Susceptibility of important fungal pathogens against some common antifungal agents.
The success rate of acute lymphoblastic leukemia (ALL) therapy has gradually increased over the past decades. With more than 80% long-term survivors, treatment of ALL in children is undoubtedly one of the great success stories of innovative study-controlled modern medicine . Attempts to boost cure rates for those who do not respond to therapy or relapse with more intense chemotherapy including allogeneic hematopoietic stem cell transplantation have further improved the outcome of patients, particularly for prognostic unfavorable subgroups . However, intensification of treatment can substantially increase morbidity, the risk for life-threatening sequelae and mortality .
Several studies address this important issue and report on the emergence of fungi. A retrospective review of ~ 5.600 patients who underwent hematopoietic stem cell transplantation at the Fred Hutchinson Cancer Research Center (Seattle) from 1985 to 1999 reports a constant increase of 3.5% in the one-year cumulative incidence of probable and proven invasive fungal infections . Investigation of autopsies, skin, and lung biopsies, and bronchoalveolar lavage fluid analyses reveal that non-fumigatus Aspergillus species, such as Fusarium and Zygomycetes have increased, especially in patients, who have received multiple transplants. These observations are particular worrisome given the increasing importance of amphothericin-B resistant organisms, resulting in a very poor one-year survival rate of ~ 20% . For those who do survive, length of hospital stay and total hospital charges are increased, compared with immunocompromised patients without fungal infection .
Despite much effort has been taken to improve therapeutic treatments and strategies, there still remains much uncertainty and controversy regarding the best method to diagnose, prevent and treat fungal infections . Practicing physicians approach this uncertainty by treating suspected infections empirically. However, researchers that conduct clinical trials tend to accept only cases in which the diagnosis is certain in order to improve clarity and uniformity of clinical trials. Therefore, members of the European Organization for Research and Treatment of Cancer / Invasive Fungal Infection Study Group (EORTC) and the National Institute of Allergy and Infectious Disease (NIAID) Mycoses Study Group (MSG) formed a consensus study group to define standard definitions of invasive fungal infections for clinical research . Practice guidelines are intended to limit practice variations towards movements such as evidence-based medicine and are primarily suggested by the European Conference of Infections in Leukemia (ECIL; http://www.ebmt.org/Contents/Resources/Library/ ECIL/Pages/ECIL.aspx). For the clinical purpose there is still a need to develop more effective prevention and treatment strategies. Such strategies may rely on newer antifungal agents that are active against amphothericin B resistant moulds and are well tolerated. Because of limited number of affected patients, multicenter collaborative trials are required.
This case-based review examines the current literature to explore basic concepts on epidemiology, diagnosis and treatment of invasive fungal infections in ALL patients. A case report will be used to illustrate these specific issues.
A systematic review of the literature for an explicit identification of major problems related to the heterogeneity of patients with acute lymphoblastic leukemia who have invasive fungal infections was undertaken. Pneumocystis infections were not considered. In brief, the abstracts of 711 articles published from 1985 through 2012 were screened. Of these, 41 articles were finally selected because these report clinical research on patients with ALL who also had deep-tissue fungal infections. The minimum diagnostic criteria used to include patients in the study were extracted from definitions devised by the investigators. Likewise, the criteria used to express different degrees of diagnostic probability were summarized, as were the terms most often used to express these levels of uncertainty.
3. Case study: A sixteen-year old patient with Ph+ ALL
A sixteen-year old adolescent was referred to the outpatient oncologic clinic with suspicion of a proliferative disease of the hematopoietic system. Two weeks prior the admission, the patient suffered from sub febrile temperatures and fatigue. At the time of the visit to the general physician scarlet was ruled out and the patient discharged. At admission, the patient’s general condition was slightly deteriorated; his physical examination revealed petechial rash over the extremities, pallor and hepatosplenomegaly. Laboratory findings showed ALL with a positive BCR/ABL result and an absolute count of 398.000 blasts per µL. He was subjected to treatment with the ALL BFM 2000 program for high-risk patients. He responded well to chemotherapy and achieved complete morphological remission on day ten of treatment. Following day fifteen, the tyrosine-kinase inhibitor Imatinib Mesylate was added to the standard treatment.
On the thirtieth day of induction chemotherapy the patient developed fever of 39.2°C. Physical examination was unremarkable. The laboratory tests showed leucopenia (0.5 x 109/L) with an absolute neutrophilic count of 19/µL, but no elevation of inflammatory proteins (CRP <0.06 mg/dL).
Empirical antibiotic regimen was initiated with a carbapenem (Meropenem) and an aminoglycoside (Gentamycin). Because of relapsing fever four days after the initiation of antibiotics, vancomycin was added. Both blood and urine cultures were aseptic.
A chest X-ray showed a distinctive and peculiar mass in the middle of the right lung. Because the radiological image was ambiguous the diagnostics were extended by a chest CT scan, which showed a large mass in the right upper lobe, surrounded by a wide zone of ground-glass attenuation demonstrating the halo sign. On the ground of the radiologic examinations, fungal infection was suspicioned and pre-emptive antifungal therapy was initiated. Fluconazole, included in the treatment as a prophylactive measure, was replaced by liposomal Amphothericin administered at a dose of three mg/kg once daily. On the next day CT-guided biopsy was planned to obtain a definitive diagnosis. Biopsy was performed with only a single puncture using a 20 G cutting needle. No pneumothorax or hemorrhage was noted after the procedure. Histological examination yielded dichotomously branching septated hyphae consistent with Aspergillus species, confirming the diagnosis of invasive fungal infection. Culture demonstrated a growth of Aspergillus flavus. Antifungal susceptibility testing with the agar-based MIC test showed good activity for Voriconazole, Posaconazole and Caspofungin, but high MIC90 for liposomal Amphothericin.
Accordingly antifungal therapy was switched to Voriconazole (6 mg/kg) for eight weeks intravenously and then orally until the twelfth week. CT imaging studies that followed confirmed a gradual recession of the lesion. The patient underwent right sided thoracotomy with wedge resection of the fungal mass. Histopathology revealed Aspergilloma with surrounding chronic granulomatous inflammation, fibrosis and sheets of macrophages. Post-operative course was uneventful and no recurrence of fungal infection over twenty four months follow up was observed. He underwent allogeneic hematopoietic stem cell transplantation and has been in complete molecular remission since.
Worldwide surveys evaluating the epidemiology of invasive fungal infections have been conducted in large center studies in North America . In European countries data is most commonly derived from single-center reports or regional reports from single countries . Though local epidemiology is a cornerstone of clinical decision making, efforts are now undertaken worldwide to start multi-national surveys on fungal infections in order to improve uniformity of clinical trials.
Until 2 decades ago, infections by Candida were the most common fungal pathogen in patients treated for ALL. However, with the introduction of Fluconazole as primary antifungal prophylaxis and the application of more aggressive treatment protocols, including allogeneic hematopoietic stem cell transplantation, a notable shift towards the advent of invasive aspergillosis has been noted . Whereas almost all of the fungal infections were attributable to candidiasis (11/11) in autopsy studies of the late seventies, mould infections were responsible for 62% of IFIs (16/26) two decades later . Concordantly, a large multi-centre report from the SEIFEM-2004 study (Sorveglianza Epidemiologica Infezioni Fungine nelle Emopatie Maligne) confirms this trend, indicating, that over half of all fungal infections (346/538) were caused by moulds, in most cases Aspergillus species (310/346) . Most importantly, such infections have become a prime cause of death in patients with hematologic malignancies. The IFI-attributable mortality rate was 39% (209/538). The highest IFI-attributable mortality rates were associated with zygomycosis (64%) followed by fusariosis (53%), aspergillosis (42%), and candidemia (33%) .
Along with the increased incidence of mould infections caused by Aspergillus species, other emerging mould opportunistics, such as Zygomycetes and Fusarium species, have progressively been noted; interestingly, frequency varies by geographical location . Another trend in changing the face of epidemiology is that infections caused by non-albicans Candida species (e.g. Candida glabrata, C. krusei, C. tropicalis, C. parapsilosis) have steadily increased, particularly in patients with ALL .
Yeast like pathogens enter the body via the gut or skin and mostly follow the pattern of fungemia and disseminated infection known from Candida species. Rarely the central nervous system, cardiovascular system or other tissues represent sites of dissemination .
Besides invasive infections of the skin and subcutaneous tissues, moulds involve, as airborne pathogens, the sino-pulmonary tract; the emerging opportunistic moulds have a higher propensity for dissemination, in particular into the central nervous system. Because of the lack of specific clinical, radiographic and histological features and the absence of diagnostic surrogate markers in blood, the diagnosis depends on the identification of the organism by means of culture based methods .
In general, diagnostic testing should begin with non-invasive methods and only approach invasive steps if needed. Diagnostic options include conventional or high-resolution CT (this has less radiation exposure and was performed in this case report), positron emission tomography (PET), magnetic resonance imaging (MRI), GM assay, 1,3-ß-D-Glucan test, Polymerase chain reaction (PCR), bronchoalveolar lavage, blood culture and tissue biopsy. At this time, MRI and PET are more research-oriented than commonly used clinical approaches. The utility of standard blood cultures is limited because of a high percentage of false-negative results, particularly in patients with disseminated aspergillosis. Of the listed options, the GM and 1,3-ß-D-Glucan serum assay, PCR and the CT scan will be described in detail.
5.1. Galactomannan (GM)
GM testing with the Platelia Aspergillus Enzyme Immunoassay (EIA; Biorad Laboratories, Redmond, WA) has been approved by the U.S. Food and Drug Administration (FDA) for Aspergillus diagnostics and is included as a mycological criterion in the revised definitions of invasive fungal disease from the EORTC/MSG consensus group . The test is based on detection of a component of the Aspergillus cell wall, Galactomannan (GM), which is released in the surrounding environment by growing Aspergillus species. Concentration of serum GM correlates with fungal burden in animals with experimental pulmonary aspergillosis – and, according to the 2011 ECIL clinical practice guidelines may be considered as surrogate marker for detection of invasive aspergillosis (http://www.ebmt.org/Contents/Resources/Library/ ECIL/Pages/ECIL.aspx). Recent data suggest that sequential measuring of GM serum levels may be used for therapeutic monitoring in children and adults with pulmonary aspergillosis. The guidelines from the Infections Disease Society of America (IDSA) state, that duration of antifungal therapy must not only rely on disappearance of GM levels, but also on resolution of clinical and radiological findings .
The GM EIA has been most studied in hematologic malignancy and bone marrow transplantation populations. Both the specificity and sensitivity of the GM EIA for invasive aspergillosis are high for infected, neutropenic adult patients from these populations. Comparison of 5 studies which use EORTC/MSG criteria and give adequate information for individual patients with results of a formal meta-analysis, indicate sensitivity, specificity of 76% to 73%, 86% to 90% in children and adults respectively . Controversy of GM testing exists about the interpretation of the assay cutoff level (0.5, 1.0), which was originally set at 1.5 and was applied in Europe but which was lowered to 0.5 after review by the FDA. Studies have shown that using an index cutoff for positivity of 0.5 versus greater indices substantially increases sensitivity, with only minimal loss in specificity . Factors, which increase false positivity and influence the specificity of the assay, include a low level of cut-off (<0.5), colonization with Bifidobacterium bifidum in the intestinal flora, which mimics the epitope recognized by the EB-A2 in the enzyme-linked immunosorbent assay kit  and invasive infections with other fungi, such as Penicillium spp., histoplasmosis, and blastomycosis . Moreover, cross-reactivity of the assay has been shown with the use of piperacillin/ tazobactam or amoxicillin/clavulanate antibiotic therapy and in infants with the nutrition of milk-based formulas .
The IDSA guidelines currently recommend using the GM EIA in conjunction with CT scans for early, noninvasive diagnosis of invasive aspergillosis in high-risk patients . The test should be performed serially, at least twice per week through the periods of highest risk, whether the periods involve neutropenia or active GVHD .
When GM in serum is used for screening for invasive mold infection in children with hematological malignancies/undergoing HSCT, data should be interpreted with caution, since the assay has a number of limitations in the sensitivity and specificity profile. Prospective monitoring of GM in serum every three to four days in children at high risk for IFD is reasonable for early diagnosis of invasive aspergillosis. Although the optimal cut-off value of GM in the serum of children is not well defined, published data support the use of a threshold of an optical density index 0.5 (http://www.ebmt.org/Contents/Resources/Library/ ECIL/Pages/ECIL.aspx).
5.2. 1,3-ß-D Glucan
1,3-ß-D Glucan (BG) is a fungal cell wall component circulating in the blood of patients with invasive aspergillosis, candidemia, but also Fusarium, Trichosporum, Saccharomyces, and Pneumocystis jirovecii. Moreover, BG is also detected in patients with infections due to bacteria such as Streptococcus pneumoniae, Pseudomonas aeruginosa and in healthy individuals. However, BG is absent in patients with cryptococcosis and zygomycosis . Antibiotics such as cefepime, piperacillin/tazobactam or meropenem may cause positive BG levels. Investigations using different BG assays in 2979 patients (594 with proven or probable IFI) have reported a pooled sensitivity of 76.8% and specificity of 85.3% . Differences in study design (population studies versus case-control, variation in the number of proven or probable IFIs, proportions of patients with candidemia and aspergillosis, case-mix of neutropenic and non-neutropenic patients, and previous antifungal prophylaxis) highlight the need for further investigations. In children, data is very limited: elevated levels of BG were reported in a case-control study of only four children with IFI (3 patients with candidemia, one patient probable aspergillosis) .
The Fungitell assay (Associates of Cape Cod) for detection of 1,3-ß-D Glucan is approved by the FDA for the diagnosis of invasive mycoses, including aspergillosis  and is included as mycological criterion in the revised definitions of invasive fungal disease from the EORTC/MSG consensus group . Unfortunately, there is no recommendation from ECIL or IDSA for clinical practice. However, BG testing in adults is considered as having good diagnostic accuracy for early diagnosis of IFD; in children, data are too limited to make any recommendations (http://www.ebmt.org/Contents/Resources/Library/ ECIL/Pages/ECIL.aspx).
Detection of antifungal DNA has been advocated as a promising, rapid and more sensitive diagnostic tool, but false-positive results can occur, and a standardized commercial method is not yet available. Several PCR assays to detect fungal DNA have been described, but most have shown that the global performance was too low to be of clinical interest. Different situations have been reported: PCR either has high sensitivity and NPV, while specificity and PPV is low, or, conversely, high specificity and PPV with low sensitivity and NPV [21-24]. These discrepancies can be due to the different technical approaches used. Indeed, a major difference is the type of PCR method used in these studies, i.e., nested PCR, PCR–enzyme-linked immunosorbent assay, or RT-PCR [22;24]. Also, the superiority of large serum volumes (> 1 ml) in comparison with conventional serum samples (100 µl to 200 µl) has clearly been shown . In view of changing epidemiology a panfungal PCR might be advantageous to permit the detection of a wide range of fungal pathogens. Its sensitivity of 96%, negative predictive values of 98%, whereas the specificity and positive predictive value were 77% and 62%, respectively is far superior to single PCR measurements .
In summary, despite ISDA and ECIL do not give any recommendations, combining non-culture based diagnostics is an important research direction that may improve the overall predictive value of these systems .
5.4. Chest CT scan
Systematic chest CT scan allows early diagnosis of invasive pulmonary aspergillosis, is more sensitive and specific than traditional chest radiographs and is a clinical criterion in the revised definitions of invasive fungal disease from the EORTC/MSG consensus group. Characteristic findings consist of nodules surrounded by the ‘halo sign’, an area of haziness or ground-glass opacity, or pleura-based, wedge-shaped areas of consolidation . These findings correspond to areas of hemorrhagic infarcts. In severely neutropenic patients, the halo sign is highly suggestive of angioinvasive aspergillosis. However, a similar appearance has been described in a number of other conditions, including infections with herpes virus or cytomegalovirus, Kaposi sarcoma, Wegener granulomatosis, and bronchiolitis obliterans organizing pneumonia . The air crescent sign, a crescent-shaped area of radiolucency in a region of nodular opacity, is usually seen during convalescence in angioinvasive aspergillosis (i.e., 2–3 weeks after initiation of treatment and concomitant with resolution of the neutropenia) . Of note, some studies suggest that cavitation and the air-crescent sign are more likely to be observed in adults, and may frequently be absent from CT scans obtained from young children with pulmonary invasive aspergillosis . When obtaining serial CT scans, it is also important to realize that irrespective of antifungal therapy, the pattern is characterized by an initial rise in number and size of lesions, followed by a plateau in lesion size, and gradual reduction . Moreover, is time until complete radiologic remission and outcome independent of initial or maximum lesion size and number in patients with invasive pulmonary aspergillosis . The appearance of cavities on serial CT scans (frequently accompanied by the appearance of the air-crescent sign as neutropenia resolves) may be indicative of patient recovery. Similarly, if antifungal therapy is initiated and subsequent scans show an increase in the number or size of lesions, this is more likely a reflection of the typical progression of disease rather than failed therapy. According to the ECIL recommendations, in high-risk patients with persistent febrile neutropenia that persists beyond 96 hours or with focal clinical findings, imaging studies (e.g., CT-scan of the lung or adequate imaging of the symptomatic region) should be performed. Further diagnostic work-up (e.g., BAL, biopsy) should be considered and mold-active antifungal treatment should be initiated.
Antifungal strategies include prophylaxis, empiric antifungal therapy, pre-emptive antifungal therapy and treatment of established invasive fungal infection (Figure 3). For individual patient populations, each strategy needs to consider the patients risk, the local epidemiology, the availability of diagnostic tools and the availability and effectiveness of antifungal agents. Last, but not least a cost – benefit analysis (i.e. toxicity, financial aspects) is mandatory. For the purpose of this textbook spectrum, potency, mode of action, and clinical indication of antifungal agents will be discussed.
6.1. Amphotericin B
Amphotericin deoxycholate (DAMB) and its lipid formulations, including amphotericin B colloidal dispersion (ABCD), amphotericin B lipid complex (ABLC), and liposomal amphotericin B (LAMB,) have a wide range of activity against most fungal pathogens. Only Aspergillus terreus and Fusarium species are less susceptible (Table 1.) .
In comparison to DAMB, nephrotoxicity is rarely seen with the use of the lipid formulations; infusion-related reactions, such as fever, chills and rigor are substantially less frequent with LAMB. Mild increases in bilirubin and alkaline phosphatase are associated with all three lipid formulations, elevation of transaminases with LAMB only. Currently, DAMB is licensed for neonatal invasive candidiasis and induction therapy for cryptococcal meningitis; LAMB is approved as first line empirical treatment of suspected invasive aspergillosis and candidiasis; ABCD is licensed for second-line treatment of patients with invasive aspergillosis, and ABLC for second-line treatment of patients with invasive Candida or Aspergillus infections .
The recommended therapeutic dosages are 0.7 to 1.0 mg/kg/day for DAMB, 3–4 mg/kg/day for ABCD, 5 mg/kg/day for ABLC, and 3 (to 5) mg/kg/day for LAMB, respectively. The available evidence does not suggest pharmacokinetic differences of LAMB between adults and children including preterm and newborn infants .
Fluconazole has a very narrow fungal susceptibility against Candida species (Candida glabrata and krusei have a high MIC index) and lacks activity against Aspergillus species and zygomycetes (Table 1.). Fluconazole is not metabolized and mainly renally excreted, and drug levels correlate with strictly with renal function . It is licensed for prophylactic use in patients at risk for IFIs and for targeted treatment of candidiasis. For pediatric patients (they show a more rapid excretion and shorter half-life), the recommended dosage is higher than for adults, 8 to 12 mg/kg/day versus 5 mg/kg/day, respectively .
The compound is active against most Candida and Aspergillus species, but the susceptibility against Candida glabrata and C. krusei is limited (Table 1). There is no activity against zygomycetes. The pharmacokinetics is characterized by inter-individual variability of gastrointestinal absorption and hepatic metabolism . Accordingly, measurement of drug levels is necessary and results of meta-analysis suggest that the trough plasma level should be higher than 0.5 µg / mL . Concerns have arisen on the interaction with drugs such as vincristine and cyclosporine, which are major components of both induction ALL therapy and prevention of GvHD in ALL transplanted patients. Moreover, 10% of patients experience gastrointestinal adverse effects, such as nausea and diarrhea, which limits its acceptance . According to the ECIL and IDSA guidelines, Itraconazole should be used as second-line therapy in the prevention of IFIs.
Voriconazole has similar activity as Itraconazole and is active against most Candida and Aspergillus species, but not zygomycetes, Candida glabrata and C. krusei (Table 1.). The effectiveness of this compound has been demonstrated in large clinical trials in both adults and children, and has led to its approval for empirical and pre-emptive antifungal therapy . Because of its wide use, breakthrough infections with zygomycetes have been reported . Additionally, breakthrough infections with susceptible strains have been noted in patients with low plasma levels, necessitating the monitoring of through plasma levels . In children, Voriconazole is more rapidly metabolized, suggesting a higher dosage of 7-8 mg/kg/b.i.d. than in adults (4-5 mg/kg/b.i.d.) . Relevant side effects of Voriconazole include elevations of liver enzymes, visual disturbances and photosensitivity skin reactions, particularly if combined with nucleoside analoga, which are commonly used in the treatment of ALL. In addition, the interaction of Voriconazole with a number of drugs (e.g. Vincristine, Cyclosporine A, and Omeprazole) has to be considered .
This compound has a potent and broad-spectrum activity against most clinically important fungal infections, including zygomycetes, distinguishing it from the other azoles . According to the ECIL/ IDSA guidelines it is recommended as second-line treatment of aspergillosis, fusariosis, chromoblastomycosis and coccidioidomycosis. In addition, Posaconazole is approved for prophylaxis in high-risk patients older than 13 years of age with ALL and in hematopoietic stem cell transplant patients with graft-versus-host disease [31;39]. The dosage for prophylaxis is 200 mg three times daily, for salvage treatment the dose is increased to 400 mg two times daily. Similar to Voriconazole, interference with cytochrome P450 dependent metabolites (e.g. Cyclosporine) need to be considered .
Caspofungin is active against Candida spp. and Aspergillus spp., but resistant against Cryptococcus species and zygomycetes (Table 1.). It is licensed for adult and pediatric patients, including neonates, for empirical antifungal therapy in persistently febrile neutropenic patients, for second-line pre-emptive therapy of suspected aspergillosis and for primary therapy in non-neutropenic patients with invasive Candida infections. The recommended dose regimen in adults consists of a single 70-mg loading dose on day 1, followed by 50 mg daily thereafter . A dosage of 1 mg/kg for children has been suggested . A favorable safety profile has been described, the most common drug-related adverse events were fever, increased ALT, and rash; few events were serious or required treatment discontinuation .
Micafungin was recently licensed for neonates, children and adults for prophylaxis and treatment of invasive candidiasis in patients with prolonged neutropenia and after hematopoietic stem cell transplantation . The spectrum of activity is similar to that of Caspofungin (Table 1). The recommended dosage is 100 mg/day for invasive candidiasis (≤40 kg body weight: 2 mg/kg) with the option of dose escalation to 200 mg/day or 4 mg/kg/day; and 50 mg/day (≤40 kg: 1 mg/kg) in the preventive indication . The most frequent adverse events include vomiting, high fever, diarrhea, nausea, and hypokalemia .
Although various guidelines on antifungal management have been published, we suggest using a simple approach, which is guided by local factors, such as the pattern of resistance and the availability of diagnostic tools. As newer strategies might soon be implemented, we are unable to assess the efficacy of our approach to date. Our report underlines that many questions regarding antifungal treatment have to be addressed in future studies, such as the duration of treatment or the benefit of costly combination antifungal therapy.