Histoplasmosis: Laboratory Diagnosis

Abstract

The diagnosis of histoplasmosis is based on clinical, imaging, and laboratory evidence of the disease. The gold standards of laboratory diagnosis are the presence of the fungus in the pathological examination of tissues and its isolation (direct microscopy, histopathology, cytopathology) in mycological culture (4–6 weeks required) by clinical specimens. The antibody test, sometimes negative in immunocompromised patients, must be performed at least four weeks after acute infection to be positive. The antibody test is most useful in subacute and chronic forms of histoplasmosis. The antigen test is the most common method for establishing the diagnosis of acute pulmonary histoplasmosis or progressive disseminated histoplasmosis. The antigen test in urine or serum has good sensitivity, better in bronchoalveolar lavage fluid. There are skin sensitivity tests with low sensitivity that are used only for epidemiologic studies and are not recommended for diagnosis. Molecular diagnosis has improved the sensitivity of clinical specimens. Laboratory PCR assays with different molecular targets have been developed. Currently, the main procedure for molecular diagnosis of histoplasmosis is the application of a rapid DNA probe on a fungus isolated from a culture. This chapter summarizes the currently available tools for the laboratory diagnosis of histoplasmosis, focusing on the complexity of the assays and their performance in different clinical contexts.

1. Introduction

In 2017, the World Health Organization (WHO) created the first list of bacterial priority pathogens (WHO BPPL) and inspired by it, the first list of fungal priority pathogens (WHO FPPL) appeared [1].

The pathogens were classified into three groups according to their ability to cause invasive systemic infections that pose challenges in treatment and/or management or drug resistance: critical, high, and intermediate. Histoplasma capsulatum belongs to the high group. Three action areas and strategies were proposed:

1. Improvement of mycological laboratory diagnostic capacity and surveillance, optimization, and standardization of diagnostic tools worldwide;

2. Investment in mycological research, diagnosis, and treatment;

3. Public health interventions with education programs and curricula focused on fungal diseases and priority pathogens.

The geographic distribution of Histoplasma capsulatum has changed. It is underestimated because of inadequate public health measures (surveillance and reporting), uneven and unequal access to health care facilities and diagnostic tests (the disease burdens resource-poor regions), climate change, environmental disturbances, improved clinical and laboratory diagnostic detection, and increases in at-risk populations (travelers, immunocompromised patients). Global annual incidence rates for this disease, its distribution, and trends in specific countries or regions are unavailable due to lack of studies. In addition, there is no vaccine, progression to the invasive form is not preventable, access to diagnostics is moderate, and diagnosis for invasive forms is challenging.

Most laboratories have basic tests and only a few have tools such as molecular testing, next-generation sequencing, or other approved tests for histoplasmosis that allow diagnosis of fungal infection when there is high suspicion of the infection.

Diagnosis of Histoplasmosis (HPM) is difficult and requires a multifactorial approach (Table 1). Identification of Histoplasma capsulatum (H. capsulatum) by direct microscopy based on characteristic intracellular yeasts and/or culture isolation of the fungus in biological specimens is the gold standard for diagnosis. However, these tests have their limitations:

1. The low sensitivity, which depends on the clinical form of HPM;

2. The time needed to culture the fungus (4 to 6 weeks), which still requires conversion to the yeast-like form;

3. The need for a level 3 safety laboratory to handle H. capsulatum.

	Antigen detection	Serologic Test	Molecular methods	Culture	Microscopy (direct and histology)
Specimen	Urine, serum, CSF, BAL. Urine antigendetection tests are more sensitive than serum detection tests.	Serum	Tissues and body fluids, BAL. Commercially available highly specific molecular tests that allow rapid identification when applied to the culture isolate.	Blood, bone marrow, liver, skin, mucosal lesion, respiratory tract products.	In the microbiology laboratory, H. capsulatum can be detected by staining and direct microscopy of body fluids or tissue samples.
Time for positive results after fungal exposure	Few weeks after exposure. Is detected in the first few weeks of illness, especially in patients exposed to high levels of fungal inoculum. Can also be used to evaluate patient response to treatment; it should be below the detection limit if antifungal therapy is successful, and an increase in antigen levels signals relapse.	2 to 6 weeks	No FDA-approved commercial PCR-based test. In the future, molecular methods will play an increasingly important role in the diagnosis of HPM.	2–3 weeks up to 8 weeks. The gold standard for diagnosis of HPM is isolation of the fungus in culture and observation of characteristic intracellular yeasts in histopathology.	The presence of H. capsulatum yeasts in certain tissues or sterile body fluids (e.g., skin lesions) is indicative of acute infection.
Available within	Few hours / MVD LFA 1 min/ Alpha Histoplasma Antigen EIA- 3 hours Some tests (MVD EIA) require specimens to be sent to central laboratory.	Depend on laboratory	Hours	Fungus requires several weeks to grow in standard culture. The laboratory should have safety level 3 to handle it.
Tests	Enzyme-linked immunosorbent assay (ELISA) technique. The EIA tests MiraVista® MVD EIA Ag detection. MVD LFA Alpha Histoplasma Antigen EIA, − only test approved by FDA and CE.	Immunodiffusion (ID)-H and M antigen precipitates. Complement fixation reaction (CF) -A CF titer ≥1:32 suggest HPM but is not diagnostic, a fourfold increase in antibody titer at least 2 wk. apart. Enzyme immunoassay (EIA). Latex agglutination. Western blot. IGRAs (Interferon-gamma release assays).	A real-time PCR assay. Loop-mediated isothermal amplification (LAMP) (urine).	Mycelial phase incubation at 25–30°C. Yeast phase incubation at 37°C. Conventional blood culture. Lysis centrifugation system.	H. capsulatum stains poorly with Gram stains, so it is rarely detected by this method. Fluorescent staining: calcofluor white, that binds chitin in the cell wall of the fungus, is useful for identifying H. capsulatum in clinical specimens.
Sensibility and specificity	Antigen test has high sensitivity and specificity, good negative predictive value. Depend on clinical form and type test.	EIA: IgM and IgG antibodies with a sensitivity of 77–96% and a specificity of 92%. Anti-H antibodies cannot distinguish between the active form and the resolved disease. EIA more sensitive than ID or CF.	More sensitive and more specific than antigen detection or serologic testing.
False-positive reactions in urine or serum samples or cross-reactivity	Cross-reactivity with other mycosis: Blastomycosis, Paracoccidioidomycosis, Penicilliosis, Talaromycosis, Aspergillosis. Diagnosis should not be based solely on a positive urine antigen test, further test.	Cross-reactivity with endemic fungi such as Blastomyces dermatitidis.	PCR or LAMP have a lower probability of false-positive.

Table 1.

Summary of laboratory diagnostic tools.

In this regard, immunologic methods of antibody and antigen detection in clinical specimens such as serum, plasma, cerebrospinal fluid, urine, and bronchoalveolar lavage (BAL), as well as fungal DNA detection, are options for presumptive diagnosis of HPM. Analytical performance of assays for the diagnosis of histoplasmosis varies depending on disease stage and clinical form (Table 2).

	Acute pulmonary histoplasmosis	Subacute pulmonary HPM	Chronic pulmonary HPM	Disseminated HPM	HIV and HPM
Antigen tests	*82.8–88.3% [2]; Mira Vista urine antigen sensitivity 83% in acute HPM. Antigen is detected in approximately 75% of patients with acute pulmonary HPM within the first few weeks of illness, especially in patients exposed to high levels of fungal inoculum.	Only 30.4% in subacute form [2].	Mira Vista urine antigen sensitivity * 87.5% [2] In patients with less severe and chronic forms of pulmonary (e.g., cavitary) HPM or in patients with local complications of pulmonary HPM (e.g., mediastinal granuloma), antigen is detected in 10–20% of patients.	Antigen can be detected in the CSF Mira Vista urine antigen sensitivity is *91.8% in urine Mira Vista antigen sensitivity in serum is 100%.	Alpha Histoplasma Antigen EIA, (result in 3 hours), has a high sensitivity of 98%, a specificity of 97%, and a negative predictive value of 100% in patients with HIV and HPM and can be performed in individual laboratories. Alpha Histoplasma Antigen detection: in urine at 90% of patients and in serum in 50%. Antigen detection assays were the most accurate at diagnosing HPM in PLHIV. MVD LFA is a promising tool for point-of-care testing in suspected HPM, especially in people living with HIV/AIDS.
Serology	*64.3–66.7% [2]; Anti-M antibodies occur in 80% of patients.; Anti-H Ab occurs in less than 20% of cases, appears in severe acute pulmonary HPM.; The combination of Ag and Ab improves sensitivity up to 96%.	*95.1% [2]	*83.3% [2]; Anti-M Aboccurs. Anti-H Ab appeared.	*75% [2]; Serologictest positive at the time of presentation. Anti-H Ab appeared in disseminated HPM. Test detection of antibodies to Histoplasma in CFS.	Cross-react with other mycoses: Blastomices, Paracoccidioides, Coccidioides. −58% sensitivity, greater than 90% specificity. Antibody detection assays have high specificity, but the sensitivity is poor.
Culture	*0–20% [2]; 42% in acute pulmonary HPM.	*53.8% [2]	*66.7% [2]	* 74.2% [2]; In disseminated HPM, specimens collected from the blood, as well as from affected organs such as bone marrow, liver, skin, and mucosal lesions, may result in isolation of H. capsulatum. CSF culture is often negative.	In PLWHA, up to 90% of respiratory cultures and 50% of blood cultures may be positive. Antigen and molecular diagnosis assays had greater sensitivity and specificity compared with culture and antibody assays.
Molecular assay			Metagenomic next-generation sequencing (mNGS) on BAL.		Specimen: respiratory, tissue biopsy, BAL. blood and bone marrow provided the highest assay sensitivity. Similar to antigen testing, presented excellent analytical performance for disseminated HPM in PLWHA.
Pathology	*0–42% [2]; The presence of H. capsulatum yeasts in certain tissues or sterile body fluids (e.g., skin lesions) is indicative of acute infection.	*42.1% [2]	*75% [2]; The histopathologic examination is more likely to be positive in the subacute or chronic form than in the acute.	*76.3% [2]; The histopathologic examination is more useful in disseminated HPM than in localized pulmonary HPM.	The combination of antigen detection and cytopathology on BAL resulted in a sensitivity of 96.8%, both being rapid diagnostic tools.

Table 2.

Diagnostic tests depending on clinical form of histoplasmosis.

*

%of diagnostic test for HPM.

2. Antigen detection

The antigen test for histoplasmosis in urine and serum was first developed in 1986 [3], and become an important method for the diagnosis of Histoplasmosis revolutionizing the diagnosis of this disease. The antigen released by the fungal cells can be detected in biological samples: urine, serum, CSF, BAL. Urine antigen detection tests are more sensitive than serum detection tests, results are available within a few hours, they have high sensitivity and specificity, and good negative predictive value.

The first test was a solid-phase radioimmunoassay with the detection of circulating polysaccharide H. capsulatum antigen, which was performed after 1989 as an enzyme immunoassay (EIA) [4], with the same sensitivity and specificity but simpler to use.

Two tests based on enzyme-linked immunosorbent assay (ELISA) technique were developed with good diagnostic results but limited availability. Their use is limited to developed endemic areas and is rarely applied in non-endemic regions, probably due to low cost-effectiveness outside these regions.

The EIA tests have been modified several times to provide a quantitative test that can be used in body fluids other than urine and serum, such as CSF or BAL [5], and to avoid exposure of laboratory personnel to radioactivity.

The first test developed was manufactured by MiraVista® (MiraVista Diagnostic, Indianapolis, IN, USA) as a second-generation semi-quantitative test, followed by a third-generation quantitative antigen test. In a multicenter study by Hage et al. [6], the sensitivity and specificity of this test were investigated in different clinical forms of histoplasmosis. They found a sensitivity of 91.8% in urine from patients with disseminated HPM, 87.5% in chronic pulmonary HPM, 83.3% in acute HPM, and only 30.4% in subacute form. In serum samples, the sensitivity of the test was 100% in disseminated HPM. The EIA MiraVista test (MVD EIA) requires specimens to be sent to a central laboratory.

The IMMY® ALPHA ELISA kit (IMMY, Norman, OK) is a two-step immunoenzymatic sandwich test using polyclonal antibodies and can be used for quantitative detection of Histoplasma antigens in urine.

Recently, MiraVista Diagnostic developed a lateral flow-based assay for the detection of Histoplasma antigens in urine (MVD LFA). It is a single-format, “pregnancy test-like”, CE labeled product, that is easy to perform (less than 1 minute to perform the test, 40 minutes to obtain the result), it does not require specialized laboratory equipment or complex infrastructure or highly trained personnel, uses urine with sensitivity and specificity greater than 90% [7, 8], and has a concordance between MVD ELISA and LFA tests of 84%. This technique was first developed for the detection of Aspergillus galactomannan with very good results, and MiraVista released a similar test for the detection of Histoplasma capsulatum antigen using an immunochromatographic sandwich dipstick assay. Thus, MVD LFA is a promising tool for point-of-care testing in suspected histoplasmosis, especially in people living with HIV/AIDS (PLWHA). A study conducted to compare MVD LFA and MVD ELISA showed a sensitivity of 96% for both tests and a specificity of 96% for LFA and 77% for ELISA [9].

The only in vitro diagnostic test approved by the FDA and CE is the Alpha Histoplasma Antigen EIA, manufactured by Immuno Mycologics (IMMY, Norman, OK, USA). This test, which uses a monoclonal antibody, lasts for 3 hours, has a high sensitivity of 98%, a specificity of 97%, and a negative predictive value of 100% in patients with HIV and histoplasmosis [8], and can be performed in individual laboratories. These rapid antigen tests are very important in low-income areas, with high mortality rates, especially PLWHA.

An ELISA test manufactured by Optimum Imaging Diagnostic for the detection of Histoplasma antigenuria was recently studied, with a good sensitivity of 92% but 68% false-positive results [10].

In 2019, WHO included the test for the detection of Histoplasma antigens in the second edition of the WHO list of essential in vitro diagnostics [11].

The goal of The International Histoplasmosis Advocacy Group (IHAG) for 2025 is that at least one laboratory in each Latin American country has a rapid test (antigen detection or molecular test) for the diagnosis of histoplasmosis [12].

These tests are very important in patients with HIV and histoplasmosis because their antibody levels are low. In patients with HIV and disseminated histoplasmosis, antigen can be detected in urine in 90% of patients and in serum in 50% [13]. Antigen detection was also useful in bronchoalveolar lavage in PLWHA with Histoplasma-related pneumonia [14]. The MVista Histoplasma antigen enzyme test was adapted for quantitative detection of antigen in BAL. The combination of antigen detection and cytopathology on BAL resulted in a sensitivity of 96.8, both being rapid diagnostic tools. However, cross-reactivity in patients with Blastomycosis was observed [15].

Antigen is detected in approximately 75% of patients with acute pulmonary histoplasmosis within the first few weeks of illness, especially in patients exposed to high levels of fungal inoculum [16]. In patients with less severe and chronic forms of pulmonary (e.g., cavitary) histoplasmosis or in patients with local complications of pulmonary histoplasmosis (e.g., mediastinal granuloma), antigen is detected in 10–20% of patients [17]. In patients with mediastinal fibrosis or granulomatous mediastinitis, Histoplasma antigen cannot be detected in urine or plasma.

In patients with Histoplasma meningitis, antigen can be detected in the CSF [18], although CSF culture is often negative. Limited data are available for the use of the antigen test in non-HIV patients with disseminated histoplasmosis, but the test appears to be sensitive for this patient population as well. 92% of patients have antigenuria, but antigen is present in serum in only half of them [17]. There are also no data on the utility of this test in BAL from non-HIV patients.

Antigen detection can also be used to evaluate patient response to treatment; it should be below the detection limit if antifungal therapy is successful, and an increase in antigen levels signals relapse [13]. However, in some patients who have been successfully treated, a low concentration of antigen in the urine may persist for many months [19].

False-positive reactions occur in the majority of urine or serum samples from patients with other mycoses: Blastomycosis (a major diagnostic problem in the United States of America because the endemic areas of Blastomycosis and Histoplasmosis are intermingled and antigen tests show reactivity for both fungi), Paracoccidioidomycosis [20], Talaromycosis, Aspergillosis [5], and less frequently in patients with Coccidioidomycosis [21]. Aspergillus galactomannan tests react with Histoplasma galactomannan and may be positive in patients with HPM, but patients with Aspergillosis do not have false-positive antigen [22].

EIA test results should be interpreted in the appropriate clinical context due to cross-reactivity with other fungal antigens. Diagnosis should not be based solely on a positive urine antigen test; further serologic or/and cultural testing should be performed to confirm the diagnosis. A suspicious false-positive reaction is a positive serum antigen test but a negative urine test. This may also occur in transplant recipients who received thymoglobulin (rabbit antithymocyte globulin) due to human anti-rabbit antibodies that developed in response to thymoglobulin in the second week after administration and disappeared by the eighth week. These antibodies resulted in a false-positive Histoplasma antigen test in serum by EIA but not in urine [23].

3. Serologic test

Although antibody detection plays a less important role than antigen detection, antibody tests are useful for diagnosing various forms of HPM. After fungal exposure, anti-H. capsulatum antibodies take 2 to 6 weeks to develop, so they are more useful in subacute or chronic HPM than in acute pulmonary HPM. Even if suspicion is high and initial antibody tests are negative, they should be repeated after 1 or 2 months, which may be helpful in diagnosing acute infection. Almost always, patients with disseminated HPM have antibodies to H. capsulatum at the time of presentation. Immunocompromised patients may have a negative antibody test (they are unable to respond to H. capsulatum antigens and produce antibodies) and may cross-react with other mycoses: Blastomices, Paracoccidioides, Coccidioides [5].

The available serological tests for the detection of H. capsulatum antibodies are immunodiffusion (ID), complement fixation reaction (CF), enzyme immunoassay (EIA), latex agglutination, and Western blot. The first three tests are the most commonly performed because of their availability, precision, and convenience. These methods are non-invasive but have their limitations: wide variation in results within a patient, long time for positive results (to develop antibodies after exposure), and cross-reactivity with other endemic fungi such as Blastomyces dermatitidis [24].

Immunodiffusion uses an antigen preparation obtained from mycelial culture of H. capsulatum, and the presence of antibodies is signaled by the appearance of H and M antigen precipitates on an agar gel. It is widely used in clinical practice, is based on a simple and reliable method, is not expensive, and has good specificity, which is 71–100% higher than the complement fixation method [25].

Anti-M antibodies appear earlier in the course of HPM and may be present for years after the infection has resolved. They occur in acute form (in approximately 80% of patients) or in chronic infection. Thus, a single positive M band cannot distinguish between the active form and the resolved disease [2, 19].

The H-band is much rarer, occurring in less than 20% of cases, and rarely found without an M-band. The H-precipitin band is seen in patients with severe acute pulmonary HPM, with disseminated disease, chronic lung disease, or mediastinal lymphadenopathy over several months. When the infection has resolved, this antibody disappears [26].

The complement fixation assay tests for both antibodies: yeast and mycelium (histoplasmin). A fourfold increase in antibody titer (either of them) is considered positive and indicates active HPM. A CF titer ≥1:32 suggests HPM but it is not diagnostic. Diagnosis should not be based on a single value, as CF antibodies are often present years after infection and a single low CF titer sometimes means that the patient has been exposed to H. capsulatum at some point. The CF assay is slightly more sensitive than ID for diagnosing HPM, especially for the yeast phase; the sensitivity of both assays (CF and ID) exceeded 90% in some studies [27]. The CF mycelial antibody is the most specific test but has low sensitivity. The sensitivity of this test is lower in hemolytic or lipemic samples [25].

The development of an easy-to-perform EIA test (MiraVista Diagnostics, Indianapolis, IN, USA) detects IgM and IgG antibodies with a sensitivity of 77–96% and a specificity of 92%.

Comparing these tests using samples from the same patients, the EIA appears to be more sensitive than ID or CF. The utility of the EIA test (or CF and/or ID) is more important in the diagnosis of Histoplasma meningitis, as detection of antibodies to Histoplasma in CFS may be the only indicator of disease [28].

H. capsulatum can be detected by the latex agglutination test; detection of antibodies to H. capsulatum is based on latex connection with histoplasmin. This method has low sensitivity and cross-reactivity with tuberculosis but is inexpensive and specific [24].

Another assay is being investigated to improve the diagnosis of active or latent forms of HPM: Interferon-gamma release assays (IGRAs) [29]. Based on the promising preliminary results, further studies are needed to validate this assay. Currently, IGRAs have several disadvantages and limitations: short time for sample processing, complex laboratory capacity, and personal experience, high cost for this test.

The combination of methods, antigen and antibody detection can improve sensitivity (up to 96%) for the diagnosis of acute HPM. Similar improved sensitivity has been noted with the combination of antigen detection and cytopathology with the presence of yeast cells consistent with H. capsulatum in BAL [30].

4. Molecular methods

For more rapid and accurate detection of H. capsulatum in tissues and body fluids, a number of polymerase chain reaction (PCR) methods have been developed: conventional, nested, and real-time PCR (targeting different regions of the H. capsulatum genome) with higher sensitivity and specificity, but many of these have been developed in-house. Although these molecular methods are more sensitive and more specific than antigen detection or serologic testing, there is no FDA-approved commercial PCR-based test.

Currently, the main molecular method for diagnosing HPM involves the use of a rapid DNA probe to identify H. capsulatum isolated from a variety of culture extracts. A real-time PCR assay has been used to identify H. capsulatum in tissue biopsies or at BAL. Other semi-nested PCR assays showed promising results in identifying H. capsulatum in blood or tissue from patients in whom HPM was detected.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used in laboratories with limited resources but has some limitations.

Nucleic acid amplification tests (NAAT) such as PCR or LAMP have a lower probability of false-positive results due to other fungi compared to conventional tests.

A reference database has been established for the identification of H. capsulatum using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), but limited data are available [31].

Metagenomic next-generation sequencing (mNGS) has been used on BAL to diagnose chronic progressive pulmonary lesions; panfungal PCR can also be used to diagnose HPM, but so far they have only been used for research purposes [32].

Although culture is considered the gold standard test for the diagnosis of HPM, molecular methods may be more sensitive. In the future, molecular methods will play an increasingly important role in the diagnosis of HPM to assist clinicians. Their results are currently limited by the heterogeneity of molecular assays, the targets used, the small number of subjects included in the studies, the different types of specimens tested, and the lack of a standard comparative method, as well as the limited presence of molecular methods in international diagnostic guidelines [33].

More robust multicenter studies including studies in large populations are needed for performance evaluation and validation of these assays in different types of patients or samples.

5. Culture and microbiology stains

The gold standard for diagnosis of HPM is isolation of the fungus in culture and observation of characteristic intracellular yeasts in histopathology.

Culture sensitivity is low, the fungus requires several weeks to grow in a standard culture, and the laboratory should have safety level 3 to handle it.

In the microbiology laboratory, H. capsulatum can be detected by staining and direct microscopy of body fluids or tissue samples. H. capsulatum stains poorly with Gram stains, so it is rarely detected by this method. Fluorescent staining: calcofluor white, that binds chitin in the cell wall of the fungus, is useful for identifying H. capsulatum in clinical specimens.

H. capsulatum can be identified in culture after specimens are inoculated on appropriate medium: Sabouraud dextrose agar, and incubated at 25°C, allowing the fungus to grow.

Growth of the mycelial phase occurs at 25 to 30°C, and colonies usually appear in 2–3 weeks but can take up to 8 weeks. The colony is white to tan in color. After identification of a colony on solid medium, a lactophenol cotton blue test can be performed to determine the morphology of the mold. Initially, septate hyphae are seen, followed by smooth-walled (or less commonly spiny) microconidia (size 2–5 μm), and finally tuberculate macroconidia (size 7–15 μm), which are characteristic of H. capsulatum and have a distinct projection on their surface; this development depends on the maturity of the mycelia. Identification of the tuberculate macroconidia strongly suggests H. capsulatum, but the fungus belonging to the genus Sepedonium may also have such a structure. Therefore, a more definitive, specific test is needed to verify that the mold is H. capsulatum before a definitive diagnosis of HPM can be made. There are commercially available, highly specific molecular tests that allow rapid identification when applied to the isolate. There are also more complicated and time-consuming methods, such as the exoantigen test, which is less practical and requires a biosafety level 3 laboratory. They are being replaced by molecular tests.

Yeast-like colonies appear when plates are originally incubated at 37°C, and at microscopy will appear small round narrow-budding yeast. In vitro, the colony is cream-colored and becomes gray with age.

Incubation of the mold at 37°C will transform the mycelia phase into a yeast phase. This has been used in the past as a method to confirm H. capsulatum due to its dimorphic nature, but the conversion rate is low and laborious, and therefore, it cannot be used as a diagnostic tool.

There are some factors that influence the sensitivity of cultures to detect H. capsulatum: clinical manifestation (the highest positivity (74%) occurs in patients with disseminated chronic cavitary pulmonary HPM followed by acute disseminated pulmonary HPM (42%)) [19], host immunity and disease burden, exposure to a large inoculum for the organism. In other forms of HPM such as mild or moderate acute pulmonary HPM, granulomatous mediastinitis, mediastinal fibrosis, and chronic meningitis, cultures are usually negative. In PLWHA, up to 90% of respiratory cultures and 50% of blood cultures may be positive [25].

In disseminated histoplasmosis, specimens collected from the blood, as well as from affected organs such as bone marrow, liver, skin, and mucosal lesions, may result in isolation of H. capsulatum. The average growth time for H. capsulatum in blood cultures is between 12 and 15 days [34] and is rarely observed in conventional blood culture systems (where blood culture bottles are incubated only for 5 days). The lysis centrifugation system (isolator tubes) was more sensitive than automated systems for growing H. capsulatum from blood [35]. Hyphal forms and large, bizarre yeast shapes are seen on smears of blood cultures, rather than typical small, oval yeasts. If sputum or BAL is sent for culture, the laboratory should be informed of the suspected diagnosis to use selective medium (which adds ammonium hydroxide to the agar surface to increase pH, which is helpful, decreases commensal fungal growth, and increases H. capsulatum growth).

6. Histopathology and cytology

The presence of yeast cells consistent with H. capsulatum in the tissues allows a presumptive diagnosis of HPM. The organism is sufficiently characteristic to allow diagnosis of proven HPM according to the European Organization for Research and Treatment of Cancer (EORTC) and Mycoses Study Group Education and Research Consortium (MSGERC) consensus guidelines [36]. H. capsulatum is a non-encapsulated organism. The “capsule” noted in the original initial description report that gave the species its name was an artifact of tissue processing.

H. capsulatum var. capsulatum appears as 2–4 μm narrow, based budding yeast with thin, non-refractile cell walls, stained with Gomori methenamine silver (GMS) or periodic acid-Schiff stains (PAS). Yeasts are typically found intracellularly, phagocytosed in macrophages and histiocytes, often in clusters of many organisms, but can also be found in extracellular spaces, free in tissues. In bone marrow samples, Giemsa staining helps visualize the yeast forms. Wright-Giemsa staining is also applied to peripheral blood and smears to identify intracellular clusters of budding yeasts in patients with disseminated disease. Hematoxylin-eosin (H&E) staining detects H. capsulatum when the organism load is very high (otherwise too insensitive).

H. capsulatum var. duboisii, the causative agent of African histoplasmosis, is larger (6–12 μm) and easily distinguished; it can be seen as short chains in tissues.

Some organisms can mimic the appearance of H. capsulatum in tissues. However, the clinical picture and specific histochemical stains that show a different appearance on histopathological examination help to distinguish H. capsulatum from other organisms, such as Cryptococcus spp., Balstomyces spp., Candida glabrata, Pneumocistis spp., Coccidioides spp., Talaromyces spp., Leishmania spp., Toxoplasma gondi, and Trypanosoma cruzi.

In an appropriate clinical context (e.g., acute pneumonia), the presence of H. capsulatum yeasts in certain tissues or sterile body fluids (e.g., skin lesions) is indicative of acute infection.

The histopathologic examination requires invasive procedures such as bronchoscopy or biopsies. It is more useful in disseminated HPM than in localized pulmonary HPM and is more likely to be positive in the subacute or chronic form than in the acute [6].

Sometimes non-viable organisms can be found in mediastinal or pulmonary granuloma tissue years after initial infection. Pathology may show incomplete granulomas and/or fibrosis rather than a well-formed pyogranulomatous reaction. Complementary tests such as negative cultures, antigenemia, and luck in symptoms can help distinguish between healed, old disease, and active infection.

Patients with severe disease with diffuse pulmonary infiltrates are likely to have organisms detected on lung biopsy. In patients with granulomatous mediastinitis, the caseous specimen collected from necrotic nodules may contain some yeast-like H. capsulatum. In biopsies from patients with fibrosing mediastinitis, organisms are not usually detected in the fibrotic tissue.

Examination of fluids or tissue aspirates for individual cells can provide narrow-based evidence for HPM. Cytologic specimens stained with GMS or PAS show closely spaced budding yeast cells, mainly in macrophages.

It is uncommon to find H. capsulatum on cytologic examination of sputum unless it is a burden infection. Cytopathologic examination of BAL has a sensitivity of 50% for acute pulmonary HPM [37]. When cytopathologic examination BAL is combined with fluid antigen testing, the sensitivity increases to 97% [15].

Fine needle aspiration is a method that can provide a cytodiagnosis of HPM when performed on lymph nodes, adrenal glands, or other tissue.

7. Skin reactivity tests

The histoplasmin skin test reagent has been essential in defining the endemic area for H. capsulatum and can be used to evaluate previous exposure or latency but not active infection. It was useful in revealing the high frequency of asymptomatic infections in endemic areas.

The skin test has not been useful for diagnosis because it cross-reacts with other fungi (especially Blastomyces dermatitidis), produces interference with complement fixation antibody tests, and is insensitive to disseminated infection.

The skin test reagents are no longer commercially available.

8. Conclusions

In North and Latin America, histoplasmosis is the most common endemic fungal infection, but a number of cases have been reported from many areas of the world. In patients who have lived outside endemic areas, a differential diagnosis with HPM should be considered if there is a history of travel or residence in endemic areas.

As a result of climate change, the epidemiology of endemic invasive fungal infections is changing. The exact incidence of HPM throughout the world is still unknown. There is an urgent need to establish a global traceability system to learn more about the diseases.

Disseminated HPM could be diagnosed at an early stage using time-saving methods without cultures, which would reduce hospitalization costs and increase patient survival.

There is an urgent need for newer, faster, and more sensitive diagnostic tools to be available in clinical laboratories around the world to enable faster diagnosis, which plays an important role in improving patient outcomes in the clinic.

Conflict of interest

The authors declare no conflict of interest.