Immunological Stability ofClostridium difficileToxins in Clinical Specimens

2018 ◽  
Vol 39 (4) ◽  
pp. 434-438 ◽  
Author(s):  
Donna M. Schora ◽  
Lance R. Peterson ◽  
Elena A. Usacheva
Keyword(s):  
Clostridium Difficile ◽  
Detrimental Effect ◽  
Quantitative Polymerase Chain Reaction ◽  
Storage Conditions ◽  
Toxin Detection ◽  
Discernible Effect ◽  
Stool Specimens ◽  
Polymerase Chain ◽  
The Impact ◽  
Good Detection

OBJECTIVEThe impact of storage on stability and detection ofClostridium difficiletoxins in feces is poorly understood. The objective of this study was to investigate the immunological stability ofC. difficiletoxins in clinical stool specimens under different storage conditions by evaluating this stability using toxin detection by enzyme immunoassay (EIA).METHODSStool specimens positive forC. difficileinfection (CDI) by quantitative polymerase chain reaction (qPCR) were used for EIA testing with theC. difficileTox A/B II kit. The EIA-positive specimens were stored aerobically under refrigerated (4–10°C) and frozen (−30°C and −80°C) conditions. Measurement of toxin quantity was conducting using optical density (OD) on days 0, 14, 30, 60, 90, and 120 of storage.RESULTSClostridium difficiletoxins demonstrated good detection in undiluted stool specimens by EIA up to 120 days of storage. Good detection of the toxins was observed in diluted samples at refrigerated and −80°C temperatures. Dilution detrimentally affected toxin detection at −30°C.CONCLUSIONStorage of undiluted clinical stool specimens at refrigerated, −30°C, and −80°C temperatures for up to 120 days has no discernible effect on the immunological stability ofC. difficilecytotoxins. However, storage at −30°C has a detrimental effect onC. difficiletoxin stability in diluted specimens.Infect Control Hosp Epidemiol2018;39:434–438

scholarly journals The Changing Landscape of Pediatric Viral Enteropathogens in the Post–Rotavirus Vaccine Era

2020 ◽  
Author(s):  
Natasha Halasa ◽  
Bhinnata Piya ◽  
Laura S Stewart ◽  
Herdi Rahman ◽  
Daniel C Payne ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Medical Care ◽  
Race And Ethnicity ◽  
Quantitative Polymerase Chain Reaction ◽  
Rotavirus Vaccine ◽  
Older Children ◽  
Chain Reaction ◽  
Stool Specimens ◽  
Polymerase Chain ◽  
To Receive

Abstract Background Acute gastroenteritis (AGE) is a common reason for children to receive medical care. However, the viral etiology of AGE illness is not well described in the post–rotavirus vaccine era, particularly in the outpatient (OP) setting. Methods Between 2012 and 2015, children 15 days through 17 years old presenting to Vanderbilt Children’s Hospital, Nashville, Tennessee, with AGE were enrolled prospectively from the inpatient, emergency department, and OP settings, and stool specimens were collected. Healthy controls (HCs) were enrolled and frequency matched for period, age group, race, and ethnicity. Stool specimens were tested by means of reverse-transcription real-time quantitative polymerase chain reaction for norovirus, sapovirus, and astrovirus RNA and by Rotaclone enzyme immunoassay for rotavirus antigen, followed by polymerase chain reaction verification of antigen detection. Results A total of 3705 AGE case patients and 1563 HCs were enrolled, among whom 2885 case patients (78%) and 1110 HCs (71%) provided stool specimens that were tested. All 4 viruses were more frequently detected in AGE case patients than in HCs (norovirus, 22% vs 8%, respectively; rotavirus, 10% vs 1%; sapovirus, 10% vs 5%; and astrovirus, 5% vs 2%; P < .001 for each virus). In the OP setting, rates of AGE due to norovirus were higher than rate for the other 3 viruses. Children <5 years old had higher OP AGE rates than older children for all viruses. Conclusions Norovirus remains the most common virus detected in all settings, occurring nearly twice as frequently as the next most common pathogens, sapovirus and rotavirus. Combined, norovirus, sapovirus, rotavirus, and astrovirus were associated with almost half of all AGE visits and therefore are an important reason for children to receive medical care.

Mo1852 The Impact of Polymerase Chain Reaction on Clostridium difficile Detection and Clinical Outcomes

2015 ◽  
Vol 148 (4) ◽  
pp. S-726-S-727
Author(s):  
Mona Akbari ◽  
Victor Novack ◽  
Ciaran P. Kelly ◽  
Daniel A. Leffler
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Difficile ◽  
Clinical Outcomes ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
The Impact

scholarly journals TEL-AML1 regulation of survivin and apoptosis via miRNA-494 and miRNA-320a

Blood ◽  
2010 ◽  
Vol 116 (23) ◽  
pp. 4885-4893 ◽  
Author(s):  
Christofer Diakos ◽  
Sheng Zhong ◽  
Yuanyuan Xiao ◽  
Mi Zhou ◽  
Gisele M. Vasconcelos ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Mirna Expression ◽  
Childhood Leukemia ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Survivin Expression ◽  
Bioinformatic Analysis ◽  
Expression Arrays ◽  
Polymerase Chain ◽  
The Impact

Abstract There is increasing evidence that miRNA and transcription factors interact in an instructive fashion in normal and malignant hematopoiesis. We explored the impact of TEL-AML1 (ETV6-RUNX1), the most common fusion protein in childhood leukemia, on miRNA expression and the leukemic phenotype. Using RNA interference, miRNA expression arrays, and quantitative polymerase chain reaction, we identified miRNA-494 and miRNA-320a to be up-regulated upon TEL-AML1 silencing independently of TEL expression. Chromatin immunoprecipitation analysis identified miRNA-494 as a direct miRNA target of the fusion protein TEL-AML1. Using bioinformatic analysis as well as functional luciferase experiments, we demonstrate that survivin is a target of the 2 miRNAs. miRNA-494 and miRNA-320a were introduced to the cells by transfection and survivin expression determined by Western blot analysis. These miRNAs blocked survivin expression and resulted in apoptosis in a similar manner as TEL-AML1 silencing by itself; this silencing was also shown to be Dicer-dependent. miRNAs-494 and -320a are expressed at lower levels in TEL-AML1+ leukemias compared with immunophenotype-matched nonTEL-AML1 acute lymphoblastic leukemia subtypes, and within TEL-AML1+ leukemias their expression is correlated to survivin levels. In summary our data suggest that TEL-AML1 might exert its antiapoptotic action at least in part by suppressing miRNA-494 and miRNA-320a, lowering their expression causing enhanced survivin expression.

scholarly journals Covid-19 deaths in Africa: prospective systematic postmortem surveillance study

BMJ ◽  
2021 ◽  
pp. n334 ◽  
Author(s):  
Lawrence Mwananyanda ◽  
Christopher J Gill ◽  
William MacLeod ◽  
Geoffrey Kwenda ◽  
Rachel Pieciak ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Tertiary Care ◽  
Threshold Value ◽  
University Teaching ◽  
Surveillance Study ◽  
Polymerase Chain ◽  
The University ◽  
The Impact ◽  
Deceased People ◽  
Underlying Risk

Abstract Objective To directly measure the fatal impact of coronavirus disease 2019 (covid-19) in an urban African population. Design Prospective systematic postmortem surveillance study. Setting Zambia’s largest tertiary care referral hospital. Participants Deceased people of all ages at the University Teaching Hospital morgue in Lusaka, Zambia, enrolled within 48 hours of death. Main outcome measure Postmortem nasopharyngeal swabs were tested via reverse transcriptase quantitative polymerase chain reaction (PCR) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Deaths were stratified by covis-19 status, location, age, sex, and underlying risk factors. Results 372 participants were enrolled between June and September 2020; PCR results were available for 364 (97.8%). SARS-CoV-2 was detected in 58/364 (15.9%) according to the recommended cycle threshold value of <40 and in 70/364 (19.2%) when expanded to any level of PCR detection. The median age at death among people with a positive test for SARS-CoV-2 was 48 (interquartile range 36-72) years, and 69% (n=48) were male. Most deaths in people with covid-19 (51/70; 73%) occurred in the community; none had been tested for SARS-CoV-2 before death. Among the 19/70 people who died in hospital, six were tested before death. Among the 52/70 people with data on symptoms, 44/52 had typical symptoms of covid-19 (cough, fever, shortness of breath), of whom only five were tested before death. Covid-19 was identified in seven children, only one of whom had been tested before death. The proportion of deaths with covid-19 increased with age, but 76% (n=53) of people who died were aged under 60 years. The five most common comorbidities among people who died with covid-19 were tuberculosis (22; 31%), hypertension (19; 27%), HIV/AIDS (16; 23%), alcohol misuse (12; 17%), and diabetes (9; 13%). Conclusions Contrary to expectations, deaths with covid-19 were common in Lusaka. Most occurred in the community, where testing capacity is lacking. However, few people who died at facilities were tested, despite presenting with typical symptoms of covid-19. Therefore, cases of covid-19 were under-reported because testing was rarely done not because covid-19 was rare. If these data are generalizable, the impact of covid-19 in Africa has been vastly underestimated.

scholarly journals A Practical Approach for Quantitative Polymerase Chain Reaction, the Gold Standard in Microbiological Diagnosis

2021 ◽  
Author(s):  
Tímea Mosolygó ◽  
Krisztián Laczi ◽  
Gabriella Spengler ◽  
Katalin Burián
Keyword(s):  
Polymerase Chain Reaction ◽  
Undergraduate Students ◽  
Quantitative Polymerase Chain Reaction ◽  
Real Life ◽  
Medical Diagnostics ◽  
Microbiological Diagnosis ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Gene Expression Studies ◽  
The Impact

From gene expression studies to identifying microbes quantitative polymerase chain reaction (qPCR) is widely used in research and medical diagnostics. In transmittable diseases like the Ebola outbreak in West Africa (2014-2016), or the present SARS-CoV2 pandemic qPCR plays a key role in the detection of infected patients. Although the technique itself is decades old with reliable approaches (eg. TaqMan essay) in the diagnosis of pathogens many people showed distrust in it during the SARS-CoV2 outbreak. This came mainly from not understanding or misunderstanding the principles of qPCR. This situation motivated us to design a simple laboratory practical class, in which students have opportunities to understand the underlying principles of qPCR and its advantages in microbiological diagnosis. Moreover, during the exercise, students can develop skills such as handling experimental assays, and the ability to solve problems, discuss their observations. Finally, this activity brings them closer to the clinical practice and they can see the impact of the science on real life. The class is addressed to undergraduate students of biological sciences.

scholarly journals Propidium Monoazide Improves Quantification of Resting Spores of Plasmodiophora brassicae with qPCR

Plant Disease ◽  
2017 ◽  
Vol 101 (3) ◽  
pp. 442-447 ◽  
Author(s):  
Fadi Al-Daoud ◽  
Bruce D. Gossen ◽  
Justin Robson ◽  
Mary Ruth McDonald
Keyword(s):  
Heat Treatment ◽  
Plasmodiophora Brassicae ◽  
Quantitative Polymerase Chain Reaction ◽  
Infested Soil ◽  
Propidium Monoazide ◽  
Spore Viability ◽  
Heat Treated ◽  
Resting Spores ◽  
Polymerase Chain ◽  
The Impact

Plasmodiophora brassicae, which causes clubroot of Brassica crops, persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) analysis is often used to quantify resting spores but does not distinguish between DNA of viable and nonviable spores. The impact of pretreating spores with propidium monoazide (PMA), which inhibits amplification of DNA from nonviable microorganisms, was assessed in several experiments. Spore suspensions from immature and mature clubs were heat treated; then, PMA-PCR analyses and bioassays were performed to assess spore viability. Prior to heat treatment, assessments comparing PMA-PCR to qPCR for mature spores were similar, indicating that most of these spores were viable. However, only a small proportion (<26%) of immature spores were amplified in PMA-PCR. Bioassays demonstrated that clubroot severity was much higher in plants inoculated with mature spores than with immature spores. Heat treatment produced little or no change in estimates of mature spores from qPCR but spore estimates from PMA-PCR and clubroot severity in bioassays were both substantially reduced. Estimates of spore concentration with PMA-PCR were less consistent for immature spores. To facilitate use of PMA-PCR on infested soil, a protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard methods.

scholarly journals Storage procedures and time influence the detectability of Clostridium difficile toxin A but not toxin B in porcine fecal specimens

2019 ◽  
Vol 32 (2) ◽  
pp. 222-225
Author(s):  
Łukasz Grześkowiak ◽  
Jonathan Riedmüller ◽  
Wilfried Vahjen ◽  
Jürgen Zentek
Keyword(s):  
Clostridium Difficile ◽  
Storage Conditions ◽  
Toxin A ◽  
Toxin B ◽  
Clostridium Difficile Toxin ◽  
Clostridium Difficile Toxin A ◽  
Swine Feces ◽  
C 30 ◽  
The Impact

Storage procedures are known to affect the detectability of Clostridium difficile toxins in equine and human feces. We assessed the impact of different storage conditions on the detectability of C. difficile toxins in swine feces. Specimens were inoculated with toxins, 112 ng/g of toxin A (TcdA) and 16 ng/g of toxin B (TcdB) and subjected to the following 3 storage treatments: 4°C, −30°C, repetitive freezing at −30°C and thawing. Toxin determination was assessed at 1, 2, 7, 14, and 21 d with ELISA. A decrease in concentrations of TcdA with time was observed for samples stored at 4°C and repetitive freezing–thawing ( p ≤0.05). On day 14, storage at 4°C resulted in decreased TcdA concentration as opposed to storage at −30°C and repetitive freezing–thawing ( p ≤0.05). On day 21, storage at 4°C resulted in decreased TcdA detectability compared with storage at −30°C ( p ≤0.05). The TcdB concentration was unaffected. These results on toxin detectability in swine feces should be carefully considered in in vitro studies on toxigenic C. difficile. Our results also offer valuable information for microbiologists and veterinarians monitoring the presence of virulent C. difficile in pigs.

scholarly journals Study of the preanalytical variables affecting the measurement of clinically relevant free-circulating microRNAs: focus on sample matrix, platelet depletion, and storage conditions

2020 ◽  
Vol 30 (1) ◽  
pp. 83-95 ◽  
Author(s):  
Martina Faraldi ◽  
Veronica Sansoni ◽  
Giovanni Lombardi ◽  
Giuseppe Banfi ◽  
Ewa Ziemann ◽  
...  
Keyword(s):  
Room Temperature ◽  
Quantitative Polymerase Chain Reaction ◽  
Venous Blood ◽  
Storage Conditions ◽  
Circulating Micrornas ◽  
Sample Matrix ◽  
Polymerase Chain ◽  
And Storage ◽  
Plasma Mirnas ◽  
Platelet Depletion

Introduction: Circulating microRNAs (miRNAs) are emerging as potential biomarkers. However, the lack of preanalytical and analytical standardization limits their use. The aim of this study was to determine the expression of different miRNAs in plasma according to different collection and storage conditions. Materials and methods: Venous blood from 10 volunteers was collected in tubes spray-coated with dipotassium salt of ethylendiaminetetraacetic acid, either with (plasma-preparation tube, PPT) or without (K2EDTA) gel separator. Platelet-poor plasma (PPP) was also obtained from K2EDTA plasma. After storage under different conditions, miRNA-enriched total RNA was isolated from plasma and reverse transcribed. A panel of 179 miRNAs was assayed by quantitative polymerase chain reaction and the results were analysed by GenEx software. Detectability and stability of miRNAs were determined. Results: The number of undetected miRNAs was: 18, 24, and 22 in PPT; 83, 43, and 20 in K2EDTA; and 76, 106, and 104 in PPP samples, for plasma immediately frozen at - 80°C and plasma stored for 24h at room temperature or 4°C, respectively. Circulating miRNA expression in PPT samples was not affected by storage delay or temperature, while the percentage of up- and down-regulated miRNA in K2EDTA and PPP samples ranged from 2%, and 1% to 7%, and 5%, respectively. Conclusions: Sample matrix, temperature and delay in storage strongly influence the expression level of plasma miRNAs. Our results indicate PPT tubes as the most suitable matrix to improve total miRNA detectability and stability, independently of temperature.

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