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.

An Overview on SARS‑CoV‑2(COVID‑19)and Other Human Coronaviruses and Their Detection Capability via Amplification Assay,Chemical Sensing,Biosensing,Immunosensing,and Clinical Assays

A novel coronavirus of zoonotic origin(SARSCoV-2)has recently been recognized in patients with acute respiratory disease.COVID-19 causative agent is structurally and genetically similar to SARS and bat SARS-like coronaviruses.The drastic increase in the number of coronavirus and its genome sequence have given us an unprecedented opportunity to perform bioinformatics and genomics analysis on this class of viruses.Clinical tests like PCR and ELISA for rapid detection of this virus are urgently needed for early identification of infected patients.However,these techniques are expensive and not readily available for point-of-care(POC)applications.Currently,lack of any rapid,available,and reliable POC detection method gives rise to the progression of COVID-19 as a horrible global problem.To solve the negative features of clinical investigation,we provide a brief introduction of the general features of coronaviruses and describe various amplification assays,sensing,biosensing,immunosensing,and aptasensing for the determination of various groups of coronaviruses applied as a template for the detection of SARS-CoV-2.All sensing and biosensing techniques developed for the determination of various classes of coronaviruses are useful to recognize the newly immerged coronavirus,i.e.,SARS-CoV-2.Also,the introduction of sensing and biosensing methods sheds light on the way of designing a proper screening system to detect the virus at the early stage of infection to tranquilize the speed and vastity of spreading.Among other approaches investigated among molecular approaches and PCR or recognition of viral diseases,LAMP-based methods and LFAs are of great importance for their numerous benefits,which can be helpful to design a universal platform for detection of future emerging pathogenic viruses.

Development and Validation of a Simoa Assay for Determination of Recombinant Batroxobin in Human Serum

Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies.Therefore,a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101.In this study,a sensitive bioanalytical method was developed and validated,using a Quanterix single molecular array(Simoa)assay.Moreover,to thoroughly assess the platform,enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed,and their performance was compared with that of this novel technology platform.The assay was validated in compliance with the current guidelines.Measurements with the Simoa assay were precise and accurate,presenting a valid assay range from 6.55 to 4000 pg/mL.The intra-and inter-run accuracy and precision were within-19.3%to 15.3%and 5.5%to 17.0%,respectively.S3101 was stable in human serum for 280 days at-20℃and-70℃,for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature(22℃-28℃),respectively,and after five and two freeze-thaw cycles at-70℃and-20oC,respectively.The Simoa assay also demonstrated sufficient dilution linearity,assay sensitivity,and parallelism for quantifying S3101 in human serum.The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.

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.

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.

Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

Response of Lymphocytes to Radiation in Untreated Breast Cancer Patients as Detected with Three Different Genetic Assays

Objective To detect the response of lymphocytes to radiation in untreated breast cancer patients with three different genetic assays. Methods Blood samples were collected from 25 untreated patients and 25 controls. Each blood sample was divided into two parts： one was irradiated by 3-Gy X-ray （irradiated sample）, the other was not irradiated （non-irradiated sample）. The radiosensitivity of lymphocytes was assessed by comet assay, cytokinesis-block micronucleus （CBMN） assay and 6-TG-resistant cells scored （TG） assay. Results The baseline values of micronucleated cell frequency （MCF） and micronucleus frequency （MNF） in the patients were significantly higher than those in the controls （P〈0.01）, and 3-Gy X-ray induced genetic damage to lymphocytes in the patients increased significantly as compared with that in the controls as detected with the three genetic assays （P〈0.01）. The proportion of radiosensitive cases in the patient group was 48% for the mean tail length （MTL）, 40% for the mean tall moment （MTM）, 40% for MCF, 44% for MNF, and 48% for mutation frequencies of the hprt gene （Mfs-hprt）, respectively, whereas the proportion of radiosensitive cases in the control group was only 8% for all the parameters. Conclusion The difference in the lymphocyte radiosensitivity between the breast cancer patients and the controls is significant. Moreover, there are wide individual variations in lymphocyte radiosensitivity of patients with breast cancer. In some cases, the radiosensitivity of the same patient may be different as detected with the different assays. It is suggested that multiple assays should be used to assess the radiosensitivity of patients with breast cancer before therapy.

Hepatitis C virus antigens enzyme immunoassay for one-step diagnosis of hepatitis C virus coinfection in human immunodeficiency virus infected individuals

BACKGROUND Current diagnosis of hepatitis C virus(HCV)infection requires two sequential steps:testing for anti-HCV followed by HCV RNA PCR to confirm viremia.We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay(HCV-Ags EIA)for one-step diagnosis of viremic HCV infection.AIM To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus(HIV)-coinfected individuals.METHODS The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera:10 without HCV or HIV infection;54 with viremic HCV monoinfection;38 with viremic HCV/HIV coinfection;and 45 with viremic HCV and non-viremic HIV coinfection.RESULTS Upon decoding,it was 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test.In five sera with HCV infection,HCV RNA was as low as 50-59 IU/mL,and four out of five tested positive for HCV-Ags EIA.Likewise,it was also 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection,regardless if HIV infection was active or not.CONCLUSION The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL.It is highly sensitive and specific in the setting of HIV coinfection,regardless of HIV infection status and CD4 count.These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.

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。

Leishmania tropica: The comparison of two frequently-used methods of parasite load assay in vaccinated mice

Objective:To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods:BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stressinducible protein-1 with/without adjuvant.After three vaccinations,mice were challenged by Leishmania tropica promastigotes.Two months after challenge,the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods.Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium.For real-time PCR,DNA of the lymph nodes was extracted,equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers.The data of the two methods were compared by appropriate statistical methods.Results:Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice.In addition,wherever parasite load of a group was estimated high(or low)by one method,the estimated parasite load by another method was the same,although statistically significant differences were found between some groups.Conclusions:Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups.However,due to the lower errors and faster process,the real-time PCR method is preferred.

Comparative Assay of Hepatitis B and C Virus Infection Markers by Different Assay Kits^1

In order to compare sensitivity of EIA and RIA assay kits for hepatitis B and C virus (HBV and HCV, respectively) infection markers, 100 serum samples in total were collected form 50 adult women each in urban and rural areas in northeast China. The number of positive cases to the three infection markers on HBV (i.e., HBsAg +, anti HBs +, and anti HBc +) and the one on HCV (anti HCV +) were examined in two laboratories, i.e., in Laboratory A with EIA kits produced in China and in Laboratory B with RIA kits. HCV infection positivity (anti HCV +) was examined by EIA kits in both laboratories, but from different sources in and outside of China, respectively. The assay in Laboratory A gave 2 HBsAg + cases out of the 100 cases examined, whereas there were 9 positive cases in Laboratory B. In contrast, 19 cases were positive to anti HCV when examined in Laboratory A, and there were 3 cases in Laboratory B. Thus, the kits used in Laboratory A gave fewer HBsAg + and more anti HCV + cases than the kits used in Laboratory B. The prevalence of anti HBs + or anti HBc + and cases did not differ when assayed in the two laboratories with EIA and RIA kits, respectively. The agreement of positive and negative findings between the two sets of testing were 93%, 93%, 93%, 86% and 82% for HBsAg, anti HBs, anti HBc, HBV (i.e., either positive to anyone of the three markers or negative to all three markers), and anti HCV, respectively. The implication of the observation on epidemiology on HBV and HCV infection prevalence was discussed.

Development of An ICR Mouse Bioassay for Toxicity Evaluation in Neurotoxic Poisoning Toxins-Contaminated Shellfish

Objective To develop an ICR （female） mouse bioassay （MBA） for toxicity confirmation and evaluation of neurotoxins （brevetoxins）-contaminated shellfish. Methods Brevetoxins （BTX-B） as a causative agent of neurotoxic shellfish poisoning （NSP） under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. Results BTX-B could be detected in shellfish tissues at concentration of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit （80 μg/100 g shellfish tissues）. The LD 50 identified was 455 g/kg for BTX-B under general shellfish matrices （excluding oyster matrices） dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. Conclusion The two ELISA analyses agree favorably （correlation coefficient, r 0.96; Student＇s t-tests, P〉0.05） with the developed bioassay.

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.