Diagnosis Of Acanthamoeba Keratitis Research Articles

Delayed diagnoses of Acanthamoeba keratitis at a tertiary care medical centre.

To determine the prevalence and reasons for delays in diagnosis in patients with Acanthamoeba keratitis (AK) presenting to Wilmer Eye Institute, Baltimore, Maryland. This retrospective study analysed all patients with culture-positive AK seen between 2012 and 2019 at a tertiary referral centre. Patient demographic information, clinical history, risk factors, symptom duration, referral patterns, slit lamp examination findings, visual acuity and need for surgery were collected. The study included 45 eyes of 43 patients. On average, patients were symptomatic for 52.6 days before culture collection. Thirty-one percent of patients were diagnosed within 28 days of symptom onset while 69% were diagnosed after 28 days. Before presentation to a tertiary care centre, 69% of patients were evaluated by an ophthalmologist outside of this institution and 27% were evaluated by a provider other than an ophthalmologist. AK was most commonly misdiagnosed as herpetic keratitis, occurring in 38% of patients. The strongest risk factor for AK was contact lens use. Only 11% of patients presented with the classic ring infiltrate and 82% had pain. Patients with an early versus late diagnosis had a mean Snellen visual acuity (VA) of 20/224 versus 20/296 at presentation (p = 0.33) and a mean Snellen VA of 20/91 versus 20/240 at final visit (p = 0.07). 11% of patients required a therapeutic penetrating keratoplasty. Delayed diagnosis of AK in our cohort occurred due to a misdiagnosis as herpetic keratitis, non-specific clinical signs including the lack of pain in a number of patients, and a delay in referral to a tertiary care centre. Any contact lens wearer with an atypical keratitis should be referred promptly for Acanthamoeba cultures.

Confocal microscopy in the diagnosis of acanthamoeba keratitis

To evaluate the capabilities of corneal confocal microscopy (CCM) in the diagnosis of acanthamoeba keratitis. We examined 33 patients (35 eyes) with suspected acanthamoeba keratitis using light and laser confocal microscopy of the cornea (ConfoScan and HRT devices, respectively). CCM was technically feasible in 23 of 35 cases (65.7%). Acanthamoeba cysts were detected in 16 cases (69.6%); trophozoites were visualized in 17.4% of cases; signs of keratoneuritis were detected in 12 eyes (52.2%); in 7 cases (30.4%), hyporeflective honeycomb-like cavities were observed in the stroma; deep stromal striae were observed in 9 cases (39.1%); activated keratocytes were detected in 11 patients (47.8%), and Langerhans cells were detected in 19 cases (82.6%). The main diagnostic value of confocal microscopy is the detection of direct markers of AK - cysts and trophozoites of Acanthamoeba. Both light and laser CCM techniques are suitable for the diagnosis of AK, but the resolution of laser confocal microscopy is higher. The non-invasive nature of this method and the possibility of multiple subsequent examinations make it possible to verify the diagnosis of acanthamoeba keratitis and monitor the treatment.

Production of a polyclonal antibody against inosine-uridine preferring nucleoside hydrolase of Acanthamoeba castellanii and its access to diagnosis of Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a rare disease but its prevalence throughout the globe continues to grow, primarily due to increased contact lens usage. Since early-stage symptoms associated with AK closely resemble those from other corneal infections, accurate diagnosis is difficult and this often results in delayed treatment and exacerbation of the disease, which can lead to permanent visual impairment. Accordingly, developing a rapid Acanthamoeba–specific diagnostic method is highly desired. In the present study, a rapid and differential method for AK diagnosis was developed using the secretory proteins derived from the pathogenic Acanthamoeba. Among the vast quantities of proteins secreted by the pathogenic Acanthamoeba, an open reading frame of the inosine-uridine preferring nucleoside hydrolase (IPNH) gene was obtained. After expressing and purifying the IPNH protein using the pGEX 4T-3 vector system, mice were immunized with the purified proteins for polyclonal antibody generation. Western blot was performed using protein lysates of the human corneal cell, non-pathogenic amoeba, pathogenic amoeba, and clinical amoeba isolate along with lysates from other causes of keratitis such as Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium solani to confirm Acanthamoeba-specificity. Western blot using the polyclonal IPNH antibody revealed that IPNH was Acanthamoeba-specific since these proteins were only observed in lysates of Acanthamoeba origin or its culture media. Our findings indicate that the IPNH antibody of Acanthamoeba may serve as a potential agent for rapid and differential AK diagnosis.

Comparison of two confocal microscopes for diagnosis of acanthamoeba keratitis

Comparison of two confocal microscopes for diagnosis of acanthamoeba keratitis

Acanthamoeba Keratitis and Acanthamoeba Conjunctivitis: A Case Report

Acanthamoeba species are vision-threatening agents by causing cornea infections known as Acanthamoeba keratitis. A 5 year-old kid with the complaints of erythema, eyelid edema, inflammation, limitation of eye movements in the right eye, and having no history of wearing contact lenses or trauma, was diagnosed of Acanthamoeba conjunctivitis through laboratory examinations in the Ophthalmology clinic. The visual sharpness of the patient improved after the treatment. A 44 year-old female patient suffering from pain, stinging, irritation, and inability to see in the left eye with the history of wearing contact lenses or trauma was diagnosed of Acanthamoeba keratitis through laboratory examinations. The agent was isolated and identified as "A. castellani" in the Genotype "T2". Examination of the left eye on the 15th day of treatment indicated that all complaints disappeared except for the cataract originated visual loss. However, the first diagnosis of Acanthamoeba keratitis appeared in the literature on a case with no history of wearing contact lenses and trauma it is found to be attention grabbing. We think that Acanthamoeba should not be ignored among microbial agents that cause eye infection with or without trauma and contact lens usage history.

A 5-Year Review of Acanthamoeba Keratitis Related to Wearing Contact Lenses in Korea.

To investigate the presentation, clinical characteristics, and outcomes of Acanthamoeba keratitis (AK) in Busan, South Korea, over a 5-year period. This retrospective study involved a review of the medical records of 16 patients (19 eyes in total) who were diagnosed with AK, related to wearing contact lenses, at the tertiary hospital, Pusan National University Hospital at Busan City, from December 2013 to December 2018. Nineteen eyes of 16 patients with a diagnosis of AK were identified. The average age of the patients was 21.1±12.6 years; there were 2 men and 14 women. The mean period from the onset of the first symptoms to diagnosis was 7.0±6.5 days. The average initial visual acuity was 0.78±0.37 (tested on a logarithm of the minimum angle of resolution chart), and the final visual acuity after treatment was 0.07±0.07, indicating a significant improvement (P=0.001). A variety of corneal lesions were identified. Early diagnosis of AK was associated with a significantly better final visual acuity. The average therapeutic period for AK, when a surface epithelial lesion of the cornea was identified, was 4 months compared with an average period of over 6 months for a deeper stromal lesion. Therefore, this study highlights the fundamental importance of early diagnosis, preventing deeper layers of the cornea from being affected, and appropriate management to ensure a favorable outcome.

Genotyping of Acanthamoeba Species Isolated from Keratitis Patients by PCR Sequencing Methods in Tehran, Iran

Background and Aims: Amoebae of the genus Acanthamoeba are unicellular amphizoic opportunistic pathogens that may cause fatal granulomatous encephalitis, eye keratitis, amebic pneumonitis and skin nodules as well as abscesses in humans and animals. Acanthamoeba keratitis is caused by trauma to the eye, contaminated cleaning solutions and the use of contact lenses. The aim of the present study was to identify the genotypes of Acanthamoeba in all patients with a clinical diagnosis of Acanthamoeba keratitis referring to eye clinic in Tehran using polymerase chain reaction (PCR). Materials and Methods: In this study, samples were collected from 35 patients who had referred to the eye clinic and were cultured on 1.5% non-nutrient agar. DNA was extracted, and then PCR amplification was performed using genus specific primers. Sequencing analysis and basic local alignment search tool search were conducted to determine the genotypes. Phylogenetic tree was generated using maximum likely algorithm in phylogenetic program MEGA version 6. Results: Eight cases were positive for Acanthamoeba using genus specific primer pairs. All specimens were reported as genotype T4. Conclusions: Determination of genotypes showed all isolates belonging to genotype T4; this abundance may be due to its higher prevalence in the environment or its greater virulence. However, further analysis of clinical and environmental samples is necessary to clarify this property.

Clinical features and outcomes of Acanthamoeba keratitis in a tertiary hospital over 20- year period

To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period. This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes. Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p=0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83mm, p=0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p=0.02) and dilated pupil/iris atrophy (p=0.01). Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma.

Clinical course of Acanthamoeba keratitis by genotypes T4 and T8 in Hungary.

Genus Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease, associated predominantly with contact lens (CL) wear; its epidemiology is related to the specific Acanthamoeba genotypes. This study reports seven CL wearer, Acanthamoeba PCR-positive patients with AK, diagnosed between January 2015 and 2018. Patients had the diagnosis of AK 1.36 months after first symptoms. Genotyping allowed the identification of six isolates of the T4 and one of the T8 genotypes. At first presentation, pseudendritiformic epithelopathy/dirty epithelium (four eyes, 57.1%), multifocal stromal infiltrates (five eyes, 71.4%), ring infiltrate (three eyes, 42.8%), and perineuritis (one eye, 14.3%) were observed. AK was healed without later recurrence in two eyes (28.5%) using triple-topical therapy, in three eyes (42.8%) following additional penetrating keratoplasty. In one patient (14.3%), AK recurred following successful application of triple-therapy and was treated successfully with repeated triple-topical therapy and in one patient (14.3%), no follow-up data were available after diagnosis. We could not observe correlation of genotype and clinical course or the necessity of corneal transplantation in our case series.

Reduction of Acanthamoeba Cyst Density Associated With Treatment Detected by In Vivo Confocal Microscopy in Acanthamoeba Keratitis.

Acanthamoeba keratitis (AK) is a severe vision-threatening ocular infection that is frequently a diagnostic challenge. Treatment course is lengthy and often not fully effective. Contact lens wear has been recognized as the prime risk factor for AK. In vivo confocal microscopy (IVCM) is a noninvasive imaging modality that allows direct visualization of potential causative pathogens in real time with an established utility in the diagnosis of AK. In this study, we aim to assess the utility of IVCM in monitoring disease progression in contact lens wearers with culture-confirmed keratitis. Fourteen eyes from 11 patients with culture-confirmed AK were included in this retrospective study. IVCM was performed during the patient's initial visit and all follow-up visits. All available confocal sequences were reviewed and graded in a masked fashion. Density of Acanthamoeba cyst infiltration and changes in the cyst density as a percentage of baseline cyst density measured at each patient's initial visit were calculated. A univariate regression analysis was performed to assess the association between treatment and changes in cyst density per month of treatment. Acanthamoeba cysts were identified by IVCM in all of these culture-confirmed cases of keratitis. Mean cyst density in the central cornea at presentation was 99 ± 64.9 cells per square millimeter (range, 38-255/mm). Cyst density in our study population significantly decreased by approximately 5.3% with each month of antiamebic treatment (P = 0.001; R = 0.41). Reduction in Acanthamoeba cyst density with treatment can be monitored by IVCM, which in turn can be used clinically in prognostication and disease monitoring of AK.

<em>Acanthamoeba</em> keratitis: a challenge in diagnosis and the role of amniotic membrane transplant as an alternative therapy

Acanthamoeba keratitis (AK) is a very rare, sight-threatening corneal disease caused by the infection of a free-living amoeba. The aim of this report was to demonstrate the challenges in the diagnosis of AK with an atypical presentation. Amniotic membrane transplantation (AMT) was done in this case to accelerate wound healing. A woman who frequently used contact lens came with redness and a whitish spot in her left eye 2 weeks before admission. There was a history of rinsing contact lens in tap water. The patient had already been on a topical steroid for 2 weeks. Visual acuity of the left eye was light perception with wrong projection. Examination of the left cornea showed a semi-circular central ulcer with a 5.8-mm area and 1/3 stromal depth. It is surrounded by a feathery-edge infiltrate with positive satellite lesions but without hypopyon. It was diagnosed as corneal ulcers due to mixed infection (bacteria and fungi). After 2 weeks of bacterial and fungal therapy, the size of the ulcer decreased, but the corneal infiltrate remained; therefore, an Acanthamoeba culture was done to determine the possibility of Acanthamoeba infection. Two weeks after the Acanthamoeba topical therapy, the visual acuity improved. AMT was performed to accelerate wound healing. Two months post-AMT, visual acuity improved to 1/60 with no conjunctival or ciliary vasodilation, and the ulcer was resolved. The patient was planned to undergo keratoplasty. The diagnosis of AK with an atypical presentation is challenging. AMT can be used as an alternative therapy to accelerate wound healing.

Risk factors and clinical signs of severe Acanthamoeba keratitis.

PurposeTo determine risk factors and clinical signs for severe Acanthamoeba keratitis (AK) by comparing severe cases with mild cases with good prognosis.Patients and methodsWe reviewed medical records of ten cases of AK (five males and five females) referred to our hospital and classified cases into two groups. One eye that required therapeutic keratoplasty and three eyes with a poor visual acuity (<0.2) on last visit were included in the severe group. Six eyes that had good prognosis with a visual acuity of 1.2 on last visit were classified as mild group. We compared patients’ age, the time required for diagnosis, visual acuity on first visit, the history of steroid eye drops use, and other clinical findings.ResultsThe average age of the severe group was older than the mild group (P=0.04). The duration between onset and diagnosis of AK and visual acuity on first visit was not statistically different. A history of steroid eye drop use was found in four eyes of the severe group (100%) and four eyes of the mild group (67%). Keratoprecipitates were found in all severe group eyes and one mild group eye during follow-up (P=0.01). One case in the severe group was diagnosed with diabetes mellitus at initial examination. We detected Staphylococcus epidermis by palpebral conjunctival culture in one case of the severe group.ConclusionAging may be a possible risk factor for severe AK. The presence of keratoprecipitates is a possible sign of severe AK. Attention is also required in patients with comorbidities such as diabetes mellitus and bacterial infection.

Effectiveness of sampling methods employed for Acanthamoeba keratitis diagnosis by culture.

This retrospective, observational study was designed to evaluate the effectiveness of the sampling methods commonly used for the collection of corneal scrapes for the diagnosis of Acanthamoeba keratitis (AK) by culture, in terms of their ability to provide a positive result. A total of 553 samples from 380 patients with suspected AK received at the Parasitology Section of the Public Health Institute of Chile, between January 2005 and December 2015, were evaluated. A logistic regression model was used to determine the correlation between the culture outcome (positive or negative) and the method for sample collection. The year of sample collection was also included in the analysis as a confounding variable. Three hundred and sixty-five samples (27%) from 122 patients (32.1%) were positive by culture. The distribution of sample types was as follows: 142 corneal scrapes collected using a modified bezel needle (a novel method developed by a team of Chilean corneologists), 176 corneal scrapes obtained using a scalpel, 50 corneal biopsies, 30 corneal swabs, and 155 non-biological materials including contact lens and its paraphernalia. Biopsy provided the highest likelihood ratio for a positive result by culture (1.89), followed by non-biological materials (1.10) and corneal scrapes obtained using a modified needle (1.00). The lowest likelihood ratio was estimated for corneal scrapes obtained using a scalpel (0.88) and cotton swabs (0.78). Apart from biopsy, optimum corneal samples for the improved diagnosis of AK can be obtained using a modified bezel needle instead of a scalpel, while cotton swabs are not recommended.

Factors Influencing the Diagnostic Accuracy of Laser-Scanning In Vivo Confocal Microscopy for Acanthamoeba Keratitis.

To determine the factors that influence the sensitivity and specificity of laser-scanning in vivo confocal microscopy (IVCM) for diagnosing Acanthamoeba keratitis (AK). This retrospective, controlled study included 28 eyes of 27 patients with AK and 34 eyes of 34 patients with bacterial keratitis (as the control group). All patients had undergone corneal imaging with a laser-scanning IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module). The IVCM images were independently evaluated by 2 experienced and 2 inexperienced masked observers. Sensitivity and specificity of IVCM for diagnosing AK and the effects of various clinical and imaging parameters on the sensitivity were then investigated. Overall, IVCM had average sensitivity and specificity of 69.7% ± 2.5% and 97.1% ± 4.2% for experienced observers and 59.0% ± 7.6% and 92.7% ± 10.4% for inexperienced observers, respectively. However, the sensitivity did not show any significant association with the duration of disease, size of ulcer, depth of involvement, culture results, or cyst morphology. Although interobserver agreement was good (κ = 0.60, P < 0.001) for the experienced observers, it was only at a moderate level (κ = 0.48, P < 0.001) for the inexperienced observers. IVCM has a moderate sensitivity and a high specificity for diagnosis of AK. Although clinical parameters do not affect this diagnostic accuracy, a higher sensitivity is seen when images are interpreted by experienced observers.

Acanthamoeba keratitis in 194 patients: risk factors for bad outcomes and severe inflammatory complications

Background/aimsTo determine demographic and clinical features of patients with Acanthamoeba keratitis (AK) that are independent risk factors both for bad outcomes and for severe inflammatory complications (SIC).MethodsA retrospective audit of...

Comparison of In Vivo Confocal Microscopy, PCR and Culture of Corneal Scrapes in the Diagnosis of Acanthamoeba Keratitis.

Acanthamoeba keratitis (AK) is an uncommon but serious corneal infection, in which delayed diagnosis carries a poor prognosis. Conventional culture requires a long incubation period and has low sensitivity. Polymerase chain reaction (PCR) and in vivo confocal microscopy (IVCM) are available alternative diagnostic modalities that have increasing clinical utility. This study compares confocal microscopy, PCR, and corneal scrape culture in the early diagnosis of AK. We reviewed the case notes of patients with a differential diagnosis of AK between June 2016 and February 2017 at the Bristol Eye Hospital, United Kingdom. Clinical features at presentation, and results of IVCM, PCR, and corneal scrape cultures were analyzed. A total of 25 case records were reviewed. AK was diagnosed in 14 patients (15 eyes). Based on the definition of "definite AK," the diagnostic sensitivities of IVCM, PCR, and corneal scrape cultures were 100% [95% confidence interval (CI), 63.1%-100%], 71.4% (95% CI, 41.9%-91.6%) and 33.3% (95% CI, 9.9%-65.1%), respectively. The 3 methods showed a specificity of 100% and a positive predictive value of 100%. Using a reference standard of only positive corneal cultures, IVCM, and PCR had a sensitivity of 100% (95% CI, 29.2%-100%) and 75% (95% CI, 19.4%-99.4%), respectively. All 3 diagnostic tests are highly specific, and a positive test result is highly predictive of disease presence. IVCM is both highly sensitive and specific when performed by an experienced operator. PCR is a useful adjunct in the diagnosis of AK because of its wider availability compared with IVCM, and it may be used in combination with IVCM for microbiologic confirmation.

Assessment of Confocal Microscopy for the Diagnosis of Polymerase Chain Reaction–Positive Acanthamoeba Keratitis: A Case-Control Study

To determine invivo confocal microscopy diagnostic criteria to diagnose Acanthamoeba keratitis (AK) using polymerase chain reaction (PCR) as the reference diagnostic technique. Retrospective case-control study. Data were recorded prospectively and analyzed retrospectively. Fifty patients with PCR-positive AK (study group) and 50 patients with bacterial, fungal, viral, or immune keratitis featuring negative Acanthamoeba PCR results (control group). Invivo confocal microscopy performed at the acute stage of keratitis. Presence of invivo confocal microscopy images suggestive of AK. Multivariate logistic regression was used to determine the relationship between types of images and presence of PCR-positive AK. The following 4 types of images were associated significantly with PCR-positive AK (P < 0.05): bright spots (round or ovoid hyperreflective objects with no double wall; diameter, <30 μm); target images (hyperreflective objects with hyporeflective halo; diameter, <30 μm); clusters of hyperreflective objects (diameter, <30 μm); and trophozoite-like objects (diameter, >30 μm). Specificity of both target and trophozoite images was 100%. This figure was 98.2% for clusters and 48.2% for bright spots. If the diagnosis of AK was made on presence of target images, clusters or trophozoite images (at least 1 of the 3 features), the positive predictive value of confocal microscopy was 87.5% and the negative predictive value was 58.5%. Acanthamoeba keratitis is a serious vision-threatening disease. Invivo confocal microscopy can help in this challenging diagnosis, especially when PCR is delayed, shows negative results, or is not available. Target images and trophozoite-like images are pathognomonic of AK. Clusters of hyperreflective objects are highly specific of AK. However, the overall sensitivity of invivo confocal microscopy features of AK is low. In addition to the clinical features, microbiological tests (direct examination and cultures of corneal scrapings), and PCR, invivo confocal microscopy allows for more rapid diagnosis and treatment initiation, potentially leading to an improved outcome.

Molecular diagnosis of Acanthamoeba keratitis: evaluation in rat model and application in suspected human cases.

Acanthamoeba keratitis (AK) is a progressive corneal infection that demands rapid and sensitive techniques for diagnosis to avoid risk of visual impairment. We evaluated two DNA extraction techniques and a semi-nested-PCR (snPCR) targeting the 18S rRNA gene to detect Acanthamoeba cysts and trophozoites. The most effective protocol was evaluated in samples of corneal scrapings and biopsies from an AK rat model and applied to diagnosis of human cases of AK. DNA extraction performed with a commercial kit based on DNA binding to magnetic beads was more efficient than a method based on alkaline lysis, allowing the detection of one trophozoite and one cyst of Acanthamoeba in samples prepared from cultures. This technique and sn-PCR were applied in corneal scrapings of rats experimentally infected with Acanthamoeba (n=6), resulting in 100% of positivity, against 16.7% (n=6) of positive identification in culture method using non-nutrient agar (NNA) with Escherichia coli. Corneal biopsies from rats were also tested (n=6) and resulted in positivity in all samples in both molecular and culture methods. Eight out of ten presumptive human cases of Acanthamoeba keratitis were also confirmed by sn-PCR of scrapping samples, while the culture method was positive in only four cases. We discuss that animal model of AK can be an efficient tool to validate diagnostic methods and conclude that DNA extraction with the kit and snPCR protocol described here is an effective alternative for diagnosis of AK.

Evaluation of Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Acanthamoeba Keratitis

Background: The clinical features of Acanthamoeba keratitis (AK) are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP) with microscopy, non-nutrient agar (NNA) culture and polymerase chain reaction (PCR) in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis), 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%), and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%). The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings

A Multiplex Dot Hybridization Assay For Detection and Differentiation of Acanthamoeba and Herpes Keratitis.

We verified a multiplex dot hybridization (MDH) assay for the rapid detection and differentiation of Acanthamoeba keratitis (AK) and herpes simplex keratitis (HSK). Molecular detection of Acanthamoeba and herpes simplex virus in corneal scrapes was performed with the MDH assay and standard diagnostic methods. The experimental group included corneal scrapes (n = 33) from patients with culture- or pathology-confirmed AK (n = 15) and real-time PCR-confirmed HSK (n = 16). The control group included 50 samples from cases of bacterial keratitis (n = 15), fungal keratitis (n = 15), and initially presumed AK (n = 5) or HSK (n = 17) which finally were excluded by culture for Acanthamoeba or by real-time PCR for herpes simplex virus, respectively. Discrepant results between methods were resolved by DNA sequencing of the PCR amplicons. After discrepant analysis, the sensitivity for the diagnosis of AK and HSK was both 93.3% by the MDH assay, while the specificity was 100% for the two types of keratitis. The turnaround time of MDH assay was within a working day using an already prepared array. Two false-negatives (one AK case and one HSK case) were obtained by the MDH assay. The MDH assay could effectively prevent missed or delayed diagnosis of AK and HSK and has a potential to be adopted in routine clinical practice if the test is commercialized.