In the current article, a developed, patented method denoted the ‘Camel Semen Collection Kit‐CSCK’, was designed to solve the problem of semen collection in dromedary camels. CSCK is composed of ...three main parts: (1) Semen collection sac: made from supersensitive flexible low‐density polyethylene‐ (LDPE); (2) Metal stainless steel applicator: designed to introduce the collection sac intravaginally and fixate it to the vaginal wall of a female camel through air insufflation; (3) Fixation sticker: a cushion sheet sticker is used to secure the outer portion of the collection sac to the female's perineal area. Semen was collected twice a week from eight dromedary bulls by using electroejaculation (EJ), artificial vagina (AV) and CSCK. Successful semen collections were 81.3%, 84.4% and 43.8% using EJ, CSCK and AV techniques respectively. Semen obtained by EJ technique showed lower semen volume, gross activity, sperm concentration, total sperm motility and percentage of live sperm cells compared to the other two techniques. Semen collected by CSCK showed a longer collection period and higher volume, gross activity, sperm motility and percentage of live spermatozoa and a lower rate of visible contamination compared to AV technique. The advantages and disadvantages of the three techniques were compared and discussed. In conclusion, CSCK represents a practical and easy method to reliably collect high‐quality semen from any untrained male dromedary camel and may facilitate the widespread application of assisted reproductive technologies (ARTs) on a large scale in this species.
We implemented multiple nucleic acid amplification test platforms because of the limited availability of test kits for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the early ...stages of the pandemic. Interpretation of results generated by different platforms and prioritization for testing algorithms required cross-comparison.
To compare the analytical sensitivity of 3 commercial SARS-CoV-2 molecular assays, selected samples were studied in parallel with Cobas SARS-CoV-2 test, NxTAG CoV Extended Panel, and ID NOW COVID-19 assays.
A total of 8043 SARS-CoV-2 tests performed from March 22 to April 19, 2020, were included in this study. For all 1794 positive specimens detected by the cobas SARS-CoV-2 assay, the cycle threshold (Ct) values were manually tracked and plotted to demonstrate the distribution of sample viral levels. Additionally, 50 and 63 low-positive specimens (Ct values >32) as well as 50 and 61 consecutive positive specimens by the cobas assay were tested with NxTAG and ID NOW, respectively, to estimate their relative sensitivities.
The Ct values of cobas SARS-CoV-2-positive samples were evenly distributed throughout ranges of 13.32 to 39.50 (mean, 25.06) and 13.60 to 42.49 (mean, 26.45) for ORF1 and E gene targets, respectively. NxTAG reliably detected only specimens with E gene Ct values lower than 33, and is estimated to detect 89.4% of positive specimens detected by cobas assay. ID NOW had performance variation independent of Ct value and is estimated to detect 83.5% of cobas positives.
Clinical specimens exhibit a wide range of viral burden, with a significant portion at low levels. Analytical sensitivity of testing platforms is critical for reliable detection of SARS-CoV-2 and uniform care to patients.
PurposeA pilot study to validate the collection of vitreous reflux (VR) after intravitreal injection using Schirmers tear strips was carried out. We assessed its efficiency for proteomics studies by ...estimating the differential expression of 27 cytokines using multiplexed bead array in diabetic macular oedema and proliferative diabetic retinopathy. To set, validate and assess the efficacy of Schirmer tear strips for collecting VR in patients undergoing intravitreal injections for diabetic macular oedema (DME).Patients and methodsVR samples were collected from 11 eyes of DME patients after intravitreal injections using Schirmer tear strips. Undiluted vitrectomy samples were obtained from six eyes of non-diabetic patients with idiopathic macular hole and seven eyes of diabetic patients with high-risk proliferative diabetic retinopathy (Hr-PDR), which were also subsampled on the Schirmer tear strips. Tear sampling was done in a subset of the DME patients. Total protein concentration between VR and vitrectomy samples was compared. Levels of the set of 27 cytokines in Schirmer tear strips samples were measured. Inter-group comparison for cytokines was done using Mann-Whitney U-test.ResultsSimilar protein concentration in VR samples and vitrectomy samples (P<0.05) was obtained. Tear protein contamination was not detected in VR samples. In comparison with no-DR patients, 25 and 20 of the measured 27 cytokines were significantly elevated (P<0.05) in the Hr-PDR and DME patients, respectively. As compared with no-DR patients, vascular endothelial growth factor was only moderately elevated in DME patients (P>0.05), but significantly elevated in Hr-PDR patients (P<0.05). Interleukin 1 receptor antagonist/interleukin 1b (IL1RA/IL1b) ratio was 13 times higher in DME patients as compared with Hr-PDR group.ConclusionWe demonstrated a simple, safe method of VR sampling. This technique provides a pure, albeit small, vitreous sample for proteomics. IL1RA/IL1b ratio was found to be 13-fold higher in the DME group as compared to the Hr-PDR.
Display omitted
•Microsampling techniques DBS and VAMS in regulated Bioanalysis.•Improved quality data in Pharmacokinetic and Toxicokinetic study.•Bioanalytical considerations, method validation and ...applications of Dried matrix.•Regulatory perspective and current status n industry.
Microsampling, a reduced volume (< 50 μl) sampling method has successfully gained attention at the International Conference on Harmonization (ICH) level. It has been reflected in a few guidelines like ICH SA3, S11 and M10. The established benefits of microsampling support its use in Toxicokinetic (TK) and Pharmacokinetic (PK) studies, clinical studies, neonate sampling and remote sampling. When designing the TK component of a juvenile animal study, microsampling and sparse sampling (if justified) are strongly encouraged from the view of 3Rs (Replace, Refine, and Reduce). The novel sampling techniques arose with benefits over conventional sampling in terms of ease of sampling, storage, and shipment. These improved sampling techniques are less invasive and preferred by patients and trial participants. For the acceptance of these techniques in regulated bioanalysis, it is essential to prove its suitability with a robust and reliable method. Though there are many opportunities for the newer and smarter microsampling devices, the major obstacles are hematocrit influence, homogeneity of samples, repeats, incurred samples reanalysis and regulatory acceptance. With the advancement in techniques, opportunities are marching ahead of obstacles. The two microsampling techniques Dried Blood Spot (DBS) and Volumetric Absorption Microsampling (VAMS) are studied and elaborated in this article with respect to bioanalytical consideration, method validation and regulatory perspectives on its acceptance in regulated bioanalysis.
Mass spectrometry (MS) has become a powerful tool for metabolome, lipidome, and proteome analyses. The efficient analysis of multi-omics in single cells, however, is still challenging in the ...manipulation of single cells and lack of in-fly cellular digestion and extraction approaches. Here, we present a streamlined strategy for highly efficient and automatic single-cell multi-omics analysis by MS. We developed a 10-pL-level microwell chip for housing individual single cells, whose proteins were found to be digested in 5 min, which is 144 times shorter than traditional bulk digestion. Besides, an automated picoliter extraction system was developed for sampling of metabolites, phospholipids, and proteins in tandem from the same single cell. Also, 2 min MS2 spectra were obtained from 700 pL solution of a single cell sample. In addition, 1391 proteins, phospholipids, and metabolites were detected from one single cell within 10 min. We further analyzed cells digested from cancer tissue samples, achieving up to 40% increase in cell classification accuracy using multi-omics analysis in comparison with single-omics analysis. This automated single-cell MS strategy is highly efficient in analyzing multi-omics information for investigation of cell heterogeneity and phenotyping for biomedical applications.
The rapid identification and isolation of infected individuals remains a key strategy for controlling the spread of SARS-CoV-2. Frequent testing of populations to detect infection early in ...asymptomatic or presymptomatic individuals can be a powerful tool for intercepting transmission, especially when the viral prevalence is low. However, RT-PCR testing-the gold standard of SARS-CoV-2 diagnosis-is expensive, making regular testing of every individual unfeasible. Sample pooling is one approach to lowering costs. By combining samples and testing them in groups the number of tests required is reduced, substantially lowering costs. Here we report on the implementation of pooling strategies using 3-d and 4-d hypercubes to test a professional sports team in South Africa. We have shown that infected samples can be reliably detected in groups of 27 and 81, with minimal loss of assay sensitivity for samples with individual Ct values of up to 32. We report on the automation of sample pooling, using a liquid-handling robot and an automated web interface to identify positive samples. We conclude that hypercube pooling allows for the reliable RT-PCR detection of SARS-CoV-2 infection, at significantly lower costs than lateral flow antigen (LFA) tests.
As part of any plan to lift or ease the confinement restrictions that are in place in many different countries, there is an urgent need to increase the capacity of laboratory testing for severe acute ...respiratory syndrome coronavirus 2 (SARS-CoV-2). Detection of the viral genome through reverse transcription-quantitative PCR (RT-qPCR) is the gold standard for this virus; however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR has resulted in a worldwide shortage of several of these supplies. Here, we show that directly lysed saliva samples can serve as a suitable source for viral RNA detection that is less expensive and can be as efficient as the classical protocol, which involves column purification of the viral RNA. In addition, it bypasses the need for swab sampling, decreases the risk of the health care personnel involved in the testing process, and accelerates the diagnostic procedure.
Background. Human papillomavirus (HPV) vaccine was recommended in 2006 for routine vaccination of US females aged 11–12 years. Most vaccine used through 2014 was quadrivalent vaccine (4vHPV), which ...prevents HPV-6, -11, -16, and -18 infection. To evaluate vaccine impact, we measured HPV prevalence in the National Health and Nutrition Examination Survey (NHANES). Methods. We analyzed HPV DNA types detected in self-collected cervicovaginal specimens and demographic, sexual behavior, and self-reported vaccination data from females 14–34 years old. We estimated HPV prevalence in the prevaccine (2003–2006) and vaccine eras (2007–2010 and 2011–2014). Results. Among 14- to 19-year-olds, 4vHPV-type prevalence decreased from 11.5% (95% confidence interval CI, 9.1%–14.4%) in 2003–2006 to 3.3% (95% CI, 1.9%–5.8%) in 2011–2014, when ≥1-dose coverage was 55%. Among 20- to 24-year-olds, prevalence decreased from 18.5% (95% CI, 14.9%–22.8%) in 2003–2006 to 7.2% (95% CI, 4.7%–11.1%) in 2011–2014, when ≥1-dose coverage was 43%. Compared to 2003–2006, 4vHPV prevalence in sexually active 14- to 24-year-olds in 2011–2014 decreased 89% among those vaccinated and 34% among those unvaccinated. Vaccine effectiveness was 83%. Conclusions. Within 8 years of vaccine introduction, 4vHPV-type prevalence decreased 71% among 14- to 19-year-olds and 61% among 20- to 24-year-olds. Estimated vaccine effectiveness was high. The decrease in 4vHPV-type prevalence among unvaccinated females suggests herd protection.
We analyzed 4,352 participant- and staff-collected respiratory specimens from 2,796 subjects in the Oregon Child Absenteeism due to Respiratory Disease Study. Trained staff collected oropharyngeal ...specimens from school-aged children with acute respiratory illness while household participants of all ages collected their own midturbinate nasal specimens in year one and anterior nasal specimens in year two. Human ribonuclease P levels were measured using RT-PCR for all staff- and participant-collected specimens to determine adequacy, defined as Cycle threshold less than 38. Overall, staff- and participant-collected specimens were 99.9% and 96.4% adequate, respectively. Participant-collected midturbinate specimens were 95.2% adequate in year one, increasing to 97.2% in year two with anterior nasal collection. The mean human ribonuclease P Cycle threshold for participant-collected specimens was 31.18 in year one and 28.48 in year two. The results from this study suggest that community-based participant collection of respiratory specimens is comparable to staff-collected oropharyngeal specimens, is feasible, and may be optimal with anterior nasal collection.
Herbarium specimens provide verifiable and citable evidence of the occurrence of particular plants at particular points in space and time, and are vital resources for assessing extinction risk in the ...tropics, where plant diversity and threats to plants are greatest. We reviewed approaches to assessing extinction risk in response to the Convention on Biological Diversity's Global Strategy for Plant Conservation Target 2: an assessment of the conservation status of all known plant species by 2020. We tested five alternative approaches, using herbarium-derived data for trees, shrubs and herbs in five different plant groups from temperate and tropical regions. All species were previously fully assessed for the IUCN Red List. We found significant variation in the accuracy with which different approaches classified species as threatened or not threatened. Accuracy was highest for the machine learning model (90%) but the least data-intensive approach also performed well (82%). Despite concerns about spatial, temporal and taxonomic biases and uncertainties in herbarium data, when specimens represent the best available evidence for particular species, their use as a basis for extinction risk assessment is appropriate, necessary and urgent. Resourcing herbaria to maintain, increase and disseminate their specimen data is essential to guide and focus conservation action.This article is part of the theme issue 'Biological collections for understanding biodiversity in the Anthropocene'.
PDF
