Pooling samples for hepatitis C RNA detection.
Ontology highlight
ABSTRACT:
SUBMITTER: Aguilera A
PROVIDER: S-EPMC8266286 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Virulent ovine foot rot is a contagious foot disease. Given the development and validation of a real-time PCR to detect Dichelobacter nodosus isolates that contain the virulence-associated protease genes aprV2 and aprB2, the diagnosis of foot rot has made considerable progress. We evaluated pooling methods to reduce the number of samples during a foot rot control program. Samples of individual feet were compared to a 4-feet sample of the same sheep. All further analyses based on 4-feet samples (pools-of-5 and pools-of-10 4-feet samples) were compared to samples of individual sheep, and a risk-based herd sampling was evaluated and compared to the whole flock. The sensitivity and specificity of the 4-feet samples for detection of aprV2-positive strains was 93.8% (CI: 87.6-97.5%) and 98.3% (CI: 96.5-99.3%), respectively. The sensitivity and specificity of the pools-of-10 was 86.7% (CI: 78.4-92.7%) and 100.0% (CI: 97.4-100%), respectively. Pools-of-5 were not significantly more sensitive than pools-of-10. The pooling of 4 individual foot samples into one 4-feet sample is an adequate method to reduce the number of samples of individual sheep. The sensitivity of pools-of-5 and pools-of-10 is too imprecise for a control program. Risk-based sampling allowed for a substantial reduction of samples to be tested, had a sensitivity of 95.8% (CI: 78.9-99.9%) and specificity of 100.0% (CI: 88.1-100.0%) when determining the foot rot flock status, and represents an adequate methodology to predict within-flock freedom from infection.
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Project description:Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g(av), where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10(4) [range, 7.9 x 10(2) to 1.0 x 10(6)] versus 2.3 x 10(4) [range, 4.0 x 10(2) to 2.2 x 10(5)]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.
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Project description:Human hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. In Italy, the true burden of HEV infection is unknown. Molecular HEV screening of raw sewage samples from 11 wastewater treatment plants yielded 19 positives (16%; 18 genotype I, 1 genotype III) evenly distributed throughout Italy. Evidence that HEV could be establishing itself in our region is accumulating and may justify more active surveillance to monitor its spread.
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Project description:PurposeThe pandemic of SARS-CoV-2 or COVID-19 has hugely created an economic imbalance worldwide. With the exponential increase in the number of cases and to keep in check on the community transmission, there is high demand and acute shortage of diagnostic kits. The pooled-sample strategy turns out to be the promising strategy intended to determine the optimal testing for specimens with limited resources and without losing the test sensitivity and specificity. The study was performed with standard molecular biology graded lab equipment, FDA-approved COVID-19 RNA extraction, and SARS-CoV-2 tests kits.Materials and methodsThe study aims to comparatively analyze the pooling strategy of the naso-oropharyngeal specimen sample and RNA extracted from the same patient samples in the pool of 3,5, and 8 with no significant loss in test usability. Another primary focus of the study was detection of low or borderline SARS-CoV-2 positives in the pooling strategy. A total of 300 samples (240 positives and 60 negatives) were tested for 3, 5, and 8 pools of specimen samples and RNA elutes.ResultsThe comparative analysis determined the sensitivity for three and five pool strategy to be above 98% and eight pool strategy to be 100%.ConclusionThe RNA elutes pooling strategy concordance rate is better than that of specimen pooling with 100% specificity. Thus, in the substantial crisis of resources with the global pandemic, pooling approaches for SARS-CoV-2 can be practical in a low prevalence rate of 5%.
| S-EPMC9870240 | biostudies-literature
Project description:BackgroundTypically, pooling of mRNA samples in microarray experiments implies mixing mRNA from several biological-replicate samples before hybridization onto a microarray chip. Here we describe an alternative smart pooling strategy in which different samples, not necessarily biological replicates, are pooled in an information theoretic efficient way. Further, each sample is tested on multiple chips, but always in pools made up of different samples. The end goal is to exploit the compressibility of microarray data to reduce the number of chips used and increase the robustness to noise in measurements.ResultsA theoretical framework to perform smart pooling of mRNA samples in microarray experiments was established and the software implementation of the pooling and decoding algorithms was developed in MATLAB. A proof-of-concept smart pooled experiment was performed using validated biological samples on commercially available gene chips. Differential-expression analysis of the smart pooled data was performed and compared against the unpooled control experiment.ConclusionsThe theoretical developments and experimental demonstration in this paper provide a useful starting point to investigate smart pooling of mRNA samples in microarray experiments. Although the smart pooled experiment did not compare favorably with the control, the experiment highlighted important conditions for the successful implementation of smart pooling - linearity of measurements, sparsity in data, and large experiment size.
| S-EPMC2900278 | biostudies-literature
Project description:We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed.
| S-EPMC4390636 | biostudies-literature
Project description:Over 15% of the data sets catalogued in the Gene Expression Omnibus Database involve RNA samples that have been pooled before hybridization. Pooling affects data quality and inference, but the exact effects are not yet known because pooling has not been systematically studied in the context of microarray experiments. Here we report on the results of an experiment designed to evaluate the utility of pooling and the impact on identifying differentially expressed genes. We find that inference for most genes is not adversely affected by pooling, and we recommend that pooling be done when fewer than three arrays are used in each condition. For larger designs, pooling does not significantly improve inferences if few subjects are pooled. The realized benefits in this case do not outweigh the price paid for loss of individual specific information. Pooling is beneficial when many subjects are pooled, provided that independent samples contribute to multiple pools.
| S-EPMC552978 | biostudies-literature
Project description:Prevalence is a common epidemiological measure for assessing soil-transmitted helminth burden and forms the basis for much public-health decision-making. Standard diagnostic techniques are based on egg detection in stool samples through microscopy and these techniques are known to have poor sensitivity for individuals with low infection intensity, leading to poor sensitivity in low prevalence populations. PCR diagnostic techniques offer very high sensitivities even at low prevalence, but at a greater cost for each diagnostic test in terms of equipment needed and technician time and training. Pooling of samples can allow prevalence to be estimated while minimizing the number of tests performed. We develop a model of the relative cost of pooling to estimate prevalence, compared to the direct approach of testing all samples individually. Analysis shows how expected relative cost depends on both the underlying prevalence in the population and the size of the pools constructed. A critical prevalence level (approx. 31%) above which pooling is never cost effective, independent of pool size. When no prevalence information is available, there is no basis on which to choose between pooling and testing all samples individually. We recast our model of relative cost in a Bayesian framework in order to investigate how prior information about prevalence in a given population can be used to inform the decision to choose either pooling or full testing. Results suggest that if prevalence is below 10%, a relatively small exploratory prevalence survey (10-15 samples) can be sufficient to give a high degree of certainty that pooling may be relatively cost effective.
| S-EPMC6445468 | biostudies-literature
Project description:BackgroundSARS-CoV-2 testing demand has outpaced its supply. Pooling samples for lower risk populations has the potential to accommodate increased demand for SARS-CoV-2 molecular testing.ObjectiveTo evaluate the sensitivity, specificity, and reproducibility of 4-way pooling of SARS-CoV-2 specimens for high-throughput RT-PCR.Study designIndividual samples were pooled 1:4 through automated liquid handling, extracted, and assayed by our emergency use authorized CDC-based RT-PCR laboratory developed test. Positive samples were serially diluted and theoretical and empirical PCR cycle thresholds were evaluated. Thirty-two distinct positive samples were pooled into negative specimens and individual CTs were compared to pooled CTs. Low positive samples were repeated for reproducibility and 32 four-way pools of negative specimens were assayed to determine specificity.ResultsFour-way pooling was associated with a loss of sensitivity of 1.7 and 2.0 CTs for our N1 and N2 targets, respectively. Pooling correctly identified SARS-CoV-2 in 94 % (n = 30/32) of samples tested. The two low positive specimens (neat CT > 35) not detected by pooling were individually repeated and detected 75 % (n=6/8) and 37.5 % (n = 3/8) of the time, respectively. All specimens individually determined negative were also negative by pooling.ConclusionWe report that 1:4 pooling of samples is specific and associated with an expected 2 CT loss in analytical sensitivity. Instead of running each sample individually, pooling of four samples will allow for a greater throughput and conserve scarce reagents.
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Project description:A total of 91 fig and 185 date samples were analyzed by reverse transcription (RT) real-time PCR for the presence of hepatitis A virus (HAV) RNA. Two batches of dates tested positive, and the HAV RNA detected was genotyped as IA. These findings warrant further development of methods applicable to food which is consumed untreated and is exported from countries in which HAV is endemic.
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