Background/Objectives

According to “Regaining Arctic Dominance: The U.S. Army in the Arctic,” published in January 2021, the Arctic is warming faster than the rest of the earth. Arctic surface temperatures have increased by as much as three times the global average during the period 1971-2019 and as centuries-old layers of permafrost thaw. Thawing permafrost is leading to profound changes in the Arctic that will significantly impact human health, ecosystems, infrastructure, communities, and economies. The scope of work for this project is to anticipate and mitigate the impact of the changing environment on military operations in the Arctic by examining potential pathogen reanimation from the thawing permafrost. The objective of this work is to provide the military with information to enable better protection of warfighters in the Arctic theater. There is evidence that thawing permafrost can release trapped pathogens, to which soldiers and civilians may not have previously been exposed. Previously frozen bacteria and viruses (e.g., Variola virus (smallpox), Brucella suis (brucellosis), and Bacillus anthracis (anthrax)) have the potential to re-emerge due to changing climate conditions. To assess potential health threats, we are identifying pathogens that may be released from thawing permafrost from soil core and water samples near military installations in Alaska.

Approach/Activities

To date, environmental sampling consisted of 24 intact soil core samples collected in winter (March 2023), and 240 surface water and soil samples, respectively, collected over 4 months during the summer (June–Sept. 2023). Samples were collected in three primary areas on military installations in Alaska, with 10 soil and water samples collected per site per month, and with stratified-random sampling locations. Intact soil cores were collected using specialized coring equipment designed specifically for sampling in frozen soils or permafrost. Four core segments (0–1 m and 1–2 m) at each location were collected. Samples were flash-frozen to -80°C immediately after collection and shipped frozen to the laboratory for microbial and genomic analyses. All samples were sequenced using three methods: 16S sequencing, whole genome sequencing (WGS), and viral RNA targeted hybridization to identify and quantify a broad view of microbial community composition. For identified pathogenic organisms of concern, attempts to culture organisms are being made to assess viability. 16S sequencing results were analyzed by QIIME2. Whole genome sequencing was analyzed by UltraSEQ. The viral hybridization results were analyzed by the OneCodex bioinformatic pipeline that is a companion software for the panel. Pathogens found by more than one sequencing method were confirmed by quantitative polymerase chain reaction (PCR) (qPCR). Culturing method development is underway to assess viability of pathogens identified by sequencing. The project is anticipated to be completed within a three-year period, with additional bouts of environmental sampling and laboratory analysis planned.

Results/Lessons Learned

Accessibility and soil characteristics of the areas played a significant role in selecting the sampling sites. One core from each sample site was characterized for soil descriptions by the Natural Resource Conservation Service (NRCS). Results indicated soils are acidic and differ per location and likely contain permafrost. To date, sequencing on the permafrost cores and summer soil and water samples has been completed and data analysis is underway. From the core samples, 13 unique human pathogens—predominantly opportunistic, Enterobacter, and respiratory infectious pathogens—were identified by at least two methods. Legionella pneumophilia (the bacteria that can cause Legionnaire’s disease) was detected in most core samples by 16S and/or WGS and confirmed by qPCR. Results from the permafrost core viral hybridization sequencing showed that only one sample contained a virus, human papillomavirus 20, above the limit of detection. Data analysis results for the summer soil and water samples are ongoing. We developed an information triage system that prioritizes which organisms, identified by genomics sequencing, should be confirmed by qPCR. This system considers properties of the pathogen (e.g., causes respiratory disease, high transmissibility) and the number of sequencing methods that detected the pathogen. Pathogens which are identified by both 16S sequencing at the genus level and WGS that cause diseases with potential for an outbreak that could affect warfighter readiness are then selected for confirmation by qPCR. Culturing work to date has focused on developing defensible methods to confidently detect viable organisms in samples where sequencing identified genetic material. It was determined that direct culturing does not provide sufficient sensitivity or specificity, as the limit of detection was far too high to be acceptable. Instead, enrichment-based viability PCR assays were developed and successfully validated for Bacillus anthracis and Yersinia pestis, two potential candidates in the Arctic environment. Enrichment-based viability PCR allows for detection of as few as 10 to 100 viable organisms in samples. Should additional organisms need to be added to the target list, the general framework of the assay is sufficiently flexible to permit this with minimal additional development work. Our research will be critical in establishing processes to mitigate health risks related to climate change and thawing permafrost to ensure sustainable military operations in Arctic environments. This process could become an operational planning and execution tool to maximize medical readiness and to assess health risks in future operational environments.

Funding Source: MTEC Medical Technology Enterprise Consortium (MTEC), Military Performance Advancement Initiative (MPAI) under the U.S. Army out of Fort Detrick Maryland. Grant/contract number: MTEC-22-02-MPAI-050. Disclaimer: The views expressed in this news release/article are those of the authors and may not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.