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Microbial Analyses of Contact Lens–Associated Microbial Keratitis

Konda, Nagaraju*; Motukupally, Swapna Reddy; Garg, Prashant; Sharma, Savitri; Ali, Mohd Hasnat§; Willcox, Mark D. P.

doi: 10.1097/OPX.0000000000000082
Original Articles

Purpose To investigate the utility of additional microbial analyses to detect the causative microorganism of microbial keratitis and to determine the strength of association between clinical variables.

Methods This retrospective study included 125 eyes of 123 people presenting with presumed infectious keratitis associated with contact lens wear, and examined between January 2001 and November 2011.

Results Sixty-three male and 60 female patients with a mean age of 24.14 ( ±6.7) and 26.7 (±10.0) years, respectively, were included in the study. Among the samples tested, 38 (40%) corneal cultures, 85 (92%) contact lens cultures, and 49 (80%) contact lens case cultures yielded positive results. In the samples for which all microbial investigations were performed and corneal culture was positive, cultures of contact lenses and corneas yielded the same microbes in 94% of cases, and for contact lens case and corneal cultures 77% of cases yielded the same microbes. Importantly, these were monocultures of the same microbe isolated from the cornea in the contact lens and lens case cultures. The odds of having a positive corneal culture was 37 times higher (p = 0.008) when patients presented to the clinic 2 weeks after the onset of symptoms. Pseudomonas aeruginosa was the predominant organism isolated, and most isolates were susceptible to commonly used antimicrobials.

Conclusions Contact lens culture might be an effective test to perform to detect and determine the causative microorganism when corneal cultures are unavailable or yield no microorganisms upon culture. A longer duration of symptoms before presenting to the clinic was significantly associated with obtaining a positive corneal culture.






Brien Holden Vision Institute (NK), Sydney, New South Wales, Australia; School of Optometry and Vision Science (NK, MDPW), University of New South Wales, Sydney, New South Wales, Australia; Jhaveri Microbiology Centre (SRM), Prof. Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, Andhra Pradesh, India; Cornea Service (PG), L. V. Prasad Eye Institute, Hyderabad, Andhra Pradesh, India; Ocular Microbiology Services (SS), L. V. Prasad Eye Institute, Bhubaneswar, Odisha, India; and Department of Biostatistics (MHA), L. V. Prasad Eye Institute, Hyderabad, Andhra Pradesh, India.

Swapna Reddy Motukupally Jhaveri Microbiology Centre Prof. Brien Holden Eye Research Centre Hyderabad Eye Research Foundation L. V. Prasad Eye Institute Hyderabad, India e-mail:

Contact lenses provide an excellent alternative to glasses for correction of refractive errors. However, contact lens use is associated with the risk of complications that may range from simple self-limiting adverse events to severe corneal infection.1–4 Microbial keratitis (MK) represents the most severe sight-threatening complication related to contact lens use. The major risk factors for contact lens–associated MK are overnight use of daily wear lenses,5 using lenses on extended wear schedule for longer duration,6 being of male gender,2 inadequate hygiene of the contact lenses,5 and poor contact lens storage case cleaning.7 Different etiological agents have been associated with contact lens–related microbial keratitis. Bacteria are prevalent in contact lens–associated MK, with the majority of them being Gram-negative bacilli, most commonly Pseudomonas aeruginosa.4–9 Fungal agents of contact lens–associated MK account for less than 5% of all cases except during a recent Fusarium outbreak.5,9,10

Unlike other cases of corneal ulceration where only corneal scraping material is available for microbiology, in contact lens–associated MK episodes other samples such as the contact lens, the contact lens case, and lens care solutions may be available. These are often cultured and the results might be used singly or in combination to help determine the causative agents of contact lens-MK and thereby to treat the patients with appropriate antibiotics.11 While many studies have assessed the prevalence,1 trends,4 risk factors,12 and etiopathogenesis13,14 of contact lens–associated MK, limited data are available relating to the usefulness of these additional microbial tests. One study reported that contact lens culture may help to detect the causative organism in contact lens–associated MK, especially where corneal scrapings were culture negative.11 The current study aimed to investigate whether culture of contact lenses or contact lens cases could be effective in determining the causative microorganism in cases of presumed infectious keratitis associated with contact lens wear (contact lens-MK), and also to study the strength of association between the clinical or patient history variables and culture results. Where the likely causative microbes were identified as being bacteria, bacterial sensitivity to antibiotics is presented.

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In this retrospective study, all the medical records and microbiology databases of patients who presented to the outpatient department of L. V. Prasad Eye Institute (LVPEI), Hyderabad between January 2001 and November 2011 and who were diagnosed as suffering from contact lens-MK were reviewed. Ethics approval from Institutional Review Board, L. V. Prasad Eye Institute was obtained in prior (Ethics Ref. No. LEC08154). Contact lens-MK in this study was defined as corneal inflammatory disease in patients with a history of contact lens wear characterized by epithelial defects associated with stromal infiltration, tissue necrosis and stromal loss, clinically diagnosed as microbial keratitis by a consultant ophthalmologist specialized in cornea and anterior segment, and the prescription of antimicrobials to treat the disease. Patients with a history of bandage contact lens use or those presenting with contact lens–induced peripheral ulcer or infiltrative keratitis were excluded from the study.

The demographic data, treatment given, and the final outcome of the patients were obtained retrospectively by review of the patients’ medical records. The microbiological data were retrieved from the hospital microbiology records. These data included the culture report from corneal scrapes, contact lenses, and contact lens cases. The contact lens culture was performed only in instances in which the lens was collected aseptically either from the eye or from the contact lens storage case of the patient at the time of presentation to the clinic. Corneal scrapings were obtained from the patients under topical anesthesia (0.5% proparacaine hydrochloride) and slit-lamp magnification. The scrapings were obtained aseptically using a sterile surgical blade no. 15 on a Bard Parker handle. Every corneal scraping was subjected to a direct microscopic examination, culture, and antibiotic susceptibility testing. Microscopic examination included Gram stain, Giemsa stain, and calcofluor-potassium hydroxide (KOH + CFW) mount. The samples were inoculated directly onto 5% sheep blood agar, chocolate agar, Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), brain heart infusion broth, and thioglycollate broth as a part of the standard institutional protocol. SDA and PDA were incubated for 14 days at 25°C to detect fungal growth, while the remaining media were incubated for 7 days at 37°C for the detection of bacterial growth. Lenses were aseptically removed from the eye with sterile gloves and sent for microbiological analysis in sterile phosphate buffered saline and cultured on a chocolate agar plate by agar sandwich technique. Plates were incubated at 37°C in the presence of 5% CO2 and observed for the presence of growth. In the presence of positive culture, colony-forming units (CFUs) were enumerated and colonies identified using Gram stain, standard microbiological techniques, and further confirmed by Vitek system (Biomerieux, Germany).4,15,16 A corneal culture was considered positive when there was growth of the same microorganism on two or more media, or a confluent growth at the site of inoculation on one medium, or growth on one media with consistent direct microscopy findings (such as Gram stain) from corneal scrapes. CFUs greater than 10 were considered significant in the case of CL cultures. Contact lens cases received in the laboratory were opened without touching the interior and 50 μL of the solution from the case was aspirated using a sterile micropipette for culture. If the contact lens case was dry, a sterile cotton swab moistened with sterile normal saline solution was used to sample the interior of the case. Chocolate agar was used for culture, which was incubated at 37°C for up to 7 days. Any growth was processed further for identification and antibiotic susceptibility testing. The growth was not quantified in terms of CFU and was reported as either present or absent (i.e., on binary scale). All cultures with a positive result were reported and were correlated with those from lenses or corneal scrapes and clinical findings.11 Antimicrobial susceptibility testing was performed for all bacterial isolates using the Kirby Bauer Disk diffusion method. The antibiotics investigated were amikacin (10 µg), cefazolin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), gatifloxacin (5 µg), gentamicin (10 µg), moxifloxacin (5 µg), ofloxacin (5 µg), tobramycin (10 µg), and imipenem (10 µg) discs (Hi-media, Mumbai). The antibiotic sensitivity tests were performed according to the Clinical and Laboratory Standards Institute guidelines.16

On the basis of the initial result of corneal smear microscopy, patients diagnosed to have bacterial keratitis were treated immediately with a combination of 5% fortified cefazolin and 1.4% gentamicin or 0.3% ciprofloxacin eye drops. Antibiotic therapy was modified when necessary on the basis of clinical response, culture, and antibiotic susceptibility reports. Subjects whose infections were determined to be of fungal etiology were started on 5% natamycin eye drops and those of Acanthamoeba infection were started on topical 0.02% polyhexamethylene biguanide and 0.02% chlorhexidine eye drops. All cases received cycloplegics (atropine sulfate) as per the standard protocol at the Institute. Therapeutic penetrating keratoplasty was performed in patients with corneal perforation, or in those who were either non-responsive or worsened on medical therapy.

Statistical analysis was performed using the R software (version 9.2). A multivariate regression analysis with forward and backward elimination using Akaike information criterion17 was used to test the strength of association between the clinical variables and positive corneal cultures. Corneal culture results were taken as a dependent variable to analyze the strength of association between the average infiltrate size, time between onset of symptoms and presentation at the hospital, infiltrate location, and positive corneal cultures using multiple logistic regressions (Table 4). For the purpose of this analysis, infiltrate size was categorized as <1, 1–2, and >2 mm. Time of presentation to the clinic from the onset of symptoms was classified into <1, 1–2, and >2 weeks, and infiltrate location as central, mid-peripheral, and peripheral. A p value of less than 0.05 was considered to be statistically significant.

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One hundred twenty-five eyes of 123 patients met the diagnostic criteria of contact lens-MK and were included in the study. Two patients had simultaneous involvement of both eyes. Of the patients, 63 (51.2%) were male and 60 (48.8%) were female, with age mean and standard deviation (SD) of 24.14 (±6.7) and 26.7 (±10.0) years, respectively. The proportion of each microbiological test and its aggregation is shown in Fig. 1. The number of samples subjected to culture and positive culture results are shown in Table 1.





Corneal culture was used as a standard test and evidence of difference or similarity of the microbiology of contact lenses and contact lens cases was measured. A paired observational analysis was performed, and both corneal culture and contact lens culture were performed on 68 patients. Of these samples, 43 (63%) did not yield any microbial growth on corneal culture; nevertheless, of these 43, 39 (91%) gave a positive result on contact lens culture. Similarly, corneal culture and contact lens case cultures were performed on 46 patients. Corneal cultures were negative from 29 (63%) patients, although of these 29 patients 21 (72%) yielded positive results when their contact lens cases were cultured. When the corneal culture was positive, 24 (96%) of contact lens and 13 (76%) of contact lens case cultures were also positive (i.e., cultured the same microbe) (Table 2). All three samples were cultured from 46 patients; of these corneal cultures were positive only in 17 (37%) cases, while contact lenses and contact lens case cultures were positive in 42 (91%) and 34 (74%) cases, respectively (Table 3), and when there was a positive corneal culture the same microbes were identified from lenses or cases as from the corneal scrapes. Out of the 17 cases that were positive on corneal culture, 16 (94%) and 13 (76%) were also positive on contact lens and contact lens case cultures. For the times when positive cultures were obtained, 96% (68 of 71) of contact lenses and 98% (46 of 47) of contact lens cases yielded monocultures of microbes.







The association between positive corneal culture results and the average infiltrate size, time between onset of symptoms and presentation at the hospital, and infiltrate location using multiple logistic regressions are presented in Table 4. There was a 37-fold higher chance of obtaining positive corneal culture results when patients presented to the clinic after 2 weeks of the start of symptoms, as compared to the those who presented within a week (p = 0.008). The other clinical variables showed no statistically significant association with corneal culture results.

Using data obtained from corneal culture, contact lens, or contact lens case microbiology to diagnose the suspected etiological agents of contact lens-MK, 83 (71%) cases were bacterial, three (2.6%) cases were fungal, one (0.9%) case was caused by Acanthamoeba, and nine (7.7%) cases yielded mixed microorganisms. In 21 (17.9%) cases, there was no growth of microbes and the remaining eight (6.4%) cases were treated empirically and not investigated further either due to either a small infiltrate size or the fact that the patient presented during the healing stage of the disease. Among the identified bacteria, Pseudomonas spp. were the predominant microorganisms in 61 (73.5%) of cases, followed by Staphylococcus epidermidis from four (4.8%) cases, Serratia spp. from four (4.8%) cases, coagulase negative staphylococci from two (2.4%) cases, Burkholderia cepacia from two (2.4%) cases, Enterobacter spp. from two (2.4%) cases, Staphylococcus haemolyticus from one (1.2%) case, Escherichia coli from one (1.2%) case, Achromobacter xylosoxidans from one (1.2%) case, Corynebacterium pseudodiphtheriticum from one (1.2%) case, Klebsiella pneumoniae from one (1.2%) case, and five (6.0%) cases yielded unidentified non-fermenting Gram-negative bacilli.

Figs. 2 and 3 show antibiotic susceptibility patterns of all the bacterial isolates identified in the study. Most of the isolates were susceptible to all groups of antimicrobials, except to chloramphenicol to which most bacterial isolates including Pseudomonas spp. showed resistance. Bacterial isolates were susceptible to ciprofloxacin (86%), ofloxacin (88%), gatifloxacin (89%), gentamicin (87%), tobramycin (80%), amikacin (79%), and cefazolin (65%). Among the 61 Pseudomonas spp. isolated, four showed in vitro resistance to multiple drugs including fluoroquinolones, aminoglycosides, and third-generation cephalosporins. Almost all cases resolved upon treatment with antibiogram-guided antimicrobial therapy. Among the four multi–drug-resistant cases of P. aeruginosa, three showed good clinical response to ciprofloxacin eye drops, while one of them did not respond to medical therapy and the patient required a therapeutic penetrating keratoplasty. Upon healing, the patients were prescribed glasses in 75 (60.0%) cases, contact lenses in 23 (18.4%) cases, penetrating keratoplasty in eight (6.4%) cases, LASIK in three (2.4%) cases, and two cases (1.6%) were referred to their local doctor, while 14 (11.2%) cases were lost to follow-up.





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This study was designed to determine whether culture of additional material that is often present in cases of contact lens-MK could aid in diagnosis of causative microorganisms in the event of negative culture of corneal scrapings or when scraping was not performed. Although microscopic examination of smears can help to inform initial antimicrobial therapy, it has been reported that corneal smear results are not very sensitive in the diagnosis of bacterial keratitis.18 Contact lens cultures were performed in 92 cases, and 85 of these (92.4%) yielded microbial isolates. Contact lens cases were cultured 61 times, and 49 of these (80%) yielded microbial isolates. Previous studies have also shown that contact lenses and contact lens cases are more likely to yield positive cultures than corneal culture.11,19,20

Currently, corneal culture is considered as a standard test to diagnose microbial keratitis, and so this was used to compare to culture results from other samples. Martins et al19 reported that bacterial and Acanthamoeba keratitis cases with negative corneal culture can have positive contact lens cultures which agreed with their clinical diagnosis of the disease. In addition, other studies have reported 67%11 or 40%21 of contact lens cultures were positive even though corneal culture was negative.

Das et al reported that 34 and 70% of corneal and contact lens cultures were positive and found a significant association between these two tests.11 Cultures of contact lenses and contact lens cases from healthy (non-infected) lens wearers can often yield mixed bacterial types ranging from 72 to 86.6%.22,23 However, in the current study, among the samples that had positive cultures, 96% (68 of 71) of contact lenses and 98% (46 of 47) of contact lens cases were monocultures, and in cases when a microbe also was cultured from the cornea, the same microbe was cultured from the contact lens or contact lens case in these instances. Thus, in cases of contact lens-MK where corneal scrapings cannot be performed or the corneal scrapes show no microbial growth, any microbe grown in monoculture found from culturing contact lenses or contact lens cases is likely to be the causative agent. A standard microbial investigation practice for contact lens–associated and non–contact lens–associated corneal ulcers is followed at LVPEI. In addition, when a patient calls in to make an appointment for consultation of a contact lens problem, as a standard protocol, the front desk manager instructs the patients to visit the hospital along with their contact lenses, lens care solution, and contact lens case as early as possible. This might have allowed us to retrieve more contact lenses aseptically from the patient’s eye than might be expected in other centers in private clinics. However, data was not collected on how many contact lenses were collected from the patients’ eyes directly and how many from the contact lens cases. In the current study, 26% of the patient’s contact lenses were not cultured because either patients did not come with their contact lenses or they were removed non-aseptically. Storage cases were retrieved from 61 out of 123 (49.5%) people with MK. There is a possibility that there was some selection bias in collection of lens storage cases as compliant wearers may be more likely to present with their storage cases. However, compliance of the people with contact lens hygiene could not be judged due to the retrospective nature of the current study. An ideal situation where all CL cases are subjected to culture and compared with corneal cultures would have removed this potential bias.

A higher rate of positive corneal culture has been shown to depend upon the infiltrate size, location, and response to initial treatment.9 Peripheral corneal ulcers have been reported to be sterile when compared with central ulcers.24,25 Das et al reported that among 22% of patients who had peripheral corneal lesions, 9% had culture positive results.11 Lam et al5 have reported that an infiltrate of diameter greater than 4 mm was more likely to be associated with a positive corneal culture, in comparison to those smaller in size, and this was supported by Bennett et al.26 In contrast, Rattanatam27 and Mah-Sarorra9 et al found no statistically significant difference in culture results between small, peripheral lesions and large or centrally located lesions. Data from the current study did not find an association between infiltrate size and culture positivity; rather, the length of time after the onset of symptoms was associated with corneal culture positivity. Patients who presented to the clinic at or after 2 weeks of onset of symptoms had a significantly higher risk (OR 37) of a positive corneal culture compared to those who presented before 1 week. Multivariate results revealed that an infiltrate size greater than 2.0 mm trends towards significance with cases healed after 2 weeks (OR, 37; p = 0.008). This could be attributed to the referral practice at our center and most of these cases with infiltrate size >2 mm are likely to have already been prescribed antibiotic treatment compared to cases with smaller infiltrate size. As a result, multivariate analysis showed a possible protective nature. However, in an ideal situation, all of these cases should carry a risk.

Reports have shown that Gram-negative bacilli are commonly isolated from contact lens-MK in tropical countries while Gram-positive bacteria are common in moderate temperatures.5 P. aeruginosa was the most predominant isolate in the current study accounting for 73.5% of bacteria isolated from contact lens-MK cases. These results were comparable to studies conducted by Sharma et al,4 Mah-Sarorra et al,9 and Rattanatam et al.27

Results of in vitro sensitivity tests from studies conducted in the USA and India of patients with culture-positive contact lens-MK have shown that the vast majority of isolates of P. aeruginosa from MK are susceptible to fluoroquinolones.4,9 In the current study, 86-89% of all the bacterial isolates were susceptible to fluoroquinolones. Among four multidrug-resistant isolates of P. aeruginosa, three showed in vivo susceptibility (i.e., topical administration of a fluoroquinolone resolved the disease) despite the in vitro resistance. One patient failed to resolve with medical therapy and required a therapeutic penetrating keratoplasty. The final outcome of all patients was good and almost all patients resolved with minimal vision loss.

This study has demonstrated that contact lens culture might be an effective test to perform to detect and determine the causative microorganism when corneal cultures cannot be performed or show no growth. None of the clinical variables tested in this study was associated with corneal culture results except the time between onset of symptoms and presentation to the clinic. Contact lens-MK was predominantly caused by Gram-negative bacteria. Most of the bacterial isolates were sensitive to common ocular antibiotics. Treatment outcome was noted to be good in almost all patients.

Swapna Reddy Motukupally

Jhaveri Microbiology Centre

Prof. Brien Holden Eye Research Centre

Hyderabad Eye Research Foundation

L. V. Prasad Eye Institute

Hyderabad, India


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Conflict of interest and source of funding: NK is funded by the Australian Government Postgraduate Award and Brien Holden Vision Institute. The author(s) have no proprietary or commercial interest in any material discussed in this article. For the remaining authors, none were declared.

Support of Hyderabad Eye Research Foundation, Hyderabad, staff at Jhaveri Microbiology Centre, and medical records departments at L. V. Prasad Eye Institute KAR-campus, Hyderabad, India is gratefully acknowledged.

Received: December 5, 2012; accepted June 26, 2013.

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contact lens wear; presumed infectious keratitis; microbial analysis

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