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. 2016 Jul 14;10(7):e0004841.
doi: 10.1371/journal.pntd.0004841. eCollection 2016 Jul.

Allovahlkampfia spelaea Causing Keratitis in Humans

Mohammed Essa Marghany Tolba  1   2 , Enas Abdelhameed Mahmoud Huseein  1 , Haiam Mohamed Mahmoud Farrag  1   3 , Hanan El Deek Mohamed  1 , Seiki Kobayashi  4 , Jun Suzuki  5 , Tarek Ahmed Mohamed Ali  6 , Sumio Sugano  2
Affiliations

Allovahlkampfia spelaea Causing Keratitis in Humans

Mohammed Essa Marghany Tolba et al. PLoS Negl Trop Dis. .

Abstract

Background: Free-living amoebae are present worldwide. They can survive in different environment causing human diseases in some instances. Acanthamoeba sp. is known for causing sight-threatening keratitis in humans. Free-living amoeba keratitis is more common in developing countries. Amoebae of family Vahlkampfiidae are rarely reported to cause such affections. A new genus, Allovahlkampfia spelaea was recently identified from caves with no data about pathogenicity in humans. We tried to identify the causative free-living amoeba in a case of keratitis in an Egyptian patient using morphological and molecular techniques.

Methods: Pathogenic amoebae were culture using monoxenic culture system. Identification through morphological features and 18S ribosomal RNA subunit DNA amplification and sequencing was done. Pathogenicity to laboratory rabbits and ability to produce keratitis were assessed experimentally.

Results: Allovahlkampfia spelaea was identified as a cause of human keratitis. Whole sequence of 18S ribosomal subunit DNA was sequenced and assembled. The Egyptian strain was closely related to SK1 strain isolated in Slovenia. The ability to induce keratitis was confirmed using animal model.

Conclusions: This the first time to report Allovahlkampfia spelaea as a human pathogen. Combining both molecular and morphological identification is critical to correctly diagnose amoebae causing keratitis in humans. Use of different pairs of primers and sequencing amplified DNA is needed to prevent misdiagnosis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Morphological characters of trophozoite and cyst.
(A) Trophozoite showing unidirectional movement (arrows) and cyst aggregating to each other (arrow head) (inverted microscope using x20 objective lens). (B) Cysts in 10 days old culture (inverted microscope using x20 objective lens). (C) Trophozoite showing filopodia (arrow) (oil immersion x100 objective lens). (D) Cysts with perinuclear clear ring (x40 objective lens)
Fig 2
Fig 2. PCR amplification of 18S ribosomal RNA gene.
(A) Lane (1) shows faint band of JDP1 & JDP2 primers, lane (2) shows band amplified using short universal primers, while lane (3) shows band specific for 5.8S ITS with auxiliary band (arrow) indicating long variant. (B) Amplification of whole 18S gene using 3 pairs of primers, bands appear at expected molecular weight calculated during primer design.
Fig 3
Fig 3. Phylogenetic tree of Allovahlkampfia spelaea Egyptian eye strain in relation to other free living amoebae.
(A) Tree created using whole length assembled sequence showing close relation to SK1 strain. (B) Tree created using 5.8S ITS sequence showing close relation to SK1 strain.
Fig 4
Fig 4. Allovahlkampfia spelaea caused definite ulcer to rabbit eye (5 days pi).
(A) Rabbit’s eye stained with methylene blue showing ulcer. (B) Rabbit’s eye examined with slit lamp after fluorescein staining to show ulcer (conjunctiva show mild congestion).
See this image and copyright information in PMC

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