Pediatric Histoplasmosis Workup

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• General considerations in histoplasmosis

  • The laboratory diagnoses of fungal disease and histoplasmosis are particularly challenging because of the nonspecific clinical findings, the difficulty of culturing organisms, and the confusing array of tests available. MiraVista Diagnostics has established a Web site to assist clinicians.

  • General laboratory findings in disseminated disease include pancytopenia, elevated liver enzyme levels, hyperbilirubinemia, and elevated serum lactate dehydrogenase (LDH) levels.

  • Silver stain of tissue sections or Wright stain of smears of peripheral blood or bone marrow aspirates are useful for diagnosing acute disseminated infection or severe pulmonary infection.

• Culturing

  • The criterion standard of diagnosis is culture of the fungus from clinical specimens.

  • H capsulatum can be recovered from sputum, bronchoalveolar lavage (BAL), skin lesions, blood, or bone marrow on routine fungal cultures, but the organism grows slowly, and plates must be kept as long as 12 weeks. A DNA probe for H capsulatum permits rapid identification.

  • Blood culture by using the lysis-centrifugation system is somewhat more rapid and increases sensitivity.

  • Cultures are positive in as many as 85% of patients with progressive disseminated histoplasmosis (PDH) or chronic pulmonary histoplasmosis (CPH), but they can be falsely negative in about 20% of disseminated cases.

  • The combination of blood and bone marrow cultures increases the likelihood of positive cultures.

  • Bronchoscopy is an important diagnostic tool, especially for PDH, with a diagnostic yield of 60% in patients from endemic areas with pulmonary infiltrates and 88% for chronic cavitary histoplasmosis.

  • Use of several specimens may increase the yield.

• Skin testing

  • Histoplasmin skin testing is not recommended for diagnostic purposes because of the high rate of positive reactions in endemic areas, because of the variable duration of responses to the skin test, and because of skin testing can affect the results of subsequent serologic tests.

  • Skin testing has been useful as an epidemiologic tool.

• Serologic testing

  • A number of tests have been developed to detect H capsulatum antigen or host antibodies to infection. Tests to detect host antibodies are those more commonly used in the clinical setting. However, test results for antibodies can be falsely negative in patients with disseminated disease because of underlying immunosuppression. On the other hand, patients with disseminated disease have a high fungal burden that enables rapid diagnosis by means of antigen detection. Because of the low fungal burden in patients with mild manifestations, the yield of antigen detection is low.

  • Antibody levels peak 6 weeks after exposure and decline over 2-5 years. Elevated antihistoplasmal antibody levels might result from a previous infection or after other types of fungal infections.

• Antibody testing

  • The standard serologic tests for histoplasmosis are the immunodiffusion (ID) test and the complement fixation (CF) test.

  • Histoplasmin, a filtrate of mycelial cultures, is the antigen used in the ID test. Two possible precipitin bands are observed: The H band reflects antibodies formed during active infection and becomes undetectable within 6 months. The M band is present in acute and chronic acute and chronic infection and remains elevated for years. This test is less sensitive than CF and should not be used for screening. M precipitins can be detected in 50-75% of patients with acute histoplasmosis and in almost 80-100% of patients with chronic pulmonary infections.

  • The CF test uses both mycelial and yeast phase antigens. An antibody to a yeast-phase CF titer of more than 1:32 is consistent with active infection in an endemic area, though an acute titer of more than 1:8 suggests infection, especially in nonendemic areas. CF has higher sensitivity than ID. In acute pulmonary histoplasmosis, the CF result is positive in 90% of patients, whereas the sensitivity of ID is as much as 75%.

  • A 4-fold rise in titer between acute and convalescent paired sera is diagnostic. Antibodies may clear within months after a brief exposure but might persist for years after a prolonged exposure.

  • Although CF and ID both are fairly specific, some cross-reactivity with other mycoses occurs.

  • Radioimmunoassay (RIA) and subsequent enzyme immunoassay (EIA) have been reported to be more sensitive than CF; however, higher background seropositivity in endemic areas and recent studies questioning the sensitivity of these assays compared with CF limit their usefulness. ^[12]

  • Antibody responses can also be measured with enzyme immunoassay or Western blot assay. ^{[13, 14]} Although antibodies can be detected faster with these methods than with standard tests, these methods are difficult to standardize and their results are hard to quantitate and interpret.

• Antigen testing

  • Detection of polysaccharide antigen in serum, urine, or BAL of patients with disseminated and acute pulmonary histoplasmosis is a rapid and specific diagnostic method. ^[15] Urine specimens have high sensitivity, as much as 90% in immunocompetent patients with disseminated or acute pulmonary disease. BAL fluid antigen levels can be higher than those in blood or urine, and matched BAL, urine, and serum specimens have the highest yield. Heat and ethylenediaminetetraacetic acid treatment of serum specimens significantly increases sensitivity without compromising specificity. ^[16]

  • The recommended approach is first to perform antigen testing with blood and urine from a patient with suspected histoplasmosis. Then, the focus in testing depends on the symptoms. For example, in patients with respiratory symptoms, obtain BAL samples; in those with CNS symptoms, obtain CSF.

  • Cross-reactivity with other endemic mycoses occurs.

  • If initial results are positive, the antigen test can be used to monitor the treatment response. Antigen levels decrease with treatment, eventually reaching undetectable levels in patients who are cured or in patients undergoing chronic maintenance treatment. Persistent antigenemia or antigenuria indicates an ongoing infection and supports the continuation of antifungal therapy. Antigen levels rise during relapse, enabling detection in patients whose antifungal treatment has been discontinued.

  • Recently, Histoplasma antigen detection by means of enzyme-linked immunosorbent assay (ELISA) has become available for different specimens, including serum, urine, BAL, and CSF samples. The sensitivity of this test is reported to be as high as 92% in urine specimens and 82% in serum specimens from patients with disseminated histoplasmosis. ^{[5, 17]} Although the sensitivity is low in self-limited and CPH, the specificity is as much as 98%.

• Molecular diagnostic testing

  • Preliminary data suggest that polymerase chain reaction (PCR) might improve the accuracy of identifying H capsulatum in tissue specimens. ^{[18, 19]} DNA probes are also commercially available and are used for definitive identification of positive cultures. DNA probes are also commercially available and are used for confirmation of positive cultures.

  • A retrospective review of pediatric patients with cancer at St Jude Children's Research Hospital demonstrated that the most rapid and specific tests for histoplasmosis were histopathologic examination of lung biopsy specimens in patients with localized pulmonary infection and Histoplasma -specific antigen detection in the urine of patients with disseminated histoplasmosis. ^[20]

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• Syndromes in immunocompetent hosts

  • Acute pulmonary syndrome: Plain chest radiography may demonstrate enlarged mediastinal lymph nodes and small reticulonodular infiltrates, with or without small bilateral pleural effusions. More severe acute pulmonary syndromes have more prominent diffuse infiltrates (see the image below).

    Acute pulmonary syndrome in a 16-year-old female adolescent.

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  • Mediastinal obstructive syndromes: Enlarged mediastinal lymph nodes or granulomas, with or without calcification, are often observed on plain chest radiographs (see the image below).

    Hilar lymphadenopathy in an 11-year-old child.

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  • In fibrosing mediastinitis, roentgenographic findings can be subtle and include superior mediastinal widening or carinal splaying (see the image below).

    Fibrosing mediastinitis with mediastinal widening and tracheal deviation.

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  • CT demonstrates the extent of mediastinal involvement better than chest radiography. Other studies, such as esophagography, vascular contrast studies, and ventilation-perfusion scanning, can be useful to determine the extent of obstructive involvement of mediastinal structures.

  • Pericarditis: Echocardiography demonstrates pericardial fluid, but findings are nonspecific.

• Syndromes in hosts with an underlying illness or immunodeficiency

  • CPH: Radiographic manifestations of CPH include apical fibronodular opacities, cavitary lesions, and pleural thickening. CT scanning may be helpful in defining lesions in the context of underlying lung disease.

  • CNS histoplasmosis: Localized enhancing lesions, single or multiple, can be observed on CT scanning or MRI.

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• Pulmonary function testing may demonstrate fixed or variable airway obstructive patterns in mediastinal obstructive syndromes.

• Acute pulmonary disease is most likely to demonstrate a restrictive pattern.

• A recent small study found that testing of fecal mucus for Histoplasma was helpful in diagnosing disseminated histoplasmosis in children.

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• Pericardiocentesis: Pericarditis may occur in 10% of patients who are symptomatic. Pericardiocentesis yields bloody sterile pericardial fluid.

• Bronchoscopy: In acute severe pulmonary syndromes, bronchoscopy with bronchial washing may be indicated to obtain diagnostic material. In chronic pulmonary forms, bronchoscopy with bronchial brushing or transbronchial biopsy may be indicated to obtain samples and to rule out malignancy. Bronchoscopy also may be useful in hemoptysis and broncholithiasis.

• Biopsy: Biopsy of the affected tissues can be performed during open procedures or thoracoscopy.

• Lumbar puncture: In CNS histoplasmosis, the results of lumbar puncture demonstrate lymphocytic pleocytosis, with elevated protein and normal or low glucose values.

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• Pulmonary histoplasmosis has a predominantly mononuclear infiltrate. Multiple granulomas, with multinucleated giant cells, are characteristic findings. Large granulomas are often caseating. The periphery of granulomas may show fibrosis, and calcification of central areas may be present. On hematoxylin and eosin staining, the yeast form of H capsulatum has a false capsule (see the image below).

  Hematoxylin and eosin stain of infected lung tissue. Histoplasma organisms appear to have a false capsule.

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• Special stains, such as Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS), may reveal budding yeast, but the organisms can be mistaken for Pneumocystis carinii and other fungal organisms. In chronic pulmonary forms, in addition to underlying lung disease, vascular involvement, tissue necrosis, and scarring are present. Extensive fibrosis with collagen deposition is observed in fibrosing mediastinitis.