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Introduction: Oral squamous cell carcinoma is a multifactorial disease that is the sixth most common cancer worldwide. MicroRNAs have been confirmed to play a role in oral squamous cell carcinoma, acting as either oncogenes or tumor suppressor genes. This study examined the expression level and role of microR-148 and microR-375 in oral cancer. Materials and Methods: In this study, we used 30 cancer samples with infection and 30 cancer samples without infection. To analyze the expression of microRNA 375 and microRNA 148, we used real-time PCR. First, we extracted total RNA from the samples. Then, we generated cDNA from it. Finally, the obtained cDNA was used in the real-time PCR technique. Results: In cancer patients with oral infection, there was an increase in microRNA-148 expression and a decrease in microRNA-375 compared to cancer patients without oral infection. Conclusion: The downregulation of microRNA-375 and upregulation of microRNA-148 can be utilized as diagnostic biomarkers and prognostic factors in oral cancer. Keywords: Oral cancer; OSCC; Realtime pcr; MiR-148; MiR-375.

Introduction: Oral squamous cell carcinoma is a multifactorial disease that is the sixth most common cancer worldwide. MicroRNAs have been confirmed to play a role in oral squamous cell carcinoma, acting as either oncogenes or tumor suppressor genes. This study examined the expression level and role of microR-148 and microR-375 in oral cancer. Materials and Methods: In this study, we used 30 cancer samples with infection and 30 cancer samples without infection. To analyze the expression of microRNA 375 and microRNA 148, we used real-time PCR. First, we extracted total RNA from the samples. Then, we generated cDNA from it. Finally, the obtained cDNA was used in the real-time PCR technique. Results: In cancer patients with oral infection, there was an increase in microRNA-148 expression and a decrease in microRNA-375 compared to cancer patients without oral infection. Conclusion: The downregulation of microRNA-375 and upregulation of microRNA-148 can be utilized as diagnostic biomarkers and prognostic factors in oral cancer. [ABSTRACT FROM AUTHOR]

Vechur is a unique but threatened indigenous cattle breed of Kerala. The use of cryopreserved semen serves long term conservation and improvement of the breed. The fertility of the cryopreserved sperm depends on the ability to withstand the freeze thaw cycle. Oxidative stress and reactive oxygen species (ROS) generated in the spermatozoa during freezing and thawing of frozen semen is considered a major factor responsible for causing infertility due to decrease in sperm quality parameters. Semen ejaculates from 20 Vechur bulls were collected. Bulls consistently producing semen with 70 per cent progressive motility were selected and analysed for cryotolerant sperm. Using conventional liquid Nitrogen method, six bulls exhibiting differential post thaw motility were grouped into Good Freezability Rate (GFR) (40 per cent) and Poor Freezability Rate (PFR) (<40 per cent) Three ejaculates from three bulls in each group were collected and cryopreserved. RNA was isolated from fresh and post thaw semen from two groups. Three candidate genes HSP90AA1, PRDX6 and SOD1 were selected for expression profiling. These genes were reported to be involved in the protection of the sperm from temperature changes, molecular folding (HSP90AA1) and oxidative stress (PRDX6 and SOD1). Relative quantification was performed by qPCR with PPIA as internal control gene. Statistical analysis revealed a significant (p<0.05) downregulation of HSP90AA1 and PRDX6 from fresh to GFR semen. While, there was no significant difference between fresh and GFR in case of SOD1 and a significant (p<0.05) downregulation in PFR from GFR for all the three selected candidate genes were revealed. The current study suggests the three candidate genes can be considered for selecting bulls based on sperm parameters. [ABSTRACT FROM AUTHOR]

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Detecting and identifying the origins of foodborne pathogen outbreaks is a challenging. The Next-Generation Sequencing (NGS) panel method offers a potential solution by enabling efficient screening and identification of various bacteria in one reaction. In this study, new NGS panel primer sets that target 18 specific virulence factor genes from six target pathogens (Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) were developed and optimized. The primer sets were validated for specificity and selectivity through singleplex PCR, confirming the expected amplicon size. Crosscheck and multiplex PCR showed no interference in the primer set or pathogenic DNA mixture. The NGS panel analysis of spiked water samples detected all 18 target genes in a single reaction, with pathogen concentrations ranging from 108 to 105 colony-forming units (CFUs) per target pathogen. Notably, the total sequence read counts from the virulence factor genes showed a positive association with the CFUs per target pathogen. However, the method exhibited relatively low sensitivity and occasional false positive results at low pathogen concentrations of 105 CFUs. To validate the detection and identification results, two sets of quantitative real-time PCR (qPCR) analyses were independently performed on the same spiked water samples, yielding almost the same efficiency and specificity compared to the NGS panel analysis. Comparative statistical analysis and Spearman correlation analysis further supported the similarity of the results by showing a negative association between the NGS panel sequence read counts and qPCR cycle threshold (Ct) values. To enhance NGS panel analysis for better detection, optimization of primer sets and real-time NGS sequencing technology are essential. Nonetheless, this study provides valuable insights into applying NGS panel analysis for multiple foodborne pathogen detection, emphasizing its potential in ensuring food safety. [ABSTRACT FROM AUTHOR]

The salamander killing fungus Batrachochytrium salamandrivorans (Bsal), recently introduced from Asia, is threatening salamander populations in different parts of Europe. In fact, this pathogen is rapidly spreading in Central Europe and has been also introduced into NE Spain. Of special concern are those regions with an exceptionally high salamander diversity such as Italy, where 19 salamander species are present most of which are strictly endemic. In this study, we update the information on the presence of Bsal in Italian wild salamanders, by adding samples from two presumptive outbreak sites, one on the island of Sardinia and one on continental Italy (Liguria). In addition, we reviewed the potential susceptibility of all the Italian salamander species on the basis of laboratory experimental infection trials, or from the tested susceptibility of the phylogenetically nearest species, according to the literature. Overall, 15 skin swabs from three species (Speleomantes sarrabusensis, Speleomantes strinatii and Salamandra salamandra) collected in the two presumptive Bsal outbreak sites were analysed by quantitative molecular methods, but none gave positive results. The majority of Italian salamander species and almost all of the endemic ones showed a high susceptibility to Bsal infection. Therefore, even if the presence of Bsal in Italian salamander populations has not been proven yet, the entire salamander fauna is highly threatened and preventive management actions should be taken. The need for strict biosecurity protocols on the international trade of captive or wild amphibians and for the implementation of preventive measures during field activities to limit the introduction and spread of the Bsal pathogen is again stressed. [ABSTRACT FROM AUTHOR]

Relevance. The use of gene therapy drugs for the treatment of genetic diseases and stimulation of regeneration processes is lengthy and involves repeated injections, which may lead to increased dissemination of gene therapy constructs from the injection site and undesirable ectopic expression of growth factors encoded in them. Existing approaches to study the pharmacokinetics of a drug to assess the dissemination of a gene therapy drug from the site of administration are not applicable. Objective: to evaluate the suitability of the real-time PCR method for studying the biodistribution of a promising gene therapy drug in mice during a course of use. Methods. Male F1 CBA×C57/Black mice after nerve injury were injected with the test plasmid into the denervated tibial muscle after nerve injury, as well as after 4, 9 and 13 days at a dosage of 60 and 120 μg/mouse. After 7, 14, and 28 days, organ and tissue samples were removed, total DNA was isolated, and plasmid DNA content was assessed by real-time PCR. Results. We have shown that the studied genetic construct is able to disseminate from the injection site. We have found that the peak of dissemination for this construct in the organs and tissues of the mouse is reached 14–28 days after the end of the course application, while ectopic expression of growth factors is not observed in them. Conclusion. The proposed method is specific, highly sensitive, and linear over a wide range of concentrations. Thus, it can be recommended for studying the biodistribution of potential gene therapy drugs in the body of experimental animals as part of a preclinical studies complex.

JC polyomavirus is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease resulting from the lytic infection of oligodendrocytes that may develop in immunosuppressed individuals: HIV1 infected or individuals under immunosuppressive therapies. Understanding the biology of JCPyV is necessary for a proper patient management, the development of diagnostic tests, and risk stratification. The review covers different areas of expertise including the genomic characterization of JCPyV strains detected in different body compartments (urine, plasma, and cerebrospinal fluid) of PML patients, viral mutations, molecular diagnostics, viral miRNAs, and disease. The implementation of molecular biology techniques improved our understanding of JCPyV biology. Deep sequencing analysis of viral genomes revealed the presence of viral quasispecies in the cerebrospinal fluid of PML patients characterized by noncoding control region rearrangements and VP1 mutations. These neurotropic JCPyV variants present enhanced replication and an altered cell tropism that contribute to PML development. Monitoring these variants may be relevant for the identification of patients at risk of PML. Multiplex realtime PCR targeting both the LTAg and the archetype NCCR could be used to identify them. Failure to amplify NCCR should indicate the presence of a JCPyV prototype speeding up the diagnostic process. [ABSTRACT FROM AUTHOR]

As antimicrobial resistance continues to pose a significant threat to global health, this study provided a focused examination of the prevalence and behavior of key antibiotic resistance genes in aquatic environments. We investigated the quantitative distribution of intI1, sul1, bla TEM , bla NDM , bla VIM , mcr-1, tetQ, and crAssphage within wastewater influents (n = 12), effluents (n = 12) and river water samples (n = 12), from three municipal wastewater treatment plants and three river locations in an urbanized watershed in Central Thailand over dry and wet seasons. The qPCR method demonstrated that intI1, sul1, bla TEM , and tetQ were the most abundant in all samples (2.71-7.89 mean log 10 copies/100 mL), with all genes exhibiting consistently uniform levels across diverse locations, suggesting the potential for any site to act as a monitoring sentinel. Although there is a significant reduction of ARG concentrations by WWTPs (0.62 - >4.05 LRV), the persistence of these genes in effluents points to the limited effectiveness of existing treatment methodologies. Temporal data indicated stable ARG concentrations over time, but tetQ levels rose during the wet season, in alignment with the monsoonal climate in Thailand. Additionally, we identified crAssphage, a marker of human sewage contamination, exhibited strong correlations with the more abundant ARGs (rho 0.65 - 0.81), implying that human waste contributes significantly to the environmental burden of ARGs. The results of this research highlight the widespread nature of ARGs in water systems and the need for improved treatment and sanitation strategies to mitigate the public health threat posed by antimicrobial resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pediococcus spp. are lactic acid bacteria which are considered spoilage in the wine making processes. Early detection can minimize the negative effects of Pediococcus spp on wine quality and prevent the development of advanced spoilage stages. The goal of this work was development and testing of a fast and efficient Real-Time Polymerase Chain Reaction (R-T PCR)-based method for easy detection of Pediococcus spp. strains in wine at the early stages of wine spoilage. As a result, primer sets for the Real time-PCR were designed to allow for correct and efficient detection of Pediococcus spp., and experimentally tested.

Streptococcus agalactiae (GBS) is the major contagious cause of early-onset sepsis in newborns. Screening and intrapartum antibiotic prophylaxis for GBS in pregnant women could effectively prevent early-onset GBS infection in newborns. The conventional method for isolation and identification of GBS on the blood plate medium is labor-intensive, time-consuming, and low sensitive. This study is aimed to optimize parameters for a realtime PCR reaction with primers and a probe designed to detect GBS-specific cfb gene. The optimized experiments were carried out on the strain S. agalactiae ATCC 13813. The specificity of the optimized procedure was tested on DNA samples of Staphylococcus aureus ATCC 25923, Gardnerella vaginalis, and Chlamydia trachomatis. In addition, the optimized reaction was tested on 30 vaginal - rectal samples from pregnant women between 35-37 weeks gestation. The optimized procedure was specific to GBS with 50 copies/reaction sensitivity, an accuracy of 99.94%, and amplification efficiency (EA%) of 94.5%. GBS was detected in ten samples among the 30 vaginal-rectal samples by the realtime PCR, while only eight samples were found to be positive in the conventional plate method.

Borreliosis or Lyme disease is a disease transmitted by ixodidae ticks during feeding on blood (Ixodes pacificus and Ixodes scapularis in the USA, Ixodes persulcatus in Asia, Ixodes ricinus in Europe) and is widespread in the entire northern hemisphere. In Romania, the geographic distribution and prevalence of Borrelia burgdorferi sensu lato was 1.4% in 41 counties, with a prevalence between 0.75-18.8%. B. burgdorferi sensu lato. had a prevalence of 3.8%, being found inside ticks in 55 of 183 localities. Successful treatment and full recovery can only be achieved through early diagnosis. The clinical and serologic diagnosis of Lyme disease is particularly difficult because of the phenotypic heterogeneity within and among spirochete species. A case study is presented in this paper: an eight-yearold male Yorkshire terrier dog, which was diagnosed positive for Lyme disease, based on a test which uses a peptide called C6 and which comes from the VlsE protein of B. burgdorferi, used to detect antibodies in dogs. The results demonstrate the reliability of the commercial SNAP 4Dx Plus Test for B. burgdorferi, which uses C6 to differentiate antibodies produced by natural infection from antibodies formed after vaccination. In addition, using realtime PCR, the diagnosis was negative, confirming the results from the literature, according to which the PCR technique is only recommended for research, the positivity percentage being low, especially when the sample is blood (0.1%). We conclude that the tests for the detection of antibodies specific to Lyme disease are recommended and useful. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria occur in sugar and alcoholised, slightly acid niches such as flowers, fruits, beer, wine, cider, vinegar, souring fruit juices and honey. On these substrates they oxidize the sugars and alcohols, resulting an accumulation of organic acids as final products. These transformations are of considerable interest for the biotechnological industry. The best known industrial application of acetic acid bacteria is vinegar production. The study used a method Real-Time PCR allows the quantitative detection of the members of the acetic acid bacteria group including the well-known Acetobacter, Gluconacetobacter and Gluconobacter species, and also other rare occurring but also acetic acid producing representatives as Acidisphaera sp., Acidocella sp., Acidomonas sp., Asaia sp., Granulibacter, Kozakia sp. and Swaminathania sp. The main objective of the present work is to test fast, sensitive and reliable technique such as Real-Time PCR and to detect acetic acid bacteria group without plating, isolated from white wine vinegar and directly from vinegar and the acetic acid bacteria grown in glucose medium (GYC) at 30°C, 48 h. The results obtained indicate that the amount of live acetic acid bacteria in vinegar during the fermentation process is not sufficient to detect them in this direct way.

Breast milk has been reported as a bacteria source that affects infant gut microbiota development. The present study utilizes a realtime PCR method to identify Lactobacillus and Bifidobacterium spp. in the breast milk of healthy women and attempts to identify factors affecting those human milk bacteria. Breast milk samples—both colostrum and mature milk—of 100 healthy women, were collected in Greece along with data about the demographic factors and nutritional habits of the volunteers. The colostrum samples were found to have higher percentages of either Bifidobacterium or Lactobacillus (76.9% and 48.6%, respectively) compared to the mature milk samples. For younger women, aged from 18 to 29 years, and women from rural areas, bacteria were detected in higher incidence than for older groups and women in urban areas, respectively. Moreover, for high-BMI women, bacteria were detected in lower incidence than for those with normal BMI. Probiotic supplements did not affect the composition of the breast milk-identified bacteria. Various factors such as lactation stage, maternal age, maternal weight, and residential location may contribute to the presence of those species in human milk. RT PCR has significant potential for the microbiological analysis of human milk. [ABSTRACT FROM AUTHOR]

In this study, the three biofilm-anoxic-oxic-MBR systems were operated in parallel using different carbon source feed types. The three systems were operated with complete sludge retention to compare microbial community composition and system efficiency. High average removal of ammonia and COD was obtained in the three reactors. However, total nitrogen and total phosphorus removal efficiency were significantly higher in the VFAs feed systems when compared with the glucose feed system. The highest and most stable BNR efficiency was observed when acetate was used as a carbon source. The qPCR analysis revealed that ammonium oxidizing bacteria, denitrifiers and total bacteria were all highest in the acetate feed system followed by the propionate feed system. Moreover, among all carbon source types, the PUS-biofilm could maintain a higher degree of abundance of total bacteria than the sludge biomass. Meanwhile, ammonium oxidizing bacteria and denitrifiers were enriched in the sludge biomass rather than in the PUS-biofilm. The results of illumina sequencing revealed that acetate followed by propionate were favourable to the growth of microorganisms that were associated with the BNR process, which was the main reason for the high efficiency of nutrient removal in the acetate and propionate feed systems. [ABSTRACT FROM AUTHOR]

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder mainly caused by homozygous deletions that include exon 7 of the survival motor neuron 1 (SMN1) gene. A nearby paralog gene, SMN2, obstructs the specific detection of SMN1. We optimized a duplexed real-time PCR approach using locked nucleic acid (LNA)- modified primers to specifically detect SMN1. Methods: An LNA-modified primer pair with 3´ ends targeting SMN1 specific sites c.835- 44g and c.840C was designed, and its specificity was examined by real-time PCR and Sanger Sequencing. A duplexed real-time PCR approach for amplifying SMN1 and control gene albumin (ALB) was developed. A randomized double-blind trial with 97 fresh peripheral blood samples and 25 dried blood spots (DBS) was conducted to evaluate the clinical efficacy of the duplexed approach. This new approach was then used to screen 753 newborn DBS. Results: The LNA-modified primers exhibited enhanced specificity and 6.8% increased efficiency for SMN1 amplification, compared with conventional primers. After stabilizing the SMN1 test by optimizing the duplexed real-time PCR approach, a clinical trial validated that the sensitivity and specificity of our new approach for detecting SMA patients and carriers was 100%. Using this new approach, 15 of the screened 753 newborns were identified as carriers via DBS, while the rest were identified as normal individuals. These data reveal a carrier rate of 1.99% in Hunan province, South Central China. Conclusions: We have developed a novel, specific SMN1 detection approach utilizing real-time PCR with LNA-modified primers, which could be applied to both prenatal carrier and newborn screening. [ABSTRACT FROM AUTHOR]

Objective. To study level and structure of microbial colonization in cancer patients before admission to a surgery department using molecular methods. Materials and Methods. The presence of DNA of methicillin-resistant Staphylococcus aureus, methicillinresistant coagulase-negative staphylococci, Candida albicans/C. glabrata/C. krusei, Acinetobacter baumannii in the clinical specimens and the presence of antimicrobial resistance genes in cultures were detected by multiplex real-time PCR. A total of 741 clinical samples and 313 cultures were studied. Results. The high level of microbial colonization, including microorganisms with antibiotic resistance genes, was found in cancer patients. Methicillin-resistant staphylococci, A. baumannii and Candida spp. were detected in 56.6%, 20% and 24.8% of cancer patients, respectively. Of patients infected with A. baumannii, 35.9% had OXA-carbapenemase genes. A. baumannii producing blaOXA40 was the most common cause of infectious complications. Conclusions. Molecular methods can be used effectively not only for the diagnosis of infectious diseases, but also in microbiological monitoring programs.

Aim. To compare the species composition of lactobacilli in women of reproductive age using real time PCR on urogenital samples before and after culturing microorganisms in Blood-Serum-Y^ast extract-Agar (BSYA). Materials and methods. Using real time PCR, we have examined the species composition of vaginal lactobacilli in 25 healthy women of reproductive age. Samples of urogenital swabs (endocervical and vaginal swabs) were studied. Species identification has been carried out twice: in the native clinical material and in the samples received after using microorganism culture technique in BSYA. Results. After culturing vaginal microorganisms in BSYA, L. iners predominance was found only in 2 cases (8%). At the same time, when using native clinical material this species predominance was discovered in 10 samples (40%). Conclusion. When culturing lactobacilli in BSYA, the growth of L. iners is stunted compared to other species of vaginal lactobacilli. The use of cultural testing turned out to be ineffective in case of predominance of L. iners, the species associated with an increased risk of vaginal dysbiosis.

Purpose: The aim of this study was to determine BRAF V600E mutation rates of malignant melanoma (MM) cases in the Diyarbakır region in this study to investigate the relationship between mutation rate of prognostic parameters. We also aimed to compare the results with values from west Turkey. Materials and Methods: Between all cases of MM, including primary MM and metastatic MM diagnosed at Dicle University Medical Faculty Pathology Department between January 2014 and July 2019 were included. The BRAF V600E mutation results, which were studied with the real-time polyemerase chain reaction (PCR) method, were compared with the prognostic parameters. Results: In 93 patients with MM, the BRAF V600E mutation r ate w as 2 1.5% (n = 20). T runk a nd h ead-neck tumors, cutaneous and mucosal tumors, Clark stage V tumors, tumors with a diameter of 0 -2 mm, Ki-67 proliferation between 11-20%, non-ulcerous tumors, chronic sun exposure in a higher rate was detected. Conclusions: BRAF mutations in Diyarbakir region were found to be lower than the value in the region in the west of Turkey. We think that the reason for this is more frequent monitoring of acral lentiginous melanoma (ALM) in our region. Although the BRAF V600E mutation was observed more in small-sized tumors, it was seen more in high Clark stage tumors, suggesting that the mutation may occur later in the advanced stages of the tumor. Studies with high case series may reveal possible correlations between BRAF and prognostic values and may be useful in patient selection for treatment. [ABSTRACT FROM AUTHOR]

This research aimed to validate real-time polymerase chain reaction using specific primer targeting on cytochrome-b 33 (cyt-b 33). The primer cyt-b 33 could specifically amplify dog meat DNA and method could detect 0.5 pg dog meat DNA in meatball formulation. The repeatability assay was acceptable with RSD value ≤ 25%. The validated method was further used for detection of meatball products commercially sold in Yogyakarta Indonesia, and results revealed that all samples (n = 14) did not contain dog meat DNA. This research finding supports the implementation of Indonesian Law Number 33 of 2014 in developing method for Halal Product Assurance. [ABSTRACT FROM AUTHOR]

Breast milk has been reported as a bacteria source that affects infant gut microbiota development. The present study utilizes a realtime PCR method to identify Lactobacillus and Bifidobacterium spp. in the breast milk of healthy women and attempts to identify factors affecting those human milk bacteria. Breast milk samples—both colostrum and mature milk—of 100 healthy women, were collected in Greece along with data about the demographic factors and nutritional habits of the volunteers. The colostrum samples were found to have higher percentages of either Bifidobacterium or Lactobacillus (76.9% and 48.6%, respectively) compared to the mature milk samples. For younger women, aged from 18 to 29 years, and women from rural areas, bacteria were detected in higher incidence than for older groups and women in urban areas, respectively. Moreover, for high-BMI women, bacteria were detected in lower incidence than for those with normal BMI. Probiotic supplements did not affect the composition of the breast milk-identified bacteria. Various factors such as lactation stage, maternal age, maternal weight, and residential location may contribute to the presence of those species in human milk. RT PCR has significant potential for the microbiological analysis of human milk.

BACKGROUND AND AIM: Silibinin (silybin) is an active component of silymarin with anti-bacterial activities. In drug resistant isolates of Pseudomonas aeruginosa has been reported overexpression of mexB gene. The aim of this study was evaluation of mexB gene expression in silibinin treated and untreated isolates of Pseudomonas aeruginosa. METHODS: In this descriptive analytical study, Pseudomonas aeruginosa isolates were obtained from hospitals and laboratories of Guilan province. After determining several antibiotics susceptibility (disc diffusion and MIC), 5 ciprofloxacin resistant isolates of Pseudomonas aeruginosa were treated by ciprofloxacin (1/2MIC) only (group1) and in the combination with silibinin-encapsulated micelles (nanoparticles) (group2). After 24h, RNA extraction and cDNA synthesis was performed in group1 and group2 and mexB gene expression was evaluated by quantitative-realtime PCR (Q-RT-PCR) and 2-ΔΔCT equation. FINDINGS: In this study from 69 isolates, 33.33% by disc diffusion test and 37.86% by MIC test were determined ciprofloxacin resistant. Our analysis showed that silibinin -encapsulated nanoparticles (400µg/ml) induced death up t0 50% in ciprofloxacin (1/2MIC) treated resistant isolates during 24h. In treated cells with silibinin and ciprofloxacin revealed downregulation of mexB gene compared to treated cells with ciprofloxacin alone. CONCLUSION: It seems that silibinin is cause of increasing ciprofloxacin effect on inhibition of growth of Pseudomonas aeruginosa through decreasing expression of genes implicated in efflux pomp systems such as mexB.

Abstract Background Recently, it is reported that asparagine synthetase (ASNS) is an independent predictor of surgical survival in hepatocellular carcinoma (HCC) patients. It is also reported that activating transcription factor 6 (ATF6) expression is decreased in HCC patients. So in the present study, we explored the relationship between ASNS and ATF6, and whether ASNS expression was associated with HCC. Methods ATF6 was over expressed in 3 HCC cell lines (HepG2, HepG2.2.15 and SMMC-7721). We then examined the mRNA levels of ASNS and ATF6 in 90 HCC patients, 77 chronic hepatitis B patients and 70 controls. We also genotyped 2 functional polymorphisms in ASNS in a case–control study. Results The expression of ASNS was significantly elevated when ATF6 was over expressed. The expressions of these 2 genes were both decreased in HCC patients, and it was more significantly with ASNS. The mRNA levels of ASNS and ATF6 were positively correlated with each other. rs34050735 was associated with HCC in the case–control study (P = 0.003) and also an independent predictor of overall survival of HCC patients (P = 0.001). Conclusions Taken together, these findings indicated that rs34050735 in ASNS may associate with HCC and may be a promising biomarker of HCC.

Contaminates such as pesticides, toxic molecules of natural origin, genetically modified organisms and others can occur in processed food, especially if the main ingredient grows in open fields exposed to the environment. In particular, some health threatening toxic compounds are natural ingredients of plants that grow wild next to vegetables intended for consumption and can therefore enter the crop yield and stay there undetected. The tropane alkaloids-containing nightshade thornapple Datura stramonium, often grows in close vicinity to millet (Panicum miliaceum) a widely cultivated cereal, representing an important nutrient source in different countries of Asia and Africa. Discriminating thornapple from millet during harvest is not easy and consequently, millet-containing food products are often contaminated with tropane alkaloids from thornapple. In this work, two DNA specific hydrolysis probe qPCR methods were developed for Datura stramonium and Panicum miliaceum in order to detect thornapple contamination in millet-containing food products. The specificity and sensitivity of the developed assay system allows for its application in screenings during food product testing.

Triple negative breast cancer is the heterogeneous and most aggressive known subtype of breast cancer due to absence of estrogen, progesterone and HER2 receptors. We aimed to identify a panel of circulating miRNAs which can act as diagnostic and prognostic biomarkers specifically for TNBC. The blood samples were collected from 37 TNBC diagnosed patients and 34 age-matched non-TNBC controls to investigate the expression of miRNA panel. Plasma was isolated by standard protocol of centrifugation and then isolation of total RNA including miRNA was done. About 100ng of isolated miRNA was converted to cDNA after addition of poly (A) tailing and then expression profiling was done by using real time-qPCR technique in which miR-specific DNA primers were used to increase sensitivity of reaction. Expression of miRNA panel was normalized with mir-16 and expression fold change was calculated by 2-ΔΔCt method. Further analysis was done by SPSS, medcalc and Graphpad prism software. Area under curve analysis was used to get diagnosis outcome of selected miRNA panel. Upregulation of miRNA panel (mir-376c, mir-155, mir-17a and mir-10b) has been seen in the plasma samples of TNBC patient's samples relative to non-TNBC control samples. The scatter plots and other statistical analysis have given differences in expression of four miRNA panel with significant p-value (p< 0.001), respectively. Kruskal Wallis test and Mann Whitney U test have clearly showed significance and association of miRNA panel with clinicopathological characteristics of breast cancer which have revealed their role as diagnostic biomarkers. AUC curve analysis also remarks their high sensitivity and specificity. We have identified panel of four miRNAs which can act as circulating diagnostic and prognostic biomarkers in triple negative breast cancer in the population of Pakistan. Especially, mir-376c, mir-155 and mir-17a may act as early stage biomarkers in TNBC and mir-10b is found to be late stage biomarker which can also act as prognostic biomarker in TNBC. [ABSTRACT FROM AUTHOR]

Huanglongbing disease (HLB) has rapidly spread in Mexico mainly affecting the Mexican lime industry (Citrus aurantifolia). Knowledge of the variation of carbohydrate metabolism caused by Candidatus Liberibacter asiaticus (CLas) in citrus fruits has allowed assessing its pathogenicity. Yet, response to the infection associated with the metabolism of sugars in Mexican lime is not characterized. The hypothesis was that the infection by CLas differentially affects carbohydrate metabolism during the pre and post-emergence phase of symptoms in Mexican lime plants. The objective was to characterize the changes in carbohydrate metabolism during the CLas infection progression in Mexican lime in greenhouse conditions. The experimental design was completely randomized with five biological subjects and two technical replications. In plants inoculated with CLas, we observed that starch content significantly increased (p ≤ 0.05) through time, up to four times at 16 months after inoculation (mpi) respect to the control. Confocal microscopy showed starch granules accumulation in vascular bundles and collapsed vascular tissue in inoculated plants. The sucrose content was higher (p£0.05) in the asymptomatic (4 mpi) and symptomatic (16 mpi) stage and the glucose and fructose content were higher (6.3 and 1.5 times) than in the non-infected plants. Photosystem II efficiency (Fv/Fm = 0.77) in symptomatic plants decreased respect to healthy ones (Fv/Fm = 0.81). These changes could affect field production. This is the first study that quantifies changes in the carbohydrate metabolism in Mexican lime plants and their relation to the disease progression. [ABSTRACT FROM AUTHOR]

• Serotypes 1, 3, 19A and 7F were the most common types in pediatric PPE in the pre-PCV13 era. • Serotypes 1 and 3 decreased by 91.8% and 31.5%, respectively, while serotypes 7F and 19A disappeared in vaccinated children. • The impact of PCV13 might be underestimated due to the improvement of surveillance and diagnostic methods. • A significant shift to non-PCV13 serotypes was not found in PPE during the 12-year follow-up. • Realtime PCR is significantly more sensitive than culture in the diagnosis of PPE. Etiology and serotyping of parapneumonic effusion (PPE) and the impact of vaccination was evaluated over a 12-year period, before and after the PCV13 introduction (2011) for Italian children From 0 to 16 years of age. Five hundred and two children were evaluated; 226 blood and 356 pleural fluid samples were obtained and tested using Realtime-PCR and culture. In the pre-PCV13 era S. pneumoniae was the most frequent pathogen identified (64/90; 71.1%) with a large predominance of serotypes 1 (42.4%), 3 (23.7%), 7F (5.1%) and 19A (11.9%). The impact of vaccination, calculated on children 0–8 years of age, demonstrated a significant reduction of PPE: with an incidence rate of 2.82 (95%CL 2.32–3.41) in the pre-PCV13 era and an age-standardized rate (ASR) of 0.66 (95% CL 0.37–1.99) in the post-PCV13 era, p < 0.0001. No increase in non-PCV13 serotypes was recorded. S. pneumoniae remained the most frequent pathogen identified in the post-PCV13 era in unvaccinated children with an unchanged serotype distribution: respectively 26/66 (39.4%), 25/66 (37.9%), 5/66 (7.6%), and 4/66 (6.1%) for 1, 3, 7F and 19A. On the other hand 7F and 19A disappeared in vaccinated children and serotype 1 and 3 decreased by 91.8% and 31.5%, respectively. Realtime PCR was significantly more sensitive than culture both in pleural fluid (79.7% vs 12.5%) and in blood (17.8% vs 7.4%). In conclusion, our findings indicate that routine immunization with PCV13 has significantly reduced the burden of childhood PPE in vaccinated children, without increasing PPE due to other bacteria and without serotype shift. Moreover, the impact of PCV13 may be underestimated due to the increase in pneumococcal surveillance in Italy. Data has also shown that Real-time PCR is an essential tool to better define the etiology of PPE and to monitor vaccination plans. Longer studies will be necessary to evaluate the role of herd protection in PPE prevention. [ABSTRACT FROM AUTHOR]

Background: Human bocavirus (HBoV) is known to cause lower respiratory tract infections (LRTI) in children and may result in substantial morbidity and mortality. The aim of this study was to determine HBoV prevalence among hospitalized infants and small children with acute LRTI in Zagreb, Croatia, as well as to evaluate HBoV DNA quantity in samples in relation to the patients' age and co-infection with other respiratory viruses. Methods: During winter season 2016/2017, a total of 295 children younger than three years of age who were admitted to hospitals with LRTI were tested for the presence of HBoV, respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV) types 1 to 3, and human metapneumovirus (HMPV). HBoV was detected with a real-time PCR method, and the other viruses were diagnosed using monoclonal antibodies in direct fluorescence assay. Results: Viral etiology was proven in 225/295 (76.3%) of patients. The most commonly diagnosed virus was RSV (59.3%), followed by HBoV (23.1%), PIVs (4.4%), ADV (3.1%), and HMPV (1.4%). HBoV-infected children were older than RSV-infected children; likewise, detection rates of HBoV infection increased with age, while RSV infection rates decreased with age. In 51% of HBoV-positive samples an additional respiratory virus was also detected. There was no difference in HBoV DNA quantity between samples with single virus detection and those with multiple virus detection (p = 0.056), although samples positive only for HBoV showed higher cycle threshold values. There was no difference in HBoV DNA quantity in samples of different age groups (p > 0.05). Conclusions: Frequent detection of HBoV in small children with LRTI, even in combination with other viruses, highlights its role in the pathogenesis of respiratory disease. [ABSTRACT FROM AUTHOR]

Introduction During 2008–2012, a total of 466 ticks Ixodes ricinus removed from humans were collected and tested for the presence of Borrelia burgdorferi sensu lato (Bbsl). Ticks were collected in all districts of the South Moravian region of the Czech Republic (CZ). Objective The aim of this study was to determine the infestation of Bbsl in ticks Ixodes ricinus and the identification of genospecies of Bbsl group by DNA sequencing. Material and Methods DNA isolation from homogenates was performed by UltraClean BloodSpin DNA kit (MoBio) and by automated instrument Prepito (Perkin-Elmer). Detection of spirochetes was carried out by RealTime PCR kit EliGene Borrelia LC (Elisabeth Pharmacon). Finally, all the positive samples were sequenced on an ABI 3130 Genetic Analyzer (Life Technologies) and identified in the BLAST (NCBI) database. Results A total positivity of the samples was 26%. For the first time in the Czech Republic, 5 of the isolated strains were genotyped as Borrelia spielmanii (7.1%). Other representatives of Bbsl were also observed: B. afzelii (70.0%), B. garinii (10.0%), B. valaisiana (8.6%), and B. burgdorferi s. s. (4.3%). Conclusions A general view of the spreading of Bbsl in the South Moravian region was demonstrated. The most interesting result of the study is the finding of B. spielmanii for the first time in this region.

Abstract Background and study aims Hepatitis C virus (HCV) infection is a severe problem among patients on maintenance haemodialysis who are at particular risk for blood-borne infections because of prolonged vascular access and potential for exposure to contaminated equipment. Occult hepatitis C virus infection (OCI) is defined as the presence of HCV RNA in liver or peripheral blood mononuclear cells (PBMCs) in the absence of detectable HCV antibody or HCV RNA in the serum. In this study, we aimed to investigate the existence of occult hepatitis C virus infection in PBMCs of haemodialysis (HD) patients in one center. Moreover, we tried to link the condition to risk factors associated with HCV infection in those patients. Patients and methods We included 40 patients with renal diseases undergoing regular haemodialysis who were repeatedly anti-HCV negative. HCV RNA detection was tested by Quantitative Real time PCR in serum and PBMCs. Results The results of this study revealed that 23% of our haemodialysis patients have occult hepatitis C virus infection. There was a highly significant increase in ALT levels in patients with OCI versus the negative group. Also, there is a significant increase of history of blood transfusion in patients with occult HCV (p = 0.03) while the duration of haemodialysis showed no statistical significant difference between both groups. The viral load of the occult hepatitis C virus infection subjects ranged from 581to 74,307 copies/ml. Conclusion These results highlight the potential risk of hepatitis C virus transmission from patients within haemodialysis units in Egypt. Isolation of patients on dialysis machines depending on the results of hepatitis serological markers is not enough. Testing for hepatitis C virus -RNA in peripheral blood mononuclear cells is more reliable in identifying patients with an OCI when a liver biopsy is not available. [ABSTRACT FROM AUTHOR]

Background: Information on the burden, characteristics and seasonality of non‐influenza respiratory viruses is limited in tropical countries. Objectives: Describe the epidemiology of selected non‐influenza respiratory viruses in Thailand between June 2010 and May 2014 using a sentinel surveillance platform established for influenza. Methods: Patients with influenza‐like illness (ILI; history of fever or documented temperature ≥38°C, cough, not requiring hospitalization) or severe acute respiratory infection (SARI; history of fever or documented temperature ≥38°C, cough, onset <10 days, requiring hospitalization) were enrolled from 10 sites. Throat swabs were tested for influenza viruses, respiratory syncytial virus (RSV), metapneumovirus (MPV), parainfluenza viruses (PIV) 1‐3, and adenoviruses by polymerase chain reaction (PCR) or real‐time reverse transcriptase‐PCR. Results: We screened 15 369 persons with acute respiratory infections and enrolled 8106 cases of ILI (5069 cases <15 years old) and 1754 cases of SARI (1404 cases <15 years old). Among ILI cases <15 years old, influenza viruses (1173, 23%), RSV (447, 9%), and adenoviruses (430, 8%) were the most frequently identified respiratory viruses tested, while for SARI cases <15 years old, RSV (196, 14%) influenza (157, 11%) and adenoviruses (90, 6%) were the most common. The RSV season significantly overlapped the larger influenza season from July to November in Thailand. Conclusions: The global expansion of influenza sentinel surveillance provides an opportunity to gather information on the characteristics of cases positive for non‐influenza respiratory viruses, particularly seasonality, although adjustments to case definitions may be required. [ABSTRACT FROM AUTHOR]

The comparatively high cost of laboratory detection methods for Clostridium difficile infection (CDI) coupled to a low prevalence rate has resulted in testing algorithms that use cheaper and relatively sensitive screening methods, followed by more specific confirmatory methods. The aim of this prospectively-conducted study from two centres in the UK, and one in the Republic of Ireland was to determine the efficacy of the EntericBio ® realtime C. difficile Assay (EBCD) for the detection of toxigenic C. difficile in stool samples. The EBCD was compared to the in-use testing methods for Clostridium difficile (CD) detection in each centre. In the two UK centres, the EBCD was compared to the C.diff Quik Chek Complete ® kit (Techlab), and discrepancies were tested further using The Xpert ® C. difficile PCR assay (Cepheid) and PCR ribotyping after cultivation using the spore culture method, respectively. In the Irish centre, EBCD comparison was to an algorithm of C. DIFF CHEK™-60 test (Techlab) for screening followed by C. difficile Premier ™ Toxins A&B assay (Meridian Bioscience ® ) in the case of positive results; discrepancies were tested using the Xpert ® C. difficile PCR assay. In a retrospective analysis of data, a total of 947 stool samples were tested, of which eight (0.8%) proved inhibitory to the EBCD assay. Of the 939 valid tests conducted, reported sensitivities of the EBCD were 94.7%, 100% and 97.9%, respectively; specificities were 99.6%, 100% and 100%, respectively; positive predictive values were 94.7%, 100% and 100%, respectively, and negative predictive values were 99.6%, 100% and 99.8%, respectively. The CD positivity rates in the current study ranged between 6.6% and 8.2%. [ABSTRACT FROM AUTHOR]

Background: Miana (Coleus scutellariodes (L) Benth) are a herbal medicine with potential anti-inflammatory properties in patients infected with vulvovaginal candidiasis. The objective of this work was to the analyze IL-37 expression following administration of miana leaf extract (MLE) in an animal model of vulvovaginal candidiasis. Material and Methods: Adult Balb/cmice, aged 8-12 weeks and weighing 30-40 g, were divided into five groups. Four groups were administered Candida albicans via intravaginal inoculation with a diluted dose of 10-2/ml and were treated with either MLE, a placebo, or ketoconazole; one group constituted the healthy control and was only treated with MLE. Real-time PCR was used to measure the expression of IL-37. Results: These findings indicated that a component within MLE may mediate its anti-inflammatory characteristics, as indicated by the increase in mRNA IL-37 expression in mice inoculated with C. albicans. The highest increase in fungal load to 101.6 CFU was observed in the placebo group at day 14. Whereas for the mice treated with MLE at 750 mg/kg b.w, the fungal load only increased to 30.0 CFU, similar to that of mice treated with ketoconazole (29.6 CFU). In the mice treated with MLE at 500 mg/kg b.w, the fungal load increased to 68.2 CFU. Conclusion: Fungiostatic effect of MLE 750 mg/kg BB is not less than ketoconazole and MLE may act as anti-inflammatory throught its role as an antioxidant so it could potentially be used as an alternative treatment in humans especially patients with vulvovaginal candidiasis. [ABSTRACT FROM AUTHOR]

Soy sauce is worldwide popular condiment of Asian origin. With the advent of GM soybean production, soy sauce drew the interest of food safety control. Samples collected for inspection are generally of industrial grade soy sauce type, which is produced from hydrolyzed soybean and grain. Following the failure to perform RealTime PCR based GMO screening on a number of submitted samples we tested our screening system on soy sauce produced following traditional method based on fermentation. Four batches of soy sauce were produced and DNA extracted. DNA concentration ranged from 32,68 to 65,36 ng/μl. Amplification of taxon specific target was successful with rather high Ct ( > 30). Promoter P-35S sequence was not detected, but T-NOS was detected in three samples with values reaching or exceeding LOD of the method. The results show that it is possible to detect transgenic elements in traditionally produced soy sauce while DNA extraction from industrial grade soy sauce is not possible.

Influenza and other acute respiratory infections are of great concern for public health, causing excessive morbidity and mortality throughout the world. Influenza virus A(H2N2), which caused a pandemic of so called “Asian flu” in 1957 was expelled from the human population by the new pandemic virus subtype H3N2 in 1968, however, influenza A(H2) viruses continue to circulate in wild birds and poultry. The lack of immunity in human population and the continued circulation of influenza A(H2) among animals makes emergence of a new pandemic virus possible. One of the basic techniques of molecular diagnostics of infectious diseases is the realtime polymerase chain reaction (PCR). The aim of this work was to design oligonucleotide primers and probes for the rapid detection of influenza A virus subtype H2 by realtime reverse transcription - polymerase chain reaction (rRT-PCR). Analysis of 539 sequences of influenza A(H2N2) virus hemagglutinin gene from GISAID EpiFlu database revealed conservative regions suitable for use as binding sites for primers and probes. 191 probes were designed and 2 sets of primers and probes (H2-1 and H2-2) were selected for further experimental evaluation. Detection limit of RT-PCR system was 50 copies of DNA per 25 μl reaction when 10-fold dilutions of pCI-neo-H2 plasmid used as template. Analytical specificity of selected sets of primers and probes were tested on wide range of influenza strains and non-influenza respiratory viruses. H2-2 set found to have insufficient specificity detecting seasonal influenza A(H1N1) viruses and was excluded from further analysis. Analytical sensitivity was further tested on vaccine strain A/17/California/66/395 (H2N2) and A/Japan/305/1957 (H2N2), limit of detection for primers-probe set H2-1 was 3.2 (CI95%: 3.07–3.48) lg EID 50 /ml. Designed primers and probes for the realtime RT-PCR universal detection of influenza A(H2) viruses could be used in clinical trials of vaccines against influenza A(H2) and screening for H2 in cases of unsubtypeable influenza A in humans. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to evaluate the efficacy of Pluchea dioscoridis leaf extract on growth, survival, morphogenesis, and virulence gene expression of Candida albicans. Methodology: Anticandidal activity was studied using the hole-plate method against four pathogenic C. albicans strains from clinical isolates. The effect of the extract on the growth profile of the yeast was also examined via a time-kill assay. Microscopic observations using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were made to determine the major alterations in the microstructure of C. albicans. Quantitative changes in phospholipase, hemolysin, and secreted aspartyl proteinase (SAP1 and SAP10) genes expression as virulence factors were analyzed using real-time polymerase chain reaction (RT-PCR). Results: The extract exhibited high anticandidal activity, with the zones of inhibition between 0.5 and 6 cm, and recorded a minimum inhibitory concentration (MIC) value of 30 mg/mL. The time-kill study suggested that the extract possessed yeasticidal properties at higher concentrations and eradicated the growth of yeast cells. The SEM and TEM micrographs exhibited major abnormalities that occurred on the yeast cells after being exposed to the extract, resulting in complete alterations in their morphology and collapse of the cells beyond repair. At MIC concentration, phospholipase, proteinase, and hemolysin gene expression was reduced to 90%, 70%, 90% for SAP1, and 40% for SAP10,respectively, compared to that obtained from untreated C. albicans, as demonstrated by quantitative reverse transcription (RT)-PCR analysis. Conclusions: The P. dioscoridis leaf extract may be an effective anticandidal agent to treat pathogenic yeast infections. [ABSTRACT FROM AUTHOR]

Currently, tuberculosis is still one of the most common infectious diseases, mainly caused by Mycobacterium tuberculosis bacterium infection. PCR is a molecular biology technique used to detect infectious agents, especially viruses or bacteria with slowly culture as in the case of Mycobacterium tuberculosis because of its fastness, accurateness and high sensitivity. We carried out this study in order to develop a protocol based on Realtime PCR using 16S rRNA gene as a target sequence which is extensively used in taxonomy, molecular evolution and medical diagnostics. We have succeeded in designing the primers/probes on the 16S rRNA gene to detect Mycobacterium tuberculosis by Realtime PCR; have identified the parameters of the specificity of the primers/probe, theoretically and experimentally; the sensitivity of the primers/probe in the Realtime PCR protocol reached to 102 copies/ml; the protocol was also tested on 30 samples given good results.

Hepatitis B is a disease caused by the hepatitis B virus (HBV), and can be acute or chronic. Infection with hepatitis B virus is the leading cause of chronic hepatitis worldwide and people with chronic hepatitis B infection are at increased risk for developing liver cancer (hepatocellular carcinoma). In addition, the hepatitis B virus is the leading cause of cirrhosis. HBV can be classified into 8 genotypes A through H based on the divergence of 8% or more in the nucleotide sequences of complete genome. The identification of HBV genotypes are more concerned as the relating of HBV genotypes to clinical manifestation, activity of liver disease as well as treatment responses of Hepatitis B. We carried out this study in order to determine the genotype of HBV-infected patients in the area of Dak Lak Province. Prevalence of HBV genotype was determined on the sample size of 100 by Realtime PCR: 63 samples infected with genotype B, 21 samples infected with genotype C and 16 samples simultaneously infected with genotype B and C. Also, a number of clinical symptoms such as the concentrations of HBV DNA, HBeAg antigen were also analyzed in each genotype.

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-4, IL-6, IL-8 and IL-10 genes in milk cells of healthy cows and cows with clinical mastitis. Gene expression was analyzed by real-time PCR.IL8 gene expression was higher than another interleukin. E. coli is among the major mastitis pathogens responsible for clinical mastitis in dairy cows, but the infection are normally cleared by the immune system within a few days. Mastitis tolerance/susceptibility is difficult to measure directly and hence milk somatic cell count (SCC) or milk somatic cell score (SCS) is used as an indicator trait for mastitis as both traits are highly positively correlated. Thus, understanding of mastitis pathogen and somatic cell count associated with cell mediated immune response useful for future control of mastitis. [ABSTRACT FROM AUTHOR]

Introduction. During 2008-2012, a total of 466 ticks Ixodes ricinus removed from humans were collected and tested for the presence of Borrelia burgdorferi sensu lato (Bbsl). Ticks were collected in all districts of the South Moravian region of the Czech Republic (CZ). Objective. The aim of this study was to determine the infestation of Bbsl in ticks Ixodes ricinus and the identification of genospecies of Bbsl group by DNA sequencing. Material and methods. DNA isolation from homogenates was performed by UltraClean BloodSpin DNA kit (MoBio) and by automated instrument Prepito (Perkin-Elmer). Detection of spirochetes was carried out by RealTime PCR kit EliGene Borrelia LC (Elisabeth Pharmacon). Finally, all the positive samples were sequenced on an ABI 3130 Genetic Analyzer (Life Technologies) and identified in the BLAST (NCBI) database. Results. A total positivity of the samples was 26%. For the first time in the Czech Republic, 5 of the isolated strains were genotyped as Borrelia spielmanii (7.1%). Other representatives of Bbsl were also observed: B. afzelii (70.0%), B. garinii (10.0%), B. valaisiana (8.6%), and B. burgdorferi s. s. (4.3%). Conclusion. A general view of the spreading of Bbsl in the South Moravian region was demonstrated. The most interesting result of the study is the finding of B. spielmanii for the first time in this region. [ABSTRACT FROM AUTHOR]

Malting barley grains are essential components in the beer production. Fusarium infection can have severe effects on malt and beer, because it may inhibit the enzymatic activity in malt and may induce the occurrence of gushing and changes in the colour and flavour of the finished beer. We examined the growth of the filamentous fungi Fusarium culmorum in artificially infected and non-infected barley malting grains during the first steps of the malting process and under the effects of fungicide pretreatment (Hutton and Prosaro 250 EC) of barley plants. Our study focused on the fungi growth in two distinct barley malting cultivars Bojos and Malz. Fusarium growth was investigated by quantitative real-time PCR using TagMan MGB probes. Furthermore, we focused on the Tri5 and Tri6 genes because they play the most important roles in trichothecene biosynthesis. Surprisingly, the higher transcription activity of the Tri genes was found in the fungicide-treated cultivar Malz as compared with untreated cultivars. [ABSTRACT FROM AUTHOR]

Background Hepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options. Objective To establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B. Study design Genotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA. Results Of 100 samples from 50 patients, ≥2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively. LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available. Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold. Conclusion Genotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA. [ABSTRACT FROM AUTHOR]