The Vaginal Microbiome: Disease, Genetics and the Environment

The vagina is an interactive interface between the host and the environment. Its surface is covered by a protective epithelium colonized by bacteria and other microorganisms. The ectocervix is nonsterile, whereas the endocervix and the upper genital tract are assumed to be sterile in healthy women. Therefore, the cervix serves a pivotal role as a gatekeeper to protect the upper genital tract from microbial invasion and subsequent reproductive pathology. Microorganisms that cross this barrier can cause preterm labor, pelvic inflammatory disease, and other gynecologic and reproductive disorders. Homeostasis of the microbiome in the vagina and ectocervix plays a paramount role in reproductive health. Depending on its composition, the microbiome may protect the vagina from infectious or non-infectious diseases, or it may enhance its susceptibility to them. Because of the nature of this organ, and the fact that it is continuously colonized by bacteria from birth to death, it is virtually certain that this rich environment evolved in concert with its microbial flora. Specific interactions dictated by the genetics of both the host and microbes are likely responsible for maintaining both the environment and the microbiome. However, the genetic basis of these interactions in both the host and the bacterial colonizers is currently unknown. _Lactobacillus_ species are associated with vaginal health, but the role of these species in the maintenance of health is not yet well defined. Similarly, other species, including those representing minor components of the overall flora, undoubtedly influence the ability of potential pathogens to thrive and cause disease. Gross alterations in the vaginal microbiome are frequently observed in women with bacterial vaginosis, but the exact etiology of this disorder is still unknown. There are also implications for vaginal flora in non-infectious conditions such as pregnancy, pre-term labor and birth, and possibly fertility and other aspects of women’s health. Conversely, the role of environmental factors in the maintenance of a healthy vaginal microbiome is largely unknown. To explore these issues, we have proposed to address the following questions:

*1.	Do the genes of the host contribute to the composition of the vaginal microbiome?* We hypothesize that genes of both host and bacteria have important impacts on the vaginal microbiome. We are addressing this question by examining the vaginal microbiomes of mono- and dizygotic twin pairs selected from the over 170,000 twin pairs in the Mid-Atlantic Twin Registry (MATR). Subsequent studies, beyond the scope of the current project, may investigate which host genes impact the microbial flora and how they do so.
*2.	What changes in the microbiome are associated with common non-infectious pathological states of the host?* We hypothesize that altered physiological (e.g., pregnancy) and pathologic (e.g., immune suppression) conditions, or environmental exposures (e.g., antibiotics) predictably alter the vaginal microbiome. Conversely, certain vaginal microbiome characteristics are thought to contribute to a woman’s risk for outcomes such as preterm delivery. We are addressing this question by recruiting study participants from the ~40,000 annual clinical visits to women’s clinics of the VCU Health System.
*3.	What changes in the vaginal microbiome are associated with relevant infectious diseases and conditions?* We hypothesize that susceptibility to infectious disease (e.g. HPV, _Chlamydia_ infection, vaginitis, vaginosis, etc.) is impacted by the vaginal microbiome. In turn, these infectious conditions clearly can affect the ability of other bacteria to colonize and cause pathology. Again, we are exploring these issues by recruiting participants from visitors to women’s clinics in the VCU Health System.

Three kinds of sequence data are generated in this project: i) rDNA sequences from vaginal microbes; ii) whole metagenome shotgun sequences from vaginal samples; and iii) whole genome shotgun sequences of bacterial clones selected from vaginal samples. The study includes samples from three vaginal sites: mid-vaginal, cervical, and introital. The data sets also include buccal and perianal samples from all twin participants. Samples from these additional sites are used to test the hypothesis of a per continuum spread of bacteria in relation to vaginal health. An extended set of clinical metadata associated with these sequences are deposited with dbGAP. We have currently collected over 4,400 samples from ~100 twins and over 450 clinical participants. We have analyzed and deposited data for 480 rDNA samples, eight whole metagenome shotgun samples, and over 50 complete bacterial genomes. These data are available to accredited investigators according to NIH and Human Microbiome Project (HMP) guidelines. The bacterial clones are deposited in the Biodefense and Emerging Infections Research Resources Repository ("http://www.beiresources.org/":http://www.beiresources.org/). 

In addition to the extensive sequence data obtained in this study, we are collecting metadata associated with each of the study participants. Thus, participants are asked to complete an extensive health history questionnaire at the time samples are collected. Selected clinical data associated with the visit are also obtained, and relevant information is collected from the medical records when available. This data is maintained securely in a HIPAA-compliant data system as required by VCU’s Institutional Review Board (IRB). The preponderance of these data (i.e., that judged appropriate by NIH staff and VCU’s IRB are deposited at dbGAP ("http://www.ncbi.nlm.nih.gov/gap":http://www.ncbi.nlm.nih.gov/gap). Selected fields of this data have been identified by NIH staff as ‘too sensitive’ and are not available in dbGAP. Individuals requiring access to these data fields are asked to contact the PI of this project or NIH Program Staff. 
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The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

A Sarcina bacterium linked to lethal disease in sanctuary chimpanzees in Sierra Leone

Human and animal infections with bacteria of the genus Sarcina (family Clostridiaceae) are associated with gastric dilation and emphysematous gastritis. However, the potential roles of sarcinae as commensals or pathogens remain unclear. Here, we investigate a lethal disease of unknown etiology that affects sanctuary chimpanzees (Pan troglodytes verus) in Sierra Leone. The disease, which we have named “epizootic neurologic and gastroenteric syndrome” (ENGS), is characterized by neurologic and gastrointestinal signs and results in death of the animals, even after medical treatment. Using a case-control study design, we show that ENGS is strongly associated with Sarcina infection. The microorganism is distinct from Sarcina ventriculi and other known members of its genus, based on bacterial morphology and growth characteristics. Whole-genome sequencing confirms this distinction and reveals the presence of genetic features that may account for the unusual virulence of the bacterium. Therefore, we propose that this organism be considered the representative of a new species, named “Candidatus Sarcina troglodytae”. Our results suggest that a heretofore unrecognized complex of related sarcinae likely exists, some of which may be highly virulent. However, the potential role of “Ca. S. troglodytae” in the etiology of ENGS, alone or in combination with other factors, remains a topic for future research.Peer Reviewe

Characterizing Phage Genomes for Therapeutic Applications

Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates

Immunological and Genetic Investigation of SARS-CoV-2 Reinfection in an Otherwise Healthy, Young Marine Recruit

We used epidemiologic and viral genetic information to identify a case of likely reinfection in an otherwise healthy, young Marine recruit enrolled in the prospective, longitudinal COVID-19 Health Action Response for Marines (CHARM) study, and we paired these findings with serological studies. This participant had a positive RT-PCR to SARS-CoV-2 upon routine sampling on study day 7, although he was asymptomatic at that time. He cleared the infection within seven days. On study day 46, he had developed symptoms consistent with COVID-19 and tested positive by RT-PCR for SARS-CoV-2 again. Viral whole genome sequencing was conducted from nares swabs at multiple time points. The day 7 sample was determined to be lineage B.1.340, whereas both the day 46 and day 49 samples were B.1.1. The first positive result for anti-SARS-CoV-2 IgM serology was collected on day 49 and for IgG on day 91. This case appears most consistent with a reinfection event. Our investigation into this case is unique in that we compared sequence data from more than just paired specimens, and we also assayed for immune response after both the initial infection and the later reinfection. These data demonstrate that individuals who have experienced an infection with SARS-CoV-2 may fail to generate effective or long-lasting immunity, similar to endemic human beta coronaviruses

An Observational Study of Sepsis in Takeo Province Cambodia: An in-depth examination of pathogens causing severe infections.

The world's most consequential pathogens occur in regions with the fewest diagnostic resources, leaving the true burden of these diseases largely under-represented. During a prospective observational study of sepsis in Takeo Province Cambodia, we enrolled 200 patients over an 18-month period. By coupling traditional diagnostic methods such as culture, serology, and PCR to Next Generation Sequencing (NGS) and advanced statistical analyses, we successfully identified a pathogenic cause in 46.5% of our cohort. In all, we detected 25 infectious agents in 93 patients, including severe threat pathogens such as Burkholderia pseudomallei and viral pathogens such as Dengue virus. Approximately half of our cohort remained undiagnosed; however, an independent panel of clinical adjudicators determined that 81% of those patients had infectious causes of their hospitalization, further underscoring the difficulty of diagnosing severe infections in resource-limited settings. We garnered greater insight as to the clinical features of severe infection in Cambodia through analysis of a robust set of clinical data

SARS-CoV-2 Infections in Vaccinated and Unvaccinated Populations in Camp Lemonnier, Djibouti, from April 2020 to January 2022

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the disparity between developed and developing countries for infectious disease surveillance and the sequencing of pathogen genomes. The majority of SARS-CoV-2 sequences published are from Europe, North America, and Asia. Between April 2020 and January 2022, 795 SARS-CoV-2-positive nares swabs from individuals in the U.S. Navy installation Camp Lemonnier, Djibouti, were collected, sequenced, and analyzed. In this study, we described the results of genomic sequencing and analysis for 589 samples, the first published viral sequences for Djibouti, including 196 cases of vaccine breakthrough infections. This study contributes to the knowledge base of circulating SARS-CoV-2 lineages in the under-sampled country of Djibouti, where only 716 total genome sequences are available at time of publication. Our analysis resulted in the detection of circulating variants of concern, mutations of interest in lineages in which those mutations are not common, and emerging spike mutations