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 ~  Abstract
 ~  Material and Methods
 ~  Results
 ~  Discussion
 ~  References

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ORIGINAL ARTICLE
Year : 2001  |  Volume : 19  |  Issue : 4  |  Page : 201-205
 

Prevalence of aspergillosis in chronic lung diseases


Department of Microbiology, JN Medical College, AMU Aligarh - 202 002, U.P, India

Correspondence Address:
Department of Microbiology, JN Medical College, AMU Aligarh - 202 002, U.P, India

 ~ Abstract 

Eighty eight patients of chronic lung diseases (CLD) attending TB and Chest department of J.N. Medical college Hospital were studied to find out the prevalence of Aspergillus in Broncho-alveolar Lavage (BAL) and anti- aspergillus antibodies in their sera. Direct microscopy and fungal culture of BAL was done. Antibodies were studied by immunodiffusion (ID) and Enzyme linked immunosorbent assay (ELISA). Dot blot assay for anti-aspergillus antibodies was also performed in sera of patients which were either positive by ID or by ELISA. Aspergillus was isolated in culture from 13(14.7%) cases of CLD, while, 30.6% cases showed anti-aspergillus antibodies by serological methods. Aspergillus fumigatus was the predominant species isolated. 17(19.3%) cases of CLD showed antibody against Aspergillus by ID, 22(25%) by ELISA, while 19 of 27 seropositive cases also showed positive results by Dot Blot assay. In cases of bronchogenic carcinoma and pulmonary tuberculosis, anti-aspergillus antibodies were detected equally by ID and ELISA in 21.42% and 21.05% cases respectively. In bronchial asthma, the antibodies could be detected in 60% cases by ELISA, while, in only 10% cases by ID. ELISA was found more sensitive than ID for detection of anti-aspergillus antibodies. The sensitivity of Dot Blot lies some what between ID and ELISA. It is concluded that prevalence of Aspergillosis is quite high in chronic lung diseases, culture and serological test should be performed in conjunction and more than one type of serological tests should be performed to establish the diagnosis.

How to cite this article:
Shahid M, Malik A, Bhargava R. Prevalence of aspergillosis in chronic lung diseases. Indian J Med Microbiol 2001;19:201-5


How to cite this URL:
Shahid M, Malik A, Bhargava R. Prevalence of aspergillosis in chronic lung diseases. Indian J Med Microbiol [serial online] 2001 [cited 2019 Mar 26];19:201-5. Available from: http://www.ijmm.org/text.asp?2001/19/4/201/8190


In the last two decades fungal infections have become important cause of respiratory tract infections.[1] The increase in frequency is mainly due to intensive cytotoxic therapy, greater use of broad spectrum antibiotics, corticosteroids and immunosuppresants.[2] Fungal infections of the lung, especially, pulmonary aspergillosis is being encountered more frequently and pose a difficult diagnostic challenge due to lack of pathognomonic clinical features and characteristic roentgenographic findings.[3] Pre-existing lung diseases acts as an important predisposing factor for pulmonary aspergillosis.[4],[5] The diagnosis of pulmonary aspergillosis is usually missed as the tests for their detection cannot be undertaken in routine diagnostic laboratories.
Since fungal infections are not notifiable diseases, there is paucity of data regarding world wide incidence and prevalence. In fact, the situation may be linked to what Ajello[6] described as the tip of the “Medical Mycological Iceberg”. There is a high prevalence of chronic lung diseases in our country and the exact incidence of pulmonary aspergillosis is not known. Therefore, the present study was designed with the following aims and objectives (a) isolation and characterisation of Aspergillus species from BAL of patients suffering from chronic lung disease (b) study of anti-aspergillus antibodies in the sera of these patients and (c) correlation of culture positivity and Aspergillus antibodies for the diagnosis of aspergillosis.

 ~ Material and Methods Top

The present study was conducted on patients of various chronic lung diseases attending the out patient department or admitted in the wards of the department of tuberculosis and chest disease of J.N. Medical College Hospital, Aligarh, during the period of one year. The cases selected for the study were patients of various lung diseases of more than one year duration on whom bronchoscopy was performed. The patients on cytotoxic drugs, radiation therapy or with other conditions known to cause immunosuppression were excluded from the study. 88 patients were included in the study group. None of the selected patients had received any anti-fungal therapy prior to the study. 12 healthy, age and sex matched individuals, not having history suggestive of any lung disease in the last one year were included as control group.
Bronchoscopy was performed and BAL was collected in a sterile vial. Three consecutive BAL specimens were collected. Blood was collected in EDTA and plain vial for routine haematological investigations and fungal serology respectively. Sera were separated and stored at -200C till tested.
BAL was homogenised and subjected to direct microscopy by using 10% KOH mount and Lacto Phenol Cotton Blue (LPCB) mount. It was streaked on two sets of Sabouraud's Dextrose Agar (SDA) plain and SDA with Chloramphenicol (0.05 mg/mL) and also on Czapek Dox agar. One set was incubated at 250C and the other at 370C. Tease Mount Preparation (TMP) of the fungal growth was made in LPCB for identification of morphology of sporing fungal heads. Micro-slide culture was performed for confirmation of fungal species[7] and all the fungal isolates were characterised according to Thom & Raper.[8]
Serological techniques
Preparation of antigens: Antigens of A. fumigatus, A. flavus and A. niger were made separately. The fungi were grown for 3 weeks in glucose asparagine broth. The mycelial mat was separated and transferred into 95% alcohol and kept for 48 hrs. It was squeezed gently between two layers of filter paper and dried in a dessicator with anhydrous calcium chloride. The dried fungal pellicles were finely pulverised and passed through sieve of 150 mesh /sq. cm. to get fine powder. This powder was defatted by shaking in the solvent acetone (3 to 4 vol. of material). The active allergenic substances from the defatted powder were extracted with highly alkaline buffer pH 8. The soluble active ingredients were filtered and dialysed against normal saline for 48 hrs. Standardisation was done by weight by volume method (1:50) i.e. 1 gram of substance extracted with 50 mL of extracting fluid (buffered saline pH 8.0) and finally compared with a commercially available antigen from Sanofi Pasteur Diagnostics, France.
Immunodiffusion: ID was carried out according to modified Ouchterlony technique.[9]
ELISA: Indirect ELISA was performed by methods described by Kauffman et al[9] with some modifications.
Dot Blot: The method for Dot Blot was developed in our own laboratory. Strips were cut from Immobilon - Polyvinylidene difluoride (PVDF) membrane, 2 µL of antigen having a concentration of 2 mg/mL were coated as drops and strips were kept at 600C for 30 min. Strips were washed with TBS and unoccupied sites were blocked by 1% bovine serum albumin for 2 hrs. Strips were washed again, a drop of 1:100 dilution individual sera were added over the antigen drop and strips were incubated at 370C for 30 min. Secondary HRP conjugate diluted in TBS was added after washing and allowed to stay for 1 hr. at room temperature. Strips were washed and HRP colour substrate system (50 mM Tris-HCl, pH 8.0, 0.48 mM 4-chloro 1 naphthol, 200 mM NaCl & 17% methanol) was added. The reaction was started with the addition of Hydrogen peroxide to the conc. of 0.01% v/v. The colour was allowed to develop for 5 minutes. The substrate was removed and the membrane was dried.

 ~ Results Top

Among the 88 patients studied, 47.7% were suffering from bronchogenic carcinoma [Table - 1] which constituted the major study group followed by pulmonary tuberculosis (22.7%) and bronchial asthma (11.3%) respectively. The mean age of the patients was 49.94 + 14.4yrs and the Aspergillus culture positivity was maximum (61.5%) in the age group 45-60 yrs. Male to female ratio was 5:1. Maximum number of patients (93.1%) presented with the history of cough followed by dyspnoea (47.72%) and febrile illness (29.54%), while only 5.68% gave the history of cough with expectoration of bronchial plugs in their sputum [Figure - 1].
Fungal culture: BAL culture of 13 (14.7%) patients showed growth of Aspergillus species at least in 3 consecutive specimens. Of these 13 cases microscopic examination revealed fungal elements in 7 cases. Presence of fungal heads was noted in 5 cases (3 of Bronchial asthma + 2 of pulmonary T.B.) while, fragmented fungal hyphae were found in 2 additional cases (one of pulmonary T.B. and other of Bronchogenic carcinoma) in the direct microscopic examination of BAL. Aspergillus fumigatus was the predominant species isolated from 9 (69.23%) cases followed by Aspergillus flavus which was isolated in 5 (38.46%) cases (one case showed combined growth of A. fumigatus and A. flavus). Taking into consideration various lung diseases, the culture positivity was found in 6(14.3%) cases of bronchogenic carcinoma, 3(15%) cases of pulmonary tuberculosis and in 3(30%) cases of bronchial asthma [Table - 1]. None among the control group showed fungal growth in the culture.
Serodiagnosis: Serodiagnosis was performed by ID and ELISA. Out of 88 patients, 27 were found positive either by ID or ELISA or by both. Dot Blot was performed only in these 27 cases. One person in healthy control group was found weakly positive for Aspergillus antibodies. 17 (19.3%) cases showed precipitating antibodies against aspergillus antigen in ID, 22 (25%) cases showed anti-aspergillus antibodies in ELISA and out of 27 seropositive cases, 19 showed anti-aspergillus antibodies by Dot Blot technique.
Among the 27 seropositive cases it was noted that 10 could not be detected by ID while they were positive by ELISA. Similarly, 5 cases were found negative by ELISA while they were detected by ID [Figure:2]. Correlation between the two was highly significant (x2 = 23.34, P<0.001). All the cases which could be detected by Dot blot technique were also positive by ELISA.
Correlation between culture positivity and serological tests: Out of 13 culture positive cases, 9 cases showed antibodies in both ID & ELISA while 2 showed antibodies in ID and the other 2 in ELISA. Among the remaining 14 cases which were culture negative but seropositive for Aspergillus, 3 cases showed precipitating antibodies in ID, 8 cases showed antibodies by ELISA, while, 3 cases showed antibodies in both the tests [Figure:2]. The difference between culture positivity and presence of Aspergillus antibodies was statistically significant (P<0.001).
Out of 42 patients of bronchogenic carcinoma, 6(14.28%) were culture positive, 9 (21.42%) showed precipitin bands in ID and also positive by ELISA. 3 of 20 patients (15%) of pulmonary tuberculosis reported positive on culture, 5(25%) showed antibody against Aspergillus by ID and also by ELISA. However, in chronic bronchial asthma, of the 10 patients, 3 (30%) were Aspergillus culture positive, 6 (60%) were positive by ELISA while only 1(10%) showed presence of precipitating antibodies by ID [Table - 1].

 ~ Discussion Top

Methods of definitive diagnosis of pulmonary aspergillosis depend upon isolation of Aspergillus in culture, demonstration of specific immunological changes in the patient's serum, or histopathological examination of the biopsy material,10 but, the isolation of Aspergillus in culture is not always achievable because of faulty collection and processing of the specimen or because of non communication of the cavity with main bronchus, while histopathological examination is very tedious and cumbersome. However, serodiagnostic tests are helpful especially when direct demonstration of Aspergillus is not achievable in the culture. In the present study the mean age for aspergillosis was found to be 46.3 + 12.4 yrs. and male to female ratio was 5:1, however, it was not found statistically significant (P>0.05). Orie et al 11 also reported almost similar results.
In our study, the prevalence of aspergillosis in BAL was 14.7%. Our findings regarding the prevalence was similar to Henderson et al[12] who reported an incidence of 11% while it was slightly higher from Pepy et al[13] and Campbell & Clayton.[14] The isolation in their studies was 8% and 8.2% respectively. Aspergillus fumigatus was the predominant species isolated from BAL in this study similar to the findings of Bordane et al.[15]
Serodiagnosis of aspergillosis by detection of antibodies against Aspergillus antigen has been extensively used, most commonly by precipitin methods.[16] However, problem with inter-laboratory reproducibility, low sensitivity in immunocompromised patients and low specificity have encouraged the development of alternative approach like ELISA for serodiagnosis.[17] The interpretation of values for specific antibody in ELISA can be given as positive or negative.[18]
Among the cases of bronchogenic carcinoma, 14.2% were culture positive while 21.4% showed anti-aspergillus antibody by ID as well as by ELISA. Although no comparative study regarding the serological evaluation in bronchogenic carcinoma is available but it was evident from our study that in bronchogenic carcinoma, the detection of anti-aspergillus antibodies by ID and ELISA showed similar sensitivity. Similar findings were seen in pulmonary tuberculosis, but, in bronchial asthma only 10% cases showed the presence of antibodies by ID while in 60% cases by ELISA which signifies the presence of reaginic antibodies in asthmatic patients.
In our study, screening of CLD patients for Aspergillus antibodies showed that ELISA was approximately 50% more sensitive than ID for the diagnosis of pulmonary aspergillosis. Similar results were shown by Marx et al.[19] The discrepant observation between ID and ELISA can be explained by (i) that by ELISA, antibodies can be detected against antigenic components that do not precipitate in ID but are immobilised on polystyrene surface (ii) not every precipitating antigenic component binds to polystyrene surface and (iii) It may also be possible that a group of precipitating antigenic components are present in the extract in a concentration too low to cause a detectable precipitate in ID but predominantly bind to polystyrene surface.[9],[19]
On correlating culture and serological methods, it was noted that Aspergillus was isolated in only 48.1% of cases which were also positive for aspergillus antibodies. Similar results were also reported by Richardson[10] who showed that BAL culture positivity was found in less than 60% of cases. It was noted in the present study that serological tests especially ELISA was useful in culture negative cases. It is concluded from the present study that prevalence of Aspergillosis in patients of chronic lung disease is quite high in our country, therefore, every patient of a chronic lung disease not responding to regular therapy should be searched for Aspergillosis. Culture along with serology should be performed as, performing single diagnostic test, either culture or serology, is inadequate for the diagnosis. It is also recommended that more than one type of serological test should be performed to establish the correct diagnosis of Aspergillosis. In our study we found the sensitivity of Dot Blot technique (70.3%) to be greater than ID (54.5%), however, it was slightly lower than ELISA which may be due to subjective error. Therefore it is suggested that peripheral laboratories, which do not have the facilities to perform ELISA, can opt for Dot Blot technique along with ID. 

 ~ References Top

1.Randhava HS. Mycology today and tomorrow. In: Proceedings of National symposium on "Recent advances in Resp. Mycosis" (Department of Microbiology V.P. Chest Institute New Delhi) 1999.1.  Back to cited text no. 1    
2.Kusne S, Torre CJ, Manez R et al: Factors associated with invasive lung aspergillosis and the significance of positive Aspergillus culture after liver transplantation. J Infect Dis. 1992;166: 1379-1383.  Back to cited text no. 2    
3.Randhava HS, Khan ZU. Acute pulmonary mycosis in India: current status and laboratory diagnostic aspects. Ind J Tub 1987; 34:3.  Back to cited text no. 3    
4.Kampmeier RH, Block HA. Pulmonary aspergillosis in association with bronchial carcinoma. Amer Rev Tuberc 1934; xxxv: 315.  Back to cited text no. 4    
5.Villar TG, Cortez Pimental J, Freitas M, Costa E. The tumour like forms of Aspergillosis of the lung (Pulmonary Aspergilloma). Thorax 1962; 17: 22-38.  Back to cited text no. 5    
6.Ajello L. The Medical mycological Iceberg: A study of incidence and prevalence. HSMHA Hlth, 1971; 86: 437-448.  Back to cited text no. 6    
7.Procop GW, Roberts GD. Laboratory Methods in Basic Mycology. In: Bailey & Scott's Diagnostic Microbiology 10th edition, Belly A. Forbes, Daniel F. Sahm, Alice S. Weissfeld (eds) Mosby (1998) pp. 953.  Back to cited text no. 7    
8.Thom C, Raper KB. A manual of The Aspergilli. (Baltimore and London) 1945, pp. 148-150.  Back to cited text no. 8    
9.Kauffman HF, Beaumont F, Meurs H, Vander HS, Vries K de. Comparision of antibody measurements against Aspergillus fumigatus by means of double diffusion and enzyme linked immunosorbent assay (ELISA). J. Allergy Clin Immunol 1983; 72: 255-261.  Back to cited text no. 9    
10.Richardson MD. Aspergillus and Penicillium species, Part vii: Monomorphic septate filamentous systemic pathogenic fungi. In: Topley & Wilson's Microbiology & Microbial infections, 9th edition, Vol. 4, Libero Ajello, Roderick J Hay (eds) 1998, pp. 298.  Back to cited text no. 10    
11.Orie NGM, de Vries GA, Kikstra A. Broncho-Pulmonary Aspergillosis. Amer. Rev Resp Dis 1960; 82: 649.  Back to cited text no. 11    
12.Henderson AH, English MP, Veeht RJ. Pulmonary Aspergillosis: A survey of its occurrence in the patients with chronic lung diseases and a discussion of the significance of diagnostic tests. Thorax 1968 ; 23: 513-518.  Back to cited text no. 12    
13.Pepys J, Riddel RW, Citron KM, Clayton YM, Short EL. Clinical and immunological significance of Aspergillus fumigatus in sputum. Am Rev Resp Dis 1959; 80: 167-180.  Back to cited text no. 13    
14.Campbell MJ, Clayton YM. Bronchopulmonary aspergillosis. Am Rev Resp Dis 1964; 89: 186.  Back to cited text no. 14    
15.Bordane EJ Jr. The clinical spectrum of Aspergillosis. Part II: Classification and description. Rc Crit Rev Clin Lab Sci 1980; 13: 85.  Back to cited text no. 15    
16.Fisher BD, Armstrong D, Yu B, Gold JW. Invasive Aspergillosis. Progress in early diagnosis and treatment. Am J Med 1981; 71: 571-577.  Back to cited text no. 16    
17.Richardson MD, Warnock DW. Enzyme linked immunosorbent assy and its application to the serological diagnosis of fungal infection. Sabouradia 1983; 21: 1-14.  Back to cited text no. 17    
18.Richardson MD, Llewellyn PA, Warnock DW. Antibody to Aspergillus fumigatus antigens in normal sera: Influence on positive - negative discrimination in ELISA. Journal of Immunoassay 1984; 5(3&4): 205-220.  Back to cited text no. 18    
19.Marx JJ Jr, Robert L, Gray BS. Comparison of the enzyme linked immunosorbent assay and double immunodiffusion test for the detection and quantitation of antibodies in farmer's lung diseases. J Allergic Clinic Immunol 1982; 70:109.  Back to cited text no. 19    
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