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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 21  |  Issue : 1  |  Page : 24-28

‘Clinicomycological correlation and antifungal susceptibility pattern of Aspergillus species’ – A retrospective and prospective study in a tertiary care centre in South India


Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India

Date of Web Publication12-Aug-2019

Correspondence Address:
Dr. Anupma Jyoti Kindo
Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai - 600 116, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_30_18

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  Abstract 


CONTEXT: Aspergillus is present ubiquitously in the environment in the form of spores. Infection due to Aspergilli is uncommon in immunocompetent individuals, unless they possess any abnormalities or have undergone any treatment with corticosteroids in which pulmonary aspergillosis is the most common. In immunocompromised individuals, the infection by this fungus is in a higher extent of risk.
AIM: A retrospective study was done and correlated with the antifungal susceptibility pattern of Aspergillus species.
MATERIALS AND METHODS: A total of 72 isolates of Aspergillus were collected and confirmed by conventional methods except Aspergillus tetrazonus was identified by DNA sequencing. Antifungal susceptibility testing (AFST) was performed by conventional broth microdilution according to the Clinical Laboratory Standards Institute M38-A2 2008 to all the isolates.
RESULTS: In our study, Aspergillus flavus was found to be the predominant followed by Aspergillus niger being the second most to be identified. AFST was performed for all the isolates, where they exhibited minimum inhibitory concentration (MIC) values within the range of 0.0078–2 μg/ml except Aspergillus terreus (4 μg/ml), which exhibited intrinsic resistance to Amphotericin B.
CONCLUSION: Aspergillus infection can be treated easily, unless they become invasive. Therefore, antifungal therapy should be started early by determining the MIC values for the isolates, especially for the systemic infections and prevent from mortality.

Keywords: Antifungal susceptibility testing, Aspergillus, azole resistance


How to cite this article:
Jishnu BT, Sriparna AT, Vichitra K, Kindo AJ. ‘Clinicomycological correlation and antifungal susceptibility pattern of Aspergillus species’ – A retrospective and prospective study in a tertiary care centre in South India. J Acad Clin Microbiol 2019;21:24-8

How to cite this URL:
Jishnu BT, Sriparna AT, Vichitra K, Kindo AJ. ‘Clinicomycological correlation and antifungal susceptibility pattern of Aspergillus species’ – A retrospective and prospective study in a tertiary care centre in South India. J Acad Clin Microbiol [serial online] 2019 [cited 2019 Oct 23];21:24-8. Available from: http://www.jacmjournal.org/text.asp?2019/21/1/24/264252




  Introduction Top


The diseases caused by Aspergillus are collectively termed aspergillosis and consists of both invasive and localised (aspergilloma) infections. Invasive infections are characterised by their entrance to the bloodstream and are nearly always fatal which suggest that most individuals inhale several hundreds of Aspergillus conidia per day where they can reach the small cavities of the lungs.[1] In healthy individuals, macrophages phagocytose the conidia, whereas neutrophils kill the developing hyphae if germinating conidia are left untreated, patients succumb to the infection in less than two weeks after diagnosis.[2] Aspergilloma infections are usually localised to pre-existing lung cavities caused by tuberculosis and other pulmonary diseases.[1] The defining characteristic of aspergilloma is the presence of a “fungus ball,” in which the hyphae form a dense network and are embedded in an extra-cellular matrix made up of proteins and monosaccharides, polysaccharides and secondary metabolites produced by the colony.[3],[4] Individuals with immunodeficiencies due to chronic granulomatous disease, AIDS, chemotherapy or organ transplantation for example, are at much higher risk of acquiring infection with this fungus. The present study was undertaken to know the epidemiology and demography of the patients infected with this organism, and the antifungal susceptibility of the Aspergilli isolated from these patients in our institute.


  Materials and Methods Top


The study period was from 2012 to 2016 August. For the retrospective study, the laboratory records were referred to find the isolates of Aspergillus. Hospital numbers were noted for the years 2012–2014 September. The pro forma was filled from the medical record section. For the prospective study, isolates and hospital numbers were taken. The Institutional Ethical Committee approval was obtained before the commencement of the study (CSP-MED/14/OCT/19/183). Informed consent was obtained from the study population.

Study population

All the isolates which came to microbiology laboratory and which grew Aspergillus were taken for the prospective study. The selected cases were studied as per the pro forma enclosed, and the detailed history of the selected cases was recorded. All the patients were followed up to the treatment level to know the clinical outcome after the treatment.

Collection of isolates

The samples were processed as per routine mycological procedures. The samples were subjected to potassium hydroxide wet mount examination and those which showed fungal elements were inoculated in Sabouraud Dextrose agar with Gentamicin (20mg/l) in duplicate. One set of tube was incubated at 37°C and another set at 25°C. Cultures were examined for growth, daily in the first week and twice a week for the subsequent period. Macroscopic and microscopic morphology of the obtained growth was studied.[5] The identification of the growth was based on growth rate, temperature requirements and colony characteristics such as colour, texture, pigment and submerged hyphae. The colony characters on the reverse were also studied for the presence or absence of pigment and whether they were diffused or localised. After observing the colony morphology, they were speciated according to the standard procedures.[5] All the isolates were systematically identified by standard techniques as follows: (1) tease mount and (2) slide culture technique. All Aspergillus species were identified by conventional method except Aspergillus tetrazonus.

Antifungal susceptibility testing

The procedure followed was according to the Clinical Laboratory Standards Institute (CLSI) M38-A2 2008 documented titled reference method for broth microdilution antifungal susceptibility testing of filamentous fungi; approved standard – Second Edition, intended for testing common filamentous fungi or moulds, including the dermatophytes.(Order no. 841-Ord-24751, CLSI). The five drugs tested were as follows: (1) triazole agents – Itraconazole (16657-100MG, Sigma-Aldrich), Voriconazole (PZ0005-5MG, Sigma-Aldrich) and Posaconazole (32103-25MG, Sigma-Aldrich); (2) polyene antifungals – Amphotericin B (CMS462-5G, HiMedia Laboratories) and (3) echinocandins – Caspofungin diacetate (SML0425-5MG, Sigma-Aldrich). Reading for Amphotericin B, Itraconazole, Voriconazole and Posaconazole was taken at 48 h, minimum inhibitory concentration (MIC) was the first well without visible growth. The quality control species of Aspergillus used was Aspergillus fumigatus ATCC 204305. Uninoculated drug control as well as Roswell park memorial institute medium (RPMI) control was used for sterility check.

In general, the cost of each isolate comes to approximately Rs. 500 for doing the fungal ID and antifungal susceptibility testing (AFST).

MIC 50 and MIC 90 of the isolates were calculated using Stastistical package for the Social Sciences computer software (IBM-SPSS no:16).


  Results Top


A total of 72 Aspergillus isolates from individual patients were collected from the Central Microbiology Laboratory and the Mycology Laboratory over a period of 22 months (October 2014–August 2016), and the archived clinical Aspergillus isolates in the mycology laboratory were included in the study.

Forty patients were male and 32 were female. The age of the patients ranged between 4 and 86 years. A majority of the patients were in the age group of 21–30 years (40.27%), with a male preponderance. Majority of the patients in this study group were above 20 years. The maximum number of specimens 29 (40.27%) were from the age group of 21–30 years with male predominance. Of the 72 patients in the study group, only 8 (11%) were immunocompromised and the remaining 64 (89%) being immunocompetent. Among the immunocompromised patients, 7 (9.72%) were diabetic and 1 (2.50%) was asthmatic. In our study, Aspergillus species were isolated from pus from the auditory canal, which was the predominant specimen (44) followed by nasal tissue (9), exudate (6), sputum (5), nasal swab (4), bronchoalveolar lavage (2), corneal scraping (2), and heel tissue 1 (1.38%). [Figure 1] shows the distribution of the isolates from various clinical sites. Apergillus flavus was found to be the most predominant and was identified in 46 (63.80%) cases of Aspergillosis. Aspergillus niger was the second most predominant species, isolated in 16 cases (22.20%). Aspergillus fumigatus, Aspergillus terreus and A. tetrazonus were the other species identified in 5 (6.94%), 4 (5.55%) and 1 (1.38%) cases, respectively. All Aspergillus isolates tested in this study exhibited MIC values within the range of 0.0078–2 μg/ml except for A. terreus (4 μg/ml), which is known to exhibit intrinsic resistance to amphotericin B. MIC50 and MIC90 of individual species are as shown in [Table 1].
Figure 1: Distribution of clinical site and species isolate

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Table 1: Minimum inhibitory concentration 50 and minimum inhibitory concentration 90 of clinical Aspergillus isolates

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Molecular identification

All Aspergillus species were identified by conventional method except A. tetrazonus. For A. tetrazonus, DNA extraction was done directly from the culture plate by an in-house column-based method which has been standardised in Mycology laboratory. PCR amplification was done using the pan-fungal primers internal transcribed spacer 1 (ITS1) and ITS4. Sequencing of the PCR product was done at SciGenom Labs Pvt. Ltd., Cochin, Kerala. Using NCBI nucleotide BLAST, the sequence was matched 100% with A. tetrazonus strain ATCC 16816. The sequence was deposited with the NCBI Genbank database with accession number KX683008.


  Discussion Top


The study had two cases of proven systemic infection for which BAL sample was sent. No galactomannan or Beta-D-glucan assay was done for the sputum or BAL samples. The fungal diagnosis was based only on culture, so according to EORTC MSG Criteria, we had only proven cases of Aspergillus. The most common isolate was Aspergillus flavus. A. tetrazonus was one unusual isolate we got which was identified by ITS sequencing. There is a wide variation in the species distribution among results of different workers in various geographical areas.[6] The variations in incidence and species distribution could be due to differences in climatic conditions (temperature and humidity) which affect the growth of Aspergillus in environment, season of the study period, environmental conditions (winds, dust particles in air) of the study area and predisposing conditions present among the patients.[6] It is a known fact that Aspergillus affects the immunocompromised patients. In our study, eight cases (11%) were immunocompromised (seven diabetes mellitus, one asthma). The remaining 64 patients (89%) were immunocompetent.

In vitro antifungal susceptibility testing of clinical Aspergillus isolates was done following the M38-A2 CLSI guidelines. While azole-resistant clinical Aspergillus isolates have been reported from many parts of the world, resistance rates remain low including our setting not influencing the primary choice of antifungal therapy. The terms susceptible and resistant can only be applied to mean lower and higher (>8 μg/ml) MICs, since clinical breakpoints have not yet been established for AFST of filamentous fungi such as Aspergillus by the CLSI for azoles. However, the breakpoints for Amphotericin B as susceptibility, intermediate susceptibility and resistance are: ≤0.5, 1 and ≥2 for A. fumigatus and ≤0.25, 0.5 and ≥1 for A. flavus and A. terreus.[7] Based on these breakpoints and previous epidemiological susceptibility data, all Aspergillus isolates tested in this study exhibited MIC values within the range of 0.0078–2 μg/ml except for A. terreus (4 μg/ml), which is known to exhibit intrinsic resistance to Amphotericin B. MIC50 and MIC90 of individual species are as shown in [Table 1] correlates with the other studies. The epidemiological cutoff values, the upper limit of wild-type MIC distributions which aid in determining the likelihood of resistance in Aspergillus spp, have been proposed by CLSI and can be used to interpret the results of AFST.[8],[9] The patients with pulmonary infection were started on Itraconazole 200 mg twice daily along with Amphotericin B (3 mg/kg/day) for two months and repeat radiology and culture was performed. All the patients were followed up for the entire period of treatment for clinical, radiological and mycological response whichever was applicable since we had isolates from non-invasive sites also. The patients in our study group belonged to low socioeconomic status and hence the clinicians had to limit the treatment with Itraconazole in few patients. The emergence of Itraconazole resistance alone is of concern,[10] fortunately in our population, the MIC to Itraconazole was low. The administration of liposomal Amphotericin B (3 mg/kg/day) can be used in combination with Caspofungin and Voriconazole as well as a second-line treatment in patients refractory to monotherapy.[9],[10] Amphotericin B and its lipid derivatives are appropriate for initial and salvage therapy of Aspergillus infections when azoles are contraindicated and no other antifungal drugs can be administered.[11] In our study, all patients with otomycosis were treated with topical Clotrimazole and oral Itraconazole (when indicated) for 21 days. There was clinical and mycological improvement in all these cases. We had one patient with invasive otitis externa which grew A. terreus from the ear which was resistant to Amphotericin B and was started on Voriconazole. Caspofungin in combination with Voriconazole is considered useful in infections with A. terreus as it is resistant to polyenes.[11],[12] In this case, the clinical and radiological improvement was observed after two months of Voriconazole therapy and no initial aggressive surgical debridement was required. The surgical removal of affected tissue is indicated in localised lesions, with a low risk of bleeding, and if the site is accessible. In cases that do not have an adequate therapeutic response, the clinician must re-evaluate the diagnosis of Aspergillus infection and should consider antifungal resistance and send for antifungal resistance testing.[13] Patients with Aspergillus sinusitis underwent functional endoscopic sinus surgery when necessary and were treated with oral Itraconazole 200 mg/bd for two months. The patients improved clinically and radiologically. Mycological clearance could not be established since no specimen was sent after two months. Patients with Aspergillus keratitis were treated with 1% topical Itraconazole till the clinical signs and symptoms totally subsided. Patients with burns and cutaneous aspergillosis underwent surgical debridement and were treated with intravenous voriconazole. Patients with pulmonary aspergillosis were treated with Amphotericin B and Itraconazole. Both the patients were discharged with clinical improvement.


  Conclusion Top


Aspergillus causes a variety of infections in both the immunocompetent and immunocompromised patient population. Speciation is possible by conventional methods (culture and microscopy) but may occasionally require molecular techniques for the rare species. Aspergillus is a fungal pathogen which is easy to treat unless it is invasive, it shows low MICs to the commonly used antifungal agents. It is important to determine MICs for isolates, especially from deep/systemic infections to treat the patient promptly with a single or combined antifungal regimen and prevent morbidity and mortality.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Latgé JP. Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 1999;12:310-50.  Back to cited text no. 1
    
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Xess I, Mohanty S, Jain N, Banerjee U. Prevalence of Aspergillus species in clinical samples isolated in an Indian tertiary care hospital. Indian J Med Sci 2004;58:513-9.  Back to cited text no. 2
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Beauvais A, Schmidt C, Guadagnini S, Roux P, Perret E, Henry C, et al. An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus. Cell Microbiol 2007;9:1588-600.  Back to cited text no. 4
    
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de Hoog GS, Guarro J, Gene J, MJ Figueras. Atlas of clinical fungi, 2nd edn, Centraalbureau voor Schimmelcultures, Utrecht/Universität Rovira I Virgill, Reus, Spain, 2000. p. 1-1126.  Back to cited text no. 5
    
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Singh R, Urhekar Ad, Singh G. Incidence of Aspergillus infections in patients in a tertiary care Hospital in Navi Mumbai. 2015. 05.10.4172/2165-8056-1000127.  Back to cited text no. 6
    
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Elefanti A, Mouton JW, Verweij PE, Zerva L, Meletiadis J. Susceptibility breakpoints for amphotericin B and Aspergillus species in an in vitro pharmacokinetic-pharmacodynamic model simulating free-drug concentrations in human serum. Antimicrob Agents Chemother 2014;58:2356-62.  Back to cited text no. 7
    
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Espinel-Ingroff A, Cuenca-Estrella M, Fothergill A, Fuller J, Ghannoum M, Johnson E, et al. Wild-type MIC distributions and epidemiological cutoff values for amphotericin B and Aspergillus spp. For the CLSI broth microdilution method (M38-A2 document). Antimicrob Agents Chemother 2011;55:5150-4.  Back to cited text no. 8
    
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Rodriguez-Tudela JL, Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Monzon A, Cuenca-Estrella M. Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus. Antimicrob Agents Chemother 2008;52:2468-72.  Back to cited text no. 9
    
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Dannaoui E, Borel E, Monier MF, Piens MA, Picot S, Persat F, et al. Acquired itraconazole resistance in Aspergillus fumigatus. J Antimicrob Chemother 2001;47:333-40.  Back to cited text no. 10
    
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Denning DW, Marr KA, Lau WM, Facklam DP, Ratanatharathorn V, Becker C, et al. Micafungin (FK463), alone or in combination with other systemic antifungal agents, for the treatment of acute invasive aspergillosis. J Infect 2006;53:337-49.  Back to cited text no. 11
    
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Pfaller M, Boyken L, Hollis R, Kroeger J, Messer S, Tendolkar S, et al. Use of epidemiological cutoff values to examine 9-year trends in susceptibility of Aspergillus species to the triazoles. J Clin Microbiol 2011;49:586-90.  Back to cited text no. 12
    
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Patterson TF, Thompson GR 3rd, Denning DW, Fishman JA, Hadley S, Herbrecht R, et al. Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of America. Clin Infect Dis 2016;63:e1-60.  Back to cited text no. 13
    


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