Functional, imaging, and respiratory evaluation (FIRE) of patients post-hospitalization for COVID-19: protocol for a pilot observational study.

INTRODUCTION: We describe a protocol for FIRE CORAL, an observational cohort study that examines the recovery from COVID-19 disease following acute hospitalization with an emphasis on functional, imaging, and respiratory evaluation. METHODS AND ANALYSIS: FIRE CORAL is a multicenter prospective cohort study of participants recovering from COVID-19 disease with in-person follow-up for functional and pulmonary phenotyping conducted by the National Heart, Lung and Blood Institute (NHLBI) Prevention and Early Treatment of Acute Lung Injury (PETAL) Network. FIRE CORAL will include a subset of participants enrolled in Biology and Longitudinal Epidemiology of PETAL COVID-19 Observational Study (BLUE CORAL), an NHLBI-funded prospective cohort study describing the clinical characteristics, treatments, biology, and outcomes of hospitalized patients with COVID-19 across the PETAL Network. FIRE CORAL consists of a battery of in-person assessments objectively measuring pulmonary function, abnormalities on lung imaging, physical functional status, and biospecimen analyses. Participants will attend and perform initial in-person testing at 3 to 9 months after hospitalization. The primary objective of the study is to determine the feasibility of longitudinal assessments investigating multiple domains of recovery from COVID-19. Secondarily, we will perform descriptive statistics, including the prevalence and characterization of abnormalities on pulmonary function, chest imaging, and functional status. We will also identify potential clinical and biologic factors that predict recovery or the occurrence of persistent impairment of pulmonary function, chest imaging, and functional status. ETHICS AND DISSEMINATION: FIRE CORAL is approved via the Vanderbilt University central institutional review board (IRB) and via reliance agreement with the site IRBs. Results will be disseminated via the writing group for the protocol committee and reviewed by the PETAL Network publications committee prior to publication. Data obtained via the study will subsequently be made publicly available via NHLBI's biorepository. STRENGTHS AND LIMITATIONS OF THE STUDY: Strengths: First US-based multicenter cohort of pulmonary and functional outcomes in patients previously hospitalized for COVID-19 infection Longitudinal biospecimen measurement allowing for biologic phenotyping of abnormalities Geographically diverse cohort allowing for a more generalizable understanding of post-COVID pulmonary sequela Limitations: Selected cohort given proximity to a participating center Small cohort which may be underpowered to identify small changes in pulmonary function.

Improved method for rapid and accurate isolation and identification of Streptococcus mutans and Streptococcus sobrinus from human plaque samples.

Mutans streptococci (MS), specifically Streptococcus mutans (SM) and Streptococcus sobrinus (SS), are bacterial species frequently targeted for investigation due to their role in the etiology of dental caries. Differentiation of S. mutans and S. sobrinus is an essential part of exploring the role of these organisms in disease progression and the impact of the presence of either/both on a subject's caries experience. Of vital importance to the study of these organisms is an identification protocol that allows us to distinguish between the two species in an easy, accurate, and timely manner. While conducting a 5-year birth cohort study in a Northern Plains American Indian tribe, the need for a more rapid procedure for isolating and identifying high volumes of MS was recognized. We report here on the development of an accurate and rapid method for MS identification. Accuracy, ease of use, and material and time requirements for morphological differentiation on selective agar, biochemical tests, and various combinations of PCR primers were compared. The final protocol included preliminary identification based on colony morphology followed by PCR confirmation of species identification using primers targeting regions of the glucosyltransferase (gtf) genes of SM and SS. This method of isolation and identification was found to be highly accurate, more rapid than the previous methodology used, and easily learned. It resulted in more efficient use of both time and material resources.

Genotypic characterization and comparison of Streptococcus mutans in American Indian and Southeast Iowa children.

Early childhood caries is a complex health care issue that has a multifactorial etiology. One aspect of this etiology is the colonization and propagation of acidogenic bacteria at an early age. There have been several bacterial species associated with caries but 1 common species is Streptococcus mutans. Here, we describe genotypic diversity and commonality of Streptococcus mutans recovered from children representing 2 groups with similar socioeconomic demographics: a Northern Plains American Indian Tribe and a Southeast Iowa population. Forty 36-month-old American Indian children were selected from a cohort of 239 mothers and children, and forty 2- to 5-year-old children from Southeast Iowa were selected to compare the genotypic profiles of Streptococcus mutans recovered from each child's plaque. S. mutans isolates were selected from whole mouth plaque samples; DNA was extracted and amplified via AP-PCR to show specific genotype patterns. These patterns were compared with GelComparIIv6.5 gel analysis software. We found 18 distinct genotypes from 524 isolates; 13 of which were common between the 2 communities. Five genotypes were unique to only the American Indian children while the Southeast Iowa children harbored no unique genotypes. Although the American Indian children had some genotypes that were not present in the Southeast Iowa children, these were not widely distributed among the community. Furthermore, the levels of genotypic diversity and commonality were similar between the 2 populations. This study sets the groundwork for a comprehensive comparison of genotypes and caries among larger subsections of both populations.

Genotypic characterization of initial acquisition of Streptococcus mutans in American Indian children.

BACKGROUND: Severe-early childhood caries (S-ECC) is one of the most common infectious diseases in children and is prevalent in lower socio-economic populations. American Indian children suffer from the highest levels of S-ECC in the United States. Members of the mutans streptococci, Streptococcus mutans, in particular, are key etiologic agents in the development of caries. Children typically acquire S. mutans from their mothers and early acquisition is often associated with higher levels of tooth decay. METHODS: We have conducted a 5-year birth cohort study with a Northern Plains Tribe to determine the temporality and fidelity of S. mutans transmission from mother to child in addition to the genotypic diversity of S. mutans in this community. Plaque samples were collected from 239 mother/child dyads at regular intervals from birth to 36 months and S. mutans were isolated and genotyped by arbitrarily primed-polymerase chain reaction (AP-PCR). RESULTS: Here we present preliminary findings from a subset of the cohort. The focus for this paper is on initial acquisition events in the children. We identified 17 unique genotypes in 711 S. mutans isolates in our subset of 40 children, 40 mothers and 14 primary caregivers. Twelve of these genotypes were identified in more than one individual. S. mutans colonization occurred by 16 months in 57.5% of the children and early colonization was associated with higher decayed, missing and filled surface (DMFS) scores (p=0.0007). Children colonized by S. mutans shared a common genotype with their mothers 47.8% of the time. While multiple genotypes were common in adults, only 10% of children harbored multiple genotypes. CONCLUSION: These children acquire S. mutans at an earlier age than the originally described 'window of infectivity' and often, but not exclusively, from their mothers. Early acquisition is associated with both the caries status of the children and the mothers.

Cariogenicity of Streptococcus mutans glucan-binding protein deletion mutants.

Streptococcus mutans is a principal etiologic agent in the development of dental caries due to its exceptional aciduric and acidogenic properties, and its ability to adhere and accumulate in large numbers on tooth surfaces in the presence of sucrose. Sucrose-dependent adherence is mediated by glucans, polymers of glucose synthesized from sucrose by glucosyltransferase (Gtf) enzymes. S. mutans makes several proteins that have the property of binding glucans. We hypothesized that three of these glucan-binding proteins (Gbps), Gbps A, C and D, contribute to the cariogenicity of S. mutans. A specific pathogen-free rat model was used to compare the cariogenicity of S. mutans UA130 and a panel of mutants with individual or multiple gbp gene deletions. The mutants were also evaluated in vitro for properties related to cariogenicity, such as acidogenicity, aciduricity, and adhesion to glucan. Only a subset of Gbp mutants were attenuated for cariogenicity, with the combined loss of Gbps A and C most affecting smooth surface caries. The attenuation of Gbp mutant strains was unlikely due to differences in acid-related properties since the mutants were at least as acidogenic and acid-tolerant as the parental strain. Additionally, loss of Gbps did not reduce adhesion to a pre-formed biofilm of S. sanguinis. Analyses of the caries data with in vitro biofilm properties previously determined for the mutant panel found correlations between cariogenicity and biofilm depth and substratum coverage. It is concluded that Gbps contribute to the cariogenicity of S. mutans through a mechanism that may involve alteration of biofilm architecture.

Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture.

Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases that catalyze the synthesis of glucan and the nonglucosyltransferase glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest that these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties were quantified by the biofilm quantification software comstat. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.