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Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay
Nazgol Khosravinia1, Abdolmajid Fata1,2, Elham Moghaddas2
1Department of Medical Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Background:
The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene.
Methods:
Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique. Sensitivity, specificity, accuracy and likelihood ratio were evaluated.
Results:
Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype.
Conclusion:
Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.
Insights
Acanthamoeba keratitis, a serious eye infection, is increasingly diagnosed using PCR, which proved more sensitive than microscopy or culture. The study identified the T4 genotype as prevalent in Mashhad, Iran.
Area of Science:
- Ophthalmology
- Infectious Diseases
- Molecular Biology
Background:
- Acanthamoeba is a protozoan parasite found in soil and water.
- Acanthamoeba keratitis (AK) is a severe corneal infection with rising incidence globally and in Iran.
- Early and accurate diagnosis is crucial for managing AK.
Purpose of the Study:
- To compare the diagnostic efficacy of direct microscopy, culture, and PCR for Acanthamoeba detection in clinical samples.
- To determine the genotypes of Acanthamoeba species causing keratitis in Mashhad, Iran, using 18S rRNA gene sequencing.
Main Methods:
- Corneal scrape specimens from 20 patients suspected of AK were analyzed.
- Samples were subjected to direct microscopy, culture on non-nutrient agar, and PCR (Nelson-PCR and JDP-PCR).
- Diagnostic performance metrics (sensitivity, specificity, accuracy) were evaluated, and 18S rRNA gene sequencing was performed for genotyping.
Main Results:
- Acanthamoeba keratitis was confirmed in 13 (69.2%) of the 20 suspected patients.
- Contact lens wear, eye trauma, and swimming in fresh water were identified as primary risk factors.
- PCR methods (Nelson-PCR and JDP-PCR) demonstrated superior sensitivity and accuracy compared to direct microscopy and culture.
- Sequencing revealed all identified Acanthamoeba strains belonged to the T4 genotype.
Conclusions:
- Amoebic keratitis is a significant cause of parasitic keratitis, particularly among contact lens users in Mashhad.
- PCR, specifically using 18S ribosomal DNA with JDP primers, is a highly sensitive and reliable diagnostic tool for AK.
- The T4 genotype is the predominant genotype associated with AK in this region.
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