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Fungal Infections

Skin testing for endemic fungi

GMS stain of histoplasmosis in tissueSkin testing is useful to know whether a person has been previously infected with an endemic fungus such as Histoplasma capsulatum, Coccidioides immitis/posadasii, Paracoccidioides brasiliensis/lutzii or Sporothrix schenckii. Unfortunately, these tests are of limited use for the diagnosis of acute disease due to one of these pathogens, unless the patient has had a recent negative test. Once the test is positive, it remains positive for the rest of the life of the patient unless the patient becomes immunosuppressed. As many of the infections are asymptomatic, skin testing is a tool mainly used for epidemiologic purposes. In particular skin test surveys in localised populations show how many patients have been infected with one of the endemic fungi. Notably patients with chronic or disseminated disease often have negative skin tests.

These tests are similar to a tuberculin skin test. If the patient has been infected with the fungus being tested and has a normal immune status, the injection of the fungal antigen in the superficial dermis elicit a delayed-type- hypersensitive response in one or two days characterized by erythema and induration. 0.1 ml of the antigen are administered intradermally in the volar surface of any of the arms using a short, beveled 24 to 26 gauge needle. The test is read after 48 hours of the inoculation measuring with a ruler the diameter of the visible and palpable induration produced at the injection point. An induration equal or above 5 mm is considered as a positive test. The response is more reproducible if the antigen is injected with a needle and syringe compared with a scratch or prick test.

The main problem with these tests is the lack of the commercial source of these reagents. Many of the studies have been done with in house preparations with little validation studies. Recently, the Food and Drug Administration (FDA) has approved a skin test for Coccidioidomycosis (Spherusol) produced by Nielsen Biosciences, San Diego, California. Due to the neglected status of endemic fungal infections and the lack of real data of the prevalence of these diseases, it would be desirable to promote the standardization and commercial availability of skin tests has now happened with coccidioidomycosis. This would be an inexpensive and appropriate approach to update our knowledge about the real burden of this disease in different localities. Public health policies for alerting physicians to the relative risk of each infection related to precise geography would help decrease the morbidity and mortality caused by these fungi.

Skin test

Positive test

Negative test

Previously exposed

Acute infection

Never exposed

Immunocompromised

Chronic or disseminated disease

Poor prognosis after new infection

Coccidioides

Yes

Yes

Yes

Yes

Yes

Yes

Histoplasma

Yes

NK

Yes

Yes

NK

NK

Paracoccidioides

Yes

NK

Yes

Yes

NK

NK

Skin test for Coccidioidomycosis

Skin test reaction for coccidioidomycosis antigenCoccidioidomycosis is an endemic disease of the America continent caused by two soil-dwelling fungi Coccidioides immitis and Coccidioides posadasii. The most affected countries are the USA, Mexico, Guatemala, Brazil, Paraguay and Argentina. It is estimated that 150,000 infections occur annually in the USA with unknown figures in other areas of Central and South America (for more precise information see: link to Coccidioidomycosis).

Fig. 1 to the left shows an example of a Coccidioides skin test result.

Coccidioidomycosis is a granulomatous disease that stimulates a delayed-type hypersensitivity response when an antigen is intradermally inoculated. This is the basic principle of the skin test used since the 1940s that has been used to know the epidemiology of the disease as well as to assist in its clinical management. Recently, the FDA has approved a new formulation of the spherule derived antigen preparation (Spherusol, Nielsen Biosciences, San Diego, California, USA) so there is available again a standardized and commercial source of a skin test for an endemic fungal infection. The indication approved by the FDA is only to test the cellular immunity in patients from 16 to 64 years old with an established history of pulmonary coccidioidomycosis. No other indications have been approved because the FDA did not consider the old studies conducted with the same antigen acceptable.

The test is contraindicated in patients with erythema nodosum because local necrosis can appear at the site of the injection. Reaction has to be read at 48 hours because some patients have immediate false positive reactions that appear between 15 min to 1 hour after the skin test injection and persist for 24 hours.

Coccidioidin microscopy (click to enlarge)From the clinical point of view, in primary pulmonary coccidioidomycosis skin testing became positive after 3 days to 3 weeks after the onset of symptoms (83% of patients positive in the first week; 99% in the third week). No interactions have seen with serological testing. Persistent positivity of the test suggests a protective cellular immune response and a good outcome for the disease. On the contrary, the cellular response can decrease with repetitive testing in patients with a poor evolution of the disease (an increase of the antibody titre in the complement-fixing test is usually seen in these cases). Finally, patients with severe coccidioidomycosis may never develop a positive skin test. In summary, a skin test helps to assess the clinical situation of the patient. In symptomatic disease, a negative skin test after three weeks rules out coccidioidomycosis, unless that person is immunocompromised or has early disseminated disease. A positive skin test in a healthy person identifies that person as previously exposed and unlikely to suffer the disease in the future. In a person with an acute infection a positive skin test is a good prognostic signal. Repetitive skin testing can help to follow-up patients with acute disease. Relapsing patients usually have negative skin tests (closer follow-up) whereas non-relapsing patients have repetitive positive results (reassuring follow-up). As this skin test is commercially available may be a cheap and useful diagnostic tool to be used in countries where this disease does not exist but there are suspected cases of patients with recent travel history to an endemic region. A negative result rules out the disease, a positive result warrants further investigation in a reference laboratory.

For epidemiological purposes, skin testing is used to know the prevalence of coccidioidomycosis in certain areas. In addition, skin testing is valuable to identify people susceptible to the disease before starting high-risk activities such as construction projects or archaeology digs, or imprisonment in a locality known as an endemic areas.

Skin test for Histoplasmosis

Histoplasmosis is worldwide disease caused by two different fungi, Histoplasma capsulatum and Histoplasma duboisii. There are endemic areas of histoplasmosis around the world, the Americas being the most prominent. African histoplasmosis is caused by H. duboisii and is limited to the area between the tropics of Capricorn and Cancer and Madagascar (link to Histoplasma capsulatum). In AIDS patients it has been estimated that there are between 150,000 to 300,000 cases of disseminated histoplasmosis annually.

Currently, there is no a commercial source for Histoplasmosis skin testing which makes difficult to compare the studies already done and to perform new ones because all antigen preparations available are from in-house source. In summary, there is a clear need of standardization of Histoplasma antigen for skin testing that would make possible a commercial source for this diagnostic tool.

As with other endemic fungal diseases, histoplasmosis stimulates a delayed-type hypersensitivity response when an antigen is intradermally inoculated. The injection of the antigen intradermally causes a response. An induration equal or above 5 mm after 48h of the inoculation is considered a positive test.

Currently, skin testing with histoplasmin antigen is not useful for diagnostic purposes unless the patient has had a recent negative result. People living in endemic areas usually have skin positive tests. In addition, there are cross-reactions between endemic fungal diseases especially blastomycosis and sometimes a skin test falsely increases the antibody titres in a fixation complement test. Finally, the skin test is negative in patients with severe histoplasmosis. Therefore, histoplasmosis skin testing is only useful to know the number of patients in certain geographical location that have been exposed to Histoplasma.

Skin test for Paracoccidioidomycosis

Micrsocopy of ParacoccidioidesParacoccidioidomycosis is an endemic fungal infection of South America. The real prevalence is not known but it seems that Brazil is the country with more cases in the world (1-3 cases per 100,000 inhabitants). Other countries with frequent cases are Argentina, Colombia, Ecuador and Venezuela. It is much more frequent in males than in females (ratio≈20:1).

Figure to the right shows an example of Paracoccidioides in tissue. (Gomori- Grocott staining, kindly supplied by Eduardo Bagagli and Marcello Franco -copyright)

Currently, there is no a commercial source for P. brasiliensis or P. lutzii skin testing antigen. There are two main antigens, Fava Netto Antigen and gp43. It seems that gp43 is superior to Fava Netto antigen because the response obtained is much higher (92.3% vs 53.8%).

As with other endemic fungal diseases, paracoccidioidomycosis stimulates a delayed-type hypersensitivity response when an antigen is intradermally inoculated. The injection of the antigen intradermally causes a response. An induration equal or above 5 mm after 48h of the inoculation is considered a positive test.


Cutaneous skin test for a delayed type hypersensitivity reaction using the antigen paracoccidioidin. Kindly supplied by Eduardo Bagagli and Marcello Franco (copyright).
Paracoccidioidomycosis skin testing has limited utility for diagnostic purposes. A positive result indicates prior exposure or an acute infection if a recent negative reaction was obtained. Conversion from negative to positive during therapy is a good prognostic signal. Many inhabitants in endemic areas have a positive reaction with no signs or symptoms of disease. However, the disease can progress slowly from a subclinical form to a more widespread and aggressive disease. Therefore, a positive test should warrant a closer follow-up of male or post-menopausal female patients.

There are probably some cross-reactions with other endemic fungi and further study is required. Skin reaction testing is useful to know the prevalence of the infection in geographical areas b but a standardized antigen is required to make the studies results comparable.

References

Skin testing image: Smith CE, Whiting EG. The use of coccidioidin. Am Rev Tuberc. 1948;57(4):330-60.

Coccidioides microscopy: Pappagianis D, Maibach H, Smith CE. Microscopic characteristics of the cutaneous reaction to coccidioidin in humans. Am Rev Respir Dis. 1967;95(2):317-9.

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