scholarly journals Effect of Incorporation of Bead-Beating during DNA Extraction for Detection of Trichuris trichiura in Stool Samples in Community Settings: A Systematic Review

2021 ◽  
Author(s):  
Divya Rana ◽  
Nischal Pokhrel
Keyword(s):  
Dna Extraction ◽  
Odds Ratio ◽  
Quantitative Polymerase Chain Reaction ◽  
Risk Of Bias ◽  
Community Settings ◽  
Real Patient ◽  
Bead Beating ◽  
Helminth Eggs ◽  
Stool Samples ◽  
Google Search

Objectives: This meta-analysis was designed to assess the effect of addition of a bead-beating step during DNA extraction to effectively isolate Trichuris trichura DNA for quantitative Polymerase Chain Reaction (qPCR)-based diagnosis. Abstract was reported according to PRISMA-DTA abstract checklist. Methods: Eligibility criteria: qPCR-based molecular studies comparing the inclusion of bead-beating step during the DNA extraction from stool samples with extraction without the step were included in the analysis. Information sources: Studies using real patient samples in community settings were included. PubMed and Google search engine were searched in December 2019. Risk of bias and applicability: Risk of bias and applicability were assessed using QUADAS-2 checklist. Synthesis of results: Odds ratio for individual studies were combined to estimate Random Effects Model odds ratio. Additional literature were searched to discuss biochemical nature of helminth eggs. Results:Included studies: A total of six independent sub-studies were gathered from two published original articles. Division of the two major studies into six sub-studies was indispensable due to natures of the study carried. 128 of total 192 samples (in all studies) were positive for Trichiuris trichiura when bead-beating was used during DNA extraction compared to 108/192 when bead-beating was excluded. Combined odds ratio was 1.66 (95% CI: 1.059 to 2.602). Biochemical nature of helminth eggs was discussed. Discussions: Strengths and limitations: Though only two article were included in the study, six exclusive individual sub-studies were analyzed. Inherent differences in the background prevalence of helminth in study population could impact sensitivity of qPCR. Interpretation: It was found that the inclusion of the bead-beating step during DNA extraction significantly increased the sensitivity of the test.

scholarly journals Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Beatrice Barda ◽  
Christian Schindler ◽  
Rahel Wampfler ◽  
Shaali Ame ◽  
Said M. Ali ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Controlled Trial ◽  
Bead Beating ◽  
Novel Treatments ◽  
Two Samples ◽  
Stool Samples ◽  
Pcr Method

Abstract Background Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections. Results Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using “bead-beating” for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively. Conclusions In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the “bead-beating protocol” should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

scholarly journals Effect of Bead-Beating on DNA Extraction from Trichuris trichiura Positive Stool Samples: A Meta-Analysis

2020 ◽  
Author(s):  
Divya Rana ◽  
Nischal Pokhrel
Keyword(s):  
Dna Extraction ◽  
Meta Analysis ◽  
Trichuris Trichiura ◽  
Diagnostic Methods ◽  
Accurate Estimation ◽  
Chain Reactions ◽  
Bead Beating ◽  
Polymerase Chain Reactions ◽  
Positive Stool ◽  
Stool Samples

Polymerase chain reactions are helpful diagnostic methods to accurately quantitate the intensities of infections of various soil-transmitted helminths, especially in the low-intensity infection samples. Method of DNA extraction hugely impacts the outcomes of the diagnostic method. Trichuris trichiura is one of the three major helminths prevalent world-wide and accurate estimation of their loads in stool is affected by the method of DNA extraction. We meta-analyze two studies by dividing them into all together 6 sub-studies. The objectives of the meta-analysis required the two studies to be divided into sub-studies as the different methods of DNA extractions could not be combined. We found that the inclusion of the bead-beating step during DNA extraction significantly increases the sensitivity of the test.

Comparison of DNA Extraction Efficiency and Reproducibility of Different Aeration Diffuser Biofilms Using Bead-Beating Protocol

2018 ◽  
Vol 28 (6) ◽  
pp. 293-304
Author(s):  
Pitiporn Asvapathanagul ◽  
Manel Garrido-Baserba ◽  
Betty H. Olson ◽  
Hee-Deung Park ◽  
Deqiang Chen ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Efficiency ◽  
Quantitative Polymerase Chain Reaction ◽  
E Coli ◽  
Bead Beating ◽  
Copy Numbers ◽  
Total Dna ◽  
Polymerase Chain ◽  
Gordonia Amarae

An existing bead-beating DNA extraction protocol was employed to compare the DNA extraction recovery and fragment quality of 6 different aeration diffuser biofilms. <i>Escherichia coli</i>, <i>Gordonia amarae</i>, and mixed liquor were used as controls. The fraction of total DNA<sub>biofilm</sub> decreased monotonically with increasing number of beat beatings (BB) when the amount of DNA present was sufficient (&#x3e;4 μg<sub>DNA</sub>/cm<sup>2</sup>), excluding the ceramic disk. While controls required only 2 BBs, 3 out of 5 BBs achieved ≥70% of total DNA (70.3 ± 1.7%) for 5 out of 6 biofilms. Quantitative polymerase chain reaction (PCR) analyses of 353 and 1,505 basepair (bp) amplicons from pure culture extracts showed target copy numbers were not degraded for the first 2 BBs, but the third BB decreased amplicon concentrations by 0.65 and 1.12 log for <i>E. coli</i>, and 0.39 and 0.40 log for <i>G. amarae</i>, respectively. The 353 bp fragment amplification from biofilm samples showed minimal degradation for the first 3 BBs. PCR and gel electrophoresis confirmed integrity of amplified 1,505 bp DNA fragments over the 5 BBs, except in the EDPM (75 mm diameter, tube) diffuser biofilm (4.98 ± 0.62 μg<sub>DNA</sub>/cm<sup>2</sup>). Taken together, this study showed type of diffuser membrane biofilms had no effects on extraction efficiency, but low DNA concentrations reduced extraction performance.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Detectability vs. time and costs in pooled DNA extraction of cutaneous swabs: a study on the amphibian chytrid fungi

2019 ◽  
Vol 40 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Joana Sabino-Pinto ◽  
E. Tobias Krause ◽  
Molly C. Bletz ◽  
An Martel ◽  
Frank Pasmans ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Pathogen Detection ◽  
Batrachochytrium Dendrobatidis ◽  
Quantitative Polymerase Chain Reaction ◽  
Large Fraction ◽  
Work Load ◽  
Detection Sensitivity ◽  
Pooled Dna ◽  
Reaction Volumes ◽  
User Friendly

Abstract Epidemiology relies on understanding the distribution of pathogens which often can be detected through DNA-based techniques, such as quantitative Polymerase Chain Reaction (qPCR). Typically, the DNA of each individual sample is separately extracted and undergoes qPCR analysis. However, when performing field surveys and long-term monitoring, a large fraction of the samples is generally expected to be negative, especially in geographical areas still considered free of the pathogen. If pathogen detection within a population – rather than determining its individual prevalence – is the focus, work load and monetary costs can be reduced by pooling samples for DNA extraction. We test and refine a user-friendly technique where skin swabs can be pooled during DNA extraction to detect the amphibian chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans (Bsal). We extracted pools with different numbers of samples (from one to four swabs), without increasing reaction volumes, and each pool had one sample inoculated with a predetermined zoospore amount. Pool size did not reduce the ability to detect the two fungi, except if inoculated with extremely low zoospore amounts (one zoospore). We confirm that pooled DNA extraction of cutaneous swabs can substantially reduce processing time and costs without minimizing detection sensitivity. This is of relevance especially for the new emerging pathogen Bsal, for which pooled DNA extraction had so far not been tested and massive monitoring efforts in putatively unaffected regions are underway.

scholarly journals Identification of Interorganellar Crosstalk Effect on Various Diseases, Including Kidney Disease: A Systematic Review and Meta-analysis

2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Fateme Shamekhi Amiri
Keyword(s):  
Relative Risk ◽  
Odds Ratio ◽  
Meta Analysis ◽  
Risk Of Bias ◽  
Data Sources ◽  
Cellular Functions ◽  
Pubmed Central ◽  
Subcellular Organelles ◽  
Age Related ◽  
Health And Disease

Context: Subcellular organelles communicate with each other via their metabolites and maintain different cellular functions. They contain nucleus, mitochondria, endoplasmic reticulum, peroxisomes, and lysosomes. Objectives: This study aimed to identify interorganellar communication (crosstalk) in physiopathological states of cells in health and disease. Data Sources: The databases including PubMed Central, Embase, Scopus, and Google Scholar were searched to extract data. For statistical analyses, percentage, relative risk, and odds ratio were used. Moreover, the risk of bias was assessed by Cochrane collaboration’s tool. Results: Out of 20 studies included in this research, 12 (60%) studies included mitochondria-ER communication, 4 (20%) studies mitochondria-lysosome communication, 2 (10%) studies mitochondria-peroxisome, and 2 (10%) studies mitochondria-nucleus. Interorganellar crosstalk between mitochondria and peroxisome or lysosome had risk and odds of 1.5 (effect) on aging and age-related disorders. There were no effects of mitochondrial communication with other organelles on certain pathologies. The relative risk of mitochondria to nucleus crosstalk on apoptosis was assessed 1.13, and relative risk of mitochondria to lysosome crosstalk was assessed 2. In addition, the odds ratio of mitochondria to lysosome crosstalk on apoptosis was assessed 5, indicating a large effect on this crosstalk. Conclusions: Recent expansion of pharmacological, molecular, and genetic tools indicated these organelles have active intracellular and extracellular communications, which is important for cells and organ homeostasis. Disruption of such communication has been associated with aging and age-related disorders in this research.

scholarly journals Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples

Diagnostics ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 36 ◽  
Author(s):  
Sören Hansen ◽  
Marco Roller ◽  
Lamia Alslim ◽  
Susanne Böhlken-Fascher ◽  
Kim Fechner ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Mycobacterium Avium ◽  
Extraction Method ◽  
Magnetic Beads ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Rapid Identification ◽  
Cold Chain ◽  
Silica Membrane ◽  
Molecular Assays ◽  
Stool Samples

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from “sample in” to “result out” was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

Public health implications of soil contaminated with helminth eggs in the metropolis of Kaduna, Nigeria

2008 ◽  
Vol 82 (2) ◽  
pp. 113-118 ◽  
Author(s):  
B.V. Maikai ◽  
J.U. Umoh ◽  
O.J. Ajanusi ◽  
I. Ajogi
Keyword(s):  
Odds Ratio ◽  
Economic Status ◽  
Soil Samples ◽  
Sample Collection ◽  
Dry Period ◽  
Rainy Period ◽  
Socio Economic Status ◽  
Cultural Variables ◽  
Dipylidium Caninum ◽  
Helminth Eggs

AbstractEnvironmental and socio-cultural variables influencing the distribution of helminth eggs in 608 soil samples were studied in 14 playgrounds that differ in socio-economic status in Kaduna metropolis, Nigeria, using a modified sieving method and a sucrose flotation medium of specific gravity 1.27. Helminth eggs were found in 62% of the soil samples and the distribution was as follows: Toxocara spp. 50.4%, Taenia spp./Echinococcus spp. 36.9%, Dipylidium caninum 26.3%, Ancylostoma spp. 9.0%, Ascaris spp. 7.2%, Trichuris spp. 3.7% and Ascaridia spp. 1.9%. A higher prevalence (68.1%) was recorded during the dry harmattan period while in the rainy period the rate was 58.1%. Mean egg densities ranged from 1.11 ± 0.32 to 3.92 ± 2.47 in areas moderately rated. Samples from site 14, which was highly rated, were more contaminated (78.1%) than those collected from other sites, while the intensity of contamination (14.0%) was more in moderately rated site 4 than in the rest of the sites. There were significant associations between the prevalence of helminth eggs and rainy period of the study (odds ratio (OR) = 0.38; 95% confidence interval (CI) on OR: 0.20 < OR < 0.70), presence of dogs (OR = 0.56; 95% CI on OR: 0.37 < OR < 0.85) and grass (vegetation) (OR = 1.44; 95% CI on OR: 1.03 < OR < 2.04) in the sites. On the other hand, there was no association between the prevalence of helminth eggs and the dry period of the study, presence of refuse in the playgrounds, topography of playgrounds, depth of sample collection and socio-economic status of people in playgrounds (P>0.05). This study shows that the period of study, the presence of dogs and vegetation influence the prevalence of helminth eggs in soil in Kaduna metropolis.

scholarly journals Association of Picornavirus Infections With Acute Otitis Media in a Prospective Birth Cohort Study

2020 ◽  
Vol 222 (2) ◽  
pp. 324-332
Author(s):  
Elina M Seppälä ◽  
Sami Oikarinen ◽  
Jussi P Lehtonen ◽  
Subas Neupane ◽  
Hanna Honkanen ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Birth Cohort ◽  
Otitis Media ◽  
Odds Ratio ◽  
Adjusted Odds Ratio ◽  
Quantitative Polymerase Chain Reaction ◽  
Population Attributable Risk ◽  
Acute Otitis Media ◽  
Attributable Risk ◽  
Prospective Birth Cohort

Abstract Background Human rhinoviruses (HRVs), human enteroviruses (HEVs) and human parechoviruses (HPeVs) have been linked to acute otitis media (AOM). We evaluated this association in a prospective birth cohort setting. Methods A total of 324 healthy infants were followed up from birth to age 3 years. Nasal swab samples were collected at age 3, 6, 12, 18, 24, and 36 months and screened for HRV and HEV using real-time reverse-transcription quantitative polymerase chain reaction. Stool samples were collected monthly and analyzed for HRV, HEV, and HPeV. AOM episodes diagnosed by physicians were reported by parents in a diary. The association of viruses with AOM was analyzed using generalized estimation equations, and their relative contributions using population-attributable risk percentages. Results A clear association was found between AOM episodes and simultaneous detection of HEV (adjusted odds ratio for the detection of virus in stools, 2.04; 95% confidence interval, 1.06–3.91) and HRV (1.54; 1.04–2.30). HPeV showed a similar, yet nonsignificant trend (adjusted odds ratio, 1.44; 95% confidence interval, .81–2.56). HRV and HEV showed higher population-attributable risk percentages (25% and 20%) than HPeV (11%). Conclusions HEVs and HRVs may contribute to the development of AOM in a relatively large proportion of cases.

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