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Aspergillus Infections in Transplant Recipients

University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

SUMMARY

INTRODUCTION

EPIDEMIOLOGY

Hematopoietic Stem Cell Transplant Recipients

Liver Transplant Recipients

Lung Transplant Recipients

Heart Transplant Recipients

Renal Transplant Recipients

PATHOPHYSIOLOGIC BASIS OF INFECTION

Host Response against Aspergillus

Biologic Basis by Which Risk Factors Confer Susceptibility

DIAGNOSIS

Diagnostic Laboratory Technology

Aspergillus galactomannan.

(i) Assay performance with various patient populations.

(ii) Other performance characteristics.

1,3-ß-D-Glucan.

Aspergillus DNA detection by PCR assays.

MANAGEMENT

In Vitro Susceptibility to Antifungal Agents

In Vitro and Animal Studies Utilizing Combinations of Antifungal Agents

Amphotericin and azoles.

Amphotericin and flucytosine, rifampin, or terbinafine.

Amphotericin and echinocandins.

Echinocandins and azoles.

Therapy of Invasive Aspergillosis

Overview.

Further clinical experience with voriconazole and posaconazole.

Clinical experience with the echinocandins.

Clinical experience with the combination regimens.

Therapy in specific situations. (i) Aspergillus tracheobronchitis.

(ii) Allergic bronchopulmonary aspergillosis.

(iii) Aspergilloma.

(iv) Infections of the sinuses.

(v) Infections of the central nervous system.

(vi) Endophthalmitis.

(vii) Infections of skin and soft tissue.

(viii) Osteomyelitis.

(ix) Infections of the heart and vascular system.

Adjunctive immunotherapy.

Surgery for invasive aspergillosis in transplant recipients.

INFECTION CONTROL MEASURES

PROPHYLAXIS

Organ Transplant Recipients

Hematopoietic Stem Cell Transplant Recipients

REFERENCES

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INTRODUCTION

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The number of patients undergoing transplantation has increased exponentially in recent years. An estimated 15,000 allogeneic and 25,000 autologous stem cell transplants are performed worldwide annually (132). Within the last 5 years (1998 to 2002), a total of 113,682 solid organ transplant surgeries (an average of 22,736 annually) were performed in the United States (319). This represents a 20.1% increase in the numbers compared to those in the previous 5 years, i.e., 1993 to 1997 (319).

Transplant recipients are among the most significant subgroups of immunosuppressed hosts at risk for invasive aspergillosis. Aspergillus infections have been reported in 2 to 26% of hematopoietic stem cell transplant (HSCT) recipients and in 1 to 15% of organ transplant recipients (Table 1). Historically, the mortality rate in transplant recipients with invasive aspergillosis has ranged from 74 to 92%. An estimated 9.3 to 16.9% of all deaths in transplant recipients in the first year are considered attributable to invasive aspergillosis (237).

This review discusses the evolving trends in the epidemiology, advances in diagnostic laboratory assays, and the approach to antifungal treatment and prophylaxis for invasive aspergillosis in transplant recipients.

EPIDEMIOLOGY

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The frequency of invasive aspergillosis in autologous transplant recipients has decreased, due largely to more rapid engraftment afforded by the use of hematopoietic growth factors and of grafts with higher stem cell content (102).

The use of peripheral stem cells compared to bone marrow cells for transplantation has been shown to lead to faster hematopoeitic cell repopulation and a reduced potential for disease recurrence (251, 305). Peripheral blood stem cells contain not only more progenitor cells, but also an approximately 1-log-unit-greater number of lymphocytes (23). The larger number of lymphocytes is beneficial in facilitating engraftment and in providing antileukemic effects; however, it may potentially confer a greater likelihood of GVHD (23). While the risk of acute GVHD does not appear to be different, that of chronic GVHD may be higher when peripheral stem cells are used for transplantation (23). Compared to marrow transplantation from an HLA-matched related donor, peripheral blood stem cell transplantation from an HLA-matched related donor was associated with a lower risk of early- but not of late-onset invasive aspergillosis (186).

There has also been an increase in the use of umbilical cord blood stem cells for transplantation, particularly in patients 20 years of age (132). The recipients of cord blood are at a notably high risk for invasive aspergillosis early after transplantation (186). Delayed reconstitution of neutrophil function and cellular immunity and the use of antithymocyte globulin as part of the conditioning regimen in these patients may account for these observations (186). Grafts selected for CD34⁺ progenitor cells have the potential to reduce tumor contamination but may delay lymphocyte reconstitution, particularly of CD4⁺ cells (215). A trend towards a higher rate of invasive aspergillosis was noted in allogeneic transplant recipients with T-cell-depleted or CD34-selected grafts (186). Invasive aspergillosis was documented in 6% of the patients after CD34-selected autologous peripheral blood stem cell transplantation and in 2% (P = 0.20) of those in a contemporaneous cohort who received unselected autologous peripheral stem cell transplantation (225).

Conventionally, the goals of conditioning regimens in HSCT recipients have been to eradicate all host-derived hematopoietic cells in order to minimize graft rejection and to eliminate residual tumor cells. However, severe and protracted neutropenia resulting from these aggressive myeloablative regimens has been a major risk factor for invasive aspergillosis and other opportunistic infections. Nonmyeloablative transplants in which engraftment is achieved with reduced-intensity conditioning regimens have now emerged as an important therapeutic modality in HSCT recipients (87, 135, 197, 208, 329). Between 1998 and 2000, there has been an approximately fivefold increase in the number of nonmyeloablative transplants; these less intensive conditioning regimens are now used in about 25% of the allogeneic transplants in some institutions (132).

Nonmyeloablative conditioning comprises a wide spectrum of reduced-intensity regimens. The risk of infection posed may therefore vary for different regimens and between institutions. Generally, these regimens have a reduced potential for causing mucosal and myelopoietic toxicity but are highly immunosuppressive. Invasive aspergillosis has been reported in 11 to 23% of the nonmyeloablative transplant recipients, with most studies reporting rates that are comparable to or somewhat higher than those after conventional myeloablative hematopoetic cell transplantation (87, 137, 329). A trend towards a higher risk of invasive aspergillosis during the first year after transplant was noted among nonmyeloablative compared to myeloablative transplant recipients (hazard ratio, 1.54; 95% confidence interval [CI], 0.96 to 2.47%; P = 0.09) (87). GVHD has been identified as the major risk factor for invasive aspergillosis in nonmyeloablative transplant recipients, with most infections occurring postengraftment (8, 87, 329). Severe acute GVHD of grade III or IV, chronic extensive GVHD, and cytomegalovirus (CMV) disease each conferred an independent risk for invasive mold infections in nonmyeloablative transplantation recipients (87). In another study, a higher grade of GVHD and a longer duration of corticosteroid therapy portended a significant risk for invasive aspergillosis in nonmyeloablative transplant recipients (329).

A majority (76%) of the invasive mold infections after nonmyeloablative transplantation occurred between days 40 and 180; only 8% occurred before day 40, and 16% developed later than 6 months posttransplantation (87). A lower risk of severe and protracted neutropenia and the retention of donor T-cell responses through mixed chimerism may have a role in preventing early infections after nonmyeloblative conditioning (137, 183).

Aggressive management of GVHD with potent immunosuppressive agents is also a major contributor to the risk for Aspergillus infections (Table 2). Alemtuzumab (Campath-1H), an anti-CD52 monoclonal antibody that depletes peripheral blood T and B cells (without affecting the stem cells), has increasingly come to be used in HSCT recipients. Campath-1H as part of the conditioning regimen conferred a significantly greater risk for early-onset invasive aspergillosis (8). Treatment of GVHD with Campath-1H was likewise associated with a higher risk for late-occurring invasive aspergillosis (8). Infliximab (Remicade) is an anti-tumor necrosis factor alpha antibody used to treat GHVD. Its administration portended a high risk of invasive filamentous fungal infections (193).

Isolation of Aspergillus species from the respiratory tract or of A. fumigatus from any site has a high predictive value (ranging from 60 to 82) for invasive infection in HSCT recipients (237, 327). A. niger appears to be less virulent than A. fumigatus and less likely to be associated with invasive infection when detected in clinical specimens (327). Overall, 8.3% of A. niger isolates compared to 80% of A. fumigatus isolates in one report were associated with invasive infection (327). The fact that A. niger was more likely to be recovered from rectal swabs suggests that its acquisition may occur via the gastrointestinal, rather than the respiratory, tract (327). An association between A. flavus and sinusitis has been noted in these patients (78, 327).

A notable trend in HSCT recipients is the increasing frequency of isolation of Aspergillus species other than A. fumigatus (18, 187). A. terreus is the most common Aspergillus species that is detectable in the bloodstream (151). Overall, A. terreus accounts for 3% of the Aspergillus infections. In a recent report, 20% of the invasive mold infections in HSCT recipients were due to A. terreus (18). From 1996 to 1998, 33.7% of positive bronchoalveolar lavage (BAL) or biopsy cultures from HSCT recipients yielded non-A. fumigatus species of Aspergillus, compared with 18.3% from 1993 to 1995 (P = 0.01). These data are worrisome given that some molds, e.g., A. terreus, are innately resistant to amphotericin B.

The mortality rate in HSCT recipients with invasive aspergillosis ranges from 66.6 to 80% and does not differ for those with early- versus late-onset infections. Invasive aspergillosis is a significant contributor to non-relapse-related mortality in nonmyeloablative HSCT recipients; 9% of overall mortality and 39% of nonrelapse mortality in these patients were due to invasive mold infections (87). The 1-year infection-related mortality rate did not differ for patients who received standard myeloablative (19%) or reduced-intensity (10%) conditioning regimens after allogeneic peripheral blood cell transplantation (190). For patients with mold infections, the 1-year mortality was 68%, a rate similar to that of the recipients of myeloablative transplantation (87).

Renal dysfunction and retransplantation are among the most significant risk factors for invasive aspergillosis in liver transplant recipients (85, 237, 290). Renal failure, particularly the requirement of renal replacement therapy, has been shown to portend a 15- to 25-fold-greater risk for invasive aspergillosis. Approximately one-fourth of the cases of invasive aspergillosis have occurred after retransplantation (85) Liver transplant recipients undergoing retransplantation have a 30-fold higher risk of invasive aspergillosis (85, 287). Fulminant hepatic failure as an indication for liver transplantation, cytomegalovirus infection, and human herpesvirus 6 infection has also been shown to be a risk factors for invasive aspergillosis in these patients (58, 77, 91, 259, 286).

Two notable changes in the epidemiology of invasive aspergillosis appear to have occurred in liver transplant recipients: the onset of invasive aspergillosis later in the posttransplant period and a decline in the incidence of disseminated and central nervous system infections (125, 291). In a study that compared a cohort of patients with invasive aspergillosis from 1998 to 2002 with those from 1990 to 1995, 55% of the infections in the later cohort compared with 23% in the earlier cohort occurred after 90 days after transplantation (291). The precise reasons for the later occurrence of invasive aspergillosis in liver transplant recipients are unclear. Technical surgical advances and better management have led to a lower frequency of organ system failures and an improved outcome in the early postoperative period. Thus, patients at risk in the early 1990s, who may otherwise not have survived, now constitute a growing group of liver transplant recipients who still develop Aspergillus infection, albeit in the later posttransplantation period (291). Cytomegalovirus infection in organ transplant recipients in the present era of ganciclovir prophylaxis is occurring later (167, 288). In this context, a risk factor for invasive aspergillosis may merely have been delayed, thus accounting for the later occurrence of opportunistic infections, such as invasive aspergillosis. In addition, hepatitis C virus infection as an underlying liver disease has become an increasingly common indication for liver transplantation in recent years (291). Indeed, the frequency of hepatitis C virus infection as an underlying liver disease in patients in the later cohort was threefold higher than that in the earlier cohort (291). Significant allograft dysfunction is a known risk for aspergillosis, and because hepatic dysfunction in patients undergoing liver transplantation for hepatitis C virus often occurs later in the posttransplantation period, another risk factor for invasive aspergillosis may now be occurring later.

Whereas disseminated and central nervous system infections were present in 61.5 and 46% of the patients, respectively, in the earlier cohort, only 30% of the patients in the later cohort had disseminated and none had central nervous system infection (291). A potential role of currently used immunosuppressive agents has been proposed as a plausible explanation for these trends. Calcineurin and target-of-rapamycin (TOR) inhibitor agents have potent in vitro activities against Aspergillus species (253, 292). These agents (by inhibition of fungal homologs of calcineurin or the target of rapamycin) have been shown to affect a variety of cellular and physiological processes in a number of pathogenic fungi, including Aspergillus. Calcineurin regulates hyphal growth and is essential for cell cycle progression in A. nidulans (253). Furthermore, calcineurin and TOR inhibitors, in particular, tacrolimus and sirolimus, were found to enhance the activities of antifungal agents in vitro and to attenuate the growth of all Aspergillus species tested (150).

The immunosuppressive activities of these agents outweigh their antifungal activities in vivo. Consequently, invasive aspergillosis continues to be observed in organ transplant recipients. However, it is plausible that the currently used calcineurin and TOR inhibitors may have an impact on the clinical manifestations, tissue tropism, or risk of dissemination associated with this fungus (292). Data from animal studies corroborate these observations. In a mouse model of invasive aspergillosis, cyclosporine, tacrolimus, and rapamycin had no impact on survival (119). However, histopathologic examination documented widely disseminated hyphae in the brains of cyclosporine-treated mice, whereas the brains of tacrolimus- and sirolimus-treated mice showed a nearly complete absence of Aspergillus hyphae (119).

The mortality rate in liver transplant recipients with invasive aspergillosis has ranged from 83 to 88% (68, 85, 237). More recent studies, however, have reported better outcomes (84, 291). A lower mortality rate in patients receiving transplants between 1998 and 2002 (60%) than in those receiving transplants between 1990 and 1995 (92%) was attributable largely to a lower incidence of disseminated infection and a lesser severity of illness in the current cohort of patients, independent of the use of lipid formulations of amphotericin B as therapy (127).

Host factors, including the underlying lung disease and the type of lung transplantation (single versus bilateral), appear to influence the risk and type of Aspergillus infection posttransplantation. Single lung transplant recipients are more likely to develop Aspergillus infections later after transplantation and to have a higher incidence of invasive pulmonary aspergillosis (as opposed to tracheobronchitis) and a higher mortality than other lung transplant recipients (293, 299, 333). In the vast majority of the single lung transplant recipients, invasive aspergillosis has been documented in the native lung, suggesting that the infection likely originates from a preexistent focus or a nidus in the native diseased lung (293, 333). Single lung transplant recipients who developed invasive aspergillosis were more likely to have chronic obstructive pulmonary disease as an underlying condition, which is known to predispose patients to airway colonization with Aspergillus (293). Using DNA primers for strain typing, a molecular epidemiologic study of lung transplant recipients documented that while the clinical strain of Aspergillus in one lung transplant recipient was identical to the one collected from home, the isolates in the other three patients were deemed more likely to be of nosocomial origin (34). A higher mortality rate in single lung transplant recipients may be related to a higher incidence of invasive pulmonary as opposed to tracheobronchial infections.

Bilateral and right lung transplant recipients had a higher incidence of bronchial anastomotic infections in one report (109). Patients undergoing bilateral lung transplantation generally have longer operations and may have a higher risk of ischemia at the site of anastomosis and greater impairment in the cough reflex and muociliary clearance that may confer a higher risk of colonization and subsequent infection of the anastomosis.

Colonization with Aspergillus is common in patients with cystic fibrosis before transplantation (221, 232). While these patients have been shown to be at risk for developing bronchial anastomotic infections, particularly within the first month of transplantation, the risk for pulmonary or disseminated infections does not appear to be higher.

Aspergillus infections in lung transplant recipients may be accompanied by fever in only 15% of the patients (293). Fever is generally absent in tracheobronchitis or bronchial anastomotic infections. A characteristic radiographic appearance is also typically lacking. Focal areas of patchy consolidation of infiltrates are the most common lesions on imaging studies. Nodular infiltrates occur in 27 to 30% of patients, and the halo sign is distinctly unusual (293).

The overall mortality rate from Aspergillus infections in lung transplant recipients is 52 to 55% (198, 293). The mortality rate ranges from 23.7 to 29% in patients with tracheobronchial infections and from 67 to 82% in those with invasive pulmonary infections. Since invasive pulmonary as opposed to tracheobronchial infections occur more commonly in single lung transplant recipients and in the later posttransplant period, the outcome has been poorer in single lung transplant recipients with invasive aspergillosis and in those developing late-onset Aspergillus infections.

Aspergillus can be detected in 10% of heart transplant recipients after transplantation (212). Recovery of Aspergillus species from respiratory tract cultures, particularly of A. fumigatus, is highly predictive of invasive aspergillosis in these patients (212). Reoperation, CMV disease, posttransplant hemodialysis, and the existence of an episode of invasive aspergillosis in the institution's heart transplant program 2 months before or after the transplantation date have been shown to be independent risk factors for invasive aspergillosis in heart transplant recipients (213).

The mortality rate in heart transplant recipients with invasive aspergillosis has ranged from 53 to 78% for invasive pulmonary aspergillosis, was 90% for disseminated infections, and was 100% for disseminated infections that involved the central nervous system (205).

PATHOPHYSIOLOGIC BASIS OF INFECTION

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A body of evidence now suggests that T-cell function and adaptive immunity characterized by a dysregulated production of T-helper (Th) cell cytokines play a pivotal role in the pathogenesis of invasive aspergillosis (49, 50, 54, 96, 113, 164, 169, 199, 262-264). T-helper CD4⁺ cells are the major effector cells responsible for cell-mediated immune responses for the eradication of pathogens (233). Upon recognition of exogenous antigen presented by major histocompatibility complex class II molecules, T-helper cells differentiate into Th1 or Th2 cells, which are morphologically indistinguishable but differ in the pattern of cytokines they produce (233). Th1 cytokines induce a predominantly cell-mediated inflammatory response, whereas Th2 cytokines facilitate antibody production (164, 233).

Th1 responses, e.g., tumor necrosis factor alpha, gamma interferon gamma, interleukin-12 (IL-12), and IL-15, have been shown to confer protection against Aspergillus. These cytokines augment superoxide production and enhance the antifungal activity of polymorphonuclear and mononuclear phagocytes against Aspergillus species. In a mouse model of invasive pulmonary aspergillosis, exposure of immunocompetent animals to a sublethal inoculum of Aspergillus conidia led to the development of resistance to subsequent local and systemic infection (49). This protective Th1 response was characterized by antigen-specific CD4⁺ T cells that produced gamma interferon and IL-2. Notably, adoptive transfer of Aspergillus-specific CD4⁺ splenic T cells from these animals conferred protection in naive animals challenged with the Aspergillus conidia (233).

Th2 responses, e.g., IL-4 and IL-10 production, by comparison have been associated with disease progression, and their neutralization has been associated with an improvement in infection. Th2 cytokines impair the microbicidal activity and hyphal damage mediated by mononuclear cells. IL-10, which is typically a Th2 cytokine, may paradoxically enhance the phagocytic activity against A. fumigatus hyphae (262). However, it had no impact on the intracellular conidiocidal activity (262). The net result of Th2 regulatory cytokines, however, is an increased susceptibility to infection.

Signal transduction mediated by Toll-like receptors (TLRs) has been shown to play a key role in immunity against aspergillosis (220). Of 10 human TLRs identified to date, TLR2 and TLR4 are involved in the regulation of cytokines that are important in the pathogenesis of A. fumigatus (220). Aspergillus conidia stimulated both TLR2 and TLR4 to induce a Th1 cytokine response (220). Germination of hyphae led to the loss of TLR4-mediated signals; however, TLR2-dependent mechanisms remained intact, leading to stimulated production of IL-10 and ultimately a predominant Th2 response (220). Thus, the switch from a proinflammatory to an anti-inflammatory cytokine profile during germination of Aspergillus hyphae may represent an escape mechanism by which the fungus evades the host defense (220). These data also suggest that Toll-like receptor-mediated signaling pathways may represent a potential target for immunomodulatory interventions against Aspergillus (220).

T-cell responses may have an important role in the host defense against aspergillosis in nonneutropenic hematopoietic stem cell transplant recipients and organ transplant recipients in whom neutropenia is not a major risk factor for invasive aspergillosis. Allogeneic stem cell transplant recipients in the late posttransplant period (median, 134 days) were shown to have suppressed Aspergillus-specific T-cell reconstitution (113). These patients also had a low gamma interferon/IL-10 ratio, which is suggestive of a Th2 response that may potentially account for their protracted susceptibility to the development of invasive aspergillosis. By comparison, healthy individuals demonstrated a predominantly Th1-type cellular response.

Finally, dendritic cells pulsed with Aspergillus conidia or transfected with conidial RNA can induce Th1-cell priming (33). Upon adoptive transfer, such cells were protective against invasive aspergillosis in mice receiving allogeneic hematopoietic stem cell transplantation (33). Thus, dendritic cells could potentially be employed as one of the vaccine strategies against invasive aspergillosis (33, 302).

Taken together, these data suggest that approaches targeted towards augmenting cellular immunity, such as neutralization of suppressive cytokines, enhancement of Th1 responses, and transfer of adoptive cellular immunotherapy, warrant future investigations as therapeutic modalities for Aspergillus infections.

DIAGNOSIS

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A substantial delay in establishing an early diagnosis remains a major impediment to the successful treatment of invasive aspergillosis. Cultures of the respiratory tract secretions lack sensitivity. Aspergillus is grown from sputum specimens in only 8 to 34%, and from BAL specimens in 45 to 62%, of patients with invasive aspergillosis (237). Thus, confirmation of the diagnosis of invasive aspergillosis has typically required histopathologic evaluation. However, profound neutropenia and thrombocytopenia often preclude the pursuit of biopsies for acquisition of tissue. Characteristic radiographic findings on high-resolution imaging studies e.g., the halo sign, have allowed earlier diagnosis of infection in neutropenic patients. The halo sign, however, is documented in 33 to 60% of patients and is short-lived (41). To be useful for the diagnosis of invasive aspergillosis, the computed tomography scan must be performed within 5 days of the onset of infection, since 75% of the initial halo signs disappear within a week (41). The "air crescent" sign does not appear until the third week of the illness, and its appearance may be too delayed to be helpful in the diagnosis of invasive aspergillosis (41).

In the recent years, efforts have been directed towards identifying noninvasive markers for rapid and reliable diagnosis of invasive aspergillosis. Those based on the detection of antifungal antibodies have proven to be unreliable in transplant recipients receiving immunosuppressive agents (51). Instead, tests based on identifying fungal antigens or metabolites released into the circulation are potentially promising.

Although galactomannan testing using latex agglutination has been available for over a decade, a high detection threshold for galactomannan with this assay has precluded its use as a diagnostic test for invasive aspergillosis (307). The galactomannan concentrations detectable by latex agglutination, radioimmunoassay, and enzyme-linked immunosorbent assay inhibition are 15, 10, and 4 to 5 ng/ml, respectively. The double-sandwich enzyme-linked immunosorbent assay, on the other hand, can detect galactomannan at concentrations of as low as 0.5 ng/ml and has proven to be a potentially promising tool for the early diagnosis of Aspergillus infection (307).

False-positive tests have been documented in up to 5 to 14% of patients with hematologic malignancy and HSCT, 13% of liver transplant recipients, and 20% of lung transplant recipients (44, 126, 162, 176, 177, 260). Cytotoxic chemotherapeutic agents and autoreactive antibodies e.g., in chronic graft-versus-host disease, have been shown to cause false-positive tests in patients with hematologic malignancies and in HSCT recipients. Liver transplant recipients undergoing transplantation for autoimmune liver disease and those requiring dialysis were significantly more likely to have false-positive galactomannan tests (162). Galactomannan is renally cleared, with excretion into the urine accounting for 35% of the dose by 24 h in an animal model study (22). However, the effect of renal failure or dialysis on the clearance of galactomannan is not known.

In a report on lung transplant recipients, false reactivity with the Aspergillus EIA was documented in 20% (14 of 70) of the patients (126). False-positive tests occurred within 3 days of lung transplantation in 43%, within 7 days in 64%, and within 14 days in 79% of the patients (126). Patients undergoing lung transplantation for cystic fibrosis and chronic obstructive pulmonary disease were more likely to have positive tests in the early posttransplant period (126). Cross-reactivity of galactomannan antibody with the lipoteichoic acid of Bifidobacterium bifidum subsp. pennsylvanicum, which is found in large inocula in the guts of breast- and formula-fed infants, may account for high false positivity of the test in premature infants and neonates (201).

Cross-reactivity of Platelia Aspergillus galactomannan EIA with Penicillium spp. has been noted (310) but is deemed to be of little clinical relevance since Penicillium spp. are rarely pathogens in humans. Ansorg et al., however, first documented that drugs of fungal origin such as antibiotics may be associated with a false-positive test; galactomannan was detected in a batch of ampicillin-sulbactam and in two batches of piperacillin (13). At least three recent reports from Europe have documented false EIA reactivity related to the administration of piperacillin-tazobactam (2, 309, 326). Over a 10-week period at one institution, receipt of piperacillin-tazobactam was documented in 67.5% (25 of 37) of patients with positive tests, compared to 5.4% (2 of 37) of those with negative tests (P < 0.001) (309). In another report, of samples from patients receiving piperacillin-tazobactam, 74% tested positive, compared to 11% of those not receiving this agent (326). Twelve of 15 batches of piperacillin-tazobactam tested yielded a positive test, with a median index value of 4.6 (326). Three of four batches of piperacillin-tazobactam were shown to test positive, with estimated amounts of 10, 2, and 30 µg of the galactomannan antigen per 4-g dose of piperacillin-tazobactam in each of the batches (2).

The reactivities of commonly used antibiotics of fungal origin (penicillins and cephalosporins), nonfungal origin (erythromycin and gentamicin), and synthetic origin (quinolones) with the Platelia Aspergillus galactomannan assay were assessed in one study (294). Undiluted samples of piperacillin-tazobactam and piperacillin tested positive, whereas those of amoxicillin, ampicillin-sulbactam, cefazolin, ceftazidime, erythromycin, gentamicin, and levofloxacin tested negative. All lots (n = 3) of piperacillin-tazobactam and all bags within each lot tested positive with an index value of greater than 5.168. At achievable concentrations in serum, however, only one of three lots of piperacillin-tazobactam yielded a positive test; concentrations of 75, 150, and 300 µg/ml tested positive, whereas lower concentrations, mimicking the trough levels (5 to 10 µg/ml) tested negative for the galactomannan. Thus, achievable concentrations of piperacillin-tazobactam in serum may potentially result in a false-positive test for galactomannan (294, 309, 326). The timing of collection of the sample may influence the test results, with reactivity being less likely in samples collected at trough levels or prior to the administration of the dose (294).

With neutropenic patients at high risk for invasive aspergillosis, the German Aspergillus PCR study group documented sensitivity and specificity rates of 63.5 and 93.5%, respectively, with nested PCR and of 14.3 and 98.9%, respectively, with galactomannan for the diagnosis of invasive aspergillosis (39). Quantifying nested PCR results with light cycler-mediated PCR assay did not add to the diagnostic utility of the test (39). When assessed simultaneously with a cohort of patients with leukemia and those undergoing bone marrow transplantation, the sensitivities of PCR, galactomannan assay, and 1,3-ß-D-glucan measurement for the detection of invasive aspergillosis were 79, 58, and 67%, respectively, and the specificities were 92, 97, and 84%, respectively (138). Other studies have reported PCR to be less sensitive than the glactomannan assay for the diagnosis of invasive aspergillosis (35, 60). PCR results were usually positive when the galactomannan sample was highly positive; 12 of 20 PCR assays that yielded a positive result were observed in association with galactomannan titers of >5 ng/ml (35). A prospective comparison of real-time PCR, galactomannan, and 1,3-ß-D-glucan assays as weekly screening for invasive aspergillosis in patients with hematologic disorders showed that the galactomannan test was most sensitive at predicting the diagnosis (142).

Although the conventional immunoassays and PCR clearly represent an advance compared to the detection of Aspergillus by culture, they largely lack optimal sensitivity, specificity, and the speed for the rapid diagnosis of infection. Rider et al. have proposed a novel pathogen sensor system for the identification of specific microorganisms that is based on the rationale that B lymphocytes as mediators of the adaptive immune system have evolved to efficiently identify pathogens (256). A biosensor comprising B-cell lines genetically engineered to express membrane-bound antibodies specific for the pathogen emits light within seconds of exposure to the targeted pathogen. A notable attribute of this pathogen identification system is that it can be tailored to detect a specific species of the microorganism (254, 256). Although this technology has not yet been applied for mycologic diagnosis, it is particularly relevant for the detection of Aspergillus, where timely and accurate diagnosis is critically important for the prompt initiation of appropriate therapy.

MANAGEMENT

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Fluconazole is essentially ineffective against Aspergillus spp. Amphotericin, the newer azoles, and the echinocandins are generally active against Aspergillus spp. (Table 4). However, resistance of Aspergillus isolates to amphotericin, itraconazole, voriconazole, and caspofungin has either been observed in isolates from patients or been created in the laboratory (71, 74, 81, 131, 180), It has been suggested that in vitro susceptibility testing of Aspergillus spp. is a predictor of clinical outcome in invasive aspergillosis (163). This statement was made on the basis of the finding that nine patients infected with amphotericin resistant-A. terreus but treated with amphotericin died (163). Itraconazole resistance in A. fumigatus has been correlated with clinical failure in both humans and experimental animal models of infection (71). However, clinical failure in invasive aspergillosis may have many causes other than drug resistance; furthermore, in patients who have died from invasive aspergillosis despite amphotericin treatment, emergence of resistance to amphotericin or itraconazole during treatment has not been detected (65, 207). In a review of 11 patients with hematologic malignancies in whom A. fumigatus or A. flavus was isolated, while on amphotericin, resistance to amphotericin was not detected, but it was thought that poor drug penetration to the site of infection may have been a contributor to failure (238).

Although amphotericin preparations have been the drugs of choice for invasive aspergillosis for many years, a recent randomized trial (116) comparing amphotericin with voriconazole appears to have placed voriconazole as the treatment of choice for invasive aspergillosis. However, it must be recognized that the clinical appearance of invasive aspergillosis may be mimicked by mucormycosis and some other fungal infections. Voriconazole lacks activity against the zygomycetes, so amphotericin should remain the drug of choice if microbiologic confirmation of Aspergillus infection is lacking.

The design of the study comparing voriconazole with amphotericin was as follows. The study was an open, randomized comparison of voriconazole and conventional amphotericin (116). Patients received conventional amphotericin (1 mg/kg/day) or voriconazole (6 mg/kg intravenously for two doses and then 4 mg/kg every 12 h intravenously, which could be followed by 200 mg every 12 h orally). Patients could be switched to other licensed antifungal therapy (for example, lipid preparations of amphotericin or itraconazole) after their initial randomized therapy. A total of 392 patients were enrolled over 3 years in 92 medical centers in 19 countries. Of these, 277 patients had confirmed invasive aspergillosis and received at least one dose of the study drug; 144 of these patients received voriconazole and 133 received conventional amphotericin. Approximately 80% of the patients had a hematologic malignancy or had undergone allogeneic bone marrow transplantation. After 12 weeks of receiving the first dose of study drug, a complete or partial response was seen in 52.8% of those who had received voriconazole and in 31.6% of those who had received amphotericin. These differences were statistically significant. Survival of patients at 12 weeks was 70.8% for those who had received voriconazole versus 57.9% for those who had received amphotericin. Again this difference was statistically significant (hazard ratio, 0.59; 95% CI, 0.40 to 0.88).

Unanswered questions include whether voriconazole versus lipid preparations of amphotericin from the outset of therapy would have provided the same results. Three lipid preparations of amphotericin are now marketed for clinical use: amphotericin liposome for injection (AmBisome; Fujisawa, Deerfield, Ill.), amphotericin colloidal dispersion (Amphotec; Sequus, Menlo Park, Calif.), and amphotericin B lipid complex (Abelcet; The Lipsome Company, Princeton, N.J.). There are conflicting data as to which lipid preparation should be preferred for treatment of invasive aspergillosis. However, a dose of 5 mg/kg/day is currently accepted as appropriate for all of these drugs in the initial treatment of invasive aspergillosis.

A single-center, retrospective study that compared the outcomes for 41 liver transplant recipients who received either conventional amphotericin or amphotericin B lipid complex documented that the 60-day mortality rate was 83% in the patients treated with conventional amphotericin versus 33% in the patients treated with amphotericin B lipid complex (168). Although this was not a randomized analysis, in multivariate analysis amphotericin B lipid complex therapy was an independent predictor of survival (168). In a randomized, double-blind, controlled trial for the treatment of invasive aspergillosis in immunocompromised patients, of whom 42% were HSCT and 5% were organ transplant recipients, amphotericin B colloidal dispersion was shown to have an efficacy equivalent to that of amphotericin B (32).

For A. terreus, voriconazole compared to other antifungal therapies was associated with reduced mortality at 12 weeks (300) and may be a better therapeutic option than a polyene.

Two practical issues arise when utilizing voriconazole in the management of transplant recipients. The first is that there is a significant interaction between voriconazole and cyclosporine, tacrolimus, or sirolimus (265, 323). In human liver microsomes, voriconazole at a concentration of 4 µg/ml inhibited the metabolism of tacrolimus by 50% (323). Indeed, all azole antifungal agents have the potential to increase the levels of cyclosporine, tacrolimus, and sirolimus via inhibition of cytochrome P450 isoenzymes (323). The rank order of potency of the azoles for the inhibition of P450 isoenzymes is ketoconazole > voriconazole > itraconazole > fluconazole (322). The second issue is that the use of intravenous voriconazole is contraindicated in patients with creatinine clearance of less than 50 ml/min. This is because of concerns regarding accumulation of voriconazole's renally excreted carrier, sulfobutyl ether ß-cyclodextrin sodium. The consequences of accumulation of sulfobutyl ether ß-cyclodextrin sodium in humans are not known. Oral administration of voriconazole in patients with moderate to severe renal dysfunction is safe.

Posaconazole is a new triazole compound that exhibits significant in vitro activity against a number of fungi, including Aspergillus (61, 62). The drug is currently in advanced stages of clinical development. It is orally bioavailable and exhibits dose-proportional pharmacokinetics up to a total dose of 800 mg/day; i.e., saturation of absorption occurs at doses of above 800 mg (61). Food increases the relative oral bioavailability of posaconazole by 400% (62). The drug is metabolized by glucoronidation, with only minor amounts of the unchanged drug excreted in the urine (158). Posaconazole inhibits CYP3A4 but not cytochrome P450 enzymes. Since cyclosporine and tacrolimus are substrates for CYP3A4 as well, coadministration of posaconazole with tacrolimus decreased tacrolimus clearance by 5-fold (272). Concomitant administration of cyclosporine with posaconazole necessitated a 14 to 29% reduction in cyclosporine dosage (62).

Posaconazole was used in the treatment of 25 patients with invasive aspergillosis refractory to conventional therapies (108). Of 15 patients who were still alive 4 weeks after use of the drug had commenced, 53% had a positive clinical response (108). The dose used was 200 mg four times per day as an oral suspension and 400 mg twice per day when the patient was discharged from hospital (108). Of note is that posaconazole, unlike other azoles, has promising activity against zygomycosis (99). In patients with proven or probable zygomycosis who received posaconazole for refractory infection or intolerance to standard therapies, a successful outcome was documented in 70% (99).

Micafungin has been successfully utilized in Japan as salvage therapy for invasive aspergillosis in patients refractory to amphotericin, but the number of published reports are few (342). An open-label multicenter study of micafungin in the treatment of 42 patients with invasive aspergillosis in Japan has been performed (147). The clinical response rate was 60% (6 of 10) in invasive pulmonary aspergillosis, 66.7% (6 of 9) in chronic necrotizing pulmonary aspergillosis, and 54.5% (12 of 22) in pulmonary aspergilloma. Unfortunately, this information has appeared only in abstract form, and the definitions of each infection type are yet to be documented. Coadministration of caspofungin and cyclosporine has resulted in increased concentrations of caspofungin in plasma (a 35% increase in the area under the curve) but no change in the amount of cyclosporine in the blood (69, 270). The mechanism of this interaction is unclear, although it has been speculated that cyclosporine limits caspofungin uptake into the liver. Healthy volunteers who received both cyclosporine and caspofungin developed raised liver function tests (14). However, in a retrospective study of 40 patients treated during market use, significant elevations in transaminases were documented infrequently with the concomitant use of caspofungin and cyclosporine (184). Most patients with liver function enzyme elevations had other causes to account for these, and discontinuations of therapy because of hepatoxicity were uncommon (184). Coadministration of caspofungin with tacrolimus has resulted in slightly reduced tacrolimus levels, but the two can be safely used together as long as tacrolimus levels are frequently monitored (69, 268).

At least two studies have now described clinical experience with caspofungin in combination with liposomal amphotericin in management of invasive aspergillosis (4, 152). This combination