Detection of anti-coccidioidal antibodies in serum

Overview: For diagnosing primary infections, serologic tests are the most commonly employed laboratory approach. Of the variety of tests available, some are highly specific for an active infection while a few have a significant frequency of false-positive results. Specific tests are typically selected by the director of the clinical laboratory. Factors involved in such selection include cost, rapidity of obtaining results, the availability of tests from specific reference laboratories that provide other testing services, and the sensitivity and specificity of the tests. Moreover, tests available to a specific provider may change over time because of renegotiated contracts and other factors. This has complicated the interpretation of coccidioidal serologic testing. Because of this, the following two general principles are useful in the primary care setting.

First, in most circumstances, a positive serologic test for coccidioidal antibodies is highly presumptive of a significant coccidioidal infection. Therefore, a report of a positive serologic test should always be reviewed by someone familiar with test interpretation. Second, a negative serologic test never excludes the presence of a coccidioidal infection. For this reason, in evaluating a possible coccidioidal infection, one or even two repeated serologic tests will increase the sensitivity. If repeated testing over the course of two months fails to produce a serologic diagnosis, further serologic testing is likely to be unrewarding.

Tube precipitin (TP) antibodies. Antibodies of this type were originally detected by the presence of a precipitin button that formed at the bottom of a test tube after overnight incubation of patient serum mixed with coccidioidal antigen. Because IgM is most adept at forming such immune precipitins and because these reactions were detected early after onset of infection, this test is now often referred to as the "IgM test." The antigen responsible for this reaction is a polysaccharide from the fungal cell wall. Up to 90% of patients will have TP antibodies detected at some time within the first three weeks of symptoms and this will decline to less than 5% 7 months after the onset of a self-limited illness.

Complement fixing (CF) antibodies. When patient serum is mixed with coccidioidal antigen, an immune complex forms which consumes complement. This event is detected by the subsequent addition of tanned red blood cells, which normally lyse in the presence of complement but remain intact if the complement is depleted.

Since IgG is the immunoglobulin class usually involved in such immune complexes, this test is often referred to as the "IgG test." Although this test was originally developed using various complex extracts 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. In early coccidioidal infections, CF antibodies are detected somewhat later and for longer periods than TP antibodies. CF antibodies can be detected in other body fluids and their detection in the cerebrospinal fluid is an especially important aid to the diagnosis of coccidioidal meningitis. Another difference between CF and TP antibodies is that CF results are expressed as titers, such as 1:4 or 1:64, indicating the greatest dilution of serum at which complement consumption is still detected. In general, higher titers reflect more extensive coccidioidal infection and rising CF antibody concentrations are associated with worsening disease. Thus serial determinations of CF antibody concentrations are or of prognostic as well as diagnostic value.

Immunodiffusion tests (IDTP, IDCF). Antibodies that were detected by the original TP or CF tests can be detected by an alternative procedure known as the immunodiffusion (IDTP and IDCF tests, respectively). Although the conduct of the IDTP and IDCF tests are quite similar, each uses a different antigen in order to measure different types of antibodies. As with the original tests, the IDTP is reported by some laboratories as the "IgM test" and the IDCF as the "IgG test" result. Both tests have been found to be at least as sensitive as their original counterparts. Moreover, immuno-diffusion tests are amenable to being manufactured and distributed as commercially prepared kits, thus allowing the tests to be performed in labs not fully dedicated to a mycology specialty.

Enzyme-linked immunoassays (EIA). An EIA test for coccidioidal antibodies is available commercially. The test kit allows for the specific detection for IgM or IgG antibodies. However, these results are not interchangeable with the "IgM test" or "IgG test" results mentioned above. Positive EIA results are highly sensitive for coccidioidal infection. However, occasionally false positive results are noted especially with the IgM EIA test. At present, EIA results should normally be confirmed with IDTP, IDCF, or CF tests before they are considered diagnostic.

Latex tests. Latex tests for coccidioidal antibodies are also commercially available. They are attractive to clinical laboratories because of their ease of use and rapidity of obtaining a result. However, there are significant numbers of false-positive reactions and therefore a positive latex test is not as reliable as any of the other tests described in this section.

Cultures for C. immitis

Overview. Isolating C. immitis from a sputum or other clinical specimen is definitive evidence of a coccidioidal infection. Despite this, early infections are usually not diagnosed by culture. The reasons why cultures are not routinely obtained in ambulatory care setting are related to several factors. First, fungal cultures are an unusual request in the ambulatory care setting. Although it would be valuable if this were to change, currently requesting fungal cultures on a sputum specimen may be disruptive to work flow. Another consideration is that patients with coccidioidal pneumonia may not be able to produce a specimen for culture. While this problem can usually be circumvented, it takes extra steps. Finally, there is a potential risk to laboratory personnel of isolating C. immitis. Laboratories handling fungal cultures should be thoroughly versed in safe-handling of such specimens and culture medium, and small outpatient laboratories may not be so equipped or trained. None of these considerations are absolute barriers to obtaining culture confirmation. Since negative serologies do not exclude the diagnosis of coccidioidomycosis, cul-tures may be the only way to obtain a timely diagnosis in some patients. As a general rule, the more serious the illness the more likely fungal cultures should be considered as an essential part of the diagnostic evaluation.

Handling of specimens. Sputum or other clinical specimens should be collected in a sterile container. This may be done in the clinic at no risk to personnel since the infection is not transmitted from the primary specimen. Patients with scant sputum can be asked to take a specimen cup home with them and collect a specimen early in the morning (when sputum is usually more readily retrievable) and then return the cup. Such specimens can be stored refrigerated until transfer to the medical facility. For more serious problems, other respiratory secretions (bronchoscopic aspirates) and tissue specimens (skin or bone biopsies) can be submitted for culture.

Evaluation by the laboratory. Direct examination of secretions can be carried out immediately or after the addition of potassium hydroxide. Although culture results are more sensitive than direct examination, identification of spherules in this way is diagnostic and very rapid. C. immitis cannot be detected by gram staining. However, spherules can be seen with cytology stains such as bronchoscopy specimens, by hematoxylin and eosin stains of tissue sections, and with other specialized stains.

C. immitis is not a particularly fastidious organism and will grow well on most mycologic and bacteriologic media. Furthermore, growth usually develops within five to seven days of incubation. Some clinical laboratories within the coccidioidal endemic region have used these characteristics to advantage by holding ALL routine bacteriologic sputum cultures for a week before discarding the plates since some patients who are thought to have a bacterial pneumonia will actually yield C. immitis. When growth occurs, it is typically as a white (non-pigmented) mould. However, there are many exceptions to this general appearance and the morphologic appearance is not reliable in determining if the fungus is or is not C. immitis. Once growth is evident on culture medium, care should be taken not to open the culture container except in an appropriate bio-containment cabinet. Cultures at this stage are highly infectious and can cause disease in persons exposed to them unless the cultures are properly handled. Since the morphologic appearance of C. immitis is not sufficient to determine the species, additional laboratory testing must be carried out for specific identification. The two most common ways for microbiologists to do this are: (1) detectection of a specific coccidioidal antigen (exoantigen) in an extract of the fungus, or (2) detection of a specific DNA sequence using a DNA probe. In place of maintaining these capabilities, some smaller laboratories opt to refer the culture to a reference laboratory where species identification is completed.

Skin testing

Overview. Dermal hypersensitivity to coccidioidal antigens is highly specific for coccidioidal infection. However, since skin tests remain positive after infection in most persons for life, it may not relate to the current illness. In addition, some of the most serious infections may be associated with selective anergy and the skin test may not demonstrate reactivity. Therefore, as useful as skin test results are for epidemiologic studies, important limitations exist when used as a screening procedure for recent infection. In patients known to have Valley Fever, skin testing may have prognostic significance since patients with well controlled infection usually display positive skin tests whereas those with progressive infections often fail to develop dermal reactivity to coccidioidal antigens.

Interpretation. Results of skin tests are measured both 24 and 48 hours after the antigen is injected intradermally. Induration of greater than 5 mm is considered reactive. Erythema at the injection site is not of diagnostic value. Coccidioidal skin testing does not influence coccidioidal serology results.