Improving the genotyping resolution of Cryptosporidium hominis subtype IbA10G2 using one step PCR-based amplicon sequencing

• Published in: Infection, genetics and evolution genetics and evolution, 2017-11, Vol.55, p.297-304
• Main Authors: Beser, Jessica, Hallström, Björn M., Advani, Abdolreza, Andersson, Sofia, Östlund, Gabriel, Winiecka-Krusnell, Jadwiga, Lebbad, Marianne, Alm, Erik, Troell, Karin, Arrighi, Romanico B.G.
• Format: Article
• Language: English
• Subjects: Cryptosporidiosis; Cryptosporidium - classification; Cryptosporidium - genetics; Genetic Markers; Genetic Variation; Genome, Protozoan; Genotyping; Molecular Typing; Multi-locus genotype (MLG); Multi-locus sequence typing (MLST); Next Generation Sequencing (NGS); Outbreak investigation; Polymerase Chain Reaction - methods; Sequence Analysis, DNA; Whole Genome Sequencing
• ISSN: 1567-1348; 1567-7257
• DOI: 10.1016/j.meegid.2017.08.035

Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype is responsible for many waterborne outbreaks as well as sporadic cases and is considered virulent and highly important in the epidemiology of cryptosporidiosis. Due to low heterogeneity within the genome of C. hominis it has been difficult to identify epidemiological markers with higher resolution than gp60. However, new markers are required in order to improve outbreak investigations and studies of the transmission dynamics of this clinically important subtype. Based on the whole genome sequences of 17 C. hominis isolates, we have identified several differential loci and developed a new sequence based typing panel with higher resolution than gp60. An amplicon sequencing method was also developed which is based on a one-step PCR which can be sequenced using a Next Generation Sequencing (NGS) platform. Such a system provides a rapid and high-throughput workflow. A panel of nine loci with 10 single nucleotide variants (SNV) was selected and evaluated using clinical IbA10G2 isolates from sporadic, cluster and outbreak associated cases. The specimens were separated into 10 different genetic profiles named sequence types (STs). All isolates within an outbreak or cluster belonged to the same ST, including several samples from the two large waterborne outbreaks which occurred in Sweden between 2010 and 2011 indicating that these outbreaks might be linked. The results demonstrate the methods suitability for improved genotyping of C. hominis IbA10G2. •Nine suitable loci were identified and a sequence based typing panel to resolve C. hominis IbA10G2 was developed.•The newly developed typing panel was evaluated on 44 clinical IbA10G2 isolates.•This method shows potential for differentiating the virulent subtype IbA10G2 with higher resolution than current methods.•Amplicon preparation based on one-step PCR for Next Generation Sequencing (NGS) provides a rapid and high-throughput method.