Section 4 -

Advanced Methods for the Diagnosis of Invasive Fungal Infections Randall T. Hayden

Case 4 - Clinical History The patient was three-year old male, diagnosed with acute myeloid leukemia, who developed fever and neutropenia after admission for his first course of induction chemotherapy. Blood cultures were negative. One week later, chest radiograph showed a right lower lobe infiltrate accompanied by a right pleural effusion. The radiologist's interpretation was pneumonia vs. atelectasis.. Antifungal therapy was added to the patient's antimicrobial regimen. Two weeks after the patient's initial presentation, he experienced a new febrile episode, (Tmax, 38.6°C). The patient improved clinically on broad-spectrum antibiotic and antifungal therapy, but one week later presented with an evolving eschar with underlying erythema over his left knee. No evidence of joint involvement was seen, and radiographic exams showed no bony involvement. One week later the patient reported bilateral ear pain with exam showing otitis externa and retracted tympanic membranes. Five days later an erythematous lesion was noted on the patient's right thigh and right external ear. An infectious disease consultation was requested. Physical exam was remarkable for a 5x5 cm eschar with underlying erythema over the left knee, a 5mm erythematous lesion over the right ventral thigh and a 7-8 mm scab lesion over the left ventral thigh. The right pinna was also erythematous, without tenderness. Laboratory results were significant for an absolute neutrophil count of 0 and several series of negative blood cultures. Chest, abdominal and pelvic CT scans were significant for cervical, submandibular, and submental lymphadenopathy. Chest radiograph showed mild peri-bronchial thickening, but no focal opacities. Shave biopsies of the patient's cutaneous lesions on the right pinna, right anterior proximal thigh, and left knee were performed. H&E and GMS-stained slides of the right pinna were provided for your examination. Results of fungal culture revealed Fusarium species. Case 4 - Figure 1 Click to view with ImageScope Click to view with a Web-Based Viewer Case 4 - Figure 2 Click to view with ImageScope Click to view with a Web-Based Viewer Fungal disease, particularly that caused by filamentous moulds, represents an increasing source of morbidity and mortality for immunocompromised patients. Transplant patients are most severely affected, with bone marrow or hematopoietic stem cell transplant recipients at highest risk. Transplant centers worldwide have seen multi-fold increases in rates of invasive fungal infections since the early 1990's, while rates of candidal disease have stabilized or dropped, in the era of prophylaxis with fluconozole. Although several new and promising mould-active anti-fungal agents have recently become available, the diagnosis of fungal infection remains a challenge. Clinical and radiographic means of diagnosis are often lacking in sensitivity or specificity. Laboratory methods, in the past limited to direct examination of cytologic or histopathologic specimens, and to culture, have also been limited in predictive value and in turnaround time. Increasingly, newer laboratory methods, relying on either immunologic or molecular diagnostic techniques, have come into use. The optimal diagnosis of invasive fungal disease may depend on one or all of these methods, as well as on more traditional means, in any given case. Immunologic methods are primarily represented by antigen detection assays. Currently, there are two commercially produced antigen detection systems on the market for detection of invasive infection with filamentous fungal organisms; both are aimed at constituents of the fungal cell wall. One of these tests, targeting galactomannan (GM), is a typical double-sandwich enzyme immunoassay (EIA), marketed for the detection of aspergillus species. The other method, the β1,3 D-glucan test, is a biochemical assay, based on the horseshoe crab limulus lysate reaction. The glucan test is a broad method, resulting in positive results with all fungal organisms, excepting the zygomycetes and Cryptococcus species. Both assays are currently marketed for testing of serum or plasma specimens and both show variable sensitivities and specificities, depending on patient population and other factors. Sensitivity may be reduced in patients with lesser degrees of immunosuppression or with concomitant mould-active antifungal therapy. Specificity and positive predictive value may be adversely affected by a number of cross-reacting substances, which differ depending on the assay used. Molecular diagnostic tests, though highly promising based on analytical assay characteristics, have yet to fully realize this promise in the clinical diagnostic laboratory. Tests often target multi-copy genes, such as the ribosomal RNA genes. Several groups have demonstrated the potential utility of signal amplification methods, particularly in situ hybridization, for the characterization of fungal elements in tissue sections. These techniques may allow the rapid differentiation of like-appearing organisms, even in paraffin embedded material, while culture-based methods may be slower, may be unavailable (if tissue was not submitted for microbiologic work-up), or may fail entirely as the sensitivity of culture has been reported by some to be less than that of morphologic examination. Target amplification methods, such as polymerase chain reaction (PCR) have been developed for detection of several fungal pathogens in specimens including blood, tissue, and respiratory samples, among others. Like other techniques, these have demonstrated variable clinical utility, sometimes with a limited clinical predictive value. Perhaps surprisingly, immunologic methods, such as antigen detection, may sometimes offer advantages over PCR in terms of sensitivity and in detecting invasive infections early in its clinical course. Despite these issues and despite the fact that no molecular diagnostic methods have been widely distributed by commercial manufacturers, increasing reports have indicated their potential utility. It may be that the optimal diagnosis of invasive fungal infection will involve a multi-phasic approach, making use of both traditional and advanced methods, including both immunologic and molecular diagnostic tools. Reference List Chen, S. C., C. L. Halliday, and W. Meyer. 2002. A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays. Med.Mycol. 40:333-357. Hayden, R. T., P. A. Isotalo, T. Parrett, D. M. Wolk, X. Qian, G. D. Roberts, and R. V. Lloyd. 2003. 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