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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgement:
 ~  References

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ORIGINAL ARTICLE
Year : 2002  |  Volume : 20  |  Issue : 3  |  Page : 141-144
 

Mycological and serological study of pulmonary aspergillosis in central India


Department of Microbiology, Government Medical College, Nagpur - 440 003, Maharashtra, India

Correspondence Address:
Department of Microbiology, Government Medical College, Nagpur - 440 003, Maharashtra, India

 ~ Abstract 

PURPOSE: To study the prevalence and predisposing factors of Aspergillus infection and correlate microscopic, culture and serological findings along with drug sensitivity. METHODS: Sputum samples from 123 patients of pulmonary disease with clinical suspicion of having fungal, especially Aspergillus infections, were examined microscopically and for culture. Minimum inhibitory concentration (MIC) of itraconazole was tested against the isolates. Serum samples from these patients were tested for precipitin against Aspergillus antigen using immunodiffusion (ID) technique. RESULTS: Aspergillus species were isolated in 20 (16.26%) cases and Aspergillus fumigatus was the predominant species isolated in 16 (80%) cases. Precipitins were detected in 29 (23.58%) cases. Serum samples collected from 50 healthy individuals to serve as controls showed no precipitin against Aspergillus antigen galactomannan. This fungus was found to be sensitive to itraconazole with MIC range 0.125-1g/mL. CONCLUSIONS: Serological tests have an edge over routine smear and culture methods for the diagnosis of pulmonary aspergillosis. Itraconazole is more effective than amphotericin B and fluconazole in the treatment of aspergillosis.

How to cite this article:
Kurhade A M, Deshmukh J M, Fule R P, Chande C, Akulwar S. Mycological and serological study of pulmonary aspergillosis in central India. Indian J Med Microbiol 2002;20:141-4


How to cite this URL:
Kurhade A M, Deshmukh J M, Fule R P, Chande C, Akulwar S. Mycological and serological study of pulmonary aspergillosis in central India. Indian J Med Microbiol [serial online] 2002 [cited 2019 Dec 11];20:141-4. Available from: http://www.ijmm.org/text.asp?2002/20/3/141/6938


Amongst the various mycoses of man, aspergillosis is one of the earliest fungal diseases to be recognized. Aspergillus spp. are ubiquitous fungi, commonly occurring in soil, water, and decaying vegetation. Aspergillus spp. have been cultured from unfiltered air, ventilation systems, contaminated dust dislodged during hospital renovation and construction, horizontal surfaces, food, and ornamental plants.[1] The most important nosocomial infection due to Aspergillus spp. is pneumonia,[2],[3] the primary route of acquiring infection being inhalation of the fungal spores. From lungs, dissemination may take place leading to systemic aspergillosis. Occurrence of Aspergillus infection in association with chronic lung diseases like pulmonary tuberculosis, lung abscess and bronchopneumonia with residual lung cavity, asthma and lung malignancy is documented.[4] The diagnosis of Aspergillus infection presents considerable difficulty. The signs and symptoms in most cases of aspergillosis are non-specific, and radiological findings are of little diagnostic help. Diagnosis of aspergillosis requires the isolation and identification of the fungus. Galactomannan antigen-based serologic assays are now being developed in an attempt to allow for the rapid and specific diagnosis of Aspergillus infections; though their clinical usefulness is presently undefined.[5],[6],[7] In addition to diagnosis, the treatment of aspergillosis is also controversial.[8],[9] The present study was undertaken to find correlation between microscopic findings, culture and serodiagnosis of Aspergillus infection. We also studied the prevalence of various species of Aspergillus and predisposing factors for Aspergillus infection and drug sensitivity.

 ~ Materials and Methods Top

Subjects
The study was carried out at the department of Microbiology, Government Medical College and hospital, Nagpur. The procedure followed was in accordance with ethical standards on human beings. A total of 123 cases of chronic pulmonary infections: Pulmonary TB (57), lung abscess (15), lung malignancy (8), chronic bronchitis (13), pleural effusion (13) and bronchopneumonia (6) with the suspicion of secondary invasion by Aspergillus on the basis of clinical and radiological findings, were included in the present study. In addition to this, 11 cases with suspected allergic bronchopulmonary aspergillosis were also included.
Tests
At least two samples of expectorated morning sputum samples on two consecutive days were collected from each patient in a wide mouthed autoclaved sterile glass bottle and examined for fungi by microscopic examination of 10% KOH mount and cultured on Sabouraud dextrose agar (SDA) medium. Isolated Aspergillus species were identified as per standard methods.[10] Susceptibility to amphotericin B, itraconazole and fluconazole was tested by determining minimum inhibitory concentration by agar dilution method at Ranbaxy Research Laboratory. Serum was collected from cases as well as age and sex matched healthy controls and tested for the presence of Aspergillus precipitin by agar gel double diffusion test, using antigen prepared from the liquid culture media.[11]
Preparation of antigen
Culture filtrate antigen of A.fumigatus and A.niger was prepared using two strains of each species. Steps employed for preparation of antigen in brief were:
[Figure]
The Aspergillus was grown for 3-4 weeks in a synthetic medium glucose asparagine broth as stationary culture. The mycelial mat were separated and transferred to 95% acetone and kept for 24 hours. Then it was squashed gently between two layers of filter paper and dried in desiccator with anhydrous calcium chloride and then finely pulverized in pestle and mortar. Solvent ether was used for defatting. The active allergenic substances from the defatted powder were extracted with highly alkaline buffer (pH 8.0) and soluble active ingredients were separated by filtration through Whattman No.1 filter paper. Allergenic extracts were sterilized by filtration through millipore filter paper.
Agar gel diffusion test[12]
The test was done in 1.5% purified agar gel in barbitone buffer (pH 8.6) on microscope slides. After pouring the gel it was allowed to set for 10 minutes then it was transferred to the moist chamber and kept at 4C for the next 45 to 60 minutes. The wells were punched in the gel and charged with Aspergillus antigen solution and test samples (sera). The standard positive sera were regularly employed as control. The slide was transferred to moist chamber and kept at 4C for 48 hours for immunodiffusion and examined under oblique illumination for immunoprecipitate between the wells of test and antigen.

 ~ Results Top

In the present study, out of 123 cases, confirmed etiology of pulmonary aspergillosis was established in 20 (16.26%) cases. Ten cases were positive by either KOH preparation or culture and were considered of doubtful etiology. In all positive cases repeat culture was done to confirm etiology [Table - 1]. A.fumigatus was the commonest species isolated; followed by A.niger and  A.flavus   [Table - 2].
All culture positive cases of pulmonary aspergillosis were also positive by agar gel double diffusion test, Well-defined intense precipitates were observed in 23.58% serum samples. Twenty-eight sera reacted with A.fumigatus antigen while only 3 reacted with A.niger antigen. A serum sample from a case with A.flavus also reacted with A.fumigatus antigen in the present study. The correlation between serological results and culture is shown in [Table - 3].
Antiaspergillus antibodies were not detected in any of the controls. All strains of Aspergillus isolated in the study were also tested for antifungal susceptibility to three drugs, amphotericin B, itraconazole and fluconazole [Table - 4]. Itraconazole was found to be more active (MIC range 0.5-2g/mL).

 ~ Discussion Top

Comparison of the prevalence of Aspergillus in sputum shows that it occurs with greater frequency in malignancy and tuberculosis patients than other pulmonary diseases. Occurrence of Aspergillus infection in association with chronic lung disease is documented.[13]
Susceptibility to Aspergillus appears to be enhanced by certain factors like therapy with antibiotics and chemotherapeutic drugs and steroids, depression of bone marrow activity, carcinoma and leukaemia. Especially antibiotics and steroids appear to be more significant as they may stimulate the growth and virulence of infecting fungus by destruction of competing bacterial flora. Common use of broad spectrum antibiotics in the pulmonary disease for long duration, for example in pulmonary tuberculosis, lung abscess, and chronic bronchitis may predispose to fungal infection.
The isolation of Aspergillus from sputum is often considered a contaminant. Whether the isolated agent is a contaminant or indeed responsible for the pathological condition is often questionable. Routine blood cultures are not much help as they are remarkably insensitive[14] and systemic antibody responses in immunocompromised patients are likely to be unreliable indicators of infection.[15],[16] However, seropositivity detected in the present study correlates with results of similar studies[17],[18],[19] and suggests that serological tests have an edge over routine culture for diagnosis of pulmonary aspergillosis. Detection of antibodies indicates that there is definite infection induced antibody production and thus rules out the presence of fungus as mere contaminant. Pulmonary aspergillosis occurs in immunocompromised hosts in whom immune response is less or may be absent.[20] In such cases serological findings may be absent. Therefore, such cases with negative serological findings may not be considered negative for infection. Thus a combination of culture and serology can be ideal to confirm the diagnosis of pulmonary aspergillosis.
There have been no convincing demonstrations that treatment failure in patients with aspergillosis can be attributed to the development of amphotericin B resistance. In the present study the results of invitro susceptibility testing of 20 isolates of Aspergillus species show that itraconazole was more active than amphotericin B (MIC range 0.5-2 g/mL) and fluconazole (MIC 0-64g/mL).

 ~ Acknowledgement: Top

The authors are thankful to Dr. Ashok Rattan, Director, Ranbaxy Research Laboratory, Department of Microbiology, for his help to test antifungal drug susceptibility of isolates of Aspergillus strains. 

 ~ References Top

1.Walsh TJ, Dixon DM. Nosocomial aspergillosis: environmental microbiology, hospital epidemiology, diagnosis and treatment. Eur J Epidemiol 1989;5:131-142.   Back to cited text no. 1    
2.Sherertz RJ, Belani A, Kramer BS, et al. Impact of air filtration on nosocomial Aspergillus infections. Unique risk of bone marrow transplant recipients. Am J Med 1987;83:709-718.   Back to cited text no. 2    
3.Young RC, Bennett JE, Vogel CL, Carbone PP, DeVita VT. Aspergillosis: the spectrum of the disease in 98 patients. Medicine 1970;49:147-173.   Back to cited text no. 3    
4.Cordonnier C, Bernaudin JF, Bierling P, Huet Y, Vernant JP. Pulmonary complications occurring after allogeneic bone marrow transplantation: a study of 130 consecutive transplanted patients. Cancer 1986;58:1047-1054.   Back to cited text no. 4    
5.Bretagne S, Costa JM, Bart-Delabesse E, Dhedin N, Rieux C, Cordonnier C Comparison of serum galac-tomannan antigen detection and competitive polymerase chain reaction for diagnosing invasive aspergillosis. Clin Infect Dis 1998;26:1407-12.  Back to cited text no. 5    
6.Talbot GH, Weiner MH, Person SL, Provencher M, Hurwitz S. Serodiagnosis of invasive aspergillosis in patients with hematologic malignancy: validation of the Aspergillus fumigatus antigen radioimmunoassay. J Infect Dis 1987;155:12-27.   Back to cited text no. 6    
7.Dupont B, Huber M, Kim SJ, Bennett JE. Galactomannan antigenemia and Antigenuria in aspergillosis: studies in patients and experimentally infected rabbits. J Infect Dis 1987;155:1-11.   Back to cited text no. 7    
8.Wierzbicka M, Wesolowski S, Podsiadlo B, Bestry I. Treatment of patients with pulmonay aspergilloma with Itraconazole (Polish) Original title: Leczenie itrakonazolem chrych z grzybniakami kropid lakozymi pluc: Pnemonologia Alergologia Polaska. 1996;64(1-2):59-63.  Back to cited text no. 8    
9.Stevens DA, Kan VL, Judson MA, Morrison VA, Dummer S, Denning DW, Bennett JE, Welsh TJ, Patterson TF, Pankey GA. Practice guidelines for diseases caused by Aspergillus. Infectious diseases society of America. Clinical Infectious disease. 2000;30(4):696-709.  Back to cited text no. 9    
10.Emmons CW, Binford CH, Utz JP. Aspergillosis in medical Mycology. (Lea and Febiger) Philadelphia 1977;3rd Edition,Chapter 19 297-303.  Back to cited text no. 10    
11.Chakrabarti A, Singh K, Jtana M. Medical Mycology Laboratory Procedure International workshop on medical mycology. Department of Medical Microbiology; PGI of Medical Education and Research Chandigarh India 1998;16-21 November,106-108.  Back to cited text no. 11    
12.Kelkar SS, Khare PM. A basic approach to immunoprecipitation Gel: Gel immunodiffusion techniques in Research and Laboratory medicine. (Popular prakashan, Mumbai) 1984; 1st Edition, 40-42.  Back to cited text no. 12    
13.Edge JR, Stanfield DS, Fletcher DE. Pulmonary aspergillosis in an unsettled hospital population. Chest 1971;59(4):407-413.  Back to cited text no. 13    
14.Kammer RB, Utz JP. Aspergillus species endocarditis: the new face of a not so rare disease. Am J Med 1974;56:506-521.  Back to cited text no. 14    
15.Holmberg K, Berdischewsky M, Young LS. Serologic immunodiagnosis of invasive aspergillosis. J Infect Dis 1980;141:656-664.  Back to cited text no. 15    
16.Burnie JP, Matthews RC, Clark I, Milne LJ. Immunoblot fingerprinting of Aspergillus fumigatus. J Immunol Methods 1989;118:179-186.   Back to cited text no. 16    
17.Shivananda PG, Rao PV, Devi JN, Survarchala M. Aspergillus in bronchial asthma. Indian J Med Sci.1983;37(9):154-155.  Back to cited text no. 17    
18.Varkey B, Rose HD. Pulmonary Apergilloma :A rational approach to treatment. Am J Med 1976;61:626-631.  Back to cited text no. 18    
19.Vijayachari P, Lukshmi N, Kumar AG. Serodiagnosis of pulmonary aspergillosis. Indian J Med Microbiology 1997;15(1):23-24.  Back to cited text no. 19    
20.Young RC, Bennett JE. Invasive aspergillosis:absence of detectable antibody response. Am J respir Dis 1971;104:710-716.  Back to cited text no. 20    
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