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During a household-transmission field study using COVID-19 antigen rapid diagnostic tests (Ag-RDT), a common test strip lot was identified among 3 participants with false-positive results. In blinded laboratory evaluation, this lot exhibited a significantly higher false-positive rate than other lots. Because a positive Ag-RDT result often prompts action, reducing lot-specific false positives can maintain confidence and actionability of true-positive Ag-RDT results.

SARS-CoV-2 viral load measurements from a single specimen type are used to establish diagnostic strategies, interpret clinical-trial results for vaccines and therapeutics, model viral transmission, and understand virus-host interactions. However, measurements from a single specimen type are implicitly assumed to be representative of other specimen types. We quantified viral-load timecourses from individuals who began daily self-sampling of saliva, anterior nares (nasal), and oropharyngeal (throat) swabs before or at the incidence of infection with the Omicron variant. Viral loads in different specimen types from the same person at the same timepoint exhibited extreme differences, up to 109copies/mL. These differences were not due to variation in sample self-collection, which was consistent. For most individuals, longitudinal viral-load timecourses in different specimen types did not correlate. Throat-swab and saliva viral loads began to rise up to 7 days earlier than nasal-swab viral loads in most individuals, leading to very low clinical sensitivity of nasal swabs during the first days of infection. Individuals frequently exhibited presumably infectious viral loads in one specimen type while viral loads were low or undetectable in other specimen types. Therefore, defining an individual as infectious based on assessment of a single specimen type underestimates the infectious period, and overestimates the ability of that specimen type to detect infectious individuals. For diagnostic COVID-19 testing, these three single specimen types have low clinical sensitivity, whereas a combined throat-nasal swab, and assays with high analytical sensitivity, were inferred to have significantly better clinical sensitivity to detect presumed pre-infectious and infectious individuals.Significance StatementIn a longitudinal study of SARS-CoV-2 Omicron viral loads in three paired specimen types (saliva, anterior-nares swabs, and oropharyngeal swabs), we found extreme differences among paired specimen types collected from a person at the same timepoint, and that viral loads in different specimen types from the same person often do not correlate throughout infection. Individuals often exhibited high, presumably infectious viral loads in oral specimen types before nasal viral loads remained low or even undetectable. Combination oropharyngeal-nasal swabs were inferred to have superior clinical sensitivity to detect infected and infectious individuals. This demonstrates that single specimen type reference standard tests for SARS-CoV-2, such as in clinical trials or diagnostics evaluations may miss infected and even infectious individuals.

BackgroundIn a recent household-transmission study of SARS-CoV-2, we found extreme differences in SARS-CoV-2 viral loads among paired saliva, anterior-nares swab (ANS) and oropharyngeal swab specimens collected from the same timepoint. We hypothesized these differences may hinder low-analytical-sensitivity assays (including antigen rapid diagnostic tests, Ag-RDTs) using a single specimen type (e.g., ANS) from reliably detecting infected and infectious individuals.MethodsWe evaluated a daily at-home ANS Ag-RDT (Quidel QuickVue) in a cross-sectional analysis of 228 individuals and in a longitudinal analysis (throughout infection) of 17 individuals enrolled early in the course of infection. Ag-RDT results were compared to RT-qPCR results and high, presumably infectious viral loads (in each, or any, specimen type).ResultsThe ANS Ag-RDT correctly detected only 44% of timepoints from infected individuals on cross-sectional analysis, and in this population had an inferred limit of detection of 7.6×106copies/mL. From the longitudinal cohort, daily Ag-RDT clinical sensitivity was very low (ConclusionNasal Ag-RDTs, even when used daily, can miss individuals infected with the Omicron variant and even those presumably infectious. Evaluations of Ag-RDT detection of infected or infectious individuals should be compared with a composite (multi-specimen) infection status to correctly assess performance.Key pointsNasal-swab rapid antigen tests have low analytical sensitivity and the sampling of only the nasal cavity hinders their ability to detect infected individuals, including those with high and presumably infectious viral loads in throat or saliva specimens.

BackgroundThe small intestine (SI) is the primary site of nutrient absorption, so its large surface area lacks the thick protective mucus that is characteristic of the large intestine. Because the SI epithelium is relatively exposed, any microbes that colonize the thin mucosa of the SI may exert a substantial effect on the host. Thus far, potential bacterial colonization of the SI mucosa has only been documented in disease states, suggesting mucosal colonization is a rare occurrence, likely requiring multiple perturbations.ResultsHere, we tested whether we could induce bacterial association with jejunum mucosa by a combination of malnutrition and oral co-gavage with a specific bacterial cocktail (E. coli and Bacteroides spp.) that has previously induced environmental enteropathy in mouse models. To overcome the current limitations in imaging and allow definite determination of whether bacterial colonization of the SI mucosa is occurring, we optimized our previously developed whole-tissue three-dimensional (3D) imaging tools with third-generation hybridization chain reaction (HCR v3.0) probes. Only in mice that were malnourished and gavaged with the bacterial cocktail did we detect dense bacterial clusters surrounding intestinal villi suggestive of colonization. Healthy mice gavaged with bacteria and malnourished mice not gavaged with bacteria showed no evidence of mucosal colonization. Furthermore, in malnourished mice gavaged with bacteria we detected villus loss, which may represent one possible consequence that bacterial colonization of the SI mucosa has on the host.ConclusionsOur results suggest that dense bacterial colonization of jejunum mucosa is possible in the presence of multiple perturbations and that villus loss may be one possible consequence to such colonization. Furthermore, our results demonstrate the utility of whole-tissue 3D imaging tools. Although 2D imaging of thin sections may have failed to detect and capture the full spatial complexity of such rare events, whole-tissue 3D imaging tools enabled their detection over large areas of intestinal mucosa and visualization of their spatial complexity in 3D.

Improving our understanding of host-microbe relationships in the gut requires the ability to both visualize and quantify the spatial organization of microbial communities in their native orientation with the host tissue. We developed a systematic procedure to quantify the three-dimensional (3D) spatial structure of the native mucosal microbiota in any part of the intestines with taxonomic and high spatial resolution. We performed a 3D biogeographical analysis of the microbiota of mouse cecal crypts at different stages of antibiotic exposure. By tracking eubacteria and four dominant bacterial taxa, we found that the colonization of crypts by native bacteria is a dynamic and spatially organized process. Ciprofloxacin treatment drastically reduced bacterial loads and eliminated Muribaculaceae (or all Bacteroidetes entirely) even 10 d after recovery when overall bacterial loads returned to preantibiotic levels. Our 3D quantitative imaging approach revealed that the bacterial colonization of crypts is organized in a spatial pattern that consists of clusters of adjacent colonized crypts that are surrounded by unoccupied crypts, and that this spatial pattern is resistant to the elimination of Muribaculaceae or of all Bacteroidetes by ciprofloxacin. Our approach also revealed that the composition of cecal crypt communities is diverse and that Lactobacilli were found closer to the lumen than Bacteroidetes, Ruminococcaceae, and Lachnospiraceae, regardless of antibiotic exposure. Finally, we found that crypts communities with similar taxonomic composition were physically closer to each other than communities that were taxonomically different.

Although three-dimensional (3D) genome organization is central to many aspects of nuclear function, it has been difficult to measure at the single-cell level. To address this, we developed 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE). scSPRITE uses split-and-pool barcoding to tag DNA fragments in the same nucleus and their 3D spatial arrangement. Because scSPRITE measures multiway DNA contacts, it generates higher-resolution maps within an individual cell than can be achieved by proximity ligation. We applied scSPRITE to thousands of mouse embryonic stem cells and detected known genome structures, including chromosome territories, active and inactive compartments, and topologically associating domains (TADs) as well as long-range inter-chromosomal structures organized around various nuclear bodies. We observe that these structures exhibit different levels of heterogeneity across the population, with TADs representing dynamic units of genome organization across cells. We expect that scSPRITE will be a critical tool for studying genome structure within heterogeneous populations.

Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and presymptomatic transmission, curb the spread of variants, and maximize treatment efficacy. Low-analytical-sensitivity nasal-swab testing is commonly used for surveillance and symptomatic testing, but the ability of these tests to detect the earliest stages of infection has not been established. In this study, conducted between September 2020 and June 2021 in the greater Los Angeles County, California, area, initially SARS-CoV-2-negative household contacts of individuals diagnosed with COVID-19 prospectively self-collected paired anterior-nares nasal-swab and saliva samples twice daily for viral-load quantification by high-sensitivity reverse-transcription quantitative PCR (RT-qPCR) and digital-RT-PCR assays. We captured viral-load profiles from the incidence of infection for seven individuals and compared diagnostic sensitivities between respiratory sites. Among unvaccinated persons, testing saliva with a high-analytical-sensitivity assay detected infection up to 4.5 days before viral loads in nasal swabs reached concentrations detectable by low-analytical-sensitivity nasal-swab tests. For most participants, nasal swabs reached higher peak viral loads than saliva but were undetectable or at lower loads during the first few days of infection. High-analytical-sensitivity saliva testing was most reliable for earliest detection. Our study illustrates the value of acquiring early (within hours after a negative high-sensitivity test) viral-load profiles to guide the appropriate analytical sensitivity and respiratory site for detecting earliest infections. Such data are challenging to acquire but critical to designing optimal testing strategies with emerging variants in the current pandemic and to respond to future viral pandemics.

Background Upper gastrointestinal (GI) disorders and abdominal pain afflict between 12 and 30% of the worldwide population and research suggests these conditions are linked to the gut microbiome. Although large-intestine microbiota have been linked to several GI diseases, the microbiota of the human small intestine and its relation to human disease has been understudied. The small intestine is the major site for immune surveillance in the gut, and compared with the large intestine, it has greater than 100 times the surface area and a thinner and more permeable mucus layer. Results Using quantitative sequencing, we evaluated total and taxon-specific absolute microbial loads from 250 duodenal-aspirate samples and 21 paired duodenum-saliva samples from participants in the REIMAGINE study. Log-transformed total microbial loads spanned 5 logs and were normally distributed. Paired saliva-duodenum samples suggested potential transmission of oral microbes to the duodenum, including organisms from the HACEK group. Several taxa, including Klebsiella, Escherichia, Enterococcus, and Clostridium, seemed to displace strict anaerobes common in the duodenum, so we refer to these taxa as disruptors. Disruptor taxa were enriched in samples with high total microbial loads and in individuals with small intestinal bacterial overgrowth (SIBO). Absolute loads of disruptors were associated with more severe GI symptoms, highlighting the value of absolute taxon quantification when studying small-intestine health and function. Conclusion This study provides the largest dataset of the absolute abundance of microbiota from the human duodenum to date. The results reveal a clear relationship between the oral microbiota and the duodenal microbiota and suggest an association between the absolute abundance of disruptor taxa, SIBO, and the prevalence of severe GI symptoms.