key: cord-0727304-5dnjafmr
authors: Ladau, J.; Abuabara, K.; Walker, A. M.; Joachimiak, M. P.; Bansal, I.; Wu, Y.; Hoffman, E. B.; Kuo, C.; Falco, N.; Streich, J.; van der Lan, M. J.; Wainwright, H. M.; Brodie, E. L.; Hess, M.; Jacobson, D. A.; Brown, J. B.
title: The impact of environmental mycobiomes on geographic variation in COVID-19 mortality
date: 2021-12-16
journal: nan
DOI: 10.1101/2021.12.14.21267549
sha: de1b0ec7c6767285cd64f9a93a11bc2d8de98aab
doc_id: 727304
cord_uid: 5dnjafmr

Mortality rates during the COVID-19 pandemic have varied by orders of magnitude across communities in the United States. Individual, socioeconomic, and environmental factors have been linked to health outcomes of COVID-19. It is now widely appreciated that the environmental microbiome, composed of microbial communities associated with soil, water, atmosphere, and the built environment, impacts immune system development and susceptibility to immune-mediated disease. The human microbiome has been linked to individual COVID-19 disease outcomes, but there are limited data on the influence of the environmental microbiome on geographic variation in COVID-19 across populations. To fill this knowledge gap, we used taxonomic profiles of fungal communities associated with 1,135 homes in 494 counties from across the United States in a machine learning analysis to predict COVID-19 Infection Fatality Ratios (the number of deaths caused by COVID-19 per 1000 SARS-CoV-2 infections; 'IFR'). Here we show that exposure to increased fungal diversity, and in particular indoor exposure to outdoor fungi, is associated with reduced SARS-CoV-2 IFR. Further, we identify seven fungal genera that are the predominant drivers of this protective signal and may play a role in suppressing COVID-19 mortality. This relationship is strongest in counties where human populations have remained stable over at least the previous decade, consistent with the importance of early-life microbial exposures. We also assessed the explanatory power of 754 other environmental and socioeconomic factors, and found that indoor-outdoor fungal beta-diversity is amongst the strongest predictors of county-level IFR, on par with the most important known COVID-19 risk factors, including age. We anticipate that our study will be a starting point for further integration of environmental mycobiome data with population health information, providing an important missing link in our capacity to identify vulnerable populations. Ultimately, our identification of specific genera predicted to be protective against COVID-19 mortality may point toward novel, proactive therapeutic approaches to infectious disease.

During the first eighteen months of the global COVID-19 pandemic, more than 176 million people were infected with SARS-CoV-2 and 3.8 million people died directly from it 13, 14 . However, the toll of COVID-19 has varied greatly through both time and across geographical locations: for instance, case-fatality ratios across counties in the United States varied by over four orders of magnitude during the first eight months of the pandemic 15, 16, 13 . Although some of this variation can be explained by demographic, climatic, or social factors, other factors likely also contribute substantially to area-level variation in COVID-19 disease outcomes 1, 17, 18 . Identifying these other factors would be of value for forecasting trajectories of this and future pandemics, informing non-pharmaceutical interventions, and potentially indicating research directions towards novel therapeutic and immunological strategies.

In addition to interactions with pathogenic microbes that cause disease, humans interact constantly throughout their lives with a myriad of non-pathogenic microbes in the environment 19, 20 . These interactions may significantly modulate disease outcomes -for instance, mitigating respiratory illnesses including asthma and allergic disease 21, 7, 22 . While the mechanisms of action vary, beneficial effects of environmental microbes on health outcomes often share the following characteristics: (i) the environmental microbes that have beneficial effects often originate from soils, freshwater environments, plants, and other non-anthropogenic sources, as opposed to the built environment, potentially reflecting a history of human evolutionary adaptation to them 11, 7, 23, 24, 25 ; (ii) environmental microbes reduce disease severity by stimulating immune system development early in life 6, 26, 11 ; and (iii) exposure to a diversity of environmental microbes rather than a single taxon is necessary to confer beneficial effects 7, 23, 25 .

Functional immune response has been reported to be essential for moderating the severity of COVID-19 infection 27, 28 . Prompted by the observations that the environmental micro-biome influences immune system development and function 29 , and that COVID-19 severity correlates with hospital microbiome composition 30 and with dysbiosis of the lung and 31 the gut microbiomes 32,33 , we hypothesized that the environmental microbiome, specifically the environmental mycobiome, is an important factor determining area-level variation in COVID-19 mortality.

To investigate the link between COVID-19 mortality and environmental fungi, we leveraged data on the taxonomic compositions of fungal communities from 1,135 homes across the United States ( Figure E1 ) 34, 29 and data on COVID-19 from across the United States 13, 35 . Each home had paired samples from indoors and outdoors, allowing comparison of the indoor and outdoor fungal communities. Using a novel approach, we also estimated SARS-CoV-2 IFR for each county across the United States, and consequently for each home for which fungal data were available (Supplementary Information). Here, we assume that the variability of the taxonomic compositions within each United States county is smaller than the variability across the entire United States. In addition, we extensively explored the influence of demographic, sociological, climate, and soil factors, some of which are known to influence IFR, fungal community composition, or both.

Most people in the United States interact primarily with microbes indoors, spending an average of 87% of their time in their homes and other constructed environments 36 . For fungi, in particular, there exist important differences between primarily non-pathogenic outdoor species (which are sometimes also found indoors), and pathogenic or opportunistic species that proliferate in damp indoor environments 37 . Hence, we hypothesized that the presence of outdoor fungi in the built environment may be associated with reduced SARS-CoV-2 IFR. To test this hypothesis, we employed a two-tiered strategy. Firstly, we looked for association between relative fungal abundances (at the level of genera) and SARS-CoV-2 IFR using a machine learning approach -iterative Random Forests (iRF). Secondly, we employed a quantile regression strategy to test the prediction that fungal indoor-outdoor beta diversity is most strongly associated with the upper quantiles of IFR -the most severely impacted counties. The iRF analysis found marginal impact of the relative abundances of fungal genera or indoor-outdoor beta diversity after taking into account 754 demographic, sociological, climate, and soil factors (Supplementary Information).

An ablation analysis revealed that after accounting for an expansive collection of other, alternative factors, the impact of fungal genera relative abundances had a statistically significant, albeit small (≈ 1%) effect on mean IFR (Table E1, Figure E7 ). However, our quantile regression analysis revealed that the 75% and all quantiles above of SARS-CoV-2 IFR, were strongly dependent on indoor-outdoor beta diversity. Indeed, indoor-outdoor beta-diversity is associated with reduced SARS-CoV-2 IFR (4.7 or fewer mortalities per 1000 infections; 90th percentile), while dissimilar indoor and outdoor communities (reduced indoor-outdoor beta diversity) are associated with elevated SARS-CoV-2 IFR (12.4 mortalities per 1000 infections; 90th percentile; Figure 1A -B). Hence, while the signal influencing mean IFR is relatively weak, the upper quantiles reveal strong and highly significant associations. These results remained qualitatively the same when the samples from each United States state and Census Division were individually omitted (Figures E2 -E4; Supplementary Information), indicating that the associations we detect are not due to any single geographic region. Therefore, when accounting for the effects of confounding variables (below), high diversity of outdoor fungi present indoors appears to be associated with suppressed COVID-19 mortality, while lower diversity does not appear to confer these benefits.

Multiple lines of evidence indicate that the association between fungal beta-diversity and SARS-CoV-2 IFR suppression is likely causal; that is, that the aforementioned associations are driven by the fungi themselves, rather than other confounding factors, such as demographic and climate variables. First, SARS-CoV-2 IFR early in the pandemic (April 2020 -November 2020) is positively associated with fungal indoor-outdoor beta-diversity -when pharmaceutical interventions were unavailable, but the association is weaker later in the pandemic (December 2020 -January 2021) when vaccination began and other pharmaceutical interventions were widely administered ( Figure 1C) . Second, although a machine learning analysis associating IFR to fungal beta-diversity and 754 climate, demographic, housing, and COVID-19 policy-related factors was able to discern only a weak effect uniquely attributable to fungal beta diversity (Table E1 , Figure E7 , and Supplementary Information), the differences between indoor and outdoor fungal communities that are associated with SARS-CoV-2 IFR is explained primarily by soil edaphic factors -particularly pH -accounting for over 40% of the variance, while other factors that are known to be related to COVID-19, such as demographic and other non-climate factors, explain less than 30% of the variance combined ( Figure 1D ). This is intriguing, as it consistent with a causal model where soil edaphic factors affect COVID-19 severity by affecting microbial distributions, which are well-documented to be driven by soil characteristics, including pH 38, 39 . Third, causal inference incorporating both fungal indoor-outdoor beta-diversity, demographic, climate, and other predictors indicate a causal link between indoor-outdoor fungal beta-diversity and SARS-CoV-2 IFR even when these other potentially confounding variables are considered (p < 0.001, Supplementary Information).

To better understand the components of the fungal communities that contribute to the signal indicating suppression of SARS-CoV-2 IFR we employed a novel, beta-diversity correlation partitioning method (Supplementary Information). At least four of the following fungal genera play a key role in suppressing SARS-CoV-2 IFR: Alternaria, Aspergillus, Epicoccum, Eurotium, Toxicocladosporium, and Wallemia spp., and a novel Mycosphaerellaceae genus (some of these genera have correlated distributions making effects of individual genera indistinguishable; Figure 2A and 2C). High relative abundance of three of these genera both indoors and outdoors is necessary to detect SARS-CoV-2 IFR suppression: high relative abundance of Alternaria, Epicoccum, and Mycosphaerellaceae spp. just indoors -consistent with a primarily indoor origin -or outdoors -consistent with low indoor exposures -is insufficient ( Figure 2B and Figure E5 ).

Our results pointing to beneficial effects of these seven genera for reducing COVID-19 mortality are novel. In many contexts, these genera are known to affect health negatively: for example, Alternaria, Wallemia, and Aspergillus spp. can be negatively associated with Irritable Bowel Syndrome, Ulcerative Colitis, and Keratitis, respectively 40, 41 (Table E2) . Moreover, two species of Aspergillus can negatively affect COVID-19 outcomes 40 . However, these same fungi can positively affect diseases as well; for example, the three aforementioned genera can improve outcomes of oral cancer, Clostridium difficile-related diseases, and Seb-4 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. , COVID-19 mortality is reduced by over a factor of two compared to homes where the communities are dissimilar (high beta-diversity). Each point represents a United States county; shading indicates point density. Although the 90th percentile is sensitive to spatial autocorrelation, these trends are not driven by a single state or region of the United States ( Figures E2 -E4) . (B) The reductions in COVID-19 mortality (as measured by the standardized effect size, SES) are greatest in the upper quantiles of the COVID-19 mortality distribution, suggesting that outdoor fungi are sufficient but not necessary to reduce COVID-19 mortality. (C) The association between COVID-19 mortality and fungal beta-diversity is strongest early in the pandemic, before December 2020 when vaccination became widespread. Circles with black outlines indicate significant associations (SES> 3). (D) Fungal beta-diversity is a strong predictor of suppression of SARS-CoV-2 IFR relative to other variables [column graph; points represent individual variables, columns show means; correlations give the association between the given variable and the windowed 75th percentile of IFR (see Supplementary Information) ]. Moreover, fungal beta-diversity is most strongly associated with soil pH and other environmental variables (inset bar graph), suggesting that it is not a proxy for demographic and other variables that are known to effect COVID-19 mortality.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted December 16, 2021. ;  orrheic dermatitis, respectively 40 (Table E2) . Environmental and endemic microbes may also positively effect COVID-19 outcomes 42, 43 . Different species from the same genus can have differential effects based on context, for example host genetics and age 44 , health status 45 , and interactions with other microbial exposures 46 . Fully understanding the context-dependence of the effects of environmental fungi on COVID-19 and other human disease outcomes is an area requiring further study.

In a general context, immune system development has been documented to be promoted by synergistic effects from the exposure to diverse microbes, as opposed to the effects of exposure to individual or a few microbes 7, 23, 25 . Consistent with these observations, the seven genera that we identified reveal positive synergistic effects: indoor-outdoor differences in relative abundance for genera individually tend to be poor predictors of SARS-CoV-2 IFR suppression (cross validation R 2 between 0 and 0.07, median 0), while the beta-diversity of these genera taken together is strongly predictive (cross validation R 2 between 0.02 and 0.43, median 0.19; Figure 2C ).

We hypothesized the association between fungal diversity and SARS-CoV-2 IFR suppression will be strongest in regions where the human population has been stable for the last ten years or more, because in these regions, people will have been most likely to have been exposed to the fungi occurring there when their immune systems were developing early in their lives 6, 11, 47 . Consistent with this prediction, in locations where the human population remained stable from 2010 to 2017, fungal beta diversity is more strongly associated with SARS-CoV-2 IFR suppression than in regions where it has fluctuated due to population turnover ( Figure 3) .

Collectively, these results accurately map locations where the occurrence of outdoor fungi in the indoor environment is forecast to most strongly suppress COVID-19 mortality in the United States: the Desert Southwest, Intermountain West, and Upper Midwest; a region that broadly corresponds with part of the country where soils are more alkaline, the primary predictor of indoor-outdoor fungal beta-diversity in our analysis (Figure 4) . Soil pH is known to correlate with microbial diversity; it is also related to the water balance of ecosystems (mean annual precipitation relative to mean annual potential evapotranspiration) 48 , which in turn predicts fine dust aerosolization 49 . Thus along with climate 32 , in the United States pH may serve as a signature of microbial composition and propensity for microbial transport into homes via dust. In fact, a correlative effect has been observed between bioaerosol counts, especially mold spores, and respiratory disease incidence. One offered hypothesis for the association between mold spores and respiratory disease incidence is competition for Toll-like receptor 4 (TLR4) binding 50 .

To reduce fatalities and limit the socioeconomic impact of the COVID-19 pandemic, and future pandemics a comprehensive understanding of factors affecting area-level variation in COVID-19 disease outcomes is essential 5 . We develop a novel strategy to address challenges in estimating SARS-CoV-2 IFR (as opposed to the case fatality ratio and other measures of COVID-19 severity), to identify factors driving SARS-CoV-2 IFR at large spatial scales. Here we find that environmental fungal communities, particularly indoor-outdoor beta diversity, are predictive of geographic variation in COVID-19 infection fatality ratios, above and beyond many other social and environmental factors. Our analyses indicate that exposure to high levels of outdoor fungi in homes is protective. If, as supported by our analysis, there is a causal relationship between long-term fungal exposures and SARS-CoV-2 IFR, 6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In regions where soils tend to be basic (red background shading), indoor-outdoor fungal beta-diversity tends to be low, and fungal suppression of SARS-CoV-2 IFR is high (red dots). By contrast, the opposite trend holds in regions with acidic soils (blue background shading and dots); here, where fungal suppression of SARS-CoV-2 IFR is lessened, SARS-CoV-2 IFR can be high or low depending on whether other factors (e.g., climate, demographics) reduce SARS-CoV-2 IFR.

8 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101/2021.12.14.21267549 doi: medRxiv preprint then the environmental mycobiome constitutes an important missing link in our capacity to identify human populations that are vulnerable to poor outcomes from COVID-19. If, on the other hand, despite our extensive survey of environmental and socioeconomic predictors, we are missing as-yet unidentified confounding factors, our study underscores the utility of the environmental mycobiome as a biosensor 51 . Widespread beneficial effects of environmental fungi may not be specific to COVID-19; limited data support similar findings for childhood allergic disease and other viral diseases 52, 53, 54 , and it may be relevant for other autoimmune and immune-mediated diseases. Our results provide a foundation for research on the role of fungi and fungal interactions on the immune system, an important addition to a body of literature that has focused primarily on bacteria to date 55, 56 . Recent advances in sequencing and classification of environmental fungi will enhance future efforts in this area 57 , and underscore the importance of biosurveillance for this and future pandemics.

Details of all methods are provided in Supplementary Information.

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The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101/2021.12.14.21267549 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101/2021.12.14.21267549 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101/2021.12.14.21267549 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 16, 2021. ;

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This manuscript has been coauthored by UT-Battelle, LLC under contract no. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan, last accessed September 16, 2020 The authors declare no competing interests.

Supplementary Information is available for this paper. Correspondence and requests for materials should be addressed to Joshua Ladau.