Slovenians Lead in Identifying Bioterrorist Agents Through Next-Generation Sequencing

Bioterrorism is a threat to national and international security. Classic methods for determining and identifying such agents (e.g. culture assays, PCR) may be rapid and reliable, however, they are targeted and, as a rule, do not allow the identification of specific genetic changes responsible for the level of isolate virulence. With the advent of next-generation sequencing (NGS), it is possible to gain insight into the entire genetic material of the studied microorganisms or samples, allowing the species (strain) to be identified and genetic changes (e.g. a dedicated virulence record) to be determined.

The NGS method, however, presents a unique challenge due to the demanding bioinformatics processing of the large amount of sequenced data obtained.

With a view to reviewing the current state of countries’ preparedness for the detection of bioterrorist agents using the NGS method – specifically, using for whole-genome sequencing (WGS) method - the United States organisation ‘Secretary-General's Mechanism for Investigation of Alleged Use of Chemical , Biological or Toxin Weapons' (UNSGM)’, in collaboration with the 'Danish Technical University' (DTU), organised an inter-laboratory control in 2018. It invited numerous reference laboratories around the world to participate.

Since the Institute of Microbiology and Parasitology of the Faculty of Veterinary Medicine has a contract with the Ministry of Defence of the Republic of Slovenia for providing professional advice and taking measures in the event of a biological weapons attack, the Laboratory for Molecular Bacteriology also participated in the aforementioned inter-laboratory control.

The aim of the inter-laboratory control was to identify microbial species, thus determining potential deliberate genetic changes (entering or deleting records for virulence factors) and assessing the laboratory's ability to assess biological risk. In a fictionalised (but realistic) scenario for inter-laboratory control, a suspected worker in a microbiology lab left 36 unmarked samples - potential bioterrorist agents. Their genomic sequences were available. What kind is it? Is this species a bioterrorist agent? Was the isolate in the sample genetically modified (GMO) by the suspect and thus affect its virulence?

The participating laboratories had to answer the above questions within one month of receiving the genomic sequences, based completely on their own ideas, and, in doing so, to use any bioinformatic tools and their own professional judgment for this purpose. The test involved public health, veterinary and general microbiology laboratories and laboratories specialising in biological defence. The participating institutions were located globally. Some countries collaborated with several laboratories. A total of 60 laboratories participated. The Institute of Microbiology and Parasitology at the Faculty of Veterinary Medicine, the University of Ljubljana (Bojan Papić, Darja Kušar, Jana Avberšek) performed best among the participating laboratories, with a success rate of 94.3%. Laboratories from China, Norway, Germany, Sweden, Portugal, the United Kingdom, Switzerland, USA, France, Singapore, Belgium, Italy and Australia also performed well with success rates of 82.3% to 91.4%. Most of the participating institutions (44/60) had less than an 80% success rate. The results show that Slovenia is very well prepared for the potential identification of bioterrorist agents using NGS. The overall poor performance of the participating institutions is also worrying and indicates the need to train staff in the field of bioterrorism and NGS bio-information processing.