Opportunistic invasive fungal infections: diagnosis & clinical management.

Invasive fungal infections are a significant health problem in immunocompromised patients. The clinical manifestations vary and can range from colonization in allergic bronchopulmonary disease to active infection in local aetiologic agents. Many factors influence the virulence and pathogenic capacity of the microorganisms, such as enzymes including extracellular phospholipases, lipases and proteinases, dimorphic growth in some Candida species, melanin production, mannitol secretion, superoxide dismutase, rapid growth and affinity to the blood stream, heat tolerance and toxin production. Infection is confirmed when histopathologic examination with special stains demonstrates fungal tissue involvement or when the aetiologic agent is isolated from sterile clinical specimens by culture. Both acquired and congenital immunodeficiency may be associated with increased susceptibility to systemic infections. Fungal infection is difficult to treat because antifungal therapy for Candida infections is still controversial and based on clinical grounds, and for molds, the clinician must assume that the species isolated from the culture medium is the pathogen. Timely initiation of antifungal treatment is a critical component affecting the outcome. Disseminated infection requires the use of systemic agents with or without surgical debridement, and in some cases immunotherapy is also advisable. Preclinical and clinical studies have shown an association between drug dose and treatment outcome. Drug dose monitoring is necessary to ensure that therapeutic levels are achieved for optimal clinical efficacy. The objectives of this review are to discuss opportunistic fungal infections, diagnostic methods and the management of these infections.

Introduction

Invasive fungal infections (IFI) have significantly increased due to advances in medical care in the at risk immunocompromised population. Fungal species are widely distributed in soil, plant debris and other organic substrates, and make up approximately 7 per cent (611,000 species) of all eukaryotic species on earth1, although only about 600 species are human pathogens2.

Major risk factors for IFI include neutropenia <500 neutrophils/ml for more than 10 days, haematological malignancies, bone marrow transplantation, prolonged (>4 wk) treatment with corticosteroids; prolonged (>7 days) stays in intensive care, chemotherapy, HIV infection, invasive medical procedures, and the newer immune suppressive agents. Other risk factors are malnutrition, solid organ transplantation, severe burns or prolonged stays in intensive care (>21 days), systemic corticosteroids for >7 days, and major surgery34. There are also reports of the presence of infection in immunocompetent patients5 without signs or symptoms of conditions associated with immunocompromised status.

Infection can be transmitted by the inhalation of spores (aspergillosis, cryptococcosis, histoplasmosis), percutaneous inoculation in cutaneous and subcutaneous infections (dermatophytosis, madura foot), penetration into the mucosa by commensal organisms such as Candida albicans, and the ingestion of a toxin in contaminated food or drink (gastrointestinal disease).

Infections may be mild and only superficial or cutaneous (e.g. dermatophytosis and Tinea versicolor) or may cause life-threatening, systemic illness (e.g. candidiasis, aspergillosis and mucormycosis). The clinical manifestations of the disease caused by a given fungal agent can be highly variable and related to host immunity and physiological condition. For example, Candida spp. can invade a local site (mucocutaneous or cutaneous candidiasis, onychomycosis) or cause systemic infections (renal, liver abscess, lung and nervous central system). Allergic symptoms were reported in infections with other fungi such as Aspergillus spp. (allergic bronchopulmonary aspergillosis). The isolation of these organisms from clinical samples may indicate colonization, infection or disease; consequently interpreting the results of diagnostic tests may be challenging for clinicians who treat these patients. Treatment requires early suspicion and is difficult because only a few antifungal agents are available, most usually have side effects, and some organisms have developed resistance6. Clinicians need drugs that are highly effective but have low toxicity.

In this review opportunistic fungal infections, diagnostic methods and the management of these infections are discussed.

Clinical manifestations and epidemiology

The standard definition of IFI was developed by members of the European Organization for Research in the Treatment of Cancer–Invasive Fungal Infection Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group7.

Among immunocompetent hosts, keratitis and onychomycosis are the most common infections. Other infections in immunocompetent patients include sinusitis, pneumonia, thrombophlebitis, peritonitis, fungemia, endophtalmitis, septic arthritis, vulvovaginitis and osteomyelitis8.

In immunocompromised patients, any fungus present in the environment may be potentially pathogenic. Aspergillus and Candida spp. are the main organisms isolated most frequently from immunocompromised patients. The other most relevant aetiologic agents are Cryptococcus spp., Fusarium spp., Zygomycete, Dematiaceous fungi and opportunistic yeast-like fungi9. Colonization of the mucosal surfaces by Candida spp. is the first step in the process of systemic candidiasis. Candida is isolated from 84-88 per cent of mucocutaneous surfaces in hospitalized patients or even healthy adults10. Because of its stronger adherence capacity, C. albicans is found more often than other Candida species10.

Central nervous system candidiasis may present with scattered brain microabscesses or larger lesions, vasculitis or thrombosis. Candida species are reportedly the third or fourth most frequently isolated organisms in nosocomial bloodstream infections, the fourth most common cause of hospital-acquired systemic infections in the USA in nosocomial candidemia11, and the fifth most common cause of bloodstream infections in paediatric intensive care units12. The crude mortality rates in the USA were reported to be up to 50 per cent13.

There are many species of Aspergillus in the environment but the most frequently recovered species depending on the geographical region are Aspergillus fumigatus, A. flavus, A. niger or A. terreus. Clinical manifestations can range from colonization in allergic bronchopulmonary disease to active disseminated infection, with mortality rates ranging from 40 to 90 per cent depending on the site of infection, host immune status, and the treatment regimen14. The crude mortality rate has been reported to be as high as 95 per cent in bone marrow transplant recipients14. Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity reaction to Aspergillus mycelia, which colonize the bronchi with a prevalence rate of 1 to 2 per cent in people with asthma, 7 to 14 per cent in steroid-dependent asthma patients, and 2 to 15 per cent in patients with cystic fibrosis15. Other clinical manifestations of aspergillosis are invasive pulmonary aspergillosis, cutaneous and wound infections, keratitis, and Aspergillus sinusitis.

Cryptococcus neoformans is a major cause of infection in immunocompromised patients16. Primary infections due to C. neoformans are common and most are asymptomatic in the lung. Cryptococcus neoformans can cause a severe form of meningitis and meningo-encephalitis in patients with AIDS. Disseminated cryptococcal infection can cause clinical manifestations in the skin, ocular, soft tissue or bones and joints16.

Zygomycosis is the most lethal opportunistic fungal infection particularly among patients with diabetes mellitus, haematological malignancies, and patients receiving deferoxamine treatment. Rhizopus, Mucor, and Rhizomucor species account for up to 75 per cent of mucormycosis cases17. Infection with Entomophthora species has been reported in immunocompetent patients18. Fusarium species cause a broad spectrum of infections in humans, including superficial and disseminated infections, the latter with a mortality rate that approaches 100 per cent19. Members of this genus may also cause allergic diseases and mycotoxicosis following the ingestion of toxin-contaminated food20.

Pathogenesis

Many factors are important in the virulence and pathogenic capability of microorganisms. Enzymes secreted from colonizing organisms (especially when C. albicans is involved), such as extracellular phospholipases, proteinases and hydrolytic enzymes, contribute to host tissue invasion21. Dimorphism in the ability of some Candida species to grow as a unicellular yeast at room temperature and in pseudohyphal and filamentous forms in the host's body or at 37 °C is another virulence factor22, because the hyphal forms are able to release hydrolytic enzymes and can specifically invade epithelial and endothelial cells23.

Conidial size and count are factors that affect virulence in some aetiologic agents. Aspergillus fumigatus, with small conidia, is the main agent in invasive pulmonary aspergillosis, whereas A. flavus, with large conidia, is an important aetiologic agent in Aspergillus sinusitis, cutaneous and wound aspergillosis24. A low conidia count may not produce infection. According to Pasqualotto et al25, Aspergillus species caused death when as few as 102 spores were inoculated. Other pathogenic factors detected in patients with invasive aspergillosis are sensory deprivation (environmental pH), mutational restriction of nutrient acquisition, siderophore and amino acid biosynthesis, extracellular elastolytic proteases, gliotoxin and hyaronic acid. Aspergillus fumigatus and C. albicans can support growth across a broad range of environmental pH values, and the biosynthesis of specific materials protects virulence factors involved in fungal pathogenicity.

The outer cell wall layer plays a major role in an organism's pathogenicity. Chitin can influence immune recognition by blocking dectin-1, leading to significant reductions in cytokine production26. In Cryptococcus species the polysaccharide capsule protects the organism against the host immune system. Other pathogenicity factors in this organism include melanin production, mannitol secretion, superoxide dismutase, proteases and phospholipases27. The synthesis of melanin and other conidial pigments in fungal organisms such as A. fumigatus reduces complement opsonization by camouflaging binding sites, and reduces the ability of C3 to bind with conidia, although this property is still controversial28.

Rapid growth and affinity to the blood stream, heat tolerance, the production of efficient proteolytic enzymes such as lipases, proteases, glycosidic and lipolytic extracellular enzymes3, siderophore production and an efficient iron transport system are pathogenic agents in members of the Zygomycetes family3. Toxin production is another pathogenic factor in Aspergillus and Fusarium species, which can contaminate food and cause mycotoxicosis20.

Immunity

The first-line anatomical barriers of defense are the skin and mucosal surfaces, which protect the human body with an acidic pH, enzymes, mucus and other antimicrobial secretions29. When these barriers are broken by surgery or indwelling catheters, radiotherapy, burns or chemotherapy, fungal agents can reach the deep tissues. The clinical forms and severity of the disease manifestations depend on the host's defenses and immune response. An active host immune response against fungal spores or hyphae leads to allergies and asthma, and to the collapse of immune system defenses in Aspergillus infection3031, which results in the invasive form of infection. Both acquired and congenital immunodeficiency may be associated with increased susceptibility to IFI, which frequently occurs in patients with phagocytic and cellular immune defects.

Once fungi enter, opsonization promotes fungal uptake and innate immune activation by a wide range of phagocytic and signaling receptors31 which include C-type lectin receptors and Toll-like receptors by host cell pattern recognition receptors32. In healthy and immunocompetent individuals, the innate immune system (neutrophils and macrophages) is an efficient sentinel that provides protection from thousands of fungal species through phagocytosis of the invading pathogens by cell surface receptors. Numerous cytokines, e.g. interleukin (IL)-1β, IL-12, IL-17, IL-23 and tumour necrosis factor (TNF)-α, are important in directing innate and adaptive responses to fungal pathogens29.

Cell-mediated immunity through Th1-based responses to fungal infections, characterized by TNF-α, IL-12 and interferon (IFN)-γ production, is a protective factor, and Th2-based responses, characterized by IL-4, IL- 6 and IL-13 production29, are maladaptive and deleterious factors3334. Uncontrolled Th2 responses lead to chronic infections or allergic responses; in this connection, IL-4 and IL-13 play important pathogenic roles in allergic bronchopulmonary mycosis3536.

Diagnosis of infections

The clinical manifestations of fungal infection are not specific, and like other infective diseases, a high degree of suspicion is required for the early diagnosis and optimal management of these infections. Systemic fungal infections, according to standard criteria7, are established when histopathologic examination with special stains confirms fungal tissue involvement or when the aetiologic agent is isolated from clinical sterile specimens by culture.

Radiological evidence from X-rays and high-resolution computed tomography is useful for the diagnosis of fungal infections. Pulmonary fungal infections such as aspergillosis, fusariosis, scedosporiosis or zygomycosis are characterized by central cavitation of pulmonary lesions, infiltration, pulmonary nodules, and halo or air-crescent signs3738. Documentation of the diagnosis of infection requires serial high-resolution computed tomography; however, the risk of radiation exposure in children must be considered3940. The specificity of these methods is lower in children than in adults39.

Conventional mycological methods include direct microscopic examination and the culture of samples in the mycology laboratory. Pathological examination and direct smears of samples with potassium hydroxide by an expert is the most rapid, cost-effective and sensitive method for the diagnosis of fungal infections. An expert can identify some genera of aetiologic agents such as yeasts or molds (with septated or nonseptated hyphae) and thus help ensure prompt, effective therapy. However, it may not be possible to identify fungal strains by this method, and fungal growth or the use of complementary methods such as hybridization in paraffin-embedded tissue may be necessary.

Culturing clinical specimens (tissue, sputum, urine, wound, or blood) to isolate the aetiologic fungal agent is the gold standard for the diagnosis of IFI, provided that the samples are from sterile sites such as blood, tissue or cerebrospinal fluid, in which fungal infection can be documented. The sensitivity of culture differs among published studies41. In cases with a specific fungal infection such as zygomycosis, the aetiologic agent loses its viability during tissue homogenization before culture42. In addition, this method can produce false negative results if the clinical specimens are obtained after treatment with antifungal agents. Collecting appropriate tissues and other sterile specimens for culture or histology from patients whose condition is unstable, especially when neutropenia is present or platelet count is low, is difficult because it requires invasive procedures. The sensitivity of blood culture for the diagnosis of fungal infection is controversial. Multiple or repeated blood cultures should be performed to increase the likelihood of detecting candidemia, and filamentous fungi are rarely isolated from blood43. Culture can be useful to determine the sensitivity of the isolated fungi to antifungal agents and identify resistant species, and are thus needed to optimize patient management6. However, given the limitations of conventional methods of diagnosis for IFI, negative result on direct or pathologic smears and cultures do not rule out infection, so it is essential to use other suggested methods.

There are many serological methods for the diagnosis of fungal infections, and the results of these tests become available sooner than culture. Skin tests and serum IgE level are suitable methods for the diagnosis of disorders such as ABPA. It should be noted that antibody assays are often negative in immunosuppressed patients. The detection of antigens in serum or cerebrospinal fluid is recommended for the diagnosis of Cryptococcus infections. In severely ill patients the detection of antigens by enzyme immunoassay or the latex agglutination test in blood, bronchoalveolar lavage (BAL) fluid or urine can be useful for a rapid diagnosis. Cross-reactions are seen in some infected patients. The cut-off values for the detection of antigens in different populations may be different44, therefore, it is important to interpret the results appropriately. The serial measurement of antigen titres is not a reliable indicator for the evaluation of antifungal therapy.

The detection of fungal cell wall markers in serum has been reported for galactomannan (GM), (1, 3)-beta-D-glucan (BDG) and mannan7. Galactomannan is relatively specific for Aspergillus species45, and can be detected in urine, bronchoalveolar lavage fluid cerebrospinal fluid and other specimens with enzyme immunoassay. Various sensitivity rates from 30 to 100 per cent, and similarly wide-ranging specificities from 38 to 98 per cent have been reported for GM4647. Factors that limit the specificity of this test are immune reactivity with other fungi such as Penicillium spp. and Paecilomyces spp., false positive results with antibacterial agents such as beta-lactam antibiotics, particularly piperacillin–tazobactam48 and amoxycillin with or without clavulanate49, and dietary GM in pasta, cereals and milk50.

1,3-Beta-D-glucan (BDG) is present in the cell wall of most pathogenic fungi, including Fusarium, Candida, Aspergillus and Trichosporon, and is not species-specific or genus-specific for each organism5152. However, Mucorales, C. neoformans and Blastomyces dermatitidis contain relatively small amounts of cell wall BDG; therefore, these assays may not be completely reliable in patients infected with these organisms53. Sensitivities of 55 to 100 per cent, specificities of 71 to 93 per cent, positive predictive values of 40 to 89 per cent and negative predictive values of 73 to 100 per cent have been reported with various cut-off values for positivity ranging from 6 to 120 pg/ml for this assay5354. Depending on the type of assay (Fungitell or Fungitec) and population, the recommended cut-off levels are 7 pg/ml55, 20 pg/ml54 or 60-80 pg/ml51. False-positive BDG findings occur in the patients with fungal colonization or mucositis who have received empirical antifungal therapy55. The combined use of a BDG assay (Glucatell) and a GM enzyme immunoassay (Platelia Aspergillus) improves the specificity of diagnosis56.

Mannan is mainly found as a characteristic cell wall component in yeasts. The detection of circulating Candida mannan and anti-mannan antibodies has been used as a diagnostic marker for invasive candidiasis or candidemia caused by the most pathogenic species of Candida in adult patients with neutropenia5758 and after myeloablative chemotherapy59. The overall sensitivity of mannan antigen detection in patients with candidemia has been reported to be between 69 and 90.9 per cent, and specificity between 89 and 46.2 per cent compared to culture as the gold standard6061. In neonates, this test has yielded promising results particularly for ruling out candidiasis, considering its high negative predictive value of 98 per cent62.

Detection of fungal DNA

The polymerase chain reaction (PCR) assay may serve as a powerful non-culture method for the diagnosis of systemic fungal infection in high-risk patients. Qualitative methods are sensitive in detecting fungal DNA in human blood samples, tissues, bronchoalveolar lavage and other body fluids63. The sensitivity and specificity vary according to type of the tests. For example, nested PCR has a sensitivity of 92.8 per cent and a specificity of 94 per cent 64, and panfungal PCR has sensitivity 80 per cent and a specificity of 95.6 per cent65. Quantitative methods include PCR ELISA, with a sensitivity of about 83.3 per cent and a sensitivity of about 91.7 per cent66, and real-time PCR, with the sensitivity about 100 per cent and a specificity about 97 per cent67. Molecular methods, which are rapid and can yield results within 6 h, have revolutionized the diagnosis of fungal infections because these enable diagnosis during the incubation period and early stage68 of infection, and prior to bone marrow transplantation67.

The diagnosis of infection based on molecular and serologic techniques can provide powerful tools for the early diagnosis of IFI. Because the fungi are common in the environment and opportunistic fungi in immunocompromised patients can cause high morbidity and mortality, the interpretation of positive or negative results with different laboratory methods is difficult for clinicians, so more than one method should be used for early diagnosis.

Prevention

To prevent infection in immunocompromised patients, exposure to fungal spores must be limited with high-efficiency particulate air filters and positive pressure in the patient's room, and high-risk patients should avoid contact with soil, tap water and shower facilities69. Decreasing the duration of neutropenia or discontinuing immunosuppressive agents should be considered in efforts to prevent fungal infection. Knowledge of the susceptibility pattern of current fungi in each region and the use of prophylactic doses of interferon-γ and azole antifungal agents can be useful to manage infection in high-risk patients; however, after long-term prophylaxis with antifungal agents, infection by resistant aetiologic agents needs to be considered. In addition, fungal colonization of different sites in the same patient (mouth, rectum, nose, urinary tract and vagina) and cutaneous infections should be evaluated before antineoplastic therapy and major surgery70.

Treatment

The timely initiation of antifungal treatment is a critical component in the outcome for the patient. Unfortunately, patients with fungal infection often die of complications attributed to the infection despite antifungal therapy7071. Delays in antifungal treatment in candidemia infections are associated with a 20 per cent increase in mortality if more than 12 h have elapsed after a positive blood culture result, and the mortality rate increases significantly on each of the following three days72.

Localized infection is usually treated with topical antifungal agents, whereas disseminated infection requires the use of systemic agents with or without surgical debridement, and in some conditions immunotherapy is also advisable. Fungal infections are difficult to treat because antifungal therapy in Candida infections is still controversial and based on clinical grounds, and in molds, the fungus isolated from the culture medium must be assumed to be the pathogen because these organisms are saprophytic in the environment. Some molds such as Aspergillus terreus, Fusarium solani and Mocuralea are intrinsically resistant to antifungal agents; therefore, mortality rates in the patients infected with these organism are high373.

The management of fungal infections is different depending on the type of infection and aetiologic agents. Antifungal agents have varying spectrums of activity, dosing, safety profiles and costs. Furthermore, many confounding factors such as the aetiologic agent, age, underlying diseases and surgical complications can influence the outcome74. For example, for invasive aspergillosis voriconazole is superior to deoxycholate amphotericin B as the primary treatment in most patients6.

Many studies have shown an association between drug dose and outcome7576. Therapeutic drug monitoring (TDM) is necessary to ensure that therapeutic levels are achieved7778. Evidence supports TDM to optimize clinical efficacy when the drug is used for prophylaxis and therapy in IFI. The criteria for treatment response include the disappearance of clinical and radiological symptoms attributed to the infection. In some cases, the interpretation of radiologic images may be problematic because lesions may remain in the lungs and sinuses. Because fungal pathogens are eukaryotes and many of their biological processes are similar to those in humans, many antifungal drugs can cause toxicity when used at therapeutic doses79. Monitoring the clinical response to individualized dose regimens can be helpful in interpreting the patient's condition with regard to treatment efficacy and toxicity, disease progression during therapy, drug absorption in patients with suspected poor oral absorption, signs or symptoms of significant toxicity or improper compliance with therapy.

Serum drug concentration is influenced by many factors. Fluconazole and flocytosin in most preparations are excreted by the kidney as active drugs, and itraconazole and voriconazole metabolism in the liver involves specific enzymes, predominantly CYP3A4, CYP2C9, CYP3A4 and CYP2C1980818283. Genetic polymorphisms of the enzyme84 involved and complications in the liver or kidney can reflect the serum level of these antifungal agents.

The absorption of some antifungal agents requires special conditions. For example, absorption of itraconazole from capsule formulations is pH-dependent and requires an acidic environment, which is favoured by a full meal or a cola drink8485. In contrast, absorption of the oral solution is enhanced in the fasted state86. Several patient-specific factors have been demonstrated to affect azole absorption including food (especially fat), gastric pH and the use of proton pump inhibitors, diseases associated with chemotherapy, and the frequency of administration (because of saturable absorption)8788. Differences in serum drug levels between healthy volunteers and patients have been reported in connection with these factors8990. Another important pharmacokinetic variable is drug interaction between azoles and rifampicin, carbamazepine, long-acting barbiturates, ritonavir, efavirenz and rifabutin91, and most notably with cytochrome P450-inducing drugs88.

The clinical use of flucytosine is limited due to its gastrointestinal, haematological and neurological toxicity, the rapid development of resistance when used as monotherapy, and the lack of parenteral formulations92. Accordingly, serum flucytosine concentration should be monitored to identify clinical indications, drug interactions and toxicity939495.

Fluconazole is primarily eliminated via renal excretion, with approximately 80 per cent of the unchanged drug appearing in the urine. Thus, dosage adjustments are warranted in patients with a creatinine clearance <50 ml/min96. When the susceptibility pattern of the drug is dose-dependent and in patients with renal dysfunction, monitoring can help improve the response.

Routine monitoring methods are available for lipophilic triazoles, itraconazole, voriconazole and posaconazole78. High performance liquid chromatography and bioassays have been developed to monitor the serum concentrations of triazoles, although the former method is the assay of choice when available97. Additional studies are needed to measure serum levels and determine the best timing to achieve optimum concentrations of different antifungal agents in different populations. The efficacy and safety of antifungal agents are influenced by serum concentrations, clinical factors and the patient's physiological condition; all these factors can therefore, provide clinically useful information to monitor steady-state concentrations in patients with serious IFI.

Conclusion

The best approach to the optimal management of fungal infection is early detection and identification of the causal agent, so that appropriate treatment can be initiated as soon as possible, especially in immunocompromised patients. Clinicians should be familiar with the use of diagnostc methods and suitable antifungal agents, because these are the factors with the greatest impact on the outcome for patients. The serum concentration of antifungal agents should be monitored to record both the efficacy and toxicity of these drugs.