Investigating metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections: protocol for a laboratory-based cross-sectional study

Background: Fungal-bacterial cocolonization and coinfections pose an emerging challenge among patients suspected of having pulmonary tuberculosis (PTB); however, the underlying pathogenic mechanisms and microbiome interactions are poorly understood. Understanding how environmental microbes, such as fungi and bacteria, coevolve and develop traits to evade host immune responses and resist treatment is critical to controlling opportunistic pulmonary fungal coinfections. In this project, we propose to study the coexistence of fungal and bacterial microbial communities during chronic pulmonary diseases, with a keen interest in underpinning fungal etiological evolution and the predominating interactions that may exist between fungi and bacteria. Objective: This is a protocol for a study aimed at investigating the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections through determining and characterizing the burden, etiological profiles, microbial communities, and interactions established between fungi and bacteria as implicated among patients with presumptive PTB. Methods: This will be a laboratory-based cross-sectional study, with a sample size of 406 participants. From each participant, 2 sputa samples (one on-spot and one early morning) will be collected. These samples will then be analyzed for both fungal and bacterial etiology using conventional metabolic and molecular (intergenic transcribed spacer and 16S ribosomal DNA–based polymerase chain reaction) approaches. We will also attempt to design a genome-scale metabolic model for pulmonary microbial communities to analyze the composition of the entire microbiome (ie, fungi and bacteria) and investigate host-microbial interactions under different patient conditions. This analysis will be based on the interplays of genes (identified by metagenomics) and inferred from amplicon data and metabolites (identified by metabolomics) by analyzing the full data set and using specific computational tools. We will also collect baseline data, including demographic and clinical history, using a patient-reported questionnaire. Altogether, this approach will contribute to a diagnostic-based observational study. The primary outcome will be the overall fungal and bacterial diagnostic profile of the study participants. Other diagnostic factors associated with the etiological profile, such as incidence and prevalence, will also be analyzed using univariate and multivariate schemes. Odds ratios with 95% CIs will be presented with a statistical significance set at P<.05. Results: The study has been approved by the Mbarara University Research Ethic Committee (MUREC1/7-07/09/20) and the Uganda National Council of Science and Technology (HS1233ES). Following careful scrutiny, the protocol was designed to enable patient enrollment, which began in March 2022 at Mbarara University Teaching Hospital. Data collection is ongoing and is expected to be completed by August 2023, and manuscripts will be submitted for publication thereafter. Conclusions: Through this protocol, we will explore the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections among patients with presumptive PTB. Establishing key fungal-bacterial cross-kingdom synergistic relationships is crucial for instituting fungal bacterial coinfecting etiology. Trial Registration: ISRCTN Registry ISRCTN33572982; https://tinyurl.com/caa2nw69 International Registered Report Identifier (IRRID): DERR1-10.2196/48014 JMIR Res Protoc 2023;12:e48014 doi:10.2196/4801

The clinical presentations, etiology and factors associated with foot mycoses among patients attending dermatology clinic at Mbarara Regional Referral Hospital, Mbarara, Uganda

Background: Foot mycosis is one of the most common fungal infections of the skin worldwide. The magnitude of the problem in terms of clinical presentations, etiology and associated factors remain as poorly established in low-income countries especially in Uganda.Objective: To study the clinical presentations, etiology and factors associated with foot mycoses among patients attending Mbarara Regional Referral Hospital, Mbarara, Uganda (MRRH). Materials and methods: This was a cross-sectional study that recruited 110 patients attending the skin clinic at MRRH between November 2018 and May 2019. The subjects were examined physically, and skin scrapings of suspected lesions were collected for laboratory processing.Results: Out of 110 suspects for foot mycoses 101 (91.8%) were diagnosed with a fungal. Fungal culture confirmed foot mycosis in 67 (60.9%) of the cases. Of the 67 fungal culture growth, dermatophytes were 39.5%, followed by yeasts with 32.6% and non-dermatophyte molds (NDMs) with 27.9%. The commonest associated factors were age and wearing occlusive shoes.Conclusion: The study reveals that foot mycosis is a common clinical entity at our dermatology clinic and interdigital was the most common clinical presentation, highlighting dermatophytes as the commonest etiological agents. Age and shoe design remain the most significant associated factors of the infection

A bacterial endosymbiont of the fungus Rhizopus microsporus drives phagocyte evasion and opportunistic virulence

This is the final version. Available on open access from Elsevier via the DOI in this recordData and code availability: Genomic sequencing data is available through the Sequence Read Archive (SRA) on NCBI and accession codes for sequences referenced in this work are: R. pickettii: SRA: SRR16523121 and R. microsporus: SRA: SRR16610381. This work did not lead to the generation of any new code.Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.Wellcome TrustBiotechnology and Biological Sciences Research Council (BBSRC)Darwin Trust of EdinburghNational Institutes of Allergy and ImmunologyMedical Research Council (MRC)Royal Societ