High Resolution Genotyping of Clinical Aspergillus flavus Isolates from India Using Microsatellites

Klaassen CHW (2011) High Resolution Genotyping of Clinical Aspergillus flavus Isolates from India Using Microsatellites. PLoS ONE 6(1): e16086. doi:10.1371/journal.pone.0016086 High Resolution Genotyping of Clinical Aspergillus flavus Isolates from India Using Microsatellites Shivaprakash M. Rudramurthy 0 Hanneke A. de Valk 0 Arunaloke Chakrabarti 0 Jacques F. G. M. Meis 0 Corne H. W. Klaassen 0 William Joseph Steinbach, Duke University Medical Center, United States of America 0 1 Mycology Division, Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research , Chandigarh , India , 2 Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital , Nijmegen , The Netherlands Background: Worldwide, Aspergillus flavus is the second leading cause of allergic, invasive and colonizing fungal diseases in humans. However, it is the most common species causing fungal rhinosinusitis and eye infections in tropical countries. Despite the growing challenges due to A. flavus, the molecular epidemiology of this fungus has not been well studied. We evaluated the use of microsatellites for high resolution genotyping of A. flavus from India and a possible connection between clinical presentation and genotype of the involved isolate. Methodology/Principal Findings: A panel of nine microsatellite markers were selected from the genome of A. flavus NRRL 3357. These markers were used to type 162 clinical isolates of A. flavus. All nine markers proved to be polymorphic displaying up to 33 alleles per marker. Thirteen isolates proved to be a mixture of different genotypes. Among the 149 pure isolates, 124 different genotypes could be recognized. The discriminatory power (D) for the individual markers ranged from 0.657 to 0.954. The D value of the panel of nine markers combined was 0.997. The multiplex multicolor approach was instrumental in rapid typing of a large number of isolates. There was no correlation between genotype and the clinical presentation of the infection. Conclusions/Significance: There is a large genotypic diversity in clinical A. flavus isolates from India. The presence of more than one genotype in clinical samples illustrates the possibility that persons may be colonized by multiple genotypes and that any isolate from a clinical specimen is not necessarily the one actually causing infection. Microsatellites are excellent typing targets for discriminating between A. flavus isolates from various origins. - Aspergillus species are known to cause a wide range of disorders including allergic, colonizing and invasive disease in immunocompromised as well as immunocompetent hosts [1]. A. fumigatus is the predominant etiological agent in patients with aspergillosis followed by A. flavus [14]. However, in certain countries such as India, Saudi Arabia and Sudan, A. flavus is the predominant etiological agent in patients with fungal rhinosinusitis and endophthalmitis [46]. A. flavus has been reported to cause outbreaks of mucocutaneous and subcutaneous aspergillosis [4,5,7,8]. In immunosuppressed mice it was observed that much lower inocula of A. flavus spores could kill animals compared to A. fumigatus spores [911]. The agent is also known to cause environmental aflatoxin contamination in crops like maize, cottonseed, almond, pistachio and peanuts, which leads to substantial economic damage worldwide [12]. Despite the growing challenge due to A. flavus, the molecular epidemiology of this fungus has not been well studied. Molecular typing of A. flavus has been hindered by lack of discriminatory and exchangeable techniques for this fungus. Several genotypic methods have been utilized for the molecular typing of this fungus which includes random amplified polymorphic DNA (RAPD) [13,14] restriction fragment length polymorphism (RFLP) [15,16] and amplified fragment length polymorphism (AFLP) [17]. These techniques all utilize complex banding patterns to discriminate between isolates. These techniques often have a poor interlaboratory reproducibility and the exchange of results between laboratories is difficult. In this context, molecular typing using microsatellites yields multiple advantages such as a high discriminatory power, high reproducibility and easy exchange of results [18]. Microsatellite based typing has been well established for A. fumigatus [1926]. The technique is based on PCR amplification of short tandemly repeated DNA motifs of 2 10 bp that are abundantly present in the genomes of fungi followed by the determination of fragment size by capillary electrophoresis (CE). Sizing by CE has a resolution below one nucleotide which is essential for the high level of reproducibility of the technique [1922,24,26]. The number of repeats is extrapolated from the fragment size. We developed a multiplex, multicolor microsatellite panel for genotyping of A. flavus and investigated whether there might be a link between A. flavus genotype and clinical presentation of the infection. This work was presented at the 4th Advances Against Aspergillosis (AAA), Rome, Italy 46 February 2010. Materials and Methods Ethics statement This study is approved by the Institutional Ethics Committee of the Postgraduate Institute of Medical Education and Research, Chandigarh, India. No informed consent was obtained. The ethics committee allowed the use of clinical isolates without informed consent provided that no individual patient would be identified. All data were analyzed anonymously. Isolates A collection of 162 clinical isolates of A. flavus isolated from the patients attending the Postgraduate Institute of Medical Education and Research, Chandigarh, India and stored at the National Culture Collection of Pathogenic Fungi (NCCPF), PGIMER, Chandigarh, India was included in the study. The isolates were recovered from clinical samples collected over a two year period (January 2007 through December 2008). PGIMER, Chandigarh is a tertiary care referral medical centre catering the need of patients from provinces (states) of north India including Punjab, Haryana, Himachal Pradesh, western Uttar Pradesh, and northern Rajasthan (covering an area over 350,000 sq km.). The strains were isolated from patients with allergic fungal rhinosinusitis (AFRS, n = 75), invasive fungal rhinosinusitis (n = 23), pulmonary aspergillosis (n = 26), keratitis (n = 23), endophthalmitis (n = 5), or others (n = 10). All isolates were from different patients. A. flavus NRRL 3357 was obtained from the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands and was used as the control strain in all experiments. The identity of the isolates were verified by taxonomic criteria [27] and further confirmed by amplified fragment length polymorphism (AFLP) analysis [22]. DNA isolation For the isolation of DNA a pre-wetted cotton swab was saturated with conidia from a freshly grown sporulating culture. Conidia were resuspended in a vial containing 350 ml lysis buffer an (...truncated)