Microbiological and epidemiological study of infectious keratitis in children and adolescents

Purpose:
To analyze epidemiological and microbiological aspects of microbial keratitis in children and adolescents.


Methods:
This retrospective cohort study was conducted at the Department of Ophthalmology and Visual Science, Escola Paulista de Medicina, Universidade Federal de São Paulo, between July 15, 1975, and December 31, 2010. We analyzed corneal samples from 859 patients with clinical suspicion of infectious keratitis, comparing epidemiological and microbiological characteristics of bacterial keratitis with those of non-bacterial and non-viral keratitis. We also compared Gram-positive and Gram-negative pathogens in patients with bacterial keratitis. We created a susceptibility profile of the bacterial microorganisms studied.


Results:
Of the 859 patients, 346 (40.3%) showed positive culture results for non-viral microorganisms. Teenagers (13-18 years) made up the group with the highest number of patients with keratitis (164, 47.4%). The most frequent risk factors for keratitis were trauma (33.5%) and previous ocular surgery (24.9%). Gram-positive bacteria (71.8%) were the most often isolated, with coagulase-negative Staphylococcus (23.8%) the most prevalent microorganism. Logistic regression analysis showed age (p=0.002), topical antimicrobial drug use (p=0.01), and trauma due to non-chemical burns (p=0.005) were risk factors for non-bacterial keratitis. Age (p=0.01) was also a risk factor for Gram-negative bacterial keratitis.


Conclusion:
Our study showed that in the age range studied, the prevalence of keratitis caused by Gram-negative bacteria or by the non-viral microorganisms evaluated increases with age. Previous use of topical antimicrobial drug and trauma due to non-chemical burns are associated with non-bacterial keratitis. Knowledge of the risk factors and the microorganisms involved may help improve treatment of keratitis in children and adolescents and minimize visual impairment.

The diagnosis of keratitis is usually clinical and the treatment is empirical, with the application of broad-spectrum topical antimicrobial agents until identification of the etiologic agent is completed.However, few studies have investigated the causative agents and risk factors of infectious keratitis in children and adolescents.
The purpose of this study was to investigate the epidemiological and microbiological findings of infectious keratitis in patients up to 18 years of age attending a tertiary referral center.In addition, the Arq Bras Oftalmol.2016;79(5):289-93 risk factors associated with the presence of bacterial keratitis and non-bacterial non-viral keratitis were analyzed, as well as the in vitro susceptibility profile of the bacterial isolates.

METHODS
This retrospective cohort study assessed microbial keratitis in patients up to 18 years old in the period from July 15, 1975, to December  31, 2010.All data included in the laboratory records of patients with clinical suspicion of keratitis were evaluated at the Ocular Microbiology Laboratory, Department of Ophthalmology, Federal University of São Paulo, São Paulo, Brazil.The variables studied included age, sex, affected eye, use of contact lenses, use of topical medications, ocular antecedents, comorbidities, and previous ocular surgery.The in vitro susceptibility profiles of the bacteria were identified, and the annual incidence of infectious keratitis and the prevalence of etiologic agents were also evaluated.The local Ethics Committee approved this study.
All subjects underwent sample collection from corneal ulcers by an ophthalmologist.The corneal scraping was performed using topical anesthesia with proxymetacaine (5 mg/ml) or, when necessary, narcosis in the operating room.We inoculated the collected material in solid (blood agar, chocolate agar and Sabouraud agar; Oxoid Ltd., Basingstoke, UK) and liquid (brain heart infusion and thioglycolate; Oxoid Ltd.) culture media (22)(23)(24) .The material was placed in a transport medium containing saline and sterile distilled water and was subsequently inoculated on plates with soy agar culture media (semi-defined), supplemented with 20 µl of inactivated Escherichia coli when an infection with the protozoan Acanthamoeba spp. was suspected (24) .In our laboratory, the routine practice of this specific culture began in 1987 with the identification and subsequent publication of the first cases of keratitis caused by Acanthamoeba spp. in Brazil (25) .Maintenance of the inoculated culture media, and isolation and identification of bacteria, fungi, and the protozoan Acanthamoeba spp.were performed according to standard microbiology procedures (22)(23)(24)26) .
The assessment of bacterial susceptibility to antimicrobial agents commonly used in ophthalmology was performed by using the Kirby-Bauer agar-diffusion test according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS; Philadelphia, PA, USA) until 2004, and, subsequently, by the Clinical and Laboratory Standards Institute (CLSI; Philadelphia, PA, USA).
Categorical variables were analyzed using the chi-square test.Con tinuous variables were analyzed using Student's t test.A p value <0.1 was considered significant.Logistic regression was used with a p value <0.05 considered significant.MedCalc software version 11.6.1 (27) (MedCalc, Ostend, Belgium) was used for the statistical analysis.

RESULTS
Between July 1975 and December 2010, a total of 859 patients with clinical suspicion of keratitis aged 0-18 years were assessed.From these patients, 346 eyes from 346 patients [197 (56.9%) boys] with positive cultures for bacteria, fungi and/or the protozoan Acanthamoeba spp.were included in the study.
Topical medication was used at the time of sample collection in 42.8% of the patients.Antimicrobial drugs accounted for 71.6% of the topical medications used (Table 1).Major antecedents included trauma in 33.5% of patients, previous eye surgery in 24.9%, and the use of contact lenses in 18.2% (Table 1).Cancer was the major comor bidity found, in seven of the patients, accounting for 2% of the study patients.
Bacterial cultures were requested for all 346 patients and 309 (89.3%) tested positive.There were 317 requests for fungal cultures, with 14 (4.4%) testing positive and 97 requests for the protozoan Acanthamoeba spp. with 23 (23.7%) of these patients testing positive.Overall, 4% and 6.6% of all patients tested positive for fungi and Acanthamoeba spp., respectively.
All of the Gram-positive cocci were vancomycin-susceptible.Methicillin-susceptible Staphylococcus showed resistance lower than 10% to first generation cephalosporins, aminoglycosides, and fluo roquinolones, and resistance lower than 20% to trimethoprim-sul famethoxazole.Methicillin-resistant Staphylococcus showed significant resistance to most antimicrobials apart from the most recent generations of fluoroquinolones.Streptococcus pneumoniae showed no resistance to penicillin.
We analyzed the annual incidence of cases of keratitis in children and adolescents and the respective identification of microorganisms over the study period.The percentage of fungal infections remained stable throughout the study period, with positivity ranging from 1.8% to 16.6%.The prevalence of infections with Acanthamoeba spp.ranged from 1.8% to 11.1%.Bacterial infections were always the most common, with the prevalence in cultures ranging from 10% to 100%.
Univariate analysis comparing patients with bacterial and non-bacterial keratitis showed the following significant variables for non-bacterial keratitis: age (p=0.001), the use of antimicrobial drugs (p=0.02), and non-chemical or physical burns (p=0.004).In the multivariate analysis, age (odds ratio [OR] 1.19; 95% confidence interval [C] I 1.09-1.31),use of antimicrobial drugs (OR 2.50; 95%CI 1.19-5.23),and physical burn injuries (OR 17.29; 95%CI 2.40-124.57)were associated with non-bacterial keratitis (fungal or Acanthamoeba spp.) (Table 3).In univariate analysis that compared patients according to whether the keratitis was caused by Gram-negative or by Gram-positive bacte ria, age (p=0.01) was found to be statistically significant.Indeed, lo gistic regression showed age (OR 1.06; 95%CI 1.01-1.11)to be a risk factor for keratitis caused by Gram-negative bacteria in the (Table 4).

DISCUSSION
Microbial keratitis and its sequelae are important causes of ocular morbidity and blindness in developing countries.Microbial keratitis is characterized by inflammation of the cornea, usually with stromal infiltration, and is considered an ophthalmic emergency that requires immediate attention.Keratitis can progress rapidly with corneal destruction that causes permanent visual dysfunction.
In the literature, childhood is defined as the period of growth from birth to puberty.However, the definition of the limits of chronological adolescence is controversial.For the World Health Organiza tion, the age is between 10 and 19 years whereas for the United Na tions Organization, it is between 15 and 24 years.In Brazil, the Child and Adolescent Statute, law 8.069 of 1990, considers a child to be a person under 12 years old and defines adolescence as the age range 12-18 years (28) .
Childhood keratitis differs from adult keratitis in several respects, including possible reluctance of the child to undergo the clinical examination and sample collection, and the information-gathering regarding triggering factors and inflammation, which is apparently more intense in children (3,15) .Among teenagers, contact lenses are beco ming increasingly popular as a refractive error correction device, for esthetic reasons.The association between the use of contact lenses and infectious ulcers is well established.
During the period analyzed, 10.1% of keratitis cases occurred in the age group up to 18 years, mostly among male adolescents.These data corroborate previous studies in which keratitis in children and adolescents accounted for 11% to 13.1% of all ophthalmologic visits (3,8) .
Our study found that age, the use of topical antimicrobial drugs, and prior physical burns were associated with fungal and Acantha moeba spp.keratitis.Age was also associated with keratitis caused by Gram-negative bacteria.As far as we know, no previous study has analyzed this subject.
In our series, the use of topical medication was present in 42.8% of patients at the time of laboratory evaluation.Other authors have already indicated the use of empirical medication at the time of sample collection in 48% to 67.1% of patients with clinical suspicion of keratitis (3,9,16) .As they are already widespread, the use of antibiotics can inhibit bacterial growth, favoring the appearance of other microorganisms.This factor was statistically significant in the group of patients with non-bacterial keratitis and may be considered as a predisposing factor.A history of trauma was the most common antecedent found in our study as cause of keratitis in the age range studied, affecting 33.5% of patients.Trauma has been reported as the major risk factor for keratitis in several published studies, accounting for 21.2% to 58.8% of cases (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)29) .
In our study, 18.2% of patients had a history of using contact lenses.Soft lenses were the most commonly used type.More than 90% of patients with a positive culture for Acanthamoeba spp.were contact lens users and all were adolescents.As has been said, the association between the use of contact lenses and infectious ulcers is well esta blished.Several authors have reported the use of contact lenses, es pecially orthokeratology, as a predisposing factor for keratitis in chil dren and adolescents (3,(8)(9)(10)14,17,19,20,30) . The use of thistype of lens is not common in Brazil.
Of the identified etiologic agents, the most common was coa gulase-negative Staphylococcus, which was isolated from 23.8% patients; this was followed by Staphylococcus aureus (20.9%) and Pseu domonas spp.(14.2%).These results were similar to those observed in the elderly population in a previous study conducted in our institution (2) .
Methicillin-resistant Staphylococcus spp.showed a trend of resistance to most antimicrobials, something that has also been observed by other authors (15,19,20,29) , whereas coagulase-negative Staphylococcus, Streptococcus spp., and enterobacteria showed good suscepti bi lity to the majority of antimicrobial drugs tested.Streptococcus pneu moniae strains showed no resistance to penicillin.Pseudomonas aeruginosa strains showed no resistance to ciprofloxacin and low resistance to polymyxin B, gentamicin, and tobramycin, in contrast to the study by Moreno-Andrade et al., who reported 100% resistance to this antimicrobial (29) .
Keratitis caused by the protozoan Acanthamoeba spp.showed 6.6% positivity in the present study, which again is similar to previous studies (3,9,12,19) .It is noteworthy that all patients with positive culture for Acanthamoeba spp.were adolescents and that 91.3% had a history of using contact lenses.It is likely that increasing the frequency of use of contact lenses by teenagers increases the risk of adolescents developing corneal ulcers by Acanthamoeba spp.
In conclusion, we found that the risk factors for infectious keratitis in children and adolescents were noticeably the same as those observed in adults.We found that age, the use of antimicrobials, and injuries from physical burns were predisposing factors for non-bacterial kera titis.Age was associated with Gram-negative keratitis.

Table 4 . Univariate and multivariate analyses comparing keratitis caused by Gram-negative bacteria with that caused by Gram-positive bacteria among patients aged from 10 days to 18 years
Data are presented as mean ± standard deviation or n (%).