# Molecular detection of Histoplasma capsulatum in environmental samples collected from South African caves

## TL;DR

This study confirms the presence of Histoplasma capsulatum in South African caves using molecular methods, highlighting a risk of histoplasmosis for cave visitors and HIV-positive individuals.

## Contribution

The study is the first to molecularly detect H. capsulatum in South African cave environments, linking environmental strains to clinical isolates.

## Key findings

- H. capsulatum was detected in five of seven caves using RT-qPCR and Hc100 PCR assays.
- Environmental strains were phylogenetically similar to clinical isolates from South African patients.
- ITS PCR failed to detect the fungus in any samples, suggesting assay limitations.

## Abstract

Histoplasma capsulatum naturally occurs in cave soil enriched by bat guano. South African caves are documented as probable sources of exposure for speleologists, casual visitors, or guano miners with several outbreaks of acute pulmonary histoplasmosis reported since 1977. Sporadic cases of disseminated histoplasmosis occur in South Africans living with advanced HIV disease. However, detection from the environment has not been confirmed. We used molecular assays to detect and confirm the presence of H. capsulatum in regularly-explored caves.

Environmental samples were collected by a speleologist from seven South African caves from December 2020 to September 2021 in the Gauteng, Northern Cape and Western Cape provinces of South Africa and stored at 2–8 °C. DNA was extracted directly from the samples using DNeasy PowerSoil Pro Kit. In-house internal transcribed spacer (ITS) panfungal polymerase chain reaction (PCR), pan-dimorphic reverse transcriptase-quantitative (RT-q) PCR and nested Hc100 PCR assays were used to detect H. capsulatum. Sequence identity was confirmed using the National Centre for Biotechnology Information (NCBI) BLAST tool following Sanger sequencing of the Hc100 nested-PCR product.

H. capsulatum was detected in five of the seven caves. Of 56 samples tested, 18 (32%) were positive from three caves in Gauteng Province [cave 1 (3/10); cave 2 (7/10); cave 3 (5/10)], one cave in the Western Cape Province [cave 4 (2/5)] and one cave in the Northern Cape Province [cave 6 (1/10)]. These samples were positive either by RT-qPCR or Hc100 PCR assays. Both RT-qPCR and Hc100 PCR assays were positive in 21% (12/56) samples. Seven percent (4/56) of samples were only RT-qPCR assay-positive and 4% (2/56) only Hc100 PCR-positive. Phylogenetic analysis of the Hc100 gene product from 10 samples (with good-quality sequences) identified four groups. Group 1 consisted of three samples from caves 1, 3, and 6 (Gauteng/ Northern Cape); Group 2 included four samples from caves 1, 2, and 3 (Gauteng); Group 3 had one sample from cave 4 (Western Cape); and Group 4 included two samples from caves 1 and 3 (Gauteng). None of the 56 samples tested positive with the ITS PCR assay.

H. capsulatum is probably present in several regularly-explored caves with bat populations. This finding should be confirmed by culture. The RT-qPCR and the Hc100 PCR assays could be useful tools for wider environmental surveillance.

Simson and Barnetson reported the first documented case of disseminated histoplasmosis in South Africa in 1942. Cases of disseminated histoplasmosis have increasingly been reported in recent decades among people living with HIV in South Africa, making histoplasmosis the third most common laboratory-confirmed endemic mycosis. In addition, several outbreaks of pulmonary histoplasmosis have occurred among cave explorers, highlighting a possible link between cave environments and Histoplasma exposure, even though the fungus has not been successfully cultured directly from soil samples. From December 2020 to September 2021, we collected environmental samples from seven bat-inhabited caves across South Africa and tested 56 samples with three molecular assays. The RT-qPCR and Hc100 PCR assays detected Histoplasma nucleic acids from 18 samples in five caves. In contrast, the ITS PCR assay was negative on all samples. Cluster analysis further revealed that the environmental Histoplasma strains were closely related to clinical isolates from South African patients. These results reinforce the importance of taking protective measures such as wearing masks or respirators when entering caves, especially during activities that may disturb guano and release fungal conidia.