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. 2022 Sep 29;22(1):757.
doi: 10.1186/s12879-022-07741-4.

Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection

Binod Rayamajhee  1 , Savitri Sharma  2 , Mark Willcox  3 , Fiona L Henriquez  4 , Raksheeth Nathan Rajagopal  5 , Gauri Shankar Shrestha  3 , Dinesh Subedi  6 , Bhupesh Bagga  5 , Nicole Carnt  3
Affiliations

Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection

Binod Rayamajhee et al. BMC Infect Dis. .

Abstract

Introduction: Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified.

Aims: This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations.

Methodology: Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH).

Results: The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively.

Conclusions: Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.

Keywords: Acanthamoeba; Endosymbionts; Genotyping; Keratitis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Map showing the states of the AK patients and LVPEI hospital in India. The map was created using ArcGIS (Esri GIS, California, USA). AP Andhra Pradesh, MA Maharashtra, RA Rajasthan, TE Telangana, TR Tripura, UP Uttar Pradesh
Fig. 2
Fig. 2
Structures of Acanthamoeba trophozoites (A) with acanthopodia, arrows indicate needle like projections on cell surface. B, confocal image of trophozoites’ nuclei stained with DAPI. C, D, phases of encystment of strain L-1326/20 from pre-cyst (C, D) to double-walled polygonal cysts (E), arrows indicate polygonal Acanthamoeba cysts. Scale bar, 10 µm
Fig. 3
Fig. 3
Cropped agarose gel image of PCR amplicons of Acanthamoeba isolates and intracellular bacteria. Bands were visualised using 1% gel electrophoresis; primer pairs JDPFw/Rv and 515Fw/806Rv yielded ~ 450 bp and ~ 293 bp amplicons, respectively. A. castellanii (ATCC 30868) and E. coli (ATCC 10798) were used as positive control for 18S rRNA and 16S rRNA PCR reactions and nuclease free water was used as negative control. Full size gel images are included in Additional file 1 (Figs. 4 and 5)
Fig. 4
Fig. 4
Phylogenetic tree inferred from the 18S rRNA sequences of Acanthamoeba isolates. The tree was created using the neighbour-joining approach with the Kimura 2-parameter based on 1,000 replicate bootstrap values. Acanthamoeba isolates (blue coloured) of this study formed two major genotypic clades; T4 was the predominant genotype (three sub-clusters: T4A, T4B, and T4F) and T12 had only one isolate, “*”denotes NCBI reference species and genotypes (purple coloured)
Fig. 5
Fig. 5
Representative images of FISH assay depicting intracellular bacteria A and fungi C of Acanthamoeba isolates. A Rod-shaped bacteria were dispersed throughout all the amoebal cells in the population which was detected using EUB338 probe (L-579/20, indicated by red arrows). B Acanthamoeba trophozoite (L-552/20) without intracellular bacteria or fungi. C Large ovoid fungal cells were observed within Acanthamoeba strain using PF2 probe (L-2429/20, indicated by yellow arrows). Scale bar, 12 µm
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