title: Genotypic Diversity Is Associated with Clinical Outcome and Phenotype in Cryptococcal Meningitis across Southern Africa.
abstract: Cryptococcal meningitis is a major cause of mortality throughout the developing world, yet little is known about the genetic markers underlying Cryptococcal virulence and patient outcome. We studied a cohort of 230 Cryptococcus neoformans (Cn) isolates from HIV-positive South African clinical trial patients with detailed clinical follow-up using multi-locus sequence typing and in vitro phenotypic virulence assays, correlating these data with clinical and fungal markers of disease in the patient. South African Cn displayed high levels of genetic diversity and locus variability compared to globally distributed types, and we identified 50 sequence types grouped within the main molecular types VNI, VNII and VNB, with 72% of isolates typed into one of seven 'high frequency' sequence types. Spatial analysis of patients' cryptococcal genotype was not shown to be clustered geographically, which might argue against recent local acquisition and in favour of reactivation of latent infection. Through comparison of MLST genotyping data with clinical parameters, we found a relationship between genetic lineage and clinical outcome, with patients infected with the VNB lineage having significantly worse survival (n=8, HR 3.35, CI 1.51-7.20, p=0.003), and this was maintained even after adjustment for known prognostic indicators and treatment regimen. Comparison of fungal genotype with in vitro phenotype (phagocytosis, laccase activity and CSF survival) performed on a subset of 89 isolates revealed evidence of lineage-associated virulence phenotype, with the VNII lineage displaying increased laccase activity (p=0.001) and ex vivo CSF survival (p=0.0001). These findings show that Cryptococcus neoformans is a phenotypically heterogeneous pathogen, and that lineage plays an important role in cryptococcal virulence during human infection. Furthermore, a detailed understanding of the genetic diversity in Southern Africa will support further investigation into how genetic diversity is structured across African environments, allowing assessment of the risks different ecotypes pose to infection.
authors.affiliation: Institute of Infection and Immunity, St. George's University London, London, United Kingdom; Department of Infectious Disease Epidemiology, Imperial College School of Public Health, London, United Kingdom.
authors.forename: Mathew A
authors.initials: MA
authors.lastname: Beale
authors.affiliation: Institute of Infection and Immunity, St. George's University London, London, United Kingdom; School of Medicine University of St. Andrews, St. Andrews, United Kingdom.
authors.forename: Wilber
authors.initials: W
authors.lastname: Sabiiti
authors.affiliation: Institute of Infection and Immunity, St. George's University London, London, United Kingdom.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Robertson
authors.affiliation: Institute of Infection and Immunity, St. George's University London, London, United Kingdom.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Fuentes-Cabrejo
authors.affiliation: Department of Infectious Disease Epidemiology, Imperial College School of Public Health, London, United Kingdom.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: O'Hanlon
authors.affiliation: Botswana-UPenn Partnership, Gaborone, Botswana; Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America; Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Jarvis
authors.affiliation: Institute of Infection and Immunity, St. George's University London, London, United Kingdom.
authors.forename: Angela
authors.initials: A
authors.lastname: Loyse
authors.affiliation: Division of Infectious Diseases and HIV Medicine, Department of Medicine, University of Cape Town, Cape Town, South Africa; Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
authors.forename: Graeme
authors.initials: G
authors.lastname: Meintjes
authors.affiliation: Institute of Infection and Immunity, St. George's University London, London, United Kingdom.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Harrison
authors.affiliation: Institute of Microbiology and Infection and the School of Biosciences, University of Birmingham, Birmingham, United Kingdom; National Institute for Health Research (NIHR) Surgical Reconstruction and Microbiology Research Centre, University Hospitals of Birmingham National Health Service (NHS) Foundation Trust, Queen Elizabeth Hospital Birmingham, Birmingham, United Kingdom.
authors.forename: Robin C
authors.initials: RC
authors.lastname: May
authors.affiliation: Department of Infectious Disease Epidemiology, Imperial College School of Public Health, London, United Kingdom.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Fisher
authors.affiliation: Institute of Infection and Immunity, St. George's University London, London, United Kingdom.
authors.forename: Tihana
authors.initials: T
authors.lastname: Bicanic
mesh_terms: D000353:Africa, Southern / Q000453:epidemiology; D003455:Cryptococcus neoformans / Q000235:genetics* / Q000472:pathogenicity*; D005787:Gene Frequency; D014644:Genetic Variation / Q000235:genetics*; D005838:Genotype; D006801:Humans; D016919:Meningitis, Cryptococcal / Q000453:epidemiology* / Q000235:genetics* / Q000473:pathology*; D008957:Models, Genetic; D010641:Phenotype; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016019:Survival Analysis; D016896:Treatment Outcome; D014774:Virulence