Adenovirus, belong to Adenoviridae family under the Preplasmiviricota, is a medium-size, non-enveloped, double-stranded DNA virus with an icosahedral nucleocapsid [1,2]. The virus first isolated from adenoids in 1953 and then, it was detected to cause a wide range of illnesses including cough (cold- or flu-like infection), fever, runny nose, pharyngitis (sore throat), conjunctivitis (pink eye), otitis media (ear infection), bronchitis and pneumonia, gastrointestinal tract infections (diarrhea, nausea, vomiting and gastroenteritis), urinary tract infections as well as encephalitis and meningitis [3-6]. Adenovirus is very contagious and can be easily spread through direct contact and exposure to respiratory droplets released in sneezes and coughs; touching of contaminated surfaces or eye, nose and mouth; washing or drinking by contaminated water and due to exposure to stool samples in particular during changing the baby’s diaper [7,8].

Ad-CS “pink eye” is a wide spread ophthalmic viral infections in several countries, which demonstrated to be highly contagious other than other forms of conjunctivitis, as a result partially for the fact that the virus has a capability to still infective for several days at room temperature [9,10]. Consequently, Ad-CS is obviously symptomatic to show the signs of decreased vision, photophobia, lid swelling, tearing and discomfort [11]. Approximately, 15-35% of diseased individuals were showed to developing an infiltration in sub-epithelial cornea, which progressing to visual and permanent damage [12]. However, there are four clinical phenotypes recognized in Ad-CS including the acute non-specific follicular conjunctivitis, chronic keratoconjunctivitis, pharyngoconjunctival fever and epidemic keratoconjunctivitis that represents the most serious of the adenoviral infections [13]. 

Studies concerned the most effective Ad-SC therapeutic schedules have seriously hampered due to an absence of active diagnostic techniques for differentiating the disease from other etiologies of conjunctivitis. Culture is the gold for identification of adenovirus infections; however, it can take up to 3 weeks to achieve culture results [14]. In addition, diagnosis is usually following the characteristically clinical symptoms; however, the accuracy of this diagnostic tool is very low [15]. Different serological tests have faster turnaround times when compared to culture. Rapid pathogen screening tests have been approved for rapid serological diagnosis of the virus antigen with less 89% sensitivity and 94% specificity when compared to molecular polymerase chain reaction (PCR) assay [16,17]. PCR offers a rapid, highly specific and more sensitive means of diagnosis by detecting the adenovirus DNA [18,19]. Hence, the current study was aimed to confirm of adenovirus in patients undergo conjunctivitis in Wasit province, Iraq.

Ethical Approval

This study was licensed by the Scientific Committee of the Department of Biology in the College of Science (University of Wasit, Wasit, Iraq).

Collection of Samples

A total of 85 patients having ophthalmic and nasopharyngeal infections whose attended to the Al-Zahraa Teaching Hospital (Kut-Wasit province) and diagnosed clinically to be infected with Ad-CS were subjected to collection of intraocular conjunctival swabs under aseptic conditions. The swabs were placed into 1 ml of basic saline solution in transport tube and kept frozen at -20ºC until be used for extraction of DNA.

Molecular Examination

Following the manufacture instructions of the Easy Pure ® Viral DNA/RNA Kit (TransGen Biotech, China), DNAs were extracted from the swab samples and tested by the Nanodrop System (Thermo-scientific, UK) to detect their purity and concentration. For preparation of Mastermix tubes at a final volume of 25µL, one set of primers were designed; (F: 5´-GCC GCA GTG GTC TTA CAT GCA CAT C-3´) and (R: 5´- CAG CAC GCC GCG GAT AAA GT-3´) based on recently study [20] and the One Taq® Quick-Load 2X Master Mix with Standard Buffer (BioLabs, England) were used following the manufacturer instructions. PCR reaction was carried out using the Thermal cycler T100 System (BIORAD, USA) at the next conditions: 1 cycle initial dentauration (95ºC/5 min); 40 cycles for denaturation (95ºC/30 sec), annealing (58ºC/30 sec) and extension (72ºC/30 sec); and 1 cycle final extension (72ºC/5 min). Electrophoresis using 1.5% agarose gel stained with Ethidium Bromide was performed at 100 Volt, 80 Am for 1 hour and amplification of the positive bands was visualized under the Ultraviolet Illuminator System (Clinx Science, China) at 300 bp.

Statistical Analysis

The t-test in the GraphPad Prism (6.0.1) Software was applied to detect significant differences at p<0.05 [21,22].

An overall 49 (57.65%) positive samples were detected among the examined study populations (Figure 1).

In Iraq, the number of studies that aimed for detection the prevalence of Ad-CS clinically (28%) [23] and immunologically 84.9% [24], or to confirm of infections using Real-Time PCR (76.66% [4] and 77.8% [25], remain limited and need to be supported. Worldwide, the prevalence of Ad-Cs was 66.7% in Vietnam [26], 50.02-60% in Brazil [27,28], 20.6-26.5% [29], 14.6% in Iran [30], 13.8% in China [31], 39.4% in UK [15], 36.1% in France [32], 65.2% in India [33] and 38% in Egypt [34]. Variation in prevalence of Ad-CS in our study and other reports might be attributed to type, sensitivity and specificity of applied diagnostic tool; type and technique of sample collection and processing and contribution of risk factors such as age, sex, health status and severity of infection of an individual, season and geographical areas. Consequently, viral conjunctivitis is usually misdiagnosed and the accuracy rates of clinical symptoms in detection of such infections having <50% as detected by many studies. In USA, although the incidence of Ad-CS is evaluated to having as high as 20 million cases per year, exact numbers remain unknown [8].

As showed by other studies, epidemic Ad-CS reported to account approximately 6-60% of all cases of infectious conjunctivitis [35] and 65-90% of cases of viral conjunctivitis [27,36,37]. PCR is usually the principle diagnostic method used and to confirm the virus more effectively than culture, enzyme immunoassays, direct immune-fluorescence and immune dot-blot tests [8]. Different studies referred that PCR is able to detect as few as 106 to 103 viral particles per ml in different swab samples [38,39,40). 

Figure 1: A Representative Image for Some Positive PCR Products in Agarose Gel Electrophoresis at 100 Volt, 80 Am for 1 Hour

Lane L: Ladder Marker (100-1500 bp), Lane N: Negative control, Lanes (1, 2, 3, 7, 8, 9, 10, 13, 14, 15, 16 and 22): Negative samples, Lanes (4, 5, 6, 11, 12, 17, 18, 19, 20 and 21): Positive samples

Ad-Cs remains one of the most widely distributed and neglected viral diseases in Iraq. Molecular techniques offer many advantages in diagnosis of disease and confirmation of clinical symptoms. Since Ad-SC is highly contagious and usually presents a considerable cost, routine molecular testing of suspected cases will provide additional insight. Furthermore studies appear necessary due to the low available data for annual incidence of infection and the worldwide absence of licensed therapy.

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