Historically, amphotericin B deoxycholate

OUR CURRENT UNDERSTANDING OF THERAPIES FOR INVASIVE FUNGAL INFECTIONS * John R. Perfect, MD ABSTRACT This article provides an update on the current state of the art for treating invasive fungal infections, with insights, based on the experience of the author, into future directions for antifungal treatment strategies. Amphotericin B deoxycholate (ambd) is a very effective and life-saving drug, but its toxicity profile has always complicated its use. Newer lipid formulations of amphotericin B show comparable efficacy to its deoxycholate predecessor but with a significantly reduced adverse side effect profile. Thus, ambd should probably be considered a second-line drug, except in a few specific cases, such as cryptococcal meningitis. Future work will focus on refining dosing regimens for these lipid formulations. Other new developments making the old polyene obsolete include the newer, extendedspectrum triazoles and echinocandins. However, despite the positive safety and efficacy profile of these new agents, mortality rates for invasive mycoses still remain unacceptably high. This article discusses developments in the major areas of treatment strategies to overcome refractory infections, which include rapid and accurate diagnosis; use of immune modulators; drug formulation and dosing; empiric, preemptive, and prophylactic strategies; and combination therapy, as well as new strategies for candidiasis, aspergillosis, and zygomycosis. (Adv Stud Med. 2004;4(4A):S276-S282) *Based on a presentation given by Dr Perfect at a symposium held in conjunction with the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy. Professor, Department of Medicine, Division of Infectious Diseases; Director, Duke University Mycology Research Unit, Duke School of Medicine, Durham, North Carolina. Address correspondence to: John Perfect, MD, Division of Infectious Diseases, Duke School of Medicine, DUMC Box 3353, Durham, NC 27710. E-mail: perfe001@mc.duke.edu. Historically, amphotericin B deoxycholate (ambd) has been the gold standard for primary treatment of most invasive fungal infections. At the time of its discovery, it was a life-saving drug with few alternatives. However, its toxicity in severely ill patients limits its use such that, in many cases, it makes the cure almost worse than the disease. With the development of lipid formulations of amphotericin B, which appear to carry at least the same efficacy with reduced toxicity, as well as newer triazoles and echinocandins, ambd is now progressing from gold standard therapy to therapy from a bygone era. For example, 3 recent landmark studies on aspergillosis, cryptococcosis, and candidiasis show the superior clinical efficacy of voriconazole, amphotericin B plus 5-fluorocytosine, and caspofungin over ambd alone (Table 1). 1-3 AmBd should now be considered essentially a drug of the past, except in a few specific cases. However, as recently reviewed by Ostrosky-Zeichner et al, ambd is still used more often than the lipid formulations for therapy and in clinical trials because of its broad range of indications and pricing. 4 Recent treatment guidelines from the Infectious Diseases Society of America mention the lipid formulations and echinocandins as first-line therapy in certain defined situations for candidiasis. 5 As we gain more experience with optimal dosing strategies with these lipid formulations, we should see an overall replacement of ambd with them, thus sparing patients complicating toxicities while offering at least the same efficacy and chance for positive outcomes. Of particular concern, however, is the still relatively low success rates even with the newer agents all below 75% in a variety of studies, with most ranging from 50% to 60% success rates (Table 1). 1-3 Thus, our current treatments options, while much safer, are not yet optimal. TREATMENT STRATEGIES TO OVERCOME REFRACTORY INFECTIONS A major concern in medical mycology is the increasing prevalence of resistant strains and refractory S276 Vol. 4 (4A) April 2004

fungal infections. There are several strategies for addressing this important clinical issue. Our current state of the art for each strategy is discussed below. ACCURATE AND RAPID DIAGNOSIS Accurate and rapid diagnosis is essential for successful treatment of invasive mycoses. Diagnostic tests are not limited to culture results. For instance, histopathology provides important information regarding morphology in tissues or fluids to help identify the fungus and can actually help point to the diagnosis at the time of biopsy, even before culture results are available. Conversely, culture results can help an examiner to distinguish fungi when their histopathologic characteristics are very similar. Cultures and histopathology are both important strategies for accurate and rapid diagnosis of fungal infections. Galactomannan testing has been under evaluation for several years, with recently defined values for sensitivity and specificity. Values for this test in adults with hematologic malignancies or allogeneic bone marrow transplants range from 80% to 95% sensitivity and 90% to 98% specificity, depending on the endpoint index of the enzyme-linked immunosorbent assay. 6-10 It is not yet clear how reliable this test will be for patients with other types of cancers or solid-organ transplants. As a result, it is still not widely available in the United States but has been approved for use. Kami et al evaluated the galactomannan test alongside real-time polymerase chain reaction and the beta-glucan test in 33 patients with invasive pulmonary aspergillosis and 89 control patients. 11 The sensitivities for all tests ranged from 58% to 79%, the specificity ranged from 84% to 97%, and the positive predictive values ranged from 61% to 86% (Table 2). These positive test results were found to be similar to the time of positive evaluation by computed tomography. 11 A series of diagnostic technologies are being studied and improved but most of these diagnostic tests have not yet reached the status of being considered a standard of care. IMMUNE MODULATION The use of immune-modulating therapy, such as recombinant cytokines, as an adjunct to antifungal chemotherapy is a theoretically reasonable approach. The studies using this approach are limited, and the results have been mixed. An important study of 123 cases of invasive aspergillosis from 20 hospitals in 8 countries showed that, at 3 months after diagnosis or first suspicion of invasive aspergillosis, the overall mortality rate was 64% and the use of growth factors on 33% of the patients appeared not to influence outcome. 12 In fact, use of immune modulators can be considered a catch-22 situation, because the return of granulocytes is essential for cure, but the rapid mobilization of granulocytes by cytokines can lead to acute respiratory distress syndrome and may ultimately require steroids to attenuate the reconstituted response. Thus, at present, immune modulation in clinical practice still lacks the precision necessary to be used consistently in many of these very fragile patients with invasive mycoses. Table 1. Recent Landmark Studies in Invasive Mycoses Success Rates (%) Patient Population Comparator Drug AmBd Comparator Cryptococcal AmBd + 5-FC 51 60 meningitis 1 Invasive Voriconazole 31.6 52.8 aspergillosis 2 Invasive Caspofungin 61.7 73.4 candidiasis 3 AmBd = amphotericin B deoxycholate; 5-FC = 5-fluorocytosine. Table 2. Comparison of Diagnostic Tests for Invasive Aspergillosis Blood Sensitivity Specificity PPV +CT (%) (%) (%) Days Real-time PCR 79 92 79 0.3-6.6 ELISA 58 97 86 2.8-4.1 (galactomannan) Beta-glucan test 67 84 61 6.5-4.9 Isolates were taken from 33 patients with invasive pulmonary aspergillosis and 89 control patients. PPV = positive predictive value; +CT days = number of days test results preceded results from computed tomography (CT) scan; PCR = polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay. Data from Kami et al. 11 Advanced Studies in Medicine S277

DRUG FORMULATION AND DOSING As stated previously, the newer lipid formulations for amphotericin B are clearly much better tolerated than conventional amphotericin B, but the precise dosing for these lipid products has not yet been defined. The triazoles and particularly echinocandins, while efficacious, might also benefit some patients through higher dosing, especially patients with refractory diseases. As Dr Pfaller discusses (see page 269), resistance can emerge through inadvertent selection of resistant mutants from suboptimal doses of fluconazole. For instance, in neutropenic patients, we occasionally see treatment failures with echinocandin treatment, and this failure is perhaps from low drug dosing. More studies are needed on echinocandins to better define maximal tolerated doses and the circumstances in which higher doses should be used. EMPIRIC, PREEMPTIVE, AND PROPHYLACTIC STRATEGIES Empiric, preemptive, and prophylactic strategies are all used in treating serious fungal infections. Prophylaxis involves administering antifungal agents to patients with very high risk of infection, before any signs or symptoms are present, and continuing the drug during the period of risk. Empiric therapy is given to high-risk patients when general signs and symptoms of infection are observed (eg, in febrile neutropenia). Therapy is considered to be preemptive when more information regarding infection and the possible early specific areas of infection are noted without specific identification of the organism (eg, a positive serology or x-ray evidence of infection). The likelihood of infection in a patient increases from preventive to empiric to preemptive strategies. There is a wealth of published studies for empiric therapy in febrile neutropenia with more than 5000 patients. Most recently, caspofungin was compared with liposomal amphotericin B as empiric therapy in persistently febrile neutropenic patients. Of the 1095 patients in the modified intent-to-treat analysis, patients on caspofungin tended to have higher response rates for baseline fungal infections (52% vs 26%) and fewer premature discontinuations (86% vs 90%). Caspofungin was considered to be comparable to liposomal amphotericin B in overall treatment success and was better tolerated. 13 With developments in diagnostic testing, the field of medical mycology hopes to be moving toward more preemptive therapeutic strategies, but much work remains to be done. Risk stratification for preventive therapy is essential and is greatly aided by the 10% rule : unless at least 10% of the patient population is at high risk for fungal infection, prophylaxis most likely will not affect outcomes in individual medical centers. Prophylactic regimens rarely decrease infection rates to zero. Furthermore, intensive care units (ICUs) have been particularly difficult environments in which to conduct studies for further verification of prophylaxis studies because of heterogeneous populations at risk. For bone marrow transplants, the most prominent issue is whether prophylaxis against molds is needed. Again, studies are under way to answer that question, but recent prophylaxis studies are starting to point toward the importance of the need for mold prophylaxis regimens in this patient population. As the adage attests, an ounce of prevention is worth a pound of cure. Table 3 summarizes the results of several key prophylaxis studies for bone marrow transplant recipients, organ (liver) transplant recipients, and patients in the surgical ICU. 14-19 The studies show significant decreases in invasive fungal disease and, in some cases, decreased mortality with prophylaxis compared with placebo. However, for these types of studies, finding the precise patient population with high risk, especially given improved infection control procedures, can be difficult. Also, the use of a placebo arm in these trials becomes ethically questionable. We can expect to see fewer studies of prophylaxis with a placebo arm in the future. Table 3. Prophylaxis: An Ounce of Prevention is Worth a Pound of Cure Failure Rates (%) Patient Population n Fluconazole Placebo P value Bone marrow 356 14 2.8 15.8 <.001 transplant recipients 300 15 7 18.004 300 16 2.6 20 <.001 Organ (liver) 212 17 6 23 <.001 transplant recipients SICU patients 260 18 8.5 15.3 NA 43 19 4 35.02 SICU = surgical intensive care unit; NA = not available. All studies were randomized, placebo-controlled trials. Failure rates indicate the number of patients to develop invasive fungal infection at study end. S278 Vol. 4 (4A) April 2004

Recent studies are focusing on alternative antifungal agents beyond fluconazole for prophylaxis, particularly in relationship to resistance patterns. In 2002, van Burik et al presented a study comparing micafungin with fluconazole as prophylaxis for hematopoietic stem cell transplantation. 20 Micafungin resulted in 80% treatment success, which was better than the 73.5% with fluconazole. Although the difference was not statistically significant (P =.07), there were more Aspergillus infections in the fluconazole group. To assess whether the broader spectrum with itraconazole will improve outcomes, Winston et al compared itraconazole with fluconazole as prophylaxis in liver transplant recipients and allogeneic hematopoietic stem cell transplant recipients. 21 The results showed equivalent or better outcomes with itraconazole but with gastrointestinal side effects. In the study of liver transplant recipients, the results were similar with both drugs. Fungal colonization decreased by week 8 from 67% to 25% with itraconazole and from 77% to 30% with fluconazole. Proven fungal infections developed in 9% and 4% of patients in the itraconazole and fluconazole treatment groups, respectively. The number of proven and superficial infections was also similar in both groups. 21 In the study of hematopoietic stem cell transplant recipients, proven infections occurred in 9% of itraconazole recipients and 25% of fluconazole recipients during the first 180 days. Superficial fungal infections occurred in 4% and 3% of patients, respectively. In fact, prophylaxis with itraconazole was associated with fewer invasive fungal infections (P =.02). 22 This area will likely continue to be studied as fluconazole resistance continues to increase in certain highrisk patient populations. SURGERY Surgery is beneficial in circumstances where there is a high burden of fungi, particularly in necrotic lesions in the lung, and in patients who are able to tolerate it. There has been no comparative study of surgery, but it produces good results if it is individualized by patients conditions. Surgery is a bedside decision and is most successful in large necrotic angioinvasive mycoses in the lung or skin. It is adjunctive therapy for the definitive treatment with an antifungal drug. COMBINATION THERAPY Combination therapy for invasive aspergillosis is gaining greater recognition, supposedly with the rationale that if 1 drug is good, 2 or 3 must be better. As recently reviewed by Steinbach et al, the results from both in vitro and in vivo studies (including animal models) of combination therapy offer results that depict interactions ranging from synergy to antagonism. 23 Most recently, Rex et al compared fluconazole plus placebo versus fluconazole plus amphotericin B in 219 nonneutropenic patients with candidemia. 24 The results showed overall success rates of 68% with combination therapy versus 56% with single therapy (P =.043). Other measures of success were similar between the 2 study groups. The combination was not antagonistic compared with fluconazole alone, and the combination trended toward more rapid clearance of infection from the bloodstream. 24 It is the opinion of this author that combination therapy may be beneficial only in select cases (eg, cryptococcal and candidal meningitis) and will not become the standard of care until more properly controlled evidence-based studies are performed. NEW STRATEGIES FOR CANDIDA INFECTIONS The evidence-based strategies for Candida infections are gaining momentum, particularly for candidemia. Two of the classic studies compared fluconazole with amphotericin B for candidemia in nonneutropenic patients. The results show clearly comparable efficacy with both drugs, (with the exception of infection by C krusei and C glabrata) but significant toxicity was noted with amphotericin B. 25,26 Importantly, the study protocols have defined a length of time of treatment for candidemia: currently, candidemia is treated for 2 weeks after the last positive blood culture, as was done in the studies. More recently, we have seen another potential change in the treatment paradigm for candidemia, as it has been suggested that caspofungin (from a new class of antifungals, the echinocandins) offers the same benefit in candidemia as do standard therapies. In a comparator trial with amphotericin B for invasive candidiasis, the overall outcome results shown in Table 4 indicate comparable efficacy between the 2 drugs (it was a noninferiority study design). 3 During the study, patients received intravenous therapy of the study drug for 10 days, then stepped down to fluconazole for a total of 2 weeks of treatment. The proportion of neutropenic patients in this trial was only 10%. In our experience, we have seen breakthrough infections with caspofungin in neutropenic patients, supporting the concept that Advanced Studies in Medicine S279

these high-risk patients may require higher doses of caspofungin. Importantly, successful response by the Candida species was comparable between caspofungin and amphotericin B (Table 5). It is interesting to note that the outcomes of treatment, as shown in Table 5, were not exactly predicted by baseline minimum inhibitory concentration (MIC) because all isolates for which the caspofungin MIC exceeded 2 µg/ml responded favorably to caspofungin. 3 First-line treatment with caspofungin for candidemia, followed by step-down to an oral triazole once the Candida species has been identified and its susceptibility to triazoles is known, is an attractive strategy for this common invasive mycosis. NEW STRATEGIES FOR ASPERGILLUS INFECTIONS Voriconazole is now the gold standard treatment for Aspergillus infections. A randomized unblinded trial compared the efficacy and safety of voriconazole with amphotericin B in 287 patients with primarily allogeneic hematopoietic-cell transplantation, acute leukemia, or other hematologic diseases and found that voriconazole was superior to amphotericin B in successful response rates (52.8% vs 31.6%), survival rates (70.8% vs 57.9%), and severe side effects. 2 In further studies, the efficacy, tolerability, and safety of voriconazole were assessed for its use as salvage treatment for 273 patients with refractory or intolerant-totreatment fungal infections and as primary treatment for 28 patients with infections for which there is no approved therapy. Response rates for the first 2 patient populations were 47% and 68%, respectively. Overall, efficacy rates with voriconazole were 43.7% for aspergillosis, 57.5% for candidiasis, 38.9% for cryptococcosis, 45.5% for fusariosis, and 30% for scedosporiosis. 27 Thus, voriconazole is a first-line therapy for aspergillosis and can be used in salvage therapies for both yeasts and other mold infections. NEW STRATEGIES FOR ZYGOMYCETES INFECTIONS Zygomycoses are devastating infections with significant morbidity and mortality. They are often seen in patients with diabetes, hematologic or other cancers, transplant recipients, trauma patients, and other immunosuppressed patients. These infections can be some of the most difficult fungal infections to treat. Initial strategies are to control the underlying disease and provide surgical debridement. Amphotericin B had been Table 4. Capofungin vs Amphotericin B for Invasive Candidiasis: Overall Efficacy Results Caspofungin Amphotericin B Estimated Difference Analysis n/total (%) n/total (%) Adjusted for Strata (%) MITT 80/109 71/115 12.7* (n = 224) (73.4) (61.7) (95.6% CI, 0.7-26.0) Evaluable 71/88 63/97 15.4 patients (80.7) (64.9) (95.6% CI, 1.1-29.7) (n = 185) *P =.0861; P =.0346. Patients in the MITT analysis received the study treatment for at least 1day. Evaluable patients were included in the MITT and had no concomitant antifungal therapy, no protocol violations that might interfere with the assessment of efficacy, an appropriate evaluation at the end of treatment, and receipt of the study treatment for at least 5 days. Patients in the caspofungin group received a 70-mg loading dose followed by 50 mg per day of caspofungin. Nonneutropenic patients in the amphotericin B group received 0.6 to 0.7 mg/kg per day; neutropenic patients in this group received 0.7 to 1.0 mg/kg per day amphotericin B. Data shown are overall responses at end of 10-day intravenous therapy (test of cure). MITT = modified intent to treat analysis; CI = confidence interval. Data from Mora-Duarte et al..3 Table 5. Caspofungin vs Amphotericin B for Invasive Candidiasis: Response by Candida Species Candida Caspofungin Amphotericin B Pathogen n/total (%) n/total (%) C albicans 23/36 (63.9) 34/59 57.6 C glabrata 10/13 (76.9) 8/10 80.0 C guilliermondii 3/3 (100.0) 1/1 100.0 C krusei 4/4 (100.0) 0/1 (0.0) C parapsilosis 14/20 (70.0) 13/20 (65.0) C tropicalis 17/20 (85.0) 10/14 (71.4) Mixed infection 3/3 (100.0) 2/4 (50.0) Patients in the caspofungin group received a 70-mg loading dose followed by 50 mg per day of caspofungin. Nonneutropenic patients in the amphotericin B group received 0.6 to 0.7 mg/kg per day; neutropenic patients in this group received 0.7 to 1.0 mg/kg per day amphotericin B. Data shown are favorable overall response rates at the end of the 10-day intravenous therapy in the modified intent-to-treat population. Data from Mora-Duarte et al. 3 S280 Vol. 4 (4A) April 2004

Table 6. Refractory Fungal Disease: Summary of Success Rates first-line therapy for Zygomycetes infections, although its toxicity profile can be prohibitive in some patients. Recently, the lipid complex of amphotericin B was used as first- and second-line treatment for invasive zygomycosis in 64 immunocompromised patients. Response rates were 80% and 69% for those receiving first- and second-line amphotericin B therapy with stabilized or improved renal function in the majority of patients, including those with preexisting renal disease, respectively. Success rates by area of infection were 67% (lung), 64% (disseminated), 92% (sinus), 100% (single-organ extrapulmonary), and 72% (total). The newer lipid formulations of amphotericin B offer an important advance in the treatment of zygomycoses. Studies of posaconazole as an alternative therapy are also under way, following positive results of in vitro experiments and animal models. Twenty-three patients with proven or probable primary zygomycosis or refractory disease were treated for a mean of 137 days with 800 mg daily posaconazole. Underlying diseases in the patients included hematologic malignancies (many with subsequent stem cell transplants), solidorgan transplants, or diabetes. Successful outcomes were observed in 70% of patients, with 9 deaths (4 of which occurred during the first 30 days). 27 These promising results suggest that further study with this drug is warranted for treatment of zygomycosis. CONCLUSION Aspergillosis Candidiasis Fusariosis Cryptococcosis n/total (%) n/total (%) n/total (%) n/total (%) Voriconazole 28 62/142 (43) 50/87 (58) 5/11 (46) 7/18 (39) ABLC 29 55/130 (42) 65/91 (71) 17/27 (63) 7/11 (64) Caspofungin 30 37/83 (45) NA NA NA ABLC = amphotericin B lipid complex; NA = not applicable. For refractory fungal diseases, the 3 newest antifungal drugs (voriconazole, lipid formulation of amphotericin B, and caspofungin) offer success rates in the 40% to 70% range (Table 6). 28-30 In this select group of patients, these results are encouraging. Landmark clinical trials with these drugs have now been published, but managing these complicated infections involves a great deal of art as well as science of medicine. As a clinician, it is important to develop the art based on the foundation of science and avoid the temptation to combine multiple therapies without any data to support the combination. Ultimately, the host is the final arbiter of the disease and his or her treatment choices. The morbidity and mortality with invasive fungal infections remain dismal, but as we gain more experience with extended-spectrum triazoles, lipid formulations of amphotericin B, and the echinocandins, we ultimately have choices and a better chance of recovery to offer these critically ill patients. REFERENCES 1. van der Horst CM, Saag MS, Cloud GA, et al. Treatment of cryptococcal meningitis associated with the acquired immunodeficiency syndrome. National Institute of Allergy and Infectious Diseases Mycoses Study Group and AIDS Clinical Trials Group. N Engl J Med. 1997;337(1):15-21. 2. Herbrecht R, Denning DW, Patterson TF, et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med. 2002;347(6):408-415. 3. Mora-Duarte J, Betts R, Rotstein C, et al. Comparison of caspofungin and amphotericin B for invasive candidiasis. N Engl J Med. 2002;347(25):2020-2029. 4. Ostrosky-Zeichner L, Marr KA, Rex JH, Cohen SH. Amphotericin B: time for a new gold standard. Clin Infect Dis. 2003:37(3):415-425. 5. Pappas PG, Rex JH, Sobel JD, et al. Guidelines for treatment of candidiasis. Clin Infect Dis. 2004;38(2):161-189. 6. Sulahian A, Boutboul F, Ribaud P, Leblanc T, Lacroix C, Derouin F. Value of antigen detection using an enzyme immunoassay in the diagnosis and prediction of invasive aspergillosis in two adult and pediatric hematology units during a 4-year prospective study. Cancer. 2001; 91(2):311-318. 7. Maertens J, Verhaegen J, Lagrou K, Van Eldere J, Boogaerts M. Screening for circulating galactomannan as a noninvasive diagnostic tool for invasive aspergillosis in prolonged neutropenic patients and stem cell transplantation recipients: a prospective validation. Blood. 2001;97(6):1604-1610. 8. 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):1297-1306. 9. Verweij PE, Dompeling EC, Donnelly JP, Schattenberg AV, Meis JF. Serial monitoring of Aspergillus antigen in early diagnosis of aspergillosis. Preliminary investigations with two examples. Infection. 1997;25(2):86-89. Advanced Studies in Medicine S281

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Update of the multicenter noncomparative study of caspofungin in adults with invasive aspergillosis refractory or intolerant to other antifungal agents: analysis of 90 patients [abstract]. Presented at: the 42nd Annual Interscience Conference of Antimicrobial Agents and Chemotherapy; September, 2002; San Diego, Calif. Abstract M-868. S282 Vol. 4 (4A) April 2004