Opportunistic Fungal Infections, Part 2: Candida and Aspergillus

Candidiasis and aspergillosis are the most common fungal infections in immunocompromised hosts. The spectrum of disease ranges from superficial and mucosal infections to invasive tissue infections. Morbidity and mortality attributed to infections with Candida and Aspergillus can be quite high; therefore, timely identification and treatment are important.

EPIDEMIOLOGY
Candida is ubiquitous, with more than 200 described species. About 10% of the species are responsible for human disease, with Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida glabrata being the most commonly isolated. Candida krusei and Candida lusitaniae are seen less frequently. Significant risk factors for candidal disease include neutropenia, use of antibiotics, the presence of an indwelling vascular device, and prolonged stay in the ICU.

Infections attributed to Candida species can be divided into 3 categories: hematogenous (eg, candidemia, hepatosplenic candidiasis, and osteomyelitis), nonhematogenous superficial (eg, cutaneous candidiasis, oropharyngeal candidiasis, and vaginitis), and nonhematogenous deepseated (eg, esophageal candidiasis, cystitis, peritonitis, and tracheitis/ bronchitis). It is noteworthy that Candida is the fourth leading cause of nosocomial bloodstream infections in hospitalized patients.¹ Immunocompromised patients are at high risk for the development of any of these infections.

In HIV-1-infected patients, oropharyngeal and esophageal candidiasis are common and are recognized as an indicator of immunosuppression. These types of candidiasis are seen most commonly in patients with CD4⁺ T-lymphocyte counts of less than 200/?L.² Other types of infections with Candida can certainly occur in this population but are usually associated with other concurrent risk factors. Although C albicans is still the predominant species that is isolated, the emergence of fluconazole- resistant species has been observed, usually in the setting of previous fluconazole exposure.

Patients with hematological malignancies and hematopoietic stem cell transplant (HSCT) recipients are at high risk for development of invasive candidiasis (IC), which is a major cause of morbidity and mortality. The portal of entry is typically the gut mucosa, and less commonly, the integument. Although oral and esophageal infections occur, fungemia is the most serious clinical syndrome observed.³ In addition, although C albicans still accounts for most invasive Candida infections, non-albicans Candida species have emerged with increasing frequency, particularly C glabrata and C krusei. Bloodstream infection with these species has been associated with higher rates of crude mortality.⁴

Most invasive fungal infections following organ transplant are also due to Candida species. These infections are typically related to complications of surgery and usually appear near the end of the first month posttransplant.⁵ Liver and pancreas transplant recipients are at especially high risk for invasive Candida infections in the first month following transplant.6

Aspergillus species are also ubiquitous in the environment and are found in soil, water, and decaying vegetation worldwide. Portals of entry include the respiratory tract and damaged skin or operative wounds. Reports suggest that Aspergillus infections occur in 4% to 20% of bone marrow transplant (BMT) recipients, with neutropenia being the most important risk factor for infection.⁷ A recent prospective trial evaluating the cumulative incidence (CI) in 4261 HSCT recipients during a 22-month period found a CI of 0.5% after autologous HSCT and 2.3% after allogeneic transplant. Mortality following the diagnosis of invasive aspergillosis ranged from 53.8% to 84.6%.⁸ The incidence of disease in HSCT recipients has a bimodal distribution, occurring in the early engraftment stage (around day 30) and then again after 180 days posttransplant, which probably reflects the presence and treatment of graft versus host disease.

In organ transplant, Aspergillus infections are most common in heart and lung transplant recipients (1% to 15% and 3% to 15%, respectively) and less common in liver transplant (1% to 8%) and kidney transplant recipients (0.7% to 4%).⁹ More than half of the Aspergillus infections in liver transplant recipients occur in the first 3 months after transplant. In contrast, 72% of cases of invasive aspergillosis (IA) are identified in the first 3 months after heart transplant, and 50% occur in the first 5 months after lung transplant.⁸

CLINICAL MANIFESTATIONS
In immunocompromised hosts, the clinical presentation of fungal infection is often nonspecific. Fever is a common symptom, as are pulmonary symptoms of cough and dyspnea. Suspicion for fungal infection should be based on host risk factors, including the underlying immunological state and current treatments (including medications), as well as on epidemiological exposures. (See Figure 1 for a diagnostic pathway.)

Figure 1 -

Diagnostic and treatment approach to immunocompromised hosts at high risk for invasive fungal  nfections.

Oropharyngeal candidiasis
Oropharyngeal candidiasis is often asymptomatic and manifests as a painless, creamy white, plaque-like lesion on the tongue or buccal or oropharyngeal mucosa. Patients who have esophageal candidiasis often present with fever, retrosternal burning pain or discomfort, and odynophagia. Endoscopic examination reveals white plaques such as those observed with oral candidiasis that may progress to superficial ulcerations of the esophagus.²

Invasive candidiasis
IC usually presents in a nonspecific manner, with fever as the major finding. Maculopapular, erythematous skin lesions may occur, but biopsy and histopathological examination are required for diagnosis.⁷ Blood cultures will be positive when the infection is associated with an endovascular catheter; however, when disease is limited to visceral organs, blood cultures are often negative.

Candida endophthalmitis as a complication of candidemia occurs in 3.7% to 25% of candidemia cases and can be detected on funduscopic examination. However, signs may not manifest in neutropenic patients until after neutrophil recovery; therefore, ophthalmological examination should be repeated after neutropenia has resolved.³

Aspergillosis
Most patients (80% to 90%) who are infected with Aspergillus will present with pulmonary disease. However, other manifestations include rhinosinusitis; disseminated cutaneous infection (especially in children with leukemia); and disseminated disease with CNS involvement-primarily brain abscess related to mycotic aneurysm. The most immunocompromised patients are the least likely to have symptoms, and their disease rapidly leads to death. The most common symptoms, seen as the disease progresses, are fever and cough. In addition, hemoptysis occasionally is present. Patients who are receiving corticosteroids may be afebrile; however, low-grade chest pain is common.¹⁰

Chest radiographs with nodular or diffuse pulmonary infiltrates are suggestive of the disease but are very nonspecific. Cavitation and wedge shaped lesions are the most distinctive signs of IA; however, other invasive fungal infections can cause similar effects. Characteristic radiographic findings such as the halo sign or crescent sign are the result of vascular invasion, which causes thrombosis, infarction, or necrosis of the surrounding tissue.¹¹

Aspergillus rhinosinusitis
Early symptoms of acute invasive Aspergillus rhinosinusitis are also nonspecific and can be mistaken for bacterial infection. Fever, cough, epistaxis, and headache are common. Findings from an examination may be unremarkable, but an eschar can sometimes be visualized using direct endoscopy. CT scans will show fluid opacification of the sinus, often with bony erosion. MRI is also diagnostically useful. Diagnosis is dependent on clinical signs concurrent with a culture positive for Aspergillus or tissue biopsy results suggestive of fungal invasion.¹⁰

Cutaneous aspergillosis
Primary cutaneous aspergillosis is most commonly seen at or around intravenous catheter insertion sites (Figure 2) or is associated with adhesives, such as tape or occlusive dressings, or infections resulting from trauma or surgical wounds. Secondary cutaneous aspergillosis is associated with disseminated disease and may be due to direct extension or embolic disease.¹² Necrosis is common, and a biopsy is often necessary to make this diagnosis.

Figure 2 -

Note necrotic center surrounded by erythema at the site of previous intravenouscatheterization in this example of a cutaneous Aspergillus fumigatus infection

.

Cerebral aspergillosis
Cerebral aspergillosis can present as cerebral hemorrhage secondary to fungal invasion of the surrounding blood vessels. It occurs in 10% to 20% of all cases of IA. A characteristic finding on a CT scan of the head is 1 or more hypodense, well-demarcated lesions. A biopsy is required for definitive diagnosis.¹⁰

LABORATORY DIAGNOSIS
Candida species grow readily in standard blood culture media, but blood cultures are negative in up to 56% of autopsy-proved cases of IC.13-15 Microscopic features show important species-related variations. Most produce pseudohyphae that are long, branched, or curved.1 On tissue histopathological examination, this feature distinguishes Candida from Aspergillus.

Pathogenic Aspergillus species generally grow easily and relatively quickly on routine bacteriological and mycological media in the laboratory; however, a higher yield of organisms is achieved on mycological media. Fluid from bronchoalveolar lavage (BAL) or endotracheal aspiration should be processed for microscopic or cytological examination and for fungal culture.¹⁰ Culture of Aspergillus from an infected sterile site provides definitive proof of disease and is important in making therapeutic decisions. Definitions of invasive fungal disease are generally based on the European Organization for Research and Treatment of Cancer/Mycoses Study Group definitions that have recently been revised.¹⁴

Serological testing using the galactomannan (GM) assay or (1->3)- ?-D-glucan (BG) assay is reviewed further on in this article. Histopathological findings of IA show filamentous septated hyaline hyphae with acute angle branching (Figure 3).

Figure 3 -

This is a Gomori methenamine-silver stain of Aspergillus in a biopsy specimen ofthe lung of an allogeneic hematopoietic stem cell transplant recipient. Note the acute angle ofbranching hyphae that is characteristic of moulds (not specific to Aspergillus). Mucor tendsto have a 90-degree branch angle, whereas Fusarium and Aspergillus tend to have a 45-degreebranch angle.

Non-culture-based diagnostics
The current conventional diagnostics for the detection of fungal infections include antigen or antibody testing as well as routine culture methods. Culture-based methods for diagnosis of Candida and Aspergillus infection can be problematic. The sensitivity and specificity of culture of Candida and Aspergillus are low, especially when the organisms are recovered from nonsterile sites.¹⁶

The diagnostic value of a culture positive for Aspergillus depends on the risk status of the patient. In a retrospective analysis of 1209 patients with Aspergillus-positive cultures, mostly from nonsterile respiratory secretions, 148 cases of definite (n = 90), probable (n = 49), or possible (n = 9) IA were identified. Patients were stratified into high risk, intermediate risk, or low risk for IA on the basis of underlying disease. Among highrisk patients (allogeneic HSCT recipients or patients who were neutropenic or were being treated for a hematological malignancy), a culture positive for Aspergillus was associated with IA in 50% to 65% of cases, whereas in those at low risk (patients with cystic fibrosis or other connective-tissue disease), a positive culture result rarely represented invasive disease. In the intermediate risk group (for example, autologous BMT or solid organ transplant recipients or patients with diabetes or HIV infection), the association between a positive culture result and IA was 8% to 28%.¹⁷

For these reasons, alternative non-culture-based diagnostic tests are being developed, including GM antigen testing, polymerase chain reaction (PCR) assay, and BG antigen testing.

GM is a highly immunogenic antigen found in the cell wall of Aspergillus species and is released by the fungus into serum during its growth in tissues.¹⁸ Enzyme-linked imunosorbent assay for GM has been validated as a surrogate marker for detection of IA in both animal models and humans.^18-23 It is reported as an index of optical density (GM index [GMI] test), and results are considered positive when the index is 0.5 or more from 2 aliquots of the same sample.²⁴ False-positive tests have been associated with the use of certain antibiotics, including piperacillin/ tazobactam and amoxicillin/ clavulanate,^25-28 and they also may be seen in patients who have histoplasmosis.²⁹

Arecent review by Miceli and colleagues²⁴ investigated the correlation between the GM assay followed sequentially and clinical outcomes in patients with hematological malignancies using data derived from 27 published studies. Their findings showed a strong correlation between GMI and aspergillosis outcome. Specifically, a normal GMI was associated with a favorable outcome, whereas a positive GMI was associated with death. Although these results are quite interesting, caution should be used in interpreting them in regard to treatment decisions.

Recently published guidelines from the Infectious Diseases Society of America regarding the treatment of aspergillosis suggest that resolution of GM antigenemia should not be used as a sole criterion for discontinuation of antifungal therapy.²³ The role of GM in BAL fluid as a tool for early diagnosis of IA has been examined, and findings are promising; however, this diagnostic method remains investigational.^23,30-32

Serial monitoring of GM in conjunction with thoracic high-resolution CT scanning in high-risk neutropenic patients also has been evaluated. The investigators found that this approach led to improved preemptive and empirical antifungal therapy in patients in whom invasive fungal infections were later diagnosed. ³³ This approach may be useful in screening patients at high risk for infection, since a routine chest radiograph alone usually will not detect disease early enough.

BG is a component of the cell wall of most fungi except for Zygomycetes and Cryptococcus neoformans.³⁴ The presence of BG in the serum is indicative of fungal invasion, including IC, IA, and other invasive fungal infections, but is not specific to 1 particular organism.²³ False-positive results for BG assays have been reported in several contexts, including patients treated with immunoglobulin, patients exposed to glucan-contaminated blood collection tubes, patients undergoing hemodialysis using cellulose membranes, and patients receiving certain antibiotics (specifically, ampicillin/sulbactam, carbapenems, and some cephalosporins).^23,33,35

The Fungitell (formerly Glucatell) BG detection assay (Associates of Cape Cod, Inc, East Falmouth, Mass) is approved by the FDA for the diagnosis of invasive mycosis.^15,23,36 Several studies have evaluated this assay in patients with acute myelogenous leukemia and myelodysplastic syndrome,³⁶ in patients with proven or probable invasive fungal infection,¹⁵ and in patients with IC.³⁴ It was found to be sensitive and specific in the early detection of fungal disease.

Results of the BG assay are usually positive in patients with invasive Fusarium infections, but the assay is not specific to this mould. However, the combination of a positive BG assay result and a negative GM test result in a highly immunocompromised patient with a mould infection is suggestive of fusariosis, although it also can be seen with IC (specifically, hepatosplenic candidiasis).³⁷

A recent study used the BG assay in the evaluation of neutropenic patients with acute leukemia.³⁸ BG was measured twice weekly in the absence of fever and daily in the presence of fever during 190 neutropenic episodes in 95 patients. An invasive fungal infection was diagnosed in 60 neutropenic episodes (9 proven, 21 probable, and 30 possible). Proven cases included 5 instances of pulmonary IA and 4 of IC. Sensitivity of the assay for diagnosis of proven or probable invasive fungal infection was 63% and 96%, respectively. The time interval between onset of fever as a first sign of invasive fungal infection and a positive BG assay result was 0.5 day, preceding positive microbiology or histopathology and positive radiological findings in most cases. More research is required to determine whether routine monitoring with this assay is appropriate.

PCR-based diagnosis of fungal infections has shown considerable promise. There have been several studies that evaluated the use of PCR in the diagnosis of fungal infections using universal fungal PCR primers that allow for the potential detection of both yeasts and moulds.^19,38-43 Sensitivity and specificity vary depending on the assay. Head-to-head comparisons of fungal PCR assays have been lacking because most of the PCR assays are developed in house and use different fungal DNA targets and different protocols for sample selection and preparation. The issue of contamination is also important given the ubiquitous nature of moulds in the environment.¹⁶ Overall, PCR-based fungal testing is investigational at this point. Its use remains limited because of lack of standardization of the assays and lack of their commercial availability.

PROPHYLAXIS AND TREATMENT OPTIONS
Esophageal candidiasis
Systemic therapy is required for effective treatment of esophageal candidiasis (EC). A14- to 21-day course of either oral or intravenous fluconazole (100 to 200 mg qd) or oral itraconazole (200 mg qd) is highly effective and is the recommended firstline treatment.³ Alternative drugs for fluconazole-resistant infections include voriconazole, posaconazole, and echinocandins (caspofungin, micafungin, and anidulafungin).

Results of a double-blind multicenter study comparing voriconazole 200 mg bid with fluconazole 200 mg/d for treatment of EC in 391 immunocompromised patients (most with AIDS) showed equivalency of treatment effect. The cure rate was 98.3% for patients who received voriconazole and 95.1% for those who received fluconazole.⁴⁴

As for the echinocandins, 3 randomized trials demonstrated the efficacy of caspofungin in the treatment of patients with mucosal candidiasis. ^45-47 The vast majority of these patients had AIDS. Amphotericin B deoxycholate was the comparator in 2 of the studies. The incidence of drug-related adverse events was significantly lower in the caspofungin arm.^45,46 Studies also showed that the efficacy of micafungin and anidulafungin was equivalent to that of fluconazole.^48,49

Invasive candidiasis
If feasible, initial nonmedical management should include removal of all indwelling central venous catheters. Initial therapy depends on the clinical status of the patient, the physician's knowledge of the epidemiology of Candida species at his or her institution, and previous antifungal exposures. Therapeutic choices include an amphotericin B preparation, fluconazole, voriconazole, or an echinocandin.³ Treatment duration should be for a minimum of 14 days from the last positive blood culture. Fluconazole dosed at 6 mg/kg intravenously or 400 mg qd orally is appropriate when concerns about non-albicans Candida species are minimal.

Although amphotericin B was the preferred initial treatment for many years because of its broad spectrum of activity, the safety and good clinical outcomes of drugs in the echinocandin class favor their use as empiric therapy.^50-53

Micafungin recently received FDA approval for the treatment of IC at the 100-mg daily dose. In an openlabel, noncomparative study of micafungin for the treatment of candidemia in 119 patients (101 adults and 18 children), overall treatment success was found in 83.2% of patients and was similar in patients with and without neutropenia.⁵¹ In a randomized double-blind study comparing micafungin 100 mg qd, micafungin 150 mg qd, and caspofungin at a conventional dosage (a 70-mg IV loading dose, then 50 mg qd IV) in 595 patients with IC, treatment success was achieved in 76.4% of patients in the 100-mg group, 71.4% in the 150-mg group, and 72.3% in the caspofungin group, demonstrating noninferiority.⁵²

A success rate of 75.6% also was achieved in patients with IC given anidulafungin in a randomized double- blind trial that compared it (at a loading dose of 200 mg followed by 100 mg qd) with fluconazole (at a loading dose of 800 mg followed by 400 mg qd) in 261 patients.⁵³ The success rate in the fluconazole arm was 60.2%.

Invasive aspergillosis
In patients with a likely diagnosis of IA, antifungal therapy should be started while diagnostic evaluations are being performed. Voriconazole is approved for the treatment of IA and is recommended as primary therapy for invasive pulmonary aspergillosis.²³

For patients who are intolerant or refractory to voriconazole, a formulation of amphotericin B is an appropriate alternative. Available data indicate that the lipid forms of amphotericin B are as effective as amphotericin B deoxycholate but are associated with less nephrotoxicity.²³ The optimal dosage for treatment of IA has not been defined for any of the lipid forms of amphotericin B. Higher doses of these formulations are often used, but data on their use are lacking. A recent study that compared 2 doses of liposomal amphotericin B (3 mg/kg/d and 10 mg/kg/d) for the primary treatment of proven or probable IA in 201 patients found similar overall response rates (50% for the 3 mg/kg/d regimen and 46% for the 10 mg/kg/d regimen; P > .05) and similar survival rates (72% and 59%, respectively; P > .05).⁵⁴

Caspofungin has been FDA-approved for salvage therapy in adults with IA on the basis of findings from a trial in which a favorable response was achieved in 44.6% patients of who were refractory or intolerant to amphotericin B, lipid formulations of amphotericin B, or triazoles.⁵⁵ Mortality directly related to aspergillosis was 12.1%.

Combination therapy for IA has not been studied in a randomized controlled fashion. In a retrospective analysis of HSCT recipients who had received either voriconazole or voriconazole and caspofungin as salvage therapy for IA, an improved survival rate was observed in the combination arm compared with the voriconazole- alone arm (hazard ratio, 0.42; P = .048).⁵⁶ Recently published guidelines for the treatment of IA do not recommend routine administration of combination therapy; however, in the context of salvage therapy, the addition of another antifungal agent may be useful.²³ Randomized clinical trials are currently being developed to address this issue.

PREVENTION
In the hospital, patients at risk for IA, including neutropenic patients, should not be given pepper or tea that has not been sterilized because standard preparations of such substances have high counts of Aspergillus. For similar reasons, fresh flowers and potted plants should not be allowed in patient care areas.¹¹

Local construction work is a major factor for the acquisition of IA and has been associated with outbreaks in transplant centers.⁵⁷ The use of high-efficiency particulate air (HEPA) filters along with positive air pressure in patient rooms reduces this risk but does not eliminate it entirely. Barriers between patient care areas and renovation or construction areas are highly recommended, along with adjunctive monitoring of the air for spore counts on the HSCT unit.⁵⁷

A vaccine for Candida infection has been evaluated in mice and appears protective against otherwise lethal disseminated candidiasis; however, development for clinical testing in humans faces many challenges, ^58-60 and it is unlikely to be available for many years. A vaccine for Aspergillus infection would be desirable, but the likelihood of one being developed at this time is low.

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