Simple and highly discriminatory VNTR-based multiplex PCR for tracing sources of Aspergillus flavus isolates

Abstract

Aspergillus flavus is second only to A. fumigatus in causing invasive aspergillosis and it is the major agent responsible for fungal sinusitis, keratitis and endophthalmitis in many countries in the Middle East, Africa and Southeast Asia. Despite the growing challenge due to A. flavus, data on the molecular epidemiology of this fungus remain scarce. The objective of the present study was to develop a new typing method based on the detection of VNTR (Variable number tandem repeat) markers. Eight VNTR markers located on 6 different chromosomes (1, 2, 3, 5, 7 and 8) of A. flavus were selected, combined by pairs for multiplex amplifications and tested on 30 unrelated isolates and six reference strains. The Simpson index for individual markers ranged from 0.398 to 0.818. A combined loci index calculated with all the markers yielded an index of 0.998. The MLVA (Multiple Locus VNTR Analysis) technique proved to be specific and reproducible. In a second time, a total of 55 isolates from Chinese avian farms and from a Tunisian hospital have been evaluated. One major cluster of genotypes could be defined by using the graphing algorithm termed Minimum Spanning Tree. This cluster comprised most of the isolates collected in an avian farm in southern China. The MLVA technique should be considered as an excellent and cost-effective typing method that could be used in many laboratories without the need for sophisticated equipment.

Figure 1. Minimum spanning tree of 91 A. flavus isolates based on categorical analysis of 8 VNTRs.

Each circle represents a unique genotype. The diameter of each circle corresponds to the number of isolates with the same genotype. Genotypes connected by a shaded background differ by a maximum of one VNTR marker and could be considered a “clonal complex”. Thick connecting lines represent one marker difference; regular connecting lines represent two or three marker differences; thin interrupted lines represent four or more differences. The length of each branch is also proportional to the number of difference. Each epidemiological situation is represented by a specific colour: red for 32 isolates collected in an avian farm (A) in Guilin, China; yellow for 23 isolates collected other avian farms (B, C, D) in Nanning, China; purple for 20 isolates collected in patients and in the hospital of Sfax, Tunisia; green for 6 isolates collected from a stock of mouldy peanuts in Benin; brown for 4 isolates collected from the fur of domestic carnivores in France and blue for 6 reference strains.