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Two non-invasive diagnostic tools for invasive aspergilosis: (1-3)-[beta]-D-Glucan and the galactomannan assay.

ABBREVIATIONS: BDG = beta-D-glucan; GM = galactomannan; IA = invasive aspergillosis; IFI = invasive fungal infections; ODI= optical density index.

INDEX TERMS: aspergillosis; galactomannan; glucan.

Invasive aspergillosis (IA) is a serious cause of morbidity and mortality among immunocompromised patients. Prompt and non-invasive methods for diagnosing IA are needed to improve the management of this life-threatening infection in patients with hematological disorders. In summary, this retrospective review of studies performed on the two assays finds that both assays have high sensitivity and specificity but are more useful when used together as a diagnostic strategy for patients with invasive aspergillosis.

Invasive aspergillosis (IA) is one of the most serious causes of morbidity and mortality among immunocompromised patients. Among several factors that contribute to the high mortality rate, difficulties in establishing a reliable diagnosis early enough for successful intervention have been reported. (1)

The crude mortality rate of IA is very high despite appropriate antifungal treatment, since the difficulty in obtaining an early diagnosis results in a delay in establishing treatment. The diagnosis of IA is frequently established postmortem. The establishment of a prompt and optimal noninvasive method for diagnosing IA is needed to improve the management of this life-threatening infection in patients with hematological disorders. (2,3)

Current conventional diagnostic methods such as histological examination and cultures of deep tissues are not only insensitive, but require an aggressive approach. This often precludes their use due to profound thrombocytopenia, hypoxemia, and the critical condition of these patients. As a result, many physicians begin empiric or prophylactic amphotericin B therapy before making a definitive diagnosis. Initiation of empiric or prophylactic therapy with amphotericin B may lead to treatment failure of a full systemic infection or risk of nephrotoxicity. (4)

Over the past decade there have been many advances made to further the options of both diagnostics and therapeutics. Diagnostic options have widened with the addition of diagnostic imaging, histopathology, and several non-invasive laboratory tests for IA. These tests include a double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) antigen (Platellia Aspergillus), tests for (1 [right arrow] 3)-[beta]-D-glucan (BDG) (Glucatell or FungiTec G test), and a number of PCR-based assay systems for Aspergillus DNA. (5)

The GM and BDG test monitors Aspergillus GM by detecting the polysaccharide cell wall component based on the use of a rat monoclonal antibody (Mab), EB-A2, that recognizes the 1-5-[beta]-galactofuranoside side chains of the GM molecule. GM is a polysaccharide that is attached to and released from Aspergillus hyphae during growth. As little as 0.5 ng to 1.0 ng of circulating GM per ml may be detected with this double-sandwich enzyme-linked immunosorbent assay. (6) The excellent sensitivity and specificity of this assay have been repeatedly demonstrated and validated in tests of patients with hematological disorders. (7)

The assay for (1 [right arrow] 3)-[beta]-D-glucan detects (BDG) glucans produced by fungi. BDG is a ubiquitous component of diverse fungal species. The assay system is currently available for the sensitive detection of circulating BDG, based on the Limulus reaction, crab coagulation cascade through Factor G. The activation events result in clot formation when co agulogen is cleaved to coagulin by the clotting enzyme. The introduction of a chromogenic peptide substrate permits spectrophotometric quantitation of the activated proclotting enzyme. (8)

RESULTS

In a study performed by Odabasi and others, the (1 [right arrow] 3)-[beta]-D-glucan detection kit by Glucatell was examined. The results of this study showed that the Glucatell and the Fungitec-G assays were found to be specific for polysaccharides com posed of, or containing, BDG sequences. Both assays were non-reactive with two other types of polysaccharides, (1 [right arrow] 4)-[beta]-D-glucan and (1 [right arrow] 6)-[beta]-D-glucan, as well as non-glucans containing (1 [right arrow] 3)-[beta]-D-glucan linkages. The determined BDG cutoff for the Glucatell assay was 60 pg/mL and was chosen as the positive cut-off for the diagnosis of invasive fungal infections (IFI). Testing was performed with serial se rum samples from 283 subjects with acute myeloid leukemia or myelodysplastic syndrome who were receiving antifungal prophylaxis. The absence of a positive BDG finding had a 100% negative predictive value, and the specificity of the test was 90% for a single positive test result and >96% for more than two sequential positive results. This study demonstrated that the Glucatell serum BDG detection assay is highly sensitive and specific as a diagnostic for IFI. (9)

A multi-center clinical evaluation of the (1 [right arrow] 3)-[beta]-D-glucan assay, Glucatell, was described in a study completed by Ostrosky-Zeichner and others as an aid to diagnose fungal infections in humans. In this study, patients at six clinical sites in the United States were enrolled as either fungal infection negative or with proven or probable IFI. Using a cutoff of 60 pg/mL, the sensitivity and specificity of the assay were 69.9% and 87.1 %, respectively. The positive and negative predictive values were 83.8% and 75.1%. Additionally, a cutoff value of 80 pg/mL, the sensitivity and specificity were 64.4% and 92.4%, respectively, with a positive and negative predictive value of 89% and 73%. Of the ten patients with aspergillosis, 80% had positive results at cutoff values of 60 pg/mL and 80 pg/mL. The study concluded that the reproducible assay results with high specificity and high positive predictive values in a multi-center setting demonstrate that use of the assay to detect serum BDG levels is a useful diagnostic adjunct for IFI. (8)

In 2005 Pazos and others completed a study on the contribution of (1 [right arrow] 3)-[beta]-D-glucan for diagnosis and therapeutic monitoring of IA in neutropenic adults in comparison with serial screening for circulating galactomannan. The two tests (Glucatell, and Platelia Aspergillus) were used retrospectively in a twice-weekly screening for IA in 40 neutropenic adult patients. The cutoff used for GM assay and BDG assay were optical density index (ODI) of 1.0 and 60 pg/mL, respectively. Out of 11 cases there were five proven cases of invasive aspergillosis, three probable cases, and three possible cases. In both assays BDG and GM were detected in 100% of patients with proven IA and in 66% of patients with probable IA. The sensitivity, specificity, and the positive and negative predictive values for GM and BDG were identical: 87.5%, 89.6%, 70%, and 96.3%, respectively. False/positive reactions did occur at a rate of 10.3% in both tests. Although both tests anticipated clinical diagnosis, initiation of antifungal therapy, and computed tomography abnormalities, BDG showed positive results earlier than GM. This study concluded that joint use of both tests is very useful to identify false-positive reactions by each. Joint use improves each individual test's specificity and positive predictive value to 100%, without affecting the sensitivity and negative predictive values. (10)

In a study performed by Kawazu and others, 149 treatment episodes in 96 consecutive patients, including nine proven IA, two probable IA, 13 possible invasive fungal infections (IFI), and 125 no-IA episodes were studied. Overall, 1,233 samples were analyzed using the ELISA for GM detection and 1,243 samples were examined using the BDG test. The GM and BDG levels in a cohort of patients at high risk for IA were measured weekly. The two different tests were examined using receiver-operating characteristic analysis. The area under the receiver-operating characteristic curve was the greatest for ELISA, using two consecutive positive results (0.97; p = 0.055 for ELISA versus BDG). The cutoff for ELISA could be reduced to an optical density index (ODI) of 0.6. With the use of this cutoff for ELISA and the cutoff for BDG, 60 pg/mL, that give a comparable level of specificity, the sensitivity, specificity, positive predictive value, and negative predictive value of the ELISA and BDG tests were 1.00/0.93/0.55/1.00 and 0.55/0.93/0.40/0.96, respectively. The conclusion of this study was that the double sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 ODI. (11)

A series of allogeneic stem cell transplant recipients were examined in a paper written by Maertens and others. The study analyzed the relationship between antigenemia and other diagnostic triggers for initiation of antifungal therapy. The sensitivity and specificity of GM detection were 94.4%, and 98.8%, respectively. The positive and negative predictive values were also 94.4% and 98.8%. This method of detection, with a cutoff value of 1.0 ODI, was found to be better statistically than other triggers, such as unexplained fever, new pulmonary infiltrates, isolation of Aspergillus species, and computed tomography imaging. The results of the study were that antigenemia preceded diagnosis on the basis of radio logic examination or Aspergillus isolation by eight and nine days in 80% and 88.8% of patients. Antigenemia detection preceded therapy in 83.3% of patients. The conclusion of the study was that detection of GM allows earlier diagnosis of aspergillosis than conventional diagnostic criteria. (12)

DISCUSSION

Diagnosis of invasive pulmonary aspergillosis still remains a challenge, mainly because of atypical clinical presentations, coexistence with other infectious and noninfectious diseases, and a relative inability to culture these organisms by standard microbiological techniques. Non-culture based techniques for diagnosing IA have improved steadily in recent years but improvements are still needed.

The development of GM antigen ELISA (Platelia Aspergillus, Bio-Rad Laboratories) tests significantly improved the quality of non-invasive diagnostics. The Bio-Rad GM assay is one such test that may be applied for these purposes; however, reported diagnostic performance has been controversial. Although specificity of the test has been high (i.e., >90%) in most studies performed, reported sensitivities have varied.

The reviewed articles discussed above also demonstrate that the (1 [right arrow] 3)-[beta]-D-glucan assay may be useful to measure serum BDG in clinical specimens with a high specificity and positive predictive values for patients with proven or probable IFI. A cutoff value of 60 pg/mL or 80 pg/mL appears to be appropriate for this test. Although the performance of this assay does not appear to be affected by the presence of antifungal therapy, it is very sensitive to glucans that naturally reside in the environment.

In summary, this large retrospective review of studies per formed on both assays has demonstrated that GM and [beta]-glucan detection assays do have high sensitivity and specificity, but have variable cutoffs that affect the use of these assays as tools for clinical diagnosis of invasive aspergillosis. Although tissue culture and radiologic examination remain the "gold standard" indicators of invasive disease, the low cost, rapid results, and non-invasive methods of the two assays make them more appealing to physicians and patients. The two assays used in combination may provide physicians and patients with a stronger diagnostic strategy for diagnosing invasive aspergillosis.

REFERENCES:

(1.) Patterson TF, Kirkpatrick WR, White M, Hiemenz J W, and others. Invasive aspergillosis. Disease spectrum, treatment practices, and outcomes. 13 Aspergillus Study Group. Medicine (Baltimore) 2000;79(4):250-60.

(2.) Large JP Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 1999;12(2):310-50.

(3.) Singh N. Invasive aspergillosis in organ transplant recipients: new issues in epidemiologic characteristics, diagnosis, and management. Med Mycol 2005;43 Suppl 1:5267-70.

(4.) Hope WW, Walsh TJ, Denning DW Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis 2005;5(10):609-22.

(5.) Dupont B, Richardson M, Verweij PE, Meis JE Invasive aspergillosis. Med Mycol 2000;38 Suppl 1:215-24.

(6.) Stynen D, Goris A, Sarfati J, Large JP A new sensitive sandwich enzyme-linked immunosorbent assay to detect galactofuran in patients with invasive aspergillosis. J Clin Microbiol 1995;33(2):497-500.

(7.) Bart-Delabesse E, Basile M, Al Jijakli A, Souville D, and others. Detection of Aspergillus galactomannan antigenemia to determine biological and clinical implications of Beta-lactam treatments. J Clin Microbiol 2005;43(10):5214-20.

(8.) Ostrosky-Zeichner L, Alexander BD, Kett DH, Vazquez J, and others. Multicenter clinical evaluation of the (1 [right arrow] 3) beta-D-glucan assay as an aid to diagnosis of fungal infections in humans. Clin Infect Dis 2005;41(5):654-9.

(9.) Odabasi Z, Mattiuzzi G, Esrey E, Kantarjian H, Saeki F, and others. Beta-D-glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic syndrome. Clin Infect Dis 2004;39(2):199-205.

(10.) Pazos C, Ponton J, Del Palacio A. Contribution of (1 [right arrow] 3)-beta-D-glucan chromogenic assay to diagnosis and therapeutic monitoring of invasive aspergillosis in neutropenic adult patients: a comparison with serial screening for circulating galactomannan. J Clin Microbiol 2005;43(1):299-305.

(11.) Kawazu M, Kanda Y, Nannya Y, Aoki K, Kurokawa M, and others. Prospective comparison of the diagnostic potential of real-time PCR, double-sandwich enzyme-linked immunosorbent assay for galactomannan, and a (1 [right arrow] 3)-beta-D-glucan test in weekly screening for invasive aspergillosis in patients with hematological disorders. J Clin Microbiol 2004;42(6):2733-41.

(12.) Maertens J, Van Eldere J, Verhaegen J, Verbeken E, Verschakelen J, Boogaerts M. Use of circulating galactomannan screening for early diagnosis of invasive aspergillosis in allogeneic stem cell transplant recipients. J Infect Dis 2002;186(9):1297-306.

Clin Lab Sci 2006;19(4):222

Amy Kelaher is a graduate student in the Department of Medical Research and Technology, University of Maryland School of Medicine, Baltimore MD.

Address for correspondence: Amy Kelaher, Department of Medical and Research Technology, University of Maryland School of Medicine, 100 Penn Street-AHB Room #435, Baltimore MD 21201. (301) 496-3049, (410) 706-0073 (fax). akela001@umaryland.edu.

The peer-reviewed Clinical Practice Section seeks to publish case studies, reports, and articles that are immediately useful, are of a practical nature, or contain information that could lead to improvement in the quality of the clinical laboratory's contribution to patient care, including brief reviews of books, computer programs audiovisual materials, or other materials of interest to readers. Direct all inquiries to Bernadette Rodak MS CLS(NCA), Clin Lab Sci Clinical Practice Editor, Clinical Laboratory Science Program, Indiana University, Clarian Pathology Laboratory, 350 West 11 th Street, 6002F, Indianapolis IN 46202. brodak@iupui.edu
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Title Annotation:CLINICAL PRACTICE
Author:Kelaher, Amy
Publication:Clinical Laboratory Science
Geographic Code:1USA
Date:Sep 22, 2006
Words:2356
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