First evidence of genetic intraspecific variability and occurrence of Entamoeba gingivalis in HIV(+)/AIDS

Abstract

Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4(+) lymphocyte count (≤200 cells/mm(3) (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(-) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4(+) lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis isolated from HIV patients.

Figure 1. LSSP-PCR. Polyacrylamide gel (7.5%) stained with silver nitrate.

Primers: A) EGO-1 and B) EGO-2. Lane 1: molecular marker 1 Kb (Invitrogen); lanes 2–25: HIV(+)/AIDS samples; lane 26: negative control.

Figure 2. Analysis of the genetic variability of the small ribosomal subunit in the 18S rRNA of E. gingivalis in clinical samples from HIV(+)/AIDS patients, detected by LSSP-PCR.

M = male; F = female; CD4 =  CD4⁺ cell count; HIV− = HIV negative; UR = unrealized.

Figure 3. Phylogenetic relationship of Entamoeba spp. from HIV infected patients.

Neighbor-joining method was performed in 24 taxa at the SSU rRNA locus. The optimal tree with the sum of branch length = 1.32 is shown and the bootstrap values were added to phylogenetic branches.

Figure 4. Single nucleotide polymorphisms in E. gingivalis isolates.