Evaluation of polymerase chain reaction (PCR) as a diagnostic technique for acanthamoebic keratitis

Laila A Aboul-Magd; Beessa E Abaza; Waleed M Nada; Faten A Mohammed; Afaf A Taha; Sabah M.A Mohamed; Elshaimaa M Ebrahim

doi:10.4103/puj.puj_13_16

ORIGINAL ARTICLE

Year : 2016 | Volume : 9 | Issue : 2 | Page : 87-94

Evaluation of polymerase chain reaction (PCR) as a diagnostic technique for acanthamoebic keratitis

Laila A Aboul-Magd¹, Beessa E Abaza¹, Waleed M Nada², Faten A Mohammed¹, Afaf A Taha¹, Sabah M.A Mohamed¹, Elshaimaa M Ebrahim¹
¹ Department of Medical Parasitology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
² Department of Ophthalmology, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Date of Submission	19-Aug-2016
Date of Acceptance	06-Dec-2016
Date of Web Publication	25-Apr-2017

Correspondence Address:
Faten A Mohammed
Department of Medical Parasitology, Faculty of Medicine, Zagazig University, Zagazig, 44519
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/puj.puj_13_16

Abstract

Background Techniques used to diagnose Acanthamoeba keratitis (AK), a sight-threatening corneal infection, are either insensitive or time-consuming. Early and accurate diagnosis and appropriate therapy are the key to a good prognosis.
Objective The objective of this study is to shed light on the efficacy of PCR in the diagnosis of AK in comparison with other diagnostic techniques.
Patients and methods Corneal swabs and scrapings from 95 cases suspected to have AK were examined by microscopy, culture on non-nutrient agar seeded with the avian fecal Escherichia coli AFEC49, and by PCR targeting the 18S rRNA gene.
Results The highest number of positive swabs and scrapings was detected by the PCR technique (5.26 and 27.37%, respectively), followed by culture (1.05 and 13.68%, respectively), and the lowest was detected by direct microscopy (0 and 7.37%, respectively). Corneal scrapings showed more positive cases than corneal swabs with statistically significant differences irrespective of the technique used. PCR results were significant versus culture (P<0.05) and direct microscopy (P<0.001). Out of the 26 positive cases, 20 were contact lens wearers, 13 were swimming pools users, five had a history of eye trauma, and five had undergone previous eye operation.
Conclusion PCR proved to be a better technique for the detection of Acanthamoeba infection compared with direct microscopy and culture techniques. Scrape specimens are more efficient than swabs in detecting Acanthamoeba by different diagnostic techniques. The important risk factors of AK were contact lens wearers, use of swimming pools, a history of previous eye operation as well as eye trauma.

Keywords: Acanthamoeba keratitis, diagnostic technique, PCR

How to cite this article:
Aboul-Magd LA, Abaza BE, Nada WM, Mohammed FA, Taha AA, Mohamed SM, Ebrahim EM. Evaluation of polymerase chain reaction (PCR) as a diagnostic technique for acanthamoebic keratitis. Parasitol United J 2016;9:87-94

How to cite this URL:
Aboul-Magd LA, Abaza BE, Nada WM, Mohammed FA, Taha AA, Mohamed SM, Ebrahim EM. Evaluation of polymerase chain reaction (PCR) as a diagnostic technique for acanthamoebic keratitis. Parasitol United J [serial online] 2016 [cited 2023 Oct 3];9:87-94. Available from: http://www.new.puj.eg.net/text.asp?2016/9/2/87/205165

Introduction

The genus Acanthamoeba is a free-living protozoon present in the environment worldwide. It pollutes air, soil, dust, air-conditioning units, treated water, bottled water, swimming pools, dialysis units, contact lenses (CLs), and lens cases. It is considered among the most prevalent eukaryotic protozoa found in the environment, providing multiple opportunities for contact with humans and animals [1],[2],[3]. Various species of Acanthamoeba have been implicated in human infections: A. castellanii, A. astronyxis, A. culbertsoni, A. polyphaga, A. hatchetti, A. rhysodes, A. lugdunensis, A. palestinensis, A. griffini, and A. quina [4].

Because flower dust showed a positive culture for Acanthamoeba, public health education advised refraining from bringing flowers while visiting a patient visit in eye surgery and recovery units to prevent contact of the eyes with flower dust. In addition, it was advised that hydrogen peroxide (3%) should be used in ophthalmology hospital units as chlorinated disinfectants cannot kill Acanthamoeba [5]. Free-living amoeba tolerate high temperatures and survive in hot water; thus, they thrive easily in natural and artificial hot water systems [6]. The life cycle consists of trophozoite and cyst stages.

These free-living amoebae have become medically important since they were discovered as a causative agent of granulomatous amoebic encephalitis, skin lesions, nasopharyngeal, pulmonary, and kidney infections, primarily in immunocompromised patients. Corneal infection causes Acanthamoeba keratitis (AK) in both immunocompetent individuals and immunocompromised patients [7], by contact with nondisinfected aquatic environments or wearing CLs or after eye trauma [8]. In the cornea, they are believed to feed on keratocytes. Individuals with normal immunological status present with severe pain and a characteristic ring-shaped corneal infiltrate [2],[9],[10]. Delayed diagnosis has been associated with severe sight-threatening outcome and more severe clinical progression [11]. AK was first recognized in the mid-1970s. Then, a marked increase in cases occurred because of the increasing use of soft CL. The improper use of CL solutions may lead to lens contamination and thus predispose toward the development of AK [12]. The use of disinfecting systems ineffective in killing Acanthamoeba cysts and trophozoites is the main risk factor for corneal infection in contact lens wearers (CLWs). Most commercial solutions proved ineffective in eliminating Acanthamoeba cysts within a reasonable time. Adequate exposure time is necessary for effective killing of different stages of this parasite [13].

Because of very frequent misdiagnosis and complicated treatment, AK often progresses to perforating keratopathy. The condition may be confused with atypical herpes simplex keratitis or fungal keratitis. Early definitive diagnosis of AK and the prompt initiation of appropriate therapy is essential for a favorable clinical prognosis [11],[14]. Direct smear examination cannot be considered a test for confirming AK infection and can lead to misdiagnosis in 60–70% of cases [15],[16]. Direct smear analysis had the poorest diagnostic sensitivity compared with culture and PCR. Agar culture was considered the mainstay for the laboratory diagnosis of Acanthamoeba. However, it requires familiarity with the morphology of its cysts and trophozoites, and a definitive result may take up to 10 days, delaying the institution of specific therapy [14].

Recent advances of PCR and confocal microscopy improved the diagnostic potential considerably. As these techniques become more recognized and available, it is hoped that they will be useful more in clinical practice. The present work aims to determine the efficacy of PCR in the diagnosis of AK in comparison with direct microscopy and culture.

Patients and methods

Type of study

This is a descriptive analytical study.

Study population

The present study included 95 patients (50 women and 45 men), 20 from Outpatient Clinics of Zagazig University Hospitals, 50 from Al-Matarya Hospital in Cairo, and 25 patients from specialized Al-Ferdaws Hospital in Zagazig, Egypt. The study was carried out during the period from April 2012 to September 2014, in Microbiology and Biochemistry Departments, Faculty of Medicine, Zagazig University, Zagazig, Egypt. Selected patients complained of resistant corneal ulcer not responding to medical treatment for more than 2 weeks. All patients provided medical history and underwent an ophthalmic examination including an external ocular and slit-lamp examination by the ophthalmologist in the ophthalmic outpatient clinics.

Specimens

Corneal swabs using sterilized cotton swab and deep scrapes of the cornea with sterile stainless steel blades (Bard-Parker blade, Aspen Surgical Products, Inc, a Hill-Rom Company, USA) were taken by the ophthalmologist from the floor of the ulcer. Samples from each patient were divided into two parts. One was suspended in Page’s saline (PAS) for direct microscopic and culture examination and the other was placed in a sterile Eppendorf tube (containing PBS) and preserved at −20°C for future testing by PCR.

Microscopic examination

Wet mount [17] and Giemsa-stained slides [18] from saline suspension of swabs and scrapes were examined for cysts and trophozoites under a standard light microscope.

Cultivation of specimens [19]

To prepare non-nutrient agar plates (NNA), 15 g of NNA was dissolved in 1000 ml of PAS solution composed of 0.12 g NaCl, 0.004 g MgSO₄· 7H₂O, 0.004 g CaCl₂· 2H₂O, 0.142 g Na₂HPO₄, and 0.136 g KH₂PO₄ (Sigma-Aldrich Corp., St. Louis, MO, USA), dissolved in 1000 ml distilled water, and then autoclaved at 121°C for 15 min. When agar temperature decreased to 50°C, 1000 IU/ml Mycostatin (GalaxoSmithKline, (GSK), Fifth district, New Cairo, Cairo, Egypt) in distilled water and 1000 IU/ml Chloramphenicol (Misr Pharmacological Company, Belbis Desert Rd., Cairo, Egypt) in distilled water were added. The medium was poured into sterile plastic plates and left to solidify at room temperature. To prepare nutrient agar (NA) plates, 23 g of NA were dissolved in 1000 ml of distilled water. The mixture was autoclaved and solidified in Petri dish ^{More Details}es. Under aseptic conditions, 20 μl of avian fecal Escherichia ^{More Details} coli AFEC49 was used (Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA). This strain was obtained from College of Veterinary Medicine, Zagazig University, and streaked on the NA plates, followed by incubation at 37°C for 24 h [20]. Growth of E. coli was harvested by adding 2 ml of PAS solution, followed by pipetting several times to detach the E. coli colonies from the NA surface. Bacterial suspension was spread on each NNA plate by a sterile spreader. At this stage, plates could be used directly for cultivation or stored at 4°C for up to 3 weeks. Acanthamoeba spp. suspected sample (resuspended in 1 ml PAS) was poured over the NNA plate seeded with E. coli. NNA was inoculated by making one or two strokes in the center of the plate without disturbing the agar to enhance the growth of Acanthamoeba spp. and then incubated at 28–30°C. The presence of amoebae was checked by an inverted microscope [21]. If Acanthamoeba trophozoites are present, they will migrate across the plate using the bacteria as a food source. Amoeba migration tracks (bacterial clearing showing where the amoeba have been feeding) were usually easily observed within 48 h, but occasionally longer incubation (up to 2 weeks) was needed. A drop of 1% Lugol’s iodine was added to confirm the cyst morphology.

PCR technique

For DNA extraction, frozen specimens were thawed at room temperature. DNA extraction was performed using the DNeasy Blood & Tissue Kit (Qiagen; QiagenStrasse 1 40724 Hilden Geschäftsführer, Germany). PCR was performed using 18S rRNA gene primers: forward primer 5′-GGCCCAGATCGTTTACCGTGAA-3′ and reverse primer 5′-TCTCACAAGCTGCTAGGGGAGTCA-3′. These primer sequences correspond to 928–949 and 1367–1390 bp, respectively. The amplification was carried out in a Biometra T Gradient Thermal cycler (Analytik Jena company, Göttingen, Germany). The reaction was carried in 40 cycles according to Booton et al. [22] as follows: thermal cycler denaturation at 94°C for 1 min, annealing at 67°C for 1 min, extension at 72°C for 1 min, and a final elongation step at 61°C for 5 min. For gel electrophoresis, PCR products (500 bp) were visualized by electrophoresis on 2% (w/v) agarose–AE-6111 (Tris–acetic acid–EDTA) gel, stained with ethidium bromide solution (0.5 µg/ml) and visualized on a UV gel documentation system [23]. DNA extracted from positive Acanthaoeba culture maintained by frequent subcultures on fresh NNA-E. coli every week was used as a positive control in gel electrophoresis. PAS was used as a negative control.

Statistical analysis

The collected data were computerized and statistically analyzed using the Statistical Package for Social Science (SPSS, version II, for Windows 7, SPSS Inc., Chicago, IL, USA) program, version 18.0 [24]. The χ²-test was used to examine the relation between qualitative variables. Significance was defined as P less than 0.05.

Ethical consideration

Ethical approval was obtained from the Committee of Research, Publications and Ethics of the College of Medicine, Zagazig University, Zagazig, Egypt. All procedures were explained to the patients and a written or thumb-printed informed consent was obtained.

Results

Out of 95 corneal swabs and scrapings, the highest number of positive cases was detected by the PCR technique (5.26 and 27.37%, respectively), followed by culture (1.05 and 13.68%, respectively), and the lowest was detected by a direct microscope (0.0 and 7.37%, respectively) ([Table 1] and [Table 2]).

Table 1 Detection of Acanthamoeba infection in corneal swabs and scrapings of 95 examined cases by different diagnostic methods

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Table 2 Distribution of Acanthamoeba infection in 95 ophthalmic scrapings examined by different techniques

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Direct microscopic examination of wet mount smears from scrapings showed Acanthamoeba trophozoites characterized by the presence of fine, tapering, and thorn-like acanthopodia arising from the surface of the body; the nucleus had a centrally placed, large, dense nucleolus. The cysts were double walled. Giemsa-stained smears showed a wrinkled outer cyst wall (ectocyst) and an inner cyst wall (endocyst) that varied in shape, being stellate, polygonal, oval, or spherical. Cysts were uninucleate and the nucleus had a centrally placed dense nucleolus ([Figure 1],[Figure 2],[Figure 3]). Acanthamoeba migrated across the plate using the bacteria as a food source. Migrations tracks by amoebae were usually easily observed within 48 h after culture ([Figure 4]). Agarose gel electrophoresis of PCR products is shown in ([Figure 5]).

Figure 1 Acanthamoeba cyst wet mount (×40).

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Figure 2 Cyst of Acanthamoeba stained by iodine (×40).

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Figure 3 Cyst of Acanthamoeba stained by Giemsa stain (×40).

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Figure 4 Migration tracks of Acanthamoeba trophozoites on NNA plates within 48 h of culture examined by an inverted light microscope (×40).

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Figure 5 Agarose gel electrophoresis of PCR products for Acanthamoeba infection. Lane 1: 100 pb DNA ladder. Lane 2: positive control. Lane 3: negative control. Lanes 4, 5, and 7: positive samples for Acanthamoeba. Lanes 6 and 8: negative sample for Acanthamoeba.

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Determination of risk factors for AK showed that Acanthamoeba infection was insignificantly higher in CLWs (34.48%) than noncontact lens wearers (NCLWs) (16.21%) (P>0.05). AK was significantly higher in swimming pool users 61.9% (P<0.001). However, the association of eye trauma (29.41%) and a history of previous eye operation (38.46%) with Acanthamoeba were insignificant (P>0.05) ([Table 3]).

Table 3 Relation between contact lens wearers, use of swimming pools, trauma, previous eye operation, and Acanthamoeba keratitis in 95 examined cases

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Discussion

In the present study, we examined corneal swabs and corneal scrapings obtained from the 95 patients suspected of having AK. Corneal scrapings showed significantly positive cases for Acanthamoeba by direct microscopy, culture, and PCR. Similarly, Anisah et al. [25] reported that swabbing is an insensitive technique for isolation of the amoeba. This was explained by Vemuganti et al. [26], who reported that trophozoites of Acanthamoeba dwell in the anterior stroma, whereas the cysts are found in the deeper stroma. Accordingly, corneal scrapings are considered the best source for the identification of the infectious organism for AK [15],[27]. However, corneal scraping is unfortunately a more complicated technique and few patients can tolerate it [2],[11].

In the present study, the highest significantly positive results were obtained by PCR in both swabbed (5.26%) and scraped (27.37%) samples, followed by culture (1.05 and 13.68%, respectively) and then by direct microscopy (0 and 7.37%, respectively). These results are in agreement a report by Niyyati et al. [28], who reported that direct smears of all prepared corneal scrapings in 14 AK patients were negative, and although culture succeeded in isolating only two (14.3%) isolates, PCR analysis showed 10 (71.4%) positive AK patients. Laummaunwai et al. [29] also reported that molecular methods have become the additional and alternative method to microscopy and culture, especially as they can enable the detection of fewer organisms per volume of sample (even a single cyst) than morphological methods. Fewer specialized technical skills are required for PCR. It also offers a much more rapid time for diagnosis than culture [2],[17]. Therefore, molecular diagnostic tests (PCR or real-time PCR) provide fast and reliable results that support the clinical diagnosis of AK [30]. Integration of a PCR method with the conventional parasitological techniques of smear examination and in vitro culture may complete the diagnostic protocol in the case of AK and provide information on the pathogenicity of the amoebic isolates [31],[32]. Wanachiwanawin et al. [33] reported positivity rates of Acanthamoeba to be 15.3% for direct microscopy, 46.1% for culture, 92.3% for conventional PCR, and 100% for real-time PCR. Corneal ulcers in our patients that proved not to be AK probably may have resulted from other infectious or noninfectious causes. Bacteria (Pseudomonas aeruginosa), fungi (Fusarium), and viruses (Herpes simplex and Varicella) have been implicated, in addition to eye trauma, chemical exposure, and ultraviolet exposure [34]. Corneal injury with vegetative matter was more often associated with fungal keratits and injury with mud was associated with AK [35].

Our results were statistically significant for PCR versus culture and highly significant versus direct microscopic examination, indicating the superiority of PCR. In another report, the sensitivity and specificity of the PCR technique for the detection of Acanthamoeba was recorded to be 71.4 and 100%, respectively [28]. Several studies [11],[28],[36],[37] reported that examination of all AK corneal scrapings was negative by direct smear. They attributed this to the lack of sensitivity of direct examination of specimens by microscopy that requires technical expertise. In one report, 50% of cases were misdiagnosed [38]. Agar culture requires familiarity with the morphology of cysts and trophozoites and it takes up to 21 days, delaying specific therapy [14], which indicates that the culture approach is unsuitable for diagnosis in urgent cases. Niyyati et al. [28] recorded the sensitivity and specificity of the culture to be 14.3 and 100%, respectively. Other researchers confirmed that culture results in high false-negativity rates, yields delayed results, and requires technical expertise [11],[17],[36],[37]. Petry et al. [39] and Xuejun et al. [40] presented other explanations attributing difficulties in cultivation to the presence of too few amoebae in the clinical sample to initiate a culture and the inhibition of Acanthamoeba growth because of previous use of antimicrobial agents. Also, the presence of Acanthamoeba in the corneal stroma renders it inaccessible to surface scraping. Another presented factor was the possibility that the infecting strain does not readily adapt to in vitro growth.

The main disadvantage of PCR is its inhibition by topical medications and dyes, which are often administered directly to the ocular surface to aid diagnosis and treatment (lissamine green, rose bengal, fluorescein dyes, the anesthetic oxy-buprocain); also, endogenous inhibitors in ocular fluids have been shown to cause PCR inhibition [41].

In the present study, Acanthamoeba infection was insignificantly higher in CLWs (34.48%) than NCLW (16.2%) (P>0.05). Wanachiwanawin et al. [33] diagnosed AK in 62.5% of CLW and in 37.5% of NCLW. Moreover, other studies [42],[43] indicated that AK is particularly present in CLWs, accounting for up to 95% of cases. Handling of the CL may cause epithelial scratches that become infected. Continued use of CLs exerts chronic hypoxic pressure on the corneal epithelium, decreasing corneal sensitivity, epithelial mitosis, and adhesion. Epithelial microcystic edema and increased epithelial fragility result in premature desquamation of epithelial cells and thinning of the epithelial cell layer [44]. It has been stipulated that the recent increase in the incidence of AK can be attributed to several factors, including the increasing number of CLW and the widespread noncompliance with the cleaning and rinsing regimens for CLs [45],[46]. In addition, the use of ineffective CL disinfectant solutions is suspected to be linked to the increase in cases of AK [47]. However, Sharma et al. [48] reported that none of the 39 diagnosed AK patients had worn CLs. In our study, 16.2% were NCLWs. In the present study, AK was significantly higher in swimming pool users, 61.9% (P<0.001). History of swimming in canals or swimming pools is considered an important risk factor for AK [49]. In agreement with this, Magliano et al. [50] reported that Acanthamoeba spp. were found in different water samples worldwide. For instance, they were present in tap water in Brazil, whereas in Iran, they were present in rivers, waterfalls, and swimming pools [27],[51],[52], and also in rivers, springs, wells, and water tanks in Nicaragua [53]. Researchers in Poland detected Acanthamoeba in 59.7% of the swimming pool samples examined [54]. Other records of Acanthamoeba species were found in 49.2% of water samples collected from 10 swimming pools in Cairo [55]. These findings confirm the risk of infection by Acanthamoeba in recreational water sources.

Association of eye trauma with Acanthamoeba (29.41%) was insignificant (P>0.05) in our study. Manikandan et al. [56] had reported that corneal injury because of exposure to foreign bodies from various sources was a predisposing factor to AK among the NCLW. Other studies [57],[58] reported that corneal trauma by either vegetative matter, stone, dust, or splashing unclean water into the eyes is a important risk factor for AK. Physical agents other than CLs, such as mud, could also cause corneal injury and carry amoebae to the cornea [59]. Bharathi et al. [35] reported that all 33 culture-proven AK had a history of corneal injury, and the association between corneal injury and AK was significant. They added that the number of patients with a history of corneal injury with mud (84.6%) was significantly higher than that of patients reporting injury because of other traumatizing agents (15.2%). A history of previous eye operation in our patients (38.46%) was insignificant (P>0.05). Shehri et al. [60] reported that in the Kingdom of Saudi Arabia, the most important risk factor for AK was penetrating keratoplasty. Previous ophthalmic operation also represented a major risk factor for AK in older individuals [5].

Conclusion

From the present study, it was clear that molecular methods have become the additional method or an alternative to microscopy and culture. Scraps are more efficient than swabs in detecting Acanthamoeba by different diagnostic techniques. The important risk factors that predispose toward AK were swimming pool users, history of a previous eye operation, CLWs, and a history of eye trauma.

Author Contribution: LA Aboul-Magd shared in the study design, research topics, and reviewed the manuscript. BE Abaza and WM Nada shared in the study design, initiated the research idea, and reviewed the manuscript. FA Mohammed, AA Taha, and SMA Mohamed analyzed the data, and wrote and reviewed the manuscript. EM Ebrahim shared in the study design, practical work, and writing the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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