Coccidioidomycosis: Laboratory diagnosis and screening

INTRODUCTION — Coccidioides spp (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 spp 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 "Management considerations, screening, and prevention of coccidioidomycosis in immunocompromised individuals and pregnant patients".)

●(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 because antibodies can take weeks to develop. Attempting to identify the organism via direct examination or through culture is 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'.)

●Characteristics of many of the tests are reduced in immunocompromised hosts. If initial serologic testing is negative but coccidioidomycosis is still suspected, polymerase chain reaction (PCR) and/or coccidioidal antigen can be sent on the samples obtained for culture and histopathology [4].

Several routine laboratory tests and imaging studies may be abnormal in patients with coccidioidomycosis and can suggest the diagnosis (especially with early coccidioidal pneumonia), but these findings are nonspecific [5]. Patients may have a slight increase in the peripheral white blood cell count, a peripheral blood eosinophilia, and/or an elevated erythrocyte sedimentation rate [6]. In addition, levels of (1,3)-beta-D-glucan are sometimes elevated in blood [7,8] or in CSF [9]. By contrast, serum procalcitonin levels are usually normal [10]. 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", section on 'Diagnosis' and "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis", section on 'Diagnosis' and "Coccidioidal meningitis", section on 'Diagnosis' and "Management considerations, screening, and prevention of coccidioidomycosis in immunocompromised individuals and pregnant patients", section on 'Approach to serologic screening'.)

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 (EIAs) 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. 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 is prone to false-positive results in some laboratories [11-13]. 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 antibody tests should be performed when an initial EIA is positive.

The nomenclature for immunodiffusion tests can be confusing [14]. The immunodiffusion test for IgM antibodies is often referred to as IDTP because it employs the heat-stable antigen preparation used for the original tube precipitin (TP) assay. The immunodiffusion test for IgG is referred to as IDCF because it uses the heat-labile antigen preparation used in the conventional complement-fixation (CF) assay.

Immunodiffusion tests are less sensitive than EIAs but are more specific [15-17]. The immunodiffusion test for IgM (IDTP) antibodies is normally done on undiluted serum; however, its sensitivity can be increased by concentrating the serum from two- to eightfold [18,19].

If the immunodiffusion test for IgG (IDCF) is qualitatively positive, the clinician should request quantification, expressed as a titer, since that is used to monitor the response to treatment. A titer can be obtained by a quantitative immunodiffusion (QID) test or by using a conventional CF assay. (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 [20], particularly in immunocompromised hosts [21,22]. 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.

Serologic testing is less sensitive in immunocompromised persons compared with immunocompetent hosts. In a study of a heterogeneous group of immunosuppressed patients with coccidioidomycosis, seropositivity with any particular method was lower than in immunocompetent individuals [22]. Among hematopoietic cell transplant (HCT) recipients, sensitivities ranged 40 to 55 percent for any particular methodology and 64 percent when methodologies were combined [23]. Results of serology improved in solid organ transplant recipients (SOT) when serologies were repeated approximately two to four weeks after the initial presentation [24].

Another area of uncertainty is how to interpret positive EIA tests that are not supported by immunodiffusion testing. 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 or in those in whom the EIA was used as a screening tool prior to starting immunosuppressive therapy, 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 "Primary pulmonary coccidioidal infection", section on 'Diagnosis' and "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis", section on 'Diagnosis' and "Coccidioidal meningitis", section on 'Diagnosis' and "Management considerations, screening, and prevention of coccidioidomycosis in immunocompromised individuals and pregnant patients", section on 'Approach to serologic screening'.)

Additional tests — Although EIA and immunodiffusion tests are the most common, older assays, such as the CF assay and TP antibodies, are still available.

●Complement-fixation assay – One of the original methods for detecting coccidioidal antibodies is the conventional complement-fixation (CF) assay. This test measures the binding of complement by IgG antibody as determined by inhibition of lysis of foreign red blood cells [18]. As mentioned above, commercial qualitative immunodiffusion kits (IDCF) are available that mimic results of this reference procedure [25] and these can also be quantitated (QID). Either the IDCF/QID or conventional CF assay is appropriate for detecting IgG antibodies in coccidioidomycosis. The QID assay must be used when the conventional CF assay is anticomplementary, that is, when a serum sample nonspecifically binds or inactivates complement, resulting in a positive result in the control sample [14]. The antigen responsible for both the conventional CF assay and the immunodiffusion assay is now known to be a chitinase that is active during endosporulation [26-28]. Recently, chitinase peptides have been used in an enzyme-linked immunosorbent assay (ELISA) to detect and quantitate IgG antibodies [29]. This test is not yet commercially available.

A CF assay may be obtained using samples from body fluids other than serum, and their detection in the CSF is an especially important aid to the diagnosis of coccidioidal meningitis. Other antibody assays 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 antibodies have prognostic as well as diagnostic value (see "Primary pulmonary coccidioidal infection", section on 'Patient monitoring'). In either the IDCF or conventional CF assay, IgG antibody concentration is estimated by performing twofold dilutions of patient sera. As a general rule, the greater the dilution that still results in complement consumption, the more likely the patient has extensive infection. There is no precise CF titer that predicts this [30], but many clinicians become concerned when it is ≥1:32. 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).

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 [18].

●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 [31]. These tube precipitin (or TP) antibodies are directed against a glycoprotein of the fungal cell wall and develop relatively early during infection [32].

The TP assay is presumed to detect IgM because these 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 [25,33]. (See 'General approach to testing' above.)

A point-of-care lateral flow assay has been developed by a commercial laboratory that detects both IgM and IgG coccidioidal antibodies. However, a prospective study found that it had only 31 percent sensitivity compared with EIA [34].

Recovery of Coccidioides from clinical specimens — Since Coccidioides spp 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 spp 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 [35]. Bronchoalveolar washings are often analyzed with Papanicolaou staining, which also stains spherules [36]. 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 [37,38]. 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 [39]. While the classic "box car" pattern of arthroconidia is often visible (picture 2), the organisms cannot be absolutely distinguished from other fungi based on morphologic appearance.

Histopathology is useful for the diagnosis of coccidioidomycosis in immunocompromised patients. Among eight transplant recipients with new coccidioidal infections who had tissue specimens obtained, histopathology was positive in six patients [24]. Because of the diminished cellular immune response, granulomata may be less well formed and contain a greater number of organisms than those seen in immunocompetent hosts [40].

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 spp becomes increasingly important for patients with more debilitating or widespread infections. (See "Primary pulmonary coccidioidal infection", section on 'Diagnosis' and "Manifestations and treatment of nonmeningeal extrathoracic coccidioidomycosis", section on 'Confirming the diagnosis' and "Coccidioidal meningitis", section on 'Diagnosis'.)

In immunocompromised patients, the yield from culture is low. Among 27 solid organ transplant recipients with active coccidioidomycosis, cultures of respiratory samples were positive in 54 percent [24].

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 spp Other specimens that are often sent for culture include CSF and bone and soft tissue.

When cultures to detect Coccidioides spp are obtained, the clinician caring for the patient should alert the microbiology laboratory that Coccidioides spp infection is a likely possibility, because appropriate biocontainment procedures are needed. Exposure to Coccidioides spp in the laboratory setting can lead to infection of laboratory staff [41,42].

Coccidioides spp may grow 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 spp 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. Confirmation of Coccidioides spp is either achieved by demonstration of alternating arthroconidia in the presence of compatible colony morphology and growth characteristics or by using molecular confirmation such as PCR or sequencing available through reference laboratories. There are no commercially available tests to distinguish between C. immitis and C. posadasii [43].

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. Among 24 patients with severe coccidioidomycosis, including 18 who were immunocompromised, the sensitivity of coccidioidal antigenuria was 70.8 percent with a specificity of 99.4 percent [2]. Detection of coccidioidal antigen in the CSF has also been found to be useful in establishing the diagnosis of coccidioidal meningitis [44]. 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 [45-48]. The sensitivity may not exceed that of culture, but the specificity is very high. In addition, it can be used for formalin-fixed tissue [45,49,50], and the result may be available before culture identification.

While there are few data specific to the immunocompromised host, this test, when available, should be used on the same samples as culture. In a study using stored lower respiratory tract samples, the sensitivity was 100 percent, and the specificity ranged between 93.8 and 100 percent when compared with culture [4].

In general, PCR testing is only available at reference laboratories, but this is changing [4,48].

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 [51-53]. 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 [54-56]. 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 [57]. However, two subsequent studies suggest that skin-test results may not always correlate with clinical outcome [58,59]. 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 pre-emptive therapy in asymptomatic immunocompromised patients who live in or are from endemic areas. Indications for screening for coccidioidomycosis in immunocompromised individuals are discussed elsewhere. (See "Management considerations, screening, and prevention of coccidioidomycosis in immunocompromised individuals and pregnant patients", section on 'Screening and prevention in immunocompromised individuals'.)

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

●General principles – Coccidioides spp (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.)

●Approach to diagnostic testing – 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 and 'General approach to testing' above.)

●Anticoccidioidal antibodies

•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 'Detection of anticoccidioidal antibodies' 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 'Detection of anticoccidioidal antibodies' above.)

●Recovery of fungus from clinical specimens – Since Coccidioides spp are 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 coccidioidal 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.)

●Antigen – 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.