TaqMan Polymerase Chain Reaction Assay Research Articles

On the relationship between tripartite motif-containing 22 single-nucleotide polymorphisms and COVID-19 infection severity

BackgroundThe tripartite motif containing (TRIM)-22 participates in innate immune responses and exhibits antiviral activities. The present study aimed to assess of the relationship between TRIM22 single-nucleotide polymorphisms and clinical parameters with the coronavirus disease 2019 (COVID-19) infection severity.MethodsTRIM22 polymorphisms (rs7113258, rs7935564, and rs1063303) were genotyped using TaqMan polymerase chain reaction (PCR) assay in 495 dead and 497 improved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive patients.ResultsIn this study, the frequencies of TRIM22 rs1063303 GG, rs7935564 GG, and rs7113258 TT were significantly higher in dead patients than in improved patients, and higher viral load with low PCR Ct value was noticed in dead patients. The multivariate logistic regression analysis revealed that the lower levels of low-density lipoprotein (LDL), cholesterol, PCR Ct value, and lower 25-hydroxyvitamin D, and also higher levels of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and TRIM22 rs1063303 GG, rs7113258 TT, and rs3824949 GG genotypes were related to the COVID-19 infection severity.ConclusionOur finding proved the probable relationship between the COVID-19 infection severity with the genotypes of TRIM22 SNPs and clinical parameters. More research is required worldwide to show the association between the COVID-19 infection severity and host genetic factors.

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.

The association of a single-nucleotide variant in the microRNA-146a with advanced colorectal cancer prognosis.

The aim of this study was to evaluate the association of single-nucleotide variant n.60G>C (rs2910164) of microRNA (miR)-146a, related to suppressing of BRCA1/2 DNA repair protein, with the risk and survival of colorectal cancer patients, as well as miR-146a and BRCA1/2 levels and miR binding efficiency. The genotypes were identified in 125 colorectal cancer patients and 276 controls using TaqMan polymerase chain reaction assay. The miR-146a and BRCA1/2 levels were assessed by quantitative-polymerase chain reaction protocols. Primary precursor of miR-146a containing G (wild-type) and C (variant) allele were cloned into pcDNA.3.3 vector and co-transfected in HT-29 colorectal cancer cell line. Luciferase reporter assay was performed to assess miR-146a binding to BRCA2 3'-untranslated region in HT-29. The differences between groups were calculated using chi-square or Fisher's exact test, logistic regression, and Mann-Whitney test. The prognostic impact of single-nucleotide variant genotypes on overall survival was evaluated by Kaplan-Meier estimate and Cox regression. The GC or CC genotypes prevalence was similar in patients and controls (50.4% vs 50.7%, p = 0.74). However, patients with tumors in advanced stage with miR-146a GG genotype had 2.41 more chance of dying than GC or CC genotypes. In addition, tumor tissues of patients with GG genotype presented higher miR-146a (p = 0.02) and lower BRCA1 (p = 0.01) and BRCA2 (p < 0.0001) levels when compared to those with GC or CC genotypes. In fact, pcDNA.3.3-miR-146a-G presented increased binding capacity to the 3'-untranslated region of BRCA2 (p = 0.001) compared to pcDNA.3.3-miR-146a-C. In addition, the G allele altered the binding affinity between miR-146a and its BRCA2 3'-untranslated region target (p < 0.001), thus enhancing suppression of BRCA2 expression. Our results suggest that single-nucleotide variant rs2910164 does not influence the colorectal cancer risk in Brazilian patients; however, the GG genotype could act as a factor of worse prognosis in patients with advanced disease due to suppression of BRCA1/2 modulated by miR-146a.

Defining the Specific Pathogen-Free State of Xenopus Using TaqMan Assays.

Colonies of valuable inbred and transgenic laboratory-reared Xenopus frogs maintained for research constitute naïve populations of animals susceptible to some opportunistic infectious diseases. Therefore, it is prudent to characterize any new animal acquisitions before introduction into an existing colony as a biosecurity measure to preclude the concurrent introduction of an infectious microorganism associated with the new animal(s). In addition, some pathogens of Xenopus, such as Chlamydia and Mycobacterium spp, are zoonotic diseases, placing frog aquarists at risk for acquiring an infection. Because it is not cost effective to test for all diseases of Xenopus frogs, we have defined a subset of prevalent infectious microorganisms and developed TaqMan polymerase chain reaction (PCR) assays to detect these agents. The specific pathogens in our test panel were selected from relatively recent publications where they reportedly caused morbidity and/or mortality in Xenopus laevis and/or X. tropicalis The assays herein do not constitute a comprehensive list of infectious diseases of Xenopus frogs. Therefore, a frog devoid of the infectious agents in our test panel are characterized as "specific pathogen-free." Three of the described quantitative polymerase chain reaction (qPCR) assays detect many species within their genus (i.e., qPCRs for ranaviruses, Chlamydia spp, and Cryptosporidia spp).

Human Herpesvirus 6 as an Indicator of Cytomegalovirus Infection and Its Attributable Disease Symptoms in Liver Transplant Recipients.

Human β-herpes viruses, including cytomegalovirus and human herpesvirus 6, can become activated in liver transplant patients. Here, we evaluated the effects of human herpesvirus 6 infection as an independent factor on cytomegalovirus infection and the occurrence of associated diseases in liver transplant patients. In this cross-sectional study, 46 patients who underwent deceased-donor liver transplant at Nemazi Hospital, Shiraz University of Medical Sciences (Shiraz, Iran) were prospectively monitored for cytomegalovirus and human herpesvirus 6 infections during 3 months posttransplant. Taq-man real-time polymerase chain reaction assay as an accurate and rapid test and pp65-anigenemia as the standard test were used to monitor cytomegalovirus infections, whereas human herpesvirus 6 infection was monitored by Taq-man real-time polymerase chain reaction assay. We also followed clinical findings from laboratory data and symptoms of cytomegalovirus infections. Active cytomegalovirus infection was detected in 23 liver transplant recipients (50%), of which 17 (74%) were diagnosed with cytomegalovirus-related diseases. Active human herpesvirus 6 infection was detected in 25 patients (54%). Thirteen of 17 cytomegalovirussymptomatic patients had coinfection with human herpesvirus 6. In 10 ofthe 13 patients with coinfection, human herpesvirus 6 DNAemia appeared significantly earlier by 9 days than cytomegalovirus infection. In the pp65 antigenemia test, the mean number of cytomegalovirus-infected polymorphonuclear cells was 42.47 ± 5.41, which was correlated with incidence of clinical presentation. In symptomatic patients, average serum and polymorphonuclear cell viral loads of cytomegalovirus were 12064.59 copies/mL and 6735 copies/2 × 105 cells,respectively, with significant differences between the loads and cytomegalovirusattributable disease symptoms. Average human herpesvirus 6 DNA burden in serum samples of symptomatic patients was 11283 copies /mL, which was statistically related to cytomegalovirus-attributable disease symptoms. We found that human herpesvirus 6 infection is often associated with cytomegalovirus reactivation and cytomegalovirus-attributable disease symptoms.

Human bocavirus detection and quantification in fecal and serum specimens from recipients of allogeneic hematopoietic stem cell transplantation: A longitudinal study.

The aim of this study was to evaluate the occurrence of human bocavirus (HBoV) and to determine viral loads in samples of patients admitted for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fecal and serum samples were collected from 19 patients, during a 24-month period. Samples were screened by quantitative polymerase chain reaction TaqMan assay, with specific probe and primers targeting the NP1 gene of all HBoVs genotypes (HBoV-1 to - 4), and viral loads were determined using serial dilutions of a recombinant plasmid. HBoV DNA was detected in 42.1% (8 of 19) of the patients in at least one type of sample (feces and/or serum) during the study period, with 75% (6 of 8) of the patients being positive in both types of sample. Viral shedding in feces had a median of 26 days (range, 5 to 121) and viremia was detected in 87.5% (7 of 8) of the patients. The HBoV loads in fecal samples were higher than in sera and, in most cases, HBoV was detected earlier in fecal than in sera samples. In six HBoV-positive patients (6 of 8) diarrhea was observed concomitantly to viral detection in fecal samples. A high frequency and loads of HBoV in allo-HSCT recipients was observed, especially in fecal samples. Positivity in fecal samples was an early predictor of HBoV presence.

Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and Soil.

Macrophomina phaseolina is a broad-host-range fungus that shows some degree of host preference on strawberry, and causes symptoms that include crown rot and root rot. Recently, this pathogen has affected strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan polymerase chain reaction assay which is able to quantify as little as 2 to 3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.

Molecular Detection of Human Astrovirus in Children With Gastroenteritis, Northern Italy.

Human astroviruses have increasingly been identified and are important agents of diarrheal disease, especially in infants and young children. This article presents the real-time polymerase chain reaction TaqMan assay for the detection and quantification of human astrovirus for clinical fecal samples collected from hospitalized children with acute gastroenteritis in Piedmont (northern Italy) from December 2014 to November 2015. A total of 159 fecal specimens from hospitalized children with acute gastroenteritis, previously screened for rotavirus, adenovirus, norovirus, human parechovirus, salivirus and sapovirus, were tested for human astrovirus. The most commonly detected virus was norovirus GII (33.8%), followed by rotavirus (21.3%), sapovirus (10.9%), human parechovirus (8%), norovirus GI (6.7%), adenovirus (1%) and salivirus (0.52%). A total of 30 of 159 (18.87%) episodes of acute gastroenteritis were associated with human astrovirus genomic detection. Our data showed that the detection rate of astrovirus in diarrheal children (18.87%) was higher than observed in other countries, where they were reported in diarrheal children in 10.3%-0.8% of patients and a mean incidence worldwide of 11%. Our data showed that the detection rate of astrovirus in pediatric gastroenteritis was greater than previously reported in Italy.

Evaluation of TRIM5 and TRIM22 polymorphisms on treatment responses in Iranian patients with chronic hepatitis C virus infection

The tripartite motif (TRIM)-5 and TRIM22 are involved in innate immune response and show anti-viral activities. The current study aimed at evaluating the association of TRIM5 and TRIM22 polymorphisms with treatment outcomes in patients with chronic hepatitis C virus (CHC). TRIM5 rs3824949 and TRIM22 polymorphisms (rs7113258, rs7935564, and rs1063303) were genotyped using TaqMan polymerase chain reaction (PCR) assay in 425 treatment-naïve CHC patients. Rapid virological response (RVR), early virological response (EVR), and sustained virological response (SVR) were found in 54.1%, 74.8%, and 67.1% of the patients, respectively. RVR and SVR were associated with TRIM5 rs3824949 (GG), TRIM22 rs1063303 (GC), and TRIM22 rs7113258 (AA), while there was a relationship between TRIM5 rs3824949 (GG) and EVR. TRIM5 and TRIM22 single nucleotide polymorphisms (SNPs) were strongly associated with increased odds of RVR, EVR, and SVR after an interferon-based therapy in patients with CHC.

Efficient Synthesis of a Wide-Range Absorbing Azaphthalocyanine Dark Quencher and Its Application to Dual-Labeled Oligonucleotide Probes for Quantitative Real-Time Polymerase Chain Reactions.

Unsymmetrical dialkylamino-substituted zinc azaphthalocyanine (AzaPc) exhibits unique spectral and photophysical properties for dark quenchers of fluorescence in DNA hybridization probes. The panchromatic light absorption of AzaPc from 300 nm up to at least 700 nm and its lack of fluorescence make it an ideal candidate for a universal dark quencher. To prove this experimentally, oligodeoxyribonucleotide probes were labeled at the 3'-end by this AzaPc and at the 5'-end by a fluorophore used in the polymerase chain reaction (PCR)-that is, fluorescein, CAL Fluor Red 610, and Cy5. AzaPc showed a significantly higher quenching efficiency compared to the commercially available dark quenchers (BHQ-1, BHQ-2, BBQ-650) in a developed model of TaqMan PCR assay. The AzaPc-labeled probe proved to also be useful in a practical PCR assay for the quantification of the SLCO2B1 transporter gene expression. The constructed calibration curves indicated linearity in the range from 102 to 107 of target copies.

Implementation of next generation sequencing technology for somatic mutation detection in routine laboratory practice

The introduction of next generation sequencing (NGS) in the routine diagnostic setting is still in the development phase and has been limited by its complexity. Targeted NGS offers an attractive alternative to performing multiple single target assays and is very useful in meeting the increasing clinical demand for testing of multiple genetic aberrations in cancer specimens. To this end, we carried out a blinded validation study on 113 tumours in a diagnostic laboratory and compared mutation results from targeted NGS with those from Sanger sequencing, pyrosequencing, competitive allele specific TaqMan polymerase chain reaction (CAST PCR) and Cobas assays. DNA was extracted from formalin fixed, paraffin embedded (FFPE) tissue samples that included core biopsies, resections and cytology samples from three common and one rare cancer types [non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM) and gastrointestinal stromal tumour (GIST)]. Libraries were prepared using the TruSight Tumour 26 gene panel and NGS was carried out on the MiSeq instrument. Results from NGS were concordant with the mutational status determined by other platforms in 107 of the 113 cases tested (94.7%). The sequencing quality for NGS failed in four of the six false negative cases, while a further two samples gave false negative results because the c-KIT mutations were located outside the range of the NGS panel. One NSCLC sample contained an EGFR mutation previously detected by the Cobas assay. Reanalysis of the NGS data for this sample using a cut-off allele frequency of 1% revealed the mutation had an allele frequency of 2%, which was below the recommended software-determined threshold of 3%. NGS detected 113 additional mutations that were not previously known from analysis by the conventional methods. Twenty-six of these have known clinical importance, 37 have potential clinical significance, while 50 were novel mutations with unknown clinical significance. NGS detected variants using inputs of 10-20ng of FFPE extracted DNA and from specimens with a tumour cell content less than 50%, for which when possible we recommend microdissection. We conclude that results from targeted NGS are highly concordant with those from other mutation testing platforms. Targeted NGS is suitable for a range of sample types received in the diagnostic pathology laboratory, including those with limited material or with low tumour cell content (TCC). This work has allowed us to determine the quality parameter settings required in order to obtain robust mutation data by NGS.

Novel duplex real-time Taqman PCR assay for rapid detection of Enterobacter sakazakii in powdered infant formula

To develop a dulplex real-time Taqman polymerase chain reaction(PCR)assay for the detection of Enterobacter sakazakii. Primers and probes were designed based on the sequences of the macromolecular synthesis(MMS)operon and outer member protein A(ompA). Sensitivity and specificity of the assay were evaluated. Detection limit of the assay in pure culture was 4.3×103 CFU/mL and as few as 2 CFU/100 g of Enterobacter sakazakii could be detected in artificially contaminated infant formula through 24 h of enrichment. All of 15 Enterobacter sakazakii strains were successfully identified,no specific amplification were presented when 24 non-Enterobacter sakazakii were tested. The duplex real time Taqman PCR assay developed in study was sensitivity,specific and rapid. It could be used for detection of Enterobacter sakazakii from infant milk powder for food safety.

TaqMan PCR assay for detection and quantification of Stagonosporopsis tanaceti in pyrethrum seed and seedlings

Pyrethrum seed has an important role in the transmission of Stagonosporopsis tanaceti, the cause of ray blight disease of pyrethrum. A TaqMan probe based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inocula in pyrethrum seed and seedlings. Primer pair (St_qF3, St_qR2) was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using St_qF3, St_qR2 and a probe St_qP was highly specific against the genomic DNA of S. tanaceti, but did not amplify DNA of 14 related Stagonosporopsis species or other foliar pathogens of pyrethrum. The sensitivity limit of this assay was measured using the cycle threshold (Ct) value which ranged from 17.59 for 10 nanograms (ng) to 36.34 for 100 femtograms (fg) genomic DNA of S. tanaceti. There was a significant negative correlation (r = −0.999, P < 0.001) between the Ct value and the percent of S. tanaceti infected seed. In addition, this TaqMan PCR assay detected latent infection within seedlings. This assay could be applied to test commercial seed and seedlings before deciding on the appropriate management practices.

Double gene siRNA knockdown of mutant p53 and TNF induces apoptosis in triple-negative breast cancer cells.

Apoptosis is the major downregulated pathway in cancer. Simultaneous inhibition using specific small interfering RNA (siRNA) of two key player genes, p53 and TNF, is an interesting and feasible strategy when it comes to investigating various molecular pathways and biological processes in triple-negative breast cancer (TNBC), which is one of the most aggressive and therapeutically unresponsive forms of breast cancers. Our present research focuses on evaluating the impact of double p53-siRNA and TNF-siRNA knockdown at a cellular level, and also evaluating cell proliferation, apoptosis, induction of autophagy, and gene expression by using reverse transcription polymerase chain reaction array approaches. Simultaneous inhibition of p53 and TNF in Hs578T TNBC human cell line revealed a panel of up- and downregulated genes involved in apoptosis. Furthermore, the effects of double gene knockdown were validated in a second TNBC cell line, MDA-MB-231, by using reverse transcription polymerase chain reaction TaqMan assay. All our findings help in understanding the functional mechanisms of extrinsic apoptosis, cell signaling pathways, and the mechanisms involved in tumor cell survival, growth, and death in TNBC.

A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato.

Bacterial spot of tomato, a major problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hrpB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB7 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB7 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB7 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.

Comparative analysis of midgut bacterial communities in three aedine mosquito species from dengue-endemic and non-endemic areas of Rajasthan, India.

Dengue viruses are transmitted to humans through the bites of infected female aedine mosquitoes. Differences in the composition and structure of bacterial communities in the midguts of mosquitoes may affect the vector's ability to transmit the disease. To investigate and analyse the role of midgut bacterial communities in viral transmission, midgut bacteria from three species, namely Stegomyia aegypti (= Aedes aegypti), Fredwardsius vittatus (= Aedes vittatus) and Stegomyia albopicta (= Aedes albopictus) (all: Diptera: Culicidae), from dengue-endemic and non-endemic areas of Rajasthan, India were compared. Construction and analyses of six 16S rRNA gene libraries indicated that Serratia spp.-related phylotypes dominated all clone libraries of the three mosquito species from areas in which dengue is not endemic. In dengue-endemic areas, phylotypes related to Aeromonas, Enhydrobacter spp. and uncultivated bacterium dominated the clone libraries of S. aegypti, F. vittatus and S. albopicta, respectively. Diversity indices analysis and real-time TaqMan polymerase chain reaction assays showed bacterial diversity and abundance in the midguts of S. aegypti to be higher than in the other two species. Significant differences observed among midgut bacterial communities of the three mosquito species from areas in which dengue is and is not endemic, respectively, may be related to the vectorial capacity of mosquitoes to carry dengue viruses and, hence, to the prevalence of disease in some areas.

Accuracy of polymerase chain reaction-restriction fragment length polymorphism for RET rs2435357 genotyping as Hirschsprung risk

BackgroundRecently, the common RET rs2435357 variant has been shown to be strongly related to Hirschsprung disease (HSCR) in the Indonesian population. This association study was conducted in developed areas using high-throughput TaqMan polymerase chain reaction (PCR) assay. Although the TaqMan method is less time-consuming, it requires a special more expensive PCR machine and a highly skilled analyst. In this study, we analyzed the usefulness of the PCR-restriction fragment length polymorphism (RFLP) method for genotyping RET rs2435357 polymorphism in Indonesian HSCR patients given the limitation of resource allocation for more expensive technologies. Materials and methodsWe compared our previous genotyping results of RET rs2435357 in 53 HSCR patients and 86 controls using the TaqMan PCR assay with the PCR-RFLP technique. Furthermore, we included an additional 40 HSCR patients and 50 controls and subsequently genotyped all subjects using the PCR-RFLP method. ResultsCompared with our previous genotyping data of RET rs2435357 using the TaqMan PCR assay, the PCR-RFLP method indicated 100% concordant results. The overall accuracy of the PCR-RFLP for RET rs2435357 genotyping was 100%. In addition, case-control analysis demonstrated that RET rs2435357 is significantly correlated with HSCR (P = 2.2 × 10−13) with an odds ratio of 5.1 (95% confidence interval = 3.2-8.1). The transmission disequilibrium test revealed that risk allele (T) at rs2435357 is significantly overtransmitted to probands at a transmission rate (τ) of 0.87 (P = 1.5 × 10−6). ConclusionsThe PCR-RFLP method is reliable and affordable for genotyping of RET rs2435357 polymorphism in developing countries. Our results strengthen the proof that the RET rs2435357 variant is a genetic risk for HSCR in Indonesia.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina.

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.

Polymorphisms of the FOXF1 and MHC locus genes in individuals undergoing esophageal acid reflux assessments.

Gastroesophageal reflux disease (GERD) may lead to Barrett's esophagus (BE). Previously, a large genome-wide association study found two germline markers to be associated with BE, FOXF1 rs9936833 (C allele) and MHC rs9257809 (A allele). This study evaluated whether these two polymorphisms are associated with gastroesphageal acid reflux as measured by 24-hour pH testing. Patients with acid reflux symptoms referred for esophageal manometry and 24-hour pH monitoring at University Health Network (Toronto, ON) were enrolled. DNA extracted from blood was genotyped using a Taqman Polymerase Chain Reaction (PCR) assay. DeMeester scores of ≥14.7 or prior evidence of reflux esophagitis on endoscopy defined individuals with esophageal acid reflux. Logistic regression analysis, adjusted for clinical risk factors, was used to calculate odds ratios with 95% confidence intervals for each polymorphism in relation to the presence of acid reflux. Of 182 patients, the median age was 50years and 62% were female; 95 (52%) met the definition of GERD. In the multivariable analysis, both FOXF1 rs9936833 (OR = 1.82; 95%CI: 1.12-2.96; P = 0.02) and MHC rs9257809 (OR = 9.36; 95%CI: 2.92-29.99; P < 0.001) remained significantly associated with presence of acid reflux. When both polymorphisms were placed in the same model, the adjusted ORs were 2.10 (95%CI: 1.24-3.53; P = 0.005) and 10.95 (95%CI: 3.32-36.09; P < 0.001), respectively. The association for risk allele C in FOXF1 rs9936833 and risk allele A in MHC rs9257809 with the presence of acid reflux suggests a potential pathophysiologic mechanism for the role of genetic influences in BE development.

EPIDEMIOLOGY STUDY OF VIRAL NERVOUS NECROSIS (VNN) IN MALAYSIAN GROUPERS: SEQUENCE ANALYSIS STRATEGIES AND QUASISPECIES DETERMINANT’S

EPIDEMIOLOGY STUDY OF VIRAL NERVOUS NECROSIS (VNN) IN MALAYSIAN GROUPERS: SEQUENCE ANALYSIS STRATEGIES AND QUASISPECIES DETERMINANT’S