*Histoplasmosis: Laboratory Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.112411*


**Table 2.**

*Diagnostic tests depending on clinical form of histoplasmosis.*

*Histoplasmosis – A Comprehensive Study of Epidemiology, Pathogenesis, Diagnosis, and Treatment*

## *Histoplasmosis: Laboratory Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.112411*

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 antirabbit 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].
