Fungal infections have recently emerged as a world-wide health care problem, owing to extensive use of broad spectrum antibiotics, immunosuppressive agents and increasing population of terminally ill and debilitated patients [1]. Aspergillus infection is the commonest invasive fungal infection which involves respiratory tract. It can be ascribed to pervasive presence of the fungal spores in soil, water, decaying vegetation, indoor environment, including hospitals [2,3]. Aspergillus was first detected in 1729 and it received its name due to its resemblance to an aspergillum which was used to sprinkle holy water [4]. Man acquires the infection through inhalation of fungal spores, though infection has also been documented following exposure to water aerosols contaminated with Aspergillus spores and Aspergillus fumigatus, in particular, is the most important air borne saprophytic fungus [5]. Inhalation being the commonest mode of infection, lung is the most common site of involvement and pneumonia is the most important nosocomial infection caused by Aspergillus [6]. Depending on the host immunity, lung structure and degree of inoculums, the spectrum of pulmonary disease ranges from a noninvasive disease (colonization or aspergilloma), or an allergic bronchopulmonary aspergillosis (ABPA), to a semi-invasive (chronic necrotizing pneumonia) or an invasive infection (invasive pulmonary aspergillosis) [7]. Dissemination of infection from lungs can lead to systemic aspergillosis, that can be a major cause of morbidity and mortality in immunosuppressed persons [6].
Pulmonary aspergillosis is seen mostly in immunosuppressed individuals. The central cause is the impairment of phagocytic system and hence, patients with haematological disorders are particularly prone to develop it. Diabetes mellitus, alcohol abuse, renal and hepatic failure, etc. which have an effect on neutrophil proliferation, maturation, function and lifespan, are the important predisposing factors [4]. The incidence of Aspergillus infection is showing a rising trend, following solid organ or bone marrow transplantation, chemotherapeutic modalities like prolonged use of corticosteroids or other immunomodulating drugs and epidemic occurrence of human immunodeficiency virus (HIV) [8]. Aspergillus fumigatus is the commonest species causing infection, though other species like Aspergillus flavus, Aspergillus niger and Aspergillus terreus can also cause the disease less frequently [7].
In India, only few studies are available, which show the comprehensive profile of cases of pulmonary aspergillosis [1,6,9]. Herein, we are presenting the detailed profiles of all the pulmonary aspergillosis cases diagnosed in our institution from 2008–2010. We also studied the prevalence of various species of Aspergillus and the predisposing factors which cause the disease.
Material and Methods
The present observational and retrospective hospital based study included all cases of pulmonary aspergillosis which were microbiologically proven in time period of three years, from Jan, 2008–Dec, 2010 at a tertiary care centre in southern India.
All culture positive patients and those fulfilling at least two of following three clinical criteria were included in the study: 1) Predisposing illnesses like tuberculosis, chronic obstructive pulmonary disease (COPD), diabetes mellitus, etc., 2) Long term steroid therapy, 3) Radiological features suggestive of Aspergillosis.
To rule out the chances of contamination, samples that showed repeated isolation of Aspergillus (minimum 2 sputum samples or 1 sputum + 1 bronchoalveolar lavage [BAL]) were included in the study. Cases were classified according to revised definitions given by EORTC/MSG consensus group [10], as follows:
Proven Invasive Aspergillosis: Presence of host factors + Clinical criteria + Positive culture + Detection of Aspergillus antigen
Probable Invasive Aspergillosis: Presence of host factor + clinical criteria + Positive culture
Possible Invasive Aspergillosis: Presence of host factor + clinical criterion but mycological criterion absent [10].
Processing of Samples
Gram staining was done for all respiratory samples like sputum, BAL or endotracheal aspirates (ETA) and the slides were microscopically checked for the presence of gram positive septate, acutely branched fungal hyphae, which were suggestive of Aspergillus. Though gram staining is not a classical staining method for identification of filamentous fungi, it was done here as a part of routine screening of respiratory specimens.
All these samples were cultured on Sabouraud’s dextrose agar (SDA) along with culturing on routine Mc Conkey’s, sheep blood and chocolate agar and these media were incubated aerobically at 37°C for 24-48 hours. Isolated colonies, suggestive of Aspergillus were speciated following standard microbiological procedures [11].
For clinico-microbiological correlation, demographic details, clinical findings and predisposing factors were noted down for all the patients. Treatment of patients with antifungal agents and their responses to treatment were also documented.
Results
There were 22 patients (M:F = 1.2:1) with proven diagnosis of bronchopulmonary aspergillosis during the study period, within the age 19–81 years (mean = 52.5 years) [Table/Fig-1]. Fever and respiratory symptoms like cough and expectoration were present in 50% and 72.7% cases of proven bronchopulmonary aspergillosis respectively. The main predisposing factors identified in our study were chronic lung disease, including COPD and bronchial asthma (50%), smoking (13.6%), diabetes mellitus and tuberculosis (each 9.1%). Some patients, however, had no apparent predisposing factor. Three (13.6%) patients required ventilator care. Chest radiography revealed consolidation/opacity in 50% of the patients, followed by findings suggestive of adult respiratory distress syndrome in 22.7% of patients. Chest X-ray was normal in 18.2% of patients with bronchopulmonary aspergillosis. Aspergillus fumigatus was the most common species isolated, accounting for 40.9% of cases, followed by A. flavus, A. niger, A. tereus and A. versicolor in decreasing order. Mortality was seen in 6/22 cases (27.3%). Comparison of present study with few studies done in recent past has been shown in [Table/Fig-2]. All our cases belonged to the category of probable invasive aspergillosis as per standard definitions.
Clinical, Demographic and Microbiological profile of pulmonary aspergillosis cases
| Parameter | Number (%) |
|---|
| Age in years | |
| 20-40 | 7 (31.8) |
| 41-60 | 6 (27.3) |
| 61-80 | 9 (40.9) |
| Clinical findings | |
| Fever | 11(50) |
| Cough/Expectoration | 16(72.7) |
| Wt. loss | 2(9.1) |
| Dyspnea | 10(45.4) |
| Predisposing factors | |
| COPD | 5(22.7) |
| Bronchial asthma | 6(27.3) |
| Steroid RX | 9(40.9) |
| Post TB | 2(9.1) |
| DM | 2(9.1) |
| Chronic smoking | 3(13.6) |
| Others | 4(18.2) |
| Chest X-ray findings | |
| Consolidation/opacity | 11(50) |
| Cavity | 1(4.5) |
| Pleural effusion | 1(4.5) |
| ARDS pattern | 5(22.7) |
| Normal | 4(18.2) |
| Outcome | |
| Improved | 16(72.7) |
| Expired | 6(27.3) |
| Culture findings | |
| A.fumigatus | 9(40.9) |
| A.flavus | 6(27.3) |
| A.niger | 2(9.1) |
| A.versicolor | 1(4.5) |
| A.terreus | 1(4.5) |
| A.species | 3(13.6) |
Comparison of Present study with Recent Case series of Broncho-Pulmonary Aspergillosis from different parts of world
| Features | Kurhade et al., [6] | Shahid M et al., [9] | David M Denning et al., [18] | PR Gupta et al., [19] | Hae-Seong Nam et al., [20] | Present study |
|---|
| Region | Nagpur, North India | Aligarh, Uttar Pradesh | Manchester, United Kingdom | Jaipur, India | Seoul, Korea | Southern India |
| No. of Patients Studied | 20 | 13 | 18 | 98 | 43 | 22 |
| Patient group studied | Chronic pulmonary infections | Chronic lung disease | Chronic pulmonary aspergillosis | Pulmonary Aspergilloma | Chronic necrotizing pulmonary aspergillosis | Chronic pulmonary infections |
| Age group of the patients | Not mentioned | 45 – 60 years | 20 – 77 years; median age – 59 yrs | Mean age – 38.0±5.8 years | Median age – 60 years | 19-81 years; mean age 52.5 years |
| Gender | Not mentioned | Predominantly Males | Predominantly Males | Predominantly Males | Predominantly Males | Predominantly Males |
| Duration of illness | Not mentioned | Prolonged – more than one year | Several months to > 12 years | ≥2 years | Not mentioned | Prolonged – usually more than 2 weeks to > one year |
| History of chronic lung disease and use of broad spectrum antibiotics and/or steroids | Seen in 13 (65%) cases | All the cases | Seen in 12 cases (66.66%) | All cases | Seen in all cases | Present in 35% of cases |
| Commonest species isolated | Aspergillus fumigatus | Aspergillus fumigatus | Aspergillus fumigatus | Aspergillus fumigatus | Aspergillus fumigatus | Aspergillus fumigatus |
| Sample for isolation | Sputum only | BAL only | Sputum Predominantly | Sputum | Respiratory specimens | Predominately Sputum followed by BAL and ET Aspirate |
| Predominant Radiographic abnormality detected | Not mentioned | Not mentioned | Left upper lobe cavity | Cavity and ball-like leisons | Cavities | ARDS like picture in 26.66% |
| Prognosis | Not mentioned | Not mentioned | Improved (71%) | Not studied | Clinical Improvement in 58% | Improved (72.22%) |
Discussion
The presence of underlying chronic lung disease (bronchial asthma, bronchiectasis or COPD) and prolonged use of broad spectrum antibiotics and steroids has been documented in association with Aspergillus infection, as the infection is attributed to alteration of pulmonary architecture and/or local immunity and is implicated in the causation of semi-invasive or chronic necrotizing disease [4,6]. Moreover, prolonged use of broad spectrum antibiotics in chronic lung disease patients may stimulate the growth and virulence of infecting fungi by destruction of competing bacterial flora [6]. Smith NL and Denning DW, in their study, have documented the presence of tuberculosis, followed by ABPA and COPD in cases of chronic pulmonary aspergillosis [12].
Clinical diagnosis of aspergillosis is often difficult to make, as clinical signs and symptoms are not specific and as they are consistent with bronchopneumonia – fever unresponsive to antibiotics, cough, sputum production and dyspnoea [4]. A majority of our patients (80%) presented with variable symptoms mimicking bacterial pneumonia, making clinical diagnosis difficult. A high index of suspicion is crucial in identification of these cases, more so, in those patients who show an atypical presentation. Presence of haemoptysis and/or pleuritic chest pain may indicate angio-invasion, which was not found in our patients [7].
Radiological findings are also of little diagnostic help, especially in early stages of disease, because of the presence of non-specific changes. Usual findings include rounded opacities, pleural-bases infiltrates and cavitations. Typical ‘halo sign’ and ‘crescent sign’ which can be seen in invasive disease are less specific and they can be found in other fungal and bacterial infections [2,7]. However, chest radiography done in our patients revealed consolidation/opacity in 50% cases, followed by ARDS in 22.7% cases and cavity and pleural effusions in 4.5% each. No radiological abnormality was observed in 18.2% of them, which could be due to minute changes in early stage of the disease, which may not have been picked up by X-rays and this limits the role of radiography in diagnosis of pulmonary aspergillosis [13]. Radiology, however, has a role in assisting the diagnosis, especially in high risk patients [7]. However, chest CT scan, especially when combined with high resolution images (HRCT), helps in early diagnosis and improved prognosis of patients [2].
Histopathological examination of lung tissue being an invasive procedure, though it is confirmatory for diagnosis, cannot be done in all cases. Histopathological confirmations were also lacking in present study. Recently, detection of Aspergillus antigens in body fluids is being considered as another mode of diagnosis of aspergillosis. Nucleic acid detection of Aspergillus can also help in early diagnosis, but it has a disadvantage, that it cannot differentiate between colonization and infection [8,14,15]. PCR detection is also limited to few laboratories and it cannot be considered as a routine diagnostic test.
Diagnosis of pulmonary aspergillosis requires repeated isolation of fungus from the airways with appropriate clinical and/or radiological features and/or a positive tissue diagnosis [4]. We isolated Aspergillus from at least 2 sputum samples in all these patients. In three patients, where BAL fluid could possibly be obtained, we substantiated our diagnosis by isolation of Aspergillus from BAL fluid. Significance of isolation of Aspergillus from sputum samples depends upon the immune status of the hosts. In immunocompetent persons, isolation is always considered as colonization and it needs no treatment [6]. On other hand, in immunodeficient hosts, isolation of Aspergillus is predictive of invasive disease and it demands initiation of antifungal treatment [1,6]. All the cases included in present study were immunocompromised in one form or other.
Aspergillus can be identified in the direct specimens microscopically on detecting the presence of hyaline, regular, slender, septate hyphae with dichotomous branching (~45°) [4]. In our study, microscopy helped us to obtain positive results in all the cases, which were later confirmed by culture. Based on our study, we emphasize the importance of microscopy in making an early provisional diagnosis of bronchopulmonary aspergillosis, which can be later confirmed by culture and other diagnostic modalities.
In the present study, A. fumigatus was responsible for almost half of proven cases of pulmonary aspergillosis. Similar observation was seen in studies done by Kurhade et al [6] and Shahid et al [9]. A. fumigatus is responsible for a majority of the invasive diseases, followed by A. flavus, A. tereus and A. niger [5,7]. One interesting feature associated with A. niger infection is formation of calcium oxalate crystals, which can be demonstrated in tissue or fluid. Presence of such crystals, though not demonstrable in our study, is usually known to indicate fungal infection [4].
According to the standard definitions, none of our cases were identified as ‘proven’ invasive pulmonary aspergillosis. Earlier studies have also reported an alarming rate of 64% patients with proven invasive aspergillosis at autopsy, who did not meet these standard criteria before death [16]. Use of these standard defin-itions for making clinical decisions for individual patients may carry a risk of undertreatment. Hence, failure in meeting these criteria clinically, which is a major limitation, doesn’t prove absence of invasive disease. It may be due to lack of sufficient evidence in support of invasiveness [10].
Treatment of pulmonary aspergillosis depends on the type and stage of disease. Depending on the involvement of lung and spread of disease, antifungal treatment or surgery can be considered [2]. All patients diagnosed with pulmonary aspergillosis in our study received specific antifungal therapy (either oral Itraconazole or intravenous Amphotericin B). A majority of our patients (72.7%) responded favourably, but remaining 27.3% of cases showed no improvement and they expired. In a study done by Jhun et al., 99% cases of chronic pulmonary aspergillosis were treated with itraconazole and mortality was reported in 14% cases [17]. Death could be attributed to underlying co-morbid conditions or advanced stage of disease. Underlying co-morbidities in our study included diabetes mellitus, chronic obstructive lung disease, tuberculosis and patients receiving steroids and broad spectrum antibiotics for longer durations.
To conclude, pulmonary aspergillosis is a devastating disease with varied clinical presentations and nonspecific radiological findings. A high index of suspicion is necessary, especially in those with chronic lung disease and in those who are on prolonged steroid or broad spectrum antibiotic therapy. Microscopy is an important technique which can be used for making an early provisional diagnosis, which can later be substantiated with other diagnostic modalities.