INTRODUCTION — Coccidioides (C. immitis and C. posadasii) are fungi endemic in desert regions of the Southwestern United States, Mexico, and Central and South America, and they are the cause of coccidioidomycosis. Coccidioidomycosis has protean manifestations and is frequently unrecognized, especially in travelers to endemic areas who return to locations where the disease is not typically encountered.

Specific tests must be performed to make a diagnosis of coccidioidomycosis since the clinical manifestations cannot be distinguished from a variety of other infections on clinical grounds alone. Conventional approaches to diagnosing coccidioidomycosis involve detection of specific anticoccidioidal antibodies and/or identification or recovery of Coccidioides from clinical specimens. This topic will review the laboratory diagnosis of coccidioidomycosis. The epidemiology, clinical manifestations, and treatment of coccidioidomycosis are discussed separately.

●(See "Primary pulmonary coccidioidal infection".)

●(See "Coccidioidal meningitis".)

●(See "Coccidioidomycosis in immunocompromised and pregnant persons".)

●(See "Management of pulmonary sequelae and complications of coccidioidomycosis".)

●(See "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis".)

DIAGNOSTIC TESTS

Overview of diagnostic tests — Confirmatory tests are nearly always needed to establish the diagnosis of coccidioidomycosis. Which test to use depends in part upon the patient's signs and symptoms. As examples:

●Most ambulatory patients who are suspected of having coccidioidomycosis are evaluated with serologic testing. (See 'Detection of anticoccidioidal antibodies' below.)

●In patients with early disease, repeated serologic testing may be needed since antibodies can take weeks to develop. Attempting to identify the organism via direct examination or through culture can also be useful in this setting, especially in those with severe disease. (See 'Recovery of Coccidioides from clinical specimens' below.)

●A coccidioidal antigen assay is available that may be positive in the urine and blood of patients with extrapulmonary disease [1,2] and in the cerebrospinal fluid (CSF) of patients with coccidioidal meningitis [3]. (See 'Antigen detection' below and "Primary pulmonary coccidioidal infection", section on 'Patient monitoring'.)

Although several routine laboratory tests and imaging studies may be abnormal in patients with coccidioidomycosis and can suggest the diagnosis (especially with early coccidioidal pneumonia), these findings are nonspecific [4]. Patients may have a slight increase in the peripheral white blood cell count, a peripheral blood eosinophilia, and/or an elevated erythrocyte sedimentation rate [5]. In addition, levels of (1,3)-beta-D-glucan are sometimes elevated in blood [6,7] or in CSF [8]. By contrast, serum procalcitonin levels are usually normal [9]. In patients with pulmonary disease, chest radiographs or other imaging procedures may reveal suggestive abnormalities, such as dense and upper lobe parenchymal infiltrates and hilar or mediastinal adenopathy.

Discussions of how to diagnose specific disease conditions are found in separate topic reviews. (See "Primary pulmonary coccidioidal infection" and "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis" and "Coccidioidal meningitis" and "Management of pulmonary sequelae and complications of coccidioidomycosis" and "Coccidioidomycosis in immunocompromised and pregnant persons".)

Detection of anticoccidioidal antibodies — Most patients who are suspected of having coccidioidomycosis are evaluated with serologic testing.

General approach to testing — Patients with coccidioidal infection develop symptoms of their acute illness 7 to 21 days after exposure, and antibodies develop against specific coccidioidal antigens in many patients during this time. Patients who demonstrate measurable anticoccidioidal antibodies are likely to have a recent illness or one that continues to be active because antibody levels decrease over time and eventually become undetectable in most patients who resolve their infection.

Enzyme-linked immunoassays (EIA) and immunodiffusion tests are the serologic tests most commonly used to make a diagnosis of coccidioidomycosis.

●Serologic tests using EIA for immunoglobulin (Ig)M and IgG should be ordered first, if possible. Several commercial EIA test kits are available to measure coccidioidal antibodies. Because specific antibodies are detected by reagents for different immunoglobulin classes, results are specific for IgM or IgG antibodies.

These tests are probably the most sensitive tests in early infection, although IgM EIA testing can be prone to false-positive results in some laboratories [10-12]. An EIA test may also be useful if there is a high clinical suspicion for coccidioidal infection and the initial test was a negative immunodiffusion assay.

●Immunodiffusion tests should be performed when an initial EIA is positive. Immunodiffusion kits measure tube precipitin-type antibodies (reported as "IgM antibodies" or as "IDTP") and complement-fixing-type antibodies (reported as "IgG antibodies" or as "IDCF"). Immunodiffusion tests are less sensitive than EIAs but are more specific [13-15]. The immunodiffusion test for IgM antibodies is normally done on undiluted serum; however, its sensitivity can be increased by concentrating the serum from two- to eightfold [16,17].

●If the immunodiffusion test for IgG is qualitatively positive, the clinician should contact the reporting laboratory for further clarification to ascertain the titer, since that is used to monitor the response to treatment. A titer can be obtained by a quantitative immunodiffusion (IDCF) test or a conventional complement fixation (CF) assay; these tests are generally performed only in reference laboratories. CF tests are discussed below. (See 'Additional tests' below.)

There are certain limitations to serologic testing with these assays. As an example, the development of serum antibodies may lag behind the onset of illness by several weeks or more [18], particularly in immunocompromised hosts [19,20]. Thus, the absence of detectable anticoccidioidal antibodies does not exclude the diagnosis of coccidioidomycosis, especially early in the course of the clinical illness. In this setting, it is reasonable to repeat the EIA test on a weekly basis for several weeks if coccidioidomycosis is highly suspected.

Another area of uncertainty is how to interpret positive EIA tests that are not supported by immunodiffusion. The EIA is likely a true positive if a subsequent serum sample is positive by immunodiffusion, but this only becomes clear with hindsight and may not occur for all patients. In this setting, when the results of serologic testing are discordant, our approach depends upon the clinical scenario. In a patient with typical clinical manifestations of coccidioidomycosis, we generally view a positive EIA IgM/negative IDTP as supporting the diagnosis. By contrast, in severely ill patients, we do not consider an EIA alone to provide sufficient evidence to support the diagnosis, and further evaluation, such as invasive procedures for fungal cultures or histology, are often needed. (See 'Recovery of Coccidioides from clinical specimens' below.)

More detailed information on the use of serologic testing for the diagnosis of specific conditions is presented elsewhere. (See "Coccidioidal meningitis" and "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis" and "Management of pulmonary sequelae and complications of coccidioidomycosis" and "Coccidioidomycosis in immunocompromised and pregnant persons".)

Additional tests — Although EIA and immunodiffusion tests are most commonly used, older assays, such as the CF assay and tube precipitin antibodies, are still available.

●Complement fixation assay – One of the original methods for detecting coccidioidal antibodies is a complement fixation (CF) assay. This CF assay is often referred to as the "IgG test." Although commercial immunodiffusion kits that can mimic the qualitative result of this reference procedure are now widely used [21], the original assay for CF-type antibodies continues to be used routinely by some reference laboratories. In addition, the CF assay may be ordered in certain settings:

•CF-type antibodies can be detected in body fluids other than serum, and their detection in the CSF is an especially important aid to the diagnosis of coccidioidal meningitis. Other serologic tests may be performed on CSF but are less useful than the CF titer. As an example, antibodies detected by a commercially available EIA may be indicative of coccidioidal meningitis; however, their significance is not well established. (See "Coccidioidal meningitis", section on 'Diagnosis'.)

•The CF test also provides a quantitative measure of antibody concentrations, and serial determinations of CF-type antibodies have prognostic as well as diagnostic value. (See "Primary pulmonary coccidioidal infection", section on 'Patient monitoring'.)

Antibody concentration using CF is determined by assaying twofold serial dilutions of patient specimens for complement consumption. As a general rule, the greater the dilution that still results in complement consumption, the more likely the patient has extensive infection. While there is no precise CF titer that predicts this, a titer of ≥1:32 has been suggested [22]. Because the exact endpoint result in relation to actual antibody concentration is difficult to standardize among laboratories, relative changes in results from the same laboratory may be more helpful in gauging whether a patient's infection is worsening (increasing titers) or improving (decreasing titers).

This test is based upon the depletion of complement that results after antibodies in a specimen from an infected patient form an immune complex when mixed with a coccidioidal antigen. IgG is the immunoglobulin class usually involved in such immune complexes. The complement depletion is assessed by the failure of tanned red blood cells to lyse when added to the mixture; these cells are normally lysed in the presence of complement. Although the coccidioidal antigen used in this procedure is a complex extract of C. immitis, it is now known that the antigen involved in this reaction is a chitinase, a protein enzyme important for the structure of the fungal cell wall [23-25]. Anti-chitinase antibodies responsible for the CF reaction are also responsible for the immunodiffusion IgG reaction referred to above. (See 'General approach to testing' above.)

Sera from patients with histoplasmosis rarely cross-react with coccidioidin in the CF test, and when such cross-reactions occur, they yield low titers compared with those produced with histoplasma antigen [16].

●Tube precipitin-type antibodies – Another one of the original methods for detecting anticoccidioidal antibodies is to demonstrate the formation of an antigen-antibody precipitin when an extract of C. immitis is mixed with serum from an infected patient [26]. These tube precipitin (or TP) antibodies are directed against carbohydrate antigens of the fungal cell wall and develop relatively early during infection.

The tube precipitin assay is considered to be an "IgM test" because IgM antibodies are large and most capable of generating a precipitin reaction. TP antibodies frequently develop early in the course of infection and resolve over weeks to months.

TP-type antibody testing has been largely supplanted by the commercial immunodiffusion kit that measures the same antigen (IDTP) and accurately parallels the results of the original assay [21,27]. (See 'General approach to testing' above.)

Recovery of Coccidioides from clinical specimens — Since Coccidioides are not part of the normal human microbiota, identifying this fungus in respiratory secretions, tissue, or other patient specimens is definitive evidence of a coccidioidal infection. This can be accomplished by either identifying the organism on direct examination or by growing it from fungal cultures.

Staining characteristics — Spherules are the most common morphologic form of Coccidioides seen in clinical specimens. However, they are not easily visible on Gram stain. The simplest method to detect spherules is to make a "wet preparation" using saline or potassium hydroxide solution. Calcofluor staining may improve direct detection of spherules [28]. Bronchoalveolar washings are often analyzed with Papanicolaou staining, which also stains spherules [29]. For tissue, hematoxylin and eosin staining are usually sufficient to detect spherules. Sometimes, special stains, such as periodic acid Schiff or Grocott- or Gomori-methenamine silver are used to clearly demonstrate the organisms (picture 1A-C).

Occasionally, mycelial elements are present in coccidioidal lesions, most frequently in samples taken from pulmonary cavities [30,31]. In one report, hyphal forms were demonstrated in the CSF or brain tissue from five patients with plastic devices present in the central nervous system [32]. While the classic "box car" pattern of arthroconidia is often visible, the organisms cannot be absolutely distinguished from other fungi based on morphologic appearance.

Culture — Positive cultures may be the earliest and, in some instances, the only means of diagnosis. Cultures are most often obtained in patients who require hospitalization, since obtaining fungal cultures in an office setting may be logistically difficult, and isolating Coccidioides becomes increasingly important for patients with more debilitating or widespread infections. (See "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis" and "Coccidioidal meningitis" and "Coccidioidomycosis in immunocompromised and pregnant persons".)

When cultures to detect Coccidioides are obtained, the clinician caring for the patient should alert the microbiology laboratory that Coccidioides infection is a likely possibility, because appropriate biocontainment procedures are needed. Exposure to Coccidioides in the laboratory setting can lead to infection of laboratory staff [33,34].

If a sputum culture is obtained, it should ideally be either the first in the morning or one that is induced. However, respiratory specimens not collected in these manners can still yield Coccidioides. Other specimens that are often sent for culture include CSF and bone and soft tissue.

Coccidioides are capable of growing on nearly any laboratory media and, therefore, even routine cultures for bacterial isolation can be used. Growth is usually evident within the first week, and often in as soon as three days, although prior receipt of antifungal agents may delay isolation.

Most strains of Coccidioides are nonpigmented. However, this is not always the case, and laboratory personnel should handle any culture in which hyphal growth becomes evident with appropriate biocontainment procedures.

To determine if an isolated fungus is within the Coccidioides genus, additional testing is necessary. This is provided by genetic probing using a commercial kit (Genprobe, San Diego, California), which detects Coccidioides-specific DNA. This test has been highly reliable and takes less than one day to complete [35]. There are no commercially available tests to distinguish between C. immitis and C. posadasii [36].

Antigen detection — A coccidioidal antigen assay is available that may be positive in the urine and blood of patients with extrapulmonary disease [1,2] and in the CSF of patients with coccidioidal meningitis [3]. (See "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis", section on 'Diagnosis' and "Coccidioidal meningitis", section on 'Diagnosis'.)

Although coccidioidal antigen can be detected in any body fluid, urine and serum are most commonly tested. This test is most useful in diagnosing infection in those who are severely immunocompromised when serologic testing is negative, and in those with extensive disease. Detection of coccidioidal antigen in the CSF has also been found to be useful in establishing the diagnosis of coccidioidal meningitis [37]. Serial testing of antigen levels is not generally done to monitor the response to therapy in patients with coccidioidomycosis given the lack of literature to support this approach.

Polymerase chain reaction — The use of real-time polymerase chain reaction (PCR) testing has been applied to the diagnosis of coccidioidomycosis in a variety of clinical situations [38-41]. While the sensitivity may not exceed that of culture, the specificity is very high. In addition, it can be used for formalin-fixed tissue [38,42,43], and the result may be available before culture identification. In general, PCR testing is only available at reference laboratories, but this is changing [41,44].

TESTS USED TO SCREEN ASYMPTOMATIC PATIENTS

Immunocompetent patients — A reformulated skin test for coccidioidomycosis (Spherusol, Nielsen BioSciences) was approved by the US Food and Drug Administration to test for a delayed-type hypersensitivity response in patients who have already been diagnosed with pulmonary coccidioidomycosis [45-47]. Although this test was not approved for screening, its use for this purpose has been supported by published evidence with spherulin and other coccidioidal skin testing preparations [48-50]. In one report, this test was used in a prison population in a highly endemic area, and the results suggested it may be useful as a strategy to stratify individuals into immune and non-immune cohorts when necessary to prevent possible exposure [51]. However, two subsequent studies suggest that skin-test results may not always correlate with clinical outcome [52,53]. The skin test should not be used to diagnose current illness since dermal hypersensitivity often remains present for life, and a reactive skin test may therefore reflect past infection unrelated to the current illness.

Immunocompromised hosts — Serologic testing can be used to guide the use of preemptive therapy in asymptomatic immunocompromised patients who live in or are from endemic areas. Indications for testing and the use of antifungal agents for immunocompromised individuals with serologic evidence of infection are discussed elsewhere. (See "Coccidioidomycosis in immunocompromised and pregnant persons".)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Coccidioidomycosis".)

INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5^th to 6^th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10^th to 12^th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on “patient info” and the keyword(s) of interest.)

●Basics topic (see "Patient education: Valley Fever (coccidioidomycosis) (The Basics)")

SUMMARY AND RECOMMENDATIONS

●Coccidioides (C. immitis and C. posadasii) are fungi endemic in desert regions of the Southwestern United States and Central and South America, and they are the cause of coccidioidomycosis. Specific tests must be performed to make a diagnosis of coccidioidomycosis since the clinical manifestations cannot be distinguished from a variety of other infections on clinical grounds alone. (See 'Introduction' above.)

●Patients who are suspected of having coccidioidomycosis are typically evaluated with serologic testing. However, antibodies can take weeks to develop. Thus, for patients with early infection, repeated serologic testing or attempting to visualize or culture the fungus may be the only way to establish the diagnosis. (See 'Overview of diagnostic tests' above.)

●Enzyme-linked immunoassays (EIAs) and immunodiffusion tests are the serologic tests most commonly used to make a diagnosis of coccidioidomycosis. EIA tests are typically used as the initial screening test since they are more sensitive in early infection than immunodiffusion tests. Immunodiffusion tests are then performed if the EIA is positive. (See 'General approach to testing' above.)

●If the immunodiffusion test is qualitatively positive, additional testing by the conventional complement fixation (CF) test or a quantitative immunodiffusion test should be carried out. These titers can be used to monitor the response to treatment. (See 'General approach to testing' above and 'Additional tests' above.)

●Since Coccidioides is never part of the normal microbiota, identifying this fungus in respiratory secretions, tissue, or other patient specimens by either direct examination or by growing it in culture is definitive evidence of a coccidioidal infection. If cultures are obtained, the clinician should alert the microbiology laboratory that Coccidioides infection is a likely possibility because appropriate biocontainment procedures are needed to prevent infection of laboratory staff. (See 'Recovery of Coccidioides from clinical specimens' above.)

●Coccidioidal antigen can be detected in urine or serum as well as other body fluids. This test may be most useful in severely immunocompromised hosts whose serologic testing is negative and in those with extensive disease. It may also be useful in the diagnosis of coccidioidal meningitis. (See 'Antigen detection' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges John Galgiani, MD, who contributed to an earlier version of this topic review.