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.

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 Urine-Based Molecular Diagnostic Tests for Leishmaniasis Infection in Human and Canine Populations: A Meta-Analysis

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 269
Author(s):  
Styliani A. Pappa ◽  
Panagiota I. Kontou ◽  
Pantelis G. Bagos ◽  
Georgia G. Braliou
Keyword(s):  
Dna Extraction ◽  
Diagnostic Tests ◽  
Extraction Method ◽  
Meta Analysis ◽  
Middle Eastern ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Leishmania Infection ◽  
Dna Extraction Method ◽  
The Impact

Leishmaniasis is a neglected tropical disease affecting humans and domesticated animals with high mortality in endemic countries. The pleiotropy of symptoms and the complicated gold-standard methods make the need for non-invasive, highly sensitive diagnostic tests imperative. Individual studies on molecular-based Leishmania diagnosis in urine show high discrepancy; thus, a data-evidenced comparison of various techniques is necessary. We performed a systematic review and meta-analysis using the bivariate method of diagnostic methods to pool sensitivities and specificities. We investigated the impact of DNA-extraction method, PCR type, amplified locus, host species, leishmaniasis form, and geographical region. The pooled sensitivity was 69.2%. Tests performed with the kit-based DNA extraction method and qPCR outweighed in sensitivity the phenol-chloroform-based and PCR methods, while their combination showed a sensitivity of 79.3%. Amplified locus, human or canine as host and cutaneous or visceral leishmaniasis revealed similar sensitivities. Tests in European and Middle Eastern countries performed better than tests in other regions (sensitivity 81.7% vs. 43.7%). A combination of kit-based DNA extraction and qPCR could be a safer choice for molecular diagnosis for Leishmania infection in urine samples in European–Middle Eastern countries. For the rest of the world, more studies are needed to better characterize the endemic parasite species.

scholarly journals SARS-CoV-2 Gastrointestinal Shedding in Hospitalized Children

2022 ◽  
Author(s):  
Ivy Tam ◽  
Courtney McNamara ◽  
Julie Dunbar ◽  
Katherine O’Connor ◽  
Gonzalo Manzano ◽  
...  
Keyword(s):  
Cross Sectional Study ◽  
Categorical Variables ◽  
Comorbid Condition ◽  
Cross Sectional ◽  
Exact Test ◽  
Chain Reactions ◽  
Positive Stool ◽  
Gi Symptoms ◽  
Stool Samples ◽  
Pcr Test

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a respiratory virus that can cause gastrointestinal (GI) symptoms, with studies demonstrating detection of stool viral RNA weeks after respiratory tract clearance. It is unknown if children who test negative for SARS-CoV-2 on a nasopharyngeal (NP) swab may be shedding the virus in their stool. OBJECTIVE: To measure the prevalence of SARS-CoV-2 stool shedding in children with positive and negative SARS-CoV-2 NP polymerase chain reactions (PCR) tests, and to determine clinical factors associated with GI shedding. METHODS: In this cross-sectional study, we enrolled hospitalized patients 0 to 21 years old with a positive or a negative SARS-CoV-2 NP PCR test who had respiratory and/or GI symptoms. Participants were surveyed, and stool samples were sent for viral PCR testing. Fisher’s exact test was used to evaluate bivariate associations of stool PCR test positivity with categorical variables. RESULTS: Sixty-seven patients were consented; 34 patients did not provide stool samples so 33 patients were included: 17 NP-positive and 16 NP-negative for SARS-CoV-2. Eight of the 17 NP-positive patients had a positive stool PCR test for SARS-CoV-2, while none of the 16 SARS-CoV-2 NP-negative patients had a positive result (P < .01). For the 17 SARS-CoV-2 NP-positive patients, GI symptoms were associated with a positive stool PCR test (P = .05) for SARS-CoV-2, but this association was not found for all 33 patients (P = .11). No associations were found with patients in an immunocompromised state or those with a comorbid condition, fever and/or chills, respiratory symptoms, headache and/or myalgias, or anosmia and/or ageusia. CONCLUSIONS: SARS-CoV-2 GI shedding is common and associated with GI symptoms in NP-positive children, with 47% having positive stool PCRs for SARS-CoV-2. GI shedding was not demonstrated in SARS-CoV-2 NP-negative children.

scholarly journals Evaluation of the Allplex™ GI-Helminth(I) Assay, the first marketed multiplex PCR for helminth diagnosis

Parasite ◽  
2021 ◽  
Vol 28 ◽  
pp. 33
Author(s):  
Brice Autier ◽  
Jean-Pierre Gangneux ◽  
Florence Robert-Gangneux
Keyword(s):  
Multiplex Pcr ◽  
False Negative ◽  
Extraction Process ◽  
Trichuris Trichiura ◽  
Diagnostic Methods ◽  
Bead Beating ◽  
Negative Results ◽  
Multiplex Pcr Assay ◽  
False Negative Results ◽  
Mechanical Lysis

Molecular biology has been gaining more importance in parasitology. Recently, a commercial multiplex PCR assay detecting helminths was marketed: the Allplex™ GI-Helminth(I) Assay. It targets Ancylostoma spp., Ascaris spp., Enterobius vermicularis, Hymenolepis spp., Necator americanus, Strongyloides spp., Taenia spp. and Trichuris trichiura, but also the two most common microsporidia genera in human health, i.e. Enterocytozoon spp. and Encephalitozoon spp. This study aimed to evaluate and compare the Allplex™ GI-Helminth(I) Assay to classical diagnostic methods, based on a cohort of 110 stool samples positive for helminths (microscopy) or for microsporidia (PCR). Samples were stored at −80 °C until analysis by the Allplex™ GI-Helminth(I) Assay. False-negatives were re-tested with bead-beating pretreatment. Without mechanical lysis, concordance and agreement between microscopy and Allplex™ GI-Helminth(I) Assay ranged from 91% to 100% and from 0.15 to 1.00, respectively depending on the target. Concordance was perfect for Taenia spp. (n = 5) and microsporidia (n = 10). False-negative results were observed in 54% (6/13), 34% (4/11) and 20% (7/35) of cases, for hookworms, E. vermicularis and Strongyloides spp. detection, respectively. For these targets, pretreatment improved the results, but only slightly. Trichuris trichiura detection was critically low without pretreatment, as only 9% (1/11) of the samples were positive, but detection reached 91% (10/11) with bead-beating pretreatment. Mechanical lysis was also needed for Ascaris spp. and Hymenolepis spp. to reduce false-negative results from 1/8 to 1/21, respectively, to none for both. Overall, with an optimized extraction process, the Allplex™ GI-Helminth(I) Assay allows the detection of numerous parasites with roughly equivalent performance to that of microscopy, except for hookworms.

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 Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites

Parasitology ◽  
2017 ◽  
Vol 144 (7) ◽  
pp. 965-974 ◽  
Author(s):  
MARIA M. M. KAISAR ◽  
ERIC A. T. BRIENEN ◽  
YENNY DJUARDI ◽  
ERLIYANI SARTONO ◽  
MARIA YAZDANBAKHSH ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Population Based ◽  
Trichuris Trichiura ◽  
Preparation Methods ◽  
Bead Beating ◽  
Faecal Samples ◽  
Stool Samples ◽  
Low Prevalence

SUMMARYFor the majority of intestinal parasites, real-time PCR-based diagnosis outperforms microscopy. However, the data for Trichuris trichiura have been less convincing and most comparative studies have been performed in populations with low prevalence. This study aims to improve detection of T. trichuria DNA in human stool by evaluating four sample preparation methods. Faecal samples (n = 60) were collected at Flores island, Indonesia and examined by microscopy. Aliquots were taken and a bead-beating procedure was used both on directly frozen stool and on material preserved with 96% ethanol. PCR on frozen samples showed 40% to be positive for T. trichiura, compared with 45% positive by microscopy. The percentage positive increased when using ethanol preservation (45·0%), bead-beating (51·7%) and a combination (55·0%) and all three methods showed significantly higher DNA loads. The various procedures had a less pronounced effect on the PCR results of nine other parasite targets tested. Most prevalent were Ascaris lumbricoides (≈60%), Necator americanus (≈60%), Dientamoeba fragilis (≈50%) and Giardia lamblia (≈12%). To validate the practicality of the procedure, bead-beating was applied in a population-based survey testing 910 stool samples. Findings confirmed bead-beating before DNA extraction to be a highly efficient procedure for the detection of T. trichiura DNA in stool.

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.

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