**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,

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

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].
