The Experimental And Clinical Study On Nontuberculous Mycobacterium Keratitis

PART I:Pathogen study of non-tuberculous mycobacterial kertitisObjectives To study the biology characteristics of mycobacterium abscessus (MA).Methods MA was choosen as the study object because it is one of the common pathogenic bacteria of non-tuberculous mycobacterial keratitis (NTMK). By bacteria culturing, staining, biochemical reaction and micro-broth dilution method to determine the minimum inhibition concentrations (MICs), we know the characteristics of morphous, staining, growth, biochemical and the in vitro antibiotic suspectibility.Results The growth period of MA is 3 to 7 days, the suitable temperiture is 35℃-37℃. It grows well on the common blood agar culture-medium and L-G medium. The colony are flavescens, rough or smooth. MA has multiple shapes observed by transmission electron microscope (TEM).The size is (0.2μm-0.5μm)×(1μm-6μm). There are no flagella, capsulae or spore. The cell wall contains plenty of lipid which is very thick. It has the ability of resist decolorization by acid following staining, Nacl (5%) and carbazotic acid (0.2%). It can produce nitrate reductase, catalase that resist heat, phenol sulfatase, urase, etc. The MICs of each antimicrobial agent against the isolations showed that amikacin, gatifloxacin, azithromycin, clarithromycin and cefoxitin had good activity, ofloxacin, tobramycin, erythromycin and minocycline had moderate activity, doxycycline and sulfamethoxazole had poor activity.Conclusions MA is a positive acid-fast staining bacteria which has multiple shapes and contains plenty of lipid in the cell wall. MA has the ability of resist high concentrations of salt and produce multiple enzyme. It is insentive to previous regular antibiotics but sentive to the new antimicrobial agents. PART II:Experimental study of the pathology of non-tuberculous mycobacterial keratitis in rabbitsObjectives To study clinical pathological changes of NTMK in rabbits.Methods Fifty rabbits with NTMK were divided into 2 groups:group A (simple inoculation of bacteria group) and group B (inoculation of bacteria and topical glucocorticoid-treated group). The corneal infilitration of all rabbits were observed. Bacterial quantitative culture and histopathological examination examine were performed on the 5th,7th, 14th,21st,28th,42nd and 63rd day, transmission electron microscope (TEM) on the 7th,14th, 21st and 28th day.Results In the earliest stage (5 days), the cornea was cloudy and edematous and a punctate infiltration were noted. Among 7 to 14 days, the infiltration enlarged and deepened into abscess and ulcer, a lot of satellite lessions and infectious crystalline keratopathy were noted. Among 14 to 21 days, the infiltration relieved and the ulcer were insteaded by scar. The infections worsened, cornea punching occurred and big ulcers were noted in some animials. After 21 days, corneal fibrosis developed and scar formed. The mean stromal infiltration areas in group B was greater than than those of group A (F=5.470, P=0.047<0.05). The number of NTM in group B was higher than than those of group A (P=0.035 or 0.04 on the 7th or 14th day). The histopathological examination, there were plenty of neutrophile granulocyte and acid-fast bacilli in the corneal stroma. Then a lot of lymphocytes were noted. In the latter stage, the fibroblast cells proliferation and corneal ulcer were cured. Granulation tissue and fibroblasts proliferation and infilitration with a plenty of neutrophile granulocyte were found in the cornea punching ones. Lots of neutrophile granulocyte and acid-fast bacilli were noted by TEM in the early stage. And in the latter, fibrous tissue proliferated.Conclusions Multifocal superficial canous stromal infilitrates is the clinical characteristic of NTMK in rabbits. In the early stage, NTMK presents acute purulent inflammatory changes, the advanced stage shows chronic and non-specificity inflammation changes. The disease is aggravated and prolonged after use of glucocorticoid.PARTⅢ:Experimental study of the treatment of non-tuberculous mycobacterial keratitis in rabbitsObjectives To study the best treatment of NTMK.Methods The unilateral cornea of 51 New Zealand white rabbits were inoculated with NTM. Eyes with NTMK were divided into 7 groups (one group with 9,7 in others 6) and topical treated with amikacin (2.5%), gatifloxacin (3%), azithromycin (1%), the combination of amikacin and gatifloxacin, the combination of amikacin, gatifloxacin and debridement, early lamellar keratoplasty and BSS was used as negative control. Topical medicine was applied 8 times per day, 4 times per day after 1 week. The clinical manifestation of each rabbit were obersered and read each week. Results All the above medicine were effective against NTM, the scores of clinical manifestation were decreased. NTMK healed after 1 to 2 weeks of treatment with early lamellar keratoplasty group and all cured after 4 weeks.Conclusions All the three antibiotic agents including amikacin, gatifloxacin and azithromyc are effective in the treatment of NTMK, but early lamellar keratoplasty is more quick and thorough.PART IV:Pathalogical study of non-tuberculous mycobacterial keratitisObjectives To study the histopathologic and microstructure changes of lesion site of non-tuberculous mycobacterial keratitis.Methods Six cases of the lesions tissues were scraped from acute phase of non-tuberculous mycobacterial keratitis after corneal foreign body removal, of which 3 cases were for Ziehl-Neelsen acid-fast staining,3 cases for TEM inspection. Two cases of biopsied corneal tissues of chronic phase of non-tuberculous mycobacterial keratitis keratoplasty in cornea respectively were examinaed by histology and TEM.Results A single corporation or fascicular block red bacilli can be observed after acid-fast staining in the four cases of corneal scrapings. From lesions smear, a large number of neutrophil leukocytes and necrotic corneal epithelium and stroma were observed. The numurous mycobacteria positive for acid-fast staining were scattered in and out of inflammatory cells. Two cases of corneal button from keratoplasty with HE staining demonstrated that corneal epithelial hyperplasia, sroma degenerate, necrosis and elcosis, neutrophil and lymphocyte infiltration, and neovascularization and fibrous hyperplasia, but no positive mycobacteria by Ziehl-Neelsen acid-fast staining. TEM in the three cases of corneal scrapings showed varied non-tuberculous mycobacteria which were phagocytized by mononuclear macrophage or neutrophils as well as many inclusion compounds included small vacuoles and metachromatic granules in the thalline. TEM in the two cases of corneal buttons both showed slender rod-shaped mycobacteria with several to dozens of finely ground particles with high electron-dense, but no cell wall (L-form) or inclusion compounds.Conclusions In the early stage, non-tuberculous mycobacterial keratitis presents acute purulent inflammatory changes, the advanced stage shows chronic and non-specificity inflammation changes. L-formed Mycobacterium abscessus make the disease protraction.PART V:The diagnosis and treatment of non-tuberculous mycobacterial keratitisObjectives To study the clinical features, diagnosis and treatment of NTMK. Methods It was retrospective case series study. Twelve eyes in 12 patients with NTMK following corneal foreign body trauma in 2007 were studied retrospectively including the case histories, clinical findings, laboratory examinations, diagnosis, treatment and prognosis. The main laboratory examination included corneal scrapings by culturing, polymerase chain reaction (PCR), (confocal microscope, CM) and TEM, corneal lesions by histopathologic examinations and TEM. The patients were given local and systemic antibiotics therapy, lesion cleaning followed by cauterization with tincture of iodine (5%) and/or keratoplasty.Results All cases had a history of corneal trauma,there was corneal metallic foreign body removal at a single hospital in 11 cases, corneal reed trauma in 1 cases. The characteristic signs involved grayish-blue crystalloid keratopathy, multifocal infiltrates, satellites, radiatiform changes in the Descement's membrane. The results of laboratory examinations of the scrapings of the cornea infection were as follows:all cultures (12/12) were positive for rapidly growing mycobacteria, and isolates from 5 patients were all diagnosed as MA. Acid-fast staining revealed positive bacilli in all the 4 patients. Seven of 8 patients were positive for bacterium by PCR. CM showed a large number of leukocytic infiltrates in the epithelium layer and substantia propria layer, deletion, edema and cornea crease, but no bacilus. TEM of all the 3 specimens showed many slender rod-shaped or short coarse-shaped bacteria which were phagocytized by monocytes, and some necrotic tissue. Infections in 10 eyes were resolved by combined treatment regimen including a combination of antimicrobial agents (amikacin, rifampin, gatifloxation, ciprofloxacin, azithromycin and/or ofloxacin,etc.) and local lesion cleaning followed by cauterization with 5% tincture of iodine within 2-5 months; two cases resolved by keratoplasty which poorly responded to antibiotic therapy for 6 months.Conclusions NTMK is a rare, recalcitrant opportunistic infection which can occur in an epidemic fashion following corneal foreign body trauma. The diagnosis of NTMK is difficult, and may easily be misdiagnosed as fungal keratitis. Acid-fast staining, TEM, especially bacterial culture can help to obtain definitive diagnosis. NTMK has a long response period to medical management.The majority of patients can be cured by local and systemic antibiotics therapy, and the recalcitrant infections could be resolved by keratoplasty.