BackgroundHistoplasmosis is acquired by inhalation of spores of the dimorphic fungusHistoplasma spp. Although this pathogen is distributed worldwide, it is more prevalent in the Americas. However, the real burden of histoplasmosis remains undefined in many endemic regions.MethodologyWe conducted a series of 61 autopsies to individuals who died in a hospital in the Brazilian Amazon focused on infectious diseases. We performed a detailed histological and microbiological evaluation with genetic characterization ofHistoplasmastrains with the aim to evaluate the contribution of histoplasmosis to morbidity and mortality. Additionally, we assessed the clinicopathological correlation.Principal findingsEvidence ofHistoplasmainfection was detected in 21 patients (34%). Eight cases were disseminated infections, all of them occurred in HIV-positive patients. Six cases were localized histoplasmosis, limited to the lungs. In seven patientsHistoplasmaDNA was detected by PCR in patients with no histological lesions.Histoplasmainfection was detected in 38% of HIV-positive patients and was a major contributor to death in 22% of them. Lungs, liver and spleen were affected in all cases of disseminated histoplasmosis. Phylogenetic analysis of the strains suggested a high diversity ofHistoplasmaspecies circulating in the Brazilian Amazon. Histoplasmosis was clinically missed in 75% of the disseminated infections.ConclusionsThe high incidence of histoplasmosis, the low index of clinical suspicion, and the severity of the disseminated disease highlight the need of proactively implementing sensitive routine screening methods for this pathogen in endemic areas. Antifungal prophylaxis againstHistoplasmashould be encouraged in the severely immunocompromised HIV patients in these areas. In conclusion, substantial mortality is associated with disseminated histoplasmosis among HIV-positive patients in the Brazilian Amazon.