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Background   Blastocystis  spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the  18S  gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as  Blastocystis , which, in other protozoa, shows more polymorphisms than the  18S  gene and thus may offer finer discrimination when trying to identify  Blastocystis  ST. Therefore, the objective of the present study was to assess the suitability of the  PFOR  gene as an additional marker to discriminate among  Blastocystis  strains or subtypes from symptomatic carrier children.    Methods  Faecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for  Blastocystis  spp.; these samples were analysed by PCR sequencing of regions of the  18S  and  PFOR  genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity.    Results  The  18S  analysis showed the following frequencies of  Blastocystis  subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed  Blastocystis  ST1 and ST2 infection. For  PFOR , Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the  18S  analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For  PFOR , a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower.    Conclusions  Although the fragment of the  PFOR  gene analysed in the present study did not allow discrimination between  Blastocystis  STs, this marker grouped the samples in three clades with strengthened support, suggesting that  PFOR  may be under different selective pressures and evolutionary histories than the  18S  gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among  Blastocystis  STs.

Can the pyruvate: ferredoxin oxidoreductase (PFOR) gene be used as an additional marker to discriminate among Blastocystis strains or subtypes?