Figure

Figure. Bayesian tree based on 210-bp partial Hcp100gene sequences obtained from 42 guano samples collected in the tunnels of the Camino de Hierro tourist route in study of molecular...

To shed light on the distribution of Histoplasma beyond traditional known endemic areas, we conducted a study in 2 bat-inhabited tunnels of the Camino de Hierro in Salamanca, northern Spain, a pedestrian route receiving >60,000 visitors since its opening as an ecotourism attraction in 2021 (Appendix). We collected 101 guano samples in the tunnels (Appendix Table 1) and found almost 42% were positive for Histoplasma by nested PCR, using previously published primers (Appendix Table 2). Specifically, the Hcp100 gene sequences we isolated (submitted to GenBank under accession nos. PP887829–78) (Table) shared high homology with GenBank sequences corresponding to H. capsulatum s.s., H. suramericanum, H. capsulatum LAm B2, and H. capsulatum var. duboisii. Moreover, our phylogenetic analyses (Appendix) indicated that the newly obtained sequences form a fully supported monophyletic group with multiple GenBank sequences of Histoplasma (posterior probability = 1; bootstrap support = 100%), without a clear geographic or host-related pattern (Figure).

To determine species of bats roosting in this area, we used BLAST (https://blast.ncbi.nlm.nih.gov) to identify cytochrome c oxidase subunit I sequences obtained from Histoplasma-positive samples. Results corresponded to different species that are mainly distributed in Europe: Myotis blythii was the most common, followed by Rhinolophus ferrumequinum and Miniopterus schreibersii (all sequences were submitted to GenBank under accession nos. PP919660–707) (Table). That finding increases the number of possible hosts and dispersers associated with this human pathogen.

The degree of positivity we observed suggests a substantial presence of Histoplasma in the tunnels studied, which seems reasonable because the moderate temperature, constant humidity, and darkness of both tunnels investigated are suitable environmental conditions for fungal growth. Of note, outside known areas of endemicity, Histoplasma has been isolated from soils contaminated with bat guano in Romania (6).

A total of 728 cases of human histoplasmosis have been diagnosed in 17 countries in Europe, of which Spain accounts for up to 60% (5). Most cases were imported from Central and South America (9), except 4 cases: 1 autochthonous laboratory-acquired case and 3 cases with no epidemiologic history in patients who had never visited endemic areas. One case was in a drug user with HIV/AIDS, another was in a patient who had disseminated histoplasmosis develop after renal transplantation, and the third was in a patient who had occasionally traveled to France, Italy, and the United Kingdom and had previously been treated with an immunosuppressant, suggesting reactivation of a latent infection (13). However, to date, Histoplasma has not been isolated from environmental samples in Spain, and no autochthonous clinical case of histoplasmosis has been associated with exposure to bats.

The results of this study indicate that Histoplasma is present in bat-inhabited tunnels at Camino de Hierro in northern Spain. That finding evidences that the geographic distribution of this genus is wider than previously thought and also reinforces the known association between Histoplasma and bats.

The risk for histoplasmosis increases with contact with guano deposited in bat roosts. Exploring caves and similar environments is a well-documented source of Histoplasma exposure; the first outbreaks of histoplasmosis were related to bat-inhabited locations dating back to the 1930s (14). For that reason, the need to assess the presence of Histoplasma in bat-inhabited places before opening them to public access has been emphasized (11). Therefore, even though no autochthonous cases of histoplasmosis have been reported in Spain, the detection of Histoplasma in such a popular tourist attraction as Camino de Hierro makes it advisable to alert local clinicians about the importance of considering histoplasmosis in the differential diagnosis of patients with community-acquired pneumonia. Clinicians can refer to available clinical diagnostic algorithms for histoplasmosis for evidenced-based testing guidance (15).

Our results provide a warning about the presence of Histoplasma in Camino de Hierro but also could stimulate further research on bat populations in Spain, opening lines of research into their role in the transmission of histoplasmosis and other airborne infectious diseases. Research on the effects Histoplasma and other pathogens on the health of bats should also be considered, and we advise carrying out a serologic study to assess possible exposure to Histoplasma among workers and susceptible persons who have visited the tunnels. Given the tourism value and high number of visitors to Camino de Hierro, health authorities should consider our findings and implement measures to prevent potential cases of histoplasmosis, in both visitors and workers of this tourist route.