Polymerase Chain Reaction Platform Research Articles

Transmission of rhinovirus in the Utah BIG-LoVE families: Consequences of age and household structure.

BackgroundCommon cold viruses create significant health and financial burdens, and understanding key loci of transmission would help focus control strategies. This study (1) examines factors that influence when individuals transition from a negative to positive test (acquisition) or a positive to negative test (loss) of rhinovirus (HRV) and other respiratory tract viruses in 26 households followed weekly for one year, (2) investigates evidence for intrahousehold and interhousehold transmission and the characteristics of individuals implicated in transmission, and (3) builds data-based simulation models to identify factors that most strongly affect patterns of prevalence.MethodsWe detected HRV, coronavirus, paramyxovirus, influenza and bocavirus with the FilmArray polymerase chain reaction (PCR) platform (BioFire Diagnostics, LLC). We used logistic regression to find covariates affecting acquisition or loss of HRV including demographic characteristics of individuals, their household, their current infection status, and prevalence within their household and across the population. We apply generalized linear mixed models to test robustness of results.ResultsAcquisition of HRV was less probable in older individuals and those infected with a coronavirus, and higher with a higher proportion of other household members infected. Loss of HRV is reduced with a higher proportion of other household members infected. Within households, only children and symptomatic individuals show evidence for transmission, while between households only a higher number of infected older children (ages 5-19) increases the probability of acquisition. Coronaviruses, paramyxoviruses and bocavirus also show evidence of intrahousehold transmission. Simulations show that age-dependent susceptibility and transmission have the largest effects on mean HRV prevalence.ConclusionsChildren are most likely to acquire and most likely to transmit HRV both within and between households, with infectiousness concentrated in symptomatic children. Simulations predict that the spread of HRV and other respiratory tract viruses can be reduced but not eliminated by practices within the home.

Role of regulatory T cells and CD8+ T lymphocytes in the dissemination of circulating tumor cells in primary invasive breast cancer.

Circulating tumor cells (CTCs) dissemination is involved in tumor metastasis and is an independent prognostic factor in patients with primary and metastatic breast cancer. Regulatory T cells (Tregs) and cluster of differentiation (CD)8+ T lymphocytes are the main types of immune cells in the tumor microenvironment and exert opposite roles on the progression and outcome of breast cancer. The aim of the present study was to evaluate the associations between CTCs and intratumoral/peritumoral Tregs and CD8+ T lymphocytes in breast cancer. Peripheral CTCs were detected by a multi-marker quantitative polymerase chain reaction platform in 167 patients with invasive breast cancer. Intratumoral/peritumoral Tregs and CD8+ T lymphocytes were assessed by immunohistochemistry in 167 patients with invasive breast cancer to establish an association between these cell types and detection of peripheral CTCs. CTCs were detected in 55% of the patients with breast cancer. The prevalence of CTCs was positively associated with the number of intratumoral (P=0.002) and peritumoral Tregs (P=0.045), and the primary tumor size (P=0.012). This result was verified by analyzing intratumoral Tregs (P=0.044) and primary tumor size (P=0.044) with multivariate analysis, which indicated that the CTC-positive rate increased with an increasing number of intratumoral Tregs and a larger tumor size In the multivariate analysis, other variables including menopause, tumor-node-metastasis stage and peritumoral Tregs were not associated with the prevalence of CTCs. The prevalence of CTCs was inversely and weakly associated with the number of peritumoral CD8+ T lymphocytes using the univariate analysis, however this result was not statistically significant (P=0.470). In conclusion, regulatory T cells and CD8+ T lymphocytes may be involved, at least in part, in the CTCs dissemination of breast cancer, whereby Tregs appear to exert the dominant effect. Furthermore, the role of Tregs in the progression of breast cancer may be mediated by suppressing the dissemination of CTCs, which is primarily determined by intratumoral Tregs.

Pigs are useful for the molecular study of bone inflammation and regeneration in humans.

Pigs are used with increased frequency to model different kinds of orthopedic surgical conditions. In order to show the full potential of porcine models in orthopedic research, it is therefore required to examine the expression of bone regulatory genes in pigs affected by orthopedic surgery and compare it to the expression in humans and mice as mice, are one of the most applied animal species in orthopedics today. In the present study, the local molecular response to drilling of a tibial implant cavity, and the subsequent insertion of a steel implant was examined in a porcine model. Pigs were euthanized five days after drilling of the bone. The molecular response of 73 different genes was analyzed using a high-throughput quantitative polymerase chain reaction platform and compared to histopathology. Histologically, it was found that bone remodeling was initiated on day 5 after surgery and was associated with upregulation of several genes involved in bone degradation and formation ( CTSK, ACP5, IBSP, RANK, RANKL and COL1A1). Interleukin-6 and several acute-phase proteins (C3, SAA and ITIH4) were significantly upregulated, indicating their importance in the initial process of healing and osseointegration. All tested bone morphogenic proteins (BMP2, -4 and -7) including their inhibitor noggin were also significantly upregulated. Surprisingly, vascular endothelial growth factor A was not found to be regulated five days after surgery while several other vascular growth factors (ANGPT1, ANGPT2 and PTN) were upregulated. The pig was found to be a useful model for elucidation of bone regulatory genes in humans.

Bayesian evidence and epidemiological implications of environmental contamination from acute respiratory infection in long-term care facilities.

Skilled nursing home facilities (SNFs) house a vulnerable population frequently exposed to respiratory pathogens. Our study aims to gain a better understanding of the transmission of nursing home-acquired viral respiratory infections in non-epidemic settings. Symptomatic surveillance was performed in three SNFs for residents exhibiting acute respiratory symptoms. Environmental surveillance of five high-touch areas was performed to assess possible transmission. All resident and environmental samples were screened using a commercial multiplex polymerase chain reaction platform. Bayesian methods were used to evaluate environmental contamination. Among nursing home residents with respiratory symptoms, 19% had a detectable viral pathogen (parainfluenza-3, rhinovirus/enterovirus, RSV, or influenza B). Environmental contamination was found in 20% of total room surface swabs of symptomatic residents. Environmental and resident results were all concordant. Target period prevalence among symptomatic residents ranged from 5.5 to 13.3% depending on target. Bayesian analysis quantifies the probability of environmental shedding due to parainfluenza-3 as 92.4% (95% CI: 86.8-95.8%) and due to rhinovirus/enterovirus as 65.6% (95% CI: 57.9-72.5%). Our findings confirm that non-epidemic viral infections are common among SNF residents exhibiting acute respiratory symptoms and that environmental contamination may facilitate further spread with considerable epidemiological implications. Findings further emphasise the importance of environmental infection control for viral respiratory pathogens in long-term care facilities.

Teaching Molecular Microbiology: Next Generation Sequencing vs Multiplex PCR

<h3>ABSTRACT</h3> Experience in Next Generation Sequencing (NGS) is a highly desirable skill for molecular technologists. Previously, our Molecular Pathology program partnered with a local laboratory affiliate to develop a hands-on NGS educational protocol. Recently, we expanded this instructional laboratory to include the BioFire FilmArray instrument, a sample-to-answer multiplex polymerase chain reaction (PCR) platform. We used these two methods to illustrate the strengths and weaknesses of each technology and to reinforce the principles of molecular microbiology. After several classroom lectures, 18 students were given a protocol to prepare an NGS DNA library from unknown bacterial isolates. Each student performed DNA isolation, PCR of 16S rRNA, and magnetic bead PCR purification. The samples were given to the affiliate laboratory for loading and analysis on the Life Technologies Ion Torrent NGS platform. Meanwhile, the students were paired up to perform the FilmArray gastrointestinal assay on their pooled bacterial isolates. Students compared the quantitative results of NGS with the qualitative results of the FilmArray, and they were able to determine which bacteria were detected by each method as compared with the key. There was high correlation between the two methods, although some false negatives and false positives were observed for each assay. These observations were used for classroom discussions about differences in microbial DNA isolation efficiencies, test menus, analytical sensitivities, and procedural limitations of each method. In sum, the integration of the NGS and multiplex PCR methods into a unified laboratory unit provided valuable hands-on technical experience while simultaneously illustrating the concepts of molecular microbiology.

Prognostic miRNA classifier in early-stage mycosis fungoides: development and validation in a Danish nationwide study

AbstractMycosis fungoides (MF) is the most frequent form of cutaneous T-cell lymphoma. The disease often takes an indolent course, but in approximately one-third of the patients, the disease progresses to an aggressive malignancy with a poor prognosis. At the time of diagnosis, it is impossible to predict which patients develop severe disease and are in need of aggressive treatment. Accordingly, we investigated the prognostic potential of microRNAs (miRNAs) at the time of diagnosis in MF. Using a quantitative reverse transcription polymerase chain reaction platform, we analyzed miRNA expression in diagnostic skin biopsies from 154 Danish patients with early-stage MF. The patients were subdivided into a discovery cohort (n = 82) and an independent validation cohort (n = 72). The miRNA classifier was built using a LASSO (least absolute shrinkage and selection operator) Cox regression to predict progression-free survival (PFS). We developed a 3-miRNA classifier, based on miR-106b-5p, miR-148a-3p, and miR-338-3p, which successfully separated patients into high-risk and low-risk groups of disease progression. PFS was significantly different between these groups in both the discovery cohort and the validation cohort. The classifier was stronger than existing clinical prognostic factors and remained a strong independent prognostic tool after stratification and adjustment for these factors. Importantly, patients in the high-risk group had a significantly reduced overall survival. The 3-miRNA classifier is an effective tool to predict disease progression of early-stage MF at the time of diagnosis. The classifier adds significant prognostic value to existing clinical prognostic factors and may facilitate more individualized treatment of these patients.

Technical Validation of a Reverse-Transcription Quantitative Polymerase Chain Reaction In Vitro Diagnostic Test for the Determination of MiR-31-3p Expression Levels in Formalin-Fixed Paraffin-Embedded Metastatic Colorectal Cancer Tumor Specimens.

MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay.

Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection.

One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia.

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Brief Report: High Sensitivity and Specificity of the Cepheid Xpert HIV-1 Qualitative Point-of-Care Test Among Newborns in Botswana.

HIV point-of-care (POC) testing allows for early infant HIV diagnosis and treatment, but POC accuracy at birth and in the setting of antiretroviral prophylaxis for the prevention of mother-to-child HIV transmission is unknown. We evaluated the Cepheid Xpert HIV-1 Qual POC test against the Roche Taqman HIV-1 DNA polymerase chain reaction (PCR) platform using dried blood spots from 15 HIV-infected and 75 HIV-exposed uninfected newborns. These infants were screened for HIV at <96 hours of life at 5 hospital maternity wards in Botswana; all infants received postexposure antiretroviral prophylaxis with single-dose nevirapine and zidovudine, and most mothers received 3-drug antiretroviral therapy in pregnancy and at delivery. Fourteen of the 15 PCR positive samples tested positive by Cepheid POC, yielding a sensitivity of 93.3% (95% confidence interval: 68.1 to 99.8). Baseline viral load among positive infants ranged from <40 to >10,000,000 copies/mL, with a median of 2403 copies/mL. The HIV RNA for the infant with false-negative POC testing was 1661 copies/mL. Of note, 2 infants with low HIV RNA (<40 and 272 copies/mL) were correctly identified as HIV positive by Cepheid POC. All the 75 PCR-negative samples tested negative by Cepheid POC, yielding a specificity of 100% (95% confidence interval: 96.1 to 100). Our study demonstrates high sensitivity and specificity for the Cepheid POC assay in the first week of life despite early infection and antiretroviral prophylaxis. This platform may be a useful approach for adding early infant HIV diagnosis to current testing programs.

Abstract 2742: Cross-platform comparison of four leading technologies for detecting EGFR mutations in circulating tumor DNA from plasma of patients with non-small cell lung carcinoma

Abstract Analysis of circulating tumor DNA (ctDNA) is emerging as a powerful tool for guiding targeted therapy and monitoring tumor evolution in patients with non-small cell lung cancer (NSCLC), particularly when fresh tissue biopsy is not available. This study compared the ability of four leading technology platforms to detect epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, T790M and G719X) in ctDNA from NSCLC patients. The platforms included two amplification refractory mutation systems (cobas-ARMS and ADx-ARMS), a droplet digital polymerase chain reaction platform (ddPCR) and a next-generation sequencing platform (Firefly NGS). Fifteen EGFR mutations across twenty NSCLC patients were identified. We observed superior sensitivity and specificity of cobas-ARMS, ddPCR and Firefly NGS platforms, while ADx-ARMS was only suitable for the qualitative detection of EGFR mutations with allele frequency higher than 1% in plasma samples. We observed high concordance between the plasma and tissue EGFR mutational profiles for three driver mutations that are known targets of the first generation EGFR-TKI therapy (L858R, E19-dels, and G719X). Discrepancies between plasma and tissue EGFR mutational profiling could be attributed to spatial and temporal tumor heterogeneity. This pilot study illustrates the promise of ctDNA analysis in the context of treatment evaluation and drug resistance detection, and results will be validated in follow-up studies. Citation Format: Ting Xu, Xiaozheng Kang, Xiaofang You, Dai Liang, Dequan Tian, Wanpu Yan, Yongbo Yang, Hongchao Xiong, Zhen Liang, Grace Q. Zhao, Shengrong Lin, Ke-Neng Chen, Guobing Xu. Cross-platform comparison of four leading technologies for detecting EGFR mutations in circulating tumor DNA from plasma of patients with non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2742. doi:10.1158/1538-7445.AM2017-2742

Pre-Exposure Gene Expression in Baboons with and without Pancytopenia after Radiation Exposure.

Radiosensitivity differs in humans and likely among primates. The reasons are not well known. We examined pre-exposure gene expression in baboons (n = 17) who developed haematologic acute radiation syndrome (HARS) without pancytopenia or a more aggravated HARS with pancytopenia after irradiation. We evaluated gene expression in a two stage study design where stage I comprised a whole genome screen for messenger RNAs (mRNA) (microarray) and detection of 667 microRNAs (miRNA) (real-time quantitative polymerase chain reaction (qRT-PCR) platform). Twenty candidate mRNAs and nine miRNAs were selected for validation in stage II (qRT-PCR). None of the mRNA species could be confirmed during the validation step, but six of the nine selected candidate miRNA remained significantly different during validation. In particular, miR-425-5p (receiver operating characteristic = 0.98; p = 0.0003) showed nearly complete discrimination between HARS groups with and without pancytopenia. Target gene searches of miR-425-5p identified new potential mRNAs and associated biological processes linked with radiosensitivity. We found that one miRNA species examined in pre-exposure blood samples was associated with HARS characterized by pancytopenia and identified new target mRNAs that might reflect differences in radiosensitivity of irradiated normal tissue.

Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

Polymerase chain reaction (PCR) has been one of the principal techniques of molecular biology and diagnosis for decades. Conventional PCR platforms, which work by rapidly heating and cooling the whole vessel, need complicated hardware designs, and cause energy waste and high cost. On the other hand, partial heating on the various locations of vessels to induce convective solution flows by buoyancy have been used for DNA amplification in recent years. In this research, we develop a new convective PCR platform, capillary loop convective polymerase chain reaction (clcPCR), which can generate one direction flow and make the PCR reaction more stable. The U-shaped loop capillaries with 1.6 mm inner diameter are designed as PCR reagent containers. The clcPCR platform utilizes one isothermal heater for heating the bottom of the loop capillary and a CCD device for detecting real-time amplifying fluorescence signals. The stable flow was generated in the U-shaped container and the amplification process could be finished in 25 min. Our experiments with different initial concentrations of DNA templates demonstrate that clcPCR can be applied for precise quantification. Multiple sample testing and real-time quantification will be achieved in future studies.

Presence of Salmonella on the Carcass, Hide And Feces of Goats and Lambs from Major Livestock Shows in Texas Collected Over 4 Yr

ObjectivesGoats and lambs raised for exhibition at major stock shows are subjected to different management techniques than animals raised for commercial production, often leading to increased stress, travel time and feeding techniques. While these stock show animals are not a primary concern relative to the U.S. lamb and goat meat supply, they are frequently raised by children, and have increased exposure to children and families at exhibitions. Ultimately these animals also end up in the food supply system. An understanding of the role of foodborne pathogens, such as Salmonella, in the feces, hide and carcasses of these animals is valuable to reducing the risk of human illness from exposure to goats and lambs. The objective of this study was to determine presence of Salmonella on the carcass, hide and feces from show goats and lambs over 4 yr in Texas.Materials and MethodsAnimals in this study were goats or lambs exhibited at 2 large stock shows in Texas (late winter and early fall), and harvested at the Gordon W. Davis Meat Laboratory in Lubbock, Texas over a 4-yr period. Carcass swabs were taken at 3 time-points during the harvest process (pre-evisceration, post-evisceration, and after organic acid intervention was applied), and hide swabs were taken using a sterile pre-moistened sponge with 25 mL of Buffered Peptone Water. Fecal samples were collected by guiding fecal pellets from the descending colon, after evisceration, into a sterile collection cup. Carcass swabs were screened using a real-time polymerase chain reaction (PCR) platform, cultured for confirmation. Hide swabs and feces were enriched in selective media, cultured using a Xylose-Lysine-Tergitol 4 (XLT-4) agar, and subjected to latex agglutination for confirmation. Isolates from all positive samples were frozen and stored with 20% glycerol in −80°C for further analysis.ResultsFrom lambs (n = 90) and goats (n = 92), over 4 yr, hide swabs (n = 182), carcass swabs at pre-evisceration (n = 182), post-evisceration (n = 182) and post-intervention application (n = 182), and fecal samples (n = 182) were collected. Frequency of Salmonella detected from goat and lamb feces was similar (6.5% and 6.7% respectively). Salmonella on the hide was more frequently detected from lambs (16.7%) than goats (7.6%). Salmonella presence on small-ruminant (goat and lamb) carcasses was 1.1% at pre-evisceration, 3.3% at post-evisceration and 0.5% at post-intervention.ConclusionSalmonella was detected on the hide and from the feces of show goats and lambs, as well as on small-ruminant carcasses. This information confirms the presence of this pathogen, and provides data to substantiate the importance of hygiene and sanitation at livestock shows and exhibitions to protect visitors and ultimately reduce the risk of pathogen contamination on the carcasses of lambs and goats.

MicroRNA Expression for Early Prediction of Late Occurring Hematologic Acute Radiation Syndrome in Baboons.

For effective medical management of radiation-exposed persons after a radiological/nuclear event, blood-based screening measures in the first few days that could predict hematologic acute radiation syndrome (HARS) are needed. For HARS severity prediction, we used microRNA (miRNA) expression changes measured on days one and two after irradiation in a baboon model. Eighteen baboons underwent different patterns of partial or total body irradiation, corresponding to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts (BCC) the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. In a two Stage study design we screened 667 miRNAs using a quantitative real-time polymerase chain reaction (qRT-PCR) platform. In Stage II we validated candidates where miRNAs had to show a similar regulation (up- or down-regulated) and a significant 2-fold miRNA expression difference over H0. Seventy-two candidate miRNAs (42 for H1-2 and 30 for H2-3) were forwarded for validation. Forty-two of the H1-2 miRNA candidates from the screening phase entered the validation step and 20 of them showed a statistically significant 2–4 fold up-regulation relative to the unexposed reference (H0). Fifteen of the 30 H2-3 miRNAs were validated in Stage II. All miRNAs appeared 2–3 fold down-regulated over H0 and allowed an almost complete separation of HARS categories; the strongest candidate, miR-342-3p, showed a sustained and 10-fold down-regulation on both days 1 and 2. In summary, our data support the medical decision making of the HARS even within the first two days after exposure where diagnostic tools for early medical decision are required but so far missing. The miRNA species identified and in particular miR-342-3p add to the previously identified mRNAs and complete the portfolio of identified mRNA and miRNA transcripts for HARS prediction and medical management.

Matrix metalloproteinase-3 gene promoter polymorphisms: A potential risk factor for pelvic organ prolapse.

Pelvic organ prolapse (POP) is a common multifactorial condition. Matrix metalloproteinases (MMPs) are enzymes capable of breaking down various connective tissue elements. Single-nucleotide polymorphisms (SNPs) in regulatory areas of MMP-encoding genes can alter their transcription rate, and therefore the possible effect on pelvic floor supporting structures. The insertion of an adenine (A) base in the promoter of the MMP-3 gene at position −1612/−1617 produces a sequence of six adenines (6A), whereas the other allele has five (5A). The aim of the present study was to investigate the possible association of MMP-3 gene promoter SNPs with the risk of POP. The patient group comprised 80 women with clinically significant POP [Stage II, III or IV; POP quantification (POP-Q) system]. The control group consisted of 80 females without any or important pelvic floor support defects (Stages 0 or I; POP-Q system). All the participants underwent the same preoperative evaluation. SNP detection was determined with whole blood sample DNA analysis by quantitative polymerase chain reaction (PCR) in LightCycler® PCR platforms, using the technique of sequence-specific hybridization probe-binding assays and melting temperature curve analysis. The results showed there was no statistically significant difference between 5A/5A, 5A/6A and 6A/6A MMP-3 gene promoter variants in the two study groups (P=0.4758). Therefore, MMP-3 gene promoter SNPs alone is insufficient to increase the genetic susceptibility to POP development.

Detection of 23 Gastrointestinal Pathogens Among Children Who Present With Diarrhea.

Diarrheal diseases are a major cause of ambulatory care visits and hospitalizations among children. Because of overlapping signs and symptoms and expensive and inefficient testing methods, the etiology of pediatric diarrhea is rarely established. We identified children <18 years of age who were evaluated for diarrhea at Primary Children's Hospital in Salt Lake City, Utah, between October 2010 and September 2012. Stool specimens submitted for testing were evaluated by using the FilmArray gastrointestinal diagnostic system, which is a rapid multiplex polymerase chain reaction platform that can simultaneously detect 23 bacterial, viral, and protozoal agents. A pathogen was detected in 561 (52%) of 1089 diarrheal episodes. The most commonly detected pathogens included toxigenic Clostridium difficile (16%), diarrheagenic Escherichia coli (15%), norovirus GI/GII (11%), and adenovirus F 40/41 (7%). Shiga toxin-producing E coli were detected in 43 (4%) specimens. Multiple pathogens were identified in 160 (15%) specimens. Viral pathogens (norovirus, adenovirus, rotavirus, and sapovirus) were more common among children <5 years old than among those 5 to 17 years old (38% vs 16%, respectively; P < .001). Bacterial pathogens were identified most commonly in children 2 to 4 years of age. Children with 1 or more underlying chronic medical conditions were less likely to have a pathogen identified than those without a chronic medical condition (45% vs 60%, respectively; P < .01). Viral pathogens were detected more commonly in the winter, whereas bacterial pathogens were detected more commonly in the summer. Toxigenic C difficile, diarrheagenic E coli, and norovirus were the leading organisms detected among these children with diarrhea. Viral pathogens are identified frequently among young children with acute gastroenteritis.

Detection of Vaccinia virus during an outbreak of exanthemous oral lesions in Brazilian equids.

In August 2014, an outbreak of oral exanthematous disease in equids was reported in Brazil, affecting 11 donkeys and 3 mules. To investigate if Vaccinia virus (VACV) was the aetiological agent in this outbreak. Investigation of clinical cases using serological, molecular and phylogenetic approaches. To analyse the presence of neutralising antibodies against VACV, samples were submitted in triplicate to a plaque-reduction neutralisation test (PRNT50% ). On the basis of previous studies which detected VACV DNA in sera, we submitted extracted DNA samples to different polymerase chain reaction (PCR) platforms targeting Orthopoxvirus (OPV) genes (C11R, A56R and A26L). The PCR products were directly sequenced in both orientations using specific primers and capillary electrophoresis. The alignment and phylogenetic analysis of the A26L and A56R nucleotide sequences (maximum likelihood) were prepared with the obtained nucleotide fragments. Serological and molecular data suggested VACV as the aetiological agent. The neutralising antibodies against OPV were detected in 5 (55.5%) of the equids, with titres ≥40 neutralising u/ml. Based on the results obtained from all PCR platforms, all samples were positive for OPV: 9 (100%) for A56R, 4 (44.4%) for C11R and 3 (33.3%) for A26L. The alignment of the nucleotide sequences of the A26L and A56R fragments revealed that the samples were highly similar to the homologous genes from other Brazilian VACV Group 1 isolates (98.8% identity on average). Furthermore, both the A26L and A56R sequences showed signature deletions also present in the sequences of Group 1 VACV isolates from Brazil. Our data raises questions about the role of equids in the chain of VACV epidemiology. The surveillance of equids in VACV-affected areas worldwide is relevant.

Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction

MicroRNAs (miRNAs) are small noncoding RNA molecules that function in RNA silencing and posttranscriptional regulation of gene expression. miRNAs in biofluids are being used for clinical diagnosis as well as disease prediction. Efficient and reproducible isolation methods are crucial for extracellular RNA detection. To determine the best methodologies for miRNA detection from plasma, the performance of four RNA extraction kits, including an in-house kit, were determined with miScript miRNA assay technology; all were measured using a high-throughput quantitative polymerase chain reaction (qPCR) platform (BioMark System) with 90 human miRNA assays. In addition, the performances of complementary DNA (cDNA) and preamplification kits for TaqMan miRNA assays and miScript miRNA assays were compared using the same 90 miRNAs on the BioMark System. There were significant quantification cycle (Cq) value differences for the detection of miRNA targets between isolation kits. cDNA, preamplification, and qPCR performances were also varied. In summary, this study demonstrates differences among RNA isolation methods as measured by reverse transcription (RT)–qPCR. Importantly, differences were also noted in cDNA and preamplification performance using TaqMan and miScript. The in-house kit performed better than the other three kits. These findings demonstrate significant variability between isolation and detection methods for low-abundant miRNA detection from biofluids.

Association of claudin2 and PRSS1‐PRSS2 polymorphisms with idiopathic recurrent acute and chronic pancreatitis: A case–control study from India

Gene polymorphisms, including those recently described in the claudin2 gene, have been implicated in recurrent acute (RAP) and chronic pancreatitis (CP). In India, RAP and CP have been associated with SPINK1 polymorphism. In this study, we evaluated the association of claudin2 and PRSS1-PRSS2 polymorphisms with idiopathic RAP and CP. We included 101 prospectively followed patients with documented idiopathic RAP (IRAP) and 96 patients who presented with idiopathic chronic pancreatitis (ICP) without previous history of AP. Controls were 156 unrelated individuals undergoing master health check or with non-specific symptoms. All the samples were genotyped for the SNPs rs7057398 in the claudin2 (CLDN2) gene and rs10273639 in the PRSS1 gene on Realtime polymerase chain reaction platform. Clinical data pertaining to patient and disease characteristics were recorded. Claudin2 and PRSS1 polymorphisms were seen in a significantly higher proportion of female patients (P = 0.01 and 0.039, respectively). Thirty-three (32.7%) patients with IRAP developed features of early CP during follow-up (mean [95% confidence interval, CI] duration of 11.3 [8.9-13.7] months). Female patients with claudin2 (rs7057398) CC genotype were at significantly higher risk for IRAP (odds ratio [OR] [95% CI] 6.75 [1.82-23.67]; P = 0.004) and progression from IRAP to CP (OR [95% CI] 7.05 [1.51-33.01]; P = 0.007). CT genotype of PRSS1 (rs10273639) was associated IRAP (OR [95% CI] 2.59 [1.1-6.13]; P = 0.030), and both CT and CC genotypes with ICP in women (OR [95% CI] 2.86 [1.12-7.31]; P = 0.033 and 3.73 [1.03-13.59]; P = 0.048, respectively). In this study, we have demonstrated the association of claudin2 (rs7057398) polymorphism with IRAP and progression of IRAP to CP, and PRSS1 (rs10273639) polymorphism with IRAP and ICP.