Fungal genomics in respiratory medicine: what, how and when?

Respiratory infections caused by fungal pathogens present a growing global healthconcern and are a major cause of death in immunocompromised patients. Worryingly,coronavirus disease-19 (COVID-19) resulting in acute respiratory distress syndrome,has been shown to predispose some patients to fungal co-infection and secondarypulmonary aspergillosis. Aspergillosis is most commonly caused by the fungalpathogen Aspergillus fumigatus and primarily treated using the triazole drug group,however in recent years, this fungus has been rapidly gaining resistance against theseantifungals. This is of serious clinical concern as multi-azole resistant forms ofaspergillosis have a higher risk of mortality when compared against azole-susceptibleinfections. With the increasing numbers of COVID-19 and other classes ofimmunocompromised patients, early diagnosis of fungal infections is critical to ensuringpatient survival. However, time-limited diagnosis is difficult to achieve with currentculture-based methods. Advances within fungal genomics have enabled moleculardiagnostic methods to become a fast, reproducible, and cost-effective alternative fordiagnosis of respiratory fungal pathogens and detection of antifungal resistance. Herewe describe what techniques are currently available within molecular diagnostics, howthey work and when they have been used

Seroprevalence of HIV in Patients Attending VCTC in a Tertiary Care Hospital and Spectrum of Opportunistic Infections and Profile of CD4 Counts among AIDS patients and Molecular Characterization of HIV.

INTRODUCTION : Infection with Human Immuno Deficiency Virus and its end stage Acquired Immuno Deficiency Syndrome are the major public health challenges of modern times. The HIV positive patients are extremely susceptible to a variety of opportunistic infections which cause morbidity and hospitalization. Though curative treatment for HIV is not available at present, we can minimize the HIV infection by early screening and health education. AIM & OBJECTIVES : To study the seroprevalence of HIV infection in Thanjavur by subjecting the serum samples to Rapid Card tests and confirm by ELISA. To determine the CD4 counts of the reactive patients. To categorise the cases according to the presenting complaints and screen for Opportunistic Infections and do Molecular Characterisation for HIV-1. MATERIALS & METHODS : All cases were screened by COMB-AIDS kit at VCTC, TMCH, Thanjavur. Those samples which test reactive to COMB-AIDS kit are subjected to HIV Triline, HIV Trispot & ELISA.CD4 counts of the reactive patients were detected. Zeihl–Neelsen staining of sputum, culture of oral swabs for Candida, Toxoplasma, HSV-2 screening by ELISA, Cryptococcal latex agglutination test were done for reactive cases.Molecular Characterisation of HIV-1 was done for 10 samples. RESULTS : Seroprevalence of HIV was 2.8%.Oral Candidiasis (39.02%) emerged as the most common Opportunistic Infection followed by Pulmonary Tuberculosis (28.03%), Herpes Simplex Virus -2 (14.45%) Toxoplasmosis (5.78%) and Cryptococcosis (3.41%). All 10 samples answered positive in PCR

Cryptococcal Meningitis in Viet Nam

Cryptococcal neoformans is the commonest cause of invasive fungal disease in the world, causing up to 1.5 million cases of meningitis each year, and 1 million deaths. The aims of this thesis were to understand the differences in cryptococcal meningitis in Viet Nam in relation to the HIV infection status of the host, focussing on disease clinical phenotype, prognostic factors and the molecular epidemiology of the infecting isolates. In addition, the thesis examines the antifungal susceptibilities of Vietnamese Cryptococcus spp, how these vary by species and time, and the clinical utility of their measurement. Patients drawn from two prospective cohorts (HIV infected and HIV uninfected) form the basis of the thesis. The HIV uninfected patients are predominantly immunocompetent. Clinical and investigational findings are compared in relation to the literature. HIV patients had shorter durations of history and more frequent headache, but there were few differences in clinical presentation. Previously published prognostic factors were reassessed; only 3 of 40 (altered consciousness, cerebrospinal fluid (CSF) white cell count, and CSF cryptococcal antigen titre) were independently associated with outcome. Mortality was significantly higher in HIV infected patients, but the shapes of the survival curves were similar for both groups, most deaths occurring in the first 4 weeks of treatment. Amplified Fragment Length Polymorphism analysis was used to define the relationships between HIV positive and negative strains - a clade of C. neoformans var grubii with increased propensity to cause disease in immunocompetent patients was identified. The Sensititre® YeastOne® system was used to determine MICs of 7 antifungal drugs. The susceptibility of C. neoformans has decreased significantly over a 13 year period. No relationship with the rate of clearance of yeast from CSF and antifungal MIC was found, but raised amphotericin B MIC after 48 hours incubation may be associated with early death and warrants further investigation

Transcriptional Profiling of Patient Isolates Identifies a Novel TOR/Starvation Regulatory Pathway in Cryptococcal Virulence.

Human infection with Cryptococcus causes up to a quarter of a million AIDS-related deaths annually and is the most common cause of nonviral meningitis in the United States. As an opportunistic fungal pathogen, Cryptococcus neoformans is distinguished by its ability to adapt to diverse host environments, including plants, amoebae, and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified high expression levels of an alternative carbon nutrient transporter gene, STL1, to be associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel multigene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the global regulator of virulence gene VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein, Ecm15, in turn required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low-nutrient, alternative carbon sources, facilitating virulence in mice. This study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host.IMPORTANCECryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of nonviral meningitis in the United States. The fungus is carried in about 10% of the adult population and, after reactivation, causes disease in a wide variety of immunosuppressed individuals, including the HIV infected and patients receiving transplant conditioning, cancer therapy, or corticosteroid therapy for autoimmune diseases. The fungus is widely carried in the soil but can also cause infections in plants and mammals. However, the mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to inhabit these diverse environments. Further studies identified a nutrient transporter gene, STL1, to be upregulated under low nutrients and to be associated with early fungicidal activity, a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in adaptation to the human host may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus

Establishment of a consensus protocol to explore the brain pathobiome in patients with mild cognitive impairment and Alzheimer\u27s disease: Research outline and call for collaboration.

Microbial infections of the brain can lead to dementia, and for many decades microbial infections have been implicated in Alzheimer\u27s disease (AD) pathology. However, a causal role for infection in AD remains contentious, and the lack of standardized detection methodologies has led to inconsistent detection/identification of microbes in AD brains. There is a need for a consensus methodology; the Alzheimer\u27s Pathobiome Initiative aims to perform comparative molecular analyses of microbes in post mortem brains versus cerebrospinal fluid, blood, olfactory neuroepithelium, oral/nasopharyngeal tissue, bronchoalveolar, urinary, and gut/stool samples. Diverse extraction methodologies, polymerase chain reaction and sequencing techniques, and bioinformatic tools will be evaluated, in addition to direct microbial culture and metabolomic techniques. The goal is to provide a roadmap for detecting infectious agents in patients with mild cognitive impairment or AD. Positive findings would then prompt tailoring of antimicrobial treatments that might attenuate or remit mounting clinical deficits in a subset of patients