利用ALFA技术与ImmunoCAP技术检测100例过敏性疾病患者血清屋尘螨、粉尘螨、热带无爪螨、猫毛皮屑、狗毛皮屑、蟑螂和艾蒿sIgE水平。结果以ImmunoCAP法为“金标准”,结果显示,ALFA检测粉尘螨的一致性最高(κ=0.56,P<0.05),其次是屋尘螨(κ=0.43,P<0.05);同时,ALFA检测粉尘螨(87.6%)、猫毛皮屑(73.7%)和屋尘螨(87.6%)的总一致率均较高。经受试者工作特征曲线分析显示,ALFA诊断螨类过敏原的曲线下面积均大于0.90(P<0.05)。对于屋尘螨、粉尘螨和猫毛皮屑过敏原,ALFA与ImmunoCAP系统的sIgE检测结果具有强相关性(r I>0.8,P<0.05)。结论ALFA具有良好的诊断效能,可满足临床检测要求,因其无须昂贵的检测仪器和检测费用,可为基层医院临床筛查应用提供新的选择。

Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization （WHO） manual-based semen parameter＇s concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay （SCSA）, a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection （ICSI）.

Predictive value of MTT assay as an in vitro chemosensitivity testing for gastric cancer:One institution's experience

AIM:To investigate the predictive clinical value of in vitro 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay for directing chemosensitivity in patients with gastric cancer. METHODS:Results of a total of 353 consecutive patients with gastric cancer treated with MTT-directed chemotherapy or physician’s empirical chemotherapy from July 1997 to April 2003 were reviewed and analyzed retrospectively. RESULTS:The overall 5-year survival rate of MTT- sensitive group (MSG) and control group (CG) was 47.5% and 45.1%, respectively. The results of subgroup analysis with Cox proportional-hazards model were favorable for the MSG-sensitive group. However, no statistically significant difference in survival rate was observed between the two groups. CONCLUSION:Individualized chemotherapy based on in vitro MTT assay is beneficial, but needs to be confirmed by further randomized controlled trials.

Xpert®Xpress Flu/RSV Assay用于临床呼吸道感染病原学诊断的价值

目的评估体外诊断试剂Xpert®Xpress Flu/RSV Assay(Xpress Flu/RSV)对临床甲型流感病毒(FluA)、乙型流感病毒(FluB)和呼吸道合胞病毒(RSV)标本的检测性能。方法选择2018年1月11日至5月9日于南方医科大学珠江医院就诊的420例具有呼吸道感染症状的患者为研究对象,采集鼻咽拭子标本,分别采用Xpress Flu/RSV和普通聚合酶链反应(PCR)测序进行检测,结果不一致标本采用自建反转录实时荧光定量聚合酶链反应(RT-qPCR)确认,以任意两种方法结果一致为标准,分析Xpress Flu/RSV的诊断性能。结果420例患者标本中Xpress Flu/RSV检出阳性213例(50.7%),灵敏度(FluA:100.0%;FluB:100.0%;RSV:100.0%)和特异度(FluA:100.0%;FluB:99.4%;RSV:99.7%)均较高;对其他呼吸道病原体(除FluA、FluB、RSV)感染标本检测结果均为阴性;95例患者标本Xpress Flu/RSV检测结果与自建RT-qPCR相比,一致性较高(Kappa值为0.949)。结论Xpress Flu/RSV检测性能较好,适用于病毒流行期的大规模筛查。

Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF

The aim of this study was to investigate whether the sperm chromatin structure assay （SCSA） results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitrofertilization （IVF）. A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index （DFI） subgroups and high DNA stainability （HDS） subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.

A rapid reporter assay for recombinant human brain natriuretic peptide(rhBNP)by GloSensor technology

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide （rhBNP）. In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGIo-G1 was constructed by transfecting pGloSensorTM 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGIo-G1 showed high precision with intraassay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R^2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay （paired t test,p = 0.630）. All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.

Cytotoxicity evaluation and hepatoprotective potential of bioassay guided fractions from Feronia limmonia Linn leaf

Objective:To evaluate the cytotoxicity and hepatoprotective potentials of extracts,fractions or isolated compound from the leaves of Feronia limonia(F.limonia).Methods:Qualitative phytochemical analysis of extracts,fractions or compound was performed by means of thin layer chromatography and spectroscopic assays.The%purity of compound was measured by analytical HPLC.Extracts,fractions or compound have been individually evaluated for their cytotoxicity effects(10,20,100,250,500,750 and 1 000 μg/mL).Based on the inhibitory concentration(IC_(50)) obtained from the cell viability assay,graded concentrations of extracts,fractions or isolated compound were assessed(10,20,50,100,200 μg/mL) for its hepatoprotective potential against CCl_4-induced hepatotoxicity by monitoring activity levels of serum glutamatic pyruvatic transaminase(SGPT) and serum glutamic oxaloacetic transaminase(SGOT).Results:Results indicated that the methanol extract of F.limonia was non-toxic and hepatoprotective in nature as compared with the petroleum ether extract.The acetone fraction of methanolic extract also showed similar properties but the subsequent two fractions were cytotoxic.However,the pure compound isolated from the penultimate fraction of methanolic extract was non-toxic and hepatoprotective in nature.Biochemical investigations(SCOT,SCPT) further corroborated these cytological observations.Conclusions:It can be concluded from this study that F.limonia methanol extract,some fractions and pure isolated compound herein exhibit hepatoprotective activity.However,cytotoxicity recorded in the penultimate fraction and investigation of structural details of pure compound warrants further study.

Science Letters:Evaluation of a kinetic uricase method for serum uric acid assay by predicting background absorbance of uricase reaction solution with an integrated method

A patented kinetic uricase method was evaluated for serum uric acid assay. Initial absorbance of the reaction mixture before uricase action （A0） was obtained by correcting the absorbance at 293 nm measured before the addition of uricase solution, and background absorbance （Ab） was predicted by an integrated method. Uric acid concentration in reaction solution was calculated from AA, the difference between A0 and Ab, using the absorptivity preset for uric acid. This kinetic uricase method exhibited CV〈4.3% and recovery of 100%. Lipids, bilirubin, hemoglobin, ascorbic acid, reduced glutathione and xanthine 〈0.32 mmol/L in serum had no significant effects. △A linearly responded to 1.2 to 37.5 μmol/L uric acid in reaction solution containing 15 μl serum. The slope of linear response was consistent with the absorptivity preset for uric acid while the intercept was consistent with that for serum alone. Uric acid concentrations in clinic sera by different uricase methods positively correlated to each other. By Bland-Altman analysis, this kinetic uricase method accorded with that by quantifying the total change of UV absorbance on the completion of uricase reaction. These results demonstrated that this kinetic uricase method is reliable for serum uric acid assay with enhanced resistance to both xanthine and other common errors, wider range of linear response and much lower cost.

Preliminary study of a dot immunogold filtration assay for rapid detection of anti-HCV IgG

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Rapid detection of potato late blight using a loop-mediated isothermal amplification assay

Potato late blight caused by Phytophthora infestans is one of the most destructive plant diseases that threaten global food security. Early and effective diagnosis of P. infestans is required before disease management decisions are made. Here, we developed a quick protocol to detect P. infestans based on a loop-mediated isothermal amplification(LAMP) assay. The P. infestans specific multiple copy DNA sequences(PiSMC), a transposon-like element, provides an ideal target for molecular detection of this pathogen. We designed highly specific and sensitive primers allowing effective LAMP detection of the pathogen at 64℃ in 70 min. In the validation assay, all 15 P. infestans isolates collected from China, Europe and South America could be positively detected, but results of the other 9 Phytophthora species infecting different plants, fungal and bacterial plant pathogens tested were negative. The detection limit of this assay is 1 pg P. infestans DNA. Moreover, the LAMP-PiSMC assay is able to detect P. infestans from infected leaves, tubers and soil. Taken together, this study reports the development of a specific and sensitive LAMP-PiSMC assay for early diagnosis of potato late blight.

Study of Low-intensity 2450-MHz Microwave Exposure Enhancing the Genotoxic Effects of Mitomycin C Using Micronucleus Test and Comet Assay in vitro

Objective To determine the interaction between 2450-MHz microwaves (MW) radiation and mitomycin C (MMC). Methods The synergistic genotoxic effects of low-intensity 2450-MHz microwave and MMC on human lymphocytes were studied using single cell gel electrophoresis (SCGE) assay (comet assay) and cytokinesis-blocked micronucleus (CBMN) test in vitro. The whole blood cells from a male donor and a female donor were either only exposed to 2450-MHz microwaves (5.0 mW/cm2) for 2 h or only exposed to MMC (0.0125 μ/mL, 0.025 μg/mL, 0.05μg/mL and 0.1 μg/mL) for 24 h; and the samples were exposed to MMC for 24 h after exposure to MW for 2 h. Results In the comet assay, the comet lengths ( 29.1 μm and 25.9 μm) of MW were not significantly longer than those (26.3 μrn and 24.1 μm) of controls (P>0.05). The comet lengths (57.4 μm, 68.9 μm, 91.4 μm, 150.6μm and 50.6 μm, 71.7μm, 100.1 μm, 145.1 μm) of 4 MMC groups were significantly longer than those of controls (P<0.01). The comet lengths (59.1 μm, 92.3 μm, 124.5 μm, 182.7 μm and 57.4 μm, 85.5 μm, 137.5 μm, 178.3 μm) of 4 MW plus MMC groups were significantly longer than those of controls too (P<0.01). The comet lengths of MW plus MMC groups were significantly longer than those of the corresponding MMC doses (P<0.05 or P<0.01) when the doses of MMC were ≥50.025 μg/mL. In the CBMN, the micronucleated cell (MNC) rates of MW were 5% and 6%, which showed no difference compared with those (4‰ and 4‰) of controls (P>0.05). The MNC rates of 4 MMC groups were 8‰, 9‰, 14‰, 23‰ and 8‰, 8‰, 16‰, 30‰ respectively. When the doses of MMC were 3≥0.05 μg/mL, MNC rates of MMC were higher than those of controls (P<0.05). MNC rates of 4 MW plus MMC groups were 12‰, 13‰, 20‰, 32‰ and 8‰, 9‰, 23‰, 40‰. When the doses of MMC were 5≥0.05 μg/mL, MNC rates of MW plus MMC groups were much higher than those of controls (P<0.01). MNC rates of 4 MW plus MMC groups were not significantly higher than those of the corresponding MMC doses. Conclusion The low-intensity 2450-MHz microwave radiation can not induce DNA and chromosome damage, but can increase DNA damage effect induced by MMC in comet assay.