Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Evaluation of pesticide residues in commercial Swiss beeswax collected in 2019 using ultra-high performance liquid chromatographic analysis

The aim of this study was to determine residue levels of pesticides in Swiss commercial beeswax. Foundation samples were collected in 2019 from nine commercial manufacturers for analysis of 21 pesticides using ultra-high performance liquid chromatography. Individual samples showed the variability and residue ranges and pooled samples represented the average annual residue values of the Swiss production. In total, 17 pesticides were identified and 13 pesticides were quantified. They included 13 acaricides and/or insecticides, two fungicides as well as a synergist and a repellent. The means calculated from individual samples were similar to the average annual residue values for most tested pesticides. Mean values of 401, 236, 106 and 3 μg·kg−1 were obtained for the beekeeping-associated contaminants coumaphos, tau-fluvalinate, bromopropylate and N-(2,4-Dimethylphenyl)-formamide (DMF; breakdown product of amitraz), respectively. For the other pesticides, the mean values were 203 μg·kg−1 (synergist piperonyl butoxide), 120 μg·kg−1 (repellent N,N-Diethyl-3-methylbenzamide, DEET), 19 μg·kg−1 (chlorfenvinphos) and 4 μg·kg−1 ((E)-fenpyroximate), while the means for acrinathrin, azoxystrobin, bendiocarb, boscalid, chlorpyrifos, flumethrin, permethrin, propoxur and thiacloprid were below the limit of quantification (< LOQ). Individual samples contained from seven to 14 pesticides. The ranges of values for coumaphos and piperonyl butoxide (from 14 to 4270 μg·kg−1; from 6 to 1555 μg·kg−1, respectively) were larger as compared to the ranges of values for DEET and tau-fluvalinate (from < LOQ to 585 μg·kg−1; from 16 to 572 μg·kg−1, respectively). In conclusion, the most prominent contaminants were the pesticides coumaphos and tau-fluvalinate, which are both acaricides with previous authorization for beekeeping in Switzerland, followed by piperonyl butoxide, a synergist to enhance the effect of insecticides. Graphical abstract

Validation and deployment of a direct saliva real-time RT-PCR test on pooled samples for COVID-19 surveillance testing

A direct, real-time reverse transcriptase PCR test on pooled saliva was validated in 2,786 participants against oropharyngeal swabs. Among asymptomatic/pre-symptomatic participants, the test was found to be in 99.21% agreement and 45% more sensitive than contemporaneous oropharyngeal swabs. The test was then used for surveillance testing on 44,242 saliva samples from asymptomatic participants. Those whose saliva showed evidence of SARS-CoV-2 within 50 cycles of amplification were referred for confirmatory testing, with 87% of those tested by nasal swab within 72 hours receiving a positive diagnostic result on Abbott ID NOW or real-time PCR platforms. Median Ct values on the saliva PCR for those with a positive and negative confirmatory tests was 30.67 and 35.92 respectively, however, binary logistic regression analysis of the saliva Ct values indicates that Ct thresholds as high as 47 may be useful in a surveillance setting. Overall, data indicate that direct RT-PCR testing of pooled saliva samples is an effective method of SARS-CoV-2 surveillance.

A validated analysis pipeline for mass spectrometry-based vitreous proteomics: new insights into proliferative diabetic retinopathy

Background Vitreous is an accessible, information-rich biofluid that has recently been studied as a source of retinal disease-related proteins and pathways. However, the number of samples required to confidently identify perturbed pathways remains unknown. In order to confidently identify these pathways, power analysis must be performed to determine the number of samples required, and sample preparation and analysis must be rigorously defined. Methods Control (n = 27) and proliferative diabetic retinopathy (n = 23) vitreous samples were treated as biologically distinct individuals or pooled together and aliquoted into technical replicates. Quantitative mass spectrometry with tandem mass tag labeling was used to identify proteins in individual or pooled control samples to determine technical and biological variability. To determine effect size and perform power analysis, control and proliferative diabetic retinopathy samples were analyzed across four 10-plexes. Pooled samples were used to normalize the data across plexes and generate a single data matrix for downstream analysis. Results The total number of unique proteins identified was 1152 in experiment 1, 989 of which were measured in all samples. In experiment 2, 1191 proteins were identified, 727 of which were measured across all samples in all plexes. Data are available via ProteomeXchange with identifier PXD025986. Spearman correlations of protein abundance estimations revealed minimal technical (0.99–1.00) and biological (0.94–0.98) variability. Each plex contained two unique pooled samples: one for normalizing across each 10-plex, and one to internally validate the normalization algorithm. Spearman correlation of the validation pool following normalization was 0.86–0.90. Principal component analysis revealed stratification of samples by disease and not by plex. Subsequent differential expression and pathway analyses demonstrated significant activation of metabolic pathways and inhibition of neuroprotective pathways in proliferative diabetic retinopathy samples relative to controls. Conclusions This study demonstrates a feasible, rigorous, and scalable method that can be applied to future proteomic studies of vitreous and identifies previously unrecognized metabolic pathways that advance understanding of diabetic retinopathy.

Alterations in brain glycogen levels influence life-history traits and reduce the lifespan in female Drosophila melanogaster

Sexual dimorphism in lifespan, wherein females outlive males, is evident across all animal taxa. The longevity difference between sexes is controlled by multiple physiological processes with complex relationships to one another. In recent years, glycogen, the storage form of glucose, has been shown to cause rapid aging upon forced synthesis in healthy neurons. Glycogen in the form of corpora amylacea in the aging brain is also widely reported. While these studies did suggest a novel role for glycogen in aging, most of them have focused on pooled samples, and have not looked at sex-specific effects, if any. Given the widespread occurrence of sex-biased expression of genes and the underlying physiology, it is important to look at the sex-specific effect of metabolic processes. In the present study, using transgenic fly lines for the human glycogen synthase, we investigated the sex-specific effect of glycogen on stress resistance, fitness, and survival. We demonstrate that Drosophila females with altered levels of glycogen in the brain display a shortened lifespan, increased resistance to starvation, and higher oxidative stress than male flies. The present study thus provides a novel insight into the sex-specific effect of glycogen in survival and aging and how differences in metabolic processes could contribute to sex-specific traits.

Development and Evaluation of AccuPower® COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples

Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower ® COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation ™48 system (comprised of ExiPrep ™48 Dx and Exicycler ™96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower ® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers` on-market PCR instruments. The limit of detection (LoD) of SCVM was 2 copies/µ L and the LoD of the NCVM was 120 copies/mL for both the gene and the SARS-CoV-2 gene (N gene and RdRp gene). The AccuPower ® kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower ® kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower ® kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD ™ M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen`s kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower ® kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower ® kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen`s kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.

Rapid, Reliable and Robust approach for extraction-free RT-PCR based detection of SARS-CoV-2 in clinical setting to expedite large scale screening

Rapid, reliable and robust method for the detection of SARS-CoV-2 is an indispensable need for diagnostics. The development of diagnostic methods will aid to address further waves of the pandemic potentially with rapid surveillance of disease and to allay the fears. To meet this challenge, we have developed a rapid RT-qPCR method for the detection of 3 target genes or confirmatory genes in less than 30 minutes. The assay showed 100% sensitivity and 100% specificity when tested on 120 samples. We compared a conventional extraction based method with extraction-free method, and then further reduced the run time of extraction free method. Additionally, we have validated our rapid RT-qPCR method for the assessment of pooled samples. We hereby propose a most reliable approach for the mass screening of samples with ease of operation at a low cost. Finally we designed a single tube analysis method which provides qualitative as well as quantitative results in minimum time.

Missing two key parameters in the pooled testing strategy

The pooled testing strategy [1] misses two key parameters, the infection prevalence p and its variance mentioned many times in the paper as the key determinants of any pooled testing strategy. For illustrating their methods, the authors used p from other studies that employed individual tests. It turned that that no statistical estimators for p and its variance have ever been derived for testing data of pooled samples since the first formulation of testing strategies based on pooled sampled in 1943 [2]. Here I derive the maximum likelihood estimators for p and its variance based on tests of pooled samples. This should result in significant saving in time, resource, and costs.

Pooled Testing and Its Applications in the COVID-19 Pandemic

When testing for a disease such as COVID-19, the standard method is individual testing: we take a sample from each individual and test these samples separately. An alternative is pooled testing (or ‘group testing’), where samples are mixed together in different pools, and those pooled samples are tested. When the prevalence of the disease is low and the accuracy of the test is fairly high, pooled testing strategies can be more efficient than individual testing. In this chapter, we discuss the mathematics of pooled testing and its uses during pandemics, in particular the COVID-19 pandemic. We analyse some one- and two-stage pooling strategies under perfect and imperfect tests, and consider the practical issues in the application of such protocols.