Oral Histoplasmosis in an Immunocompetent Male Diagnosed by Culture, Histopathology, and MetaCAP

Abstract

Background

Histoplasmosis is a systemic fungal infection caused by the dimorphic fungus Histoplasma capsulatum, commonly found in soil contaminated by bird or bat excrement. Oral granulomatous histoplasmosis is a relatively rare presentation that often lacks typical clinical features. Patients may experience persistent oral pain or lesions lasting several weeks, and clinical presentations can mimic malignant tumors or other infectious pathogens, leading to diagnostic challenges.

Case Presentation

We report a case of oral granulomatous histoplasmosis in a 53-year-old immunocompetent male, initially suspected of having lymphoma or tuberculosis. To our knowledge, this is an exceptional case of oral histoplasmosis diagnosed in an immunocompetent patient through a combination of tissue fungal culture, pathological biopsy, and metagenomic capture sequencing (metaCAP).

Conclusion

The case highlights the importance of considering fungal infections in persistent oral lesions of immunocompetent patients. It also demonstrates that metaCAP, alongside conventional culture and histopathology, can facilitate a definitive diagnosis in challenging cases.

Introduction

Histoplasma capsulatum (H. capsulatum) is a thermophilic, dimorphic ascomycetous fungus that exists in both yeast and hyphal phases. The yeast phase predominates at 35–37°C and is characterized by thin-walled, oval cells measuring 2–5 μm in diameter. At temperatures between 26–28°C, the hyphal phase emerges, with hyphae measuring 1.25–2 μm in diameter and producing two types of spores: thick-walled macroconidia (8–15 μm) and microconidia (2–5 μm). Microconidia serve as the primary infectious propagules, readily aerosolized and inhaled.¹ H. capsulatum thrives in nitrogen-rich soil, often contaminated by bird or bat droppings. Inhalation of airborne spores from disturbed soil allows the fungus to reach the respiratory tract, including the lungs.^2,3 An increase in temperature within the host triggers a transition from the hyphae to the yeast phase.⁴

Histoplasmosis, a disease caused by H. capsulatum with a worldwide distribution, is most prevalent in Central and South America, particularly Ohio and Mississippi River valleys, as well as parts of Africa and Asia.^3,4 Sporadic cases have been reported in China, primarily in the Yangtze River Basin.⁵ A survey of 735 volunteers in China using the histoplasmin skin test showed a higher positive rate in southern compared to northern regions.⁶ Histoplasmosis can manifest as acute, subacute, chronic, or disseminated disease. Although severe, life-threatening infections can occur, approximately 90% of cases are asymptomatic or subclinical. Disseminated histoplasmosis primarily affects immunosuppressed populations, including patients with Acquired Immune Deficiency Syndrome (AIDS) (CD4+ <75/mm3, age >54 years), organ transplant recipients, and those with chronic kidney disease requiring long-term corticosteroids or TNF antagonists. It is rare in immunocompetent individuals.⁷ Compared to acute histoplasmosis, chronic histoplasmosis progresses slowly, often in elderly individuals unaware of their immune deficiency, but can still be fatal.^1,8

Oral histoplasmosis often mimics malignancies or other chronic ulcerative conditions, making prompt diagnosis challenging, especially in immunocompetent individuals, in whom such presentations are uncommon and frequently overlooked.^3,9 Oral histoplasmosis is typically considered a manifestation of disseminated infection rather than an isolated organ infection. Diagnosis can be challenging, with frequent misdiagnosis as squamous cell carcinoma, lymphoma, tuberculous granuloma, candidal thrush, or erosive lichen planus.^3,7 The incidence of oral histoplasmosis is relatively low. A Chinese study analyzing 225 cases of H. capsulatum infection over 10 years found only one case (0.4%) involving oral infection.⁵ Among them, multiple sites are affected in 50% of these cases. Risk factors are not well-established, but cell-mediated immune suppression is a contributing factor.⁷

This case report describes a unique presentation of oral granulomatous histoplasmosis in an immunocompetent male, diagnosed using a combination of traditional methods (culture and histopathology) and advanced molecular diagnostics: metagenomic capture sequencing (metaCAP), which enables comprehensive and rapid pathogen detection directly from clinical samples, compared to conventional molecular methods, metaCAP offers superior sensitivity and broad-spectrum detection, facilitating timely and accurate identification in complex cases. To date, reports of oral histoplasmosis in immunocompetent patients diagnosed with metaCAP are lacking, representing a significant gap in the literature. Here, we discuss a rare case of oral granulomatous histoplasmosis in an immunocompetent male, diagnosed using culture, pathology, and metaCAP, highlighting the diagnostic value and clinical advantages of this integrative approach.

Case Presentation

A 53-year-old male, with no significant past medical history, presented with a three-month history of gingival lesions. He resided in Shenzhen, China. Initial symptoms included gingivitis, which improved with metronidazole. Ulcers subsequently developed on the gingiva and upper labial mucosa, without fever, following farm work involving climbing lychee trees and turning soil near a chicken coop. The patient was initially evaluated at other hospitals, where lymphoma was suspected, and he received a short course of methylprednisolone, with temporary improvement. However, symptoms worsened within two months, prompting referral to our clinic.

The patient exhibited gingival congestion, ulcers, mucocutaneous depressions, and granulomatous growths on the right lower gingiva and mucosa, left upper gingiva, as well as the left upper labial mucosa (Figure S1a and b). These growths displayed granular proliferation with yellowish-white pseudomembranes. Additionally, mild bilateral mandibular lymphadenopathy was observed. The patient denied any history of exposure to infectious diseases, use of immunosuppressants, or animal contact. The patient had good compliance and was previously healthy based on medical examination reports from the past three years. His family history did not indicate any hereditary condition.

Laboratory investigations revealed the following results (Table 1). Immunoglobulin and complement levels were within the normal range. Lymphocyte subset analysis indicated a decrease in CD4+ T cells and an inverted CD4/CD8 ratio. Both liver and kidney function tests were normal. Chest CT scan (performed at other tertiary hospital before) revealed emphysema and scattered chronic inflammation in both lungs, accompanied by multiple nodules.

Table 1.

Laboratory Investigation Results

Items	Results	Reference
White blood cell count	4.71 x 10^9/L	Normal
Neutrophils count	3.22 x 10^9/L	Normal
Lymphocyte count	0.76 x 10^9/L	Low
Red blood cell count	4.46 x 10^12/L	Normal
Hemoglobin level	140 g/L	Normal
Platelet count	229 x 10^9/L	Normal
C-reactive protein level	5.57 mg/L	High
Serological tests for HIV, HCV, HBsAg, and Treponema pallidum	Negative	Normal
Serological tests for 1,3-β-D-glucan	456.43 pg/mL	High

Based on the patient’s reaction to initial treatment in other tertiary hospitals, the abnormalities of blood cells and the clinical presentation, the differential diagnosis included carcinoma, blood tumors (especially lymphoma), and atypical tuberculosis infection, none of which could be ruled out. Therefore, to establish a definitive diagnosis, tissue samples were obtained from the oral ulcer surfaces for fungal culture, histopathology, and metaCAP analysis simultaneously.

Fungal cultures were conducted on Sabouraud’s Dextrose Agar (SDA) at 25°Cand Brain-Heart Infusion Blood Agar at 35°Cwith 5% CO2. After 8 days of fungal culture on SDA at 25°C, white, fluffy-like colonies were observed (Figure 1). On Brain Heart Infusion Blood Agar, 1mm milky-white colonies were observed (Figure 2). After 20 days, white, fluffy, dense colonies were observed on SDA. Fungal culture on Potato Dextrose Agar (PDA) also demonstrated white, fluffy, dense colonies after 15 days at 25°C (Figure 3A and B). Microscopically, elongated, transparent, septate branching hyphae, microconidia, and round, thick-walled macroconidia with spiny protrusions were visible, consistent with H. capsulatum (Figure 4). Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) identified the isolate as H. capsulatum (confidence level 99.9%). MetaCAP analysis detected H. capsulatum (98,410 sequence reads, confidence level 99%).

Figure 1.

Fungal culture on SDA showing fluffy white colonies at 8 days at 25°C.

Figure 2.

Fungal culture on brain-heart infusion blood agar at 35°C with 5% CO2 showing pinpoint colonies after 8 days.

Figure 3.

(A) Culture on PDA showing white, fluffy, dense colonies after 15 days at 25°C; (B) Brownish-yellow colonies on the back of PDA after 15 days at 25°C.

Figure 4.

Microscopic morphology of the isolate stained with Lactophenol cotton blue (400×). Elongated, transparent, septate branching hyphae, microconidia, and characteristic round, thick-walled macroconidia with spiny protrusions are visible.

The histopathological examination of the right lower gingiva and upper labial mucosa revealed the presence of chronic suppurative inflammation accompanied by granuloma formation. Fungal spores were identified within the infected tissue, exhibiting non-caseating granulomatous inflammation. Narrow, oval yeast-like spores were primarily observed within macrophages and monocytes (Figure 5A and B). Both Grocott–Gomori methenamine silver (GMS) and periodic acid-Schiff (PAS) stains yielded positive results (refer to Figure 5C and D). Based on these findings, the patient was diagnosed with chronic oral granulomatous histoplasmosis caused by H. capsulatum. The patient was treated with oral itraconazole 0.2 g once daily, with gradual improvement observed. After 3 months of treatment, fungal cultures were negative. The 1,3-β-D-glucan level decreased to 84 pg/mL and white blood cell count returned to normal, indicating a significant therapeutic effect. The ulcers on the right lower mucosa, left upper gingiva, and left upper labial mucosa healed (Figure S2a and b). The patient is currently undergoing outpatient follow-up.

Figure 5.

Histopathological findings. (A) Narrow, oval yeast-like spores within macrophages and monocytes (HE stain 100×); (B) Higher magnification showing intracellular spores (HE stain 400×); (C) Spores demonstrating positive staining with periodic acid-Schiff (PAS stain 100×); (D) Spores are highlighted by Grocott-Gomori methenamine silver staining (GMS stain 100×).

Discussion

Oral histoplasmosis is an uncommon manifestation of H. capsulatum infection, rarely observed in immunocompetent individuals. In previously reported cases, oral lesions often occurred in patients with disseminated disease, HIV infection, or other immunosuppressive conditions. The incidence of oral lesions in disseminated histoplasmosis ranges from 10% to 60%.⁹ Due to its atypical presentation, oral histoplasmosis is often misdiagnosed as malignancy or other granulomatous diseases.^7,9 This case, however, occurred in an immunocompetent individual, representing an unusual presentation of localized oral histoplasmosis, and was diagnosed using a combination of traditional methods – culture and histopathology – and advanced molecular diagnostics – metaCAP.

The patient initially presented with gingivitis, which was followed by the development of oral ulcers and granulomas, leading to suspicion of lymphoma or tuberculosis. However, the temporary improvement observed after treatment with methylprednisolone further complicated the diagnostic picture. The preliminary histopathological report revealed significant inflammatory cell infiltration, prompting consideration of pathogenic infection. The differential diagnosis encompassed bacterial, viral, parasitic, and fungal etiologies. Through immunohisto- chemistry, tissue culture, and serological tests, bacterial infections such as sexually transmitted diseases were ruled out. Additionally, common viruses like herpes simplex virus types I and II were also excluded based on pathology. Parasitic infections like leishmaniasis were considered but were deemed unlikely due to the patient’s lack of relevant epidemiological history. Other fungal infections, such as blastomycosis and cryptococcosis, were also taken into account. Blastomyces species exhibit a larger diameter (8–20 μm) with broad-based budding, whereas Cryptococcus species have a diameter of 4–6 μm and are surrounded by a thick capsule, which can be excluded through cryptococcal antigen detection.³

The histopathological findings, including the presence of oval spores enclosed by a halo in HE staining and positive GMS and PAS staining, were suggestive of H. capsulatum. Cultivation of typical thick-walled macroconidia with spiny protrusions, combined with MALDI-TOF identification and metaCAP results, confirmed the diagnosis. It is well established that H. capsulatum culture is challenging due to its specific requirements. While histopathology can suggest a fungal infection, it often cannot identify the precise species. This case contributes to the literature by integrating metaCAP to confirm the diagnosis alongside traditional approaches. MetaCAP, compared with conventional mNGS (metagenomic next-generation sequencing), offers a sensitive and specific method for detecting pathogens in clinical samples. It involves capturing target nucleic acids through probe hybridization, allowing for the detection of over 3,000 pathogens, including both DNA and RNA viruses. By removing human host nucleic acids and using probes to capture pathogen nucleic acids, metaCAP offers high sensitivity, particularly suitable for samples with low pathogen content, such as tissues and sterile body fluids.¹⁰ In this case, metaCAP rapidly identified H. capsulatum with 99% confidence and more than 98,000 unique reads, supporting its complementary role when culture or histopathologic findings are inconclusive.

A pivotal question arises: Why did the patient develop oral histoplasmosis without any immunocompromising condition? A possible explanation is that the patient’s exposure to a poorly hygienic environment contaminated with chicken coop feces in the mountain and history of gingivitis likely contributed to his infection. This condition may have allowed the fungus to breach the mucosal immunological barrier and establish a chronic infection. The patient had no history of immunosuppression in medical examination reports from the past three years. However, his lymphocyte count was decreased upon presentation, indicating temporary immune suppression by the fungus. This transiently compromised status enabled a sustained infection. The elevated serum level of 1,3-β-D-glucan supports the possibility of a deep fungal infection. Culture remains the gold standard for diagnosis, although culturing H. capsulatum can be challenging. The sensitivity of culture depends on the patient’s clinical presentation, immunosuppressive status, and overall disease burden, with the highest positive rates observed in immunosuppressed patients and those with disseminated infections. Combining serum histoplasma antigen and antibody detection can improve the detection rate. Antigen detection identifies polysaccharide-galactomannan on the fungal cell wall in blood, urine, or other sterile body fluids, exhibiting high sensitivity in disseminated infections. Antibody formation takes 4–8 weeks and may be negative early in infection or in immunocompromised patients. The antibody sensitivity in solid organ transplant recipients is 36%, and in people living with HIV (PLWH), it ranges from 38% to 63%. Antibody testing is suitable for immunocompetent patients with symptoms lasting over 4 weeks or chronic histoplasmosis. Routine antigen screening is recommended for PLWH. Combining serum antigen and antibody tests can enhance the detection rate, especially in immunocompetent individuals.^1,11

H. capsulatum grows slowly, and inoculation on brain-heart infusion blood agar can aid its growth.¹² It exhibits a yeast-like phase, producing budding yeast-like cells at 35–37°C. At 26–28°C, it shows a mycelial phase with two spore morphologies: macroconidia and microconidia. Macroconidia, with spines and gear-like structures on the cell wall surface, represent the most important and typical morphological features of H. capsulatum and carry substantial diagnostic value. Microconidia possess smooth surfaces devoid of spines and are primarily responsible for human respiratory infections. Several characteristics of the fungus promote disease occurrence and development, especially in immunocompetent individuals: (1) heat resistance, enabling growth and reproduction at human body temperature; (2) ability to escape or overcome host barriers to reach internal tissues; (3) capacity to evade and tolerate the immune system; (4) production of enzymes for nutrient uptake, hydrolyzing and consuming human tissue components.²

Traditional methods, including histopathology, culture, serological testing and MALDI-TOF each has specific strengths and limitations. Histopathology can suggest fungal infection, but cannot precisely identify the species. Culture remains the gold standard but is often slow and insensitive. Serological tests and MALDI-TOF are valuable but depend on immune response and successful culture, respectively. In contrast, metaCAP offers higher sensitivity and specificity through targeted probe capture, allowing for rapid, economical and definitive identification. Integrating metaCAP into the diagnostic workflow increases accuracy and improves the detection rate, particularly in ambiguous or complex cases.

The treatment plan is contingent upon the patient’s immune status and clinical symptoms, while also taking into account the potential side effects of antimycotic medications. This particular patient responded effectively to oral itraconazole treatment. During the follow-up period, the patient continued to take the prescribed antifungal medication, and all tests for liver and kidney function, as well as blood cell counts, were found to be normal. It is important to note that once a fungus invades the host, its spores often selectively proliferate and reproduce within phagocytic cells, spreading to various organs or transitioning into a dormant phase to form granulomas.² Upon the patient’s initial presentation, a lung CT scan revealed emphysema, scattered chronic inflammation, and multiple nodules. Regular follow-up visits are essential to evaluate whether the chronic lung infection is related to the inhalation of H. capsulatum spores. If necessary, antimycotic therapy should be continued in accordance with established guidelines.¹

The Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have not yet established clinical and epidemiological breakpoints for H. capsulatum. Given that the yeast phase is the primary infectious and toxic agent, some researchers propose incorporating yeast-phase spores along with macrophages into antimicrobial susceptibility testing (AST) to create a more accurate and physiologically relevant condition for the fungus.¹³ Raimunda et al have established epidemiological cutoff values (ECVs) for amphotericin B (AMB) and itraconazole at 0.5 μg/mL and 0.013 μg/mL, respectively. These strains were isolated from immunocompromised patients infected with HIV,¹² and AST requires further exploration. Recommended antifungal medications include AMB-related agents and itraconazole. For mild to moderate histoplasmosis, itraconazole serves as the first-line treatment for a duration of 6–12 weeks. However, for severe cases and immunocompromised patients, liposomal amphotericin B (LAMB) is preferred over deoxycholate AMB for an initial period of 1–2 weeks, followed by 12 months of itraconazole treatment.¹

Dogenski et al described an oral manifestation of disseminated histoplasmosis in an immunosuppressed patient with systemic lupus erythematosus.¹⁴ Similar to our case, however, two key distinctions highlight our report’s significance. First, their patient was immunocompromised due to systemic lupus erythematosus, a known risk factor, whereas our patient was immunocompetent, making this presentation exceptionally rare. Second, our diagnostic approach demonstrates the clear advantage of metaCAP. The comparator case relied on a prolonged workup with repeat biopsy and serology. In contrast, metaCAP provided a rapid, unbiased, and definitive identification of H. capsulatum from the initial tissue sample. This was crucial, as the primary suspicion of lymphoma meant targeted fungal tests would not have been ordered. By delivering a precise diagnosis in under 48 hours, metaCAP circumvented the delays of culture and directly guided timely therapy, showcasing its power to resolve complex diagnostic dilemmas and improve patient outcomes.

This case highlights the diagnostic dilemma of chronic oral ulcers. Tuberculosis, lymphoma, deep fungal infection, and chronic inflammatory diseases were initially considered; however, histopathological features and metaCAP confirmation excluded these differential diagnoses. Multimodal confirmation provided robust diagnostic confidence and guided appropriate antifungal therapy. However, this single-case report cannot establish generalizable conclusions on the prevalence or prognosis of oral histoplasmosis in immunocompetent populations. Furthermore, the absence of long-term follow-up precludes evaluation of potential recurrence or late complications.

Conclusion

This case report delineates a rare presentation of oral granulomatous histoplasmosis in an immunocompetent male. It underscores the importance of considering fungal infections in the differential diagnosis of oral lesions and highlights the utility of metaCAP, which facilitated rapid and definitive pathogen identification in diagnostically challenging scenarios. Based on this single case, integrating advanced molecular diagnostics with traditional methods may improve diagnostic accuracy. Future research should clarify how metaCAP compares with established techniques in routine clinical practice and whether its use can meaningfully influence patient management and outcomes.

Funding Statement

This work was supported by “Chronic Disease Management Research Project of National Health Commission Capacity Building and Continuing Education Center (Grant No.GWJJMB202510025126)” and “Shenzhen Highlevel Hospital Construction Fund”.

Ethics and Consent

This was a retrospective case report study conducted in a tertiary hospital and was approved for publication of case details by the Clinical Research and Ethics Committee of Peking University Shenzhen Hospital (No. [2020] 023-2). The authors declared that they had obtained a written and signed consent from the patient to use his medical files in the context of bio-medical research. The patient provided consent for the publication of the case details.

Disclosure

The authors report no conflicts of interest in this work.