Food for thought: Eating before saliva collection and interference with SARS-CoV-2 detection

BackgroundSaliva is an optimal specimen for detection of viruses that cause upper respiratory infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its cost-effectiveness and non-invasive collection. However, together with intrinsic enzymes and oral microbiota, childrens unique dietary habits may introduce substances that interfere with diagnostic testing. MethodsTo determine whether childrens dietary choices impact SARS-CoV-2 detection in saliva, we performed a diagnostic study that simulates testing of real-life specimens provided from healthy children (n=5) who self-collected saliva at home before and at 0, 20, and 60 minutes after eating from 20 foods they selected. Each of seventy-two specimens was split into two volumes and spiked with SARS-CoV-2-negative or -positive standards prior to side-by-side testing by reverse-transcription polymerase chain reaction matrix-assisted laser desorption ionization time-of-flight (RT-PCR/MALDI-TOF) assay. ResultsDetection of internal extraction control and SARS-CoV-2 nucleic acids was reduced in replicates of saliva collected at 0 minutes after eating 11 of 20 foods. Interference resolved at 20 and 60 minutes after eating all foods except hot dog in one participant. This represented a significant improvement in detection of nucleic acids compared to saliva collected at 0 minutes after eating (P=0.0005). ConclusionsWe demonstrate successful detection of viral nucleic acids in saliva self-collected by children before and after eating a variety of foods. Fasting is not required before saliva collection for SARS-CoV-2 testing by RT-PCR/MALDI-TOF, but waiting 20 minutes after eating is sufficient for accurate testing. These findings should be considered for SARS-CoV-2 testing and broader viral diagnostics in saliva specimens.

RT-PCR/MALDI-TOF diagnostic target performance reflects circulating SARS-CoV-2 variant diversity in New York City

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern (VOC) have emerged. New variants pose challenges for diagnostic platforms since sequence diversity can alter primer/probe binding sites (PBS), causing false-negative results. The Agena MassARRAY(R) SARS-CoV-2 Panel utilizes reverse-transcription polymerase chain reaction and mass-spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we utilize a dataset of 256 SARS-CoV-2-positive specimens collected between April 11, 2021-August 28, 2021 to evaluate target performance with paired sequencing data. During this timeframe, two targets in the N gene (N2, N3) were subject to the greatest sequence diversity. In specimens with N3 dropout, 69% harbored the Alpha-specific A28095U polymorphism that introduces a 3-mismatch to the N3 forward PBS and increases risk of target dropout relative to specimens with 28095A (relative risk (RR): 20.02; p<0.0001; 95% Confidence Interval (CI): 11.36-35.72). Furthermore, among specimens with N2 dropout, 90% harbored the Delta-specific G28916U polymorphism that creates a 3-mismatch to the N2 probe PBS and increases target dropout risk (RR: 11.92; p<0.0001; 95% CI: 8.17-14.06). These findings highlight the robust capability of Agena MassARRAY(R) SARS-CoV-2 Panel target results to reveal circulating virus diversity and underscore the power of multi-target design to capture VOC.

Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multi-target approach

The COVID-19 pandemic sparked rapid development of SARS-CoV-2 diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY(R) SARS-CoV-2 Panel combines RT-PCR and MALDI-TOF mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified dataset of 1,262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 through April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly-specific for the alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Comparison of SARS-CoV-2 detection in Saliva by real-time RT-PCR and RT-PCR/MALDI-TOF Methods

The coronavirus disease 2019 (COVID-19) pandemic has accelerated the need for rapid implementation of diagnostic assays for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in respiratory specimens. While multiple molecular methods utilize nasopharyngeal specimens, supply chain constraints and need for easier and safer specimen collection warrant alternative specimen types, particularly saliva. Although saliva has been found to be a comparable clinical matrix for detection of SARS-CoV-2, evaluations of diagnostic and analytic performance across platforms for this specimen type are limited. Here, we compared two methods for SARS-CoV-2 detection in saliva: the Roche cobas(R) 6800/8800 SARS-CoV-2 real-time RT-PCR Test and the Agena Biosciences MassARRAY(R) SARS-CoV-2 Panel/MassARRAY(R) System. Overall, both systems had high agreement with one another, and both demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of each platform and determined the limit of detection of the Roche assay was four times lower than that of Agena for saliva specimens (390.6 v. 1,562.5 copies/mL). Furthermore, across individual target components of each assay, T2 and N2 targets had the lowest limits of detection for each platform, respectively. Together, we demonstrate that saliva represents an appropriate specimen for SARS-CoV-2 detection in two technologies that have high agreement and differ in analytical sensitivities overall and across individual component targets. The addition of saliva as an acceptable specimen and understanding the sensitivity for testing on these platforms can further inform public health measures for screening and detection to combat the COVID-19 pandemic.

Comparison of KLF4, SP1, and Cyclin D1 expressions between ad-enocarcinanoma of the esophagogastric junction and distal gastric adenocarcinoma / 中国肿瘤临床

Objective:Recent studies have shown that in contrast to decrease in distal gastric adenocarcinoma (DGA), incidence of adenocarcinoma of the esophagogastric junction (AEG) has increased noticeably in numerous counties. However, the reasons remain unclear. This study evaluated the possible differences in the expression of KLF4, SP1, and Cyclin D1 in AEG and DGA, and explored the potential carcinogenesis of AEG. Methods:Immunohistochemistry was performed on paraffin-embedded tissues to evaluate the pu-tative differences in the expressions of KLF4, SP1, and Cyclin D1 at protein level between AEG (n=58) and DGA (n=47). The patholog-ical significance of these markers between the two groups was also compared and analyzed. Results:The percentage of positive KLF4 expression was significantly lower in DGA than in AEG (P<0.05). Lower KLF4 expression was found both in well-or moderately dif-ferentiated cases and in poorly differentiated cases with DGA compared with their AEG counterparts (P<0.05). However, positive stain-ing for SP1 was significantly higher in DGA (P<0.05). No significant difference was found in the expression of Cyclin D1 between the two groups. Further analysis showed that in DGA, the positive expression of KLF4, SP1, and Cyclin D1 were significantly correlated with lymph node metastasis. In AEG, only Cyclin D1 expression was correlated with lymph node metastasis (P<0.05). No correlation was found among the expression of KLF4, SP1, and Cyclin D1 in AEG. In DGA, KLF4 was inversely correlated with SP1 and Cyclin D1 (r=-0.334 and r=-0.341, respectively, P<0.05), and SP1 was positively correlated with Cyclin D1 expression (r=0.340, P<0.05).Conclusion:Different expression patterns and clinicopathological significance of KLF4, SP1, and Cyclin D1 were observed between AEG and DGA, suggesting the putative difference in the carcinogenesis and progression of AEG and DGA.

Statistical method for mapping QTLs for complex traits based on two backcross populations.

Most important agronomic and quality traits of crops are quantitative in nature. The genetic variations in such traits are usually controlled by sets of genes called quantitative trait loci (QTLs), and the interactions between QTLs and the environment. It is crucial to understand the genetic architecture of complex traits to design efficient strategies for plant breeding. In the present study, a new experimental design and the corresponding statistical method are presented for QTL mapping. The proposed mapping population is composed of double backcross populations derived from backcrossing both homozygous parents to DH (double haploid) or RI (recombinant inbreeding) lines separately. Such an immortal mapping population allows for across-environment replications, and can be used to estimate dominance effects, epistatic effects, and QTL-environment interactions, remedying the drawbacks of a single backcross population. In this method, the mixed linear model approach is used to estimate the positions of QTLs and their various effects including the QTL additive, dominance, and epistatic effects, and QTL-environment interaction effects (QE). Monte Carlo simulations were conducted to investigate the performance of the proposed method and to assess the accuracy and efficiency of its estimations. The results showed that the proposed method could estimate the positions and the genetic effects of QTLs with high efficiency.