Real-time TaqMan Research Articles

PPARα polymorphisms association with total cholesterol and LDL-C levels in a Mexican population.

Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear transcription factor with a role in gene expression changes associated to lipid metabolism. PPARα polymorphic variants have been previously correlated to serum lipid profile but in Mexico, there is no previous report about that association. For this reason, the aim of this study was to investigate the relationship between PPARα polymorphic variants and lipids level in serum in a Mexican population. Two-hundred and forty women from the Northeast region of Mexico were included in the study. Anthropometric characteristics and serum lipid profile (such as triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol) were evaluated. Genomic DNA extraction and purification were made from blood samples. Real-time PCR and TaqMan probes were used for genotyping of rs1800206 and rs4253778 single nucleotide polymorphisms (SNPs). Linear regression analysis (adjusted by age and body mass index (BMI)) showed a significant statistical association of rs4253778 with total cholesterol (p=0.034) and LDL cholesterol (p=0.037). Any significant association was found between rs1800206 and lipid levels. These results suggested that rs4253778 (C allele) is associated with high levels of total cholesterol and LDL in a Mexican women population.

Enhanced expression of endogenous retroviruses and of TRIM28 and SETDB1 in children with food allergy.

BackgroundHuman endogenous retroviruses (HERVs) represent 8% of our genome. They originate from ancestral infections and although no longer contagious they can regulate transcription of adjacent cellular genes, produce viral RNAs sensed as non‐self by pattern recognition receptors, and encode viral proteins, such as Syncytin (SYN) 1 and 2, that exhibit potent immunomodulatory properties. Based on this, HERVs have been studied and proposed as relevant cofactors in several chronic inflammatory and immune‐mediated diseases. HERV transcription is regulated by host TRIM28 and SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), which in turn exert crucial regulatory functions on the host immune system. No studies explored the expression of HERVs, TRIM28, and SETDB1 in allergic patients.MethodsWe assessed, through a polymerase chain reaction real time Taqman amplification assay, the transcription levels of pol genes of HERV‐H, HERV‐K, HERV‐W, and of env genes of SYN1 and SYN2, as well as of TRIM28 and SETDB1 in whole blood from 32 children with IgE‐mediated food allergy, 19 with food protein‐induced enterocolitis syndrome (FPIES), and in healthy control children.ResultsThe expression levels of pol genes of HERV‐H, ‐K, and ‐W were significantly enhanced in patients with IgE‐mediated FA or FPIES as compared to control subjects, while the mRNA concentrations of SYN1 and SYN2 were comparable in each group of children. Both TRIM28 and SETDB1 mRNA levels were significantly higher in allergic patients.ConclusionsGiven the influence of HERVs and of TRIM28 and SETDB1 on innate and adaptive immune responses, their transcriptional activation in children with food allergies suggest that they might play important roles in the development of these diseases.

Abstract P1-08-30: Validation of the AAGAB gene as a novel on-treatment biomarker predicting pCR in patients receiving NACT for early breast cancer

Abstract Background: Neoadjuvant chemotherapy (NACT) for breast cancer is given prior to surgery, aiming to reduce tumour burden and provide early information on response to treatment. Tumours with a pathological complete response (pCR) following NACT are less likely to recur than those in patients with residual disease following NACT, suggesting that pCR following NACT may be a surrogate of good prognosis. In addition, NACT provides a “window of opportunity” where sequential sampling of a tumour enables observation of changes occurring in response to treatment. We previously demonstrated that on-treatment biomarkers improve the accuracy of predicting response before resection, by performing differential gene expression analysis across sequential samples from 50 patients, across 3 time points; pre NACT, mid chemo and post NACT (Bownes et al, 2019). A single gene marker (AAGAB) was identified as a novel potential on-treatment biomarker for pCR, with greater predictive accuracy than currently established prognostic tests, supporting the potential value of on-treatment biomarker monitoring in breast cancer to improve the accuracy of tumour response prediction. Aim: To validate and test the finding that increased expression of AAGAB at mid-chemotherapy associates with pCR in ER+ve and HER+ve breast cancer patients receiving NACT. Method: mRNA extracted from tumour from the mid-chemotherapy time point during NACT, from 28 breast cancer patients, was analysed using real-time reverse transcription-PCR (real-time RT-PCR). Firstly, mRNA was reverse transcribed using Invitrogen SuperScript IV Reverse Transcriptase (RT), then cDNA was used in an Applied Biosystems real-time RT-PCR Taqman Gene Expression Assay to measure AAGAB gene expression. GAPDH and SYMPK Taqman assays were used as endogenous controls, to allow calculation of (ΔΔCt) using the Comparative CT Method. Quantitative PCR was carried out on the Applied Biosystems StepOnePlusTM Real-Time PCR System and Relative Quantitation (RQ) was calculated using 2^-(ΔΔCt). For each patient RCB (residual cancer burden) was plotted against AAGAB expression and statistical significance calculated using the Wilcoxon rank sum exact test. Results: As a group, patients with RCB scores of 0 and I demonstrated a significantly higher level of expression that patients with RCB II and RCB III. We also observed that patients with pCR after NACT demonstrated higher levels of AAGAB expression. This increased expression was also seen in patients with a decrease in tumour cellularity of more than 85%. Conclusion: Our data suggest that measurement of AAGAB expression may provide an informative on-treatment biomarker of response to NACT. Citation Format: Fiona Semple, Alastair Ironside, Richard Bownes, Carlos Martinez-Perez, Natalie Wilson, Arran Turnbull, Olga Oikonomidou. Validation of the AAGAB gene as a novel on-treatment biomarker predicting pCR in patients receiving NACT for early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-30.

Cytauxzoon europaeus infections in domestic cats in Switzerland and in European wildcats in France: a tale that started more than two decades ago

BackgroundCytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing.MethodsThe overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995–1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing.ResultsIn study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0–0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0–0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0–14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0–4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2–44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses.ConclusionsThe study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species.Graphical

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays.

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected ≤ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

Detection of Male Genital Schistosomiasis (MGS) by Real-Time Taqman® PCR of Semen from Fishermen Along the Southern Shoreline of Lake Malawi

Detection of Male Genital Schistosomiasis (MGS) by Real-Time Taqman® PCR of Semen from Fishermen Along the Southern Shoreline of Lake Malawi

Performance characteristics of two real-time TaqMan polymerase chain reaction assays for the detection of WSSV in clinically diseased and apparently healthy prawns.

This study aimed to generate data on performance characteristics for 2 real-time TaqMan PCR assays (CSIRO and WOAH WSSV qPCRs) for the purposes of (1) detection of white spot syndrome virus (WSSV) in clinically diseased prawns and (2) detection of WSSV in apparently healthy prawns. Analytical sensitivity of both assays was 2 to 20 genome copies per reaction, and analytical specificity was 100% after testing nucleic acid from 9 heterologous prawn pathogens and 4 prawn species. Results obtained after testing more than 20 000 samples in up to 559 runs with the CSIRO WSSV qPCR and up to 293 runs with the WOAH WSSV qPCR demonstrated satisfactory repeatability for both assays. Both assays demonstrated median diagnostic sensitivity (DSe) 100% (95% CI: 94.9-100%) when testing clinically diseased prawns. When 1591 test results from apparently healthy prawns were analysed by Bayesian latent class analysis, median DSe and diagnostic specificity (DSp) were 82.9% (95% probability interval [PI]: 75.0-90.2%) and 99.7% (95% PI: 98.6-99.99%) for the CSIRO WSSV qPCR and 76.8% (95% PI: 68.9-84.9%) and 99.7% (95% PI: 98.7-99.99%) for the WOAH WSSV qPCR. When both assays were interpreted in parallel, median DSe increased to 98.3 (95% PI: 91.6-99.99%), and median DSp decreased slightly to 99.4% (95% PI: 97.9-99.99%). Routine testing of quantified positive controls by laboratories in the Australian laboratory network demonstrated satisfactory reproducibility of the CSIRO WSSV qPCR assay. Both assays demonstrated comparable performance characteristics, and the results contribute to the validation data required in the WOAH validation pathway for the purposes of detection of WSSV in clinically diseased and apparently healthy prawns.

Predictive value of vascular endothelial growth factor polymorphisms for maintenance bevacizumab efficacy in metastatic colorectal cancer: an ancillary study of the PRODIGE 9 phase III trial.

Several studies have reported the impact of single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor (VEGF) pathway genes on the efficacy of bevacizumab in metastatic colorectal cancer (mCRC), but results are still inconsistent. The PRODIGE 9 phase III study compared bevacizumab maintenance versus observation alone after induction chemotherapy with FOLFIRI plus bevacizumab. We evaluated the impact of SNPs of VEGF-A, VEGF receptors (VEGFR-1, VEGFR-2), and hypoxia inducible factor-1α (HIF-1α) on tumor control duration (TCD), overall survival (OS), progression-free survival (PFS), and duration of first chemotherapy free-intervals (CFI). We included 314/491 patients from PRODIGE 9 with a DNA blood sample available. Nine SNPs were genotyped on germline DNA using real-time Polymerase Chain Reaction TaqMan TM (Thermo Fisher Scientific, Waltham, MA , USA 02451). In the bevacizumab arm, patients with the VEGFR-1 rs9582036 CC genotype (n = 14) had significantly longer TCD [22.4 months (95% confidence interval (CI): 14.75-not reached)] than patients with the AA or CA genotype [14.4 months (95% CI: 11.7-17.1)] (p = 0.036), whereas there was no significant difference in the observation arm. In the bevacizumab arm, no significant difference was found between the CC, and AA or CA genotype for OS [28.2 (95% CI: 18.1-42.8) versus 22.5 (95% CI: 18.6-24.6) months, p = 0.5], PFS [9.4 (95% CI: 7.2-11.3) versus 9.2 (95% CI: 8.71-10.1)], and duration of the first CFI [4.6 (95% CI: 1.6-13.3) versus 4.14 (95% CI: 0.5-29.0) months, p = 0.3]. Among mCRC patients treated with bevacizumab maintenance, those with the VEGFR-1 rs9582036 CC genotype experienced longer TCD. The presence of this genotype may thus predict a benefit of bevacizumab maintenance in mCRC.

Cytokine gene polymorphisms of TNF, IFN-γ, and IL-12 as potential predictors in the onset of cervical disease in HR HPV-positive women with behavioral risk cofactors

Introduction/Objective. The aim of this study was to investigate the distribution of genotypes and alleles of proinflammatory cytokines TNF, IFN-?, and IL-12 and their effect on the development of a cervical illness and also to determine their associated influence with cofactors in HR HPV-positive women in Serbia. Methods. We have investigated 24 women and based on the cytological findings they were classified into four groups: PAP II, ASCUS, LSIL, and HSIL. Analysis of TNF, IL-12, and IFN-? polymorphisms was performed using the real-time PCR TaqMan method. Statistical analysis was performed using parametric and non-parametric tests and correlation and multiple regression analysis. Results. Significantly higher frequency of high production-related TNF AA genotype was observed in severe dysplasia. The correlation between TNF gene polymorphism and cervical findings were highly significant. There was a moderate, significant correlation between low production IFN-? AA genotype and earlier cervical infections. There was a significant correlation between the IL-12 polymorphism of the low production IL-12 AA genotype and cervical lesions. Conclusion. Results of this study show that HSIL is associated with significantly higher frequency of high production TNF AA genotype. It is known that polymorphisms of certain cytokine genes encoding proteins involved in Th1 and Th2 cellular responses may be associated with better or worse prognosis of cervical disease in women with persistent HR HPV infection. Therefore, they may be considered as biomarkers that may have a predictive role in the development of cervical cancer.

First report on the detection of Japanese encephalitis virus in fruit bats from India.

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

Serum Glycoproteomics and Identification of Potential Mechanisms Underlying Alzheimer's Disease.

Objectives This study compares glycoproteomes in Thai Alzheimer's disease (AD) patients with those of cognitively normal individuals. Methods Study participants included outpatients with clinically diagnosed AD (N = 136) and healthy controls without cognitive impairment (N = 183). Blood samples were collected from all participants for biochemical analysis and for Apolipoprotein E (APOE) genotyping by real-time TaqMan PCR assays. Comparative serum glycoproteomic profiling by liquid chromatography-tandem mass spectrometry was then performed to identify differentially abundant proteins with functional relevance. Results Statistical differences in age, educational level, and APOE ɛ3/ɛ4 and ɛ4/ɛ4 haplotype frequencies were found between the AD and control groups. The frequency of the APOE ɛ4 allele was significantly higher in the AD group than in the control group. In total, 871 glycoproteins were identified, including 266 and 259 unique proteins in control and AD groups, respectively. There were 49 and 297 upregulated and downregulated glycoproteins, respectively, in AD samples compared with the controls. Unique AD glycoproteins were associated with numerous pathways, including Alzheimer's disease-presenilin pathway (16.6%), inflammation pathway mediated by chemokine and cytokine signaling (9.2%), Wnt signaling pathway (8.2%), and apoptosis signaling pathway (6.7%). Conclusion Functions and pathways associated with protein-protein interactions were identified in AD. Significant changes in these proteins can indicate the molecular mechanisms involved in the pathogenesis of AD, and they have the potential to serve as AD biomarkers. Such findings could allow us to better understand AD pathology.

Investigation of Tomato ringspot virus (ToRSV) by Real-Time TaqMan RT-PCR in Hakkari province, Turkey

Tomato ringspot virus (ToRSV) belongs to the Nepovirus genus in the family Secoviridae. It has a wide host range and is listed as a quarantine virus in Turkey. In this study, 80 leaf samples were collected from tomato, pepper, cucumber and grapevine cultivation sites located in three different parts of Hakkari province: Şemdinli, Çukurca and Center districts. Real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) method was used for the detection of the virus. Amplification was carried out in reaction mix including QuantiNova Probe RT-PCR kit (Qiagen, Germany) using primers and TaqMan probe based on 3'-UTR (untranslated region) of virus, which amplified a 182 bp product of the genome. ToRSV was detected in 13 of the 80 samples and threshold cycle (CT) values ranged from 23.9 to 37.4. It was found that 16.25% of the samples collected from the districts of Hakkari province were found to be infected with ToRSV whereas no ToRSV was detected in the samples collected from the center of the city. The virus was detected on pepper and cucumber samples in Çukurca district, and it was also detected in tomato, pepper, cucumber and grapevine samples in Şemdinli district. To our knowledge, this study is the first report of molecular detection of ToRSV by real-time TaqMan RT-PCR in Turkey.

Abstract LBA043: mRNA expression of mTOR signaling components as potential predictive biomarker in pancreatic neuroendocrine neoplasms

Abstract Medical therapeutic options for the treatment of well-differentiated pancreatic neuroendocrine neoplasms (pNENs) include the mTOR pathway inhibitor everolimus (in combination with 5-Fluorouracil). Criteria to assess prognosis in pNEN patients are still limited, as biomarkers to predict the therapeutic response are still unknown. Several studies could show aberrant mTOR signaling pathway activity at different levels of the signaling cascade in pNENs. In this study, we analyzed the mRNA expression of different mTOR pathway components in well-differentiated pNENs, in order to identify potential prognostic and predictive biomarkers. In this retrospective multicenter study, real-time quantitative TaqMan reverse transcriptase-polymerase chain reaction was used to analyze mRNA expression of eleven different mTOR pathway genes (IGF-1, IGFBP-3, PIK3CA, AKT-1, MTOR, MLST8, DEPTOR, RAPTOR, RICTOR, 4EBP1 und VEGF-A), in tumor tissue of 75 patients with well-differentiated pNENs in comparison to normal pancreatic tissue. Relative gene expression was determined according to the 40-ΔCT method using CALM2 as housekeeping gene. The analysis of mRNA expression levels of the aforementioned eleven genes indicated an increased mTOR signaling activity in pNENs. A distinct linear correlation only occurred between MTOR and AKT-1. Clear association between mRNA expression and a clinical feature was only found for RAPTOR: High RAPTOR mRNA expression was significantly correlated with better overall survival in unselected patients. No prediction to everolimus treatment response could be deduced by mRNA expression analysis in a small subgroup of 21 patients. Even though mRNA expression of different mTOR pathway components indicated an enhanced activity of this signaling cascade in tumor tissue of pNEN patients, we could not identify a statistical significant parameter useful for prediction of everolimus treatment response. The predictive value of this study is limited due to the low number of patients who received treatment with everolimus. Further prospective studies analyzing the predictive value of the expression of mTOR pathway genes are desirable. Citation Format: Sebastian Wolfshöfer, Adrian C Lock, Daniel Kaemmerer, Ralph M Wirtz, Franziska Briest, Sven P Haugvik, Malte Buchholz, Anja Rinke, Ruza Arsenic, Marianne Pavel, Dieter Hörsch, Arend Koch, Patricia Grabowski. mRNA expression of mTOR signaling components as potential predictive biomarker in pancreatic neuroendocrine neoplasms [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA043.

Metal-Stimulated Interleukin-6 Production Through a Proton-Sensing Receptor, Ovarian Cancer G Protein-Coupled Receptor 1, in Human Bronchial Smooth Muscle Cells: A Response Inhibited by Dexamethasone.

PurposeHuman bronchial smooth muscle cells (BSMCs) contribute to airway obstruction and hyperresponsiveness in patients with bronchial asthma. BSMCs also generate cytokines and matricellular proteins in response to extracellular acidification through the ovarian cancer G protein-coupled receptor 1 (OGR1). Cobalt (Co) and nickel (Ni) are occupational agents, which cause occupational asthma. We examined the effects of Co and Ni on interleukin-6 (IL-6) secretion by human BSMCs because these metals may act as ligands of OGR1.MethodsHuman BSMCs were incubated in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM) for 16 hours and stimulated for the indicated time by exchanging the medium with 0.1% BSA-DMEM containing any of the metals or pH-adjusted 0.1% BSA-DMEM. IL-6 mRNA expression was quantified via reverse transcription polymerase chain reaction (RT-PCR) using the real-time TaqMan technology. IL-6 was measured using an enzyme-linked immunosorbent assay. Dexamethasone (DEX) was added 30 minutes before each stimulation. To knock down the expression of OGR1 in BSMCs, small interfering RNA (siRNA) targeting OGR1 (OGR1-siRNA) was transfected to the cells and non-targeting siRNA (NT-siRNA) was used as a control.ResultsCo and Ni both significantly increased IL-6 secretion in human BSMCs at 300 μM. This significant increase in IL-6 mRNA expression was observed 5 hours after stimulation. BSMCs transfected with OGR1-siRNA produced less IL-6 than BSMCs transfected with NT-siRNA in response to either Co or Ni stimulation. DEX inhibited Co- and Ni-stimulated IL-6 secretion by human BSMCs as well as pH 6.3-stimulated IL-6 secretion in a dose-dependent manner. DEX did not decrease phosphorylation of ERK1/2, p38 MAP kinase, and NF-κB p65 induced by either Co or Ni stimulation.ConclusionCo and Ni induce secretion of IL-6 in human BSMCs through activation of OGR1. Co- and Ni-stimulated IL-6 secretion is inhibited by DEX.

Vitamin D Receptor Gene Polymorphisms and the Risk of Chronic Hepatitis C Related Hepatocellular Carcinoma in Egyptian Population

Background: Small percentage of hepatitis C (HCV) patients develop hepatocellular carcinoma (HCC) during their lifetime, suggesting that genetic factors might modulate HCC development. Numerous variations on the vitamin D receptor gene (VDR) have been recognized in human cancers. The majority of them cause VDR to be unable to bind to 1, 25-OH-D. The aim of the present work was to investigate the relation of VDR FokI (rs2228570), BsmI (rs3782905) and ApaI (rs7975232) gene polymorphisms and the risk of HCC development in chronic HCV Egyptian patients. Methods: A total of 311 Egyptian patients were enrolled for this study. They were divided into 3 groups: 103 patients with liver Cirrhosis, 107 patients with HCC and 101 normal healthy subjects as the control group. Human genomic DNA Extraction was carried out using QIAamp® DNA Blood Mini Kit (QIAGEN) Genotyping of VDR ApaI (rs7975232) single nucleotide polymorphism (SNP) was carried out using real-time PCR TaqMan allelic discrimination assay with allele-specific designed fluorescent MGB probes. Results: Patients with HCC had a higher frequency of ApaI CC genotype (P=0.035) CI (0.031-0.038). Patients with HCC carried a higher ratio of ApaI CC genotype compared to those with liver cirrhosis (x2=5.4 and P = 0.03) or controls (x2=6.8 and P = 0.01). Univariate analysis revealed that age, lower platelet count (<150×103/μL), higher AFP (>100 ng/ml), and ApaI CC genotype were the factors significantly associated with the development of HCC. Stepwise logistic regression analysis showed that all were independent predictors. Conclusion: ApaI CC VDR gene mutation is an independent risk factor for HCC development in Egyptian Cirrhotic HCV patients.

First Report of 'Candidatus Phytoplasma trifolii' Related Strain Associated with Yellowing and Witches'-Broom of Industrial Hemp (Cannabis sativa) in Arizona.

In Arizona, industrial hemp (Cannabis sativa) is a newly cultivated crop for fiber, oil, cosmetic products, and health food. During July to September 2020, two fields of industrial hemp crops were identified in southern Arizona with 10 to 30% incidence of plants showing witches' broom. Disease incidence was assessed by counting symptomatic plants in 4 randomly selected rows of 25 plants in each field. Symptoms ranged from leaf mottling and yellowing on mildly affected plants to leaf curling and shortened internode length of stem on severely affected plants (Fig. 1). Shoots were randomly collected from eight symptomatic plants and three asymptomatic plants in the same area. Genomic DNA was extracted from 200 mg of each sample using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Phytoplasma was tested by a real-time PCR assay and TaqMan probe targeting the 23S ribosomal RNA gene that detects a wide range of known Phytoplasmas (Hodgetts et al., 2009). Beet curly top virus (BCTV) was targeted using BCTV-specific primers BCTV1 and BCTV2 following a method by Rondon (Rondon et al., 2016). BCTV was not detected in the plants, but Phytoplasmas were detected in all eight symptomatic plants, but not in the three control plants. The positive DNA samples were used to identify the phytoplasma by nested PCR using universal phytoplasma-specific primer pairs P1/P6 (Deng, S. et al. 1991) and R16F2n/R16R2 (Gundersen et al., 1996) targeting the 16S rRNA gene and the resulting 1.25 kb fragment in 4 positive samples was subjected to Sanger sequencing (Eton Bioscience, San Diego). All 4 sequences were identical and deposited in GenBank under accession MW981356. BLASTn results indicated 100% identity with that of several 'Candidatus Phytoplasma trifolii' strains on potato (KR072666, KF178706) in Washington and chile peppers (HQ436488) in New Mexico. It also shared 99.84% identity with the sequence of the reference strain of Candidatus Phytoplasma trifolii' (AY390261) that caused clover proliferation. The phytoplasma AZH1 was classified as a member of subgroup A within group16SrVI using iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment (Zhao et al., 2013). Phylogenetic analysis revealed that the phytoplasma AZH1 clustered with other isolates of 'Candidatus Phytoplasma trifolii' (Fig. 2), including the strain NV1 associated with witches' broom on C. sativa in Nevada (Feng et al. 2019). This is the first report of 'Candidatus Phytoplasma trifolii' related strain associated with yellowing and witches' broom on hemp in Arizona. This finding is significant as the observation of symptoms at 30% incidence in one field suggested that the identified pathogen may pose a significant threat to the production of industrial hemp production in Arizona.

Prevalence of invA Gene of Salmonella spp. in Fish and Fishery Resources from Manila Bay Aquaculture Farms Using Real-Time PCR

Manila Bay contributes significantly to the Philippines’ economy through industry, trade, tourism, and agriculture. However, microbiological contamination in the bay is putting it at risk in the present. Pathogen contamination of the water poses a serious threat to food safety, implicating public health. To address these concerns, the present study examined the prevalence of Salmonella in six different aquaculture commodities, Perna viridis (mussel), Crassostrea iridalei (oyster), Scylla serrata (crab), Penaeus spp. (shrimp), Oreochromis niloticus (tilapia), and Chanos chanos (milkfish), as well as in environmental water from growing areas. A tandem approach of culture-based and real-time PCR methods for the isolation and identification of Salmonella was carried out. To accurately identify the isolates, a real-time PCR Taqman assay based on the invA gene was used. Among the fishery resources examined, no positive samples were observed from C. chanos and O. niloticus. In addition, Salmonella was found in twenty (20) samples, representing 16.26% of all aquaculture commodities collected. Furthermore, Salmonella was found in 13.58% of the 81 environmental water samples examined. P. viridis had the highest prevalence of 55.55% out of 18 samples examined. Samples contaminated with Salmonella failed to meet the regulatory limits set by BFAR FAO 210 series 2001 and EC No. 2073/2005. In addition, it was observed that the sample matrix had a significant impact on the presence of Salmonella (p < 0.05). However, the spatial and temporal distribution of Salmonella did not vary greatly (p > 0.05). This study underscores the importance of imposing strict policies by regulatory bodies to prevent diseases, thus avoiding severe health implications.

Development and Validation of Two Diagnostic Real-Time PCR (TaqMan) Assays for the Detection of Bordetella avium from Clinical Samples and Comparison to the Currently Available Real-Time TaqMan PCR Assay

Bordetella avium (BA) is one of many pathogens that cause respiratory diseases in turkeys. However, other bacterial species can easily overgrow it during isolation attempts. This makes confirming the diagnosis of BA as the causative agent of turkey coryza more difficult. Currently, there are two PCR assays for the molecular detection of BA. One is conventional gel-based PCR and the other is TaqMan real-time PCR (qPCR) assay. However, multiple pitfalls were detected in both assays regarding their specificity, sensitivity, and efficiency, which limits their utility as diagnostic tools. In this study, we developed and validated two TaqMan qPCR assays and compared their performance to the currently available TaqMan qPCR. The two assays were able to correctly identify all BA isolates and showed negative results against a wide range of different microorganisms. The two assays were found to have high efficiency with a detection limit of approximately 1 × 103 plasmid DNA Copies/mL with high repeatability and reproducibility. In comparison to the currently available TaqMan qPCR assay, the newly developed assays showed significantly higher PCR efficiencies due to superior primers and probes design. The new assays can serve as a reliable tool for the sensitive, specific, and efficient diagnosis of BA.

IL1B rs1143634 (+3953) variant associated with severe dengue in Brazilian children

<p class="abstract">Several variants involved in the expression of proteins related to immunopathological mechanisms have been associated with dengue, but few specifically related to severe dengue. In addition, children are a group with a high incidence and mortality rate due to dengue. Several immunopathological processes developed specifically in children affected with severe dengue partially explain this risk condition. This case-control retrospective study aimed to examine the association of variants in <em>CD209, NFKBIA, IL12B,</em> and <em>IL1B </em>genes with severe dengue. These genes encode proteins involved in one of the main pathways of the immune response against dengue virus infection. The study population included affected children and householding controls from Brazil. Genotyping was performed with Real-Time TaqMan assays, and the association analysis was performed with conditional logistic regressions. We found the <em>IL1B</em> rs1143634 genotypes carrying the T allele to be associated with susceptibility to severe dengue in children. The lower circulating levels of IL-1β associated with carrying the T allele of the <em>IL1B</em> rs1143634 suggest a role of this cytokine in the impairment of the immune response and plasma leakage, and, consequently, in the development of severe dengue.</p>

Annual presence and distribution of human-pathogenic {Acanthamoeba} spp. in River Soar, Leicester, UK

BACKGROUND AND AIM: {Acanthamoeba} spp. can act as opportunistic parasites and are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions and water treatment practices. Owing to the lack of knowledge about {Acanthamoeba}'s epidemiology in England, the aim of the present work was to study, over one year, the presence and circulation of this opportunistic parasite in the River Soar, which crosses Leicester city (England) and attracts large numbers of users. METHODS: Water samples were collected from nine different locations following the course of the river through the city per season (n=45) from Summer 2017-Summer 2018, following protocol 1623 described by US EPA. Samples were concentrated using IDEXX® Filta Max system following manufacturer's instructions. DNA extraction from concentrated water was performed with FastDNA® Spin Kit. A triplex real-time TaqMan PCR assay was performed to detect {Acanthamoeba} spp., using established methodologies. RESULTS:{Acanthamoeba} spp. was detected in 17 water samples: 4 in each season from Summer to Winter 2017, two in Spring 2018 and three in Summer 2018. These free-living amoebas were discovered in each sampled location, which highlights a moderate to high circulation of {Acanthamoeba} in the River Soar throughout a year, without seasonality, despite significant changes in the water flow rate and temperatures recorded in the river and in Leicestershire. This river is rich in wildlife, which might play a role in the presence/circulation of {Acanthamoeba} spp. CONCLUSIONS:To our knowledge, this is the first report reporting the presence and circulation of {Acanthamoeba} spp. in an English river throughout a complete year. The presence of {Acanthamoeba} spp. in this river could represent a risk for human health, for example rowers, which is present throughout the whole year. Therefore, this risk should be tackled as the incidence of {Acanthamoeba} keratitis has increased in recent years in England. KEYWORDS: {Acanthamoeba} spp., River Soar, environmental presence, human risk.