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

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.

Introduction

The genus Acanthamoeba is a free-living amoeba distributed ubiquitously in various environments such as soil, air and fresh water sources (1). Even, it can be found frequently in artificial habitats among the public swimming pools, surgical instruments, and contact lens solution. Some species of Acanthamoeba can cause potentially sight-threatening infection due to Acanthamoeba keratitis [AK] (2-4). Up to now, more than 20 genotypes of Acanthamoeba have been identified based on 18S ribosomal DNA (rDNA) sequencing method (designated T1 to T20) (3). Of these, 8 species of Acanthamoeba have been implicated in eye ocular infections: A. castellanii, A. polyphaga, A. hatchetti, A. culbertsoni, A. rhysodes, A. lugdunensis, A. quina, and A. griffini that most of keratitis causing isolates belongs to type T4 (1, 5).

The gradual increases in wearing contact lenses, misdiagnosis of AK and its slow response to medical treatment due to its encystation in harsh conditions are the most important factors that make Acanthamoeba critical for human health (3, 6). Furthermore, Acanthamoeba can play the role of carrier for pathogenic bacteria (e.g. Legionella, Pseudomonas and Helicobacter) that these bacterial pathogens lead to severe human infections (5, 7). Usually, an early and reliable AK diagnosis does not occur because it is easily confused with other infectious keratitis. Actually, primary clinical signs of AK are nonspecific and indistinguishable from Herpes simplex virus infection or fungal keratitis (8, 9). Because of AK delayed diagnosis, it is associated with poor prognosis and subsequently prolonged course of treatment, as far as Acanthamoeba spp. has been able to infiltrate deeply into the corneal stroma (10–12).

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. (14, 15). In addition, these assays are limited by poor sensitivity. Therefore, the sensitivity of direct smears for diagnosis of AK can be less than 50% (16). Recently, DNA-based methods including polymerase chain reaction (PCR) has been applied as alternative diagnostic modalities to detect Acanthamoeba spp. in corneal specimens with high sensitivity ranging from 77 to 88% (15, 16). PCR analysis in order to amplify of 18S rDNA in diagnostic regions is appropriate for distinguishing most Acanthamoeba genotypes (17). In addition to being more rapid compared to culture based-methods, this procedure also donates the valuable phylogenetic information (18).

We aimed to detect Acanthamoeba spp. in corneal scrapings samples of patients clinically suspected to AK by Nelson-PCR, JDP-PCR, direct microscopic examination, culture on non-nutrient medium and to evaluate these three different methods.

Martials and Methods

Ethical approval

The study was approved by the Ethics Committee, Mashhad University of Medical Sciences with the ID code: IR.MUMS.FM.REC.1396.92.

Sampling

During 2017-2018, twenty specimens of corneal scrapes were obtained from patients suspected to AK, who referred to the Khatam-al-Anbia Eye Hospital, Mashhad University of Medical Sciences, Mashhad, Iran. All clinical samples were obtained using special eye scalpels, by an ophthalmologist. The specimens were transferred to phosphate buffer saline (PBS) in order to do fresh smear, culture and PCR examination according to previous studies.

Direct microscopic examination

Direct microscopic examination was performed by putting one-third part of the specimen into a drop of PBS solution on a glass slide. Smears were investigated under a standard light microscope with × 40 magnifications to identify of Acanthamoeba trophozoites or cysts based on morphological features (19).

Culture

Briefly, 100 micro liters of each sample were inoculated onto 1.5 % non-nutrient agar (NNA) overlaid with E. coli, and incubated at room temperature. In order to detect the outgrowth of the Acanthamoeba trophozoites and/or cysts, Plates were checked every 2 days for approximately 1 month by using an inverted microscope (20, 21). To achieve a media culture of Acanthamoeba from fungal or bacterial contamination, a piece of agar was cultivated on a fresh plate (5, 22).

DNA extraction, PCR and DNA sequencing

The third part of the specimens stored at −20 °C, was used for the molecular technique. Primarily, the samples were melted at room temperature then DNA extraction was performed by QIAamp DNA Mini kit (Cat. no. 51304) according to the manufacturer’s protocol. Two specific primer pairs were used for detection of Acanthamoeba spp. A pair of primers JDP1 (fwd) (5′-GGCCCAGA TCGTTTACCGTGAA-3′) and JDP2 (rev) (5′-TCTCACAAGCTGCTAGGGAGTCA-3′) was applied to amplify a fragment of 423 to 551 bp of ASA.S1 region in18S rRNA gene (23, 24). The second primer set was Nelson primers (fwd) (5′-GTTTGAGGCAATAACAGGT-3′) and (rev) (5_ GAATTCCTCGTTGAAGAT-3′) for amplification of a 229 pbs fragment of genomic rRNA (12). The conventional PCR reaction by both primer pairs was performed on the thermo cycler (Bio-RAD, T100 thermo cycler) using a ready-made mixture of Amplicone (Taq DNA Polymerase Master Mix RED, Denmark) in a 30-μl final volume containing 5μl of DNA and 0.3μM concentration of each primer in accordance with standard procedure. Thermal cycling conditions were as follows initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 35 S, annealing at 56 °C for 45 S and extension at 72 °C for 60 S and a final extension step at 72 °C for 10 min (6, 25). To visualize PCR-products, they were electrophoresed on 1% (w/v) agarose gel, stained with Green viewer substance (1 μl/10ml) and then the presence of bands (nearly 500bp) were confirmed under UV light using a Gel-doc Image Analyzer.

Sequencing analysis

To determine the genotype, a 500 bps fragment of ASA.S1 region was Amplified from 18s rRNA gene of Acanthamoeba strains by JDP primers, and then gel purified (PCR products purification kit, parstous Corporation, Iran, Cat. No. A101221) and finally sequenced by Macrogen Company (Korea). The DNA sequences were edited manually with MEGA6.06 software and compared with BLAST Gen Bank database.

Pathogenicity tests

Osmotolerance assay

Two concentrations of mannitol (0.5 M and 1 M) in a non-nutrient agar medium used for each strain and incubation were done at the room temperature. Growth of Acanthamoeba were followed up (were assessed) daily for a period of 7 days (26, 27).

Thermotolerance assay

Acanthamoeba isolates were obtained from non-nutrient agar containing E. coli, inoculated into new culture medium at two different temperatures 37 °C and 40 °C. Growth of amoebae was evaluated by invert microscopy every 24 h for a period of 1 week. The positive control was used concomitant with each culture for elimination of any probable error (28, 29).

Statistical analysis

One specimen was considered positive for Acanthamoeba sp. when the results for any two direct examinations, culture, or PCR with either primer set were positive. We defined this reference standard upon which each of the methods was compared for the statistical analysis. Statistical tests were performed using Med Calc ( https://www.medcalc.org/calc ) and SPSS (Chicago, IL, USA) software (version 11.5). P value <0.05 was considered significant.

Results

Among 20 patients suspected to AK, 13 (65%) samples of 4 (30.8%) males and 9 (69.2%) females were positive based on defined gold standard (positive results for two of four tests). Most of the patients were in the age group 25-35-yr-old. The results showed that all of the patients had history of at least one of the following risk factors: wearing contact lens, eye trauma due to entrance of foreign particles or swimming inside fresh water. Only soft lenses have been used by those patients. Among the patients, 2 (15.4%) individuals had only history of swimming inside private garden pool sand, in 4 (30.8%) persons, infection started after an eye trauma (Table 1). Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings.

Table 1:

The frequency of risk factors among the patients with Acanthamoeba keratitis

No. of patients	Risk factors
	Contact lens	Trauma	Swimming
2			+
4		+
3	+		+
4	+	+

Based on the defined gold standard diagnostic method, cultures were positive in 6 of 13, while only 5 samples obtained positive result by direct examination (Fig. 1). The results of Nelson-PCR were positive for 14 cases (one false positive) (Fig. 2). Finally, JDP-PCR detected Acanthamoeba parasite in 13 of 13 (Fig. 3). Among diagnostic methods, direct examination had the lowest sensitivity (38.46% with 95% confidence interval 13.86% to 68.42%). The sensitivity of culture was 46.15% (CI 74.87% to 19.22%) and the sensitivity of the Nelson-PCR and the JDP-PCR methods was equal to 100% (CI 75.29% to 100%). In fact, all positive samples of Acanthamoeba were detected by PCR method with 2 primer pairs used in this study. The specificity of JDP-PCR, Nelson-PCR, direct examination and culture was 100% (CI 59% to 100%), 85.71% (CI 42.13% to 99.64%), 100% (CI 29.04% to 96.33%) and 100% (CI 59.04% to 100%) respectively (Table 2).

Fig. 1:

Invert microscopy photographs of Acanthamoeba trophozoite (A) and cysts (B), cultured on non-nutrient agar

Fig. 2:

Agarose gel electrophoresis of PCR products using Nelson primers: lanes CS1-CS4: corneal scrape, Lane PC: Positive control, lane PN: lane Negative control, lane M: standard DNA marker (100 bp DNA size)

Fig. 3:

Agarose gel electrophoresis of PCR products using JDP primers: lanes CS1-CS4: corneal scrape, Lane PC: Positive control, lane PN: lane Negative control, lane M: standard DNA marker (100 bp DNA size)

Table 2:

Sensitivity, specificity, NPV and PPV of four diagnostic methods compared to defined gold standard

Assay	No. positive	No. negative	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
Direct –smear	5	15	38.46	100	100	46.7
Culture	6	14	46.15	100	100	50
JDP_PCR	13	7	100	100	100	100
Nelson_PCR	14	6	100	85.71	92.86	100

PPV, positive predictive value; NPV, negative predictive value

Sensitivity: number of true positives divided by number of true positives plus false negatives ×100. Specificity: number of true negatives divided by the number of true negatives plus false positives ×100

The existence of one false positive case in the Nelson-PCR indicates a lower performance than other compared diagnosis methods. Agreement between PCR method with each of the Nelson and JDP primers compared to gold standard was calculated as 100% (P value <0.001).

Although it was for direct examination 83% but there was no statistically significant relationship (P value = 0.65). A very low agreement between the methods of culture and Gold standard was observed (37% with P value = 0.03). The accuracy of the methods was calculated using measuring the area under the curve of ROC (Receiver Operating Characteristic) diagram. The analysis of these results shows the accuracy of the Nelson-PCR and JDP-PCR tests was excellent (ACU). Accuracy of culture test was acceptable and direct examination had a low accuracy (Fig. 4).

Fig. 4:

ROC analysis of molecular and traditional methods used in this study compared to gold standard for the laboratory diagnosis of Acanthamoeba keratitis

The results of sequencing identified that all strains belonged to T4 genotype with a more than 95% similarity. Sequencing of isolates was deposited in Gen Bank database under accession numbers MK297910.1 to MK297916.1 and MK297918.1 (Fig. 5).

Fig. 5:

Phylogenetic tree of Acanthamoeba species analyzed by Neighbour-joining method based on 18S rRNA gene sequences using MEGA6.06 software. Number sat the nodes represent Bootstrap values based on 1000 replicates (MK297910.1 to MK297916.1 and MK297918.1 are Genbank accession numbers of 18s rDNA sequences of the Acanthamoeba isolates in this study)

Positive Likelihood Ratio for JDP-PCR and culture were calculated unlimited while this ratio for the Nelson-PCR was calculated as 7. Negative Likelihood Ratio for PCR with both Nelson and JDP primers were equal to zero. NLR of direct examination and culture were 0.62 and 0.54, respectively (Table 3).

Table 3:

Positive and Negative likelihood ratio of four diagnostic methods compared to defined gold standard

Assay	PLR(95%CI)	NLR(95%CI)
Direct – smear	∞	062(0.4c0.9)
Culture	∞	0.54(0.33–0.89)
JDP_PCR	∞	0
Nelson_PCR	7(1.14–42.97)	0

Positive likelihood ratio: ratio between the probability of a positive test result given the presence of the disease and the probability of a positive test result given the absence of the disease (True positive rate / False positive rate = Sensitivity / (1-Specificity))

Negative likelihood ratio: ratio between the probability of a negative test result given the presence of the disease and the probability of a negative test result given the absence of the disease (False negative rate / True negative rate = (1-Sensitivity) / Specificity)

Discussion

This is the first study on accurate isolation and diagnosis of Acanthamoeba keratitis in Mashhad, Iran. The present study compared and evaluated conventional methods with molecular technique for detection of Acanthamoeba in specimens obtained from patients suspected to AK, based on JDP-PCR and Nelson-PCR. The results showed that molecular methods were the most sensitive and direct microscopic examination was the least sensitive one. Niyyati et al obtained similar results, whereas Boggild et al stated that culture method is more sensitive than JDP-PCR (12, 15). In spite of the high specificity of direct examination and culture, they have a high false negative rate; therefore, the mentioned conventional methods are less valuable than molecular methods in diagnosis of AK. The low number of positive cases detected in the direct examination can be due to little sampling obtained from cornea, because of possible danger of pathology lesion and new infection. The number of high false negative cases in the culture method is a result of superficial sampling. Because Acanthamoeba penetrates in to deeper layers of the eye and superficial sampling decrease the chance of amoeba isolation (14). However, the proper sampling for isolation of Acanthamoeba spp. from the cornea is necessary for molecular methods. But here is one point to mention that molecular techniques with high sensitivity can detect small number of amoeba (less than 1 amoeba per micro liter), while the culture method in such situation is not so successful to proliferate of Acanthamoeba trophozoites, which is a slow-growing protozoan and requires long incubation period (8).

Regarding ROC chart, this study showed that JDP-PCR has higher accuracy than Nelson-PCR, which contradicted the study, performed by Boggild et al (12). On the other hand, molecular method is fast, reducing the response time to the patient thus rapid patient treatment, decreases complications. The results of present study indicated most of our patients were young women and more than half of the patients who suffered from AK were the contact lens wearers, which corresponds with others studies (21, 30). The difference between the results of this study and other studies could be related due to sample size and sampling time. In general, women are more susceptible to AK due to the greater use of cosmetic or so-called decorative contact lenses in order to have more beautiful and eliminate eyes imperfections. Contact lenses are one of the main risk factors in AK and even fungal keratitis due to use of home remedies or boiling water for lenses maintenance and the ineffectiveness of the disinfection solution of contact lens in removal of Acanthamoeba cysts (11). The contact lenses, especially the soft type, are completely attached to corneal and decrease oxygenation of the cornea cells, which lead to desquamation of superficial cell of cornea, provide easier penetration of Acanthamoeba into the corneal stroma. Since the prevalence of a disease in a population can affect some of the characteristics of diagnostic tests, especially sensitivity and specificity; the positive and negative Likelihood Ratio was measured. The Likelihood ratio is one of the most important characteristics of diagnostic tests that help to accept or reject the presence of a disease in a person. This study revealed that all species of Acanthamoeba isolated from patients with AK belonged to the genotype T4.

According to previous studies in Iran, T4 genotype has been identified as the predominant type among patients with AK (31). However, Niyyati et al and Haji-Alilu et al reported genotypes of T3, T11 and T9 as causal agents of AK (6, 19). It seems that the high frequency of T4 genotype is due to its high virulence leading to high transmission and low susceptibility to chemical agents (32). T4 genotypes in northeastern Iran had 100% homology with recorded genotypes from north, central, west and south of this country (Table 4). Pathogenicity test showed that only one strain had a high pathogenic ability. It seems that the use of more pathogenic tests such as in vivo tests is more accurate (6).

Table 4:

BLAST Results of 18S rRNA of Acanthamoeba isolated from nine keratitis patients, Northeast of Iran

Accession numbers in this study	Accession numbers in Gene bank	Country	References
MK297910 (100%)	MG917706.1	Iran	6
MK297911 (100%)	MF139792	Iran	Unpublished
MK297912 (100%)	LC373015	Iran	Unpublished
MK297913 (100%)	MK192801	Iran	Unpublished
MK297913 (100%)	MH539781	Iran	Unpublished
MK297914 (99%)	KY514425	Iran	Unpublished
MK297915 (99%)	MG825466	Brazil	35
MK297916 (99%)	MH100806	Chile	36
MK297917 (95%)	MG969816	Iran	Unpublished
MK297918 (99%)	MH100806	Chile	36

Conclusion

Amoebic keratitis is a sporadic parasitic eye infection, which is mainly seen in contact lens wearers in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of AK in clinically suspected cases.

Therefore, the molecular PCR test with JDP primers is the most accurate and sensitive method in the diagnosis of AK in clinically suspected cases.