Approach Considerations
Because most patients recover spontaneously, pursuing documentation of coccidioidal infection is not imperative unless the patient is immunocompromised or has signs of severe progressive disease or dissemination. Diagnosis requires isolation of the organism in culture, identification on histologic specimens, or serologic testing.
The diagnostic evaluation is guided by the patient's clinical presentation and the clinician’s index of suspicion. General laboratory tests include a complete blood count (CBC) and erythrocyte sedimentation rate (ESR). Typical results are a normal white blood cell count or mild lymphocytosis, monocytosis, and/or eosinophilia (>5%) and an elevated ESR.
The specific laboratory tests include the following:
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Immunoglobulin testing
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Culture
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Polymerase chain reaction (PCR) testing
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Skin testing
Observation of Coccidioides in a clinical specimen establishes the diagnosis. Specimens may include any of the following:
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Sputum
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Bronchoalveolar lavage fluid [51]
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Blood
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Urine
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Bone marrow
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Lymph nodes
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Skin lesions
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Cerebrospinal fluid
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Biopsy specimens
Coccidioides urinary antigenemia has been found positive in more than 70% of immunocompromised patients with HIV/AIDS or solid organ transplant; no reports on its sensitivity in less compromised or immunocompetent patients are available. [52] One study has suggested an association between low serum mannose-binding lectin (MBL) levels and symptomatic coccidioidomycosis. [53]
Chest radiography is indicated, with further imaging studies as appropriate. Lumbar puncture is mandatory in patients with suspected meningitis.
Serologic Studies
For more than half a century, detection of antibodies to coccidioidal antigens has been used to establish the diagnosis of coccidioidomycosis and to monitor patients undergoing therapy. [5, 6, 7, 9, 10, 54, 55] As false positives are rare, a positive serologic result is very likely to be clinically relevant in the appropriate clinical setting; however, a negative result does not exclude the diagnosis. Repeat testing following a negative result improves sensitivity.
Serologic tests for coccidioidomycosis measure titers of immunoglobulin M (IgM) or immunoglobulin G (IgG). These tests can also be performed on cerebrospinal fluid (CSF) upon suspicion of coccidioidal meningitis.
Immunoglobulin M
The appearance of immunoglobulin M (IgM) or precipitin antibody against Coccidioides is the most sensitive serologic indication of early infection. IgM is detected in approximately half of all coccidioidal infections within the first week and in approximately 90% by 3 weeks.
The IgM antibody fades over several weeks. In most patients, these antibodies dissipate within 6 months. However, IgM may persist and/or reappear under certain circumstances (eg, chronic cavitary coccidioidomycosis or systemic reinfection associated with ventriculoperitoneal shunt placement).
Methods of IgM detection are as follows:
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Tube precipitin - Older test, not currently in use
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Immunodiffusion tube precipitin - Better specificity; requires more time to perform than latex agglutination or enzyme immunoassay
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Latex agglutination - Rapid and highly sensitive but lacks specificity; higher false-positive rate and positive results require confirmation with another test (ie, immunodiffusion tube precipitin)
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Enzyme immunoassay (EIA) - Highly sensitive but lacks specificity; positive result requires confirmation with immunodiffusion tube precipitin
IgM has only qualitative significance, as the magnitude does not correlate with dissemination or extent of disease. Also, a high rate of false-positives is noted, especially in conditions that stimulate humoral immunity.
Immunoglobulin G
Immunodiffusion and complement fixation (CF) methods can detect coccidioidal immunoglobulin G (IgG). IgG antibodies detected via CF become positive in 85-90% of patients by 3 months after infection onset, persist 6-8 months, and disappear as infection resolves. However, in some cases IgG can persist for years.
In contrast to IgM, for which quantification is uninformative, the CF titer is useful as a quantitative measure of the extent and progression of disease. The CF IgG titer may be low or absent in mild or asymptomatic disease or in immunosuppressed patients. Approximately 95-100% of patients with titers of 1:16 or less do not have disseminated disease. High titers (ie, 1:32 or higher) persist in severe, untreated extrapulmonary or disseminated disease.
Coccidioidal CF titers in the serum and cerebrospinal fluid can be followed to monitor the effect of treatment on disease and predict relapses.
Cultures
The most definitive method for diagnosis is isolation of the organism from clinical specimens. The fungus grows well on most common laboratory media within 3-5 days of inoculation on laboratory media, even when spherules are not present on direct examination. However, the morphology of the colonies (white and cottony mold) is not adequate for identification because other organisms have similar mycelial forms.
Observation of typical arthroconidia may be used to identify the Coccidioides organism.
Identification can be confirmed with a commercially available nucleic acid (gene) probe. Confirmation with exoantigen testing may also be performed, although this test has been replaced by nucleic acid probes. Culture of the organism and definitive identification takes up to 3 weeks.
In culture, Coccidioides spherules can convert to arthroconidia, which are highly contagious. For that reason, cultures should be performed only in Biosafety level 3 laboratories.
Polymerase Chain Reaction Testing
PCR assays are used to detect a target gene after DNA extraction from biopsy specimens. [56] In the clinical setting, Coccidioides PCR serum testing was found to have very high specificity and negative predictive value. [57] PCR testing is a safer and faster alternative to handling highly virulent cultures of Coccidioides.
PCR amplification has been used successfully to identify the highly specific Ag2/PRA antigen gene of C posadasii in appropriate samples of sputum. [58] This technique can be applied to both clinical specimens and cultures. [59, 60] The MBP-1 gene for both Coccidioides species and the SOW-gp82 gene for C posadasii have also been identified with high sensitivity and high specificity via PCR. [61]
Skin Testing
Skin testing for diagnosis of coccidioidomycosis involves the intradermal injection of a coccidioidal antigen preparation (eg, coccidioidin, spherulin). The induration of the skin is measured at 24 hours and 48 hours after the injection. An induration greater than 5 mm is considered reactive. Erythema at the injection site does not aid in the diagnosis of coccidioidomycosis. The skin test becomes positive 10-45 days after infection or 2-21 days after symptom onset, preceding the appearance of serologic markers.
Coccidioidin and spherulin are no longer available in the United States. However, Ampel et al found that archived coccidioidin from the 1970s retains its potency and specificity. [62]
The assessment of cutaneous reactivity to coccidioidal antigens has limited diagnostic utility due to low sensitivity and specificity in endemic areas. [7] A dermal delayed-type hypersensitivity reaction to coccidioidin is highly specific for coccidioidal infection. However, a positive result may not be related to current disease because, in most persons, this skin test result remains positive for life after infection. In addition, a low level of cross-reactivity with blastomycosis and histoplasmosis occurs.
Results in infected individuals may be falsely negative because of a lack of immune response. Anergy is common in patients with disseminated disease, even without underlying immunosuppression.
Although skin testing has important limitations when it is used as a screening procedure for recent infections with C immitis, cutaneous reactivity to coccidioidal antigens has epidemiologic and prognostic implications. In patients in whom coccidioidomycosis is diagnosed with the help of other tests, the results on skin testing of a lack of delayed-type hypersensitivity is a negative prognostic factor.
Radiography and Other Imaging Studies
Obtain chest radiography in all patients with suspected or confirmed coccidioidomycosis, to check for signs of pulmonary infection. Radiographs may be normal or may show a variety of nonspecific changes [5, 6, 7, 8, 9, 10, 11, 44, 63, 64] :
Computed tomography or magnetic resonance imaging scanning, and possibly positron-emission tomography (PET) scanning may also be valuable. For discussion of imaging studies in coccidioidomycosis, see Imaging Studies in Coccidioidomycosis.
Lumbar Puncture
Perform lumbar puncture in patients with fever, headache, nuchal rigidity, meningismus, mental status changes, or ataxia. [12, 63] Many physicians perform lumbar puncture in all patients with extrapulmonary disease or significantly elevated CF IgG titers.
CSF analysis typically reveals a lymphocytic pleocytosis with elevated protein levels and hypoglycorrhachia. In as many as 70% of patients, coccidioidal meningitis is associated with eosinophils in the CSF. Coccidioidomycosis is the most common cause of eosinophilic pleocytosis in the United States.
The diagnosis is aided by the detection of complement-fixating antibodies in the CSF. Coccidioidal meningitis preferentially involves the basilar meninges.
Demonstration of elevated CF titers in CSF establishes the diagnosis of coccidioidal meningitis. CF IgG is present in 90% of patients with coccidioidal meningitis. Elevated CSF CF titers can also be seen with isolated epidural coccidioidal involvement, but only the CSF total protein value is elevated in that situation.
False-positive CSF CF titers are rare but can occur in patients with very high serum CF titers and no meningeal involvement. Rarely, patients with meningitis as their only site of coccidioidal infection have positive CSF CF titers with negative serum CF titers.
Bronchoscopy
Bronchoscopy is a useful diagnostic procedure in suspected coccidioidal infection if results from other studies (eg, sputum, serologies) are not diagnostic. Successful identification of C immitis (and Mycobacterium tuberculosis) using endoscopic ultrasonography has been reported. [65]
Bronchoscopy may be able to double the yield (compared with sputum) in patients with parenchymal infiltrates and cavitary lesions. In one small series, bronchoscopy established a diagnosis of coccidioidal infection in almost 70% of such cases. [66]
Bronchoscopy is especially useful in patients with tracheal or endobronchial coccidioidal infection, in whom the appearance of lesions can range from erythematous plaques to submucosal nodules to endobronchial masses, and diagnosis is established histologically or from culture.
Bronchoscopy with bronchoalveolar lavage, needle aspiration, and/or lung biopsy may be indicated with persistent or progressive infections, especially in hosts who are immunocompromised.
Bronchoscopy has a very low yield in solitary pulmonary nodules secondary to coccidioidomycosis and is not recommended in these patients.
Biopsy
Peripheral solitary pulmonary nodules secondary to coccidiomycosis are especially amenable to diagnosis by percutaneous transthoracic needle biopsy. Most percutaneous transthoracic needle biopsies are CT guided, which allows direct visualization of the needle into the lesion. Specimen fungal stains demonstrating spherules or culture growing C immitis are diagnostic. Cytology of the specimen should be obtained to rule out malignancy.
Closed pleural biopsy may be diagnostic in patients with coccidioidal pleural effusions. Identification of spherules infiltrating the pleural is diagnostic. Culture of pleural biopsy specimens has the highest yield, with isolation of C immitis of in all cases in one small series. The typical pleural effusion is exudative and lymphocytic with modest eosinophilia. Pleural fluid cultures have a low yield, with isolation of C immitis in less than 20% of patients.
Surgical biopsy may be required if the diagnosis cannot be established using the aforementioned approaches. Surgical biopsy is best suited for sampling lymph nodes or parenchymal lung disease. Cervical mediastinoscopy can access most mediastinal lymph nodes and video-assisted thoracoscopy can be used to obtain parenchymal lung tissue.
In extrapulmonary coccidiomycosis, fine-needle aspiration provides a quick and less invasive diagnosis if easily accessible subcutaneous lymph nodes are noted on examination. [67]
Synovial biopsy may be needed to document coccidioidal dissemination to a joint.
Histologic Findings
The diagnosis of coccidioidomycosis can be made by observing spherules (≤70 μm in diameter) that contain endospores in specimens of any body fluid, including sputum or lesion smears and biopsy material. (See the images below.) Direct examination of sputum is less sensitive than cultures for the identification of spherules.
Sputum smears can be stained with potassium hydroxide [KOH]. Spherules are also identified on smears by using calcofluor white or cytologic stains. Spherules are identified in biopsy specimens using standard stains such as hematoxylin and eosin or Papanicolaou stains. Other useful stains for tissue specimens include periodic acid-Schiff stain (PAS) or Gomori methenamine silver stain.
The predominant tissue reaction is granulomatous. In acute lesions, macrophages and polymorphonuclear neutrophils may be numerous. As lesions become chronic, fibrosis ensues. Caseation and, rarely, calcification may occur.
Coccidioidal spherules rupturing and releasing endospores. Gomori methenamine silver (GMS) stain. Photograph by Joseph Rabban, MD.
Gomori methenamine silver stain of Coccidioides immitis spherule (pretracheal lymph node biopsy).
Periodic acid-Schiff stain of Coccidioides immitis spherule (pretracheal lymph node biopsy).
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Soft tissue abscess due to cocci.
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Pulmonary cocci spherule (Hematoxylin-eosin stain).
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Pulmonary cocci spherule, periodic acid-Schiff stain.
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Erythema nodosum can be observed in coccidioidomycosis, tuberculosis, histoplasmosis, drug reactions, and streptococcal infections.
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A Coccidioides immitis spherule containing endospores. Courtesy of Thomas Matthew.
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Arthroconidia become airborne and infect the human host to begin the parasitic phase of its life cycle. The arthroconidia develop into spherules containing endospores, which propagate infection in human tissues. Courtesy of Thomas Matthew.
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A granuloma with coccidioides immitis spherule (pretracheal lymph node biopsy).
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A ruptured Coccidioides immitis spherule (pretracheal lymph node biopsy).
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Gomori methenamine silver stain of Coccidioides immitis spherule (pretracheal lymph node biopsy).
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Periodic acid-Schiff stain of Coccidioides immitis spherule (pretracheal lymph node biopsy).
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Coccidioidal spherules rupturing and releasing endospores. Gomori methenamine silver (GMS) stain. Photograph by Joseph Rabban, MD.
