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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.

With advances in metagenomic sequencing, the microbial composition of diverse environmental systems has been investigated, providing new perspectives on potential ecological dynamics and dimensions for experimental investigations. Here, we characterized exocellular free DNA via metagenomics, using a newly developed method that separates free DNA from cells, viruses, and vesicles, and facilitated the independent characterization of each fraction.

Phytoplankton communities residing in the open ocean, the largest habitat on Earth, play a key role in global primary production. Through their influence on nutrient supply to the euphotic zone, open-ocean eddies impact the magnitude of primary production and its spatial and temporal distributions. It is important to gain a deeper understanding of the microbial ecology of marine ecosystems under the influence of eddy physics with the aid of advanced technologies. In March and April 2018, we deployed autonomous underwater and surface vehicles in a cyclonic eddy in the North Pacific Subtropical Gyre to investigate the variability of the microbial community in the deep chlorophyll maximum (DCM) layer. One long-range autonomous underwater vehicle (LRAUV) carrying a third-generation Environmental Sample Processor (3G-ESP) autonomously tracked and sampled the DCM layer for four days without surfacing. The sampling LRAUV's vertical position in the DCM layer was maintained by locking onto the isotherm corresponding to the chlorophyll peak. The vehicle ran on tight circles while drifting with the eddy current. This mode of operation enabled a quasi-Lagrangian time series focused on sampling the temporal variation of the DCM population. A companion LRAUV surveyed a cylindrical volume around the sampling LRAUV to monitor spatial and temporal variation in contextual water column properties. The simultaneous sampling and mapping enabled observation of DCM microbial community in its natural frame of reference.

Microbial communities are critical to ecosystem dynamics and biogeochemical cycling in the open oceans. Viruses are essential elements of these communities, influencing the productivity, diversity, and evolution of cellular hosts. To further explore the natural history and ecology of open-ocean viruses, we surveyed the spatiotemporal dynamics of double-stranded DNA (dsDNA) viruses in both virioplankton and bacterioplankton size fractions in the North Pacific Subtropical Gyre, one of the largest biomes on the planet. Assembly and clustering of viral genomes revealed a peak in virioplankton diversity at the base of the euphotic zone, where virus populations and host species richness both reached their maxima. Simultaneous characterization of both extracellular and intracellular viruses suggested depth-specific reproductive strategies. In particular, analyses indicated elevated lytic interactions in the mixed layer, more temporally variable temperate phage interactions at the base of the euphotic zone, and increased lysogeny in the mesopelagic ocean. Furthermore, the depth variability of auxiliary metabolic genes suggested habitat-specific strategies for viral influence on light-energy, nitrogen, and phosphorus acquisition during host infection. Most virus populations were temporally persistent over several years in this environment at the 95% nucleic acid identity level. In total, our analyses revealed variable distributional patterns and diverse reproductive and metabolic strategies of virus populations in the open-ocean water column.

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.

Significance Sinking particles composed of both organic and inorganic material feed the deep-sea ecosystem and contribute centrally to ocean carbon sequestration. Despite their importance, little is known about the biological composition of sinking particles reaching the deep sea. Time-series analyses of sinking particles unexpectedly revealed bacterial assemblages that were simple and homogeneous over time. Particle-associated eukaryote assemblages, however, were more variable and complex. Several modes of export were observed, including summer inputs from the surface, more stochastic export of surface-derived protists and animals, and contributions from midwater animals and deep-sea bacteria. In summary, sinking particles exhibited temporally variable, heterogeneous biological sources and activities that reflected their important roles in the downward transport and transformation of organic matter in the deep sea., Sinking particles are a critical conduit for the export of organic material from surface waters to the deep ocean. Despite their importance in oceanic carbon cycling and export, little is known about the biotic composition, origins, and variability of sinking particles reaching abyssal depths. Here, we analyzed particle-associated nucleic acids captured and preserved in sediment traps at 4,000-m depth in the North Pacific Subtropical Gyre. Over the 9-month time-series, Bacteria dominated both the rRNA-gene and rRNA pools, followed by eukaryotes (protists and animals) and trace amounts of Archaea. Deep-sea piezophile-like Gammaproteobacteria, along with Epsilonproteobacteria, comprised >80% of the bacterial inventory. Protists (mostly Rhizaria, Syndinales, and ciliates) and metazoa (predominantly pelagic mollusks and cnidarians) were the most common sinking particle-associated eukaryotes. Some near-surface water-derived eukaryotes, especially Foraminifera, Radiolaria, and pteropods, varied greatly in their abundance patterns, presumably due to sporadic export events. The dominance of piezophile-like Gammaproteobacteria and Epsilonproteobacteria, along with the prevalence of their nitrogen cycling-associated gene transcripts, suggested a central role for these bacteria in the mineralization and biogeochemical transformation of sinking particulate organic matter in the deep ocean. Our data also reflected several different modes of particle export dynamics, including summer export, more stochastic inputs from the upper water column by protists and pteropods, and contributions from sinking mid- and deep-water organisms. In total, our observations revealed the variable and heterogeneous biological origins and microbial activities of sinking particles that connect their downward transport, transformation, and degradation to deep-sea biogeochemical processes.

Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic transmission, curb the spread of variants by travelers, and maximize treatment efficacy. Low-sensitivity nasal-swab testing (antigen and some nucleic-acid-amplification tests) is commonly used for surveillance and symptomatic testing, but the ability of low-sensitivity nasal-swab tests to detect the earliest stages of infection has not been established. In this case-ascertained study, 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 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, high-sensitivity saliva testing detected infection up to 4.5 days before viral loads in nasal swabs reached the limit of detection of low-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-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 design optimal testing strategies in the current pandemic and will be required for responding to future viral pandemics. As new variants and viruses emerge, up-to-date data on viral kinetics are necessary to adjust testing strategies for reliable early detection of infections.

The deep chlorophyll maximum (DCM) layer is an ecologically important feature of the open ocean. The DCM cannot be observed using aerial or satellite remote sensing; thus, in situ observations are essential. Further, understanding the responses of microbes to the environmental processes driving their metabolism and interactions requires observing in a reference frame that moves with a plankton population drifting in ocean currents, i.e., Lagrangian. Here, we report the development and application of a system of coordinated robots for studying planktonic biological communities drifting within the ocean. The presented Lagrangian system uses three coordinated autonomous robotic platforms. The focal platform consists of an autonomous underwater vehicle (AUV) fitted with a robotic water sampler. This platform localizes and drifts within a DCM community, periodically acquiring samples while continuously monitoring the local environment. The second platform is an AUV equipped with environmental sensing and acoustic tracking capabilities. This platform characterizes environmental conditions by tracking the focal platform and vertically profiling in its vicinity. The third platform is an autonomous surface vehicle equipped with satellite communications and subsea acoustic tracking capabilities. While also acoustically tracking the focal platform, this vehicle serves as a communication relay that connects the subsea robot to human operators, thereby providing situational awareness and enabling intervention if needed. Deployed in the North Pacific Ocean within the core of a cyclonic eddy, this coordinated system autonomously captured fundamental characteristics of the in situ DCM microbial community in a manner not possible previously.

Transmission of SARS-CoV-2 in community settings often occurs before symptom onset, therefore testing strategies that can reliably detect people in the early phase of infection are urgently needed. Early detection of SARS-CoV-2 infection is especially critical to protect vulnerable populations who require frequent interactions with caretakers. Rapid COVID-19 tests have been proposed as an attractive strategy for surveillance, however a limitation of most rapid tests is their low sensitivity. Low-sensitivity tests are comparable to high sensitivity tests in detecting early infections when two assumptions are met: (1) viral load rises quickly (within hours) after infection and (2) viral load reaches and sustains high levels (>105– 106 RNA copies/mL). However, there are no human data testing these assumptions. In this study, we document a case of presymptomatic household transmission from a healthy young adult to a sibling and a parent. Participants prospectively provided twice-daily saliva samples. Samples were analyzed by RT-qPCR and RT-ddPCR and we measured the complete viral load profiles throughout the course of infection of the sibling and parent. This study provides evidence that in at least some human cases of SARS-CoV-2, viral load rises slowly (over days, not hours) and not to such high levels to be detectable reliably by any low-sensitivity test. Additional viral load profiles from different samples types across a broad demographic must be obtained to describe the early phase of infection and determine which testing strategies will be most effective for identifying SARS-CoV-2 infection before transmission can occur.One sentence summaryIn some human infections, SARS-CoV-2 viral load rises slowly (over days) and remains near the limit of detection of rapid, low-sensitivity tests.

The overarching logistical challenge in microbial oceanography is acquiring enough samples to provide meaningful scientific interpretation. The number of samples collected during ship expeditions is limited by weather, time on station, and budget. Here we describe a robotic, autonomous vehicle platform equipped with a unique sampling instrument that mitigates some of these constraints. In a joint cruise on the R/V Falkor, the Monterey Bay Aquarium Research Institute and the University of Hawaii deployed two of these vehicles in a mesoscale eddy north of the island of Maui. One vehicle collected contextual measurements while circling a freely drifting sampling vehicle. On the sampling vehicle we implemented several behaviors, including sampling every three hours for a 4-day underwater drift while maintaining position within the deep chlorophyll maximum layer (~100m). Results demonstrate the ability to remain with features of interest and point to an exciting future of long-term, directed, persistent sampling.

The temporal dynamics of phytoplankton growth and activity have large impacts on fluxes of matter and energy, yet obtaining in situ metabolic measurements of sufficient resolution for even dominant microorganisms remains a considerable challenge. We performed Lagrangian diel sampling with synoptic measurements of population abundances, dinitrogen (N2) fixation, mortality, productivity, export and transcription in a bloom of Crocosphaera over eight days in the North Pacific Subtropical Gyre (NPSG). Quantitative transcriptomic analyses revealed clear diel oscillations in transcript abundances for 34% of Crocosphaera genes identified, reflecting a systematic progression of gene expression in diverse metabolic pathways. Significant time-lagged correspondence was evident between nifH transcript abundance and maximal N2 fixation, as well as sepF transcript abundance and cell division, demonstrating the utility of transcriptomics to predict the occurrence and timing of physiological and biogeochemical processes in natural populations. Indirect estimates of carbon fixation by Crocosphaera were equivalent to 11% of net community production, suggesting that under bloom conditions this diazotroph has a considerable impact on the wider carbon cycle. Our cross-scale synthesis of molecular, population and community-wide data underscores the tightly coordinated in situ metabolism of the keystone N2-fixing cyanobacterium Crocosphaera, as well as the broader ecosystem-wide implications of its activities.