Browsing Category

Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field

Deconstructing and Reconstructing Cheese Rind Microbiomes for Experiments in Microbial Ecology and Evolution Mass Spectrometry and Neurodegenerative Disease Research Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field

Saliva RT-PCR Sensitivity Over the Course of SARS-CoV-2 Infection


While real-time reverse transcriptase–polymerase chain reaction (RT-PCR) on nasopharyngeal swabs is the current standard for SARS-CoV-2 detection, saliva is an attractive alternative for diagnosis and screening due to ease of collection and minimal supply requirements. Studies on the sensitivity of saliva-based SARS-CoV-2 molecular testing have shown considerable variability.We conducted a prospective, longitudinal study to investigate the testing time frame that optimizes saliva sensitivity for SARS-CoV-2 detection.Methods

Between June 17, 2020, and February 15, 2021, a convenience sample of individuals exposed to a household member with RT-PCR–confirmed SARS-CoV-2 within 2 weeks were recruited from Children’s Hospital Los Angeles and nearby community testing sites into the Household Exposure and Respiratory Virus Transmission and Immunity Study (HEARTS). Paired nasopharyngeal and saliva samples were collected every 3 to 7 days for up to 4 weeks or until 2 negative nasopharyngeal test results. RT-PCR for SARS-CoV-2 N1 and N2 genes was performed; cycle threshold less than 40 defined a positive result. A nasopharyngeal N1 cycle threshold of 34 or less was defined as high viral load. Detailed specimen collection and RT-PCR methods are reported in the eMethods in the Supplement.

Saliva sensitivity was calculated using nasopharyngeal-positive RT-PCR as the reference standard. COVID-19 onset was defined as the earlier date between first symptom (collected by questionnaire daily) or first RT-PCR positivity. Pre- and postsymptomatic were defined as asymptomatic time points before and after a symptomatic interval, respectively.

Saliva sensitivity by week of collection and between symptomatic and asymptomatic individuals were compared using the χ2 test or the Fisher exact test. Generalized estimating equations were used to determine clinical characteristics (Table) associated with saliva sensitivity in nasopharyngeal-positive pairs while accounting for repeated samples from the same individuals. Analyses were performed using SPSS version 27.0 (IBM Corp) with a 2-sided P < .05 considered significant. Written informed consent was obtained from participants. The study was approved by the institutional review board of Children’s Hospital Los Angeles.Results

We tested 889 paired nasopharyngeal swab-saliva samples from 404 participants, of which SARS-CoV-2 was detected in 524 nasopharyngeal (58.9%) and 318 saliva (35.7%) specimens. SARS-CoV-2 was detected in both specimens in 258 pairs (29.0%). Of the 256 nasopharyngeal SARS-CoV-2–positive participants (63.4%), the mean age was 28.2 years (range, 3.0-84.5 years); 108 (42.2%) were male. Participants returned for a median of 3 visits (interquartile range, 2-4). Ninety-three participants (36.3%) were asymptomatic throughout their infection; 126 (77.3%) of 163 symptomatic individuals reported mild severity.

Saliva sensitivity was highest in samples collected during the first week of infection at 71.2% (95% CI, 62.6%-78.8%) but decreased each subsequent week . Participants who presented with COVID-19–associated symptoms on the specimen collection day during week 1 of infection had significantly higher saliva sensitivity compared with asymptomatic participants (88.2% [95% CI, 77.6%-95.1%] vs 58.2% [95% CI, 46.3%-69.5%]; P < .001).

Saliva sensitivity remained significantly higher in symptomatic participants in week 2 (83.0% [95% CI, 70.6%-91.8%] vs 52.6% [95% CI, 42.6%-62.5%]; P < .001). No difference was observed more than 2 weeks after COVID-19 onset . Sensitivities did not significantly differ for never-symptomatic (34.7% [95% CI, 27.3%-42.7%]), presymptomatic (57.1% [95% CI, 31.7%-80.2%]), and postsymptomatic (42.9% [95% CI, 36.8%-49.1%]) time points (P = .26).

For each day after COVID-19 onset, the odds ratio for saliva detection was 0.94 (95% CI, 0.91-0.96) compared with the previous day (P < .001) . Participants presenting with COVID-19–associated symptoms at the time of specimen collection or with high nasopharyngeal viral loads had 2.8 (95% CI, 1.6-5.1; P < .001) and 5.2 (95% CI, 2.9-9.3; P < .001) higher odds of having a saliva-positive RT-PCR result compared with those with asymptomatic presentation or low nasopharyngeal viral loads, respectively.


Saliva was sensitive for detecting SARS-CoV-2 in symptomatic individuals during initial weeks of infection, but sensitivity in asymptomatic SARS-CoV-2 carriers was less than 60% at all time points. As COVID-19 testing strategies in workplaces, schools, and other shared spaces are optimized, low saliva sensitivity in asymptomatic infections must be considered. This study suggests saliva-based RT-PCR should not be used for asymptomatic COVID-19 screening.

This study has limitations. Samples were collected following household exposure; therefore, pretest probability was high. Nasopharyngeal swab testing was the reference standard, but this is not a perfect test for SARS-CoV-2 infection, and a positive RT-PCR result from any sample past 10 days of infection may not be predictive of viral replication or infectivity.


Our study is concordant with multiple published works supporting saliva as an alternative sample for COVID-19 screening and diagnosis, and one of a minority where saliva was shown to be more sensitive than the corresponding NP swab8,9,13, although the results by Leung et al. (53.7% saliva vs. 47.4% NP swab, 95 subjects) were not statistically different8. Several reasons may account for this difference in the studies, including enrichment from nasal and oropharyngeal secretions, where the viral load is potentially higher8,9, or a higher volume of samples collection, where approximately 10 mL of saliva was collected for testing13. Steps were taken to minimize biases and errors—NP swabs performed by trained healthcare staff, environmental testing of CAP-accredited laboratory (no evidence of contamination), conduction of tests for most of the samples in the same laboratory, and pre-processing of saliva samples with dithiothreitol before RNA extraction to resolve the issues of saliva specimen viscosity, which can lead to false negatives.


Interestingly but perhaps unsurprisingly, the use of different RT-PCR kits in the present study resulted in different test-positive rates in saliva, suggesting that this can potentially be an important consideration for clinical laboratories, where more sensitive laboratory protocols should be deployed for clinical diagnosis as opposed to mass screening for low-prevalence populations. More validation would be required to confirm this finding.

SN swabs, however, appeared less sensitive compared to both saliva and NP swabs for the diagnosis of COVID-19. Although it was convenient, less time-consuming to perform relative to saliva collection, and caused less discomfort compared to NP swabs, the markedly lower sensitivity should preclude its use where other sample types can be collected.

In our study, NGS provided efficient whole-genome profiling of SARS-CoV-2 for phylogenetic analysis directly from the clinical samples without culture. NGS detection sensitivity was excellent with a threshold of 1.7% genome coverage or 5 amplicons targets, confirming all CDC-LDT positives tested. Other groups have reported highly sensitive performance for NGS with limits of detection ranging between a threshold of 5% genome coverage or 84 genome-equivalents per mL21, or at least 5 SARS-CoV-2 targets for detection22. The phylogeny results were consistent with the virus belonging to a viral type (Clade O, lineage B.6) known to be circulating in the geographical regions of Singapore and India.

There are several limitations to our work. Firstly, the study population was confined to young and middle-aged men who were either asymptomatic or had mild disease. The results cannot be extrapolated to other populations (e.g., paediatric), where there is a clear need for alternate sample types to NP swabs. Secondly, we did not extend the follow-up testing sufficiently to determine when saliva viral shedding stopped for the majority of subjects, although this has been explored in other studies. Thirdly, we did not test for the difference, if any, between saliva obtained from naso-oropharyngeal or the mouth alone, although it is biologically plausible that the latter would result in lower sensitivity for COVID-19 diagnosis.

In conclusion, our study adds to the body of evidence supporting saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further defines the role of naso-oropharyngeal secretions and the impact of different RT-PCR kits in increasing the sensitivity of testing. In our study, SN swabs were inferior to both saliva and NP swabs. Our study also provides evidence to support NGS in challenging samples for sensitive COVID-19 molecular diagnosis. Such an NGS workflow can also provide direct-from-sample phylogenetic analysis for public health decision-making, such as contact tracing.

Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field

Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field

Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field

Using a field to lab method, mature deep-rooting traits in wheat had been correlated to root phenotypes measured on young plants from controlled situations. Mature deep-rooting root traits of twenty wheat genotypes at maturity had been established by way of coring in three field trials throughout two years. Field traits had been correlated to phenotypes expressed by the 20 genotypes after progress in 4 commonly-used lab screens:

Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field
Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field

i. soil tubes for root emergence, elongation, size and branching at 4 ages to 34 days after sowing (DAS); ii. paper pouches 7 DAS and iii. agar chambers for major root (PR) quantity and angles at Eight DAS; and iv. soil baskets for PR and nodal root (NR) quantity and angle at 42 DAS.

Correlations between lab and field root traits (r2 = 0.45 – 0.73) had been extremely inconsistent, with many traits uncorrelated and nobody lab phenotype correlating equally throughout three field experiments. Phenotypes most positively related with deep field roots had been: longest PR and NR axiles from soil tube display at 20 DAS; and slim PR angle and extensive NR angle from soil baskets at 42 DAS. Paper and agar PR angles had been positively and considerably correlated to one another, however solely extensive outer PRs in paper display correlated positively to shallower field root traits.

NR phenotypes in soil baskets weren’t predicted by PR phenotypes in any display, suggesting impartial developmental controls and worth in measuring each root varieties in lab screens.Strong temporal and edaphic results on mature root traits, and an absence of understanding of root trait adjustments over plant improvement, are main challenges in creating controlled surroundings root screens for mature root traits in the field.

Genetic Diversity and Virulence Characteristics of Staphylococcus aureus Isolates from Cases of Bovine Mastitis

Staphylococcus aureus is one of the main bacterial mastitis pathogens with important results on animal and human well being. Some research confirmed that S. aureus strains that infect completely different host species are genetically distinct, though most strains can infect a variety of host species. However, there aren’t any clearly outlined clonal patterns of S. aureus strains which can be identified to contaminate a selected host.

The aims of this research had been to guage the clonal variety and virulence traits of S. aureus isolates from instances of bovine mastitis. Bacteriological exams had been carried out on milk samples from instances of bovine mastitis from 11 dairy farms together with some milk samples from unknow farms in Eastern Tennessee. Overall, a complete of 111 S.

aureus had been remoted and recognized, and additional evaluated for his or her genetic variety by pulsed-field gel electrophoresis (PFGE) and virulence traits by PCR. Genotypic virulence components together with staphylococcal enterotoxins, and poisonous shock syndrome toxin 1 (tsst-1) had been examined by PCR. In addition, the affiliation amongst a number of identified virulence components of these isolates primarily based on our present and former research in our lab had been evaluated.

Previously generated information that had been included in the evaluation of affiliation amongst virulence components had been the presence of biofilm manufacturing related genes in the ica operon resembling icaA, icaD and icaAB, and phenotypic virulence traits resembling hemolysis on blood agar, slime manufacturing and resistance or susceptibility to 10 generally used antimicrobials in dairy farms.

The PFGE outcomes confirmed the presence of 16 PFGE varieties (designated A – P) all through farms, of which three pulsotypes, I, M and O had been the most often remoted PFGE varieties from most farms. The PFGE sort M was the most prevalent of all 16 PFGE varieties, with 64 isolates being current amongst 9 farms.

The PCR outcomes of enterotoxin genes confirmed that out of the complete 111 examined 84 (75.7%) had been destructive whereas 13 (11.7%), 2 (1.8%), 3 (2.7%), 1 (0.9%) and eight (7.2%) had been optimistic for seb, seb and sec, sec, see, and tsst-1, respectively. All 111 isolates had been destructive for sea and sej. Results of the analysis of I, M and O strains adhesion to and invasion into mammary epithelial cells confirmed that the complete rely of every pressure of micro organism adhered to and invaded into mammary epithelial cell line (MAC-T cells) was not considerably completely different (P>0.05).

This could also be a sign that there is no such thing as a important distinction in their capability to ascertain early host-pathogen interplay and colonization of the host.outcomes. There had been no statistically important associations amongst PFGE varieties and different identified virulence components of these strains.

However, PFGE varieties O and M are likely to cluster with β-hemolysin, absence of enterotoxins and susceptibility to antimicrobials. In conclusion, we didn’t discover any affiliation of pulsotype with genotypic and phenotypic virulence components and S. aureus isolates from instances of mastitis included in this research had numerous genotypes that possessed variable virulence components.

Quinoa, a dietary grain, can be utilized as an ingredient in gluten-free sourdoughs. This research characterizes quinoa flour spontaneous fermentation with emphasis in the isolation of exopolysaccharide (EPS) producer micro organism. Real, pink and black grains had been studied.

Dough yield, microbiota composition and fermentation biochemistry had been decided for a complete of 36 quinoa flour fermentations. The fermentation biochemistry was monitored by high-performance liquid chromatography (HPLC) evaluation, pH measurement and titratable acidity. Changes in the microbiota had been monitored by plating on deMann Rogosa and Sharp 5 agar (MRS5) and yeast and mildew agar (YMA) plates and with metagenetic evaluation.

The capability to supply exopolysaccharides was screened in chosen lactic acid micro organism (LAB) isolates. Production of natural acids in the spontaneous fermentation dropped the pH to 4.0 ± 0.3. The group of presumptive LAB reached 8.37 ± 0.01 log colony forming models (CFU)/mL by day 8 of back-slopped fermentations.

The microbiota was composed of LactobacillusEnterococcus, LeuconostocLactococcusPediococcus and WeissellaP. pentosaceous,L. citreum and W. cibaria had been capable of produce EPS in a starch-rich medium. P. pentosaceous confirmed larger exopolysaccharide yield, speedy acidifying kinetics and was capable of drop the dough broth pH to values under 4.Zero and a optimistic fermentation quotient after 24 h of incubation. Therefore, the bacterium could be a possible candidate for quinoa sourdough manufacturing.