Investigating diagnostic methods for histoplasmosis and the ecological niche and comprehensive population structure genomic analysis of African Histoplasma capsulatum

Abstract

Background: Histoplasmosis, emergomycosis, sporotrichosis and blastomycosis are endemic to southern Africa; the first two are also AIDS-related mycoses. Histoplasmosis is caused by pathogenic Histoplasma capsulatum fungi. Since rapid accurate diagnostic tests for disseminated histoplasmosis are not widely available in African countries, the prevalence among adults living with advanced HIV disease is mostly unknown. Knowledge about the genetic relationships of H. capsulatum isolates from the African continent to other global isolates and probable sources of exposure is scarce. H. capsulatum naturally occurs in caves occupied by bats and South African caves have been documented as probable sources of exposure for speleologists, casual visitors, or guano miners with several outbreaks of acute pulmonary histoplasmosis being reported. Materials and methods: We analysed electronic pathology laboratory data from all public and some private facilities in South Africa from 2010-2020 to identify the number of endemic mycoses cases reported. A case was defined as a patient of any age with any specimen type submitted to a laboratory from which a thermally dimorphic fungus was cultured or detected by histopathology, Histoplasma antigen enzyme immunoassay (EIA), pan-dimorphic reverse- transcriptase quantitative polymerase chain reaction (RT-qPCR) and pan-dimorphic fungal PCR/sequencing. We used stored H. capsulatum isolates from South Africa for phenotypic description, antifungal susceptibility testing and whole genome studies of African isolates compared to other H. capsulatum isolates from the Americas and India. We also validated three different methods for histoplasmosis diagnosis and used them to study the prevalence in South African patients living with HIV (PLHIV), with positive results for cryptococcal antigenaemia and CD4 cell count ≤100 cells/μl. Finally, we used three molecular assays to detect H. capsulatum directly from soil samples collected from South African caves. Results: We identified 682 cases of endemic mycoses reported between 2010 and 2020. Sporotrichosis, emergomycosis and histoplasmosis were the most common mycoses identified. Sequences from African isolates formed a single clade that was different from previously described H. mississippiense, H. ohiense, H. suramericanum, H. capsulatum sensu stricto and Indian clades. When screening for Histoplasma antigen, the EIA and lateral flow assay (LFA) yielded positive results in 6/194 (3%) and 12/194 (6%) of urine samples from PLHIV respectively and there was no reference method. The positivity rate of the RT-qPCR assay when viii testing stored blood samples from PLHIV was 10/533 (2%). From the 56 soil samples collected from South African caves, H. capsulatum was detected in five of the seven caves included. Conclusions: There is a need to increase awareness of histoplasmosis among healthcare workers and to develop rapid point-of-care diagnostic tools and make these widely accessible. Globally, systematic analysis of isolates from different regions is necessary to increase knowledge on the evolution and speciation of H. capsulatum. A novel distinct African clade was described and named Histoplasma africanus sp. nov, which warrants further studies to determine whether it is necessary to have specific diagnosis and treatment methods specific to this clade. Our study of the evaluation of easy-to-perform diagnostic assays for histoplasmosis on retrospective cohort samples yielded preliminary data on the disease prevalence, which is important, but larger prospective studies are now needed. The screening for H. capsulatum nucleic acids in soil from various caves in South Africa showed that the fungus is present in several regularly explored caves with bat populations and the methods used could now be used for further studies in understanding the broader distribution of H. capsulatum in the environment.