Keratitis is a major cause of visual morbidity worldwide, responsible for an approximately 2 million unilateral blindness cases per year. Aetiological agents of infectious keratitis can be bacterial, fungal, parasitic, and viral in nature [27]. The course of NTM keratitis can mimic fungal, herpes simplex virus or Acanthamoeba infection, which are often also associated with ocular trauma. Moreover, fungal and Acanthamoeba keratitis, as well as M. chelonae, are considered as pathogens involved in postoperative infections [28]. The diagnosis of NTM keratitis is, thus, often delayed, which leads to the insufficient antibiotic treatment and slow response to therapy. In the case described herein, M. chelonae was revealed after more than 6 months after the onset of symptoms and admission to hospital (Fig. 5). This delay could be caused by several reasons: insufficient time of culture for 4 days; poor sample collection in the beginning (conjunctival and corneal swabs); antibiotic therapy; and, in addition, late superinfection with M. chelonae. We can hypothesize, that it was the combination of initial poor sampling and insufficient culture time. The identification of NTM is critically dependent on correct sampling. It is generally recommended to collect corneal scraping from the leading edges and the base of the ulcer or abscess sample if formed [29]. Ocular swabs are likely insufficient for the diagnosis of NTM keratitis as showed in our report. For ophthalmologists it is generally uneasy to identify the causative infectious organism [30], which is clearly demonstrated by the case of M. chelonae keratitis presented here. The clinical course showed a subtle improvement at the beginning, but afterwards an obvious deterioration outweighed. This led to the overuse and frequent changing of antibiotic therapy without a proper detection of the causative agent. A combination therapy with corticosteroids in order to control inflammation resulted in an escalation of the NTM infection. Until an intrastromal abscess formed, satisfactory samples to determine the aetiological agent were not obtained. Being aware of this fact, corneal biopsy should have been certainly considered. It is considered superior to the corneal scrape despite the risk of corneal perforation, especially when the therapy and pathogen identification fail [27].

Later, even if corneal scraping were performed, the unsuccessful detection of M. chelonae could be caused by partially effective antibiotic therapy at that point. Repeated culture of corneal re-scrapings succeeded after voriconazole monotherapy was administered. The later superinfection could not be completely excluded, but M. chelonae belongs among pathogens that are involved in postpoperative infections [28]. According to our analysis of previously published studies, the average length from the onset of symptoms to M. chelonae diagnosis is about 11 weeks, ranging from 1 week to 9 months [29].

There are several methods for NTM identification including Ziehl-Neelsen staining with lower sensitivity but with rapid results [27]. The success of culture techniques is clearly dependent on a suspicion of NTM aetiology and therefore extension of incubation time for at least 8 weeks for slow growing species. The culture methods allow for the identification of NTM and antibiotic susceptibility and should combine both culture on solid media and in broth to enhance the yield [29]. PCR methods are used mostly for the precise identification of NTM DNA from colonies or positive Mycobacteria growth indicator tube (MGIT), and involve sequencing of the rpoB or hsp65 genes. Nevertheless, a suspicion of NTM keratitis should indicate to perform PCR detection of NTM DNA directly from clinical specimens in order to accelerate the diagnosis. The advantage of PCR methods lies mainly in their high sensitivity, but also brings a potential risk of false positive results, since NTM occurs ubiquitously. To minimize this risk, well trained and specialized laboratory technicians are required and the PCR results should always be evaluated in a clinical context [27].

The cornea is poorly vascularized leading to the generally imperfect drug distribution in the stroma and, thus, it is difficult to achieve drug concentrations exceeding minimum inhibitory concentration at the site of infection. Mycobacterial biofilms unfortunately preclude conventional antibiotic treatment and, along with diminished host immune responses, might lead to chronic infections [31]. The topical antimicrobial treatment with eye drops then usually requires a high loading dose with subsequent frequent application regimens. Topical antibiotic eye drops for severe keratitis mandate the initial loading dose of one drop every 5–15 min within the first hour followed by an intensive hourly application [32]. There is obviously a risk of patient non-adherence to the treatment plan due to drop application fatigue [30]. Antibiotic ointments represent are not suitable in these cases due to their poor solubility and low penetration into the cornea. Subconjunctival injections of antibiotics represent treatment modality of bacterial keratitis in those cases, where there is a risk of a poor effect of the topical treatment [32].

In this case, the initial empirical therapy regimen in November 2018 included tobramycin plus dexamethasone and ofloxacin eye drops with their corresponding ointments. Due to the minimal improvement after two months, the therapy was changed to neomycin, polymyxin B and dexamethasone. Poor outcome of keratitis therapy with ofloxacin and tobramycin was also reported in study by Van Der Beek et al. 2008 [33]. Subsequently, the suspicion on the mycotic infection was raised and voriconazole eye drops were applied, but without any success. Finally, in the middle of June 2019, corneal scrape and abscess samples were obtained and mycobacterial aetiology was determined. An intensive anti-mycobacterial therapy administered topically, intravitreally, and orally was initiated immediately and continued for 4–5 weeks (Fig. 5). It is therefore obvious, that the correct identification of the particular mycobacterial species and its susceptibility to antibiotics are critical for the selection of the appropriate therapy.

In this case, clarithromycin was selected, because M. chelonae is usually susceptible to macrolides, to which M. abscessus is frequently resistant [34]. When using the “classical” identification of mycobacteria and the sensitivity of NTM to clarithromycin, the culture needs to be extended to 14 days to obtain valid results. However, the susceptibility results can be obtained in an expedite way by the sequence analysis of rrl gene for the constitutive macrolide resistance and erm [41] gene for inducible macrolide resistance [35]. According to the American Thoracic Society/Infectious Diseases Society of America [20], clarithromycin alone can be used for mild localized skin infections. In cases of serious skin, bone, and soft tissue diseases this monotherapy should be combined with additional antibiotics based on the susceptibility testing for a minimum of 4 months. The survey of previous reports on therapy of M. chelonae linked keratitis in European countries from 1987 supports the use of combinatory antibiotic therapy (Table 2, 3, references [11, 13, 14, 16, 28, 33, 36,37,38,39]). In the majority of these studies, combination therapies consisting of three and more (7 studies) or two antibiotics (2 studies) were used. Only one study reported the successful use of antibiotic monotherapy. Macrolides were used in 9 studies, fluoroquinolones in 7 studies, aminoglycosides in 7 studies and imipenem or linezolid in two studies. Interestingly, topical linezolid was successfully used in the monotherapy of keratitis after LASIK in 33-year-old man [14]. This therapy should be accompanied with local antiseptics and surgical debridement and draining of abscesses where applicable. Oral clarithromycin is known to possess good distribution characteristics in the eye, however its bacteriostatic nature and time-dependent activity towards mycobacteria, as well as AUC/MIC dependent cure rate are the reasons, why the monotherapy for NTM keratitis should be avoided [40, 41]. In this case, once the correct diagnosis was made, clarithromycin was applied systemically with amikacin, which is the most common first-line antibiotic for the atypical mycobacterial keratitis. However, its poor penetration through epithelium of cornea and moderate activity against the clinically significant NTM species requires a combination with another antibiotic. It acts synergistically with ethambutol, clofazimine and, most importantly, with macrolides. Combinations of these antibiotics were proven to be effective in animal models. Amikacin exhibits two modes of actions - it inhibits proteosynthesis and, in the rapid growing mycobacteria it also damages bacterial cell wall [42].

Fluoroquinolones also play an important role in the successful treatment of atypical mycobacterial keratitis [31]. Moxifloxacin, fourth-generation fluoroquinolone, is superior in ophthalmology to other fluoroquinolones, because it can achieve higher concentrations in the conjunctiva or cornea. Moreover, moxifloxacin is more effective against Gram-positive bacteria than other generations of fluoroquinolones, since it inhibits both the bacterial DNA gyrase and topoisomerase IV [43]. The antibiotics suitable for the treatment of keratitis can also possess notable side effects and good care must be taken in case of a long-term fortified eye drop usage. Thus, aminoglycosides have been reported to exert a negative effect on corneal epithelium and ciprofloxacin precipitation might impair epithelial healing. Painful sensations were reported with some topical antimicrobial eye drops [44]. Precautions should be taken during the use of topical corticosteroids in patients with corneal ulcerations, where it should be minimized due to the risk of corneal melting and perforation [30]. The outcome of therapy for this infection is often not satisfactory as many patients require corneal surgery or transplant for vision recovery [20]. Antibacterial agents utilized for the treatment of ocular infections caused by nontuberculous mycobacteria are summarized in Table 3. The most common antibiotics used for topical administration were fluoroquinolones (mostly ciprofloxacin) and aminoglycosides (mostly amikacin). Clarithromycin was the most prevalent orally used antibiotic. In other study, four patients, who developed a keratitis after LASIK, were successfully treated using a 6-week long therapy with topical ciprofloxacin, amikacin and azithromycin, with and addition of oral doxycycline [38].

However, as a result of the combination therapy administered in our patient, ocular findings stabilized and a dense corneal scar formed. There have been no signs of infection recurrence following the optical penetrating keratoplasty at the time of this report.

In the case described here, an additional interesting side finding of the repeated EBV DNA detection in vitreous fluid in the quantity of 1660 copies/mL using qPCR was made. In general, herpetic and other viruses represent significant causative agents of endophthalmitis, mostly of an endogenous origin. However, other EBV diagnostic markers in this case support this finding as a sign reactivation of the latent EBV infection. These included serological markers of EBV reactivation and were further supported by the finding of EBV viremia of 6680 copies/mL. Previous reports have established, that EBV spreading by the hematogenous route within lymphocytes may cause although rarely ocular inflammation, mostly necrotizing retinitis in patients with B and NK/T-cell lymphoid tumors, other malignancies, or possibly HIV [45,46,47]. Nevertheless, in such cases, the quantity of EBV DNA is significantly higher in ocular samples than in blood [47]. In addition, among herpetic viruses HSV is the more typical causative agent of keratitis [46].

Taken together, the treatment of eye infections caused by NTM tends to be cumbersome and requires an aggressive approach. Despite the fact, that M. chelonae accounts for about half of all NTM keratitis cases described, the management of such eye infections is poorly established [12]. Therefore, all clinical isolates should be tested for their antimicrobial susceptibility. Subsequently, a combination therapy is recommended in order to prevent bacterial resistance [3]. Thus, based on our experience, we can recommend to treat complicated keratitis with abscess formation using the combination of oral and topical clarithromycin, intravitreal, topical and intracameral amikacin, and oral and topical moxifloxacin. Moreover, such combination approach for NTM ocular infections along with discontinuation of corticosteroids is strongly recommended by Kheir et al. 2015 [48].