Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Untargeted metagenomics protocol for the diagnosis of infection from CSF and tissue from sterile sites.

Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from sterile sites. We use this method routinely in our clinical service, processing 178 specimens over the past 2.5 years in a laboratory that adheres to ISO:15189 standards. We have successfully used this protocol to diagnose multiple cases of encephalitis and hepatitis.

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Comparison of SARS-CoV-2 N gene real-time RT-PCR targets and commercially available mastermixes.

BACKGROUND: This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000-1 copy/µl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. RESULTS: Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). CONCLUSIONS: This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.

Oxidative discolouration in whole-head and cut lettuce: biochemical and environmental influences on a complex phenotype and potential breeding strategies to improve shelf-life.

Lettuce discolouration is a key post-harvest trait. The major enzyme controlling oxidative discolouration has long been considered to be polyphenol oxidase (PPO) however, levels of PPO and subsequent development of discolouration symptoms have not always correlated. The predominance of a latent state of the enzyme in plant tissues combined with substrate activation and contemporaneous suicide inactivation mechanisms are considered as potential explanations for this phenomenon. Leaf tissue physical properties have been associated with subsequent discolouration and these may be influenced by variation in nutrient availability, especially excess nitrogen and head maturity at harvest. Mild calcium and irrigation stress has also been associated with a reduction in subsequent discolouration, although excess irrigation has been linked to increased discolouration potentially through leaf physical properties. These environmental factors, including high temperature and UV light intensities, often have impacts on levels of phenolic compounds linking the environmental responses to the biochemistry of the PPO pathway. Breeding strategies targeting the PAL and PPO pathway biochemistry and environmental response genes are discussed as a more cost-effective method of mitigating oxidative discolouration then either modified atmosphere packaging or post-harvest treatments, although current understanding of the biochemistry means that such programs are likely to be limited in nature and it is likely that they will need to be deployed alongside other methods for the foreseeable future.

An intra-specific linkage map of lettuce (Lactuca sativa) and genetic analysis of postharvest discolouration traits.

Minimally processed salad packs often suffer from discolouration on cut leaf edges within a few days after harvest. This limits shelf life of the product and results in high wastage. Recombinant inbred lines (RILs) derived from a cross between lettuce cvs. Saladin and Iceberg were shown to be suitable for genetic analysis of postharvest discolouration traits in lettuce. An intra-specific linkage map based on this population was generated to enable genetic analysis. A total of 424 markers were assigned to 18 linkage groups covering all nine chromosomes. The linkage map has a total length of 1,040 cM with an average marker distance of 2.4 cM within the linkage groups and was anchored to the ultra-dense, transcript-based consensus map. Significant genetic variation in the postharvest traits 'pinking', 'browning' and 'overall discolouration' was detected among the RILs. Seven significant quantitative trait loci (QTL) were identified for postharvest discolouration traits providing markers linked to the QTL that can be used for marker-assisted selection. Phenotypic stability was confirmed for extreme lines possessing the corresponding QTL parental alleles and which had shown transgressive segregation. This study indicates that a desired phenotype with reduced levels of postharvest discolouration can be achieved by breeding using natural variation.

Quantification of the relationship between glycemia and beta-cell mass adaptation in vivo.

Beta-cell mass dynamics play an important role in the adaptation to obesity, as well as in the pathogenesis of type 2 diabetes. Here we used a 24-hour modified hyperglycemic clamp protocol to investigate the effect of increasing glucose concentrations (15, 20, 25, or 35 mmol/L) on beta-cell mass and rates of beta-cell replication, death, and neogenesis in 6-week-old Sprague Dawley rats (n = 40). During the first 4 h of glucose infusion, plasma insulin levels rose to an approximate steady state in each group, but by the end of 24 h, there was no difference in insulin levels between any of the groups. There was also no difference in beta-cell mass between groups. Mean beta-cell replication rates displayed a linear relationship to mean plasma glucose levels in all hyperglycemic animals (r(2) = 0.98, p < 0.05). Relative to the uninfused basal control animals, replication rates were significantly reduced in the 15 mmol/L glucose group. The percentage of TUNEL-positive beta-cells was not different between groups. There was also no significant difference in markers of neogenesis. Thus, these data demonstrate that hyperglycemia for 24 h had no effect on beta-cell mass, death, or neogenesis in 6-week-old Sprague Dawley rats. We demonstrate a linear relationship, however, between hyperglycemia and beta-cell replication rates in vivo.

Dynamics of insulin sensitivity, -cell function, and -cell mass during the development of diabetes in fa/fa rats.

Both male Zucker Fatty (mZF) and lower-fat-fed female Zucker diabetic fatty (LF-fZDF) rats are obese but remain normoglycemic. Male ZDF (mZDF) and high-fat-fed female ZDF rats (HF-fZDF) are also obese but develop diabetes between 7 and 10 wk of age. Although these models have been well studied, the mechanisms governing the adaptations to obesity in the normoglycemic animals, and the failure of adaptation in the animals that develop diabetes, remain unclear. Here we use quantitative morphometry and our recently developed coupled beta-cell mass (beta(m)), insulin, and glucose model to elucidate the dynamics of insulin sensitivity (S(I)), beta-cell secretory capacity (beta(sc)), and beta(m) in these four animal models. Both groups that remained normoglycemic with increasing obesity (mZF, LF-fZDF) exhibited increased beta(m) and constant beta(sc) in response to a falling S(I). In rats that developed hyperglycemia (mZDF, HF-fZDF), there was a greater reduction in S(I) and slower expansion of beta(m), with constant beta(sc). beta(sc) decreased after glucose levels rose above 20 mM. Taken together, these data suggest that excessive insulin resistance and insufficient beta(m) adaptation play a primary role in the pathogenesis of diabetes.

A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes.

We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samples collected in endemic areas in Egypt and Papua New Guinea. Although the two methods had comparable sensitivity for detecting filarial DNA in reference samples, real-time PCR was more sensitive than C-PCR in practice with field samples. Other advantages of real-time PCR include its high-throughput capacity and decreased risk of cross-contamination between test samples. We believe that real-time PCR has great potential as a tool for monitoring progress in large-scale filariasis elimination programs.

Brugia malayi: effects of gamma radiation on adult worms and their intracellular Wolbachia bacteria.

Prior studies have shown that intracellular Wolbachia endobacteria are necessary for the normal development, reproduction, and survival of filarial nematodes. The purpose of this study was to examine effects of gamma radiation on Wolbachia and reproduction in Brugia malayi adult worms. Worms were exposed to 0, 10, 25, 45, 75, and 105 krad of gamma radiation from a 137cesium source and cultured in vitro for 10 days. Irradiation reduced production of microfilariae in a dose-dependent manner. Embryograms of irradiated female worms showed dose-related abnormalities with arrested development at the early embryo stage. Irradiation reduced the viability of adult worms in a dose-dependent manner, but no lethal effect was observed. Electron microscopy studies showed that irradiation cleared Wolbachia from worm tissues. Real-time polymerase chain reaction studies demonstrated greatly reduced Wolbachia DNA in irradiated worms. These effects are essentially the same as those observed in adult worms treated with doxycycline. These studies suggest that effects of irradiation on reproduction in Brugia malayi may be caused by effects of irradiation on Wolbachia.

Pyruvate prevents cardiac dysfunction and AMP-activated protein kinase activation by hydrogen peroxide in isolated rat hearts.

Ischemia-reperfusion injury in the heart results in enhanced production of H2O2 and activation of AMP-activated protein kinase (AMPK). Since mutations in AMPK result in cardiovascular dysfunction, we investigated whether the activation of AMPK mediates the H2O2-induced reduction in cardiac mechanical function. Isolated working rat hearts were perfused at 37 degrees C with Krebs-Henseleit solution. Following a 20-minute equilibration period, a single bolus of H2O2 (300 micromol/L) was added and the hearts were perfused for an additional 5 min. H2O2 induced a dramatic and progressive reduction in cardiac function. This was accompanied by rapid and significant activation of AMPK, an increase in Thr-172 phosphorylation of AMPK, and an increase in the creatine to phosphocreatine (Cr/PCr) ratio. Addition of pyruvate (5 mmol/L) to the perfusate prevented the H2O2-mediated reduction in cardiac mechanical dysfunction, activation of myocardial AMPK activity, increase in AMPK phosphorylation and the increase in the Cr/PCr ratio. Hearts challenged with H2O2 (300 micromol/L) in presence of either AMPK inhibitor Compound C (10 micromol/L) or its vehicle (dimethyl sulfoxide (DMSO), 0.1%) showed reduced impairment in cardiac mechanical function. Compound C but not its vehicle significantly inhibited myocardial AMPK activity. Thus, H2O2 induces cardiac dysfunction via both AMPK-dependent and independent mechanisms.

Potential mechanisms and consequences of cardiac triacylglycerol accumulation in insulin-resistant rats.

The accumulation of intracellular triacylglycerol (TG) is highly correlated with muscle insulin resistance. However, it is controversial whether the accumulation of TG is the result of increased fatty acid supply, decreased fatty acid oxidation, or both. Because abnormal fatty acid metabolism is a key contributor to the pathogenesis of diabetes-related cardiovascular dysfunction, we examined fatty acid and glucose metabolism in hearts of insulin-resistant JCR:LA-cp rats. Isolated working hearts from insulin-resistant rats had glycolytic rates that were reduced to 50% of lean control levels (P < 0.05). Cardiac TG content was increased by 50% (P < 0.05) in the insulin-resistant rats, but palmitate oxidation rates remained similar between the insulin-resistant and lean control rats. However, plasma fatty acids and TG levels, as well as cardiac fatty acid-binding protein (FABP) expression, were significantly increased in the insulin-resistant rats. AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid and glucose metabolism. When activated, AMPK increases fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels, and it decreases TG content by inhibiting glycerol-3-phosphate acyltransferase (GPAT), the rate-limiting step in TG synthesis. The activation of AMPK also stimulates cardiac glucose uptake and glycolysis. We thus investigated whether a decrease in AMPK activity was responsible for the reduced cardiac glycolysis and increased TG content in the insulin-resistant rats. However, we found no significant difference in AMPK activity. We also found no significant difference in various established downstream targets of AMPK: ACC activity, malonyl-CoA levels, carnitine palmitoyltransferase I activity, or GPAT activity. We conclude that hearts from insulin-resistant JCR:LA-cp rats accumulate substantial TG as a result of increased fatty acid supply rather than from reduced fatty acid oxidation. Furthermore, the accumulation of cardiac TG is associated with a reduction in insulin-stimulated glucose metabolism.

Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis.

Leptin regulates fatty acid metabolism in liver, skeletal muscle, and pancreas by partitioning fatty acids into oxidation rather than triacylglycerol (TG) storage. Although leptin receptors are present in the heart, it is not known whether leptin also regulates cardiac fatty acid metabolism. To determine whether leptin directly regulates cardiac fatty acid metabolism, isolated working rat hearts were perfused with 0.8 mm [9,10-(3)H]palmitate and 5 mm [1-(14)C]glucose to measure palmitate and glucose oxidation rates. Leptin (60 ng/ml) significantly increased palmitate oxidation rates 60% above control hearts (p < 0.05) and decreased TG content by 33% (p < 0.05) over the 60-min perfusion period. In contrast, there was no difference in glucose oxidation rates between leptin-treated and control hearts. Although leptin did not affect cardiac work, oxygen consumption increased by 30% (p < 0.05) and cardiac efficiency was decreased by 42% (p < 0.05). AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels. Leptin has also been shown to increase fatty acid oxidation in skeletal muscle through the activation of AMPK. However, we demonstrate that leptin had no significant effect on AMPK activity, AMPK phosphorylation state, ACC activity, or malonyl-CoA levels. AMPK activity and its phosphorylation state were also unaffected after 5 and 10 min of perfusion in the presence of leptin. The addition of insulin (100 microunits/ml) to the perfusate reduced the ability of leptin to increase fatty acid oxidation and decrease cardiac TG content. These data demonstrate for the first time that leptin activates fatty acid oxidation and decreases TG content in the heart. We also show that the effects of leptin in the heart are independent of changes in the AMPK-ACC-malonyl-CoA axis.

MEDICA 16 inhibits hepatic acetyl-CoA carboxylase and reduces plasma triacylglycerol levels in insulin-resistant JCR: LA-cp rats.

Intracellular triacylglycerol (TG) content of liver and skeletal muscle contributes to insulin resistance, and a significant correlation exists between TG content and the development of insulin resistance. Because acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme for liver fatty acid biosynthesis and a key regulator of muscle fatty acid oxidation, we examined whether ACC plays a role in the accumulation of intracellular TG. We also determined the potential role of 5'-AMP-activated protein kinase (AMPK) in this process, since it can phosphorylate and inhibit ACC activity in both liver and muscle. TG content, ACC, and AMPK were examined in the liver and skeletal muscle of insulin-resistant JCR:LA-cp rats during the time frame when insulin resistance develops. At 12 weeks of age, there was a threefold elevation in liver TG content and a sevenfold elevation in skeletal muscle TG content. Hepatic ACC activity was significantly elevated in 12-week-old JCR:LA-cp rats compared with lean age-matched controls (8.75 +/- 0.53 vs. 3.30 +/- 0.18 nmol. min(-1). mg(-1), respectively), even though AMPK activity was also increased. The observed increase in hepatic ACC activity was accompanied by a 300% increase in ACC protein expression. There were no significant differences in ACC activity, ACC protein expression, or AMPK activity in the skeletal muscle of the 12-week JCR:LA-cp rats. Treatment of 12-week JCR:LA-cp rats with MEDICA 16 (an ATP-citrate lyase inhibitor) resulted in a decrease in hepatic ACC and AMPK activities, but had no effect on skeletal muscle ACC and AMPK. Our data suggest that alterations in ACC or AMPK activity in muscle do not contribute to the development of insulin resistance. However, increased liver ACC activity in the JCR:LA-cp rat appears to contribute to the development of lipid abnormalities, although this increase does not appear to occur secondary to a decrease in AMPK activity.