Pediatric Aspergillosis Workup

Laboratory studies in patients with aspergillosis include the following:

• Microscopic examination and culture

  • The most common and definitive methods for the diagnosis of aspergillosis are direct microscopic examination and isolation of organism by culture; whenever possible, use both approaches because combining these techniques increases the yield by 15-20% over culture alone.

  • Microscopic examination of potassium hydroxide wet preparations or Gomori methenamine silver histopathological stains provide presumptive evidence of aspergillosis if they reveal dichotomously branched and septate hyphae. Stains such as calcofluor white may increase the sensitivity for microscopy.

  • Other fungi, such as Pseudallescheria boydii and Scopulariopsis species may appear similar to Aspergillus species under microscopy and may be mistaken for aspergillosis. Hence, definitive diagnosis relies on isolating the organism from culture media. Aspergillus can be isolated from culture of specimens (eg, blood, spinal fluid, bronchoalveolar fluid, bronchial lavage fluid, endotracheal aspirates), other fluids (eg, synovial, pleural, peritoneal), bone marrow, and from biopsies of internal organs and nonsterile specimens (eg, skin scrapings, nail, hair, urine, mucosal surfaces).

  • Aspergillus is a thermophilic species that grows easily on most mycologic media. The use of fungal media, such as Sabouraud agar, increases the likelihood of isolation. Fluffy white colonies appear after 36-90 hours on the agar surface.

  • Specialized media, such as Czapek-Dox and malt extract, may be required to confirm the species' identity. Agar dilution and microtiter methods have been used for susceptibility testing against azoles.

  • Definitive diagnosis sometimes is difficult, especially in patients whose invasive aspergillosis (IA) involves nonsterile sites, such as sinuses and the respiratory tract. The reason for this difficulty is that the culture sensitivity of sputum and respiratory tract secretions obtained by bronchoalveolar lavage (BAL) and bronchial washings may vary 50-60%. A positive culture result may also represent simple colonization. Nevertheless, in the appropriate clinical setting, repeated positive BAL culture findings have proven reliable evidence for diagnosis.

  • The isolation of Aspergillus from BAL fluid in patients with immunosuppression is highly indicative of invasive aspergillosis and has a specificity of 97%. For these patients, histological analysis must be combined with culture to confirm a definite diagnosis. For example, BAL culture findings may be combined with positive transbronchial biopsy specimen findings obtained at bronchoscopy.

  • Recovery of Aspergillus species from a sterile site, such as the brain (eg, brain abscess), or from the eye provides a valuable tool for definitively diagnosing invasive aspergillosis. Blood culture results are seldom positive.

• Serology/antigen and polymerase chain reaction (PCR)

  • Although serological tests are not widely advocated to help diagnose invasive aspergillosis, these tests have a role in diagnosing aspergillosis in immunocompetent patients ^[3] , including those with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma. In this group of patients, the most commonly used techniques to detect anti-Aspergillus antibodies are double immunodiffusion and counter immunoelectrophoresis. In contrast, results from these tests are unlikely to be positive in an immunocompromised host because neither can elicit a sufficient antibody response.

  • Circulating antigens in the biological fluids, such as galactomannan (GM) and 1-3-beta-glucan, have been used to help diagnose invasive aspergillosis. ^{[4, 5, 6]} The US Food and Drug Administration has approved a commercial assay for the diagnosis of invasive fungal infections called the Fungitell assay. The limitations of this assay are that it can be positive in several other invasive fungal infections, including candidiasis and Pneumocystis jiroveci infection. ^{[7, 8]}

  • The sandwich enzyme-linked immunosorbent assay (ELISA) test detects as much as 0.5 ng of GM per mL of serum and is currently the most sensitive method. ^[9] This test has been recently approved by the US Food and Drug Administration (FDA) and is available for use in the United States. A meta-analysis has shown moderate accuracy in immunocompromised patients, especially for those patients with hematological malignancies or hematological transplant recipients. ^[10]

  • GM is a heat-stable heteropolysaccharide released from aspergillus hyphae during periods of active growth. The negative predictive value of the ELISA test is very good and allows the diagnosis to be made prior to clinical manifestations

  • Aquino et al have described the reasons for false-positive and false-negative results, which are the primary limitation for this test. ^[9] False-positive results could be attributed to use of antibiotics in the pediatric population, infections caused by penicillin species, autoantibodies, contaminated swabs, bacteremia, and airway colonization by Aspergillus. The quantity of GM released can vary according to the species involved and several non-Aspergillus species can give rise to false-positive GM ELISA reactions. False-negative results could be ascribed to prior exposure to antifungal drugs or a cut-off that is too high.

  • EB-A2 rat monoclonal antibody is used in the presently available commercial ELISA testing for serum; this has detection limit of 0.5-1 ng/mL (10-15 times lower than the previously used tests).

  • GM can also be recovered from urine, cerebrospinal fluid, BAL samples but further testing is warranted to compare sensitivity and specificity prior to widespread use.

  • Animal studies performed by Francesconi et al have shown that combining GM and quantitative PCR (qPCR) may lead to a better diagnostic outcome than either alone. ^[11] Further studies are warranted in humans though to reach similar conclusions. It could be used as a prognostic indicator; titers of GM have been shown to fall with clinical response to therapy. However, in patients receiving caspofungin, the titers may not be in concordance with response and may be paradoxically high.

  • A probable diagnosis of invasive aspergillosis can be made in a patient with profound neutropenia, radiological evidence, and 2 consecutive positive serum GM test results. Further studies are needed prior to the use of GM assay as a surveillance tool in diagnosing invasive aspergillosis. ^[12] Use of PCR to detect these antigens in specimens of blood or BAL is also under investigation. One study showed high sensitivity and specificity and good agreement of PCR with the GM assay.

• Other laboratory tests

  • A diagnosis of ABPA often requires fulfillment of the following criteria:

    • Asthma

    • Elevated total serum immunoglobulin E (IgE) level

    • Peripheral blood eosinophilia

    • Precipitating serum antibodies against A fumigatus

    • Proximal bronchiectasis

    • Immediate cutaneous reactivity to A fumigatus antigens or specific serum IgE to A fumigatus, based on radioallergosorbent test (RAST) results

  • ABPA is suggested by an unexplained exacerbation of bronchial asthma. In patients with cystic fibrosis, wheezing, failure of antibiotherapy, and changes in the radiographic findings may suggest ABPA. A possible diagnosis of ABPA may be made with the presence of asthma or eosinophilia. Fleeting pulmonary infiltrates make the diagnosis of probable ABPA, whereas central bronchiectasis makes the diagnosis almost certain. Serologic assessment consistent with ABPA includes precipitins against A fumigatus, positive IgE antibody more than 2 times asthma control, immunoglobulin G (IgG) antibody more than 2 times asthma control, and total serum IgE more than 1000 ng/mL. Diagnosis is established if all 4 serology test results are positive; 3 positive serology test results make the diagnosis very likely. A decline in the serum IgE levels by 50-75% after treatment with prednisone is consistent with the diagnosis of ABPA.

  • Allergic fungal rhinosinusitis is diagnosed by the demonstration of type 1 hypersensitivity, presence of nasal polyps, characteristic CT scan findings (central areas of hyperattenuation within the sinus cavity), positive fungal stain or culture, and allergic mucin with fungal elements and no tissue invasion.

See the list below:

• Pulmonary

  • For patients with immunocompromise, perform a detailed radiographic evaluation, consisting of chest radiography and CT scans, within 24 hours after suspecting an invasive aspergillosis diagnosis.

  • Plain film chest radiography may reveal pleural-based wedge-shaped lesions and cavitations, which are characteristic of invasive pulmonary aspergillosis.

  • CT scanning is a valuable adjunct for early diagnosis of invasive aspergillosis. A characteristic early finding on CT scan is the halo sign, which consists of ground glass attenuation surrounding a soft tissue nodule. The halo sign is caused by hemorrhage around the central necrotic nodule. With cavitation of this nodule, an air crescent sign may subsequently develop. A halo sign on CT scan, combined with positive Aspergillus antigen test results, supports a diagnosis of invasive pulmonary aspergillosis.

• Sinuses

  • Characteristic CT scan findings include unilateral involvement of several sinuses, absence of air fluid levels, and smooth thickened sinus linings.

  • MRIs of the sinuses are a useful adjunct tool for early invasive aspergillosis diagnosis. Fluid opacification of the sinuses is characteristic.

• Cerebral: CT scans may reveal brain abscess with ring enhancement or infarction with contrast enhancement.

See the list below:

• If invasive pulmonary aspergillosis is strongly suspected in patients with diffuse disease and bilateral consolidation, bronchoscopy and bronchial biopsy can be used to help establish the diagnosis.

• In patients with peripheral pulmonary lesions, consider a needle biopsy or surgical resection because of difficult access through bronchoscopy.

• Perform invasive procedures with care in patients who are debilitated and neutropenic.

See the list below:

• Histological section of tissues obtained by biopsy reveals a characteristic pattern that may help diagnose aspergillosis. Fungal mycelia usually have a radiating pattern with invasion of the surrounding blood vessels. Features suggestive of Aspergillus species in biopsy specimens include dichotomously branching hyphae at 45° angles, uniform mycelial width, and septate hyphae.

• Extensive tissue necrosis with hemorrhagic infarction may be apparent at the infection site.

A study by Koo et al reported that an Aspergillus secondary metabolite signature in the patients’ breath can identify individuals with invasive aspergillosis in patients suspected of fungal pneumonia. ^{[13, 14]}