Identifying and quantifying genotypes in polyclonal infections due to single species

Abstract

Simultaneous infection with multiple pathogens of the same species occurs with HIV, hepatitis C, Epstein-Barr virus, dengue, tuberculosis, and malaria. However, available methods do not distinguish among or quantify pathogen genotypes in individual patients; they also cannot test for novel insertions and deletions in genetically modified organisms. The strategy reported here accomplishes these goals with real-time polymerase chain reaction (PCR) and capillary electrophoresis. Real-time PCR with allotype-specific primers defines the allotypes (strains) present and the intensity of infection (copy number). Capillary electrophoresis defines the number of genotypes within each allotype and the intensity of infection by genotype. This strategy can be used to study the epidemiology of emerging infectious diseases with simultaneous infection by multiple genotypes, as demonstrated here with malaria. It also permits testing for insertions or deletions in genetically modified organisms that may be used for bioterrorism.

Figure 1

Use of capillary electrophoresis to identify multiple genotypes within single allotypes amplified by real-time polymerase chain reaction. Panel A shows the relative fluorescence values for 3 samples from infected patients by using primers specific for the K1 allotype of merozoite surface protein 1 (msp1). Panels B, C, and D show that those samples contained 3, 4, and 1 different K1 genotype parasites, respectively, identified by amplicons of 106, 124, and 142 bp (panel B), 105, 124, 142, and 160 bp (panel C), and 160 bp (panel D), respectively. The first and last peaks on each electropherogram are the 15- and 600-bp standard markers used to define the sizes of the unknown amplicons.

Figure 2

Copy numbers for genotypes of the K1 allotype in 10 field samples. Distribution of K1 genotypes within the 8 patients whose samples yielded amplicons with K1-specific primers (Table 4). These results indicate that most infected persons had >2 allotypes. In addition, persons with K1 allotype parasites had a high degree of genotypic complexity, that is, capillary electrophoresis showed up to 4 distinct K1 genotypes in the blood of individual patients at the same time.

Figure 3

Capillary electrophoresis separation of polymerase chain reaction amplicons differing by 5 bp.

Figure 4

Comparison of amplicon concentration based on relative fluorescence from real-time polymerase chain reaction with peak area from capillary electrophoresis.