Introduction

Viral infections are responsible for causing numerous diseases in humans, and keratitis is no exception 1. Several viruses have been identified as causative agents of viral keratitis, including herpes simplex virus (HSV) and varicella- zoster virus (VZV). These viruses can infect the cornea either through direct contact or by spreading from other areas of the body. Viral keratitis is significant concern because it can result in severe corneal damage, leading to long-term vision problems 2,3,4. Additionally, there are various studies on the role of HSV-2 and AdV in the occurrence of ocular infections, indicating that the inclusion involves ocular infections and is not limited to keratitis 5,6,7.

The association between keratitis and viral infections underscores the importance of understanding the interaction between the two phenomena. Viruses have evolved various mechanisms to evade the immune system and establish persistent infections in the cornea. This persistence can lead to chronic inflammation and recurrent episodes of keratitis 1,8. Additionally, the immune response triggered by viral infections can cause collateral damage to the cornea, further exacerbating the inflammation and tissue damage. Therefore, it is crucial to study investigate the underlying pathogensesis and molecular mechanisms underlying responsible for viral keratitis to develop effective preventive measures, diagnostic tools, and treatment strategies 9,10,11,12.

Furthermore, the combined significance of keratitis and viral infections extends beyond the realm of ophthalmology. The cornea serves as a unique model for studying viral pathogenesis, as it is easily accessible and provides an ideal environment for viral replication 13,14. Understanding the association between keratitis and viral infections can lead to advancements in preventive measures, diagnostics, and treatments, improving patient care and potentially benefiting the management of other viral diseases.

Recent breakthroughs in molecular biology, particularly in the ability to identify a wide range of pathogens in a single test, known as multiplexing, have revolutionized the diagnosis of both common and rare infectious agents across various clinical samples. This multiplexing technique is often referred to as syndromic testing and is commonly employed for respiratory, gastrointestinal, and central nervous system infections. Unfortunately, its potential remains largely untapped in the field of ophthalmology, primarily due to the limited awareness of its benefits in diagnosing viral eye infections. In cases of keratitis, it is crucial to underscore the importance of promptly diagnosing underlying infections to prevent further complications and ensure appropriate treatment.

The objectives of the study were to evaluate the prevalence of HSV-1, HSV-2, AdV and VZV in patients suspected to have ocular infections.

Material and methods

Study population

This research, carried out between 2020 and 2022 at the Clinical Virology Research Center, which is affiliated with Tehran University of Medical Sciences, had the primary objective of examining the prevalence of viruses among 162 patients with keratitis. 83 (51%) patients were female and 79 (49%) were male among these 162 (100%) patients. Patients suspected of ocular infection were included in the study. These patients exhibited various clinical manifestations indicative of ocular pathology, such as infectious keratitis, corneal scar, endogenous endophthalmitis, panuveitis, endothelitis, stromal edema, and other relevant conditions.

Sample collection and genome extraction

Samples were collected from patients who were suspected for ocular infection presented with symptoms requiring microbiological diagnosis. Ophthalmologists obtained samples by performing deep scraping of the cornea with sterile stainless-steel blades 20, under a slit lamp. Four types of samples were collected for analysis: tear fluid was collected as eye wash by rinsing the ocular surface with 500 µL of sterile saline three times, and 500 µL was collected from each subject. For epithelial keratitis, the scraped corneal epithelium was collected by debriding the edge of an ulcer with plastic swabs. For stromal or endothelial keratitis, 0.1 µL of aqueous humor were collected by anterior chamber paracentesis with a 27-gauge needle from the affected eye, and vitreous humor samples were also taken. All eye specimens were transferred to the laboratory under appropriate conditions inside the tubes contained 2 mL of phosphate buffer saline (PBS), as transfer medium. For further genome extraction and infectious agent detection.

Viral nucleic acids were extracted from 100 µL of each specimen using the Qiagen Mini Blood Kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations. An internal control was added to each specimen before loading the trays to exclude false negatives due to nonspecific inhibitors of the PCR enzymes. Also, extracted genome concentration were assessed by nanodrop at A 260/280 (with A ratio of ~ 1.8).

Multiplex real-time PCR

We performed Multiplex Real-Time PCR on 5 µL of extracted materials using the Fast-track diagnostics/SIEMENS eye kit (Esch-sur-Alzette, Luxembourg) for the detection of HSV-1, HSV-2, AdV and VZV. The assay was conducted according to the manufacturer's instructions.

Statistical analysis

In this study we used IBM SPSS package (v.27). R programming language (v 4.2.3) was used to test equality of infected proportions. To summarize our data, we performed median, mean and standard deviation for continues variable age, and frequency and percentage for categorical variables such as sex and age. Kolmogorov–Smirnov test was applied to check normality. We used Mann–Whitney U Test to compare age between two sex groups. To investigate any relationship between sex and clinical tests results, we performed Fisher’s exact test. In order to investigate any relationship between age and clinical tests results, first we categorized age according to the important age groups. Then we applied Kendall’s tau-b test to find any relationship. Significance level was considered 0.05 for all statistical tests.

Ethics approval and consent to participate

The study was performed in accordance with the Declaration of Helsinki and the present study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences, Tehran, Iran.

Consent to participate

Informed consent for publication of identifiable information/ images in open access journal was obtained from all study participants.

Results

Demographic data

In this scientific study, a total of 162 individuals having ocular infection were included based on eye redness, eye pain, excess tears, difficulty opening eyelid because of pain or irritation, blurred vision and decreased vision. Among the participants, 83 (51%) were female and 79 (49%) were male. The age range of the participants was between 17 and 77 years, with a mean age of 45.3 ± 18.11 and a median age of 48. The average age for females was 48.3 ± 17.27, while for males it was 42.7 ± 18.66. The difference in average age between males and females was not found to be statistically significant (P-value = 0.219) (Table 1). Among the 162 patients, 48 (29.6%) tested positive for viral infection; of whom, 24 (50%) were female and 24 (50%) were male. There was no significant difference observed between the two groups in terms of positivity (P-value = 0.615) (Table 2).

Table 1 The effect of age group on each virus infection.
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Table 2 The effect of sex on each virus infection in positive cases (63 patients).
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Viruses distribution

The most frequently detected viruses were VZV with a prevalence of 12.3%, HSV-1 with a prevalence of 11.7% followed by AdV (4.9%) and HSV-2 (0.6%). The least frequent virus detected was HSV-2 with only one infected individual. Among female patients, VZV had the highest frequency of viral infection, while HSV-2 was not detected in any female patients. Among male patients, HSV-1 and VZV were the most frequent viruses (Table 1, Fig. 1). However, the sex of the patients did not significantly influence the occurrence of viral infections (all P-value > 0.05). The frequencies of the different viruses are reported in Table 1. Also, the effect of sex on prevalence of each virus is show in Table 2. For VZV infection, there is no statistically significant difference between females and males based on the P-value of 0.241. Similarly, for HSV-1, there is no significant difference between females (47.4% positive) and males (52.6% positive) with a P-value of 0.553. The same is observed for AdV and HSV-2, where there are no significant differences in infection rates between females and males. HSV-2 shows a difference, with 100% of males testing positive compared to none of the females (0%) (Table 2).

Figure 1
figure 1

Prevalence of infection among positive patients divided by viruses.

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The incidence of viral infections in different age groups is presented in Table 3. Among patients under 18 years old, VZV was detected in 4 patients (80%). In the age group of 18 to 49, VZV was found in 5 patients (18.5%), while among patients aged 50 and above, VZV was positive in 11 patients (35.5%) (Fig. 1). VZV was the most frequent virus observed in patients under 18 years old. AdV and HSV-2 were not detected in this young group of patients. Among patients aged 50 and above, VZV had the highest frequency at 35.5%, followed by HSV-1, AdV and HSV-2. There were 4 (8.3%) coinfections within the samples which were all VZV and HSV-1 coinfection.

Table 3 Report on the status of virus identification by sampling method from patients (overall, 64 samples from total of 48 patients were positive).
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Sample type and the actual percentage of infection

Among four types of collected samples, aqueous humor sample had the highest number of positives cases (50 positive) (P-value < 0.05) (Fig. 2). Vitreous humor, tear fluid and scraped corneal epithelium samples had 7, 3 and 4 positive cases respectively. Table 3 presented the characteristics of positive patients according to the type of sample and virus.

Figure 2
figure 2

Ocular specimen type and its percentage viral load.

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Discussion

These results obtained by this investigation are consistent with previous studies that have reported a similar distribution of corneal infections among genders. The mean age of the cases was 43.6 years, with a wide range from 8 months to 87 years. Notably, the highest prevalence was observed in the age range of 21 to 40 years, while the lowest prevalence was seen in patients below under 20 years and above 61 years. This age-related variation in infection rates may be attributed to differences in immune status, environmental exposure, and risk factors associated with specific age groups.

The analysis of sample types and respective infection rates revealed that the aqueous humor samples had the highest percentage of positive cases, followed by vitreous humor, tear fluid, and scraped corneal epithelium. These findings suggest that the aqueous humor sample probably is the most reliable for detecting viruses and chlamydia trachomatis in corneal infections. Clinicians should prioritize obtaining and analyzing aqueous humor samples for accurate diagnosis and appropriate treatment selection.

In terms of infectious agent prevalence, the survey identified VZV as the most prevalent virus. HSV-1 was the second most prevalent virus, with a higher frequency of morbidity in females. Other viruses, including AdV and HSV-2, were also detected but with lower prevalences rates. Additionally, 4 cases of coinfections were reported, with VZV coinfections being the most prevalent.

A review article by Ting et al. provides an update on the epidemiology, causative microorganisms, risk factors, and antimicrobial resistance of infectious keratitis (IK), which can be caused by bacteria, fungi, virus, parasites and polymicrobial infection. The authors report that in developed countries, corneal blindness can result from less frequent but serious conditions such as viral and Acanthamoeba keratitis. The main factors that increase the risk of IK include wearing contact lenses, injuries to the eye, diseases affecting the ocular surface or the eyelids, and complications after eye surgery 15. A study published in experimental eye research summarized the prevalence, diagnosis, and pathobiology of viral keratitis. The authors reported that herpesviruses were the predominant etiologic agent of viral keratitis, and that virus-mediated immunomodulation of host innate and adaptive immune components was critical for viral persistence and disease progression. They also discussed current and emerging therapies to treat viral keratitis, such as antiviral drugs, immunomodulators and gene therapy 16. In research conducted by remeijer et al., HSV-1 was the most prevalent herpesvirus in the cornea tissues, followed by VZV and HSV-2 with both prevalence and the corneas of patients with HK was related to several factors, such as age, time since last recurrence, new blood vessels in the cornea, steroid use before penetrating keratoplasty (PKP), and how severe the disease was. Only 2 out of 273 corneoscleral rims had herpesvirus DNA, which suggest that testing donor corneal tissues for herpesviruses is not needed to prevent HK after PKP. Graft survival, which is the success rate of the transplantation, was inversely correlated with the corneal HSV-1 viral load in patients with HK, indicating that high levels of HSV-1 in the cornea tissues may impair the graft outcome. They used Real-time PCR and virus culture to detect the presence of HSV-1, HSV-2, and VZV in the cornea tissues 17. Compared to our study, we also observed a high prevalence of HSV-1 and VZV, but in contrast to the mentioned report, we observed a low prevalence of HSV-2.

In another study by Pramod et al. to evaluated the prevalence of HSV-induced keratitis (HSK), which is an inflammation of the cornea caused by HSV, among 3,000 patients attending a corneal clinic in South India. The results were shown that HSK accounted for 5.4% of all corneal diseases seen at the clinic, indicating that HSV is a significant cause of corneal morbidity in South India 18. In a study, Mokhtari et al. determined the prevalence of HSK among 307 patients with clinical keratoconjunctivitis who attended Feiz ophthalmology centre, Isfahan, Iran, between 2012 and 2013. The authors used PCR method to detect HSV DNA in tear samples from the patients. HSV DNA was detected in 13.4% of the patients with keratoconjunctivitis, indicating that HSV is a common cause of ocular infection in Iran and also HSV-1 is more prevalent than HSV-2, accounting for 11.4% and 2% of the cases, respectively. The most common clinical manifestations of HSK were dendritic ulcer, geographic ulcer, and disciform keratitis 19. In a study by Omidian et al. investigated the frequency of EBV infection using PCR method in serum and ocular swab samples from patients with inflammatory eye diseases, in comparison with healthy controls. The authors designed primers for conserved regions of the EBV genome and performed PCR on 150 serum samples and 20 ocular swab samples. EBV DNA was detectable in 3.84% of the control serum samples, 5% of the serum samples from patients with inflammatory eye disease, and none of the serum samples from patients with eye infection. Also, EBV DNA was detectable in 10% of the ocular swab samples from patients with inflammatory eye disease, and none of the ocular swab samples from patients with eye infection or healthy controls 20. In a study by Alatawi et al. estimated the prevalence of AdVs as causative agents of ocular diseases in Saudi Arabia. The authors collected serum and ocular swab samples from 100 patients with different types of ocular diseases, such as conjunctivitis, keratitis, and uveitis, and performed PCR method to detect AdV DNA. AdV DNA was detected in 9% of the serum samples and 11% of the ocular swab samples, indicating that AdVs are prevalent in Saudi Arabia and can cause various ocular diseases. Also, viral genome was detected in 13% of the patients with conjunctivitis, 7% of the patients with keratitis, and 10% of the patients with uveitis, suggesting that AdVs can infect different parts of the eye and cause inflammation. Additionally, AdV DNA was detected in both serum and ocular swab samples in 4% of the patients, indicating that AdVs can cause systemic and local infections simultaneously 21.

Viral keratitis is a common and potentially sight-threatening ocular infection caused by various types of viruses, such as HSV, VZV, AdV, and CMV. Early detection by PCR has transformed infectious disease diagnosis by enabling the amplification of target sequences. To address cost limitations and boost diagnostic capabilities, multiplex PCR has emerged, amplifying multiple target sequences with various primer pairs in a single reaction. While early studies uncovered challenges in developing sensitive and specific multiplex assays, recent research has introduced systematic protocols and technical enhancements, including the selection of oligonucleotide primers and the use of hot start-based PCR techniques. These advancements, combined with efforts to enhance sensitivity, specificity, and automation, have led to a surge in publications exploring multiplex PCR applications, particularly for detecting clinically and epidemiologically significant viruses 22.

Early detection of viral infections is crucial for timely intervention and effective treatment. Multiplex PCR plays a pivotal role in this by enabling the simultaneous detection of multiple viral pathogens in a single test. This approach is essential in diagnosing emerging viral diseases, monitoring outbreaks, and preventing their spread. Multiplex PCR not only saves time and resources but also enhances the accuracy of diagnosis, making it a valuable tool for public health and clinical settings where rapid and precise identification of viral infections is necessary. While positive multiplex PCR results indicate the presence of viral genetic material, they do not necessarily confirm active infection. This distinction is crucial for accurate diagnosis and treatment planning. Therefore, future studies should consider utilizing diagnostic methods that can specifically detect active infections to provide a clearer understanding of the clinical relevance of the detected viruses.

According to study limitations, our study had proper number of patients in the context of statistics. However, further researches with higher population rate are recommended. Additionally, it's important to take into account other constraints such as geographical restrictions. The study's participants may be primarily from a specific geographic region (Tehran, Iran), potentially affecting the representation of viral strains and infection rates.

Conclusion

The findings of this survey have important implications for the diagnosis and management of keratitis. Accurate and timely diagnosis is crucial for appropriate treatment selection, especially considering the varying susceptibilities of different pathogens to antiviral and antiparasitic agents. The high prevalence of VZV and HSV-1 underscores the importance of considering these viruses as potential causative agents in corneal infections, particularly in cases presenting with characteristic clinical manifestations. Early diagnosis and treatment are essential to prevent complications and improve visual outcomes.