Emergence of fungal resistance

The decline in invasive candidiasis affected by the use of the triazole prophylaxis was accompanied by reports of a rise in 'difficult to treat' and more virulent colonizations and break-through IFIs.^{12, 13, 14} Other investigators, however, showed that a short course of triazole prophylaxis is unlikely to induce resistance or to select for resistant organisms (Table 2).^{15, 16, 17} Such an apparent discrepancy could be attributed to study design, patient heterogeneity regarding malignant disorder under treatment, previous therapy and chemotherapy or transplant conditioning regimens, specific institutional infections, and duration of antifungal prophylaxis (Table 3). In a meta-analysis that included 16 randomized, controlled trials (3734 patients) designed to evaluate oral fluconazole prophylaxis efficacy in neutropenic patients, C. krusei and C. glabrata were detected more frequently in patients receiving fluconazole than in the control groups (odds ratio 2.1 and 2.18, respectively).¹⁸

Table 2 - Reports of azole-resistant yeast in patients receiving anti-infective prophylaxis.

Full table

Table 3 - Difficulties encountered in designing fungal infection prophylaxis trials.

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On the other hand, Pfaller and associates, reporting a longitudinal review of fungemia due to Candida spp. infections reported from many institutions over a broad geographic area during a prolonged period of time, noted fluconazole resistance among C. albicans isolates to be quite uncommon (0–1%). The proportion of candidemia infections due to C. krusei remained unchanged during the period 1993–2001 (0–2%); however, blood stream infections caused by C. glabrata increased substantially (6–24%), without a change in the proportion of isolates resistant to fluconazole (7–10%).¹⁹

These data show that at least for now that short courses of antifungal prophylaxis are not a major threat in selecting for a resistant fungi, however close monitoring, frequent testing for fungal susceptibility and early changing antifungal agents with reduced fungal susceptibility with a more potent ones are all required to prevent the emergence of resistant microorganisms.

Emerging fungal pathogens

Aspergillus fumigatus and flavus and Candida sp are the most common causes of IFIs. During the past decade, other pathogens such as A. terreus, due in part to its innate relative resistance to AmB, have emerged as a more frequent cause of IFIS in HSCT recipients.^{5, 20} A. terreus is susceptible to eradication by voriconazole, other extended spectrum triazoles, and the echinocandins.^{21, 22} Ubiquitous fungal species previously considered as colonizers or contaminants including Trichosporon, Fusarium, and Scedosporium have also been observed as invasive pathogens with increasing frequency in HSCT recipients (Table 4).

Table 4 - Emerging fungal pathogens of clinical importance in HSCT recipients.

Full table

Rationale and type of prophylaxis

The approach to implementing prophylactic agents in the course of a HSCT to prevent or to reduce IFIs is confounded by many factors (Table 1). A recently published summary of available data on antifungal prophylactic therapy in neutropenic patients, however, showed a significant reduction in the incidence of IFIs and rates of colonization but no statistically significant lowering of mortality.²³ On the other hand, Bow et al reported a meta-analysis of 37 randomized trials that involved 7014 patients, many receiving HSCT as well as subjects who experienced prolonged neutropenia from other causes. They demonstrated that parenteral antifungal prophylaxis significantly reduced fungal-related mortality, P=0.017 (odds ration, 0.58; 95% CI, 0.41–0.82, with a relative risk reduction of 47%).²⁴

Fluconazole

As depicted in Table 5, fluconazole has been used extensively in HSCT receipts due to its excellent bio-availability when given either via the oral or i.v. route as well as its excellent safety profile. Two randomized, placebo-controlled allogeneic bone marrow transplant trials demonstrated a benefit, although the duration of chemoprophylaxis differed. Goodman et al²⁵ employed prophylaxis only during the period of neutropenia, while Slavin et al^{16, 25} extended the coverage through the first 70 days after transplantation. In both trials, fluconazole was effective in reducing the rates of colonization and superficial fungal infections (P<0.001), empiric use of AmB, and incidence of IFIs (P<0.001). The latter study also noted a survival advantage by 110 days after transplant (P=0.004), likely a result of longer prophylaxis.¹⁶ Following their initial report, this group demonstrated persistent protection against disseminated candidiasis, reduced death rate, and an overall survival benefit.²⁶ Conversely, the benefits of fluconazole were less conclusive in high-risk adult patients undergoing chemotherapy for acute leukemia.^{15, 27} The underlying reason for the lack of efficacy in this group of patients is unclear. Postulated explanations include the tendency to discontinue antifungal prophylaxis early, initiation of systemic AmB sooner, and declaration of failure in neutropenic patients who had persistent fever despite the lack of evidence of IFIs.

Table 5 - Prospective studies evaluating fluconazole prophylaxis in neutropenic patients.

Full table

The optimal fluconazole dose, duration of prophylaxis and likelihood of developing late sequelae (emergence of azole-resistant yeast) after long-term prophylaxis, however, remain to be determined. MacMillan et al²⁸ recently reported a study in which 253 HSCT recipients (29% were <19 year of age) were randomized to receive daily high- (400 mg) vs low-dose fluconazole (200 mg) during the neutropenic period. After engraftment, patients were rerandomized to receive fluconazole 100 mg/day or clotrimazole troches for 100 days. Fluconazole given at 400 vs 200 mg had equivalent efficacy in reducing the incidence of Candida colonization, superficial infections, and systemic infection during neutropenia. There was no difference, however, between fluconazole vs clotrimazole maintenance in reducing yeast colonization or infection.²⁸

Fluconazole therapy usually is very well tolerated with few side effects (dose-related gastrointestinal intolerance, headache, and rash) but may require frequent monitoring of blood concentration of cyclosporin and tacrolimus due to drug–drug interactions, especially in renal insufficiency.²⁹

Fluconazole as shown in many randomized trials has decreased rates of fungal colonization, superficial and invasive fungal infection, with a survival benefit reported in one study. Based on these data, The Center for Disease Control and Prevention recommends that all allogeneic transplant recipients receive fluconazole prophylaxis (400 mg/day) from the day of transplantation until engraftment.³⁰ However, fluconazole does not provide coverage against filamentous fungi, including Aspergillus, and the optimal prophylactic doses as well as duration of prophylaxis remain controversial.

Itraconazole

Itraconazole is another triazole derivative that possesses potent antifungal activity against a wide range of fungi.³¹ Despite erratic absorption associated with the capsular formulation, when used in neutropenic patients; itraconazole reduced both the incidence as well as the mortality of IFIs (P=0.005).³² The new oral solution formulation possesses enhanced bioavailability and when given at a dose of 7.5 mg/day to 124 HSCT recipients during the first +120 days after transplantation resulted in adequate (>0.25 g/ml) serum concentration in 96% of the patients.³³ Itraconazole oral solution was compared to placebo,³⁴ fluconazole (100 mg/day),³⁵ and AmB oral^{36, 37} capsules in randomized, controlled studies conducted in neutropenic patients that included HSCT recipients (Table 6). The incidence of IA in most of these studies was small so that no significant effect on eradication of invasive mould infections could be detected. Winston et al³⁸ and Marr et al³⁹ reported two randomized trials comparing itraconazole vs fluconazole as prophylaxis in high-risk HSCT recipients. In the first trial, the investigators compared itraconazole 200 mg/day vs fluconazole 400 mg/day in allogeneic HSCT recipients for the first 100 days after transplantation. Proven IFIs were fewer in the itraconazole arm (P=0.01) with a trend for lower fungal-related mortality. This is the first study to show that the broad-spectrum triazole may reduce the incidence of invasive mould in high-risk patients.³⁸ Marr et al³⁹ reached a similar conclusion in a study of 257 allogeneic HSCT recipients randomized to receive either itraconazole (7.5 mg/kg/day) oral solution (or 200 mg i.v./day) or fluconazole (400 mg oral or i.v./day) for the first 180 days after transplantation. In the intent-to-treat analysis, there was no difference in the incidence of proven or probable IFIs between the two groups (fluconazole 16% vs itraconazole 12%, P=0.23). When considering breakthrough infection, however, itraconazole provided better protection against invasive mould infections (fluconazole 12% vs itraconazole 5%, P=0.03) that was seen in the secondary analysis of patients on therapy. There was no significant difference in survival after 250-day follow-up (fluconazole 69% vs itraconazole 61%, P=0.110). More patients in the itraconazole arm developed liver dysfunction. Higher numbers of patients were discontinued from itraconazole due to toxicities or gastrointestinal intolerance than in the fluconazole group (36 vs 16%, P<0.001), most likely due to a higher dose of itraconazole used in the study than similar studies (7.5 mg/kg/day vs 5 mg/kg/day).³⁹

Table 6 - Prospective studies evaluating itraconazole prophylaxis in neutropenic patients.

Full table

In a recent meta-analysis that included 13 randomized, controlled clinical trials (3597 patients), itraconazole reduced the incidence of invasive fungal infection (mean relative risk reduction, 40%; P=0.002), the incidence of invasive yeast infections (mean, 53%; P=0.004), and the mortality from invasive fungal infections (mean, 35%; P=0.04). The incidence of invasive Aspergillus infections was only reduced in trials using the itraconazole cyclodextrine solution (mean, 48%; P=0.02) and not itraconazole capsules. The effect of prophylaxis was clearly associated with a higher bioavailable dose of itraconazole.⁴⁰

Itraconazole is generally well tolerated; gastrointestinal symptoms are the most frequently reported side effects, including nausea, abdominal pain, vomiting, and diarrhea. Rash and headache also are relatively common. The intravenous formulation of itraconazole should be avoided in patients with reduced renal function (creatinine clearance <30 ml/min), due to the nephrotoxicity of its carrier cyclodextrin.⁴¹ In the randomized trial that compared itraconazole to fluconazole's safety and efficacy as a prophylaxis in allogeneic bone marrow recipients, Marr et al reported a higher incidence of drug interaction between cyclophosphamide and itraconazole than with fluconazole. Among 209 patients who received azole antifungals concurrent with conditioning therapy as part of the randomized trial, there was a trend to higher mean serum bilirubin levels in those who received itraconazole compared to fluconazole (medians: 1.49 mg/dl vs 1.32 mg/dl), and there also were more itraconazole recipients whose baseline creatinine increased at least two-fold (34% itraconazole vs 20% fluconazole, P=0.03).⁴²

Itraconazole oral solution (5 mg/kg/day) is evolving into an optimal option for antifungal prophylaxis: this agent can achieve appropriate serum concentration, absorption is independent of gastric pH or food intake, drug interactions are infrequent and it has a broad-spectrum of activity. The intravenous formulation of itraconazole now is available and can be substituted in patients who are unable to tolerate the oral solution due to poor taste and diarrhea.

Amphotericin B

For many years deoxycholate AmB has been the apparent 'gold standard' for the treatment of most serious fungal infections. New formulations designed to reduce toxic effects (especially nephrotoxicity) have been developed and include parenteral lipid formulations and aerosolized products in an attempt to supplant the toxicity of the parent compound. Wolff and co-workers prospectively compared fluconazole vs low-dose intravenous AmB in patients undergoing bone marrow transplantation (29% of patients received allogeneic HSCT and 71% were autologous recipients). There were no significant difference in the incidence of proven IFIs (4.1 vs 7.5% in fluconazole and AmB-treated patients, respectively); however, AmB was significantly more toxic than fluconazole, especially in allogeneic HSCT recipients, in whom 19% of patients developed toxicity vs 0% in the fluconazole recipients, P<0.05.⁴³

Schwartz et al evaluated the benefit of aerosolized AmB vs placebo in 382 patients who developed prolonged neutropenia after chemotherapy or autologous HSCT transplantation. The incidence of proven or suspected IA was 4 vs 7% (P=0.37) and the incidence of infection-related mortality was 8 vs 7% (P=0.79), in patients who received the inhalational prophylaxis vs the control group.⁴⁴

In an attempt to improve both efficacy and tolerability, three lipid formulations have been developed and are now available: AmB colloidal dispersion (ABCD), AmB lipid complex (ABLC), and liposomal AmB (AmBisome). Although, open label studies demonstrated these preparations to be as effective as conventional AmB in patients with neutropenic fever, data supporting their role as fungal prophylaxis, however, are limited (Table 7). In a randomized, prospective trial conducted in neutropenic, allogeneic, and autologous HSCT recipients, liposomal AmB (AmBisome, 1 mg/kg/day) reduced the rate of fungal colonization but did not decrease autopsy-proven fungal infection rates.⁴⁵ Further, liposomal AmB (AmBisome) was ineffective as IFIs prophylaxis in another study involving 161 patients receiving chemotherapy or HSCT randomized to receive placebo or liposomal AmB (AmBisome, 2 mg/kg/3 times weekly).⁴⁶ Finally, Mattiuzzi and co-workers recently published an open label, randomized trial performed in 137 newly diagnosed acute myeloid leukemia or myelodysplastic disorder patients. In this trial, amphotericin lipid complex (AmBisome, 3 mg/kg/3 times/week) was compared to a combination of fluconazole (400 mg/day) plus itraconazole (400 mg/day) as a prophylaxis against IFIs during neutropenia. Proven IFIs developed in 4% of the amphotericin lipid complex group and in 5% of the azole combination's recipients during a median of 12 days of therapy.⁴⁷ These data do not support the routine use of AmB and its lipid formulation as antifungal prophylaxis in this group of patients.

Table 7 - Prospective studies evaluating amphotericin B and lipid-formulation preparation as prophylaxis in neutropenic patients.

Full table

Nonabsorbed-oral therapy

Such agents include clotrimazole troches, miconazole (capsule and buccal gel), oral polyenes (AmB tables and suspension), and nystatin (suspension). Earlier reports have shown that these compounds may reduce superficial mycoses yet lack sufficient potency to prevent emergence of an IFI when given as prophylaxis.⁴⁸ This observation was recently confirmed in a study of 106 neutropenic patients who were randomized to receive either oral itraconazole solution vs AmB suspension. Itraconazole was superior in preventing both colonization and invasive fungal infection (P<0.05).⁴⁹

New agents

Several new agents that possess enhanced potency and broad-spectrum antifungal activity against yeasts and filamentous fungi, including triazoles and echinocandins, are now available or are in the final stages of clinical development.^{10, 21}

New triazoles

Voriconazole, available in both oral and parenteral formulations, is the first of these new drugs to be released. This triazole derivative exhibits a wide spectrum of activity against clinically important fungal pathogens and is 4–16-fold more active than fluconazole and 2–8-fold more active than itraconazole against Candida spp., including C. krusei and C. glabrata. Voriconazole exhibits potent fungicidal activity against moulds, including Aspergillus. Voriconazole was approved by the Food and Drug Administration for the treatment of IA and infections caused by Scedosporium apiospermum (asexual form of Pseudallescheria boydii) and Fusarium spp. as a primary therapy (Table 8).⁵⁰

Table 8 - New antifungals.

Full table

Voriconazole undergoes extensive hepatic metabolism by the cytochrome P-450 system; as such, it has the potential for numerous drug–drug interactions. HSCT recipients are particularly at high-risk for such interaction and serum concentration monitoring and dose adjustment of certain immunosuppressant agents is warranted. Available phase I and pharmacokinetic studies using healthy volunteers as well as transplant recipients have shown that concomitant use of voriconazole increases the serum concentrations of tacrolimus and cyclosporin. These data have led to the recommendations of reducing the dose of cyclosporin by 50% and tacrolimus by 33% when used concurrently with voriconazole. No such reduction is required for prednisolone or mycophenolate; however, concurrent use of voriconazole and sirolimus is contraindicated, mainly because the extreme potentiation of sirolimus by voriconazole.^{51, 52}

Some of the relevant adverse effects of voriconazole include transient and dose-related visual disturbances (photopsia), hepatotoxicity, and mild rash. In general, most studies have shown this agent to be safe and well tolerated.^{53, 54} Dosage adjustment in patients with liver dysfunction is recommended, and intravenous administration should be avoided if the creatinine clearance is less than 50 ml/min, due to impaired excretion of its vehicle cyclodextrin.⁵²

The availability of voriconazole in oral and intravenous formulation makes it appealing for long term use as chemoprophylaxis. A multicenter study conducted through the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) recently has been launched in allogeneic HSCT patients in which voriconazole is compared to fluconazole for the prevention of IFIs during the first 6 months after transplant.

Posaconazole is a congener of itraconazole with an excellent safety profile. Compared with currently available triazoles, posaconazole possesses activity similar to that of itraconazole and at least eight-fold greater than that of fluconazole against Candida spp. (Table 8).⁵⁵ In a phase I study, pharmacokinetics, safety, and tolerability of posaconazole were evaluated after the administration of single and multiple oral doses in 103 healthy adults. Posaconazole dosing was well tolerated and no serious toxic effects were reported. The most commonly reported adverse events were headache, somnolence, dizziness, and fatigue and were mild to moderate in intensity.⁵⁶ In a multicenter study in which 25 patients with refractory IFIs or who were intolerant to standard therapy, posaconazole was well tolerated and effective in 53% of patients at week 4, and 85% of patients by week 8.²²

The new triazoles are attractive alternatives for antifungal prophylaxis as all have enhanced potency, a broad-spectrum of activity and are available in oral formulation.

Echinocandins

The echinocandins are a new class of broad-spectrum antifungals that inhibit the synthesis of (1,3) beta -D-glucan, a critical fungal cell wall component.^{21, 57} Caspofungin, the first of this group, recently was evaluated in 124 acute myeloid leukemia and myelodysplastic syndrome patients receiving induction chemotherapy (Table 8). Patients were randomized to receive either i.v. caspofungin (50 mg/day) or i.v. itraconazole (200 mg/day). At a median of 23 days on the study, both drugs provided comparable antifungal prophylaxis and were well tolerated.⁵⁸

Unlike the triazoles, caspofungin is neither a substrate nor an inhibitor of the P-450 hepatic cytochrome enzymes system. Preclinical and pharmacokinetic studies have shown an increase in plasma concentration of caspofungin when cyclosporine was administered concomitantly, possibly the result of reversible inhibition of uptake into hepatic tissue. Pending further data analysis, it is recommended to avoid caspofungin use in patients receiving cyclosporine.⁵⁹ Coadministration of caspofungin with tacrolimus results in reduction of tacrolimus level by 20%, but concomitant use with mycophenolate seems to be well tolerated.⁵⁷

In phase I studies comprising 274 normal volunteer subjects, i.v. caspofungin generally was well tolerated in single and multiple doses, the most common clinical adverse effects of headache and phlebitis. Similar results were reported in phase II and III clinical trails conducted in 349 immunocompromised patients; there were no serious clinical or laboratory side effects reported from caspofungin use.⁵⁹

Other echinocandins under investigation include micafungin and anindulafungin. Van Burik and associates randomized 882 HSCT recipients between micafungin (50 mg/day) and fluconazole (400 mg/day) as IFIs prophylaxis. At a median duration of therapy of 18 days, fewer patients assigned to micafungin therapy required the addition of empiric antifungal therapy (P=0.018). Both drugs were effective in preventing candidiasis (0.9% MI, 0.4% FL); however, micafungin was more effective in preventing IA (P=0.07). Fewer patients given micafungin discontinued study drug due to an adverse event (P=0.058).⁶⁰

The data summarized above indicate that the echinocandins, especially the FDA-approved caspofungin, are appealing therapeutic options for antifungal prophylaxis as a result of their excellent safety profile, long half-live, potency against yeast and most Aspergillus species, and their minimal drug–drug interactions. The main drawback is they are not available in oral formulation.

Diagnostic advances

IA remains a difficult antemortem diagnosis, as a blood or tissue culture may take days to weeks to provide a positive result; histopathological evidence of tissue invasion also may be challenging because profound cytopenias often precludes biopsy. ⁶¹ Efforts to develop new diagnostic techniques for detecting IFIs have been intensified.

Galactomannan (GM) is a fungal cell wall constituent released by pathogenic Aspergillus species during growth. Several assays were developed to detect GM in the serum or other body fluids at early stages of infection before the onset of clinical symptoms.⁶² Several European groups have advocated a sandwich enzyme-linked immunosorbent assay (ELISA) to detect as low as 0.5–1 ng of GM/ml with a sensitivity of 65–100% and a specificity of 81–100% as a screening strategy in high-risk patients. Table 9 summarizes data from trials using GM in early detection of IA in neutropenic recipients. GM is not only useful in the early diagnosis of IA but also as a prognostic tool, Patterson et al⁶³ reported a strong correlation between the level and duration of GM antigenemia and the clinical outcome in patients with proven or probable IA. Similar conclusions were reported by Boutboul and co-workers who monitored 37 allogeneic HSCT recipients for IA. Serial blood samples were collected before and during therapy. An increase in the GM index value of 1.0 over the baseline value during the first week of observation was predictive of treatment failure with a sensitivity of 44%, a specificity of 87%, and a positive predictive value of 94%.⁶⁴ Based on the available data, screening for GM has been adopted by many European centers as part of the management plan for allogeneic stem cell transplant recipients and was recently approved by the FDA (May 2003) as aid to diagnosis of Aspergillus invasive infection in adult neutropenic patients and in HSCT recipients. The assay, however, was approved in the US as a different threshold was used than that in Europe.

Table 9 - Utility of serum galactomannan antigen detection in the diagnosis and prognosis of invasive aspergillosis.

Full table

The assay sensitivity ranges from 93% in those patients at high-risk for IA to values as low as 28% in those subject at low-risk for IA.⁶⁵ The high false-positive rate results mainly during the early periods after HSCT, and high variations in sensitivity and specificity been reported in several studies (Table 9). Several authors have advocated using a decrease in the cutoff value of the assay from 1.5 to as low as 0.7 to improve its sensitivity and specificity in HSCT recipients.^{66, 67, 68} As the GM polysaccharide is eliminated rapidly from blood, a series of repeated samples are required for optimizing diagnosis. The assay is species –specific, and non-Aspergillus fungal pathogens are not detected by the test. Further, false positive tests have been reported in patients who developed antibodies against Aspergillus, mainly those patients in whom mucositis develops.⁶⁶

In summary, this test, although not perfect, has clinical utility in detecting the often evasive invasive aspergillosis at a preclinical stage, allowing early intervention, It also has a valuable prognostic value, many studies demonstrate a quantitative reduction in the GM level in patients who are successfully responding to anti-Aspergillus therapy.

The G test is another assay that detects a cell wall component of yeast and filamentous fungi by relying on factor G, a horseshoe crab coagulation factor. This sensitive assay has a detection limit of 1 pg/ml and a reported sensitivity and specificity of 78–100 and 88–100%, respectively. Unfortunately, however, this test does not differentiate fungal colonization from IFIs, and does not detect cryptococcosis.^{11, 4, 69}

Another detection method uses polymerase chain reaction (PCR) technique to target the highly conserved area in the fungal 18S ribosomal DNA genes. Hebart et al⁷⁰ reported a PCR assay with a sensitivity of 100% and a specificity of 65% and it was even used to predict IA 2 days prior to clinical symptoms and 9 days prior to clinical diagnosis. The sensitivity of PCR assays in detection of fungal DNA in different body fluids also has been tested. In a study of 134 allogeneic HSCT recipients, Einsele et al⁷¹ examined the bronchoalveolar (BAL) fluid for evidence of Aspergillus DNA. Five of the seven patients who had a positive BAL developed IA at a median time of 64 days after transplantation. New quantitative assays using real-time PCR also have been developed for the diagnosis of invasive pulmonary aspergillosis.⁷² Drawbacks to these systems include lack of specificity and the inability to develop universal standardizations.

Summary

A number of important diagnostic and therapeutic advances have been made recently in an effort to prevent and treat IFIs in HSCT recipients. These encouraging data coupled with new diagnostic techniques may allow changing current prophylactic paradigm to a new pre-emption strategy such the one used to prevent the CMV infections in HSCT recipients. Meanwhile, decisions regarding prophylaxis should be individualized based on the risk of infection (prolonged neutropenia, allogeneic HSCT, GVHD). Other factors include availability of oral formulation, cost, interaction with other drugs concomitantly used in HSCT recipients, safety for prolonged use, and tolerability. As new reports indicate a trend toward late aspergillosis infections and data from randomized trials show improved survival with prolonged prophylaxis, it is reasonable to advocate long-term antifungal prophylaxis beyond the period of neutropenia in high-risk patients.

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Acknowledgements

We gratefully acknowledge Dr John Wingard for his critical review of this article.