https://orcid.org/0000-0001-8726-3994
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Abstract
This observational autopsy-based case series characterized the tissue distribution of Histoplasma capsulatum in deceased people living with HIV (PLHIV) using minimally invasive tissue sampling (MITS). This study included 59 deceased PLHIV admitted to a tertiary referral hospital in the Brazilian Amazon between October 2020 and June 2022. Tissue and body fluid samples were analyzed using fungal culture, histopathology, conventional polymerase chain reaction (PCR), and quantitative PCR (qPCR). H. capsulatum was detected in 27 of 59 (45.8%) patients, including nine (33.3%) with multi-organ detection of H. capsulatum. The liver (9/27, 33.3%) and lungs (8/27, 29.6%) were the most frequently involved organs in this series. qPCR amplification was detected across multiple tissues, with lower median Ct values in the lung (40.3, range 37.0–45.0) and liver (41.0, range 38.0–44.0) than in cerebrospinal fluid (43.5, range 40.0–48.0) and brain tissue (44.0, range 41.0–47.0), suggesting greater fungal DNA detection in the lungs and liver. Molecular methods detected fungal DNA in tissue samples that were negative by culture, although several amplification signals occurred near the assay detection limit. H. capsulatum showed heterogeneous tissue distribution in deceased PLHIV, with the liver and lungs being the most frequently involved organs. Molecular methods complemented conventional diagnostic techniques by identifying fungal DNA in culture-negative tissues, providing a more comprehensive assessment of tissue involvement. These findings improve understanding of disseminated histoplasmosis in advanced HIV infection and may inform future tissue-based diagnostic strategies.
Author summary
Histoplasmosis is a serious fungal infection that causes significant morbidity and mortality in people living with the human immunodeficiency virus, especially in endemic regions. Diagnosis remains challenging because its symptoms resemble those of other opportunistic infections, and sensitive laboratory tests are often unavailable. In this study, we analyzed postmortem tissue samples collected via a minimally invasive procedure to investigate the distribution of the fungus throughout the body. Almost half of the patients presented with histoplasmosis, with the lungs and liver being the most frequently affected organs. Molecular methods detected fungal DNA in additional tissue samples that were negative by fungal culture, particularly in samples with low fungal DNA burden. Although based on postmortem samples, our findings contribute to understanding disease dissemination and reinforce the complementary role of molecular techniques in diagnosis in endemic areas.
Citation: Solórzano-Chavarría KJ, Graciliano TS, Lacerda IPS, Silva-Neto AV, Ferreira L, Santana MF, et al. (2026) Tissue distribution of Histoplasma capsulatum in deceased people living with HIV assessed by minimally invasive tissue sampling: An autopsy-based case series in the Brazilian Amazon. PLoS Negl Trop Dis 20(8): e0014545. https://doi.org/10.1371/journal.pntd.0014545
Editor: Marcio L. Rodrigues, FIOCRUZ: Fundacao Oswaldo Cruz, BRAZIL
Received: November 3, 2025; Accepted: July 2, 2026; Published: August 6, 2026
Copyright: © 2026 Solórzano-Chavarría et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are within the manuscript and/or Supporting Information files.
Funding: ISGlobal acknowledges support from grant CEX2023-0001290-S funded by MCIN/AEI/10.13039/501100011033 (to QB) and from the Generalitat de Catalunya through the CERCA Program (to QB). This study received additional funding from the MITS Alliance (to QB). This work was also supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) through Edital No. 038/2022 – PDPG/CAPES – Coordenador/Auxílio Financeiro, and through the CAPES Graduate Program Support Programme (PROAP), grant No. 1247/2022, which covered the article processing charge (APC). KJSC received a doctoral scholarship from CAPES, and DCBS receives a Research Productivity Fellowship from the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM); under Brazilian regulations, these fellowships are research stipends and do not constitute an employment relationship or salary. No author received a salary from any of the funders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Histoplasmosis, an AIDS-defining opportunistic infection, is a systemic fungal infection primarily caused by Histoplasma capsulatum var. capsulatum, with H. capsulatum var. duboisii occurring mainly in African settings [1]. The fungus thrives in soil enriched with bird or bat droppings, which explains its broad geographic distribution across endemic regions, including Latin America (e.g., Brazil, Colombia, Venezuela, and Guatemala) and parts of Africa and Asia [2–5]. Despite this distribution, histoplasmosis remains underrecognized in many endemic settings, and underdiagnosis may contribute to increased morbidity and mortality, particularly among people living with HIV (PLHIV), in whom the disease often presents in disseminated forms [4,6,7].
One of the main challenges in the clinical management of histoplasmosis is its frequent misdiagnosis as tuberculosis, given the substantial overlap in clinical and radiological characteristics [5,8]. This diagnostic difficulty is further compounded by limited access to specific diagnostic tools in many endemic settings [9]. Although urine or blood antigen detection assays for Histoplasma offer high sensitivity, especially in disseminated disease, these methods are rarely available within public health systems in many endemic countries [10,11].
In immunocompetent individuals, histoplasmosis is often asymptomatic or self-limiting. In contrast, in immunosuppressed individuals, especially PLHIV, the infection frequently disseminates, affecting multiple organs and leading to severe clinical outcomes [12,13]. Disseminated histoplasmosis (DH) is associated with rapid clinical deterioration and high mortality rates when diagnosis and treatment are delayed [14,15]. Accurate diagnosis requires a combination of histopathology, culture, and molecular methods, which often require invasive procedures to obtain tissue samples [16].
These diagnostic challenges highlight the importance of understanding the distribution of H. capsulatum across different organs. This is particularly relevant in resource-limited settings, where access to diagnostic tools is often restricted. However, evidence remains limited to which tissues are more likely to yield detectable H. capsulatum DNA, particularly with minimally invasive postmortem approaches.
Minimally Invasive Tissue Sampling (MITS) is a postmortem technique for collecting tissue samples from key organs [17,18]. It allows sampling without the need for a full autopsy or imaging guidance. This method has emerged as a valuable tool for determining the cause of death in infectious diseases [19]. It is particularly useful in low-resource and culturally sensitive settings due to its feasibility and acceptability [19,20]. In addition, MITS provides an opportunity to systematically investigate tissue involvement in disseminated infections [19,21].
To fill this knowledge gap, we conducted a systematic post-mortem investigation describing the tissue distribution of H. capsulatum in deceased HIV-positive individuals using MITS. Unlike previous studies that focused primarily on clinical presentation or diagnostic performance evaluation, our work integrates fungal culture, histopathology, conventional PCR, and quantitative PCR to provide a comprehensive characterization of fungal dissemination across multiple organs. By combining complementary diagnostic methods within a standardized MITS protocol, this research generates new pathological evidence on disseminated histoplasmosis in the Brazilian Amazon, establishing a solid foundation for future tissue-based diagnostic and mortality surveillance studies in endemic regions.
Methods
Ethical aspects
This study was approved by the Research Ethics Committee of Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), Brazil (CAAE: 34846620.8.0000.0005). All procedures were conducted in accordance with national and international ethical standards, including the Declaration of Helsinki and applicable Brazilian regulations. Written informed consent for the MIT sampling procedure was obtained from each deceased participant’s next of kin prior to postmortem sample collection, in accordance with institutional protocols. All data were anonymized prior to analysis to ensure participant confidentiality.
Study setting
This is an observational autopsy-based case series study conducted at FMT-HVD, a tertiary referral hospital specializing in the diagnosis and treatment of tropical diseases, including fungal infections [22]. The hospital is in Manaus, in the state of Amazonas, in Brazil’s Northern Region. The city has an estimated population of 2 million, with the majority residing in urban and peri-urban areas [23]. A previous study conducted at this hospital showed a high incidence of histoplasmosis (34%) among autopsied patients [14]. This substantial burden highlights the need to better characterize the tissue distribution of H. capsulatum in this setting.
Patients included
Patients who died at the FMT-HVD between October 2020 and June 2022 were included if they met the following criteria: (1) age ≥ 18 years with a confirmed diagnosis of HIV; (2) written informed consent for MITS provided by relatives; and (3) a postmortem interval of less than 48 hours. Cases with a postmortem interval exceeding 48 hours or with insufficient tissue quality due to advanced autolysis were excluded. No exclusions were made based on the presence of coinfections, as these were considered part of the clinical spectrum of advanced HIV infection. HIV infection was confirmed based on documented medical records and laboratory diagnostic criteria in accordance with national guidelines [24].
Demographic, epidemiological, and clinical information
Demographic, epidemiological, and clinical information were extracted from electronic medical records using a standardized data collection form. Variables collected included age, sex, HIV-related parameters (CD4 ⁺ cell count, viral load, and antiretroviral therapy use), presence of opportunistic infections, prior or inpatient antifungal treatment, comorbidities, and selected laboratory parameters (e.g., hematological and biochemical profiles), when available. Clinical data were obtained at the time of HIV diagnosis, before hospitalization, and during the hospital stay to capture each patient’s clinical trajectory.
Epidemiological data included place of residence (e.g., urban or peri-urban areas) and, when available, geographic origin. Information on occupational exposure, travel history, and specific environmental factors was not systematically recorded in the medical records, preventing a consistent analysis of these aspects.
Data extraction was performed by trained researchers following a standardized protocol. A subset of the cases was independently reviewed to ensure consistency of information, and any discrepancies were resolved by consensus or, when necessary, by consulting a third reviewer. Missing data were recorded as unavailable and were not imputed.
Minimally invasive tissue sampling procedure
Participants underwent tissue collection using MITS, as previously described [20,25]. The protocol included a detailed external macroscopic examination followed by percutaneous sampling using a 14-gauge needle. Samples were collected from the lungs, liver, brain, ileum, blood, and cerebrospinal fluid (CSF), as well as pleural and ascitic fluid when present.
Sampling was performed using a standardized protocol, with needle insertions guided by anatomical landmarks specific to each organ. Whenever feasible, 2–4 punctures per organ were performed, with sampling depth adjusted to reach the target parenchyma. Tissue fragments of approximately 25–50 mg were obtained for solid samples, while liquid samples had a minimum volume of approximately 200 µL. To minimize cross-contamination, a new sterile needle was used for each organ, and strict aseptic technique was maintained throughout the procedure.
Each sample was assigned a unique study code and, when sufficient material was available, divided into three analytical pipelines:
- (i). mycological analysis (Mycology Laboratory, FMT-HVD);
- (ii). molecular analysis (stored in DNase-free tubes and transported under cold chain at 2–8 °C);
- (iii). histopathological analysis (fixed in 10% neutral buffered formalin).
Although multiple samples per organ were obtained whenever possible, the amount of tissue collected may have been limited by post-mortem factors, including tissue autolysis, anatomical accessibility, and the inherent constraints of the MITS procedure, which prioritizes minimal invasiveness while maintaining adequate tissue representativeness. When sample volume or weight was limited, a predefined prioritization strategy (histopathology → mycology → molecular analysis) was applied and documented for each case.
All samples were coded, recorded on a tracking form, and transported at a controlled temperature (2–8 °C), processed on the same day whenever possible, or stored according to each analytical method’s requirements.
Mycological analysis
Mycological analyses were performed according to established mycological criteria [26]. Solid tissue samples were fragmented under sterile conditions and inoculated onto culture media, including Sabouraud agar, Mycosel agar, brain–heart infusion (BHI) agar, and Niger seed agar. Approximately 40 µL of the liquid samples were also inoculated onto the same media.
Cultures were incubated at 25–30 °C for filamentous growth and at 37 °C for yeast-phase growth and monitored for 30–45 days. Fungal growth compatible with H. capsulatum within this incubation period was considered positive. Repeat cultures were not performed systematically and depended on sample availability.
Identification of H. capsulatum was based on classical macroscopic and microscopic morphological characteristics, including microscopic examination using lactophenol cotton blue staining and demonstration of thermal dimorphism through conversion between mycelial and yeast phases, in accordance with standard mycological criteria [27,28]. Molecular detection by PCR was performed in parallel but was not used as a confirmatory method for culture results.
All procedures followed biosafety recommendations for handling dimorphic pathogenic fungi and were conducted in laboratory facilities equipped to safely manipulate Histoplasma spp. (biosafety level 3 with enhanced precautions, according to institutional protocols).
Molecular methods
Conventional PCR and qPCR were performed independently as complementary molecular methods. Molecular detection was defined as a positive result by either conventional PCR or qPCR. Molecular analyses were performed according to the methodology described by Silva et al. [29]. DNA was extracted from clinical samples using the Invitrogen Blood & Tissue Kit from 200 µL of body fluid or 25–50 mg of tissue, and eluates (50 µL) were stored at −80 °C.
A TaqMan-based qPCR assay targeting the ITS1 region of H. capsulatum was performed using primers HcITS-106F and HcITS-205R and probe HcITS-127P, as previously described by Buitrago et al. [30], with oligonucleotide conditions optimized by Silva et al. [29]. Reactions were carried out in a final volume of 20 µL containing 1 × TaqMan Master Mix, 300 nM of each primer, and 200 nM probe. Amplification was performed under the following conditions: 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 60 seconds.
Samples with Ct values ≤38 were considered positive. Amplification signals with Ct values >38 were classified as low-level molecular detections and interpreted with caution, as they occurred near the analytical detection limit and may reflect low fungal DNA burden, residual DNA, or post-mortem degradation. Ct values were interpreted as qualitative or semi-quantitative indicators of fungal DNA detection and should not be considered direct measures of viable fungal burden or clinical disease severity. Ct values were not used as a direct measure of diagnostic accuracy.
All reactions were performed in duplicate and included positive and no-template (negative) controls. An internal β-actin target was included to monitor DNA extraction efficiency and potential PCR inhibition. Samples with absent β-actin amplification or β-actin Ct > 35 were re-tested after 1:5 dilution to assess inhibition. Discordant molecular results between conventional PCR and qPCR may reflect differences in analytical sensitivity, stochastic amplification near the detection limit, DNA degradation in postmortem samples, or variability in the distribution of fungal DNA in tissue [31–34].
Laboratory procedures followed a unidirectional workflow with physically separated areas for DNA extraction, reagent preparation, and amplification to minimize contamination.
The analytical performance of the qPCR assay, including limit of detection, sensitivity, specificity, and amplification efficiency, has been previously described by Silva et al. [29] and was not re-evaluated in this study. Validation in post-mortem samples was made by Silva et al. [29]. Although the assay has demonstrated high analytical specificity, potential cross-reactivity with phylogenetically related fungi cannot be completely excluded.
Histopathological methods
After fixation in neutral buffered formalin for 24 hours, tissue samples were routinely processed with paraffin embedding and stained using hematoxylin and eosin (H&E) as well as Grocott–Gomori methenamine silver (GMS). When indicated, additional staining with periodic acid–Schiff (PAS) was also performed. Histopathological identification of H. capsulatum relied on characteristic morphological features observed in tissue sections, including small yeast forms measuring approximately 2–5 µm in diameter, predominantly intracellular localization within macrophages, and narrow-based budding. Special stains, particularly GMS and PAS, were used to enhance visualization of fungi and facilitate morphological identification. All histopathological assessments were conducted by an experienced pathologist.
Attribution of the cause of death
After completion of the pathological examination, an infectious disease pathologist reviewed all pathological findings obtained from the MITS procedure. Based on these findings, the contribution of H. capsulatum to death was classified as primary, contributory, or unrelated. When necessary, individual cases were discussed with the study investigators to resolve uncertainties and reach a consensus.
Histoplasmosis classification
Histoplasmosis cases were classified using operational categories developed for this descriptive postmortem study to facilitate characterization of tissue distribution. The predefined patterns of tissue involvement were classified as: (i) multi-organ detection, defined as detection of H. capsulatum in two or more organs and/or body fluids; (ii) lung-restricted detection, defined as detection restricted to the lungs; and (iii) single-organ/body fluid detection, defined as detection confined to a single non-pulmonary organ or body fluid. These categories were intended to describe patterns of tissue involvement rather than to establish clinical diagnostic criteria.
Statistical analysis
Given the case series design, analyses were primarily descriptive and exploratory. Categorical variables were summarized as absolute and relative frequencies [n/N (%)], while continuous variables were described using median and interquartile range (IQR) or mean ± standard deviation, as appropriate. The distribution of H. capsulatum detection across tissues was described according to predefined patterns of tissue involvement (multi-organ detection, lung-restricted detection, and single-organ/body-fluid detection). Ct values were interpreted descriptively and compared across tissues to characterize patterns of fungal DNA distribution, without assessing diagnostic accuracy. According to the standard curve, the assay showed reliable amplification up to approximately Ct 37.6. Therefore, amplification detected at Ct values ≥38 was considered close to the technical detection limit and interpreted cautiously as low-level. Missing data were recorded as “not available” and were not imputed. Statistical analyses were performed using R software (version 3.6.0; R Foundation for Statistical Computing, Vienna, Austria).
Results
Study population
A total of 59 deceased PLHIV were included in the study. The median age was 40 years (IQR 31–47), and most participants were cisgender men (42/59, 71%). The majority resided in urban areas (55/59, 93%), particularly in the metropolitan region of Manaus (49/59, 83%). Advanced HIV disease was common. Among patients with available data, 20/24 (83%) had CD4 + T-cell counts <200 cells/µL at diagnosis, 13/23 (57%) had HIV viral loads ≥100,000 copies/mL, and 22/46 (48%) were receiving antiretroviral therapy at hospital admission. Respiratory symptoms (42/53, 79%), weight loss (36/52, 69%), and fever (33/53, 62%) were the most frequent clinical manifestations. Severe illness was reflected by high rates of ICU admission (26/55, 47%), invasive mechanical ventilation (29/47, 62%), and vasoactive drug use (30/43, 70%). Opportunistic infections were identified in 20/36 (56%) patients, most commonly tuberculosis and pneumocystosis (Table 1).
Histoplasmosis was identified in 27/59 (45.8%) patients. Compared with those without histoplasmosis, affected individuals more frequently reported alcohol use (72% vs 22%) and recreational drug use (39% vs 12%), had a shorter median time since HIV diagnosis (3 vs 57 months), and more often presented with lower hemoglobin concentrations and ferritin levels >3000 ng/mL. Additional demographic, clinical, and laboratory characteristics according to histoplasmosis status are presented in Tables 1 and 2.
Tissue distribution of Histoplasma capsulatum
H. capsulatum was detected in 27 of 59 patients (45.8%). According to the predefined classification, 9 patients (33.3%) had multi-organ detection of H. capsulatum, 1 had lung-restricted detection, and 17 had single-organ/body-fluid detection. Liver and lungs were the most frequently involved organs, with fungal detection in 9/27 (33.3%) and 8/27 (29.6%) patients, respectively. Brain tissue, blood, and cerebrospinal fluid were less frequently involved.
Distinct patterns of tissue involvement were observed according to the predefined tissue-distribution categories. Multi-organ with frequent involvement of the liver (9/9, 100%), lungs (8/9, 88.9%), blood (6/9, 66.7%), and cerebrospinal fluid (4/9, 44.4%). In contrast, single organ/body fluid detection was predominantly detected locally, most commonly in blood (12/17, 70.6%), whereas liver and cerebrospinal fluid involvement were less frequent (2/17, 11.8% each). The single patient with Lung-restricted detection had fungal detection restricted to the lungs (Table 3).
Contribution of laboratory methods to tissue detection of H. capsulatum
Molecular assays detected fungal DNA in 23 of 27 patients (85.2%), whereas fungal culture and histopathology identified fungal infection in 8 (29.6%) and 5 (18.5%) patients, respectively. Culture most frequently yielded positive results from liver (5/27) and blood (6/27) samples, whereas PCR-based methods detected fungal DNA across multiple tissues. Molecular methods detected fungal DNA in tissues from 16 patients, while their corresponding culture samples were negative.
In several instances, molecular detection occurred in the absence of fungal growth in culture, particularly in samples with low fungal DNA burden. qPCR amplification was observed in multiple organs. Median Ct values were lower in lung tissue (40.3, range 37.0–45.0) and liver (41.0, range 38.0–44.0) than in cerebrospinal fluid (43.5, range 40.0–48.0) and brain tissue (44.0, range 41.0–47.0), indicating stronger molecular amplification signals in those organs (Table 4).
Histopathological findings
Histopathological examination demonstrated small intracellular and extracellular yeast forms morphologically compatible with H. capsulatum, measuring approximately 2–4 μm. GMS and PAS stains enhanced the visualization of fungi.
In the lungs, organisms were observed within alveolar spaces and macrophages, with variable inflammatory responses ranging from minimal inflammation to necrotizing granulomatous lesions. Liver specimens showed granulomatous inflammation centered on portal tracts, with fungal organisms located within macrophages and portal granulomas. Brain tissue demonstrated scattered yeast forms without a significant inflammatory or granulomatous response (Fig 1).
A) Histoplasma inside the alveolar spaces. The yeasts are small, oval, with a clear halo surrounding them, which is compatible with Histoplasma capsulatum. They are isolated and not associated with inflammatory reactions (Grocott silver methenamine stain). Magnification = 400X. B) Lung tissue shows areas of necrosis and granulomatous reaction composed of epithelioid cells (Periodic acid-Schiff stain). Magnification = 200X C) Granulomas and histoplasmosis in the portal space in liver tissue (Grocott silver methenamine stain). Magnification = 100X. D) Brain with histoplasma without granulomatous reaction (Grocott silver methenamine stain). Magnification = 400X.
Discussion
Histoplasmosis remains one of the leading neglected infectious diseases and an important cause of mortality among PLHIV in Latin America [35]. In this study, we characterized the postmortem tissue distribution of H. capsulatum in deceased PLHIV using MITS integrated with conventional microbiological, histopathological, and molecular methods. Histoplasmosis was identified in 27 of 59 patients (45.8%), and one-third of these cases (33.3%) showed multi-organ fungal detection, underscoring the substantial burden of fatal histoplasmosis among individuals with advanced HIV disease in the Brazilian Amazon. By integrating complementary diagnostic approaches, our study provided a more comprehensive assessment of tissue involvement than fungal culture alone, particularly in patients with disseminated disease affecting multiple organs.
The observed frequency of histoplasmosis confirms previous reports highlighting the substantial contribution of this infection to AIDS-related mortality in Latin America. Among the 59 evaluated individuals, 45.8% showed evidence of the disease, and in 16.9% of the cases, histoplasmosis was considered the primary cause of death. These results are consistent with data from Guatemala [36], Colombia [37], French Guiana [38], and the Brazilian Amazon [14,39]. Although the prevalence observed in our study was slightly higher than that reported by Rakislova et al. [14], who identified histoplasmosis in 38% of HIV-associated deaths in the Brazilian Amazon, the proportion of deaths directly attributed to histoplasmosis was lower in that series. On the other hand, mortality in this study was higher than that reported by Souza et al. [40], possibly reflecting the low frequency of antiretroviral therapy at hospital admission and the profound immunosuppression observed in our patients.
Unlike previous studies that focused primarily on the clinical characteristics, therapeutic outcomes, mortality, coinfections, or diagnostic performance of disseminated histoplasmosis, our research systematically characterizes the anatomical distribution of H. capsulatum across multiple tissues through MITS integrated with fungal culture, histopathology, conventional PCR, and quantitative PCR. This integrated approach provides novel pathological evidence regarding tissue involvement in fatal HIV-associated histoplasmosis and highlights the importance of combining complementary laboratory methods to enhance the characterization of disseminated fungal infections.
The strength of the study is the use of MITS, which enabled a systematic postmortem evaluation of tissue involvement. Although MITS does not provide the comprehensive anatomical view of a complete autopsy, previous validation studies have demonstrated good agreement between the two approaches in determining infectious causes of death, especially in low- and middle-income settings [19,41,42]. Beyond its value as a pathological research tool, MITS has important implications for public health surveillance [41,43]. Facilitating standardized postmortem tissue collection with relatively simple infrastructure can promote recognition of fatal histoplasmosis and other opportunistic infections, strengthen mortality surveillance, and generate more accurate estimates of disease burden in regions where conventional autopsies and advanced diagnostic methods remain limited [18,43–46].
From a clinical perspective, histoplasmosis manifests with non-specific symptoms, including fever, weight loss, respiratory symptoms, and diarrhea, which resemble those of tuberculosis and other opportunistic infections [7,47–49]. Consequently, only a minority of patients received a diagnosis and antifungal therapy prior to death. Similar results have been observed in other endemic regions [35,49], highlighting the persistent underdiagnosis of histoplasmosis among PLHIV. Although the disease has historically been considered predominantly a Latin American problem, growing evidence indicates that it is also a relevant, yet underrecognized, cause of HIV-associated morbidity and mortality in Sub-Saharan Africa and Southeast Asia [50–54]. Limited access to Histoplasma antigen detection, molecular diagnostics, and pathological investigations contributes to delayed diagnosis, frequent confusion with tuberculosis, and the underestimation of the disease burden [10,35,55,56]. Thus, the challenges identified in the Brazilian Amazon reflect a broader global reality, in which disseminated histoplasmosis remains a neglected opportunistic infection, despite high mortality among immunocompromised individuals [57].
The liver and lungs had the highest positivity rates for H. capsulatum, followed by blood and cerebrospinal fluid. Multiorgan detection was common, whereas detection restricted to the lungs was relatively rare. These findings expand current knowledge on tissue involvement in fatal HIV-associated histoplasmosis and corroborate previous postmortem observations made in the Brazilian Amazon [14]. Although the relatively small number of histoplasmosis cases precludes definitive conclusions regarding preference for specific organs, the observed distribution provides important pathological evidence concerning the anatomical dissemination of H. capsulatum in advanced stages of HIV infection.
The predominance of fungal detection in the lungs and liver is consistent with the established pathogenesis of DH. Following inhalation, the lungs constitute the primary portal of infection, where microconidia transform into yeast forms and are rapidly phagocytosed by alveolar macrophages [58,59]. Instead of being eliminated, H. capsulatum survives and replicates intracellularly, thereby interfering with phagolysosomal maturation and evading the host’s antimicrobial mechanisms [60,61]. Infected macrophages then disseminate through the lymphatic system and bloodstream to organs rich in mononuclear phagocyte system cells, such as the liver, spleen, bone marrow, and lymph nodes [62–65]. The high frequency of hepatic involvement observed in our study likely reflects the abundance of resident macrophages (Kupffer cells), which provide a favorable niche for fungal intracellular persistence, especially in advanced HIV-associated immunosuppression.
The heterogeneous patterns of tissue involvement indicate that DH does not constitute a uniform pathological process. Although the cross-sectional postmortem design precludes conclusions regarding disease progression, the three predefined patterns of tissue distribution, multiorgan detection, detection restricted to the lungs, or single organ/body fluid detection, may reflect different stages or trajectories of fungal dissemination [1,66]. Detection restricted to the lungs may indicate infection confined to the primary site of inoculation or incomplete dissemination, whereas multiorgan detection is compatible with widespread hematogenous dissemination mediated by infected macrophages [1,2,66,67]. In turn, detection limited to a single extrapulmonary organ or body fluid may indicate initial dissemination, localized persistence, or heterogeneous fungal distribution that is only partially captured by MITS [68,69]. These hypotheses require validation in prospective studies with antemortem sampling and longitudinal clinical follow-up.
Although our observations are based exclusively on postmortem samples, they provide valuable insights into future research and diagnostic strategies. The predominance of fungal detection in the liver and lungs suggests that these organs may be higher-yield targets when tissue collection is clinically indicated, especially in settings lacking access to Histoplasma antigen detection or advanced molecular diagnostics. However, such findings should not be interpreted as direct clinical recommendations but rather as a pathological framework to guide prospective studies evaluating the diagnostic yield of different biopsy sites and to refine sampling protocols for antemortem investigations and standardized MITS procedures. Integrating anatomical distribution with complementary laboratory methods may ultimately contribute to the development of evidence-based diagnostic strategies for DH in endemic and resource-limited environments.
The conventional diagnosis of histoplasmosis relies on fungal culture and the histopathological demonstration of yeast forms using PAS or GMS stains [70]. However, both methods present significant limitations. Culture is slow, often requiring weeks for fungal growth, has reduced sensitivity in disseminated disease [16,70], and demands Biosafety Level 3 laboratory infrastructure due to the infectious nature of H. capsulatum [71]. Histopathological confirmation can also be limited by a low fungal burden or the small tissue fragments obtained through MITS. In our series, histopathology confirmed only a minority of cases, despite molecular detection across multiple tissues, highlighting the complementary rather than competing role of these diagnostic approaches. Together, the focal distribution of the fungus, prior antifungal therapy, and advanced immunosuppression can reduce microscopic visualization, while fungal DNA remains detectable by molecular methods.
Molecular methods should be considered complementary diagnostic tools, rather than substitutes. In this postmortem series, PCR-based assays detected fungal DNA in culture-negative tissues, expanding the characterization of tissue involvement without implying diagnostic superiority. While fungal culture confirms the presence of viable organisms and histopathology provides direct evidence of tissue invasion and host response, molecular methods increase the likelihood of detecting fungal DNA in samples with low burden or after prior exposure to antifungals [72–74]. The integration of these approaches allowed for a more complete assessment of the anatomical distribution of H. capsulatum than would be possible with any single method.
Although some qPCR-positive samples had Ct values close to the analytical limit of detection, these results should be interpreted with caution as indicative of fungal DNA presence rather than definitive confirmation of viable organisms or active infection. However, when analyzed alongside culture, histopathology, tissue distribution, and the overall pathological context, molecular methods strengthened the characterization of disseminated histoplasmosis, identifying DNA in tissues that would otherwise have remained undiagnosed. Since the evaluation of diagnostic accuracy was not the focus of this descriptive postmortem study, these findings should be viewed primarily as complementary evidence of tissue involvement.
The contribution of H. capsulatum to death was evaluated based on pathological findings obtained through MITS. Although this technique provides a standardized assessment of tissue involvement, it does not fully capture the clinical course prior to death and therefore should be interpreted as a pathological analysis of histoplasmosis’s role rather than a definitive determination of the clinical cause of death. Even so, the high proportion of patients for whom histoplasmosis was considered a primary or contributing cause highlights the significant impact of this neglected mycosis among PLHIV in endemic areas.
Beyond the pathological implications, this study is relevant to public health. The high frequency of histoplasmosis identified among deceased PLHIV reinforces the persistent underdiagnosis of the disease and indicates the need to strengthen diagnostic capacity in HIV care programs. Expanding access to Histoplasma antigen detection, molecular diagnostics, and standardized postmortem investigations can facilitate early diagnosis, improve mortality surveillance, and provide more accurate estimates of the burden of HIV-associated histoplasmosis. These results support efforts to integrate neglected fungal diseases into HIV control programs and public health surveillance systems, especially in low- and middle-income countries, where late diagnosis remains a relevant factor in preventable mortality.
This study has significant limitations. The relatively small sample size, the single-center design, and the exclusive inclusion of deceased PLHIV introduce a selection bias towards cases of advanced disease and fatal clinical manifestations, which restrict the generalizability of the results to other endemic settings. Therefore, the patterns observed in tissue distribution should not be extrapolated to patients diagnosed in early stages of infection, those with less severe immunosuppression, or individuals who respond adequately to antifungal treatment. Furthermore, as this is a post-mortem investigation, it was not possible to establish longitudinal clinical correlations between fungal tissue distribution, disease progression, and therapeutic response. Detection of Histoplasma antigen, an important diagnostic tool for disseminated histoplasmosis, was also unavailable for comparison with microbiological, histopathological, and molecular findings.
Other methodological limitations also deserve consideration. Several positive samples showed Ct values close to the analytical detection limit, requiring careful interpretation, as this may indicate a low fungal DNA load or post-mortem DNA degradation rather than viable organisms or active infection in the tissues. Post-mortem autolysis, prior antifungal use, and the limited amount of tissue obtained by MITS may also have interfered with fungal detection. Variability in access to different organs may have impacted sample representativeness. The absence of an independent gold standard prevented a formal evaluation of diagnostic performance. Therefore, the proposed categories for tissue distribution should be understood as a pathological reference for HIV-associated histoplasmosis in fatal cases, and not as clinical classifications or guidelines for sample collection. Prospective, multicenter studies integrating Histoplasma antigen detection, longitudinal clinical data, and standardized tissue sampling are needed to validate and improve these results.
Conclusion
The combination of MITS with conventional microbiological, histopathological, and molecular methods enabled comprehensive characterization of the postmortem distribution of H. capsulatum in PLHIV with advanced disease. By integrating complementary diagnostic approaches, this study expands current knowledge on fungal dissemination, provides novel pathological evidence regarding tissue involvement in fatal HIV-associated histoplasmosis, and supports the use of MITS as a viable platform for pathological investigations and mortality surveillance in endemic areas. Although these findings should not be directly extrapolated to clinical practice, they establish a solid foundation for future prospective studies evaluating tissue-based diagnostic strategies and reinforce the potential of MITS to strengthen surveillance of neglected fungal diseases, support evidence-based public policies, and reduce HIV-associated mortality.
Supporting information
S1 Table. Clinical and demographic characteristics at hospital admission of people living with HIV (PLHIV) included in the MITS study.
https://doi.org/10.1371/journal.pntd.0014545.s001
(DOCX)
S2 Table. Characteristics at moment of HIV diagnosis.
https://doi.org/10.1371/journal.pntd.0014545.s002
(DOCX)
Acknowledgments
We thank the Dr. Heitor Vieira Dourado Tropical Medicine Foundation for its institutional support and the infrastructure provided for this study. We also express our gratitude to the healthcare professionals and laboratory technicians involved in sample collection and analysis. We have used ChatGPT (version 4, from OpenAI, San Francisco, California, USA) and Grammarly to help us refine and summarize language. We have not used these tools to generate scientific content or interpret results. We have reviewed, edited, and validated all AI-assisted information to confirm accuracy and clarity.
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