Dr. Nancy Magdalene,
Dr. Padma Paul, Dr. Thomas Kuriakose,
Dr. Anika Amritanand
Nancy MAGDALENE, Padma PAUL, Thomas KURIAKOSE, Anika AMRITANAND
Department of Ophthalmology, Christian Medical College, Vellore, India
ABSTRACT
Objectives:
- To study the prevalence of HSV 1&2 in culture negative, suppurative keratitis by Duplex Real time Polymerase chain reaction (PCR).
- To calculate the healing time of Herpes simplex virus (HSV) positive ulcers.
Methodology:
A prospective observational pilot study was conducted on patients with suppurative corneal ulcer who presented between March and October 2015. They underwent corneal scraping to identify bacteria, fungus and protozoa. PCR for HSV 1 & 2 was performed on the culture and smear negative cases. Acyclovir (topical and oral) and topical steroids were started on PCR positive patients. The Prevalence of HSV in culture negative suppurative keratitis, ulcer healing time and clinical variables associated with HSV positivity wereanalyzed.
Results:
Seventy-one patients presented with suppurative keratitis during the study period. 43 (61%) were culture positive. Twenty-Eight (39%) were culture negative, of which HSV PCR showed 3 positives. The prevalence of HSV1 in culture negative suppurative keratitis was 10.7% (95% CI 2.3% – 28.2%). Past history of red eye (p=0.009), corneal ulcers (p=0.006) and antiviral intake (p=0.001) were associated with viral etiology. Presence of corneal scar (p=0.001) more than endothelial involvement (p=0.630) suggested viral etiology. The healing time in HSV positive ulcers were 52 days (range 47-58).
Conclusions:
Viral keratitis can present as suppurative keratitis. Certain factors like past history of red eyes, corneal ulcers and antiviral use, presence of corneal scar and endothelial involvement had a higher prediction for HSV positivity. A prevalence of 10.7% in our pilot study needs further exploration with a higher sample size.
Introduction:
Microbial keratitis is a leading cause of ocular morbidity and blindness worldwide. Most centers use corneal scraping and culture sensitivity for the etiology, the positivity ranging from 35-55 % while others base treatment on clinical findings alone and patients are started on empirical treatment. Minimizing corneal scar is the priority in management of corneal ulcers. But the increasing number of culture negative specimens limits our treatment options. There may be multiple factors leading to this culture negativity but as a first step we would like to focus on identifying organisms other than bacteria and fungi, especially viruses and their contribution in suppurative corneal ulcers. With this background, we decided to investigate the contribution of viral etiology in the culture negatives as appropriate diagnosis may initiate earlier healing and thus improve visual outcomes.
To the best of our knowledge, there is no data on the presence of Herpes simplex viruses in culture negative suppurative keratitis. In our study we will be looking into the prevalence of HSV-1&2 in culture negative suppurative keratitis using qualitative Real time Duplex polymerase chain reaction (PCR).
Materials and Methods
Study Design:
This was a Prospective observational case series conducted as a pilot study on all patients with suppurative keratitis presenting to the Department of Ophthalmology, Christian Medical College Vellore, who fulfill the inclusion criterion (as described below).
Inclusion criteria:
Patients
- With suppurative corneal ulcer amenable to corneal scraping.
- In whom the corneal scraping specimens were smear and culture negative on microbiological examination.
- Who were willing to provide an informed consent.
Patients with a dendritic corneal ulcer, children, pregnant women and on whom scraping was not indicated (impending perforation or ulcer less than 1 mm due to inadequacy of infective material) were excluded.
Methodology:
An informed consent was obtained and a detailed patient questionnaire was obtained. Corneal ulcer scraping was performed with a cataract knife. The smear (Gram stain for bacteria and Lacto phenol cotton blue for fungi) and culture plates (blood agar, chocolate agar and Sabouraud Dextrose Agar) were sent. A waiting period of 5 days was set before declaring that the corneal ulcer was culture negative.
Patients already started on antibacterial or antifungal drops elsewhere were given a 6-hour drop free interval before subjecting them to scraping.Simultaneously corneal scrapings were obtained from all cases for PCR analysis. After 5 days, preserved samples of both smear and culture negative cases were processed for virus isolation using Real time Duplex PCR.
Patients with culture negative corneal ulcers were treated empirically with anti-bacterials (Cefazoline 5% and Fortified Gentamycin 1.4% eye drops) and/or antifungals (Natamycin 5% eye drops) based on clinical signs and symptoms.Patients with positive PCR for HSV-1&2 showing no improvement or worsening with empirical treatment were treated additionally with Tab Acyclovir 400 mg 5 times a day and Ointment Acyclovir 3% five times a day with tapering based on clinical response. Topical steroid drops (Prednisolone acetate 1%)
Results:
Of the 71 cases that were included in the study, 43 cases (60.5%) were either smear and or culture positive while the remaining 28 (39.5%) were both smear and culture negative.
Microbial profiles in smear and culture positive cases:
43 patients with suppurative keratitis showed positive smear or/and culture for bacteria, fungus or protozoa. There was no discrepancy in organism between the smear and culture reports. Among the 43 patients, 31 patients were positive for fungus (72%), 11 patients showed bacteria (26%) and 1 patient grew Acanthamoeba (2%).
POLYMERASE CHAIN REACTION FOR HSV 1 AND 2:
The 28 smear and culture negative samples were processed for Duplex PCR (both HSV 1 & 2). Of these, 25 cases were negative for both HSV 1 & 2 while 3 cases were positive for HSV1. None of the cases were positive for HSV2.Ours was a pilot study and we have identified 3 cases with HSV positivity, which thus contributes to a prevalence of 10.7 % (95% CI 2.3% – 28.2%) using Binomial (Clopper-Pearson) ‘exact’ method.
The microbial culture positive rate in our study was 60%. With the addition of 3 HSV positive cases to the infectious keratitis, the microbial positivity in infectious keratitis increases to 64.8% in our hospital based set up.
All patients with culture negative suppurative keratitis had both superficial and deep stromal involvements. Endothelial involvement along with superficial and deep stromal involvement was seen in 2 out of 3 HSV positive cases, but this was also seen in 13 out of 25 HSV negative patients. Thus the HSV positive group had a higher percentage of endothelial involvement than in the HSV negative group. (66.7% as compared to 52%), but the difference was not statistically significant. (P=0.630). A corneal scar was noted in 2 out of 3 patients who were HSV positive (66.7%) but only 1 out of 25 HSV negative patients had a corneal scar (4%) thus making presence of a corneal scar a predictor of HSV positivity with a p value of 0.001.
Discussion
This study was a hospital-based study that looked into the microbial etiology of corneal ulcers, where we found that 39.5% of the patients who presented to the eye department with suppurative keratitis, did not have a specific causative organism identified. We tried to investigate this subset of patients to see if there was a viral etiology especially Herpes Simplex Virus (HSV 1&2) through Polymerase chain reaction (PCR).
The definition of suppurative keratitis varied in different articles. Most studies have defined suppurative keratitis as a stromal infiltrate of cells with an overlying epithelial defect with or without a hypopyon1,2.Almost all patients with corneal ulcers had presenting complaints of pain, redness and defective vision during the current episode. But recurrent episodes of pain, redness and decreased vision have been noted in patients with viral keratitis and in immune keratitis3.
We encountered 53.1% of patients with suppurative keratitis who were started on some kind of topical medication from elsewhere. Overall, only 16% have been started on appropriate treatment considering the culture and or smear report to be a gold standard as compared to 48.4% in a study from India4. Thus our system of empirical treatment especially in primary and private health sectors in our region is quite poor. In the west as bacterial corneal ulcers predominate, use of empirical treatment with broad-spectrum antibiotics has been recommended5. But the same cannot be applied to our population considering the high fungal load. According to an Indian study, with empirical treatment 83% of suppurative keratitis healed while the rest worsened needing surgical interventions5 but the corresponding smear and cultures were not looked at.
In our study we have excluded clinically viral ulcers and have calculated the prevalence of HSV in culture and smear negative cases of suppurative keratitis. Viral corneal ulcers have specific presenting features and thus most studies have data based on clinical findings alone. Most studies on etiological agents in suppurative keratitis have done so after excluding the clinically viral ulcers. However one study done in India has noted a clinical prevalence of 7.4% of HSV keratitis among patients presenting to a cornea specialty clinic6.
Herpetic keratitis can have varying presentations and is more prevalent in the developed countries. Their incidence ranges from 8.4 – 13.2 new cases per 100000 person years in the west7. We have found a prevalence of 10.7 % (95% CI 2.3% – 28.2%). To the best of our knowledge, presence of virus in culture / smear negative suppurative keratitis has not been reported to make comparisons.
Considering the high sensitivity of PCR in virus isolation (85-100%)and a specificity of 70-75%[60], the culture negative scrapings were subjected to HSV 1 & 2 PCR. There is no sensitivity/ specificity profile documented in our lab for technique used in HSV PCR[48]. Our virology lab is certified by UK NEQAS which is about more than monitoring technical accuracy and precision of results.
Herpes keratitis can present as suppurative keratitis without classical clinical characteristics of a viral ulcer. It appears to be present in as high as 10% of smear/culture negative patients. Looking for viral etiology in all culture negative cases may not be feasible. The suspicion should be high if there was a past history of red eyes or corneal ulcer or both. Past history of antiviral use also has a strong association but not other topical medication. Presence of a corneal scar and the ulcer involving the corneal endothelium also makes it more likely to be a viral etiology.
Studies have reported healing rates of viral keratitis with oral and topical antivirals to range from 6-8 weeks (42-56 days)7. In our study the time taken to heal was 47 – 51 days in the HSV positive group after starting on oral and topical Acyclovir along with steroid drops which seems comparable.
Our study does seem to be a representative sample of suppurative keratitis presenting to a tertiary care institution. The results will however need to be studied with an appropriate sample size to make more meaningful comparisons.
Conclusion:
Herpes keratitis can present as suppurative keratitis without classical clinical characteristics of a viral ulcer. Thus culture negative corneal ulcers with the above-mentioned clinical history and features should have a high index of suspicion for the presence of viral etiology.Scraping the ulcer for microbiological analysis may worsen the already compromised corneal stroma. Prompt treatment with antivirals and steroids must be considered for faster healing rather than starting empirical treatmentthus reducing the burden of corneal blindness.
References
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- Leck AK, Thomas PA, Hagan M, Kaliamurthy J, Ackuaku E, John M, Newman MJ, Codjoe FS, Opintan JA, Kalavathy CM, Essuman V, Jesudasan CA, Johnson GJ. Aetiology of suppurative corneal ulcers in Ghana and south India, and epidemiology of fungal keratitis. Br J Ophthalmol 2002;86-11:1211-5.
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