A novel AllGlo probe-quantitative PCR method for detecting single nucleotide polymorphism in CYP2C19 to evaluate the antiplatelet activity of clopidogrel.

CYP2C19 gene has multiple single nucleotide polymorphism (SNP), which is the major determinant for clopidogrel treatment responses. Therefore, CYP2C19 SNP detection is essential for predicting clopidogrel efficacy. Currently, there is still no quick and effective method for routine detection of common CYP2C19 SNPs in clinical laboratories, which is critically needed prior to clopidogrel treatment. AllGlo™ based quantitative PCR was used to develop a novel genotyping method for CYP2C19 SNP detection, termed CyPAllGlo. The performance of CyPAllGlo was compared with that of the commonly used fluorescence in situ hybridization (FISH) method, and the data was verified by DNA sequencing. CyPallGlo was used to identify CYP2C19 polymorphisms in 363 patients with coronary heart disease. The univariate analysis was used to access the antiplatelet efficacy of clopidogrel in patients. The associations between CYP2C19 polymorphisms and clopidogrel efficacy were analyzed. Using CyPAllGlo to detect CYP2C19*2 and CYP2C19*3 alleles was highly specific and fast. The detection limit was approximately 0.07 µg/µl and 0.7 µg/µl for CYP2C19*2 and CYP2C19*3, respectively. The consistency between FISH and CyPAllGlo were 98.07% for CYP2C19*2 and 99.17% for CYP2C19*3. DNA sequencing showed that the accuracy of CyPAllGlo was 100%. The analysis time for the whole CyPAllGlo procedure was approximately 60 min. Univariate analysis showed that the anticoagulation efficacy of clopidogrel was related to patient age, CYP2C19 genotype, metabolic phenotype, and LDL level. The logistic regression analysis showed that the genotype of CYP2C19 and metabolic phenotype was the two risk factors for clopidogrel antiplatelet ineffectiveness. This novel CyPAllGlo is a rapid and accurate method for detection of CYP2C19 SNP. The specificity and consistency of CyPAllGlo are comparable with that of widely used DNA sequencing. These findings provide valuable rapid method for predicting clopidogrel efficacy, which can be quickly translated to improve personalized precision medicine for coronary heart disease treatment.

Correction: Association of hypertension with helicobacter pylori: A systematic review and meta­analysis.

[This corrects the article DOI: 10.1371/journal.pone.0268686.].

Association of hypertension with helicobacter pylori: A systematic review and meta­analysis.

BACKGROUND AND AIMS: The number of hypertensive population rises year by year recently, and their age becomes more youthful. For a long time, hypertension has long been regarded as a multi-factorial disease. In addition to smoking, genetics, diet and other factors, helicobacter pylori (H. pylori) had been regarded as a potential risk factor for hypertension in recent years. However, most studies had certain limitations and their results were inconsistent. Thus, it is necessary for us to assess the impact of H. pylori on hypertension through meta-analysis. METHODS: We searched all published relevant literature through multiple databases by July 23, 2021. Pooled results were calculated under the random effect model. Heterogeneity was evaluated by the Q statistic and the I2 statistic. The risk of bias was evaluated via ROBINS-I tool. Publication bias was evaluated by the Egger test and Begg funnel plot. RESULTS: 6 eligible studies involving 11317 hypertensive patients and 12765 controls were selected from 20767 retrieval records. Our research confirmed that H. pylori significantly increased the probability of suffering from hypertension in the random effect model (OR:1.34, 95% CI:1.10-1.63, P = 0.002, I2 = 74%). The same results were also found in both Asian population and developing country (OR:1.28, 95%CI:1.05-1.55, P = 0.003, I2 = 78.5%). CONCLUSIONS: Our results confirmed that H. pylori was a vital risk factor for hypertension. H. pylori-infected people were 13.4% higher risk for hypertension than uninfected individuals. In addition, it will be a new method to prevent and treat hypertension by eradicating H. pylori. TRIAL REGISTRATION: The registration number for systematic review in PROSPERO CRD42021279677.

A one-step potentiometric immunoassay for plasma cardiac troponin I using an antibody-functionalized bis-MPA-COOH dendrimer as a competitor with improved sensitivity.

Herein, we have reported a new one-step potentiometric immunoassay for the sensitive and specific detection of human plasma cardiac troponin I (cTnI), a biomarker of cardio-cerebrovascular diseases. Initially, the cTnI biomolecules were immobilized on the surface of a gold nanoparticle-functionalized screen-printed graphite electrode (SPGE). Thereafter, rabbit polyclonal antibodies to cTnI were covalently conjugated to the bis-MPA-COOH dendrimers through typical carbodiimide coupling. The introduction of the target analyte caused a competitive immunoreaction between the immobilized cTnI on the electrode and the conjugated antibody on the dendrimers. The potentiometric measurement was mainly derived from the change in the surface charge on the surface of the modified electrode due to the negatively charged bis-MPA-COOH dendrimers after the immunoreaction. On increasing target cTcI, the number of charged dendrimers on the immunosensor decreased, resulting in a change in the electric potential. Under optimum conditions, the potentiometric immunosensor exhibited good potentiometric responses for the detection of cTcI and allowed the determination of the target analyte at a concentration as low as 7.3 pg mL-1. An intermediate precision of ≤8.7% was accomplished with batch-to-batch identification. Meanwhile, the potentiometric immunosensor showed good anti-interfering capacity and selectivity against other proteins and biomarkers. Importantly, our system displayed high accuracy for the analysis of human plasma serum samples containing target cTcI relative to commercial human cTcI enzyme-linked immunosorbent assay (ELISA) kits.

Effect of Helicobacter pylori infection on the risk of acute coronary syndrome: A systematic review and meta-analysis.

BACKGROUND: Numerous studies have illustrated the association between Helicobacter pylori (H pylori) infection and acute coronary syndrome (ACS). However, the results are contradictory. Therefore, we conducted the meta-analysis to identify the association between H pylori and ACS. METHODS: We performed a systematic search through electronic databases (Excerpta Medica Database, PubMed, Cochrane Library, and Web of Science). Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated with a random effect model. We also carried out the sensitivity analysis and publication bias. RESULTS: Forty-four eligible studies involving 7522 cases and 8311 controls were included. The pooled result showed that H pylori infection was associated with an increase risk of ACS (OR = 2.03, 95% CI 1.66-2.47). In addition, similar results were obtained in subgroups of study quality, area, human development index, and H pylori detection method. The OR for developing countries was significantly higher than developed countries (OR = 2.58 vs OR = 1.69). Moreover, H pylori with cytotoxin-associated antigen A was also significantly associated with an increase risk of ACS (OR = 2.39, 95% CI 1.21-4.74). CONCLUSION: The meta-analysis suggested that H pylori infection was associated with an increased risk of ACS, especially in developing countries. H pylori is easily screened and can be treated with a wide range of drugs. Thus, more high-quality and well-designed studies are needed to confirm whether the treatment of H pylori is an effective way to reduce ACS risk.

Association between IL-1ß + 3954C/T polymorphism and myocardial infarction risk: A meta-analysis.

BACKGROUND: Many studies have reported that the IL-1ß + 3954C/T polymorphism (rs1143634) is related to myocardial infarction (MI). To classify the association between IL-1ß + 3954C/T and MI susceptibility, we performed a meta-analysis. METHODS: We retrieved relevant literature from electronic databases (Embase, PubMed, Cochrane, and Web of Science). Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated with a fixed effect model or a random effect model. Sensitivity analysis and publication bias results are also presented. RESULTS: Nine eligible studies (2299 controls and 2203 cases) were included. The pooled results showed a significant relationship between MI and IL-1ß + 3954C/T in an allelic comparison (T vs C: OR = 1.13, 95% CI 1.02-1.25, I = 0%, PH = .448) and in a dominant model (TC + TT vs CC: OR = 1.15, 95% CI 1.02-1.30, I = 0%, PH = .880). Ethnic subgroup analysis showed similar results in Caucasian populations: an allelic comparison (T vs C: OR = 1.16, 95% CI 1.04-1.29, I = 0%, PH = .701), homozygote model (TT vs CC: OR = 1.36, 95% CI 1.04-1.79, I = 0%, PH = .673), and dominant model (TC + TT vs CC: OR = 1.17, 95% CI 1.02-1.33, I = 0%, PH = .851). In addition, similar effects remained in subgroups analyses of high-quality studies and PCR-RFLP (restriction fragment length polymorphism) data. CONCLUSION: Our meta-analysis proved that IL-1ß + 3954C/T is associated with MI susceptibility, especially among Caucasian populations.

Performance evaluation of a chemiluminescence microparticle immunoassay for CK-MB.

BACKGROUND: To verify and evaluate the performance characteristics of a creatine kinase phosphokinase isoenzymes MB (CK-MB) assay kit, which produced by Xiamen Innodx Biotech Co. Ltd. METHODS: Evaluation was carried out according to "Guidelines for principle of analysis performance evaluation of in vitro diagnostic reagent." The performance parameters included detection limit, linearity range, reportable range, recovery test, precision verification, interference test, cross-reactivity, matrix effect, and method comparison. RESULTS: The detection limit was 0.1 ng/mL. The assay had clinical linearity over range of 0.1 ng/mL-500 ng/mL. Reportable range was from 0.1 ng/mL to 1000 ng/mL. The average percent of recovery was 99.66%. The coefficient of variation (CV) for within-run and between-run of low CK-MB sample was 5.55% and 6.16%, respectively. As for high-level sample, it was 7.88% and 7.80%. In medical decision level, the relative deviation (Bias) of all interference tests was lower than 15%. When the sample had mild-hemolysis; hemoglobin ≤15 g/L; triglyceride ≤17 mmol/L; bilirubin ≤427.5 µmol/L; rheumatoid factor ≤206U/mL, there was no significant interference to be found. Moreover, assay kit had no cross-reaction with CK-MM and CK-BB. At last, total diagnostic accuracy of kit was 93.24%, when compared with refer kit. CONCLUSION: Overall the results of the verification study indicated the performance of kit is met the requirements of the clinical test.

Detection of Salmonella Infection in Chickens by an Indirect Enzyme-Linked Immunosorbent Assay Based on Presence of PagC Antibodies in Sera.

The outcomes of infection of humans and animals with Salmonella range from a persistent asymptomatic carrier state to temporal mild gastroenteritis or severe systemic infection. A rapid and accurate diagnostic test would help formulate strategies for effective prevention of their infections in the animal population. Current sequencing data predict that the outer membrane protein, PagC, is present in all common Salmonella serovars with sequence similarities of more than 98%. PagC sequences in other bacterial species are less than 65% similarity at the amino acid level to those of Salmonella PagC. We hypothesized that PagC could be immunogenic and detection of antibodies to this protein could be an accurate indicator of Salmonella infection. The pagC gene from Salmonella enterica serovar Typhimurium CVCC542 was expressed in Escherichia coli. The purified recombinant PagC protein was immobilized in microtiter plate wells. Sera from SPF chickens infected with Salmonella or other non-Salmonella pathogens by injection were added and binding of PagC protein was detected by the horseradish peroxidase (HRP)-labeled goat anti-chicken antibody. Sera from Salmonella-infected chickens showed high specificity in contrast to the sera from chickens infected with other bacteria. When 87 Salmonella antibody-positive sera from Salmonella Pullorum orally infected SPF chicken and 93 negative sera from uninfected SPF chicken were tested, 98.3% agreement was detected. The rPagC enzyme-linked immunosorbent assay (ELISA) and agglutination had 80.6% agreement in detecting 252 clinical chicken sera samples. These results suggest that PagC antibody-based indirect ELISA can serve as a convenient and novel method for the diagnosis of Salmonella infection.

Evaluation of a newly developed chemiluminescence immunoassay for detecting cardiac troponin T.

BACKGROUND: To evaluate the performance of a chemiluminescence detection kit for cardiac troponin T (cTnT). METHODS: According to the "Guiding principles on performance analysis of diagnostic reagents in vitro" and the Clinical and Laboratory Standards Institute (CLSI) Guidelines, we assessed the detection limit, linear range, reportable range, accuracy, precision, cross reactivity, interference factors, and matrix effect of the kit, and compared these parameters with that of the commercial electrochemiluminescence detection kit for cTnT (Roche). RESULTS: The minimum detection limit of the kit was 0.01 ng/mL. The linear range was 0.01 ng/mL-25 ng/mL. The reportable range was from 0.01 ng/mL to 100 ng/mL. Precision within the batch was 2.9%-6.4%, and precision between batches was 6.0%; the accuracy was good and the recovery rate reached 96.2%. The cross-reaction test demonstrated that there was no reactivity between cTnT and a variety of troponins. Test results deviated by less than ±10% in the presence of hemoglobin ≤1000 µg/mL, bilirubin ≤250 µg/mL, triglycerides ≤11.25 mg/mL, and rheumatoid factor ≤206 U/mL, suggesting that kit results were not significantly interfered with these factors. Results from the matrix-effect assessment revealed absence of a matrix effect in the tested serum samples. Correlation study revealed that the performance of the kit was very consistent with that of the Roche electrochemiluminescence detection kit (Kappa = 0.900, P < .001) with a high correlation (r = .903, P < .001) and a total matching rate of 95.00%. CONCLUSION: The performance of the evaluated chemiluminescence immunoquantitation kit for cTnT detection was acceptable in all tested parameters, fulfilling requirements for clinical applications.

Plasma microRNA-19a as a potential biomarker for esophageal squamous cell carcinoma diagnosis and prognosis.

AIM: To investigate whether plasma miR-19a can serve as a biomarker for esophageal squamous cell carcinoma (ESCC) diagnosis and prognosis. MATERIALS & METHODS: Plasma samples from 89 ESCC, 45 benign lesion patients and 80 healthy controls were subjected to RT-qPCR analyses for miR-19a. In addition, plasma samples from 30 patients were collected before and after surgery for the same analyses. RESULTS: Plasma miR-19a was significantly increased in ESCC patients compared with healthy controls. The sensitivity of miR-19a for early stages of ESCC was 68.09%. Combination of miR-19a and cytokeratin 19 fragment 21-1 (Cyfra21-1) further improved the sensitivity to 78.70%. Moreover, plasma miR-19a level was decreased in patients after surgery. CONCLUSION: Plasma miR-19a may serve as a potential biomarker that complements Cyfra21-1 in detecting early stages of ESCC.

DHX32 Promotes Angiogenesis in Colorectal Cancer Through Augmenting ß-catenin Signaling to Induce Expression of VEGFA.

We previously reported that overexpression of DHX32 contributes to the growth and metastasis of colorectal cancer (CRC). However, the underlying mechanism is not largely characterized. Herein, we reported that DHX32 in CRC cells upregulated expression of vascular endothelial growth factor A (VEGFA) at the transcription level through interacting with and stabilizing ß-catenin. This promoted the recruitment of host endothelial cells to the tumor, and therefore, formation of microvessel in the tumor. Xenograft model revealed that depletion of DHX32 in CRC cells significantly reduced the microvessel density in the grafts and suppressed the growth of grafts. Furthermore, the expression level of DHX32 was positively associated with microvessel density in human CRC and poor outcome of CRC patients. Therefore, the report demonstrates that DHX32 is a pro-angiogenic factor, that inhibition of DHX32-ß-catenin pathway can provide a strategy for CRC treatment, and that the expression level of DHX32 has the potential to serve as a biomarker for CRC diagnosis and prognosis.

Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Outer Membrane Protein L of Salmonella.

Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wildtype SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpLspecific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPVOmpL- vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpLvaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.