Utility of Polymerase chain reaction in diagnosis of Acanthamoeba and Microsporidial keratitis

Bhosale, Namrata K.; Mandal, Jharna; Sc, Parija; Ahuja, Shashi

doi:10.1016/j.ijid.2016.02.764

Cited by 3 publications

(4 citation statements)

References 15 publications

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“…Standard laboratory method for the diagnosis of AK is mainly based on direct microscopy of corneal scrape smears and contact lens solution. Culture of these specimens on non-nutrient agar overlaid with Escherichia coli can be successful ( 13 ). In spite of high specificity of conventional methods; it requires technical experience with morphology of cysts and trophozoites of Acanthamoeba spp .…”

Section: Introductionmentioning

confidence: 99%

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay

Khosravinia

Fata

Moghaddas

et al. 2021

IJPA

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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.

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Section: Introductionmentioning

confidence: 99%

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay

Khosravinia

Fata

Moghaddas

et al. 2021

IJPA

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“…Microscopic-based detection methods are widely used in many countries to detect Acanthamoeba ; however, these assays are labour intensive, time-consuming, and require Acanthamoeba culturing for up to several days [ 2 , 3 , 4 ]. Recent advances in the Acanthamoeba detection technique employs polymerase chain reaction (PCR) assay for the sensitive and rapid detection of Acanthamoeba parasites at the genotypic-level [ 5 , 6 ]. PCR technique involves three major steps: denaturation, annealing, and extension to amplify DNA.…”

Section: Introductionmentioning

confidence: 99%

“…The DNA polymerase is the crucial ingredient in PCR to synthesize new DNA strands. Despite 90% sensitivity and 90.8% specificity of PCR assay in Acanthamoeba detection [ 3 , 4 , 5 , 6 ], the challenges of PCR technique include insufficient target DNA yield and non-specific DNA amplification leading to possible band smearing on gels [ 7 ]. Previously, Acanthamoeba detection used hot-start DNA polymerase enzymes to modify the conventional PCR techniques, avoiding non-specific amplification, and increasing the target DNA yield by inactivating the enzyme at lower temperatures [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

hBN Nanoparticle-Assisted Rapid Thermal Cycling for the Detection of Acanthamoeba

et al. 2020

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Acanthamoeba are widely distributed in the environment and are known to cause blinding keratitis and brain infections with greater than 90% mortality rate. Currently, polymerase chain reaction (PCR) is a highly sensitive and promising technique in Acanthamoeba detection. Remarkably, the rate of heating–cooling and convective heat transfer of the PCR tube is limited by low thermal conductivity of the reagents mixture. The addition of nanoparticles to the reaction has been an interesting approach that could augment the thermal conductivity of the mixture and subsequently enhance heat transfer through the PCR tube. Here, we have developed hexagonal boron nitride (hBN) nanoparticle-based PCR assay for the rapid detection of Acanthamoeba to amplify DNA from low amoeba cell density. As low as 1 × 10−4 wt % was determined as the optimum concentration of hBN nanoparticles, which increased Acanthamoeba DNA yield up to ~16%. Further, it was able to reduce PCR temperature that led to a ~2.0-fold increase in Acanthamoeba DNA yield at an improved PCR specificity at 46.2 °C low annealing temperature. hBN nanoparticles further reduced standard PCR step time by 10 min and cycles by eight; thus, enhancing Acanthamoeba detection rapidly. Enhancement of Acanthamoeba PCR DNA yield is possibly due to the high adsorption affinity of hBN nanoparticles to purine (Guanine—G) due to the higher thermal conductivity achieved in the PCR mixture due to the addition of hBN. Although further research is needed to demonstrate these findings in clinical application, we propose that the interfacial layers, Brownian motion, and percolation network contribute to the enhanced thermal conductivity effect.

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“…In addition, amoeba culture is only positive in around 55% of the cases, and the quantity of the obtained sample could be not enough to see the protozoa. PCR (polymerase chain reaction) testing is a rapid detection technique of Acanthamoeba parasites at the genotypic-level (T1-T23) [ 10 ]. Acanthamoeba genotyping is gaining importance at the treatment level because different genotypes show variation in symptoms and response to the treatment [ 11 ].…”

mentioning

confidence: 99%

New Insights in Acanthamoeba

et al. 2022

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Utility of Polymerase chain reaction in diagnosis of Acanthamoeba and Microsporidial keratitis

Cited by 3 publications

References 15 publications

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay

hBN Nanoparticle-Assisted Rapid Thermal Cycling for the Detection of Acanthamoeba

New Insights in Acanthamoeba

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