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Human milk is a complex mix of nutritional and bioactive components that provide complete nourishment for the infant. However, we lack a systematic knowledge of the factors shaping milk composition and how milk variation influences infant health. Here, we characterize relationships between maternal genetics, milk gene expression, milk composition, and the infant fecal microbiome in up to 310 exclusively breastfeeding mother-infant pairs. We identified 482 genetic loci associated with milk gene expression unique to the lactating mammary gland and link these loci to breast cancer risk and human milk oligosaccharide concentration. Integrative analyses uncovered connections between milk gene expression and infant gut microbiome, including an association between the expression of inflammation-related genes with milk interleukin-6 (IL-6) concentration and the abundance of Bifidobacterium and Escherichia in the infant gut. Our results show how an improved understanding of the genetics and genomics of human milk connects lactation biology with maternal and infant health., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus that is often transmitted to the neonate via breast milk. Postnatal CMV transmission can have negative health consequences for preterm and immunocompromised infants, but any effects on healthy term infants are thought to be benign. Furthermore, the impact of CMV on the composition of the hundreds of bioactive factors in human milk has not been tested. Here, we utilize a cohort of exclusively breastfeeding full-term mother-infant pairs to test for differences in the milk transcriptome and metabolome associated with CMV, and the impact of CMV in breast milk on the infant gut microbiome and infant growth. We find upregulation of the indoleamine 2,3-dioxygenase (IDO) tryptophan-to-kynurenine metabolic pathway in CMV+ milk samples, and that CMV+ milk is associated with decreased Bifidobacterium in the infant gut. Our data indicate two opposing CMV-associated effects on infant growth; with kynurenine positively correlated, and CMV viral load negatively correlated, with infant weight-for-length at 1 month of age. These results suggest CMV transmission, CMV-related changes in milk composition, or both may be modulators of full-term infant development., (© 2024. The Author(s).)

Background: An ageing prison population with complex health needs combined with punitive sentencing practices means palliative care for incarcerated individuals is increasingly important. However, there is limited evidence regarding the models of care delivery in high-income countries, and their associated challenges and benefits., Aim: To develop a typology of models of palliative care provision for incarcerated individuals, synthesise evidence of their outcomes and describe facilitators of and challenges in delivering different models of palliative and end-of-life care in prisons., Design: Scoping review following Arksey and O'Malley, with narrative synthesis. The protocol was registered prospectively (reviewregistry1260)., Data Sources: MEDLINE, EMBASE, CINAHL, PsycINFO, the Social Sciences Citation Index and grey literature were searched on 15th March 2023. The Mixed Methods Appraisal Tool (MMAT) was used for quality appraisal., Results: A total of 16,865 records were screened; 22 peer-reviewed articles and 18 grey literature sources met the inclusion criteria. Three models were identified: Embedded Hospice, Outsourcing Care and Community Collaboration. The Embedded Hospice model shows potential benefits for patients and prisons. Outsourcing Care may miss opportunities for comprehensive care. Collaborative Care relies on proactive prison-community relationships that could be formalised for improvement. Psychosocial and bereavement needs of those dying in prison and their caregivers lack sufficient documentation., Conclusion: Further research is needed to evaluate prison hospice costs and examine how prison hospices impact compassionate release usage. Beyond the USA, policies might formalise care pathways and recognise best practices. Further investigation to address psychosocial needs of people in prison with life-limiting illnesses and post-death bereavement support is required., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Motivation: Differentiating ecosystems poses a complex, high-dimensional problem constrained by capturing relevant variation across species profiles. Researchers use pairwise distances and subsequent dimensionality reduction to highlight variation in a few dimensions. Despite popularity in analysis of ecological data, these low-dimensional visualizations can contain geometric abnormalities such as "arch" and "horseshoe" effects, potentially obscuring the impact of environmental gradients. These abnormalities appear in ordination but are in fact a product of oversaturated large pairwise distances., Results: We present Local Manifold distance (LMdist), an unsupervised algorithm which adjusts pairwise beta diversity measures to better represent true ecological distances between samples. Beta diversity measures can have a bounded dynamic range in depicting long environmental gradients with high species turnover. Using a graph structure, LMdist projects pairwise distances onto a manifold and traverses the manifold surface to adjust pairwise distances at the upper end of the beta diversity measure's dynamic range. This allows for values beyond the range of the original measure. Not all datasets will have oversaturated pairwise distances, nor will capture variation that resembles a manifold, so LMdist adjusts only those pairwise values which may be undervalued in the presence of a sampled gradient. The adjusted distances serve as input for ordination and statistical testing. We demonstrate on real and simulated data that LMdist effectively recovers distances along known gradients and along complex manifolds such as the Swiss roll dataset. LMdist enables more powerful statistical tests for gradient effects and reveals variation orthogonal to the gradient., Availability and Implementation: Available on GitHub at https://github.com/knights-lab/LMdist., (© The Author(s) 2023. Published by Oxford University Press.)

Immigration to a highly industrialized nation has been associated with metabolic disease and simultaneous shifts in microbiota composition, but the underlying mechanisms are challenging to test in human studies. Here, we conducted a pilot study to assess the differential effects of human gut microbiota collected from the United States (US) and rural Thailand on the murine gut mucosa and immune system. Colonization of germ-free mice with microbiota from US individuals resulted in an increased accumulation of innate-like CD8 T cells in the small intestine lamina propria and intra-epithelial compartments when compared to colonization with microbiota from Thai individuals. Both TCRγδ and CD8αα T cells showed a marked increase in mice receiving Western microbiota and, interestingly, this phenotype was also associated with an increase in intestinal mucus thickness. Serendipitously, an accidentally infected group of mice corroborated this association between elevated inflammatory response and increased mucus thickness. These results suggest that Western-associated human gut microbes contribute to a pro-inflammatory immune response.

We previously reported that autologous-fecal-microbiota-transplantation (aFMT), following 6 m of lifestyle intervention, attenuated subsequent weight regain and insulin rebound for participants consuming a high-polyphenol green-Mediterranean diet. Here, we explored whether specific changes in the core (abundant) vs. non-core (low-abundance) gut microbiome taxa fractions during the weight-loss phase (0-6 m) were differentially associated with weight maintenance following aFMT. Eighty-two abdominally obese/dyslipidemic participants (age = 52 years; 6 m weightloss = -8.3 kg) who provided fecal samples (0 m, 6 m) were included. Frozen 6 m's fecal samples were processed into 1 g, opaque and odorless aFMT capsules. Participants were randomly assigned to receive 100 capsules containing their own fecal microbiota or placebo over 8 m-14 m in ten administrations (adherence rate > 90%). Gut microbiome composition was evaluated using shotgun metagenomic sequencing. Non-core taxa were defined as ≤ 66% prevalence across participants. Overall, 450 species were analyzed. At baseline, 13.3% were classified as core, and Firmicutes presented the highest core proportion by phylum. During 6 m weight-loss phase, abundance of non-core species changed more than core species ( P  < .0001). Subject-specific changes in core and non-core taxa fractions were strongly correlated (Jaccard Index; r  = 0.54; P  < .001). Following aFMT treatment, only participants with a low 6 m change in core taxa, and a high change in non-core taxa, avoided 8-14 m weight regain (aFMT = -0.58 ± 2.4 kg, corresponding placebo group = 3.18 ± 3.5 kg; P  = .02). In a linear regression model, low core/high non-core 6 m change was the only combination that was significantly associated with attenuated 8-14 m weight regain ( P  = .038; P  = .002 for taxa patterns/treatment intervention interaction). High change in non-core, low-abundance taxa during weight-loss might mediate aFMT treatment success for weight loss maintenance. ClinicalTrials.gov: NCT03020186.

The microbiome is known to play a role in many human diseases, but identifying key microbes and their functions generally requires large studies due to the vast number of species and genes, and the high levels of intra-individual and inter-individual variation. 16S amplicon sequencing of the rRNA gene is commonly used for large studies due to its comparatively low sequencing cost, but it has poor taxonomic and functional resolution. Deep shotgun sequencing is a more accurate and comprehensive alternative for small studies, but can be cost-prohibitive for biomarker discovery in large populations. Shallow or moderate-depth shotgun metagenomics may serve as a viable alternative to 16S sequencing for large-scale and/or dense longitudinal studies, but only if resolution and reproducibility are comparable. Here we applied both 16S and shallow shotgun stool microbiome sequencing to a cohort of 5 subjects sampled twice daily and weekly, with technical replication at the DNA extraction and the library preparation/sequencing steps, for a total of 80 16S samples and 80 shallow shotgun sequencing samples. We found that shallow shotgun sequencing produced lower technical variation and higher taxonomic resolution than 16S sequencing, at a much lower cost than deep shotgun sequencing. These findings suggest that shallow shotgun sequencing provides a more specific and more reproducible alternative to 16S sequencing for large-scale microbiome studies where costs prohibit deep shotgun sequencing and where bacterial species are expected to have good coverage in whole-genome reference databases., (© 2023. The Author(s).)

Background: Hmong men in Minnesota exhibit a high prevalence of gout and hyperuricemia. Although evidence of vitamin C's effectiveness as a treatment for gout is mixed, analysis of therapeutic benefit based on an individual's multiomic signature may identify predictive markers of treatment success., Objectives: The primary objective of the Hmong Microbiome ANd Gout, Obesity, Vitamin C (HMANGO-C) study was to assess the effectiveness of vitamin C on serum urate in Hmong adults with and without gout/hyperuricemia. The secondary objectives were to assess if 1) vitamin C impacts the taxonomic and functional patterns of microbiota; 2) taxonomic and functional patterns of microbiota impact vitamin C's urate-lowering effects; 3) genetic variations impact vitamin C's urate-lowering effects; 4) differential microbial biomarkers exist for patients with or without gout; and 5) there is an association between obesity, gut microbiota and gout/hyperuricemia., Methods: This prospective open-labelled clinical trial was guided by community-based participatory research principles and conducted under research safety restrictions for SARS-CoV-2. We aimed to enroll a convenient sample of 180 Hmong adults (120 with gout/hyperuricemia and 60 without gout/hyperuricemia) who provided medical, demographic, dietary and anthropometric information. Participants took vitamin C 500mg twice daily for 8 weeks and provided pre-and post- samples of blood and urine for urate measurements as well as stool samples for gut microbiome. Salivary DNA was also collected for genetic markers relevant to uric acid disposition., Expected Results: We expected to quantify the impact of vitamin C on serum urate in Hmong adults with and without gout/hyperuricemia. The outcome will enhance our understanding of how gut microbiome and genomic variants impact the urate-lowering of vitamin C and associations between obesity, gut microbiota and gout/hyperuricemia. Ultimately, findings may improve our understanding of the causes and potential interventions that could be used to address health disparities in the prevalence and management of gout in this underserved population., Trial Registration: ClinicalTrials.gov NCT04938024 (first posted: 06/24/2021)., Competing Interests: D.K. serves as CEO and holds equity in CoreBiome, a company involved in the commercialization of microbiome analysis. The University of Minnesota also has financial interests in CoreBiome under the terms of a license agreement with CoreBiome. These interests have been reviewed and managed by the University of Minnesota in accordance with its Conflict-of-Interest policies. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Wen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The composition and function of early life gut bacterial communities (microbiomes) have been proposed to modulate health for the long term. In addition to bacteria, fungi (mycobiomes) also colonize the early life gut and have been implicated in health disorders such as asthma and obesity. Despite the potential importance of mycobiomes in health, there has been a lack of study regarding fungi and their interkingdom interactions with bacteria during infancy. The goal of this study was to obtain a more complete understanding of microbial communities thought to be relevant for the early life programming of health. Breastmilk and infant feces were obtained from a unique cohort of healthy, exclusively breastfeeding dyads recruited as part of the Mothers and Infants Linked for Healthy Growth (MILk) study with microbial taxa characterized using amplicon-based sequencing approaches. Bacterial and fungal communities in breastmilk were both distinct from those of infant feces, consistent with niche-specific microbial community development. Nevertheless, overlap was observed among sample types (breastmilk, 1-month feces, 6-month feces) with respect to the taxa that were the most prevalent and abundant. Self-reported antibacterial antibiotic exposure was associated with micro- as well as mycobiome variation, which depended upon the subject receiving antibiotics (mother or infant), timing of exposure (prenatal, peri- or postpartum), and sample type. In addition, birth mode was associated with bacterial and fungal community variation in infant feces, but not breastmilk. Correlations between bacterial and fungal taxa abundances were identified in all sample types. For infant feces, congruency between bacterial and fungal communities was higher for older infants, consistent with the idea of co-maturation of bacterial and fungal gut communities. Interkingdom connectedness also tended to be higher in older infants. Additionally, higher interkingdom connectedness was associated with Cesarean section birth and with antibiotic exposure for microbial communities of both breastmilk and infant feces. Overall, these results implicate infant age, birth mode, and antibiotic exposure in bacterial, fungal and interkingdom relationship variation in early-life-relevant microbiomes, expanding the current literature beyond bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Heisel, Johnson, Gonia, Dillon, Skalla, Haapala, Jacobs, Nagel, Pierce, Fields, Demerath, Knights and Gale.)

While gut microbiome and host gene regulation independently contribute to gastrointestinal disorders, it is unclear how the two may interact to influence host pathophysiology. Here we developed a machine learning-based framework to jointly analyse paired host transcriptomic (n = 208) and gut microbiome (n = 208) profiles from colonic mucosal samples of patients with colorectal cancer, inflammatory bowel disease and irritable bowel syndrome. We identified associations between gut microbes and host genes that depict shared as well as disease-specific patterns. We found that a common set of host genes and pathways implicated in gastrointestinal inflammation, gut barrier protection and energy metabolism are associated with disease-specific gut microbes. Additionally, we also found that mucosal gut microbes that have been implicated in all three diseases, such as Streptococcus, are associated with different host pathways in each disease, suggesting that similar microbes can affect host pathophysiology in a disease-specific manner through regulation of different host genes. Our framework can be applied to other diseases for the identification of host gene-microbiome associations that may influence disease outcomes., (© 2022. The Author(s).)

Background: Dietary fiber is an integral part of a healthy diet, but questions remain about the mechanisms that underlie effects and the causal contributions of the gut microbiota. Here, we performed a 6-week exploratory trial in adults with excess weight (BMI: 25-35 kg/m 2 ) to compare the effects of a high-dose (females: 25 g/day; males: 35 g/day) supplement of fermentable corn bran arabinoxylan (AX; n = 15) with that of microbiota-non-accessible microcrystalline cellulose (MCC; n = 16). Obesity-related surrogate endpoints and biomarkers of host-microbiome interactions implicated in the pathophysiology of obesity (trimethylamine N-oxide, gut hormones, cytokines, and measures of intestinal barrier integrity) were assessed. We then determined whether clinical outcomes could be predicted by fecal microbiota features or mechanistic biomarkers., Results: AX enhanced satiety after a meal and decreased homeostatic model assessment of insulin resistance (HOMA-IR), while MCC reduced tumor necrosis factor-α and fecal calprotectin. Machine learning models determined that effects on satiety could be predicted by fecal bacterial taxa that utilized AX, as identified by bioorthogonal non-canonical amino acid tagging. Reductions in HOMA-IR and calprotectin were associated with shifts in fecal bile acids, but correlations were negative, suggesting that the benefits of fiber may not be mediated by their effects on bile acid pools. Biomarkers of host-microbiome interactions often linked to bacterial metabolites derived from fiber fermentation (short-chain fatty acids) were not affected by AX supplementation when compared to non-accessible MCC., Conclusion: This study demonstrates the efficacy of purified dietary fibers when used as supplements and suggests that satietogenic effects of AX may be linked to bacterial taxa that ferment the fiber or utilize breakdown products. Other effects are likely microbiome independent. The findings provide a basis for fiber-type specific therapeutic applications and their personalization., Trial Registration: Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract., (© 2022. The Author(s).)

Probiotics are increasingly administered to premature infants to prevent necrotizing enterocolitis and neonatal sepsis. However, their effects on gut microbiome assembly and immunity are poorly understood. Using a randomized intervention trial in extremely premature infants, we tested the effects of a probiotic product containing four strains of Bifidobacterium species autochthonous to the infant gut and one Lacticaseibacillus strain on the compositional and functional trajectory of microbiome. Daily administration of the mixture accelerated the transition into a mature, term-like microbiome with higher stability and species interconnectivity. Besides infant age, Bifidobacterium strains and stool metabolites were the best predictors of microbiome maturation, and structural equation modeling confirmed probiotics as a major determinant for the trajectory of microbiome assembly. Bifidobacterium-driven microbiome maturation was also linked to an anti-inflammatory intestinal immune milieu. This demonstrates that Bifidobacterium strains are ecosystem engineers that lead to an acceleration of microbiome maturation and immunological consequences in extremely premature infants., Competing Interests: Declaration of interests T.A.T. was the Research Director at Lallemand Health Solutions, the manufacturers of FloraBABY. The other authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Wood decomposer fungi are grouped by how they extract sugars from lignocellulose. Brown rot fungi selectively degrade cellulose and hemicellulose, leaving lignin intact, and white rot fungi degrade all components. Many trees are susceptible to both rot types, giving carbon in Earth's woody biomass, specifically lignin, a flexible fate that is affected not only by the fungal decomposition mechanism but also the associated microbial community. However, little is understood about how rot type may influence the microbial community in decaying wood. In this study, we quantified bacterial communities associated with Fomes fomentarius (white rot) and Fomitopsis betulina (brown rot) found on a shared tree host species, birch (Betula papyrifera). We collected 25 wood samples beneath sporocarps  of F. fomentarius (n = 13) and F. betulina (n = 12) on standing dead trees, and coupled microbial DNA sequencing with chemical signatures of rot type (pH and lignin removal). We found that bacterial communities for both fungi were dominated by Proteobacteria, a commonly reported association. However, rot type exerted significant influence on less abundant taxa in ways that align logically with fungal traits. Amplicon sequence variants (ASVs) were enriched in Firmicutes in white-rotted wood, and were enriched in Alphaproteobacteria, Actinobacteria and Acidobacteria in lower pH brown rot. Our results suggest that wood decomposer strategies may exert significant selection effects on bacteria, or vice versa, among less-abundant taxa that have been overlooked when using abundance as the only measure of influence., (© 2022. The Author(s).)

Background: Previous studies have linked the Mediterranean diet (MED) with improved cardiometabolic health, showing preliminary evidence for a mediating role of the gut microbiome. We recently suggested the Green-Mediterranean (Green-MED) diet as an improved version of the healthy MED diet, with increased consumption of plant-based foods and reduced meat intake. Here, we investigated the effects of MED interventions on the gut microbiota and cardiometabolic markers, and the interplay between the two, during the initial weight loss phase of the DIRECT-PLUS trial., Methods: In the DIRECT-PLUS study, 294 participants with abdominal obesity/dyslipidemia were prospectively randomized to one of three intervention groups: healthy dietary guidelines (standard science-based nutritional counseling), MED, and Green-MED. Both isocaloric MED and Green-MED groups were supplemented with 28g/day walnuts. The Green-MED group was further provided with daily polyphenol-rich green tea and Mankai aquatic plant (new plant introduced to a western population). Gut microbiota was profiled by 16S rRNA for all stool samples and shotgun sequencing for a select subset of samples., Results: Both MED diets induced substantial changes in the community structure of the gut microbiome, with the Green-MED diet leading to more prominent compositional changes, largely driven by the low abundant, "non-core," microorganisms. The Green-MED diet was associated with specific microbial changes, including enrichments in the genus Prevotella and enzymatic functions involved in branched-chain amino acid degradation, and reductions in the genus Bifidobacterium and enzymatic functions responsible for branched-chain amino acid biosynthesis. The MED and Green-MED diets were also associated with stepwise beneficial changes in body weight and cardiometabolic biomarkers, concomitantly with the increased plant intake and reduced meat intake. Furthermore, while the level of adherence to the Green-MED diet and its specific green dietary components was associated with the magnitude of changes in microbiome composition, changes in gut microbial features appeared to mediate the association between adherence to the Green-MED and body weight and cardiometabolic risk reduction., Conclusions: Our findings support a mediating role of the gut microbiome in the beneficial effects of the Green-MED diet enriched with Mankai and green tea on cardiometabolic risk factors., Trial Registration: The study was registered on ClinicalTrial.gov ( NCT03020186 ) on January 13, 2017., (© 2022. The Author(s).)

The particularly interdisciplinary nature of human microbiome research makes the organization and reporting of results spanning epidemiology, biology, bioinformatics, translational medicine and statistics a challenge. Commonly used reporting guidelines for observational or genetic epidemiology studies lack key features specific to microbiome studies. Therefore, a multidisciplinary group of microbiome epidemiology researchers adapted guidelines for observational and genetic studies to culture-independent human microbiome studies, and also developed new reporting elements for laboratory, bioinformatics and statistical analyses tailored to microbiome studies. The resulting tool, called 'Strengthening The Organization and Reporting of Microbiome Studies' (STORMS), is composed of a 17-item checklist organized into six sections that correspond to the typical sections of a scientific publication, presented as an editable table for inclusion in supplementary materials. The STORMS checklist provides guidance for concise and complete reporting of microbiome studies that will facilitate manuscript preparation, peer review, and reader comprehension of publications and comparative analysis of published results., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background & Aims: The gut virome includes eukaryotic viruses and bacteriophages that can shape the gut bacterial community and elicit host responses. The virome can be implicated in diseases, such as irritable bowel syndrome (IBS), where gut bacteria play an important role in pathogenesis. We provide a comprehensive and longitudinal characterization of the virome, including DNA and RNA viruses and paired multi-omics data in a cohort of healthy subjects and patients with IBS., Methods: We selected 2 consecutive stool samples per subject from a longitudinal study cohort and performed metagenomic sequencing on DNA and RNA viruses after enriching for viral-like particles. Viral sequence abundance was evaluated over time, as well as in the context of diet, bacterial composition and function, metabolite levels, colonic gene expression, host genetics, and IBS subsets., Results: We found that the gut virome was temporally stable and correlated with the colonic transcriptome. We identified IBS-subset-specific changes in phage populations; Microviridae, Myoviridae, and Podoviridae species were elevated in diarrhea-predominant IBS, and other Microviridae and Myoviridae species were elevated in constipation-predominant IBS compared to healthy controls. We identified correlations between subsets of the virome and bacterial composition (unclassifiable "dark matter" and phages) and diet (eukaryotic viruses)., Conclusions: We found that the gut virome is stable over time but varies among subsets of patients with IBS. It can be affected by diet and potentially influences host function via interactions with gut bacteria and/or altering host gene expression., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Although nasopharyngeal samples have been considered the gold standard for COVID-19 testing, variability in viral load across different anatomical sites could cause nasopharyngeal samples to be less sensitive than saliva or nasal samples in certain cases. Self-collected samples have logistical advantages over nasopharyngeal samples, making them amenable to population-scale screening. To evaluate sampling alternatives for population screening, we collected nasopharyngeal, saliva, and nasal samples from two cohorts with varied levels and types of symptoms. In a mixed cohort of 60 symptomatic and asymptomatic participants, we found that saliva had 88% concordance with nasopharyngeal samples when tested in the same testing lab ( n  = 41) and 68% concordance when tested in different testing labs ( n  = 19). In a second cohort of 20 participants hospitalized for COVID-19, saliva had 74% concordance with nasopharyngeal samples tested in the same testing lab but detected virus in two participants that tested negative with nasopharyngeal samples on the same day. Medical record review showed that the saliva-based testing sensitivity was related to the timing of symptom onset and disease stage. We find that no sample site will be perfectly sensitive for COVID-19 testing in all situations, and the significance of negative results will always need to be determined in the context of clinical signs and symptoms. Saliva retained high clinical sensitivity for early-stage and presymptomatic COVID-19 while allowing easier collection, minimizing the exposure of health care workers, and need for personal protective equipment and making it a viable option for population-scale testing. IMPORTANCE Methods for COVID-19 detection are necessary for public health efforts to monitor the spread of disease. Nasopharyngeal samples have been considered the best approach for COVID-19 testing. However, alternative samples like self-collected saliva offer advantages for population-scale screening. Meta-analyses of recent studies suggest that saliva is useful for detecting SARS-CoV-2; however, differences in disease prevalence, sample collection, and analysis methods still confound strong conclusions on the utility of saliva compared to nasopharyngeal samples. Here, we find that the sensitivity of saliva testing is related to both the timing of the sample collection relative to symptom onset and the disease stage. Importantly, several clinical vignettes in our cohorts highlight the challenges of medical decision making with limited knowledge of the associations between laboratory test data and the natural biology of infection.