الفهرس 
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Abstract
Acanthamoeba keratitis is a sight-threatening infection of the ocular surface that is produced by several free living amoeba of the genus Acanthamoeba Patients often present with pain, photophobia, unilateral red eye with epiphora, and irritation. Acanthamoebae have two stages in their life cycle: a vegetative or trophozoite stage that reproduces by binary fission and feeds on bacteria and detritus present in the environment, and a non dividing cyst stage with a double cyst wall, providing it with a high resistance to unfavoured and adverse environmental conditions, desiccation and disinfecting compounds, e.g. chlorine
Diagnosis of AK is based on both clinical features and laboratory tests. Definitive diagnosis of AK relies on the demonstration of trophozoite or cyst isolated from cultures of corneal scrapings, lenses and lens cases solutions on non-nutrient agar overlaid with E. coli
The current study aims at assessing the role of laboratory tests including direct smear, staining, culture and PCR in diagnosis of acanthamoebiasis in clinically suspected cases of keratitis including contact and non contact lens wearers and its correlation with different risk factors.
The study was carried out on 110 patients attending the ophthalmology outpatient clinic of Benha University Hospital who were clinically suspected to have Acanthamoeba keratitis and samples wear examined in research institute of ophthalmology in Giza (as the scraped material was placed in a transport medium (Page’s saline) before submitted to the culture). They were divided into 2 groups according to contact lens use as contact lens wearers (CLW) 63 cases (55 females and 8 males, and their age ranged from 14 to 40 years) and non contact lens wearers (non CLW) 47cases (21 females and 26 males, and their age ranged from 2 to 58 years).
The obtained samples including corneal scrapings from all patients (110), 32contact lenses, 32 contact lens storage cases and solutions belonging to symptomatic CLW were subjected to direct examination, cultivation on non nutrient agar seeded with E-coli, staining methods using trichrome and Giemsa stains and direct amplification of Acanthamoeba DNA bypassing nucleic acid extraction using a commercial KAPA PCR kit.
Based on the results of the present study, it can be summarized into:
1- The results of the present study showed that Acanthamoeba infection was detected in 21 (19.1%) of clinically suspected cases (110); 17 (81%) of them were CLW and the remaining 4 (19%) positive cases were NCLW. These results revealed a significant association between Acanthamoeba keratitis and wearing of contact lenses (P <0.05) which appears to be an important risk factor in infection.
2- Regarding to different samples obtained from CLW, 63 corneal scrapings, 32 contact lenses, 32 contact lens storage cases and 32 contact lens solutions, there were 17(27%), 4(12.5%), 3 (9.4%), 3 (9.4%) positive samples for Acanthamoeba respectively. The difference between sources of sampling and detection of Acanthamoeba was statistically highly significant (P =0.001).
3- The results revealed that the correlation between host factors (age, sex and residence) and Acanthamoeba infection among keratitic patients was statistically insignificant. The highest Acanthamoeba infection occurred in female keratitic patients aging 20- 30 years (47.6%) as most of them were CLW. Acanthamoeba keratitis is more prevalent in patients from urban areas (66.7%) than those from rural areas (33.3%).
4- Different risk factors are associated with Acanthamoeba keratitis including trauma (4.76%), foreign body (4.76%), abrasion (4.76%) , history of swimming in swimming pools or canals (4.76%) and contact lens which is the commonest factor (80.95%).
5- Regarding the diagnostic methods used in this study for detection of Acanthamoeba in corneal scrapings of CLW & NCLW patients, Out of total 110 cases, one case was positive for Acanthamoeba (0.9%) by direct smear, 19 cases were positive by culture (17.3%). While on using PCR, there were 21 positive cases (19.1%). The differences in detection rates between culture and direct smear were highly statistically significant (P= 0.001).On the other hand, the differences in detection rates between culture and PCR were not statistically significant (P=0.86). The variability of detection rates may be attributed to variable sensitivity of different diagnostic methods and technical expertise of the personnel performing the diagnosis.
6- When comparing between direct smear and PCR in detection of Acanthamoeba in keratitic patients. It was found that 20 false negative cases by direct smear that were positive by PCR. The high differences in detection rates between direct smear and PCR might be due to superficial sampling of cornea as well as invasion of amoeba to deeper corneal layer.
7- The most accurate technique for diagnosis of acanthamoebiasis, still requires in vitro cultivation which normally takes a few days for trophozoites and one to 2 weeks for encystations. Moreover, in vitro culture amplified the parasite number, thus making microscopic detection less time-consuming. However, culture may produce false negative results due to low parasitic load, small sample volumes, the use of antiseptic or antibiotics prior to sampling or weakness of the Acanthamoeba strains.
8- The long waiting time for the results obtained by cultivation may lead to a delay in proper treatment and, ultimately, worsening of the disease. Therefore, researchers have used many techniques, including a molecular approach, to detect Acanthamoeba in corneal specimens in order to reduce the time for diagnosis.
9- The cyst form of Acanthamoeba is resistant to chemical reagents, temperature, and pH. For this reason, a diversity of reagents including ammonia, lysis buffer, proteinase K and as well as increased duration times for incubation have been utilized to extract DNA for molecular studies. So in this study, we performed a direct amplification of Acanthamoeba DNA bypassing nucleic acid extraction using a new commercial KAPA PCR kit. This technique is a simple and efficient method for detection of Acanthamoeba and does not require high-cost reagents or complicated procedures to extract DNA using 18S rRNA gene primers.
10- Our results proved that genus specific PCR was superior to microscopy and culture. However, the false-negative results obtained by PCR may be due to insufficient DNA material on the corneal scraping, and the possibility of laboratory contamination.
11-Using culture as gold standard in this study, direct smear showed 18 false-negative samples that were positive by culture giving a lower sensitivity of (5.3%), specificity (100%), positive predictive value (100%), negative predictive value (83.5%) and diagnostic accuracy (83.6%). At the same time, PCR showed 3 false-positive samples that were negative by culture and one false-negative results giving a higher sensitivity (94.7%), specificity (96.7%), positive predictive value (85.7%), negative predictive value (98.9%) and diagnostic accuracy (96.4%).