Antimicrobial resistance and AmpC production in ESBL-producing Klebsiella pneumoniae and Klebsiella quasipneumoniae: A retrospective study in Japanese clinical isolates.

INTRODUCTION: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. METHODS: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. RESULTS: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. CONCLUSION: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.

Clinical and microbiological characteristics of Ruminococcus gnavus bacteremia and intra-abdominal infection.

OBJECTIVES: Ruminococcus gnavus is a rare human pathogen, and clinical data on R. gnavus infection are insufficient. This retrospective study aimed to investigate the clinical characteristics of R. gnavus infections. METHODS: This study included 13 cases of bacteremia and three cases of non-bacteremia infections caused by R. gnavus. We evaluated the patient data, infection source, clinical outcomes, and antimicrobial susceptibility of R. gnavus isolates for these cases. RESULTS: The median age of patients was 75 years (range 47-95), and eight patients were female. Twelve cases were presumed to have an intra-abdominal infection source, and the remaining four cases had an unknown infection source. The most common underlying conditions were immunosuppression (seven cases), solid tumors (seven cases), and history of gastrointestinal surgery (five cases). Thirteen patients exhibited gastrointestinal problems (dysfunction, bleeding, intra-abdominal infection, or inflammation). Multiple pathogens were observed in six cases, and fatal outcomes were recorded in three cases. Antimicrobial susceptibility data were available for eight isolates, all of which exhibited low minimum inhibitory concentrations to penicillin (≤0.03 µg/mL), ampicillin-sulbactam (≤0.5 µg/mL), piperacillin-tazobactam (≤4 µg/mL), and metronidazole (≤0.5-1 µg/mL). CONCLUSION: Ruminococcus gnavus is frequently associated with an intra-abdominal infection source, and treatment strategies should consider the possibility of multiple pathogens.

Time to positivity of Corynebacterium in blood culture: Characteristics and diagnostic performance.

The presence of Corynebacterium in blood samples can indicate true bacteremia or contamination, thus complicating the diagnosis of true bacteremia. We aimed to evaluate the usefulness of time to positivity (TTP) in diagnosing true bacteremia and contamination in cases where Corynebacterium was isolated from blood samples. We compared the TTP of the true-bacteremia group (n = 77) with that of the contamination group (n = 88). For the true-bacteremia cases that had only one set of positive blood cultures (n = 14), considering clinical and bacteriological data, additional cultures were performed on blood or other specimens. The same Corynebacterium spp. as in blood were isolated from these specimens. Receiver operating characteristic curves were generated, and the sensitivity and specificity of TTP were calculated for diagnosing true bacteremia. The median TTP of the true-bacteremia group (26.8 h) was shorter than that of the contamination group (43.3 h) (P < 0.0001). When considering TTP ≤ 25.0 h as true bacteremia, the sensitivity and specificity were 44.2% and 95.5%, respectively. Moreover, when considering TTP ≤ 69.4 h as true bacteremia, the sensitivity and specificity were 96.1% and 20.5%, respectively. Among the true-bacteremia groups with one set of positive blood cultures (n = 14), no case exhibited a TTP > 69.4 h. Only three cases showed TTP ≤ 25.0 h in the true-bacteremia group with one set of positive blood cultures. TTP > 69.4 h is likely to indicate contamination and may be useful to exclude true bacteremia in cases with one set of positive blood cultures. Meanwhile, diagnosing true bacteremia using the threshold of TTP 25.0 h would be difficult. Therefore, the clinical and bacteriological data are important for diagnosing bacteremia, especially in cases with TTP ≤ 69.4 h.

Characterization of the Emerging Enteropathogen Escherichia Albertii Isolated from Urine Samples of Patients Attending Sapporo Area Hospitals, Japan.

Recently, Escherichia albertii has been identified as a causative agent of diarrhea in humans and is often misidentified as diarrheagenic Escherichia coli (DEC), a lactose-nondegrading bacterium. In this study, we performed biochemical characterization, gene possession status, drug susceptibility testing, and sequencing analysis of the strains detected in urine samples. One urea-degrading strain was detected in terms of biochemical characteristics, but was found to be nonurea-degrading by another method, leading to conflicting results. All target strains possessed the E. albertii-specific gene, the DEC common gene eae, and the E. coli 16S rRNA gene. In the drug susceptibility test, all urine-derived strains were sensitive to tetracycline (TC), whereas the JCM 17328 strain was resistant to TC, suggesting that TC is effective against urine-derived E. albertii strains. In 16S rRNA sequencing analysis, the E. albertii strains were ranked at the top of homology, but not in the top one, making it difficult to differentiate them from other strains. In summary, if a suspected lactose-nondegrading E. coli strain was isolated from a urine sample, it could be differentiated from E. albertii by the presence of E. albertii-specific genes.

The Effect of Data Augmentation in Deep Learning Approach for Peripheral Blood Leukocyte Recognition.

Data augmentation is reported as a useful technique to generate a large amount of image datasets from a small image dataset. The aim of this study is to clarify the effect of data augmentation for leukocyte recognition with deep learning. We performed three different data augmentation methods (rotation, scaling, and distortion) as pretreatment on the original images. The subjects of clinical assessment were 51 healthy persons. The thin-layer blood smears were prepared from peripheral blood and stained with MG. The effect of data augmentation with rotation was the only significant effective technique in AI model generation for leukocyte recognition. On contrast, the effect of data augmentation with image distortion or image scaling was poor, and accuracy improvement was limited to specific leukocyte categories. Although data augmentation is one effective method for high accuracy in AI training, we consider that a highly effective method should be selected.

Clinical characteristics and antimicrobial susceptibility of Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae isolated from human urine in Japan.

Introduction. The three Klebsiella species K. pneumoniae, K. variicola and K. quasipneumoniae are difficult to distinguish, owing to their similar biochemical properties, and are often confused in medical practice.Gap statement. There is a scarcity of data comparing the clinical characteristics and antimicrobial susceptibility of K. pneumoniae, K. variicola and K. quasipneumoniae. We believe that knowledge of the characteristics of each species will help in their better identification. Further, knowing the antimicrobial susceptibility of the species will help physicians in prescribing an effective treatment course for Klebsiella infections.Aim. This study aimed to determine the clinical characteristics and antimicrobial resistance of K. pneumoniae, K. variicola and K. quasipneumoniae isolated from human urine samples.Methodology. This study included 125 K. pneumoniae strains isolated from human urine samples. Multiplex polymerase chain reaction was performed to identify K. pneumoniae, K. variicola and K. quasipneumoniae. We retrospectively investigated the patient background, complications of bacteraemia, antimicrobial susceptibility and extended-spectrum ß-lactamase (ESBL).Results. We identified 84 (67.2 %), 31 (24.8 %) and 10 strains (8 .0%) of K. pneumoniae, K. variicola and K. quasipneumoniae, respectively. There was no difference in patient background and frequency of bacteraemia complications among these species. K. pneumoniae was significantly less susceptible than K. variicola to ampicillin/sulbactam (P=0.03) and piperacillin (P<0.01). Furthermore, K. pneumoniae (79.8 %) was less susceptible to trimethoprim/sulfamethoxazole than K. variicola (96.8 %) and K. quasipneumoniae (100 %). There were nine ESBL-producing strains (7.2 %), all of which were K. pneumoniae.Conclusion. There was no difference in patient background and frequency of bacteraemia complications between K. pneumoniae, K. variicola and K. quasipneumoniae isolated from urine. The three Klebsiella species showed a varying extent of antimicrobial susceptibility and ESBL production, and accurate identification is needed to understand the epidemiology of these species.

Psychological and Antibacterial Effects of Footbath Using the Lindera umbellata Essential Oil.

Lindera umbellata (Lu) essential oil primarily contains linalool and has relaxation properties. We investigated the psychological and antibacterial effects of footbath with Lu essential oil. The participants included 20 women without medical history and received two intervention plans: footbath without any essential oil and footbath using Lu essential oil. Next, questionnaires regarding impressions and mood states were provided for them to answer. In addition, their autonomic nervous system activity was measured, and the aerobic viable of count on the feet was determined. The high-frequency value reflecting the parasympathetic nervous system activity significantly increased after footbath using Lu essential oil. In the questionnaire about the mood states, the subscale scores of tension-anxiety, depression, fatigue, and confusion after intervention were lower than those before intervention regardless of the use of the essential oil. Conversely, the anger-hostility score decreased only in the group using Lu essential oil. Furthermore, the decrease in aerobic viable count after intervention was not significantly different between the two groups. Footbath using Lu essential oil increased the parasympathetic nervous system activity and relieved anger. Taken together, we suggest that footbath using Lu essential oil has a relaxation effect.

Comparison of Chemical Composition between Kuromoji (Lindera umbellata) Essential Oil and Hydrosol and Determination of the Deodorizing Effect.

Kuromoji (Lindera umbellata) is a tree that grows throughout Japan. The components of kuromoji essential oil have antitumor and aromatherapy effects. However, the composition of the hydrosol, obtained as a by-product of the essential oil process, is unknown. Furthermore, it is unknown whether kuromoji essential oil has a deodorizing effect. Therefore, the purpose of the current study was to compare the chemical composition of kuromoji essential oil and hydrosol, as well as evaluate the deodorizing effect of the former. The chemical composition of samples was evaluated using gas chromatography-mass spectrometry (GC-MS). Additionally, the deodorizing effect of Kuromoji essential oil was investigated with the detector tube method using ammonia, hydrogen sulfide, methyl mercaptan, and isovaleric acid. Linalool was the most abundant component in both the essential oil and hydrosol; however, its proportion was higher in the hydrosol (57.5%) than in the essential oil (42.8%). The hydrosol contained fewer chemical components, but higher proportions of trans-geraniol and ethanol. Moreover, the essential oil eliminated 50% of ammonia and 97.6% or more of isovaleric acid. Interestingly, linalool was soluble in the hydrosol and did not irritate the skin. This suggests that the hydrosol may be an effective foot care product.

A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli.

Escherichia coli that causes diarrhea in humans is referred to as diarrheagenic E. coli (DEC), and has been categorized into the following 5 groups: shigatoxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), and enterotoxigenic E. coli (ETEC). In this study, we developed a novel one-step multiplex polymerase chain reaction (mPCR) for the rapid detection of 10 pathogenic genes (stx1, stx2, eae, bfpA, invE, aggR, esth, estp, elt, and astA) of DEC. Five categorized strains were used as positive controls for DEC harboring each pathogenic gene, and 828 DEC-like strains, isolated from diarrheal stool samples and assumed to be DEC on the basis of serotyping, were used in the mPCR-based detection of the pathogenic genes. To demonstrate the utility of mPCR, the 828 strains were subjected to our optimized protocol, and the results obtained were compared with those obtained by monoplex PCR. The results showed agreement for all strains. Using mPCR, we also detected 65 DEC and 41 astA-positive E. coli, and 7 of these DEC strains were "O antigen untypable" (OUT). This novel mPCR protocol allowed for rapid, convenient, and economical pathogenicity-based identification of the DEC.

Rapid diagnostic method for the detection of diarrheagenic Escherichia coli by multiplex PCR.

Two novel multiplex polymerase chain reaction (mPCR) assays were originally developed for detecting nine targeted virulence-associated genes of five categorized diarrheagenic Escherichia coli (DEC). The mPCR assay 1 included five primer sets (stx1, eaeA, invE, STp gene, and astA), and assay 2 included four primer sets (stx2, aggR, STh gene, and LT gene). The two mPCRs showed 100% specificity in identifying the reference strains without nonspecific bands, and 51 DEC and 38 astA gene-positive E. coli strains from 683 E. coli-like isolates. Our mPCR methods showed high sensitivity and specificity for detecting nine virulence genes of DEC strains. We proved that these methods will contribute to reducing the cost for the reagents of mPCR reported elsewhere and could, therefore, contribute to the diagnosis of DEC in clinical laboratories.

Comparison of mean deviation with AGIS and CIGTS scores in association with structural parameters in glaucomatous eyes.

OBJECTIVE: To evaluate which of the 3 clinically used visual field indices including mean deviation (MD), Advanced Glaucoma Intervention Study (AGIS) score, and Collaborative Initial Glaucoma Treatment Study (CIGTS) score are best in evaluating functional damage of glaucomatous optic neuropathy. METHODS: In 213 glaucomatous eyes, peripapillary retinal nerve fiber layer thickness (RNFLT) and optic disc configuration were measured with Stratus optical coherence tomography and Heidelberg Retina Tomograph-2, respectively. Visual field was measured with standard automated perimetry 30-2. Correlations of the structural parameters compared with the 3 VF indices using second polynomial regression were calculated. In addition, these correlations were analyzed among eyes of 3 different stages of glaucoma, as defined by MD score (early, MD> or =-6 dB; moderate, -12 dB< or =MD<-6 dB; advanced, MD<-12 dB). RESULTS: Among structure-function relationships in all subjects, the highest correlation determination (R) was MD with RNFLT (=0.298). CIGTS score showed better R than MD or AGIS score with rim area, but these values were not higher than any R with RNFLT. In analyses of 3 groups depending on MD, statistically significant structure-function correlations were observed only in patients with an advanced stage. CONCLUSIONS: No clear difference was found among MD and AGIS/CIGTS scores in expressing functional damage of glaucomatous eyes. MD is suggested to be no worse than others in monitoring glaucoma in clinical setting.

Intraocular pressure lowering effect of once daily versus once weekly latanoprost instillation in the same normal individuals.

This study tested whether once weekly instillation of latanoprost exerts the similar IOP lowering effect as once daily instillation in the same individuals. Latanoprost was administered on right eyes of eight healthy male volunteers once daily first for 24 days, which was followed by the 31-day washout and the subsequent once weekly instillation for another 24 days. The mean baseline IOP was 12.06 +/- 1.50 (range, 10.0 to 14.0) mmHg, whereas the mean IOP during once daily treatment was 9.87 +/- 1.71 mmHg, which was significantly lower than the former (p=0.025). The mean washout IOP was 12.56 +/- 2.16 mmHg, which was similar to the baseline IOP. The mean IOP during once weekly instillation of latanoprost was 11.34 +/- 1.51 mmHg, which was not statistically different from the washout IOP. Four of the 8 subjects showed 15% or more reduction in IOP both during once daily and once weekly instillations, two of whom were overlapped. Since the magnitude of the IOP reduction with once daily use was higher than that with once weekly use even in the responders, the current protocol of once daily instillation should be respected.

Long-term results of amniotic membrane transplantation-assisted bleb revision for leaking blebs.

BACKGROUND: To report long-term results of amniotic membrane transplantation (AMT)-assisted bleb revision for intractable late-onset bleb leak. METHODS: Retrospective medical record review of six consecutive patients who underwent AMT-assisted bleb revision for late-onset bleb leak at the Kobe University hospital between December 2001 and March 2004. RESULTS: The median (range) of age was 60 (20-77) years. All patients had a history of trabeculectomy with adjunctive use of 0.4 mg/ml mitomycin C. The median (range) of interval between the prior trabeculectomy and bleb leak was 15 (4-54) months. Preoperative intraocular pressure (IOP) was lower than 10 mmHg in two cases, while it was 25 mmHg or higher in three patients, who had a localized, ischemic, thin-wall leaking bleb circumscribed with extensively scarred tissue. The median (range) of follow-up after the AMT-assisted bleb revision was 49 (41-67) months. Postoperative IOP was well controlled in all patients without medication or with a maximum of three glaucoma medications. No patients presented recurrence of the bleb leak or AMT-related complications during entire follow-up. All patients showed at final visit a diffuse bleb which extended posteriorly beyond the conjunctival incision line. CONCLUSION: AMT-assisted bleb revision successfully treated intractable late-onset bleb leak. Further comparative studies are needed to confirm the present result.

Comparison of Pentacam Scheimpflug camera with ultrasound pachymetry and noncontact specular microscopy in measuring central corneal thickness.

PURPOSE: Although central corneal thickness (CCT) can be measured by several methods, interchangeability of different modalities has not been fully investigated. CCT is known to correlate with intraocular pressure (IOP). The aim of this study was to evaluate the agreement of Pentacam Scheimpflug system with noncontact specular microscopy (NCSM) and ultrasound (US) pachymetry in measuring CCT and the relation between IOP taken with Goldmann applanation tonometer (GAT) and the CCT measured with these three methods. METHODS: The right eyes of 135 enrolled persons without antiglaucoma drug use (100 females and 35 males), who comprised 32 patients with primary open-angle glaucoma, 14 with ocular hypertension, 45 with primary angle-closure glaucoma, and 44 controls, were studied. Intermethod comparison of CCT was made by the 95% limits of agreement analysis according to Bland and Altman. Linear regression analysis was used to assess the relationship between IOP and CCT taken with each modality. RESULTS: The mean CCT (+/-SD) taken with Scheimpflug, US, and NCSM was 559.49 +/- 38.44 microm, 553.01 +/- 39.33 microm, and 552.04 +/- 42.95 microm, respectively. The average values of CCT taken with the three instruments were not significantly different (one-factor ANOVA; p = 0.26), although the marginal mean difference between Scheimpflug and US or NCSM was statistically significant (paired t test; p = 0.0009 and 0.005, respectively). The 95% limits of agreement were 6.47 +/- 43.21 microm between Scheimpflug and US, 7.45 +/- 58.86 microm between Scheimpflug and NCSM, and 0.98 +/- 51.69 microm between US and NCSM. There was a positive association between IOP and CCT measured with US or NCSM, whereas there was no correlation between IOP and CCT measured with Scheimpflug. CONCLUSIONS: Although CCT values measured with Scheimpflug, US, and NCSM are closely similar, clinicians should keep in mind that these methods are not simply interchangeable.

Quantification of retinal nerve fiber layer thickness reduction associated with a relative afferent pupillary defect in asymmetric glaucoma.

AIM: The relative afferent pupillary defect (RAPD) is an important clinical sign of asymmetrical retinal ganglion cell and axonal damage. Although glaucoma essentially affects bilateral eyes, a subset of patients manifests asymmetrical glaucomatous optic neuropathy (GON), which exhibits an RPAD in the more advanced eyes. However, the degree to which axonal loss occurs before an RAPD is clinically detectable has not been substantiated. The purpose of this study is to assess the relationship between the depth of a clinically detectable RAPD and the reduction ratio of retinal nerve fiber layer (RNFL) thickness in the more advanced eyes relative to that in the contralateral less advanced eyes of patients with asymmetrical GON. METHODS: Enrolled were 29 consecutive glaucoma patients with the clinically detectable RAPD. An RAPD was quantified by placing log-scaled neutral density filters over the less advanced eyes while performing the swinging flashlight test. Average RNFL thickness was determined using the Fast RNFL thickness programme of optical coherence tomography 3000. Correlation coefficient and Linear regression analyses were used in assessing the relationship between the RAPD and the ratio of RNFL thickness in the more advanced eyes relative to that in the less advanced. RESULTS: RAPD ranged from 0.6 to 2.4 log units. The log-scaled RAPD had a statistically significantly inversed correlation with the average RNFL thickness ratio (r(s) = -0.729, p<0.0001). Linear regression analysis found an equation that the average RNFL thickness ratio in the more affected eyes relative to that in the less advanced (%) = (0.827-0.169xRAPD (log units))x100 (R(2) = 0.557, p<0.0001). CONCLUSIONS: When an RAPD is clinically detected, the RNFL thickness in the more advanced eyes was in average reduced to about 73% of that in the less advanced.

Agreement of rebound tonometer in measuring intraocular pressure with three types of applanation tonometers.

PURPOSE: To evaluate the agreement of iCare rebound tonometer in measuring intraocular pressure (IOP) with Goldmann applanation tonometer (GAT), Tonopen XL, and noncontact tonometer, and the influence of the central corneal thickness (CCT) on IOP measurements made with these four tonometers in 45 (12 control and 33 glaucomatous or ocular hypertensive) eyes. DESIGN: Clinically relevant experimental study. METHODS: Tonometer intermethod agreement was assessed by the Bland-Altman method. The relations of CCT with absolute IOP values and intertonometer differences were analyzed by linear regression. RESULTS: The mean differences (95% limits of agreement) in IOP readings between iCare and GAT, Tonopen XL, and noncontact tonometer were 1.40 +/- 4.29, 0.00 +/- 4.78, and 2.22 +/- 4.19 mm Hg, respectively. All tonometries had a marked association with CCT. As the CCT got thicker, iCare considerably overestimated GAT and Tonopen XL. CONCLUSIONS: Although influenced by CCT, iCare agrees well with applanation tonometers.

[Correlations among GDx-variable corneal compension, optical coherence tomography, and Heidelberg retina tomograph and relationships between these structural parameters and visual field indices].

PURPOSE: In glaucoma patients, to compare the correlations among parameters measured by Heidelberg Retina Tomography(HRT) and retinal nerve fiber layer thickness (RNFLT) measured by GDx-variable corneal compensation (GDx-VCC) and optical coherence tomography (OCT), and to assess the relationship among these structural parameters, and visual field damage. METHODS: A unilateral eye of 110 patients with open-angle glaucoma were included in this study. Each individual was analyzed by HRT (version 3.04), GDx-VCC (version 5.3.2), and OCT-1 (version A6 X1). The correlations among these parameters regarding global area, superior sector, and inferior sector were obtained from the 3 instruments and compared. The correlations among the mean deviation (MD) in the Humphrey field analysis and the parameters in global area from the 3 instruments were calculated. RESULTS: The MD showed quadratic regressions to each global parameter (GDx-VCC; 0.625 (nerve fiber indicator): OCT 0.616 (average RNFLT); HRT, 0.501 (rim area)). Most parameters in OCT and GDx-VCC had highly significant correlations. The rim area showed the highest correlation in each sector to RNFLT with GDx-VCC or OCT among HRT parameters. CONCLUSIONS: These structural parameters derived from GDx-VCC, OCT and HRT analyses had bilinear correlation to the MD. Retinal nerve fiber loss detected on GDx-VCC and OCT correlated linearly with the neuroretinal rim area measured by HRT. These instruments may be useful in monitoring structural changes in glaucoma.

[A case of conjunctival malignant melanoma treated with subconjunctival injection of interferon beta--efficacy and side effects].

BACKGROUND: The management of conjunctival malignant melanoma remains controversial. Interferon-beta (IFN-beta) is a well-known antineoplastic agent against cutaneous malignant melanoma. CASE: A 44-year-old man was referred to Kobe University, Hospital for treatment of pigmented lesions in the corneal limbus of his right eye, first recognized in 2000 and growing gradually. FINDINGS: There were two pigmentary neoplastic lesions in the conjunctiva of his right eye, one at 9 o'clock in the limbus and the other at the inferior bulbar conjunctiva close to the fornix. Primary acquired melanosis (PAM) extended widely over the bulbar conjunctiva and the corneal surface. These findings led to the clinical diagnosis of conjunctival malignant melanoma. Systemic work-up did not detect any other neoplastic lesion. COURSE: The melanotic lesions were resected and histopathologically malignant melanoma was diagnosed. Microscopically, the tumor cells were present at the surgical margin. Melanoma recurred a half-year later at 3 o'clock in the limbus of the right eye. IFN-beta (3 million units/) was injected subconjunctivally 22 times. Side effects observed were as follows: corneal epithelial erosion, increase of the corneal thickness, lid swelling, conjunctival congestion, subconjunctival hemorrhage, and liver dysfunction. These findings were transient. Melanotic lesions, including PAM, diminished 6 months after the end of treatment. CONCLUSION: This therapeutic trial of local therapy using the subconjunctival administration of IFN-beta demonstrated excellent efficacy for the treatment of conjunctival malignant melanoma. Local and systemic side effects were seen, though transient, suggesting the necessity of long-term follow-up study.