CLINICAL SCIENCE
Clinical Characteristics and Bacteriological Profile of Moraxella Keratitis Hidenori Inoue, MD,*† Takashi Suzuki, PhD, MD,*† Tomoyuki Inoue, PhD, MD,*† Takaaki Hattori, PhD, MD,*‡ Ryohei Nejima, MD,*§ Daisuke Todokoro, PhD, MD,*¶ Saichi Hoshi, PhD, MD,*k Hiroshi Eguchi, PhD, MD,*** Hitoshi Miyamoto,†† and Yuichi Ohashi, PhD, MD†
Purpose: Moraxella species are rare causative pathogens of severe sight-threatening keratitis. The aim of this study was to analyze the clinical presentation, predisposing risk factors, in vitro antimicrobial susceptibility, and treatment associated with Moraxella keratitis.
Methods: We retrospectively reviewed 30 culture-proven cases of Moraxella keratitis from multiple centers in Japan.
Results: The mean age of the patients was 58.4 6 23.4 years. The most common ocular conditions were contact lens wearing (5 patients, 16.7%) and trauma (3 patients, 10.0%). Seven patients had diabetes mellitus. Sixteen patients exhibited hypopyon in association with the corneal focus. Ring-shaped infiltration was found in 9 patients (30.0%), and irregular or amoebic-shaped infiltration was observed in 13 patients (43.3%). Eight patients (26.7%) showed small round infiltrates. All Moraxella isolates were sensitive to fluoroquinolones and aminoglycosides. All were treated with a combination ophthalmic solution containing a fluoroquinolone, tobramycin, and cefmenoxime. Although no patients developed corneal perforation, the response to treatment was slow in all cases; the mean treatment period was 41.9 days.
Conclusions: In Japan, Moraxella keratitis occurs in patients with contact lens wear, trauma, and diabetes mellitus. It presents as a small, round, ring-shaped, irregularly shaped, or amoebic-shaped focus. Moraxella species exhibit good susceptibility to fluoroquinolones and aminoglycosides. Because the treatment response may be very slow, these agents should be continued for a long period of time. Received for publication February 19, 2015; revision received March 24, 2015; accepted April 1, 2015. Published online ahead of print May 13, 2015. From the *OMIC (Ocular Microbiology and Infection Conference) Working Group; †Department of Ophthalmology, Ehime University Graduate School of Medicine, Toon, Japan; ‡Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; §Miyata Eye Hospital, Miyakonojo, Japan; ¶Department of Ophthalmology, Gunma University School of Medicine, Maebashi, Japan; kDepartment of Ophthalmology, National Center for Geriatrics and Gerontology, Obu, Japan; **Department of Ophthalmology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan; and ††Department of Clinical Laboratory, Ehime University Hospital, Toon, Japan. Supported by the Department of Bioscience, INCS, Ehime University. The authors have no conflicts of interest to disclose. Reprints: Takashi Suzuki, PhD, MD, Department of Ophthalmology, Ehime University, Graduate School of Medicine, Shitsukawa, Toon, Ehime 7910295, Japan (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cornea Volume 34, Number 9, September 2015
Key Words: Moraxella, contact lens, diabetes mellitus, keratitis (Cornea 2015;34:1105–1109)
K
eratitis is a corneal infection that occurs after an injury or in association with contact lens wearing. It can progress rapidly with devastating consequences, including corneal scarring and loss of vision. Thus, prompt identification of this condition and commencement of an aggressive course of therapy are imperative to limit tissue damage. Because the severity of keratitis and response to treatment depend on the virulence or drug sensitivity of the causative agents, it is important to understand the various clinical and pathophysiological aspects of infectious keratitis caused by each pathogen. Common bacterial species that cause keratitis have been described in various reports.1–3 Moraxella species are rare causative pathogens of keratitis, and National Surveillance of Infectious Keratitis in Japan demonstrated that 3.8% of culture-proven organisms with keratitis specimens, including bacteria, fungi, and Acanthamoeba, were Moraxella species.1 Another surveillance performed in the United States and India showed that Moraxella species were detected in 3.0% of culture-proven bacterial keratitis specimens.3 Moraxella species are short gram-negative diplobacilli that are commonly found as commensals in the oropharynx, mucous membranes, skin, and genital tract and can cause certain diseases in humans.4,5 Moraxella catarrhalis is the most commonly isolated species and is responsible for acute otitis media in children, chronic and severe otitis media, acute and chronic sinusitis, and upper and lower respiratory tract infections. Moraxella osloensis, Moraxella nonliquefaciens, and Moraxella lincolnii are part of the normal flora of the human respiratory tract. These and other Moraxella species are rare infectious agents.4,5 Moraxella lacunata can rarely cause conjunctivitis and blepharitis along with endocarditis.6–8 Most laboratories cannot identify Moraxella species other than M. catarrhalis because most Moraxella species exhibit similar biochemical reactions. Thus, genetic methods and mass spectrometry are sometimes necessary to identify Moraxella species. Various case reviews of keratitis caused by Moraxella species have been reported from the United States, United Kingdom, Australia, and India.9–15 In the 1980s, Moraxella keratitis was often associated with chronic alcoholism, www.corneajrnl.com |
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Cornea Volume 34, Number 9, September 2015
Inoue et al
malnutrition, diabetes, and poor sanitary habits, all of which were considered to be important predisposing factors.12,14,15 Reports published after 1990 demonstrated other predisposing risk factors for Moraxella keratitis, including penetrating keratoplasty, herpes keratitis, and diabetes mellitus.10,11,13 Predisposing risk factors for bacterial keratitis may differ according to the geographic area and time period. Likewise, clinical findings and treatment responses may differ among areas. No reviews of Moraxella keratitis have been reported from East Asia, including Japan. The aim of this study was to analyze the clinical presentation, predisposing risk factors, patient backgrounds, in vitro antimicrobial susceptibility, and treatment period of Moraxella keratitis in Japan. We also identified Moraxella isolates from keratitis specimens.
MATERIALS AND METHODS This multicenter retrospective study was approved by the Ehime University Review Board and conformed to the tenets of the Declaration of Helsinki. The clinical and microbiological records of all culture-proven cases of Moraxella keratitis seen at Ehime University Hospital, Miyata Eye Hospital, Tokyo Medical University, Gunma University, and Machida Hospital from January 2005 to February 2014 were retrospectively reviewed. The microbiology tests performed in all participating centers were the same. Corneal scrapings on sheep blood agar and chocolate agar plates were incubated at 35°C. Bacterial identification was performed by Gram staining and biochemical testing. Antimicrobial susceptibility testing was performed using a broth microdilution assay. All results were interpreted using cut-off values for susceptibility and resistance according to the criteria by the Clinical and Laboratory Standards Institute or the Japanese Society of Chemotherapy. Tested antibiotics were different among the participating centers. Along with the bacterial culture, direct microscopy was performed. We considered Moraxella sp as a causative agent of keratitis when the number of colonies grown on a plate were large and direct microscopy showed a lot of gram-negative rods with neutrophils. The collected data included age, sex, medical and ocular history, presenting signs and symptoms, systemic and local predisposing factors, antibiotic susceptibility, treatment, and length of follow-up. The size, shape, and location of corneal infiltrates and the presence of hypopyon were documented. We identified 9 stored isolates in Ehime University Hospital. To identify the isolates, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) using the default settings (positive linear mode; laser frequency, 60 Hz; ion source 1 voltage, 20 kV; ion source 2 voltage, 16.7 kV; lens voltage, 7.0 kV; and mass range, 2000–20,000 Da) according to the manufacturer instructions. Bacteria were applied as a thin film onto a 48-spot steel plate (Bruker Daltonics) and allowed to dry at room temperature. Each spot was overlaid with 1 mL of alpha-cyano-4-hydroxycinnamic acid as a matrix and allowed to dry. The target plate was then inserted into the Bruker Microflex LT MALDI-TOF MS system for analysis. The composite profile was analyzed using Biotyper RTC 3.1
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software with reference database version 4.0.0.1 (Bruker Daltonics), which queried a reference bank of 2290 spectra and returned the top 10 matches along with confidence scores ranging from 0.0 to 3.0. The identification criteria used in our analysis, as outlined by the manufacturer, were as follows: a score of $2000 indicated species-level identification, a score of 1700 to 1999 indicated genus-level identification, and a score of ,1700 was interpreted as unidentified. Along with MALDI-TOF MS, molecular identification by polymerase chain reaction amplification and sequencing analysis of the 16S rRNA gene using the universal primers 8UA (59-AGAGTTTGATCMTGGCTCAG-39) and 1485B (59ACGGGCGGTGTGTRC-39) were performed as described previously.16 The prevalence of predisposing risk factors was compared between patients aged ,60 and $60 years using a x2 test. P , 0.05 was considered to indicate statistical significance.
RESULTS Predisposing Factors In total, 30 cases of Moraxella keratitis were evaluated in this study. The age of the patients ranged from 7 to 94 years (mean, 58.4 6 23.4 years). There were 11 male patients and 19 female patients. A predisposing systemic or ocular condition was identified in 21 patients (70.0%) (Table 1). Seventeen patients (56.7%) had an ocular predisposing factor, and the most common ocular conditions were contact lens wearing (5 patients, 16.7%) and trauma (3 patients, 10.0%). Systemic risk factors were identified in 12 patients, with diabetes mellitus being present in 7 patients (23.3%). Seven patients had both ocular and systemic factors, whereas no predisposing risk factor could be identified in 9 patients (30.0%), and none of the patients were alcoholic or malnourished. There was a higher prevalence of ocular predisposing factors in patients aged ,60 years than in those aged $60 years (P , 0.030). Diabetes mellitus was found more frequently in patients aged $60 years than in those aged ,60 years, but the difference was not statistically significant (P , 0.053).
Clinical Findings
The patients’ ophthalmic microscopy findings were documented at the first visit to the hospital. The infiltrate was found in the central cornea in 9 patients (30.0%), paracentral cornea in 12 patients (40.0%), and peripheral cornea in 9 patients (30.0%). Fifteen patients (50.0%) had infiltrates of more than one fourth the corneal diameter, and the other 15 patients (50.0%) had infiltrates of less than one fourth the corneal diameter. With respect to shape, ringshaped infiltration was found in 9 patients (30.0%), and irregular or amoebic-shaped infiltration was observed in 13 patients (43.3%). Eight patients (26.7%) showed small round infiltrates. A representative slit-lamp photograph of each infiltrate shape is shown in Figure 1. Sixteen patients (53.3%) had hypopyon. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Cornea Volume 34, Number 9, September 2015
Profile of Moraxella Keratitis
TABLE 1. Predisposing Factors for Moraxella Keratitis Age Ocular predisposing factors Contact lens wear Trauma Others
Total Systemic predisposing factors Diabetes mellitus Others
Total
0–59 yrs (n = 16), n (%)
‡ 60 yrs (n = 14), n (%)
Total (n = 30), n (%)
0–59 yrs vs. ‡ 60 yrs, P*
4 (25.0) 1 (6.3) 7 (43.8) Conjunctivitis Corneal amyloidosis Bullous keratopathy Dry eye Phlyctenular keratitis Lagophthalmos Steroid eye drops 12 (75)
1 (7.1) 2 (14.3) 1 (7.1) Herpes simplex keratitis
5 (16.7) 3 (10.0) 8 (26.7)
0.413 0.903 NT
4 (28.6)
16 (53.3)
0.030
1 (6.3) 3 (12.5) Sjögren syndrome Dysautonomia Systemic lupus erythematosus 4 (31.3)
6 (42.9) 2 (21.4) Allergic purpura Basedow disease
7 (23.3) 5 (16.7)
0.053 NT
8 (64.3)
12 (46.7)
0.156
*x2 test. NT, Not tested.
Treatment Management procedures for Moraxella keratitis were identified in 29 cases (Table 2). A topical fluoroquinolone (levofloxacin, gatifloxacin, or moxifloxacin) was used in all 29 cases. A topical aminoglycoside ophthalmic solution (tobramycin, dibekacin, arbekacin, or sisomicin) and cefmenoxime were combined with the fluoroquinolone in 19 and 3 patients, respectively. One patient was treated with a combination of a fluoroquinolone and erythromycin. Five patients were treated with a combination of 3 antibiotics (a fluoroquinolone, an aminoglycoside, and cefmenoxime). Systemic antibiotics were used in 6 patients. No patients required surgical interventions such as penetrating keratoplasty. The
mean time required for complete closure of the epithelial defect was 23.4 days (range, 3–70 days) in 17 patients whom that in their clinical records was confirmed. The mean healing time was 41.9 days (range, 9–105 days) in 18 patients whom that in their clinical records was confirmed.
Bacteriological Profile Moraxella species were isolated from all 30 cases, and other organisms (Corynebacterium sp, Haemophilus influenzae, Staphylococcus aureus, Staphylococcus mitis, Staphylococcus epidermidis, Staphylococcus lugdunensis, G streptococci, and Enterobacter aerogenes) were detected along with Moraxella species in 6 cases. The clinical records
FIGURE 1. Representative slit-lamp photograph of Moraxella keratitis. A, Photograph showing a ring abscess with hypopyon. B, Photograph showing an irregular and amoebic infiltrate. C, Photograph showing a small round infiltrate. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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TABLE 2. Management Procedures for Moraxella Keratitis Topical Management
n (%)
Fluoroquinolone Levofloxacin Gatifloxacin Moxifloxacin Total Aminoglycoside* Tobramycin Dibekacin Arbekacin Sisomicin Total Cephalosporin* Cefmenoxime Macrolide* Erythromycin/colistin
14 9 6 29
(48.2) (31.0) (20.7) (100)
18 4 1 1 24
(62.1) (13.8) (3.4) (3.4) (82.8)
8 (27.6) 1 (3.4)
*These antibiotics were combined with a fluoroquinolone.
of 26 cases demonstrated in vitro antimicrobial susceptibility (Table 3). All test isolates were susceptible to levofloxacin, tobramycin, and chloramphenicol. Three Moraxella isolates were resistant to cefazolin. Identification of 9 stored Moraxella isolates from keratitis specimens was performed using mass spectrometry and genetic methods. Both MALDI-TOF MS and 16S rRNA gene sequencing produced the same results, and 2 and 7 isolates were identified as M. lacunata and M. nonliquefaciens, respectively.
DISCUSSION In this study, 30 cases of Moraxella keratitis from 2005 to 2014 were analyzed, and the predisposing factors, clinical findings, treatment, and bacteriological profiles of all cases were demonstrated. This is the first review of Moraxella keratitis in Japan. The age of the patients ranged from 7 to 94 years (mean, 58.4 years). The mean ages of the patients in the reports by Das et al10 and Mian and Malta13 were 70.0 and 59.9 years, respectively; thus, the age of our patients was similar to these reports. Moraxella species are commensals in the oropharynx, mucous membranes, skin, and genital tract. Therefore, they could be opportunistic pathogens that cause TABLE 3. Antimicrobial Susceptibility of Moraxella Isolates for Keratitis No of Isolates Antibiotics Levofloxacin Tobramycin Cefazolin Ceftazidime Chloramphenicol Ciprofloxacin
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Sensitive
Resistant
Sensitivity Test Not Done
26 14 21 11 7 5
0 0 3 0 0 0
4 16 6 19 23 25
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keratitis in patients of all ages. In the present study, contact lens wearing was a predominant ocular predisposing factor in patients aged ,60 years. However, it was not a prominent predisposing factor in other reports.10,13 Contact lenses are now used not only by young people, but also by middle-aged and older people, and commensal Moraxella species could opportunistically infect the cornea if it has become damaged by contact lenses. Along with contact lens wearing, trauma and other ocular surface diseases were also predisposing risk factors in the present study, similar to previous reports.10,11,13 Although Das et al10 demonstrated that herpes simplex keratitis and penetrating keratoplasty were predominant ocular predisposing risk factors, our cases included 1 patient with herpes simplex keratitis and no patients with penetrating keratoplasty. Diabetes mellitus was the predominant systemic predisposing factor, similar to previous reports.10,13 However, none of our patients had chronic alcoholism or malnutrition. As Cobo et al9 demonstrated, Moraxella keratitis can occur even among healthy people. Moraxella species could cause opportunistic keratitis in people of a wide age range with ocular surface damage. The clinical findings of Moraxella keratitis have been characterized as an indolent paracentral or peripheral ulcer that is usually oval in shape with hypopyon or hyphema.10 In our study, the clinical findings were divided into the following 3 types: ring abscess in the central cornea (9 patients), irregular amoebic-shaped infiltrate overlaying the whole cornea (13 patients), and small round infiltrate (8 patients). The first 2 types could be similar to the clinical findings in previous reports.10,11,13 The ring abscess type could have less intense inflammation in the center of focus, and be different from the ring infiltration seen in Acanthamoeba keratitis or anesthetic abuse keratopathy. We should suspect Moraxella keratitis in patients with ring abscess or irregular amoebic-shaped infiltration. Our series included patients with small round infiltrates. Keratitis with small round infiltrates can be found until the infiltrate progresses. These findings demonstrate that Moraxella keratitis can have various clinical presentations. In the present study, a combination of a topical fluoroquinolone and aminoglycoside (or cefmenoxime) was widely used to treat Moraxella keratitis. Because of their stability and broad spectra, fluoroquinolones are often used for empirical treatment or prevention of keratitis.17–20 Like fluoroquinolones, aminoglycosides exhibit good susceptibility to gram-negative rods such as Pseudomonas aeruginosa and Moraxella species.20 The present study also demonstrated that Moraxella isolates show susceptibility, and no resistance, to both fluoroquinolones and aminoglycosides. Although some isolates showed resistance to cefazolin, they were susceptible to other cephalosporins. Similarly, Das et al10 noted that Moraxella isolates showed good susceptibility to fluoroquinolones, aminoglycosides, and cephalosporins. Thus, Moraxella isolates from keratitis could have good susceptibility to antibiotics typically used for topical treatment. Indeed, our cases treated with topical antibiotics healed without corneal perforation and did not require surgical intervention. All cases in a series by Stern21 and 50% of cases in a series by Marioneaux et al12 required surgical Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Cornea Volume 34, Number 9, September 2015
interventions. Furthermore, 3 blind eyes in a series by Das et al10 were enucleated. In contrast to these previous reports, our cases were cured by topical treatment without surgical intervention. One possible reason for this good response to treatment is that our cases were usually treated with a combination of antibiotics such as a fluoroquinolone and aminoglycoside. Our previous study showed that a combination of a fluoroquinolone with tobramycin or cefmenoxime had synergistic effects and increased the antibacterial activity of each agent against gram-negative rods in vitro.20 Thus, a combination of a fluoroquinolone with tobramycin or cefmenoxime may be a feasible option for improving the effects of fluoroquinolones in the treatment of keratitis. Another reason for the good response to treatment could be that the treatment began before the keratitis progressed and had dense infiltration. Because our cases included small foci of keratitis, topical treatment was effective. Along with these reasons, some Moraxella isolates could have low virulence. Moreover, Japanese genetic variability could be associated with variation in clinical findings or treatment outcomes. However, the time required for complete closure of the epithelial defects ranged from 3 to 70 days, and the mean healing time was 41.9 days. The mean healing time in the case series by Das et al10 was 35 days; thus, Moraxella keratitis may respond slowly to treatment. Moraxella keratitis should be carefully monitored and treated for a long period of time. Although little is known about the exact reasons for the relatively long time to cure, Moraxella sp may induce persistent inflammation in the cornea and delay epithelial healing. We identified Moraxella sp isolates from keratitis using MALDI-TOF MS and genetic techniques. MALDI-TOF MS can rapidly determine the unique molecular fingerprint of an organism.22 The characteristic spectrum pattern of this molecular fingerprint is used to reliably and accurately identify a particular microorganism by matching it. Additionally, MALDI-TOF MS–based identification does not have the limitations of biochemical testing and can be used to analyze species that have been difficult to identify by biochemical testing, such as Moraxella species. Thus, it offers a safe, cost-effective, and adaptable system for rapid identification of bacteria and fungi. In this study, M. lacunata and M. nonliquefaciens were identified as causative pathogens of keratitis. M. lacunata has been considered as the cause of acute conjunctivitis or angular blepharoconjunctivitis.7 Along with these infections, M. lacunata could cause keratitis. Our study showed that more isolates were identified as M. nonliquefaciens. As Cobo et al9 demonstrated, M. nonliquefaciens could be an important cause of keratitis. Although Heidemann et al15 reported in 1987 that M. catarrhalis could cause keratitis, we did not detect M. catarrhalis in the present study. We speculate that the species of Moraxella causing keratitis could be dependent on the time period and geographical location. It is likely that M. lacunata and M. nonliquefaciens are causative pathogens for keratitis in Japan. Although we could not compare exactly the clinical findings of keratitis caused by M. lacunata and M. nonliquefaciens because of few cases, the M. lacunata cases had bigger focus size and healed slower, compared with M. nonliquefaciens keratitis cases. However, the number of Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Profile of Moraxella Keratitis
M. lacunata cases were few, and statistics could not be performed. Further investigation of the pathogenesis of Moraxella keratitis is needed. In summary, contact lens wearing and trauma could be important predisposing factors for Moraxella keratitis, which shows various clinical findings. Rapid diagnosis and the use of topical antibiotic combinations may be needed to treat keratitis. REFERENCES 1. National Surveillance of Infectious Keratitis in Japan–current status of isolates, patient background, treatment [in Japanese]. Nihon Ganka Gakkai Zasshi. 2006; 110:961–972. 2. Bourcier T, Thomas F, Borderie V, et al. Bacterial keratitis: predisposing factors, clinical and microbiological review of 300 cases. Br J Ophthalmol. 2003;87:834–838. 3. Srinivasan M, Mascarenhas J, Rajaraman R, et al. The steroids for corneal ulcers trial: study design and baseline characteristics. Arch Ophthalmol. 2012;130:151–157. 4. Schreckenberger PC, Daneshvar MI, Weyant RS, et al. Acinetobacter, achromobacter, chryseobacerium, moraxella and other nonfermentative gram-negative rods. In: Murray PR, ed. Manual of Clinical Microbiology. 8th ed. Washington, DC: ASM Press; 2003:749–779. 5. Whitman WB, Parte A, Goodfellow M, et al. Bergey’s Manual of Systematic Bacteriology: Volume 5: The Actinobacteria. New York: Springer; 2012. 6. Bharathi MJ, Ramakrishnan R, Shivakumar C, et al. Etiology and antibacterial susceptibility pattern of community-acquired bacterial ocular infections in a tertiary eye care hospital in south India. Indian J Ophthalmol. 2010;58:497–507. 7. Ringvold A, Vik E, Bevanger LS. Moraxella lacunata isolated from epidemic conjunctivitis among teen-aged females. Acta Ophthalmol (Copenh) 1985;63:427–431. 8. Nakayama A, Yamanaka K, Hayashi H, et al. Moraxella lacunata infection associated with septicemia, endocarditis, and bilateral septic arthritis in a patient undergoing hemodialysis: a case report and review of the literature. J Infect Chemother. 2014;20:61–64. 9. Cobo LM, Coster DJ, Peacock J. Moraxella keratitis in a nonalcoholic population. Br J Ophthalmol. 1981;65:683–686. 10. Das S, Constantinou M, Daniell M, et al. Moraxella keratitis: predisposing factors and clinical review of 95 cases. Br J Ophthalmol. 2006;90: 1236–1238. 11. Garg P, Mathur U, Athmanathan S, et al. Treatment outcome of Moraxella keratitis: our experience with 18 cases–a retrospective review. Cornea. 1999;18:176–181. 12. Marioneaux SJ, Cohen EJ, Arentsen JJ, et al. Moraxella keratitis. Cornea. 1991;10:21–24. 13. Mian SI, Malta JB. Moraxella keratitis: risk factors, presentation, and management. Acta Ophthalmol. 2011;89:e208–e209. 14. Baum J, Fedukowicz HB, Jordan A. A survey of Moraxella corneal ulcers in a derelict population. Am J Ophthalmol. 1980;90:476–480. 15. Heidemann DG, Alfonso E, Forster RK, et al. Branhamella catarrhalis keratitis. Am J Ophthalmol. 1987;103:576–581. 16. Masaki T, Ohkusu K, Ezaki T, et al. Nocardia elegans infection involving purulent arthritis in humans. J Infect Chemother. 2012;18:386–389. 17. Kowalski RP, Kowalski TA, Shanks RM, et al. In vitro comparison of combination and monotherapy for the empiric and optimal coverage of bacterial keratitis based on incidence of infection. Cornea. 2013;32:830–834. 18. Loh RS, Chan CM, Ti SE, et al. Emerging prevalence of microsporidial keratitis in Singapore: epidemiology, clinical features, and management. Ophthalmology. 2009;116:2348–2353. 19. Ly CN, Pham JN, Badenoch PR, et al. Bacteria commonly isolated from keratitis specimens retain antibiotic susceptibility to fluoroquinolones and gentamicin plus cephalothin. Clin Exp Ophthalmol. 2006;34:44–50. 20. Suzuki T, Ohashi Y. Combination effect of antibiotics against bacteria isolated from keratitis using fractional inhibitory concentration index. Cornea. 2013;32:e156–e160. 21. Stern GA. Moraxella corneal ulcers: poor response to medical treatment. Ann Ophthalmol. 1982;14:295–298. 22. Patel R. MALDI-TOF MS for the diagnosis of infectious diseases. Clin Chem. 2015;61:100–111.
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Clinical Characteristics and Bacteriological Profile of Moraxella Keratitis Hidenori Inoue, MD,*† Takashi Suzuki, PhD, MD,*† Tomoyuki Inoue, PhD, MD,*† Takaaki Hattori, PhD, MD,*‡ Ryohei Nejima, MD,*§ Daisuke Todokoro, PhD, MD,*¶ Saichi Hoshi, PhD, MD,*k Hiroshi Eguchi, PhD, MD,*** Hitoshi Miyamoto,†† and Yuichi Ohashi, PhD, MD†
Purpose: Moraxella species are rare causative pathogens of severe sight-threatening keratitis. The aim of this study was to analyze the clinical presentation, predisposing risk factors, in vitro antimicrobial susceptibility, and treatment associated with Moraxella keratitis.
Methods: We retrospectively reviewed 30 culture-proven cases of Moraxella keratitis from multiple centers in Japan.
Results: The mean age of the patients was 58.4 6 23.4 years. The most common ocular conditions were contact lens wearing (5 patients, 16.7%) and trauma (3 patients, 10.0%). Seven patients had diabetes mellitus. Sixteen patients exhibited hypopyon in association with the corneal focus. Ring-shaped infiltration was found in 9 patients (30.0%), and irregular or amoebic-shaped infiltration was observed in 13 patients (43.3%). Eight patients (26.7%) showed small round infiltrates. All Moraxella isolates were sensitive to fluoroquinolones and aminoglycosides. All were treated with a combination ophthalmic solution containing a fluoroquinolone, tobramycin, and cefmenoxime. Although no patients developed corneal perforation, the response to treatment was slow in all cases; the mean treatment period was 41.9 days.
Conclusions: In Japan, Moraxella keratitis occurs in patients with contact lens wear, trauma, and diabetes mellitus. It presents as a small, round, ring-shaped, irregularly shaped, or amoebic-shaped focus. Moraxella species exhibit good susceptibility to fluoroquinolones and aminoglycosides. Because the treatment response may be very slow, these agents should be continued for a long period of time. Received for publication February 19, 2015; revision received March 24, 2015; accepted April 1, 2015. Published online ahead of print May 13, 2015. From the *OMIC (Ocular Microbiology and Infection Conference) Working Group; †Department of Ophthalmology, Ehime University Graduate School of Medicine, Toon, Japan; ‡Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; §Miyata Eye Hospital, Miyakonojo, Japan; ¶Department of Ophthalmology, Gunma University School of Medicine, Maebashi, Japan; kDepartment of Ophthalmology, National Center for Geriatrics and Gerontology, Obu, Japan; **Department of Ophthalmology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan; and ††Department of Clinical Laboratory, Ehime University Hospital, Toon, Japan. Supported by the Department of Bioscience, INCS, Ehime University. The authors have no conflicts of interest to disclose. Reprints: Takashi Suzuki, PhD, MD, Department of Ophthalmology, Ehime University, Graduate School of Medicine, Shitsukawa, Toon, Ehime 7910295, Japan (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cornea Volume 34, Number 9, September 2015
Key Words: Moraxella, contact lens, diabetes mellitus, keratitis (Cornea 2015;34:1105–1109)
K
eratitis is a corneal infection that occurs after an injury or in association with contact lens wearing. It can progress rapidly with devastating consequences, including corneal scarring and loss of vision. Thus, prompt identification of this condition and commencement of an aggressive course of therapy are imperative to limit tissue damage. Because the severity of keratitis and response to treatment depend on the virulence or drug sensitivity of the causative agents, it is important to understand the various clinical and pathophysiological aspects of infectious keratitis caused by each pathogen. Common bacterial species that cause keratitis have been described in various reports.1–3 Moraxella species are rare causative pathogens of keratitis, and National Surveillance of Infectious Keratitis in Japan demonstrated that 3.8% of culture-proven organisms with keratitis specimens, including bacteria, fungi, and Acanthamoeba, were Moraxella species.1 Another surveillance performed in the United States and India showed that Moraxella species were detected in 3.0% of culture-proven bacterial keratitis specimens.3 Moraxella species are short gram-negative diplobacilli that are commonly found as commensals in the oropharynx, mucous membranes, skin, and genital tract and can cause certain diseases in humans.4,5 Moraxella catarrhalis is the most commonly isolated species and is responsible for acute otitis media in children, chronic and severe otitis media, acute and chronic sinusitis, and upper and lower respiratory tract infections. Moraxella osloensis, Moraxella nonliquefaciens, and Moraxella lincolnii are part of the normal flora of the human respiratory tract. These and other Moraxella species are rare infectious agents.4,5 Moraxella lacunata can rarely cause conjunctivitis and blepharitis along with endocarditis.6–8 Most laboratories cannot identify Moraxella species other than M. catarrhalis because most Moraxella species exhibit similar biochemical reactions. Thus, genetic methods and mass spectrometry are sometimes necessary to identify Moraxella species. Various case reviews of keratitis caused by Moraxella species have been reported from the United States, United Kingdom, Australia, and India.9–15 In the 1980s, Moraxella keratitis was often associated with chronic alcoholism, www.corneajrnl.com |
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malnutrition, diabetes, and poor sanitary habits, all of which were considered to be important predisposing factors.12,14,15 Reports published after 1990 demonstrated other predisposing risk factors for Moraxella keratitis, including penetrating keratoplasty, herpes keratitis, and diabetes mellitus.10,11,13 Predisposing risk factors for bacterial keratitis may differ according to the geographic area and time period. Likewise, clinical findings and treatment responses may differ among areas. No reviews of Moraxella keratitis have been reported from East Asia, including Japan. The aim of this study was to analyze the clinical presentation, predisposing risk factors, patient backgrounds, in vitro antimicrobial susceptibility, and treatment period of Moraxella keratitis in Japan. We also identified Moraxella isolates from keratitis specimens.
MATERIALS AND METHODS This multicenter retrospective study was approved by the Ehime University Review Board and conformed to the tenets of the Declaration of Helsinki. The clinical and microbiological records of all culture-proven cases of Moraxella keratitis seen at Ehime University Hospital, Miyata Eye Hospital, Tokyo Medical University, Gunma University, and Machida Hospital from January 2005 to February 2014 were retrospectively reviewed. The microbiology tests performed in all participating centers were the same. Corneal scrapings on sheep blood agar and chocolate agar plates were incubated at 35°C. Bacterial identification was performed by Gram staining and biochemical testing. Antimicrobial susceptibility testing was performed using a broth microdilution assay. All results were interpreted using cut-off values for susceptibility and resistance according to the criteria by the Clinical and Laboratory Standards Institute or the Japanese Society of Chemotherapy. Tested antibiotics were different among the participating centers. Along with the bacterial culture, direct microscopy was performed. We considered Moraxella sp as a causative agent of keratitis when the number of colonies grown on a plate were large and direct microscopy showed a lot of gram-negative rods with neutrophils. The collected data included age, sex, medical and ocular history, presenting signs and symptoms, systemic and local predisposing factors, antibiotic susceptibility, treatment, and length of follow-up. The size, shape, and location of corneal infiltrates and the presence of hypopyon were documented. We identified 9 stored isolates in Ehime University Hospital. To identify the isolates, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) using the default settings (positive linear mode; laser frequency, 60 Hz; ion source 1 voltage, 20 kV; ion source 2 voltage, 16.7 kV; lens voltage, 7.0 kV; and mass range, 2000–20,000 Da) according to the manufacturer instructions. Bacteria were applied as a thin film onto a 48-spot steel plate (Bruker Daltonics) and allowed to dry at room temperature. Each spot was overlaid with 1 mL of alpha-cyano-4-hydroxycinnamic acid as a matrix and allowed to dry. The target plate was then inserted into the Bruker Microflex LT MALDI-TOF MS system for analysis. The composite profile was analyzed using Biotyper RTC 3.1
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software with reference database version 4.0.0.1 (Bruker Daltonics), which queried a reference bank of 2290 spectra and returned the top 10 matches along with confidence scores ranging from 0.0 to 3.0. The identification criteria used in our analysis, as outlined by the manufacturer, were as follows: a score of $2000 indicated species-level identification, a score of 1700 to 1999 indicated genus-level identification, and a score of ,1700 was interpreted as unidentified. Along with MALDI-TOF MS, molecular identification by polymerase chain reaction amplification and sequencing analysis of the 16S rRNA gene using the universal primers 8UA (59-AGAGTTTGATCMTGGCTCAG-39) and 1485B (59ACGGGCGGTGTGTRC-39) were performed as described previously.16 The prevalence of predisposing risk factors was compared between patients aged ,60 and $60 years using a x2 test. P , 0.05 was considered to indicate statistical significance.
RESULTS Predisposing Factors In total, 30 cases of Moraxella keratitis were evaluated in this study. The age of the patients ranged from 7 to 94 years (mean, 58.4 6 23.4 years). There were 11 male patients and 19 female patients. A predisposing systemic or ocular condition was identified in 21 patients (70.0%) (Table 1). Seventeen patients (56.7%) had an ocular predisposing factor, and the most common ocular conditions were contact lens wearing (5 patients, 16.7%) and trauma (3 patients, 10.0%). Systemic risk factors were identified in 12 patients, with diabetes mellitus being present in 7 patients (23.3%). Seven patients had both ocular and systemic factors, whereas no predisposing risk factor could be identified in 9 patients (30.0%), and none of the patients were alcoholic or malnourished. There was a higher prevalence of ocular predisposing factors in patients aged ,60 years than in those aged $60 years (P , 0.030). Diabetes mellitus was found more frequently in patients aged $60 years than in those aged ,60 years, but the difference was not statistically significant (P , 0.053).
Clinical Findings
The patients’ ophthalmic microscopy findings were documented at the first visit to the hospital. The infiltrate was found in the central cornea in 9 patients (30.0%), paracentral cornea in 12 patients (40.0%), and peripheral cornea in 9 patients (30.0%). Fifteen patients (50.0%) had infiltrates of more than one fourth the corneal diameter, and the other 15 patients (50.0%) had infiltrates of less than one fourth the corneal diameter. With respect to shape, ringshaped infiltration was found in 9 patients (30.0%), and irregular or amoebic-shaped infiltration was observed in 13 patients (43.3%). Eight patients (26.7%) showed small round infiltrates. A representative slit-lamp photograph of each infiltrate shape is shown in Figure 1. Sixteen patients (53.3%) had hypopyon. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Cornea Volume 34, Number 9, September 2015
Profile of Moraxella Keratitis
TABLE 1. Predisposing Factors for Moraxella Keratitis Age Ocular predisposing factors Contact lens wear Trauma Others
Total Systemic predisposing factors Diabetes mellitus Others
Total
0–59 yrs (n = 16), n (%)
‡ 60 yrs (n = 14), n (%)
Total (n = 30), n (%)
0–59 yrs vs. ‡ 60 yrs, P*
4 (25.0) 1 (6.3) 7 (43.8) Conjunctivitis Corneal amyloidosis Bullous keratopathy Dry eye Phlyctenular keratitis Lagophthalmos Steroid eye drops 12 (75)
1 (7.1) 2 (14.3) 1 (7.1) Herpes simplex keratitis
5 (16.7) 3 (10.0) 8 (26.7)
0.413 0.903 NT
4 (28.6)
16 (53.3)
0.030
1 (6.3) 3 (12.5) Sjögren syndrome Dysautonomia Systemic lupus erythematosus 4 (31.3)
6 (42.9) 2 (21.4) Allergic purpura Basedow disease
7 (23.3) 5 (16.7)
0.053 NT
8 (64.3)
12 (46.7)
0.156
*x2 test. NT, Not tested.
Treatment Management procedures for Moraxella keratitis were identified in 29 cases (Table 2). A topical fluoroquinolone (levofloxacin, gatifloxacin, or moxifloxacin) was used in all 29 cases. A topical aminoglycoside ophthalmic solution (tobramycin, dibekacin, arbekacin, or sisomicin) and cefmenoxime were combined with the fluoroquinolone in 19 and 3 patients, respectively. One patient was treated with a combination of a fluoroquinolone and erythromycin. Five patients were treated with a combination of 3 antibiotics (a fluoroquinolone, an aminoglycoside, and cefmenoxime). Systemic antibiotics were used in 6 patients. No patients required surgical interventions such as penetrating keratoplasty. The
mean time required for complete closure of the epithelial defect was 23.4 days (range, 3–70 days) in 17 patients whom that in their clinical records was confirmed. The mean healing time was 41.9 days (range, 9–105 days) in 18 patients whom that in their clinical records was confirmed.
Bacteriological Profile Moraxella species were isolated from all 30 cases, and other organisms (Corynebacterium sp, Haemophilus influenzae, Staphylococcus aureus, Staphylococcus mitis, Staphylococcus epidermidis, Staphylococcus lugdunensis, G streptococci, and Enterobacter aerogenes) were detected along with Moraxella species in 6 cases. The clinical records
FIGURE 1. Representative slit-lamp photograph of Moraxella keratitis. A, Photograph showing a ring abscess with hypopyon. B, Photograph showing an irregular and amoebic infiltrate. C, Photograph showing a small round infiltrate. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Cornea Volume 34, Number 9, September 2015
Inoue et al
TABLE 2. Management Procedures for Moraxella Keratitis Topical Management
n (%)
Fluoroquinolone Levofloxacin Gatifloxacin Moxifloxacin Total Aminoglycoside* Tobramycin Dibekacin Arbekacin Sisomicin Total Cephalosporin* Cefmenoxime Macrolide* Erythromycin/colistin
14 9 6 29
(48.2) (31.0) (20.7) (100)
18 4 1 1 24
(62.1) (13.8) (3.4) (3.4) (82.8)
8 (27.6) 1 (3.4)
*These antibiotics were combined with a fluoroquinolone.
of 26 cases demonstrated in vitro antimicrobial susceptibility (Table 3). All test isolates were susceptible to levofloxacin, tobramycin, and chloramphenicol. Three Moraxella isolates were resistant to cefazolin. Identification of 9 stored Moraxella isolates from keratitis specimens was performed using mass spectrometry and genetic methods. Both MALDI-TOF MS and 16S rRNA gene sequencing produced the same results, and 2 and 7 isolates were identified as M. lacunata and M. nonliquefaciens, respectively.
DISCUSSION In this study, 30 cases of Moraxella keratitis from 2005 to 2014 were analyzed, and the predisposing factors, clinical findings, treatment, and bacteriological profiles of all cases were demonstrated. This is the first review of Moraxella keratitis in Japan. The age of the patients ranged from 7 to 94 years (mean, 58.4 years). The mean ages of the patients in the reports by Das et al10 and Mian and Malta13 were 70.0 and 59.9 years, respectively; thus, the age of our patients was similar to these reports. Moraxella species are commensals in the oropharynx, mucous membranes, skin, and genital tract. Therefore, they could be opportunistic pathogens that cause TABLE 3. Antimicrobial Susceptibility of Moraxella Isolates for Keratitis No of Isolates Antibiotics Levofloxacin Tobramycin Cefazolin Ceftazidime Chloramphenicol Ciprofloxacin
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Sensitive
Resistant
Sensitivity Test Not Done
26 14 21 11 7 5
0 0 3 0 0 0
4 16 6 19 23 25
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keratitis in patients of all ages. In the present study, contact lens wearing was a predominant ocular predisposing factor in patients aged ,60 years. However, it was not a prominent predisposing factor in other reports.10,13 Contact lenses are now used not only by young people, but also by middle-aged and older people, and commensal Moraxella species could opportunistically infect the cornea if it has become damaged by contact lenses. Along with contact lens wearing, trauma and other ocular surface diseases were also predisposing risk factors in the present study, similar to previous reports.10,11,13 Although Das et al10 demonstrated that herpes simplex keratitis and penetrating keratoplasty were predominant ocular predisposing risk factors, our cases included 1 patient with herpes simplex keratitis and no patients with penetrating keratoplasty. Diabetes mellitus was the predominant systemic predisposing factor, similar to previous reports.10,13 However, none of our patients had chronic alcoholism or malnutrition. As Cobo et al9 demonstrated, Moraxella keratitis can occur even among healthy people. Moraxella species could cause opportunistic keratitis in people of a wide age range with ocular surface damage. The clinical findings of Moraxella keratitis have been characterized as an indolent paracentral or peripheral ulcer that is usually oval in shape with hypopyon or hyphema.10 In our study, the clinical findings were divided into the following 3 types: ring abscess in the central cornea (9 patients), irregular amoebic-shaped infiltrate overlaying the whole cornea (13 patients), and small round infiltrate (8 patients). The first 2 types could be similar to the clinical findings in previous reports.10,11,13 The ring abscess type could have less intense inflammation in the center of focus, and be different from the ring infiltration seen in Acanthamoeba keratitis or anesthetic abuse keratopathy. We should suspect Moraxella keratitis in patients with ring abscess or irregular amoebic-shaped infiltration. Our series included patients with small round infiltrates. Keratitis with small round infiltrates can be found until the infiltrate progresses. These findings demonstrate that Moraxella keratitis can have various clinical presentations. In the present study, a combination of a topical fluoroquinolone and aminoglycoside (or cefmenoxime) was widely used to treat Moraxella keratitis. Because of their stability and broad spectra, fluoroquinolones are often used for empirical treatment or prevention of keratitis.17–20 Like fluoroquinolones, aminoglycosides exhibit good susceptibility to gram-negative rods such as Pseudomonas aeruginosa and Moraxella species.20 The present study also demonstrated that Moraxella isolates show susceptibility, and no resistance, to both fluoroquinolones and aminoglycosides. Although some isolates showed resistance to cefazolin, they were susceptible to other cephalosporins. Similarly, Das et al10 noted that Moraxella isolates showed good susceptibility to fluoroquinolones, aminoglycosides, and cephalosporins. Thus, Moraxella isolates from keratitis could have good susceptibility to antibiotics typically used for topical treatment. Indeed, our cases treated with topical antibiotics healed without corneal perforation and did not require surgical intervention. All cases in a series by Stern21 and 50% of cases in a series by Marioneaux et al12 required surgical Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Cornea Volume 34, Number 9, September 2015
interventions. Furthermore, 3 blind eyes in a series by Das et al10 were enucleated. In contrast to these previous reports, our cases were cured by topical treatment without surgical intervention. One possible reason for this good response to treatment is that our cases were usually treated with a combination of antibiotics such as a fluoroquinolone and aminoglycoside. Our previous study showed that a combination of a fluoroquinolone with tobramycin or cefmenoxime had synergistic effects and increased the antibacterial activity of each agent against gram-negative rods in vitro.20 Thus, a combination of a fluoroquinolone with tobramycin or cefmenoxime may be a feasible option for improving the effects of fluoroquinolones in the treatment of keratitis. Another reason for the good response to treatment could be that the treatment began before the keratitis progressed and had dense infiltration. Because our cases included small foci of keratitis, topical treatment was effective. Along with these reasons, some Moraxella isolates could have low virulence. Moreover, Japanese genetic variability could be associated with variation in clinical findings or treatment outcomes. However, the time required for complete closure of the epithelial defects ranged from 3 to 70 days, and the mean healing time was 41.9 days. The mean healing time in the case series by Das et al10 was 35 days; thus, Moraxella keratitis may respond slowly to treatment. Moraxella keratitis should be carefully monitored and treated for a long period of time. Although little is known about the exact reasons for the relatively long time to cure, Moraxella sp may induce persistent inflammation in the cornea and delay epithelial healing. We identified Moraxella sp isolates from keratitis using MALDI-TOF MS and genetic techniques. MALDI-TOF MS can rapidly determine the unique molecular fingerprint of an organism.22 The characteristic spectrum pattern of this molecular fingerprint is used to reliably and accurately identify a particular microorganism by matching it. Additionally, MALDI-TOF MS–based identification does not have the limitations of biochemical testing and can be used to analyze species that have been difficult to identify by biochemical testing, such as Moraxella species. Thus, it offers a safe, cost-effective, and adaptable system for rapid identification of bacteria and fungi. In this study, M. lacunata and M. nonliquefaciens were identified as causative pathogens of keratitis. M. lacunata has been considered as the cause of acute conjunctivitis or angular blepharoconjunctivitis.7 Along with these infections, M. lacunata could cause keratitis. Our study showed that more isolates were identified as M. nonliquefaciens. As Cobo et al9 demonstrated, M. nonliquefaciens could be an important cause of keratitis. Although Heidemann et al15 reported in 1987 that M. catarrhalis could cause keratitis, we did not detect M. catarrhalis in the present study. We speculate that the species of Moraxella causing keratitis could be dependent on the time period and geographical location. It is likely that M. lacunata and M. nonliquefaciens are causative pathogens for keratitis in Japan. Although we could not compare exactly the clinical findings of keratitis caused by M. lacunata and M. nonliquefaciens because of few cases, the M. lacunata cases had bigger focus size and healed slower, compared with M. nonliquefaciens keratitis cases. However, the number of Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Profile of Moraxella Keratitis
M. lacunata cases were few, and statistics could not be performed. Further investigation of the pathogenesis of Moraxella keratitis is needed. In summary, contact lens wearing and trauma could be important predisposing factors for Moraxella keratitis, which shows various clinical findings. Rapid diagnosis and the use of topical antibiotic combinations may be needed to treat keratitis. REFERENCES 1. National Surveillance of Infectious Keratitis in Japan–current status of isolates, patient background, treatment [in Japanese]. Nihon Ganka Gakkai Zasshi. 2006; 110:961–972. 2. Bourcier T, Thomas F, Borderie V, et al. Bacterial keratitis: predisposing factors, clinical and microbiological review of 300 cases. Br J Ophthalmol. 2003;87:834–838. 3. Srinivasan M, Mascarenhas J, Rajaraman R, et al. The steroids for corneal ulcers trial: study design and baseline characteristics. Arch Ophthalmol. 2012;130:151–157. 4. Schreckenberger PC, Daneshvar MI, Weyant RS, et al. Acinetobacter, achromobacter, chryseobacerium, moraxella and other nonfermentative gram-negative rods. In: Murray PR, ed. Manual of Clinical Microbiology. 8th ed. Washington, DC: ASM Press; 2003:749–779. 5. Whitman WB, Parte A, Goodfellow M, et al. Bergey’s Manual of Systematic Bacteriology: Volume 5: The Actinobacteria. New York: Springer; 2012. 6. Bharathi MJ, Ramakrishnan R, Shivakumar C, et al. Etiology and antibacterial susceptibility pattern of community-acquired bacterial ocular infections in a tertiary eye care hospital in south India. Indian J Ophthalmol. 2010;58:497–507. 7. Ringvold A, Vik E, Bevanger LS. Moraxella lacunata isolated from epidemic conjunctivitis among teen-aged females. Acta Ophthalmol (Copenh) 1985;63:427–431. 8. Nakayama A, Yamanaka K, Hayashi H, et al. Moraxella lacunata infection associated with septicemia, endocarditis, and bilateral septic arthritis in a patient undergoing hemodialysis: a case report and review of the literature. J Infect Chemother. 2014;20:61–64. 9. Cobo LM, Coster DJ, Peacock J. Moraxella keratitis in a nonalcoholic population. Br J Ophthalmol. 1981;65:683–686. 10. Das S, Constantinou M, Daniell M, et al. Moraxella keratitis: predisposing factors and clinical review of 95 cases. Br J Ophthalmol. 2006;90: 1236–1238. 11. Garg P, Mathur U, Athmanathan S, et al. Treatment outcome of Moraxella keratitis: our experience with 18 cases–a retrospective review. Cornea. 1999;18:176–181. 12. Marioneaux SJ, Cohen EJ, Arentsen JJ, et al. Moraxella keratitis. Cornea. 1991;10:21–24. 13. Mian SI, Malta JB. Moraxella keratitis: risk factors, presentation, and management. Acta Ophthalmol. 2011;89:e208–e209. 14. Baum J, Fedukowicz HB, Jordan A. A survey of Moraxella corneal ulcers in a derelict population. Am J Ophthalmol. 1980;90:476–480. 15. Heidemann DG, Alfonso E, Forster RK, et al. Branhamella catarrhalis keratitis. Am J Ophthalmol. 1987;103:576–581. 16. Masaki T, Ohkusu K, Ezaki T, et al. Nocardia elegans infection involving purulent arthritis in humans. J Infect Chemother. 2012;18:386–389. 17. Kowalski RP, Kowalski TA, Shanks RM, et al. In vitro comparison of combination and monotherapy for the empiric and optimal coverage of bacterial keratitis based on incidence of infection. Cornea. 2013;32:830–834. 18. Loh RS, Chan CM, Ti SE, et al. Emerging prevalence of microsporidial keratitis in Singapore: epidemiology, clinical features, and management. Ophthalmology. 2009;116:2348–2353. 19. Ly CN, Pham JN, Badenoch PR, et al. Bacteria commonly isolated from keratitis specimens retain antibiotic susceptibility to fluoroquinolones and gentamicin plus cephalothin. Clin Exp Ophthalmol. 2006;34:44–50. 20. Suzuki T, Ohashi Y. Combination effect of antibiotics against bacteria isolated from keratitis using fractional inhibitory concentration index. Cornea. 2013;32:e156–e160. 21. Stern GA. Moraxella corneal ulcers: poor response to medical treatment. Ann Ophthalmol. 1982;14:295–298. 22. Patel R. MALDI-TOF MS for the diagnosis of infectious diseases. Clin Chem. 2015;61:100–111.
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