Determination of Iplex Massarray as a Method for Genetic Typing and Characterization of Staphylococcus Aureus Isolates In Kenya
Nyasinga, Justine Tirimba
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Staphylococcus aureus is a clinically important pathogen in Kenya and globally owing to its widespread occurrence, virulence potential and multidrug resistance. Adequate surveillance is required to track the emergence and spread of hypervirulent and multidrug resistant strains such as methicillin and vancomycin resistant S. aureus (MRSA and VRSA). Conventional methods for S. aureus characterization include Staphylococcal protein A (spa) gene typing, multi-locus sequence typing (MLST) and Pulsed Field Gel Electrophoresis. To overcome the limitations of high costs, long turnaround times and technical complexity associated with these methods, alternative genotyping approaches are required. This study evaluated mass spectrometry-based iPlex massARRAY as a method for genetic characterization of Kenyan S. aureus isolates by comparing it to spa, MLST and conventional PCR methods. Fifty four clinical S. aureus isolates from three hospital sites in Kenya (Kisumu, Nairobi and Kericho) were analyzed. Ten single nucleotide polymorphisms (SNPs) derived from six MLST loci and 13 binary gene markers for virulence and resistance were analyzed by iPlex massARRAY in a multiplexed assay. The isolates were also characterized using MLST, spa typing and conventional PCR assays for five selected staphylococcal virulence genes (icaA, clfA, pvl, tsst and sea). IPlex massARRAY grouped 44/54 isolates into 14 SNP genotypes with 9 of the 10 MLST SNPs showing high identification rates (average 89%). MLST typing identified 21 sequence types (STs) in 49/54 isolates while spa typing identified 22 spa types in 50/54 isolates. MLST and spa typing results were generally congruent. Sequence type 152 (t355) was the most predominant type overall and among MRSA strains, ST 241 (t037/t2029) predominated. Other strains included ST 5 (t002), ST 8 (t064), ST 22 (t005), ST 30 (t318/t021), ST 39 (t007) and ST 80 (t13194). Compared to spa and MLST typing, iPlex massARRAY had 83% and 82% accuracy respectively in unambiguously resolving this collection of isolates. IPlex massARRAY showed reduced turnaround times and reagent and consumable costs per isolate (<12 hrs, 17 USD) in relation to both spa typing (4 days, 30 USD) and MLST (20 days, 126 USD) for the isolates analyzed. The gene for icaA was the most abundant (92.5%) whereas tsst was the least (4%). Chi-squared tests of association between the presence of individual virulence genes and selected markers of severity of infection did not show significant associations (p>0.05). IPlex massARRAY showed moderate accuracy in binary gene detection (43%-100%) with minor gene-specific discrepancies. IPlex massARRAY unambiguously classified a heterogeneous collection of Kenyan S. aureus isolates from 3 sites into groups consistent with spa and MLST classifications. The technique identified all MRSA isolates and resolved 2 novel spa types. IPlex massARRAY offers a faster and more affordable approach to genotyping Kenyan staphylococcal isolates. Additional informative SNPs and improved assay design for a larger isolate collection would enhance the overall performance of the assay both in typing and gene detection. Creation of a SNP database to enable interlaboratory data comparability is recommended.