New highly divergent rRNA sequence among biodiverse genotypes of Enterocytozoon bieneusi strains isolated from humans in Gabon and Cameroon

Abstract

Intestinal microsporidiosis due to Enterocytozoon bieneusi is a leading cause of chronic diarrhea in severely immunocompromised human immunodeficiency virus (HIV)-positive patients. It may be a public health problem in Africa due to the magnitude of the HIV pandemic and to poor sanitary conditions. We designed two prevalence studies of E. bieneusi in Central Africa, the first with HIV-positive patients from an urban setting in Gabon and the second with a nonselected rural population in Cameroon. Stool samples were analyzed by an immunofluorescence antibody test and PCR. Twenty-five out of 822 HIV-positive patients from Gabon and 22 out of 758 villagers from Cameroon were found to be positive for E. bieneusi. The prevalence rates of the two studies were surprisingly similar (3.0% and 2.9%). Genotypic analysis of the internal transcribed spacer region of the rRNA gene showed a high degree of diversity in samples from both countries. In Gabon, 15 isolates showed seven different genotypes: the previously reported genotypes A, D, and K along with four new genotypes, referred to as CAF1, CAF2, CAF3, and CAF4. In Cameroon, five genotypes were found in 20 isolates: the known genotypes A, B, D, and K and the new genotype CAF4. Genotypes A and CAF4 predominated in Cameroon, whereas K, CAF4, and CAF1 were more frequent in Gabon, suggesting that different genotypes present differing risks of infection associated with immune status and living conditions. Phylogenetic analysis of the new genotype CAF4, identified in both HIV-negative and HIV-positive subjects, indicates that it represents a highly divergent strain.

FIG. 1.

Sequence alignment of the ITS region of the rRNA gene of E. bieneusi genotypes using the Multalin software. New sequences from Gabon and Cameroon (CAF1 to CAF4) are compared to sequences representative of each group. (Representative sequences are the following: group 1, subgroup 1a, genotype D; group 1, subgroup 1b, genotype Peru6; group 1, subgroup 1c, genotype K; group 1, subgroup 1d, genotype E; group 1, subgroup 1e, genotype O; group 1, subgroup 1f, genotype C; group 2, genotype N; group 3, genotype WL4; group 4, genotype WL1; and the outlier sequence EntCanA). Dots indicate identity to CAF1. Point mutations of the new sequences CAF1, CAF2, and CAF3 compared to their closest homologues, K and D, are shaded gray (for accession numbers, see Table 2) (note that the upper ruler indicates the true sequence position for group 1 genotypes [243 bp], and the lower ruler indicates the Multalin alignment position number, with gaps counted).

FIG. 2.

Unrooted phylogram inferred by the maximum-likelihood analysis of the ITS of E. bieneusi genotypes. The phylogram was constructed using the Phyml software. Groups and subgroups are indicated in parentheses. New genotypes from this study are in boldface (for accession numbers, see Table 2).

FIG. 3.

Representation of group and host associations of the phylogenetic tree inferred by the maximum-likelihood analysis of the ITS of E. bieneusi genotypes. The tree was constructed using the Phyml software. Groups are circled with a solid line; subgroups composing group 1 are delineated by a dotted line. New genotypes from this study are denoted by an open circle. Only a few genotype names are indicated for clarity and to permit comparison with the positions in the phylogram shown in Fig. 2. Abbreviations: CanA, EntCanA; Pig7, EBITSPig7. For accession numbers, see Table 2. Hosts were classified as HIV-positive or HIV-negative humans, commensal mammals (cat, dog, cattle, swine, and zoo animals), commensal birds (chicken, urban park pigeon, pet, and zoo birds), and wild mammals (beaver, otter, muskrat, fox, and raccoon).