I. Overview In 1882, Koch first identified Mycobacterium tuberculosis, and soon it was discovered that in addition to Mycobacterium tuberculosis and Mycobacterium leprae, non-tuberculous mycobacteria (NTM) also existed. In the past, it was thought that NTM was rarely clinically pathogenic, however, in recent years, with the large number of corneal surgeries, especially laser in situ keratomileusis (LASIK), postoperative complications of NTM corneal infections have become a high priority in the ophthalmology community. According to statistics, two million LASIK procedures were performed worldwide in 1998, and the incidence of postoperative infectious keratitis was 0.1%~0.2%, with non-tuberculous mycobacteria (NTM) being one of the most common causative organisms. Recent studies have found that with the increase of AIDS and the use of hormones and immunosuppressants, the infection of this opportunistic pathogenic bacteria will increase. For this reason, our laboratory was the first in China to conduct research on NTM keratitis and successfully isolated and identified the first strain of Mycobacterium non-tuberculosis in corneal tissue. NTM, also known as atypical mycobacterium, is also known as acid-resistant bacterium because of its positive acid-resistant staining characteristics. It is widely present in the external environment (including soil, milk, sterile water, animal body surfaces and body fluids) and can contaminate a variety of water sources, especially reagents and rinses in hospitals, and therefore has become one of the most common bacteria in hospital infections. NTM is an elongated or slightly curved rod, 1-4um long and 0.3-0.5um wide, without flagella and pods, not forming budding spores, non-motile and slow growing. In the smear in the presence of a single bacteria, bacteria in large quantities can be aggregated into bundles or clumps. In culture, there is often a tendency to aggregate, so the pure culture of the smear is often in the form of cords. Under electron microscopy, it was found to be polymorphic and sometimes branched such as V, Y or herringbone arrangement. It stained with fluorescent dye gold ammonium and showed golden fluorescence under fluorescence microscope. NTM is an opportunistic pathogen and is susceptible to infection under conditions of decreased ocular resistance. the route of transmission of NTM has not been determined, and contact infection with NTM in the external environment is considered to be the primary route, in addition to surgical instruments contaminated by mycobacteria. The cell wall of NTM organisms is covered with a large amount of fatty acids and glycolipids, which gives them the ability to survive for a long time without being killed by phagocytic vesicles inside the host phagocyte, thus evading defense mechanisms. In addition, due to relative hypoxia in the corneal stroma, NTM can enter a non-replicative dormant state and survive by using its own acetate and fatty acids as carbon sources for lipid metabolism and nitrogen respiration. It has been shown that after mycobacteria enter the dormant phase, their virulence factor expression is down-regulated and the rate of bacteriophage metabolism decreases, allowing NTM to survive in the eye for a long time without causing disease; however, once the body resistance decreases or topical hormones are used, the cell-mediated immune response is suppressed and the dormant NTM may be transferred to the proliferative phase at any time. It has been found that the replication cycle of NTM is long, usually about 20 hours, and the growth is slow, so it leads to a long incubation period of NTM ophthalmopathy, a continuous bacterial state, and a slow pathogenesis. The modern view of immunology is that the disease caused by Mycobacterium is an immune disorder in which the bacteria induce a granulomatous immune response in the sensitive organism, leading to an imbalance in immune homeostasis and a progression toward a pathological immune response. It was found that IFN-γ plays an important role in the protective immune response caused by NTM, while TNF-α and IL-1 are closely related to tissue necrosis. Clinical diagnosis 1. Clinical manifestations: The onset of NTM keratitis after LASIK is chronic, with an average incubation period of 2~3 weeks. Early symptoms of infection include photophobia, lacrimation, eye redness, and some patients have eye pain, but it is not obvious, which is related to LASIK surgery that damaged the sensory nerve fibers of the corneal flap; later, vision loss occurs. Ocular signs: ciliary congestion, grayish infiltration of the corneal stroma, starting as a small subepithelial infiltrate, followed by a gradual expansion of the infiltrate with irregular feathery borders, and the lesions may fuse to form subvalvular abscesses, which in severe cases may produce corneal flap necrosis and free floating; some patients develop satellite lesions, annular infiltrates, coin-shaped keratitis, or crystalline keratopathy. Severe patients can cause anterior chamber inflammation. 2, diagnosis: a history of hormone use after LASIK, 2-3 weeks after the corneal infiltrative lesions or crystalline keratitis under the corneal flap, NTM keratitis should be highly suspected, etiological diagnosis must rely on laboratory tests. If the infiltrate is superficial, the corneal epithelium or superficial stroma can be scraped for stain microscopy and culture; if the infiltrate is deep or subflap infiltrate, the corneal flap should be lifted to take specimens for culture. Smear staining microscopy, culture and animal testing are the common diagnostic and identification techniques for NTM. Smear antacid staining microscopy can provide a preliminary laboratory diagnosis, its advantages are simple and fast, but the sensitivity is low. NTM culture time is longer than normal bacteria, and it usually takes 7-60 days to determine the results, often using Roche (or modified Roche) medium, Middlebrook 7H 9, 10, 11 medium or Tween ovalbumin liquid medium. Molecular biology techniques allow for a rapid, sensitive and specific diagnosis of NTM. The method is mainly PCR technique, and the target gene sequences used for NTM taxonomic identification are 16S rDNA, 16S ~23S rDNA, IS6110 and hsp65. PCR-RFLP is a technical tool that combines PCR and restriction endonuclease analysis, and PC-RFLP of 16S ~23S rDNA IGS can be used for rapid and accurate strain identification. Nucleic acid hybridization technique is a complementary binding of 125I-labeled DNA probes to rRNA of Mycobacterium avium, and the detection can be completed in 2 hours with high specificity and sensitivity. Because NTM is less virulent than Mycobacterium tuberculosis, histopathologic examination reveals NTM keratitis may show a non-caseous necrotic histiocytic reaction; neutrophilic or eosinophilic increase; and macrophage intracellular phagocytosis with acid-resistant bacteria. Clinical treatment The treatment of NTM keratitis includes pharmacological and surgical treatment. The principles of treatment are: combination of pharmacological and surgical treatment, combination of local and systemic treatment, and prohibition of hormones. 1, drug treatment: NTM has strong hydrophobicity, the outer membrane pore protein channel of the bacterial cell wall is very narrow, making it difficult for drugs to enter the body of NTM; and fast-growing NTM has strong β-lactamase activity, which can make β-lactam antibiotics lose their anti-microbial activity and produce resistance, so Penicillin and cephalosporin antibiotics are ineffective in the treatment of NTM. For early NTM keratitis, amikacin eye drops, which belongs to the aminoglycoside class of antibiotics, are preferred in most cases. At low concentrations, amikacin inhibits the growth of NTM, and at high concentrations, it has a strong bactericidal effect. The concentration of amikacin eye drops is usually 1% to 2% every 30 minutes, and the dose is reduced after 48 hours of continuous use; moderate and severe patients can be given another subconjunctival injection of 4% amikacin 0.5ml; it is not usually administered systemically. Although amikacin has been the first-line anti-NTM drug, in recent years it has been found to have significant toxic effects on the corneoconjunctival epithelium and to be prone to the development of drug-resistant strains, so Ford proposed the topical use of new macrolides such as clarithromycin (CAM), roxithromycin (RTM) or azithromycin (ATM). Azithromycin (ATM) as the first choice of treatment. These drugs have strong anti-NTM activity, high tissue concentration, long half-life and few adverse effects. In vitro experiments of clarithromycin can kill 100% of NTM, and the concentration of eye drops is usually 1%. The use of clarithromycin in this disease is limited because of the strong local irritation, and systemic clarithromycin can be chosen for moderate and severe patients. Azithromycin is easy to bind to tissues in the body, and is most sensitive to NTM infection. Its intravenous preparation is stable, and the prepared eye drops can be stored in 4℃ refrigerator for 7 days, with less irritation, and the general concentration is 0.2%~2%. For moderate and severe patients, azithromycin can be given intravenously, 500mg/day. Fluoroquinolone antibiotics have strong antibacterial activity against NTM. Among the fourth generation fluoroquinolones, Gatifloxacin (Gatifloxacin) has the best effect with an ophthalmic concentration of 0.3%. And the corneal toxicity is lower than that of aminoglycoside antibiotics. Due to the long treatment time of this disease, in order to prevent drug resistance, it is necessary to combine the drugs, usually with 0.8% amikacin ophthalmic solution or 1% clarithromycin ophthalmic solution and 0.3% levofloxacin ophthalmic solution, which can show a significant synergistic effect. Most of the literature suggests that hormone use is one of the key factors contributing to NTM keratitis. Hormones can thin the corneal stroma, prolong the course of the disease, aggravate it, and predispose to recurrent disease. Recent studies have shown that a few inflammatory cells and a large number of NTM were seen on corneal specimens after hormone use in NTM keratitis, and a large number of inflammatory cells and a small amount of NTM were seen on corneal specimens in the non-hormone use group. Hu inoculated NTM into rabbit corneas in 1998, and after 4 weeks of observation, keratitis remitted in both the antibiotic-treated and non-treated groups, but the corneal infection spread after hormone use, and the mechanism may be that hormone inhibited granulomatous inflammation, resulting in easy spread of NTM, therefore, hormones should be disabled in the treatment. 2, corneal flap irrigation: for those who are not treated with the above drugs or moderately severe patients, Karp believes that the best treatment is to lift the corneal flap at an early stage, immediately take a smear microscopy to identify the pathogenic bacteria, if it is NTM, then use 0.8% amikacin or 1% clarithromycin solution to irrigate the corneal flap and the basal bed (5 minutes); those who do not have the conditions to take a smear microscopy can use broad-spectrum antibiotics such as tobramycin or vanguardin for rinse. 3, surgical treatment: despite aggressive local treatment, 64% of patients still need surgery due to drug insensitivity, surgery includes corneal flap resection and lamellar corneal transplantation. The surgical indications for flap removal are that the lax corneal flap cannot attach to the corneal stroma; clinical symptoms are not relieved, and the response to drug therapy is poor. Flap resection both increases the local drug concentration and disrupts the survival environment of NTM, eliminating the small lumen between the flap and the stroma and allowing inflammation to be controlled. Corneal lamellar transplantation is feasible in flap resection patients to improve corrected visual acuity after inflammation has been controlled. In addition, it has been reported in the literature that direct lamellar corneal transplantation along with pharmacological control of infection can also achieve better results.