Aspergillosis is a multifaceted disease that mainly affects immunocompromised patients. Invasive aspergillosis is mainly a consequence of impaired host defense system, whereas in allergic bronchopulmonary aspergillosis (ABPA) the allergic reaction occurs between immunity of host and Aspergillus antigen, which leads to lung inflammation. The main causative agent of this disease is A. fumigatus and emergence of drug resistance, especially to azoles, has become a serious health problem in many countries. Considering this fact, identification of pathogen is very much important morphologically, as well as by using molecular markers. The present chapter highlights, the classification of pulmonary aspergillosis, molecular and morphological identification of Aspergillus strains, antifungal drugs used for treatment of pulmonary aspergillosis and multidrug resistance problem.
Chapter 12
Pulmonary aspergillosis: diagnosis and treatment
S. Quereshi*
P. Paralikar**
R. Pandit**
M. Razzaghi-Abyaneh†
K. Kon‡
M. Rai**
* Department of Microbiology and Biotechnology, Indira Priyadarshini College, Chhindwara, Madhya Pradesh, India
** Nanobiotechnology Laboratory, Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India
† Department of Mycology, Pasteur Institute of Iran, Tehran, Iran
‡ Department of Microbiology, Virology and Immunology, Kharkiv National Medical University, Kharkiv, Ukraine
Abstract
Keywords
Aspergillosis
identification
multidrug resistance
Voriconazole
Aspergillus fumigatus
1. Introduction
Aspergillus is a cosmopolitan fungus that usually occurs on organic debris, compost, food, stored grain, compost heaps, air vents, and airborne dust or in other decaying vegetation. It is aerobic and grows mostly on a high carbon source such as monosaccharides and polysaccharides. It belongs to Deutromycetes, which is devoid of a sexual reproductive growth phase. The species of Aspergillus which are associated with human illness are Aspergillus fumigatus and Aspergillus niger and less frequently, Aspergillus flavus and Aspergillus clavatus. The term “aspergillosis” indicates, an infection of the airways, which involves the respiratory tract. The cutaneous infection is caused by Aspergillus species.1 Other sites of Aspergillus infection present in human include auditory canal, skin, nails, eyes, sinuses, meninges, and bones. The central nervous system, cardiovascular system, and other tissues may be infected. The majority of Aspergillus species can tolerate temperature up to 49°C. Nearly about 200 species of Aspergillus are known, but it is estimated that around 40 Aspergillus species are responsible to cause infections in humans. Aspergillus reproduces by means of conidia. They are approximately 2–4 μm in diameter. As conidia are airborne, they can be easily inhaled and cause infections in the lungs.2 Gomori methanamine silver stain can be used to observe sputum or mucus hyphae.3,4
Aspergillosis of the head and neck region affects the nasal and paranasal sinuses. Aspergillus is the most common fungus responsible for the paranasal sinus involvement. Generally A. fumigatus is the most predominant causative agent followed by A. flavus. It is a type of invasive aspergillosis, that affects the patients with chronic nasal nodule and mucoid impaction of the sinuses. The immunological pathogenesis is not fully understood, but it is assumed that continuous inhalation of Aspergillus spores results in the colonization in the sputum plugs and leads to minute tissue damage. Spores of Aspergillus secret certain proteolytic enzymes, as a result bronchial wall invasion takes place and antigen absorption increases. Antigen release results in the production of IgE, IgG, and IgA. Hypersensitivity reaction occurs and results in tissue inflammation. Mucus plugs consist of Charcot-Leyden crystals, eosinophils, and hyphae of A. fumigatus.1,4,5
The most frequently affected sinus is the maxillary sinus. It is categorized into invasive and noninvasive types on the basis of the fungal invasion of the bones and the mucosal layer.6 Chronic invasive sinonasal aspergillosis is mostly noticed in immunocompetent patients, residing in dry air climate countries such as India, Saudi Arabia, and Sudan. In Sudan area, invasive sinonasal aspergillosis occurs due to A. flavus. Further progress in this type of aspergillosis for few months leads to granulomatous reactions. Invasive aspergillosis is further subdivided into three types: namely acute, fulminant, chronic invasive, and granulomatous invasive. Noninvasive aspergillosis is classified into two types allergic rhinosinusitis and fungal ball rhinosinusitis. The common symptoms of noninvasive aspergillosis are the invasion of sinus mucosa and bone atrophy.1–4
Aspergilloma is defined as the condition in which accumulation of Aspergillus hyphae, cellular debris, and fibrin are present within a pulmonary cavity. Aspergilloma is a fungus ball mycetoma that develops in a preexisting cavity in the lung parenchyma. Underlying causes of this cavity may include treating tuberculosis or other necrotizing infections, sarcoidosis, CF and emphysematous bullae. The ball of fungus may translocate within the cavity, but does not invade the cavity wall. However, as a complication, hemoptysis (the coughing of blood) may occur. Patients with a previous history of such lung diseases as tuberculosis, sarcoidosis, cystic fibrosis, or other are most susceptible to an aspergilloma. Usually, there are no specific symptoms in aspergilloma, but in many patients the haemoptysis may be infrequent and in small quantity, but can be severe and require urgent medical intervention.7
2. Classification and microbiology of pulmonary aspergillosis
Pulmonary aspergillosis is a disease of the lungs, which is characterized by the colonization of Aspergillus spores on the invasive infection.4 Aspergillus species, such as A. fumigatus, A. niger, A. terreus, and A. flavus have been known to exhibit many life-threatening diseases in humans, especially in the immunocompromised patients. Generally, these are present in the intrapulmonary region, nasal cavity, auditory canal and cornea. These species generate allergies, chronic and saprophytic conditions and result in different forms of aspergillosis. A. fumigatus is commonly found in the patients suffering from invasive aspergillosis. Other predominantly occurring types of Aspergillus species are A. flavus, A. niger and A. terreus.8–11 Generally, Pulmonary aspergillosis can be subdivided into ABPA, chronic pulmonary aspergillosis (CPA), invasive pulmonary aspergillosis (IPA), and simple pulmonary aspergilloma (SPA).12,13
2.1. Allergic bronchopulmonary aspergillosis
ABPA is a lung mycosis that has been recorded in people who are allergic to the Aspergillus. In a case of ABPA, the allergic reaction takes place between the immune system of the host and Aspergillus, which rises into lung inflammation.14 Common problems associated with ABPA are bronchospasm and mucus build up, which eventually lead to the breathing problem, cough, and airway obstruction.15 Bronchiectasis is the disease characterized by damage in the airways, which affects lungs badly and has been observed in the few patients of ABPA. ABPA is a disorder which is caused by A. fumigatus.16 Aspergillus sensitization is the first progression step that results in the advancement of ABPA.17 From clinical findings, it was found that A. flavus, A. niger and A. fumigatus are responsible for ABPA.18 Aspergillus sensitization is defined as an immediate hypersensitivity reaction against antigens of A. fumigatus.19,20 Another disease called allergic bronchopulmonary mycosis ABPM is a disease which is similar to ABPA like syndrome, but it is affected by fungi other than A. fumigatus. Allergic brochopulomonary fungosis occurs, when A. fumigatus is present in association with other fungi, such as, Helminthosporium species, Stemphylium lanuginosum, Fusarium vasinfectum, Dreschslera hawaiiensis, Candida species, Curvularia species, Schizophyllum commune, and species of Aspergillus such as A. nidulans, A. niger, A. flavus and A. oryzae.21
In 1952, ABPA was first reported in England by Hinson et al.22 and it has now emerged all over the world. Bronchial colonization of A. fumigatus results in a hypersensitive lung disease ABPA. This is a disease which generally affects patients of asthma and cystic fibrosis. It is estimated that 1–2% of asthma patients, 7–14% steroid-dependent patients, and 2–15% of cystic fibrosis patients are supposed to be the sufferer of ABPA.15 This disease is characterized by asthma, eosinophilia and pulmonary infiltrates. The actual susceptibility of ABPA in asthma patient is not exactly known. It is reported that high concentration of spore exposure increases the chances of ABPA. It is estimated that defects in adaptive and innate immunity may result in the persistence of A. fumigatus.19 Some researchers have stated that exposure to the spores of A. fumigatus may be responsible for ABPA in asthmatic patients. The environment is not considered as the reason for the development of ABPA. Although all asthma patients are exposed to the same environment, very few of them are prone to the ABPA. The outcome of inhalation of A. fumigatus conidia may give rise to hyphal growth. Many types of proteins are released by A. fumigatus particularly pro-inflammatory cytokines by the airway epithelium. Certain proteases released by Aspergillus are directly toxic to the pulmonary epithelial cell and causes cell detachment and cell death.23 Depending on radiological images, ABPA has been classified into three groups, which are: ABPA Seropositive (ABPA-S), ABPA-Central Bronchiectasis (ABPA-CB) and ABPA Other Radiological Findings (ABPA-CB-ORF).19
Stage 1: This is also known as acute stage. In this stage, the level of IgE and IgG specific to A. flavus is increased. Pulmonary infiltrate was observed in the middle and lower lobe of the lungs along with increase in the level of IgE.
Stage 2: This is called remission. In this stage, stage I condition persists for more than 6 months. Ig G level in the serum may be normal or slightly higher than the normal range. If stage 2 persists for longer duration, it may result in the development of stage 3.
Stage 3: This is known as exacerbation. In this stage, relapse of stage 2 is observed. The in-filtrates of Ig E are observed in the middle and lower lobes of lungs. At this level patient required proper medications for ABPA.
Stage 4: This is known as corticosteroid-dependent asthma. At this level, patients are supposed to suffer from constant cough and wheezing like symptoms. Ig E level is increased in the blood even on the oral corticosteroid consumption. Symptoms become more critical if corticosteroids consumption is stopped.
Stage 5: This is the last stage, which is known as end stage. In this stage, the patients whose diagnosis was missed during first stage and those patients who had taken treatment for steroids for the treatment of asthma may become prone to bronchiectasis and fibrosis.
2.2. Chronic pulmonary aspergillosis
CPA is a gradual and progressive inflammatory pulmonary syndrome. CPA is also named as semi-invasive aspergillosis or subacute invasive aspergillosis. The names chronic necrotizing pulmonary aspergillosis (CNPA) and semi-invasive aspergillosis were given by Gefter et al.24 According to recent guidelines from the Infectious Diseases Society of America (IDSA), there are three major subtypes of chronic types of pulmonary aspergillosis: (CNPA), chronic cavitary pulmonary aspergillosis (CCPA) and aspergilloma.25 CCPA is characterized by the occurrence of a large number of cavities with the presence or absence of fungal ball along with Aspergillus antibodies and increased in the number of inflammatory markers. Aspergilloma is defined as the condition, in which accumulation of Aspergillus hyphae, cellular debris, and fibrin are present within a pulmonary cavity.7 CNPA progresses gradually in a few months or even in a year, which results in lung destruction, such as, progressive cavitation, fibrosis. Common symptoms associated with CNPA are weight loss, productive cough, chronic sputum, hemosputum, or hemoptysis.26
From the previous case reports, it was found that A. fumigatus is supposed to be the most common causative agent present in the CPA patients. CNPA is observed in middle-aged people and elderly patients.25 Patients with chronic lung disease, such as, pulmonary tuberculosis, cystic fibrosis, chronic obstructive lung disease, pneumoconiosis, lung infarction and sarcoidosis are susceptible to CNPA.27 Immunocompromised patients, such as, suffering from diabetes mellitus, chronic liver disease, malnutrition, and alcoholism are prone to CNPA.7 The pulmonary diseases that are responsible for cavitation and which may result in the CPA are emphysematous bullae, chronic obstructive pulmonary disease, bronchiectasis, histoplasmosi, and rheumatoid nodules.28 CNPA is histologically characterized by necrosis of lung tissue followed by acute or chronic inflammation of the cavity wall. Criteria for the detection of CNPA are divided into three parts: clinical detection, radiological detection and laboratory detection. Clinical symptoms of CNPA are weight loss, productive cough, and hemoptysis. Radiological criteria include the symptoms like cavitary pulmonary lesions, paracavitary infiltrates, new cavity formation or expansion of cavity size. Laboratory criteria include increased levels of inflammatory markers. Laboratory criteria are supposed to be helpful for the early detection and therapy of aspergillosis.7 CNPA differential diagnosis is linked with dreadful lung diseases such as infectious actinomycosis, necrotizing pneumonias tuberculosis, and lung abscesses. Other noninvasive diseases associated with lung cavities are Wegener’s granulomatosis, sarcoidosis, and lymphomatoid granulomatosis. Lung diseases with primary tumors in the lungs are also related with differential diagnosis, including lymphoma and Kaposi’s sarcoma, Langerhans’ cell histiocytosis, and autoimmune diseases such as ankylosing spondylitis, rheumatoid arthritis, systemic lupus erythematosus, and primary amyloidosis.28
2.3. Tracheopulmonary aspergillosis
Tracheopulmonary aspergillosis is an unusual form of Aspergillus infection, that is confined entirely or predominantly to the bronchitis. Tracheopulmonary aspergillosis is characterized by thick mucus in bronchitis. Patients who are susceptible to tracheopulmonary aspergillosis are lung transplant recipients, patients with AIDS and generally patients with cancer.7 According to bronchoscopic appearance, tracheopulmonary aspergillosis is classified as pseudomembranous, ulcerative and obstructive. Pseudomembranous tracheopulmonary aspergillosis is the excessive invasion of tracheobronchial tree along with mucosa compromising Aspergillus species. It is the most dreadful as compared to other types. In case of obstructive tracheopulmonary aspergillosis, thick mucus and inflammation of tracheobronchial tree is observed. Ulcerative type is generally observed in the recipients of lung transplantation.7 Presence of Aspergillus colonization in the cavities or the airways in the bronchitis leads tracheopulmonary aspergilloma. It is widely spread in the immunocompromised patients, such as, in case of AIDS and those who have had lung transplantation. The most common symptoms of this aspergillosis are fever, cough, chest pain and hemoptysis.29
2.4. Invasive aspergillosis or Invasive pulmonary aspergillosis
Invasive aspergillosis or invasive pulmonary aspergillosis (IA or IPA) is an infection of the pulmonary, parenchyma, which is affected by the growing hyphae of Aspergillus. Furthermore, the term invasive aspergillosis can be modified as angioinvasive aspergillosis, if there is vascular invasion by the hyphae of aspergillosis. A. fumigatus is the most common aetiological agent of IPA. A. fumigatus is an omnipresent fungus and its conidia are airborne, so, its exposure is constant and universal to almost all human beings.30 IPA is one of the most widespread forms of invasive disease.16 A defect in the host defense mechanism is the main reason of invasive aspergillosis. The phagocytic function is greatly affected; hence, it is more in case of patients with a haematological patients like acute leukemia and in anaemic patients.7 It is a mycotic infection of immunosuppressed patients who have undertaken chemotherapy and in those hosts who have taken the doses of steroids for malignant or haemopoietic diseases. IPA mainly causes infection in the lungs, which is important reason for the mortality of patients associated hematological malignancy and in solid organ and stem cell transplant recipients,30 allogeneic bone marrow transplantation, and in HIV infected patients with its last stage, chronic granulomatosis.20,31 Patients, who have undergone bone marrow transplantation are susceptible to IPA and in those cases 90% of the patients die.32 Depending on the situation of the patients, it was found that 24% of patients with acute leukemia and 7% of patients with lymphoid malignancies are susceptible to IPA.33 Traditionally, it is reported that the patients who had undergone hematopoietic stem cell transplantation IPA was observed after 10–14 days.34 In the last few years, it has been reported that, IPA is increasing in the patients with chronic obstructive pulmonary disease (COPD).35,36 IPA has been observed in a patient with neutropenia, having neutrophil less than 500/μL or less.36
Various immunological problems are reported for immunocompromised patients who are associated with diabetes mellitus and with alcohol abuse or who may be patients with renal failure, such as neutrophil proliferation, maturation and its life span is affected very badly and these all are the characteristic features of patients with invasive aspergillosis.37 Various clinical and pathological forms of IPA occur, which include acute bronchopneumonia, angioinvasive aspergillosis, acute tracheobronchitis, and pleural aspergillosis.7 Clinical staging of IPA comprises of pleuritic chest pain, dry cough, fever, and dyspnea. Severe and pleuritic chest pain are the symbols of angioinvasive aspergillosis.29 The most frequently observed radiological imaging symbols of IPA include: diffuse pulmonary nodular infiltrates, cavitary invasions, and pleural effusions along with pleural wedge-shaped densities in the pulmonary nodules. One of the main signs of IPA is the low attenuation around the pulmonary nodule which gradually gives rise to cavities. Hitopathologically, a large number of fungal hyphae invasion and discrete nodular destruction are observed along the blood vessels.36
Diagnostically, as per Japanese guidelines, IPA is categorized into three types: “proven infection”, “clinically documented infection or probable infection” and “possible infection”. Proven infection is positive, when presence of Aspergillus hyphae infection is found mycologically and histopathologically. The detection of Aspergillus infection can be done using the sputum and bronchoalveolar lavage specimen. Probable or clinically documented infection is diagnosed when galactomannon antigen and β-d glucan is present in serum or gene is diagnosed by using polymerase chain reaction and such diagnosis is positive. Clinically documented infection is known as probable infection by the European Organization for Research and Treatment of Cancer (EORTC). Possible infection is positive when serum, genetic, and imaging finding is positive.7
3. Relationship of pulmonary aspergillosis with immunocompromised patients
Immunocompromised patients are prone to pulmonary aspergillosis. IPA is the most dreadful type of pulmonary aspergillosis, which results in high mortality and morbidity in immunocompromised patients.38 IPA is observed most frequently in immunocompromised patients with neutropenia, organ transplantation, hematopoieitc stem cell transplant, hematological malignancy, chemotherapy and corticosteroid therapy, chronic granulomatous disease (CGD), advanced stage of AIDS, acute leukemias and in anaemic patient, diabetes mellitus and with alcohol abuse with renal failure.37 From the previous report, it was found that the mortality risk was 50% in patients with neutropenia and 90% mortality risk in patients with hematopoietic stem cell transplants.37 When the neutrophil count is less than 500 cells/mm3, it results in neutopenia as the number of neutrophil is decreased the immunity is decreased, and it results in the susceptibility to IPA. As far as the risk of IPA in neutropenia is concerned, it is estimated to be 1% until the first 3 weeks and then it is 4% per day. Organ transplantation, especially the lungs, hematopoietic stem transplant is also risk issue for IPA.36 When allogeneic and autologous hematopoietic transplants are compared, it was found that allogeneic transplants have a higher risk for IPA, as compared to autologous transplants. For allogeneic transplants, the risk factor is 2.5–15%, whereas, for autologous transplants it is 0.5–4%.18 From the previous study, it was found that the risk factor is higher in the case of allogenic transplants and the percent of the risk factor in such patients increase with time. It is 5% at 2 months, near about 9% at 6 months, whereas 10% in a year and after 3 years of transplantation risk percentage rises to 11%.18 Reports have revealed that the patients with severe COPD are prone to IPA. Long-lasting use of sterocorticoids, changes in the structural architecture of the lungs, antibiotic doses and repeated hospitalization along with other associated diseases with COPD, such as diabetes mellitus, alcoholism. All these reasons are responsible for the increase of the susceptibility of COPD patients.37
Patients suffering from immunocompromised disease such as HIV, lung transplant recipient and those with nutropenia are prone to tracheobronchial pulmonary aspergillosis.3 ABPA is the most common type of pulmonary aspergillosis and is observed in patients suffering from cystic fibrosis and in asthma. About 1–2 % of asthma patients, 7–14% in steroid dependent patients, and 2–15 % cystic fibrosis patients are supposed to be susceptible to ABPA.17
4. Morphological and molecular identification of Aspergilli
Even though many novel methods are available for identification of different species of Aspergillus, morphological features are essential tools in the identification of Aspergilli.39 Morphological characteristics are subdivided into two sections: macro-morphological and micro-morphological characteristics. The micro-morphological features, which are used to distinguish Aspergillus species involve, the shape of the conidia head, vesicle shape and diameter, stipe length, width, texture and color, conidial size, shape, texture and color, seriation, size. Macromorphological characteristics depend on various features such as colony color, texture, production of soluble pigments by the fungi in the media, formation of sclerotia, cleisthethocia, and reverse color of the plate. Depending on the seriation genus Aspergillus is either uniseriate or biseriate. The morphological characteristics of Aspergillus vary with the use of growth media.40 Culture and microscopy are preliminary factors which are useful in the morphological studies. For the identification of the fungus color of the colonies is very significant. A section of the colony color can be used in the identification of Aspergillus species. Yellowish-green, green, and deep-green color colony sections were observed for sections of Flavi, Fumigati, and Nidulantes, respectively. A. niger showed a black or brownish shade.39,41
Morphological characteristics of Aspergillus in culture are very important for the identification and classification up to the genus level (Fig. 12.1). Microscopic characteristics for the identification is based on conidial heads, stipes, color and length vesicles shape and seriation, metula covering, conidia size, shape, and roughness, also colony features including diameter after 7 days, color of conidia, mycelia, exudates and reverse, colony texture, and shape. Morphological characters of the isolate are compared with morphological characters of the pure Aspergillus culture. After morphological classification of the isolates, pure cultures of Aspergillus isolates can be maintained on potato dextrose agar. After proper growth and sporulation cultures of Aspergillus can be examined using lactophenol cotton-blue mount for the sporulation after 10, 20, and 30 days. All the morphological and microscopic identification of Aspergillus species can be confirmed by comparing with the characters given in the Atlas of Clinical Fungi.42
Figure 12.1 Structure of Aspergillus
The molecular diagnosis technique is excellent because it facilitates easy, early, and correct identification of aspergillosis which can help in the antifungal treatment. Single nucleotide polymorphism multiplex (SNaPAfu) assay is a novel technique which involves identification, detection, and genotyping of clinical isolates of Aspergillus species. In this assay, only Single Nucleotide Polymorphism (SNP) gene present in Multilocus sequence typing MLST is amplified and targeted in a multiplex reaction and its analysis depends on the mini-sequence reaction. Detailed information of clinical sample can be obtained from the single amplification reaction. MLST alignment created by the multiplex reaction is required for the SNaPfu assay. SNaPfu assay requires less than 8 hours to get the results of clinical samples and the cost per sample of the assay is very low. The significant advantage of SNaPAfu assay is that, it can identify the genotype of A. fumigatus in a single step reaction.43
A real time PCR-based innovative commercialized technique known as MycAssay is developed for the detection of Aspergillus DNA in case of lower respiratory disease. This assay is generally performed in bronchoalveolar lavage (BAL) samples from patients without hematological cancer. MycAssay™ Aspergillus is a potential technique that requires less time for the diagnosis as compared to the conventional fungal culture. It provides much more accurate and additional information related to the fungal species responsible for the aspergillosis.44 A 2-PCT assay system was used to target 18 S ribosomal DNA of Aspergillus. In this assay, a tissue sample of the fungi was embedded in paraffin wax to detect ribosome of A. fumigatus. This technique involves the use of deparaffinized tissue section. Specific primers are used for the detection of aspergillosis. New molecular diagnostic techniques are potential and promising, which can improve the detection of fungal infection in critically ill patients.45 Random amplification of polymorphic DNA (RAPD-PCR) is a simple, rapid, and useful tool used for the detection of Aspergillus clinical isolates and can be useful in the identification of aspergillosis. This method is crucial for the detection of hospital isolates of Aspergillus.46
5. Clinical diagnosis of aspergillosis
5.1. Microscopy
For clinical diagnosis of aspergillosis, clinical sample like sputum is collected with precautionary measures. After the collection of sample, direct microscopy is performed. This is the simplest approach to diagnose the clinical samples. Gram staining of the sample should be performed. Usually, 10% KOH is used for wet mounting and the sample is examined under microscope. This method does not specifically differentiate Aspergillus species because the hyphae of many filamentous fungi seem to be similar and it is difficult to differentiate between them.30,47
5.2. Histopathology
This clinical identification is noticed in the case of IA. Histopathological study is performed in case of biopsy or autopsy sample of IA. This study is used to demonstrate the tissue invasion by filamentous fungi, but it is not possible to identify the mycelium of the fungus merely by observing it until the time the positive culture of fungus used is not available.47 A few clinical tests that can be used in the identification of Aspergillus species will be described in the following sections.
5.3. Galactomannam antigen test
The cell wall of Aspergillus spp. is composed of galactomannam (GM) antigens. This assay gives information about the antigen present in the clinical samples. Serum and plasma samples of patients are required for performing this assay for the clinical testing of invasive aspergillosis patients. The specificity of the results obtained by serum and plasma is up to 55–95% to 75–96%, for serum and plasma, respectively.
5.4. Beta-d-Glucan test
Beta-d-Glucan is one of the components of fungal cell walls. This assay identifies the presence of Aspergillus and Candida merely by the presence of beta-d-glucan in the patient’s serum. Antigen present on the surface of Aspergillus sp. can be determined by this assay.30,50
The FDA has approved another diagnosis assay for determining invasive fungal infection, which is known as “Fungitell”. The specificity of the results changes with the variation of clinical settings. This is one of the assays used for the clinical testing of Aspergillus sp.51
5.5. Chest radiography and computed tomography
Chest radiography is the basic and predominant method for the detection of aspergllosis in the early stages of disease. Generally, ABPA and IA affected patients are examined by using chest radiography. Rounded densities, accumulation of infiltration and cavitation can be investigated by chest radiography. By using chest radiography, tuberculosis like symptoms which are seen similar to aspergillosis can be differentiated on the basis of the chest radiograph. Although this is an accurate method of identification, at the chronic stage it cannot be differentiated perfectly. This gives better results in ABPA patient along with asthma. Computed tomography (CT) scan examination gives the basic information regarding the aspergillosis. It detects the early stage development of diseases. When a CT scan is performed on an IA patient, it shows a characteristic “halo” sign. The halo sign is a special type of chest imaging seen in IA affected patients. This is a feature characterized by invasion of hemorrhagic nodules, tumors, and other inflammatory processes. Most often the halo sign is observed in neutropenic individuals. A CT scan, when used in combination with high resolution images (HRCT), provide us with much clearer and better results. In general, plain chest radiography does not give a clear picture about the inflammation, but HRCT can. By using HRCT, a mucous plug may be noted very clearly in ABPA patients.47,52
5.6. Lateral flow device
This is an innovative technique which uses JF5, the monoclonal antibodies from mice. A lateral flow device (LFD) detects a glycoprotein and antigen present in the blood sample and BAL of IA patients. This device is a new and potent tool, which can be used in the clinical analysis of the IA patient. The main advantages of this technique are that it requires less time to perform and no expertise is required for handling it. The specificity of the result is much higher in LFD as compared to the other results. A recent study has shown that in the case of BAL, it has a specificity up to 100% and false results can be interpreted in case of Penicillium spp.48,49
5.7. Volatile organic compounds
Recently, it has been discovered that patients suffering from IA infection exhale a few organic compounds that are volatile in nature. Such volatile compounds can be detected when a patient infected with A. fumigatus breathes. These volatile compounds are called as volatile organic compounds (VOC).53 Individuals infected with IA produce 2-pentylfuran at the time of breathing and such compounds can be easily and rapidly detected. This technique shows accuracy until 81% in case of critically ill IA patients.54 This is supposed to be one of the most promising tools that can be used for early detection of Aspergillus spp. However, more study needs to be done on the concept of the volatile compounds released by infected patients.49
6. Treatment of aspergillosis
A number of antifungal drugs are available for the treatment of aspergillosis. Various antifungal compounds have been clinically active against Aspergillus spp. and are approved for treatment of pulmonary aspergillosis. The antifungal drugs include Amphotericin B and its lipid formulations, itraconazole, voriconazole, posaconazole, and caspofungin.3,55,56 Voriconazole and Amphotericin B are mostly preferred for primary treatment of IPA with a recommended dose 1–1.5 mg/kg per day.7,57 Voriconazole is a promising alternative to Amphotericin B 58 and is currently the most frequently recommended therapy for invasive aspergillosis specially.59 Posaconazole has been used for prophylaxis of IA in neutropenic patients with leukemia and myelodysplasia. Posaconazole is also recommended for treatment of IA that is refractory to an Amphotericin B formulation or to itraconazole. Micafungin and anidulafungin, members of the class of echinocandins have significant activity against aspergillosis. Antifungal drug treatment for pulmonary aspergillosis is summarized in Table 12.1.
Table 12.1
Antifungal Agents Used for the Treatment of Pulmonary Aspergillosis
| Disease | Antifungal Drug | References |
| Invasive pulmonary aspergillosis | Voriconazole Liposomal Amphotericin B Itraconazole Posaconazole | [3,60–63] |
| Aspergilloma | Itraconazole Voriconazole | [64,65] |
| Chronic necrotizing aspergillosis | Voriconazole Itraconazole Amphotericin B or Amphotericin lipid formulation Posaconazole | [17,56,66,67] |
| Allergic bronchopulmonary aspergillosis | Itraconazole Voriconazole Fluconazole | [68–70] |
| Tracheobronchial aspergillosis | Itraconazole Intravenous Amphotericin B Voriconazole | [71,72] |
6.1. Amphotericin B
Amphotericin B binds to ergosterol, which is a primary sterol content in the fungal cell membrane. It is released from the phospholipids, when it is in close proximity to ergosterol, allowing for delivery of enough amphotericin to the site of infection.29 Amphotericin B activity has been demonstrated against most of the Aspergillus species, which is a common causative agent of pulmonary aspergillosis. The side effects of use of Amphotericin B include fever, chills, rigors, myalgias, arthralgias, bronchospasm, nausea, and vomiting, have been observed in infusion-related reactions. The nephrotoxic side effects of Amphotericin B include azotemia, renal tubular acidosis, hypokalemia, and hypomagnesemia. The morbidity and mortality associated with the use of Amphotericin B have prompted the search for improved antifungal agents with activity against Aspergillus.73 Previous research has shown Amphotericin B used for the treatment of invasive aspergillosis given in doses of 5 mg/kg per day. It has been used for salvage therapy of IA at doses of 3–5 mg/kg per day, or 3 mg/kg per day.3,74 A recent study has found that the efficacy and toxicity of drug showed similar results in both, suggesting that in this specific population of patients with early pulmonary aspergillosis higher doses are not beneficial when compared with initial therapy.
6.2. Voriconazole
Voriconazole is attractive as an efficient antifungal agent and is considered to be the treatment of choice for IA and ABPA.75,76 It has high efficacy against CPA, and is available in oral and intravenous formulation.77 In vitro study has revealed that voriconazole is effective at lower dosages as compared with Amphotericin B against Aspergillus spp. In addition, voriconazole exhibits superior fungicidal activity against A. fumigatus, the leading cause of aspergillosis.77 Voriconazole penetrates into tissues and body fluids, including the lung tissue and the pulmonary epithelial lining fluid.78 Treatment of IA with voriconazole is initiated with a loading dose of 6 mg/kg IV every 12 h for two doses, followed by 4 mg/kg every 12 h. Oral voriconazole used 200 mg twice a day for CPA for a period of 6–12 months.79
6.3. Itraconazole
Itraconazole is lipophilic compound available in form of capsules, oral formulations. It is metabolized in liver and its metabolite forms are excreted into urine and biles. These excreted metabolites possess antifungal activity and effective against Aspergillus infections.80 Itraconazole is most preferably used for IA treatment specifically in standard antifungal therapy intolerant patients. It plays an adjective role in management of allergic bronchopulmonary aspergillosis.81 Recommended doses of itraconazole for IA are 400 mg/day in case of capsules and 2.5 mg/kg two times a day in case of oral formulations. Intravenous formulation of itraconazole dose of 200 mg/day is preferably recommended.82
6.4. Posaconazole
Posaconazole is given for treatment of IA to patients with aplastic anemia and CPA.83 Posaconazole was also approved for treatment of invasive aspergillosis that is resistance to Amphotericin B formulation and to itraconazole. It is administrated orally in dose of 200 mg, four times a day.3,84 Felton et al.67 reported the efficacy and safety of posaconazole for the treatment of choronic pulmonary aspergillosis and described response rates of 61% at 6 months and 46% at 12 months, with a relatively low incidence of side effects.
7. Multidrug resistance
Drug resistance to azoles is most common in pulmonary aspergillosis cases as compared with other antifungal drugs used for treatment. Azole resistance in patients has been developed, either during treatment or due to resistant spores present in environment. Although there are many antifungal agents available for the treatment of pulmonary aspergillosis, the patients still developed resistance. In order to develop new antifungal treatment we need to understand how antifungal drug resistance occurs. Antifungal drug resistance is detected in fungal cell, when it remains unaffected by antifungal drug even at curative concentration. The antifungal drug resistance might be due to alteration in target site of drug, biofilm formation, and increased efflux of drug and may be due to mutation that lowers the toxic effect of drug. Patients suffering from IPA, aspergilloma, chronic necrotizing aspergillosis, ABPA, tracheobronchial aspergillosis acquired drug resistance because of long-term exposure to the antifungal drugs.
Infections caused by Aspergillus spp. are responsible for a high rate of morbidity and mortality in patients at risk. Mostly azoles are used for the treatment and prevention of Aspergillus infections. Increasing study on pulmonary aspergillosis showed that Aspergillus has developed resistance against antifungal agents specially azoles.85 In A. fumigatus, azole resistance occurs due to mutation of genes leads to alteration in target protein.86
Resistance to triazole azole antifungal drugs in A. fumigatus is now a major clinical problem in a number of European locations, in China, Canada, and the US, with particularly high frequencies from the North-West of the UK, and the Netherlands.87 The treatment failure and continuous increase in occurrence of azole-resistant A. fumigatus which is a causative agent of pulmonary aspergillosis has been reported through many studies.21,88–92
The clinical study of van der Linden et al.93 showed that A. fumigatus is highly resistant to voriconazole. They screened clinically isolated A. fumigatus species for azole resistance in different patient types in the Netherlands. The results reveal that voriconazole followed by itraconazole and posaconazole showed resistance in patients who receive azole as primary treatment for invasive aspergillosis. Now, Itraconazole resistance is also increasing in India, Canada, China, United States, and so on. Patients treated with itraconazole also showed cross resistance to voriconazole and posaconazole.94 The survey on development of azole resistance in Aspergillus reveals severity of the resistance particularly in patients residing in Japan 11%, China 4%, United States 3.6% and India 2%.
Chowdhary et al.95 studied the widespread of resistance in clinical Aspergillus isolates in a referral chest hospital in Delhi, India during 4 years. About 1.75% of clinical isolates of Aspergillus were found resistant to azoles. One year nationwide survey carried out by Vermeulen et al.96 to study Aspergillus resistance among patients suffering from aspergillosis in Belgium. The epidemiological data was evaluated up to one year after incorporation of drug in patients. Azole resistance was observed in 7% of patients suffering from aspergilloma or chronic aspergillosis, 5.5% in patients suffering from ABPA, and 4.6% in patients with IA. A study carried out by Choukri et al.97 in France reported emerging problem of azole resistance in A. fumigatus. Their study observed azole resistance among the tested isolates of A. fumigatus was 1.8% among total patients enrolled.
Voriconazole is still primary treatment of choice for pulmonary aspergillosis. The high level of resistance was observed in patients treated with azoles, particularly itraconazole, and in some patients cross resistance was observed between itraconazole and posaconazole.98
8. Conclusions
Aspegillus-related infections are increasing day by day, causing many diseases including classic aspergillosis, infections of the auditory canal, skin, nails, eyes, sinuses, meninges and bones, etc. It is the most common fungus causing allergic sinusitis. Among these, pulmonary aspergillosis is predominant. ABPA has been reported in people who are allergic to the Aspergillus. In IPA, pulmonary parenchyma is affected by Aspergillus sp. It has been reported that pulmonary aspergillosis is common in immunocompromised hosts. Considering this fact, it is imperative to identify the pathogens by morphological and molecular markers in order to diagnose the authentic pathogen responsible for the diseases. In addition, clinical identification of pulmonary aspergillosis is also important, which is commonly based on histopathology, galactomannam antigen test, Beta-d-Glucan test, chest radiography, CT, volatile compound and LFD. Many drugs are available for the treatment of aspergillosis including Amphotericin B and its lipid formulations, itraconazole, voriconazole, posaconazole, and caspofungin. Unfortunately, the multidrug resistance problem has been increasing rapidly; for example, azole resistance is common in pulmonary aspergillosis. In fact, there is a greater need to study the reasons and mechanism of drug resistance in pulmonary aspergillosis. It has been found that A. fumigatus is highly resistant to voriconazole. The drug resistance in fungi, particularly in Aspergilli, is a matter of great concern and warrants thorough investigation to understand the real mechanism involved in drug resistance. The emergence of nanotechnology has promise to tackle this problem by using metal nanoparticles in combination with antifungal agents like azoles.
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