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The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage‐specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates. see also the Perspective by Claudio Azat [ABSTRACT FROM AUTHOR]

Our understanding of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii has been greatly enhanced by use of genome sequencing technologies. Found ubiquitously as saprotrophs in the environment, inhalation of infectious spores from these pathogens can lead to the disease cryptococcosis. Individuals with compromised immune systems are at particular risk, most notably those living with HIV/AIDS. Genome sequencing in combination with laboratory and clinical studies has revealed diverse lineages with important differences in their observed frequency, virulence and clinical outcomes. However, to date, genomic analyses have focused primarily on clinical isolates that represent only a subset of the diversity in the environment. Enhanced genomic surveillance of these yeasts in their native environments is needed in order to understand their ecology, biology and evolution and how these influence the epidemiology and pathophysiology of clinical disease. This is particularly relevant on the African continent from where global cryptococcal diversity may have originated, yet where environmental sampling and sequencing has been sparse despite harbouring the largest population at risk from cryptococcosis. Here, we review what scientifically and clinically relevant insights have been provided by analysis of environmental Cryptococcus isolates to date and argue that with further sampling, particularly in Africa, many more important discoveries await. [ABSTRACT FROM AUTHOR]

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. Author Summary: Cryptococcus neoformans (Cn) is a yeast that commonly causes meningitis in HIV infected individuals in Africa, where it may account for up to 500,000 deaths every year. In this highly translational and multidisciplinary study, we used genetic analysis techniques to show that Cryptococcus found in Southern Africa represents a hotspot of genetic diversity. We combined this data with the results of microbiological techniques that assess the natural virulence traits that the yeast uses to survive and infect humans to further show that genetic diversity is associated with differences in cryptococcal phenotype. Finally, we analysed detailed clinical data on patients to investigate the clinical effects of infection with different lineages, and showed that one genetic lineage (VNB) is significantly associated with worse survival. Whilst much of our prior knowledge regarding the genetic basis of virulence is derived from studies on laboratory-adapted cryptococcal strains, our findings from this large and comprehensive MLST genotyping study of clinical isolates—linking genotype, phenotype, clinical presentation and outcome—provide direct insights into the contribution of pathogen lineage to virulence in human cryptococcal meningitis. [ABSTRACT FROM AUTHOR]

The global burden of HIV-associated cryptococcal meningitis is estimated at nearly one million cases per year, causing up to a third of all AIDS-related deaths. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this understudied, yet increasingly important, group of pathogenic fungi. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly-virulent emerging lineages are changing patterns of human disease both temporally and spatially. Cryptococcus neoformans variety grubii (Cng, serotype A) constitutes the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some regions experiencing significant burdens of disease. We compared 183 clinical and environmental isolates of Cng from one such region, Thailand, Southeast Asia, against a global MLST database of 77 Cng isolates. Population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only ten nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating our sample. This population contains significantly less diversity when compared against the global population of Cng, specifically Africa. Genetic diversity in Cng was significantly subdivided at the continental level with nearly half (47%) of the global STs unique to a genetically diverse and recombining population in Botswana. These patterns of diversity, when combined with evidence from haplotypic networks and coalescent analyses of global populations, are highly suggestive of an expansion of the Cng VNI clade out of Africa, leading to a limited number of genotypes founding the Asian populations. Divergence time testing estimates the time to the most common ancestor between the African and Asian populations to be 6,920 years ago (95% HPD 122.96 - 27,177.76). Further high-density sampling of global Cng STs is now necessary to resolve the temporal sequence underlying the global emergence of this human pathogen.