The molecular epidemiology, genotyping, and clinical manifestation of prevalent Adenovirus infection during the epidemic keratoconjunctivitis, South of Iran

doi:10.21203/rs.3.rs-1762528/v1

Purpose

Adenoviral mediated keratoconjunctivitis is among the emergency diseasees of ophthalmology with long-term sequels. The role of adenovirus infection, ocular-related genotypes, and association with ocular symptoms needs to be investigated for epidemiological as well as clinical purposes.

Methods

The affected patients from two close keratoconjunctivitis epidemics were included in the study. The swab samples were taken from patients; total DNA was extracted and then was introduced into an in-house Real-Time PCR reaction. Besides, partial Hexon genes of 11 adenovirus positive samples were amplified and submitted to sanger sequencing. Moreover, they were finally evaluated by phylogenetic analysis.

Results

Of the 153 patients, 92 (60.1%) were males and 47 cases (30.7%) had a history of eye infection in the family or colleagues. A Real-time PCR test of 126 samples (82.4%) was positive for adenovirus, and all eleven cases that underwent sequencing analysis were determined to be serotype 8 (HAdV-D8). Adenovirus infection has a significant relationship with infection among family or colleagues (p = 0.048), membrane formation (p = 0.047), conjunctival bleeding (p = 0.046), and pain.

Conclusions

The results indicated that Adenovirus is the major cause of keratoconjuctivitis and HAdV-D8 was the most common genotype in the area. There were some clinical manifestations associated with Adenovirus infection of the conjunctiva.

Adenovirus

Genotype

Epidemic Keratoconjunctivitis

Adenovirus is the leading cause of Epidemic Keratoconjuctivitts (EKC) worldwide. Conjunctivitis accounts for a large number of visits to ophthalmology clinics, and imposes a heavy financial burden on the health system. According to this, 1% of all doctor visits in the United States are related to conjunctivitis [1]. Most cases of conjunctivitis are viral-based, and discrimination from a bacterial basis is of great importance. In addition, 65–90% of all viral conjunctivitis is caused by adenovirus, which makes it the most important agent [2].

Adenovirus is a naked double-stranded DNA virus with a special projection, named fiber [2]. Human adenoviruses are divided into 7 groups from A to G and 68 serotypes based on biological properties [3]. Among all groups, members of group D, including genotype 8, 19, and 37, are the major causes of ECK, followed by other serotypes such as 2, 4, 7, 11, 14, 16, and 29 [3, 4].

Adenoviral conjunctivitis is diagnosed by cell culture, Polymerase chain reaction (PCR), enzyme immunoassays, direct immunofluorescence tests, immune dot-blot tests, and point of care tests [5]. PCR is now known as the "gold standard" for diagnosing viral conjunctivitis. Studies suggest that PCR is more sensitive for detecting adenoviruses than other virological methods [6]. Adenovirus serotyping can be simply performed by surface antigen sequencing. Identification of the adenovirus genotypes which are involved in EKC is required to understand the geographical distribution and to draw a relation between a specific genotype and clinical features [7].

A study at Wills Ophthalmology Hospital in Philadelphia found that the prevalence of adenoviral conjunctivitis was 62% [8]. In a large study in Turkey, adenoviral DNA was detected by PCR in 213 of 488 samples (43%), collected from patients with viral conjunctivitis. The genotypic analysis revealed that seven genotypes, including 3, 4, 8, 11, 19, 37, and 53, were circulating during the epidemic episode while genotypes 4 and 8 were the dominant genotypes found in 66.3% and 24.7% of the samples, respectively [7]. In addition, the prevalence of EKC does not appear to be related to gender, race, social status, or nutritional status [9].

In Iran, studies regarding adenovirus prevalence and genotyping are limited to a few molecular epidemiology studies that indicate the dominant prevalence of adenovirus and genotype HAd8 [10] [11].

Viral conjunctivitis is highly contagious and is transmitted through infected personal items, direct contact with infected fingers, eye secretions, medical equipment, and swimming pools. In addition, tears, saliva, respiratory droplets, and feces are involved in transmission [12]. Other ways that EKC can spread are through tonometry, slit-lamp exams, putting in contact lenses, and contact with infected doctors [13].

Although adenoviral conjunctivitis is commonly a self-limiting disease with no long-term complications, in severe cases of EKC, significant chronic complications can occur which affect life. Severe EKC can lead to secondary dry eye, fibrosis of glands and lacrimal ducts, corneal scarring, the development of irregular astigmatism, and even permanent vision loss. Conjunctival scarring can occur in patients with severe disease after the formation of a membrane [14]. The impact of adenovirus infection on the severity of ocular disease and its correlation with viral genotype is a matter of doubt. Actually, differential diagnosis of each EKG-related infection is hard to achieve, but finding the specific clues for each agent is practical. The traumatic/inflamamtory extent of the involved conjunctiva and following sequels of adenovirus infection could be differentiated from others.

Owing to the complications of EKC, the present study aimed to determine the frequency of adenovirus infection, circulating dominant genotype, and the correlation of infection with symptoms during two close epidemics, in a reference clinical center, Shiraz, the biggest city in the south of Iran in 2019.

Patients and clinical evaluation

In this cross-sectional and descriptive study, using an easy sampling method, 165 eligible patients referred to the ophthalmology emergency department of referral Khalili Teaching Hospital were included in the study during two close epidemics in the summer and autumn seasons of 2019. The confirmed bacterial conjunctivitis cases were excluded from the study. Also, patients with long-term use of eye drops, glaucoma, history of herpes simplex keratitis, eye surgery in the past week, use of contact lenses in the past few days, and history of allergic conjunctivitis were excluded.

After obtaining informed consent, patients' information was collected in the form of a questionnaire provided by the examiner physician.

The study was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.MED.REC.1399.507).

Any patient with conjunctivitis whose symptoms started two weeks ago was asked to be included in the study. Their clinical pictures were evaluated by an ophthalmologist based on history and ophthalmological examinations. Inclusion criteria include referring to the hospital with symptoms of conjunctivitis, such as redness, pain, tearing, follicular reaction, eye discharge, and preauricular lymphadenopathy. Also, exclusion criteria were including history of long-term use of eye drops, glaucoma , history of herpes simplex keratitis, eye surgery in the past week, use of contact lenses in the past few days , and history of Allergic conjunctivitis.

Sampling

The samples were collected from the lower palpebral conjunctiva using a sterile Dacron swab with a plastic shaft, which was subsequently introduced into a transport medium (DMEM culture media, PH=7.3) supplemented with Pen-Strep antibiotics and amphotrypcin B anti-fungal. Specimens were transported on ice to the lab and stored at -70°C until used.

Real-Time PCR development

An in-house Real-Time PCR assay targeting a small part of the Hexon gene was developed for the detection of the adenoviral genome. Real-time PCR was performed in a SYBR-Green based method using a pair of primers to detect all human-related adenoviral serotypes (Table 1).

Real-time PCR was performed using the RealQ plus master mix Green (Ampliqon Inc.) mixture containing 5 μL template, 1X master mix, heat-labile uracil-N-glycosylase, and 300 nM of each specific Adenovirus primer. The reaction was done by using the ABI7500 instrument under the thermal cycling condition of an initial denaturation step at 95°C for 10 min, followed by 40 amplification cycles of denaturation at 95°C for 20s, annealing and elongation at 60°C for 15s. At the end of thermal cycling, melting temperature (Tm) analysis was also applied in order to avoid false positive interpretation by primer dimers and non-specific bands.

In order to confirm the test validity and specificity, four negative controls, including staphylococcus aureous extracted genome, human herpes simplex virus-1 (HSV-1) extracted genome, a plasmid expressing eGFP (Q.Biogene Inc.), as well as total DNA extracted from human cells were also included. Besides, Human Adenovirus serotype 36, recombinant plasmid containing adenovirus backbone type 5 (Q.biogene Inc.), and a clinical serotype 8 (that had been confirmed by sequencing, HAdD8) were also included as positive controls, to investigate the specificity.

The detection limit of the method was investigated on a diluted standard sample of the recombinant plasmid containing an Adenovirus type 5 genome backbone. The concentration of plasmid was measured using Nanodrop^TMabsorbance reading and the conversion of mass to copy number was done by online convertor tools. A serial dilution of up to 10^-9 was prepared and introduced into the experiment to find out the lowest copy number at which the developed test works well.

Molecular Phylogenetic analyses and serotyping

Total DNA was extracted from samples using the DNP Nucleic Acid Isolation Kit (CinnaGen Inc., Tehran, Iran) according to the manufacturer’s instructions. The extracted DNA was stored at -70 until Real-Time PCR.

In order to reveal the prevalent genotype, a fragment of the hexon gene was amplified using a specific Ad-Hex primer set (Table 1). The primer pairs were designed using the NCBI primer designing tool and were evaluated on positive clinical samples as well as available positive controls.

The thermal cycling conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of: 30 s at 95 °C, 30 s at 55 °C, and another 30 s at 72 °C. In this study, 12 samples from different time-points of epidemics were randomly selected to be sent for sequencing. PCR products were purified using the MN gel purification kit (Macherey-Nagel Inc.) before being sent for Sanger sequencing.

All the sequences were retrieved from provided files and were screened by BLAST online software to see the similarity with the relative sequence. Then, they underwent multiple sequence alignment with numerous reference adenovirus sequences from different groups with the help of Mega10 software. The confirmed reference/clinical strains included human adenoviruses group A, B, C, D, E, and F. Afterward, a phylogenetic tree was constructed by Clustal-X using the distance-based method, Neighbor-Joining, and was then validated following 1000 bootstrap replicates.

Statistical analysis

Data were statistically defined in terms of median and range or mean and standard deviation (± SD), frequencies. For comparing categorical data, the Chi-square test and Fisher’s exact test were done. p-value <0.05 was considered statistically significant. All statistical calculations were done using SPSS version 22 (SPSS Inc., Chicago, IL, USA).

Demographic data and history

Out of the 153 patients included in the study, 92 (60.1%) were males and 61 (39.3%) were females. Participants had a mean age of 32.8 years and an age range of 0.5 to 72 years. Based on the history of contact with the infected person or possibly environmental causes, the results showed that 47 patients (30.7%) had a history of eye infection in family members or colleagues during the last 2 weeks before the referring time and 106 patients (69.3%) had no history of contact with the patients or possible contaminated environment.

Out of 153 patients, 51 patients (33.3%) had infection in left eye, 54 (35.3%) in right eye, and 48 (31.4%) in both eyes. The most of patients were in the age group of 30 to 39 years old (44, 28.8%) and 1 patient (0.77%) was over 70 years old.

Analytical specificity and detection limit of the Real-Time PCR

In silico analysis of primers by the primer BLAST tool revealed a highly specific pattern for the recognition of all human adenovirus serotypes. On the other hand, no sign of possible OFF-target for the human genome or other viral sequences, especially Herpesvirus and Enterovirus, has been detected. The in silico application of the detection primer pair on multiple sequences alignment also showed their suitable strength and specificity for a wide range of adenovirus types. The recent in-house Real-time PCR method was determined to be able to detect all serotypes classified in serogroups A to F. The test showed high specificity when it was tested on several irrelevant samples, while it could detect positive DNA samples. The detection limit of the test is estimated to be 50-100 copies/ml as compared to the serially diluted plasmid containing an adenovirus type 5 backbone.

Molecular detection by Real-Time PCR

The results of Real-Time PCR showed that 126 samples (82.4%) were positive for adenovirus and 27 samples (17.6%) were negative. Investigation of the relationship between demographic characteristics of studied patients and the Real-Time PCR results showed that there was a significant relationship between a positive test in terms of conjunctivitis and gender (P=0.023) and a history of infection in family or colleagues (P=0.048), but this relationship in terms of involved eyes (P=0.637) was not significant. Also, the most positive cases of conjunctivitis were in the age group of 30 to 39 (26.2%), and there was no significant relationship between age and the infection rate (P=0.224). Only one patient was over 70 years old.

Clinical Manifestations

On clinical examination, patients were assessed based on the frequency of Punctate Epithelial Erosions (PEE), corneal epithelial defects (CED), subepithelial infiltration (SEI), Punctate Epithelial Keratitis (PEK), conjunctival injection (CI), chemosis, discharge, conjunctival hemorrhage, existence of membranes, follicular reaction, eyelid inflammation, eyelid redness, photophobia, blurred vision, foreign body sensation (FBS), tearing, itching, pain, cold symptoms, and lymphadenopathy (LAP). The most common clinical findings in patients were conjunctival injection (90.8%), eye discharge (86.6%), conjunctival hemorrhage (85%), tearing (80.4%), follicular reaction (75.8%), and foreign body sensation (67.3%), respectively. The detailed clinical findings are summarized in table 2.

Also, there was a relationship between some clinical findings and a positive Adenovirus genome test. Overall, the results revealed that positive PCR results have a significant relationship with the history of infection in family or colleagues (p = 0.048), gender, pain, tearing, membrane formation (p = 0.047), conjunctiva bleeding (p = 0.046), and pain as demonstrated in tables 3, 4, and 5. Moreover, The sensation of a foreign thing was on the border of significancy (p = 0.51).

Phylogenetic analysis and tree

The fragment encompassing near 700 bp of the Hexon gene was amplified to determine the genotype of viruses. The screening of sequences in BLAST deeply showed the similarity of all the sequences to adenovirus type 8. In addition, the phylogenetic analysis by Neighbor-Joining showed that all 12 sequenced samples were placed in the same cluster of adenovirus type 8, which were nearly identical together with no clear distance on branches, as shown in figure1. As the sequences were too similar to each other, owing to the outbreak, selected sample sequences were deposited in GenBank under the accession numbers MW354697 (Ad-Shrz-S4) and MW354698 (Ad-Shrz-S10).

The pattern of the phylogenetic tree also showed a suitable differentiation among the investigated reference sequences, which was a marker of tree reliability created by this part of the Hexon gene.

Outbreaks of adenoviral keratoconjunctivitis are serious public health concern in ophthalmology departments[15]. In addition, adenovirus-mediated conjunctivitis is a highly contagious infection with a transmission rate of up to 50% by estimation [16]. The virus is transmitted through contaminated personal things, infected fingers, discharges from the eyes, medical equipment, and swimming pools [12]. The comparison of different laboratory studies of acute conjunctivitis showed 40–75% accuracy of diagnostic methods and a reliable place of molecular testing in the clinical setting [6].

There are multiple adenovirus genotypes/serotypes which are associated with different types and severity of infection [12]. Group D, including types 8, 19, 37, and 11 (in group B), are the predominant viruses of keratoconjunctivitis outbreaks from different regions worldwide [17, 18], however, the study considering the prevalent serotypes in the Middle-East is limited. In our region, some studies were conducted to determine the prevalence of adenoviruses in patients with acute conjunctivitis [19]. Also, Shafiei et al. in a similar study demonstrated adenovirus serotype 8 as the most common cause of conjunctivitis in Ahvaz, southwest of Iran [11]. In this regard, the current study aimed to determine the prevalence of adenovirus and related serotypes during two close long-period EKC at Khalili referral Hospital, in the biggest city in the south of the country,

The results showed that out of 153 patients, most of them were males. The Real-Time PCR showed that 82.4% of samples were adenovirus positive. The age group of 30–39 years old was the most affected population, while a rare case of > 65 years old was diagnosed. In a study in Iraq, most of the subjects were women, and the most affected age group with HAdVs was 30–49 y/o (34%), and finally the age over 50 was the least infected one. Because the elderly are more likely to stay at home, they are more likely to have neutralizing antibodies, and their innate immunity is not as strong as at younger ages, the risk of severe inflammatory conjunctivitis acquisition is less than at younger ages, according to theory. However, AL-Mousawi study in Iraq is different as it showed the female gender as an animportant risk factor in groups [20]. Li et al. also revealed that the incidence was significantly higher among male students, in agreement with our study. They pointed out that the largest incidence of adenoviral keratoconjunctivitis infection was detected among 30-39-year-old (17.05%) people [21]. However, Shafiei et al. determined that the lowest incidence rate was in the age group of 10 years, which was different from our study [11]. In this regard, the Iranian study showed that 94.4% of cases based on PCR had an adenoviral origin, and there was no significant difference in terms of gender, age, and income level with the disease. These results are inconsistent with our study because some demographic characteristics, like gender and direct contact with an infected person in the family or friends, were risk factors for EKC [10].

There is a significant relationship between positive adenovirus tests and the history of infection in family or colleagues. In this regard, Li et al. reported that the main risk factor for infection was close contact with the patients or contaminated equipment [21]. Besides, the current study showed that conjunctival injection, eye discharge, conjunctival hemorrhage, tearing, follicular reaction, and foreign body sensation are the most common symptoms. Das and Basu found redness (63.7%) and watery discharge (42.1%) as the commonest signs [22]. Epidemic keratoconjunctivitis is also the most severe form of conjunctivitis, and it presents with watery discharge, hyperemia, chemosis, and ipsilateral lymphadenopathy [23]. Preauricular lymphadenopathy or tenderness was documented in 1406 (7.3%) cases. Other studies pointed out that common manifestations of EKC were follicular hyperplasia, pseudomembrane formation, preauricular lymphadenopathy, corneal involvement, and blurred vision, which were different from the clinical findings in our study [21, 24]. Aoki et al. found that the presence of chemosis, sub-epithelial infiltration, and pre-auricular lymphadenopathy were more associated with EKC than with other ocular infections, such as bacterial or other viral agents involved in ocular infections. [25]. They also reported pre-auricular lymphadenopathy and SEI in 23.5% and 43% of 68 cases, respectively, in adenoviral keratoconjunctivitis, which was due to several adenovirus types [25]. However, in the current study, there was no significant relationship between adenovirus detection and conjunctival injection, eye discharge, preauricular lymphadenopathy [25], and those symptoms which Lee et al mentioned [26]. Also, there was no significant relationship between adenovirus genome detection and preauricular lymphadenopathy inconsistent with Aoki et al [25].

On the other hand, there is no significant relationship between clinical signs of the cornea and PCR results. In a study, there was a correlation between clinical manifestations of EKC and viral infection [27]. The current results also showed that among clinical signs related to conjunctiva and eyelid, conjunctival bleeding and membrane formation had a significant relationship to positive PCR results. Aoki et al. and Hou et al. found different findings that there was no significant relationship between PCR results and conjunctival bleeding and membrane formation signs [25, 28]. Also, pain and tearing had a significant relationship to PCR results, which was inconsistent with Aoki et al [25]. However, Lee et al. showed that there was a significant relationship between tearing and the existing adenovirus genome as in our study [26].

Uemura et al. also didn’t find a significant difference in clinical scores between human groups and several factors, such as days after onset, sex, HAdV DNA copy number on a logarithmic scale, and age [29]. Overall, we also didn’t find any related evidence regarding the association of other clinical manifestations with the presence of adenovirus in the molecular assessment. The inconsistency of the current study results with other reports on the relationship between clinical symptoms and positive molecular tests may be explained by some difference in sample size, the method of detection, or non-random sampling. Also, it was a single-center study from a referral center and needed more investigation in a larger population. Of our important limitations was the absence of other adenovirus types as positive controls and relevant enterovirus genus during the test set-up.

The phylogenetic analysis showed that all of the detected viruses were type D8 adenoviruses. The HAdV8 was also detected as the major cause of many conjunctivitis outbreaks in different populations all over the world, such as Iran [11], Japan [30], Spain [31], Vietnam [32], Tunisia [33], China [34], and Turkey [7]. Sammons et al. showed that HAdV-8 is also the commonest genotype for epidemic conjunctivitis in neonatal patients [35]. Nguyen et al. also demonstrated that HAdV8 in Hanoi (VN2017) belongs to a subgroup of HAdV8 which is circulating in many parts of the world and their genomes are highly conserved [32].

Other studies have shown that the type of adenovirus might contribute to the severity of disease as HAdv-8, HAdv-37, HAdv-53, HAdv-54, and HAdv-57 induce more inflammatory responses and illness than other types [36]. However, in our epidemics, the comparison between types was not applicable. Anyway, these results emphasized a critical need for the use of dignostic methods to find more dangerous types and limit their spread. [37]).

In conclusion, the current study suggests that adenovirus is the most common cause of epidemic keratoconjunctivitis. Moreover, HAdV-D8 was the predominant circulating type in our area. In addition, demographic characteristics and clinical symptoms such as pain, tearing, membrane formation, lymphadenopathy around the ear, and conjunctival bleeding have been shown to be associated with the presence of an adenoviral genome.

Conflicts of interest/Competing interests: The authors declare no conflicts of interest.

Funding The study was supported by grants from shiraz university of medical sciences (Grants No: 22044, 18556, and 18713)

Ethics approval: The study was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.MED.REC.1399.507)

Acknowledgment

The study was supported by grants from shiraz university of medical sciences (Grants No: 22044, 18556, and 18713)

Author contributions:

J.S and M.N suggested the idea and concept. V.R.A., S.Y.H, R.G, N.J, and M.G were invovolve into design and performing of experiments. V.R.A, S.H, and M.G provided samples and releveant questionary from patients in clinic. The real-time PCR was performed by R.G, F.N and N.J. The manuscript has provided by J.S, M.N, VRA and SYH. However, it has sientifically edited by all authors.

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Table 1: The primer list employed in the study

Primer pair

Primer sequences

Length of product

Hexon detection

Forward :5'-TGCAACATGACMAARGACTGG-3'

Reserve: 5'- RCTCATRGGCTGGAAGTT-3'

132 bp

Hexon sequencing

Forward: 5'- GACAACCGGGTGCTAGACATG-3'

Reserve: 5'-GTAGTTGGGTCTGTTGGGGCAT-3'

690 bp

Table 2. The clinical findings in history and physical exam related to cornea, conjunctiva, and eyelid

Patients’ history		Cornea		conjunctiva and eyelid
Clinical finding	Frequency (%)	Clinical finding	Frequency (%)	Clinical finding	Frequency No (%)
Tearing	123(80.4%)	PEE	39(25.5%)	CI	139(90.8%)
FBS	103(67.3%)	PEK	12(7.8%)	Follicular reaction	116(75.8%)
Itching	83(54.2%)	CED	8(5.2%)	Petechia	107(69.9%)
Cold symptoms	51(33.3%)	SEI	7(4.6%)	Purulent eye Discharge	79(51.6%)
Blurred vision	47(30.7%)	-	-	Eyelid swelling	76(49.7%)
Photophobia	37(24.2%)	-	-	Chemosis	63(41.2%)
pain	35(22.9%)	-	-	Non-purulent eye Discharge	53(34.6%)
-	-	-	-	Red eyelids	41(26.8%)
-	-	-	-	LAP	38(24.8%)
-	-	-	-	Pseudo membrane	32(20.9%)
-	-	-	-	Sub Conjunctival hemorrhage	23(15%)
-	-	-	-	True membrane	8(5.2%)

Table 3. Relationship between corneal clinical findings and Adenovirus genome detection

Real-Time PCR
P-Value *	Total	Negative	Positive	Results	Clinical finding
0.58	39	8	31	Yes	Punctate Epithelial Erosions (PEE)
0.58	114	19	95	No	Punctate Epithelial Erosions (PEE)
1	8	1	7	Yes	corneal epithelial defects (CED)
1	145	26	119	No	corneal epithelial defects (CED)
1	7	1	6	Yes	subepithelial infiltration (SEI)
1	146	26	120	No	subepithelial infiltration (SEI)
1	12	2	10	Yes	Punctate Epithelial Keratitis (PEK)
1	141	25	116	No	Punctate Epithelial Keratitis (PEK)

Table 4. Relationship between conjunctiva and eyelid clinical manifestation and Real-Time PCR results

Real-Time PCR
P-Value *	Total	Negative	Positive	Results		Clinical findings
0.71	139	24	115	Yes		Conjunctival Injection (CI)
	14	3	11	No
0.96	63	11	52	Yes		Chemosis
	90	16	74	No
0.86	116	21	96	Yes		Follicular reaction
	36	6	30	No
0.80	76	14	62	Yes		Eyelid inflammation
	77	13	64	No
0.28	41	5	36	Yes		Eyelid redness
	112	22	90	No
0.25	38	2	36	Yes		Lymphadenopathy (LAP)
	115	25	90	No
0.97	79	14	65	Purulent	Yes	Discharge
	53	9	44	Non-purulent
	21	4	17	No
0.046	107	17	90	Petechiae		Yes	Conjunctival bleeding
	23	2	21	Sub. Conj. Hemorrhage
	23	8	15	No
0.047	8	0	8	True		Yes	Membrane
	32	2	30	Pseudo
	113	25	88	No

Table 5. The relationship between clinical findings and Real-Time PCR results

Real-Time PCR
P-Value *	Total	Negative	Positive	Results	Clinical finding
0.088	37	3	34	Yes	Photophobia
0.088	115	24	91	No	Photophobia
0.28	47	6	41	Yes	Blurred vision
0.28	105	21	84	No	Blurred vision
0.051	103	14	89	Yes	Foreign body sensation (FBS)
0.051	49	13	36	No	Foreign body sensation (FBS)
0.048	123	18	105	Yes	Tearing
0.048	30	9	21	No	Tearing
0.91	83	15	68	Yes	Itching
0.91	69	12	57	No	Itching
0.042	35	2	33	Yes	Pain
0.042	117	25	92	No	Pain
0.65	51	10	41	Yes	Cold symptoms
0.65	102	17	85	No	Cold symptoms

No competing interests reported.

The molecular epidemiology, genotyping, and clinical manifestation of prevalent Adenovirus infection during the epidemic keratoconjunctivitis, South of Iran

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Abstract

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Introduction

Materials And Methods

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Discussion

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Additional Declarations

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