Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains

Abstract

Toxoplasma gondii is an important food- and waterborne opportunistic pathogen that causes severe disease in immunocompromised patients. T. gondii has an unusual clonal population structure consisting of three widespread lineages known as I, II, and III. To establish the genotypes of strains of T. gondii associated with human toxoplasmosis, we have developed a set of four highly sensitive and polymorphic nested PCR markers. Multiplex nested PCR analysis was used to genotype parasites in cerebral spinal fluid samples from 8 of 10 human immunodeficiency virus-positive patients. Remarkably, a majority of these patients had infections with type I strains or strains containing type I alleles, despite the fact that this lineage is normally uncommon in humans and animals. Multiplex analysis of these four unlinked makers was able to distinguish all three common genotypes and also detected two strains with mixed genotypes. Further analysis based on sequencing of a polymorphic intron revealed that one of these recombinant strains was an exotic lineage distinct from the archetypal clonal lineages. The multiplex nested PCR analysis described here will be useful for analyzing the contribution of parasite genotype to toxoplasmosis.

FIG. 1.

Nested PCR amplification of markers (5′-SAG2, 3′-SAG2, SAG3, BTUB, and GRA6) from control CSF spiked with parasites. The center four lanes of each group represent the samples spiked with 1, 2.5, 5, and 10 freshly isolated parasites added to negative CSF, respectively. The negative controls (−) correspond to water blanks (first and last lane of each group). Products were resolved on a 1% agarose gel stained with ethidium bromide. Lanes M, molecular mass markers from φX174 digested with HaeIII.

FIG. 2.

RFLP analysis of type strains and a mixed clinical isolate of T. gondii using 4 nested PCR markers. Agarose gel electrophoresis of undigested and restriction-digested products for type strains (type I RH, type II Me49, and type III CTG; lanes I, II, and III, respectively) and a mixed-genotype isolate (lanes WU05) is shown. Products were resolved on 3% agarose gels stained with ethidium bromide. Lanes: Neg, negative PCR control; M, molecular mass markers corresponding to φX 174 digested with HaeIII. SAG2 typing was based on a previously reported method (13). GRA6 typing was adapted from a previously reported method (8). SAG3 typing was adapted from a previously reported method (11).