A. castellanii and P. aeruginosa mutually exacerbate damage to corneal cells during coinfection.

IMPORTANCE: At the National Cheng Kung University Hospital, numerous cases of amoebic keratitis had been identified with concurrent bacterial infections. Among these bacterial coinfections, Pseudomonas aeruginosa accounted for 50% of the reported cases. However, the impact of pathogenic bacteria on amoeba-induced corneal damage remains unclear. In our study, we successfully demonstrated that P. aeruginosa accumulated on the Acanthamoeba castellanii surface and caused more severe corneal damage. We also indicated that the exposure of P. aeruginosa to amoeba-soluble antigens enhanced its adhesion ability, promoted biofilm formation, and led to more severe corneal cell damage. These findings significantly contributed to our understanding of the risk associated with P. aeruginosa coinfection in the progression of amoeba keratitis.

Impact of cornea recovery training and certification on ophthalmology residents.

Background: In Asian countries, such as Taiwan, social taboos regarding organ and tissue donation decreases the prevalence of organ and tissue transplants. This also applies to cornea recovery, which is a skill that requires precision and practice to perform well. In Taiwan, to ensure the maintenance of high-quality corneas, a comprehensive training program and certified examination has been implemented. This study aims to investigate the impact of these programs and examinations on cornea recovery. Methods: Researchers evaluated the efficiency of the training and certified examination process by comparing the corneoscleral rim width, Descemet's membrane folds, endothelial layer stress lines, and endothelial cell density performed by ophthalmology residents in 2018 and 2019. Results: After training and certification, the Descemet's membrane folds rate decreased from 14.3 % to 2.0 % and endothelial layer stress lines rate decreased from 22.5 % to 5.0 %. The endothelial cell density of donor grafts significantly improved from 2681.9 cells/mm2 to 2869.7 cells/mm2 (p < 0.001). Conclusions: This study used objective data to evaluate cornea recovery quality after training and certification. The training and certified examination significantly improved the surgical skills of ophthalmology residents and could be applied in other tissue or organ recovery procedures to maintain and improve quality.

Inhibitory effect of host ocular microenvironmental factors on chlorhexidine digluconate activity.

Acanthamoeba spp. are free-living protozoan that cause a serious human eye disease called Acanthamoeba keratitis (AK). Several new and effective medical therapy for AK patients remains highly debated and therefore, CHG is still considered one of the first lines of treatment for AK patients. We hypothesized that ocular microenvironmental factors are responsible for Acanthamoeba drug resistance and clinical AK treatment failure. To investigate the influence of the ocular surface on CHG treatment, we tested the effect of several ocular elements on the anti-amoeba activity of CHG. The suspected inhibitory elements, including mucin, albumin, human and amoeba cell lysates, live and heat-killed bacteria, and cornea, were added to the amoebicidal activity platform, where amoeba was incubated with CHG at varying concentrations. Mucin showed a significant inhibitory effect on CHG activity against Acanthamoeba castellanii In contrast, albumin did not affect CHG treatment. Furthermore, human and amoeba cell lysates as well as live and heat-killed bacterial suspensions also significantly inhibited CHG activity. Additionally, we found that pig corneas also reduced CHG activity. In contrast, dry eye drops and their major component, propylene glycol, which is commonly used as eyewash material, did not have an impact on CHG activity. Our results demonstrate the effect of ocular microenvironmental factors on CHG activity and suggest that these factors may play a role in the development of amoeba resistance to CHG and treatment failure.

Comparison of Microbial Sampling Sites and Donor-Related Factors on Corneal Graft Contamination.

Grafts used for corneal donation should be sterile to avoid transplantation failure and secondary infection. However, there are no clear and globally accepted specifications from eye banks on microbial sampling sites. The objective of this study was to analyze microbial contamination of corneal grafts collected from different sampling sites. We found that the contamination rates and strain compositions significantly differed at different sampling sites. To clarify the effect of the microbial sampling site on corneal graft contamination, microbial sampling was conducted using 30 corneal grafts at the extraocular and intraocular sides of the graft in 2020 from the National Eye Bank of Taiwan. Microbial contamination significantly differed (p < 0.05) between the different sampling sites on the graft according to McNemar's test. Although the two sampling sites showed the same specificity (33.33%), the sensitivity of sampling on the extraocular side (82.35%) was higher than that on the intraocular side (17.65%) of the graft. Donor-associated factors, including the cause of death, operating place, and cold compression, were analyzed using chi-square statistics, which revealed no significant differences in microbial contamination. Thus, our data provide evidence for the microbial sampling site of donated grafts and clear specifications for maintaining the quality of corneal grafts.

Association between sodium glucose co-transporter 2 inhibitors and incident glaucoma in patients with type 2 diabetes: A multi-institutional cohort study in Taiwan.

PURPOSE: Type 2 diabetes (T2D) is an important risk factor for glaucoma, and sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to protect the optic nerves. We therefore aimed to evaluate the association between SGLT2 inhibitors and incident glaucoma. METHODS: This retrospective cohort study analyzed the largest multi-institutional electronic medical records database in Taiwan, containing data of over a million individuals. We included T2D patients newly prescribed SGLT2 inhibitors or glucagon-like peptide-1 receptor agonists (GLP-1 RAs) from 2016 to 2018. Our primary outcome was incident glaucoma diagnosis between initiation of SGLT2 inhibitors or GLP-1 RAs, and 31st March 2021. After applying inverse probability of treatment weighting (IPTW) to increase homogeneity between the two treatment groups, we estimated hazard ratios (HR) with 95% confidence intervals (CI) for the risk of glaucoma, based on Cox proportional hazards regression models. RESULTS: We included 9,927 and 1,065 T2D patients who had been newly prescribed SGLT2 inhibitors or GLP-1 RAs, respectively. Lower risk of incident glaucoma was observed in patients receiving SGLT2 inhibitors (7.9 events per 1,000 person-years), compared to those receiving GLP-1 RAs (10.0 events per 1,000 person-years), with an HR of 0.81 (95% CI: 0.69-0.95). Multiple sensitivity analyses and a negative control outcome analysis confirmed the robustness of our main findings. CONCLUSION: This study suggests that T2D patients newly prescribed SGLT2 inhibitors have a reduced risk of incident glaucoma, compared to those prescribed GLP-1 RAs, in clinical practice. Future prospective studies are suggested to confirm this association.

Cytochrome P450 monooxygenase of Acanthamoeba castellanii participates in resistance to polyhexamethylene biguanide treatment.

Acanthamoeba spp. are free-living parasites that can cause severe infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK). Polyhexamethylene biguanide (PHMB) is a topical application for AK treatment. However, PHMB is not entirely effective against all Acanthamoeba strains or isolates. The mechanisms by which Acanthamoeba protects itself against extreme drug conditions without encystation are still unknown. According to a previous study, cytochrome P450 monooxygenase (CYP450MO) plays an important role in the oxidative biotransformation of numerous drugs related to metabolism. In this study, a CYP450MO fragment was inserted into the pGAPDH-EGFP vector and transfected into Acanthamoeba castellanii. We found that CYP450MO-overexpressing Acanthamoeba had higher survival rates than those of the control cells after PHMB treatment. Moreover, we also found that encystation-related genes such as cellulose synthase I (CSI), encystation-mediating serine proteinase (EMSP), and autophagy-related protein 8 (ATG8) expression levels were not significantly different between Acanthamoeba transfected by pGAPDH-EGFP or pGAPDH-EGFP-CYP450MO. We suggest that Acanthamoeba transfected by pGAPDH-EGFP-CYP450MO may not induce encystation-related genes to resist PHMB treatment. In conclusion, these findings indicate that CYP450MO may be an additional target when PHMB is used for treatment of amoebic keratitis.

TITLE: La monooxygénase du cytochrome P450 d'Acanthamoeba castellanii participe à la résistance au traitement par le polyhexaméthylène biguanide. ABSTRACT: Les Acanthamoeba spp. sont des parasites libres qui peuvent provoquer des infections graves telles que l'encéphalite amibienne granulomateuse (EAG) et la kératite amibienne (KA). Le polyhexaméthylène biguanide (PHMB) est une application topique pour le traitement de la KA. Cependant, le PHMB n'est pas entièrement efficace contre toutes les souches ou isolats d'Acanthamoeba. Les mécanismes par lesquels Acanthamoeba se protège contre des conditions médicamenteuses extrêmes sans enkystation sont encore inconnus. Selon une étude précédente, la monooxygénase du cytochrome P450 (CYP450MO) joue un rôle important dans la biotransformation oxydative de nombreux médicaments liés au métabolisme. Dans cette étude, un fragment CYP450MO a été inséré dans le vecteur pGAPDH-EGFP et transfecté dans Acanthamoeba castellanii. Nous avons constaté que les Acanthamoeba surexprimant le CYP450MO avaient des taux de survie plus élevés que ceux des cellules témoins après un traitement au PHMB. De plus, nous avons également constaté que les gènes liés aux enkystations tels que la cellulose synthase I (CSI), la sérine protéinase médiatrice de l'enkystation (EMSP) et les niveaux d'expression de la protéine 8 liée à l'autophagie (ATG8) n'étaient pas significativement différents entre les Acanthamoeba transfectés par pGAPDH-EGFP ou par pGAPDH-EGFP-CYP450MO. Nous suggérons que les Acanthamoeba transfectés par pGAPDH-EGFP-CYP450MO ne peuvent pas induire les gènes liés à l'enkystation pour résister au traitement PHMB. En conclusion, ces résultats peuvent indiquer que la monooxygénase du cytochrome P450 peut être une cible potentielle pour le traitement par PHMB de la kératite amibienne.

Intracellular Microbiome Profiling of the Acanthamoeba Clinical Isolates from Lens Associated Keratitis.

Acanthamoeba act as hosts for various microorganisms and pathogens, causing Acanthamoeba Keratitis (AK). To investigate the association between endosymbionts and AK progression, we performed a metagenomics study to characterize the intracellular microbiome from five lenses associated with AK isolates and standard strains to characterize the role of ocular flora in AK progression. The used clinical isolates were axenic cultured from lenses associated with AK patients. AK isolates and standard controls such as 16S ribosomal RNA sequencing techniques were used for analysis. The microbiome compositions and relative abundance values were compared. The orders of Clostridiales and Bacteroidales presented major populations of intracellular microbes belonging to all isolates. Comparison of the different source isolates showed that most of the abundance in keratitis isolates came from Ruminococcus gnavus (121.0 folds), Eubacterium dolichum (54.15 folds), Roseburia faecis (24.51 folds), and Blautia producta (3.15 folds). Further analysis of the relative abundance data from keratitis isolates showed that Blautia producta was positively correlated with the disease course. In contrast, Bacteroides ovatus was found to be abundant in early-stage keratitis isolates. This study reveals the abundant anaerobic Gram-positive rods present in severe keratitis isolate and characterize the association between Acanthamoeba and ocular flora in AK progression.

Risk of Glaucoma in Patients Receiving Hemodialysis and Peritoneal Dialysis: A Nationwide Population-Based Cohort Study.

This paper investigated the incidence and risk of newly diagnosed glaucoma after the initiation of maintenance dialysis in Taiwan. A case-control study was conducted using the National Health Insurance Research Database (NHIRD) in Taiwan. There were 3949 patients with dialysis in the study group and 78,980 non-dialysis subjects matched by age and sex in the comparison group. The incidence of newly diagnosed glaucoma after the initiation of maintenance dialysis was analyzed based on the diagnostic code for glaucoma. Patients with dialysis had a higher risk of glaucoma (adjusted hazard ratio (aHR): 1.270; 95% confidence interval (CI): 1.035-1.560) than patients without dialysis. The incidence rate of glaucoma was 8.18 per 10,000 person months in the dialysis group, which was higher than that in the non-dialysis group (5.01 per 10,000 person months). Patients with dialysis exhibited a significantly higher risk of angle-closure glaucoma (ACG) (aHR: 1.550; 95% CI: 1.074-2.239). In contrast, there was no significant risk of developing open-angle glaucoma or normal-tension glaucoma in dialysis patients. Our data suggest that dialysis patients are more susceptible to ACG. Regular ophthalmic examinations may be useful in patients with dialysis to identify high-risk individuals with glaucoma, and preventive measures can be applied to avoid permanent vision loss as soon as intraocular pressure (IOP) elevation is identified.

Trend of Neovascular Glaucoma in Taiwan: A 15-year Nationwide Population-based Cohort Study.

PURPOSE: To determine the trends in epidemiological data in patients with neovascular glaucoma (NVG) in Taiwan. METHODS: The data were obtained from the 2016 version of the Longitudinal Health Insurance Database, which contains data on two million randomly sampled patients. Patients with NVG diagnostic codes were enrolled in this study, after which we separated the study population according to different time periods. The demographic data, systemic and ocular comorbidities and treatments that were applied to manage NVG were collected from the database. RESULTS: The overall age-standardized prevalence was 52.48 per 100,000 individuals, and the age-standardized incidence was 4.33 per 100,000 person-years in patients with NVG. In general, men had a higher prevalence and incidence, and the incidence was observed to fluctuate. The patients with the highest educational levels accounted for less than 5% of the NVG patients, and the patients with the highest income levels accounted for less than 15% of the NVG patients. Systemic comorbidities in NVG patients, especially metabolic syndrome, were observed to increase. The percentage of the patients receiving anti-vascular endothelial growth factor treatments increased by more than two-fold after 2008, whereas reductions in trabeculectomy and cyclodestruction procedures were observed. CONCLUSION: The prevalence of NVG was observed to increase in men, and the incidence fluctuated during the study period. Furthermore, the systemic comorbidities, and the use of anti-vascular endothelial growth factor treatment increased; the latter may be associated with a decrease in the use of incisional glaucoma surgery for NVG in recent years.

Microsporidial stromal keratitis: characterisation of clinical features, ultrastructural study by electron microscopy and efficacy of different surgical modalities.

AIMS: To report the clinical manifestations, ultrastructure and evaluate the efficacy of therapeutic lamellar keratectomy (TLK) and penetrating keratoplasty (PK) for microsporidial stromal keratitis (MSK). METHODS: Fourteen MSK cases between 2009 and 2018 were recruited. Each patient's clinical presentation, light microscopy, histopathology, PCR and electron microscopy (EM) of corneal samples were reviewed. RESULTS: The patients were 70.0±4.7 years old (average follow-up, 4.5 years). Time from symptoms to presentation was 10.6±13.0 weeks. The corneal manifestations were highly variable. Corneal scrapings revealed Gram stain positivity in 12 cases (85.7%) and modified Ziehl-Neelsen stain positivity in 9 (64.3%). Histopathology revealed spores in all specimens, while sequencing of small subunit rRNA-based PCR products identified Vittaforma corneae in 82% of patients. EM demonstrated various forms of microsporidial sporoplasm in corneal keratocytes. All patients were treated with topical antimicrobial agents or combined with oral antiparasitic medications for >3 weeks. As all patients were refractory to medical therapy, they ultimately underwent surgical intervention (TLK in 7, PK in 6 and 1 received TLK first, followed by PK). Postoperatively, the infection was resolved in 78.6% of the patients. Nevertheless, a high recurrence rate (21.4%) was noted during 3-year follow-up, with only two patients retained a final visual acuity ≥20/100. CONCLUSION: MSK usually presents with a non-specific corneal infiltration refractory to antimicrobial therapy. The diagnosis relies on light microscopic examinations on corneal scrapings and histopathological analyses. Surgical intervention is warranted by limiting the infection; however, it was associated with an overall poor outcome.

Postoperative Mycobacteroides abscessus subsp. abscessus endophthalmitis: Clinical analysis of 12 clustered adults and a proposed therapeutic algorithm.

PURPOSE: Postoperative endophthalmitis caused by nontuberculous mycobacterium is a rare but devastating complication after intraocular surgery. However, optimal treatment strategies remain undetermined in view of its rarity. METHODS: We investigated the cases of culture-proven postoperative Mycobacteroides abscessus subsp. abscessus endophthalmitis in southern Taiwan, focusing on clinical manifestations and microbiological study, and aimed to describe clinical staging and to propose a therapeutic modality for this disease. RESULTS: Twelve cases, including two published cases, were treated in two medical centers in southern Taiwan between Aug. 2011 and Dec. 2016, and all ever received cataract surgery at one clinic. Their disease courses could be categorized into four distinct stages, i.e., the initial, quiescent, recurrent, and end stage, and some cases experienced 1-4 cycles of quiescent-recurrent stages. Although all eyes ended up with phthisis or were eviscerated, the affected eyes receiving pars plana vitrectomy (PPV) tended to become quiescent and survived longer than those without PPV (adjusted hazard ratio: 13.9, p < 0.05). Eight isolates of eight patients were available for microbiological study. All isolates were susceptible to amikacin, and inducible clarithromycin resistance was observed in 100% of isolates. CONCLUSION: Despite the preservation of vision in postoperative M.abscessus endophthalmitis remained a challenge, a stage-based approach is proposed, which may facilitate decision-makings for the future study.

Characterisation of the ß-lactam resistance enzyme in Acanthamoeba castellanii.

ß-Lactams are well known as the best antibiotics for inhibiting the cross-linking between adjacent polysaccharide chains and peptides in the peptidoglycan layer of bacterial cell walls, causing bacterial cell lysis. There are no reports on the action of and resistance mechanisms to ß-lactams in protozoa. Acanthamoeba castellanii is a free-living protozoan pathogen capable of causing blinding keratitis and fatal granulomatous encephalitis. When Acanthamoeba is exposed to harsh conditions, it differentiates into the cyst stage to avoid environmental stresses, such as drug treatment. In this study, it was shown that the mature encystation rate of A. castellanii is decreased by treatment with cefotaxime (CTX) and clavulanic acid (CLA); however, the drugs do not kill the amoeba. We hypothesise that ß-lactam antibiotics may disturb synthesis of the double cell wall during the encystation process of Acanthamoeba. Interestingly, CTX is considered a powerful ß-lactam, whereas CLA is considered a weak ß-lactam but an efficient ß-lactamase inhibitor. It was demonstrated that Acanthamoeba expresses ß-lactamases to prevent inhibition of the encystation process by ß-lactams. To reveal the functions of Acanthamoeba ß-lactamases, a recombinant Acanthamoeba ß-lactamase was produced in Escherichia coli that conferred resistance to ß-lactams such as CTX, cefuroxime, penicillin and meropenem. Consequently, we suggest that Acanthamoeba produces enzymes similar to ß-lactamases to avoid interference from the environment. Here we provide a new point of view on an important gene responsible for drug resistance and advocate for the development of more efficient treatment against Acanthamoeba infection.

Ocular characteristics in a variant microcephalic primordial dwarfism type II.

BACKGROUND: Microcephalic osteodysplastic primordial dwarfism, type II (MOPD II) is a rare disease that is assumed to be caused by a pericentrin (PCNT) gene mutation. Clinical manifestations have been reported in pediatrics and neurology; however, only a few ocular findings have been documented. CASE PRESENTATION: We present three unrelated cases of MOPD II with similar facial features and short stature. Unlike the cases described in the literature, all subjects had normal birth weight and height but their growth was retarded thereafter. In addition to delayed milestones, they have a broad forehead, maxillary protrusion, long peaked nose, high nasal bridge, low-set large ears, extreme reromicrogenia, and normal-sized teeth. These three patients had similar ocular manifestations with the short axial length associated with high hyperopia more than + 9 diopters (D) and macular scarring. The oldest subject was a 20 year-old male without neurological symptoms. One female subject had developed alopecia during the previous 2 years. The other female subject had moyamoya disease, but a genetic study revealed a normal PCNT gene. CONCLUSION: This is the first report of MOPD II focusing on ocular findings, suggesting that macular dystrophy and high hyperopia are the common ocular characteristics of MOPD II. Prompt referral to an ophthalmologist is essential. Although refractive amblyopia can be treated with optical correction, visual prognosis may be poor due to maculopathy.

Identification and characterization of a secreted M28 aminopeptidase protein in Acanthamoeba.

Acanthamoeba is a free-living pathogenic protozoan that is distributed in different environmental reservoirs, including lakes and soil. Pathogenic Acanthamoeba can cause severe human diseases, such as blinding keratitis and granulomatous encephalitis. Therefore, it is important to understand the pathogenic relationship between humans and Acanthamoeba. By comparison of systemic analysis results for Acanthamoeba isolates, we identified a novel secreted protein of Acanthamoeba, an M28 aminopeptidase (M28AP), which targets of the human innate immune defense. We investigated the molecular functions and characteristics of the M28AP protein by anti-M28 antibodies and a M28AP mutant strain generated by the CRISPR/Cas9 system. Human complement proteins such as C3b and iC3b were degraded by Acanthamoeba M28AP. We believe that M28AP is an important factor in human innate immunity. This study provides new insight for the development of more efficient medicines to treat Acanthamoeba infection.

Author Correction: Major ocular trauma in Taiwan: 2002-2004 versus 2012-2014.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Effect of ethanol pretreatment in Acanthamoeba keratitis: a long-term follow-up study.

PURPOSE: The aim of this study was to evaluate the long-term outcomes of ethanol pretreatment in Acanthamoeba keratitis (AK). PATIENTS AND METHODS: This single-center, retrospective, interventional study included 22 patients (24 eyes) who developed AK and underwent ethanol pretreatment between 2009 and 2015. Samples for smears, polymerase chain reaction, and culture for evidence of Acanthamoeba were collected. After ethanol pretreatment, the patients were treated with corneal epithelial debridement, topical 0.02% polyhexamethylene biguanide, and 0.1% propamidine isethionate. The primary outcomes were a clinically stable ocular surface, complete recovery from corneal infection, and acceptable corneal haze. The secondary outcome measure was improvement in best-corrected visual acuity. Complications and predictors of the visual outcome were also recorded. RESULTS: Ethanol pretreatment was successful in 20 (83.3%) of the 24 eyes, and no further optical keratoplasty was required. Four eyes required rescue therapeutic keratoplasty because of rapid progression of AK. Patients in whom ethanol pretreatment was successful achieved good final visual outcomes regardless of sex, age, or causative Acanthamoeba species. Patients with worse initial best-corrected visual acuity and rigid gas permeable lens-related AK had better improvement in vision. CONCLUSION: Ethanol as a pretreatment for AK is safe and effective. Combined with corneal epithelial debridement, ethanol pretreatment may preclude the need for optical and therapeutic keratoplasty. This technique is suitable for all stages of AK presenting within 3 weeks of symptom onset and achieves favorable results especially in early AK.

Major ocular trauma in Taiwan: 2002-2004 versus 2012-2014.

We investigated the temporal changes in major eye injuries in Taiwan by reviewing the medical records of all patients with ocular trauma hospitalized at the National Cheng Kung University Hospital during 2002-2004 and 2012-2014. A total of 169 eyes (161 patients) during 2002-2004 and 121 eyes (120 patients) during 2012-2014 were enrolled (mean ± SD age: 41.9 ± 20.8 years in 2002-2004, and 51.8 ± 19.3 years in 2012-2014). Males accounted for ~75% of patients. The most frequent injury-causing object was metallic material (~24%), and blunt traumas were most frequently attributable to traffic accidents and falls. The most common locations of injuries for males and females were the workplace and home, respectively. Open-globe injuries occurred in ~70% of eyes, requiring primary repair, cataract extraction, and/or intraocular lens implantation. The frequencies of fall injury, lacrimal system laceration, lens injury, corneal/scleral foreign bodies, and use of intracameral antibiotics increased from 2002-2004 to 2012-2014, while those of closed-globe injury, vitreous haemorrhage, optic nerve injury, and medical treatment decreased. The final visual acuity remained poor (≤20/200) in >1/3 of injured eyes. Despite therapeutic advancements, major eye injuries still pose a high risk for poor visual outcome.

A DNA dot hybridization model for molecular diagnosis of parasitic keratitis.

PURPOSE: Developing a DNA dot hybridization model for diagnosing parasitic keratitis. METHODS: Newly designed oligonucleotide probes for detecting Acanthamoeba and microsporidia were tested with target reference strains of Acanthamoeba (n = 20) and microsporidia (n = 3), and non-target microorganisms, including bacteria (n = 20) and fungi (n = 20). These probes, which had passed the preliminary tests, were then assembled as a parasite dot hybridization (PDH) model for assessing 33 clinical samples from patients with clinically suspected Acanthamoeba and microsporidia keratitis, including eight positives for Acanthamoeba, 13 positives for microsporidia, and 12 negatives for both pathogens. RESULTS: Two probes for detecting Acanthamoeba and two for detecting microsporidia passed the tests using target and non-target strains and then were assembled in the PDH model. For clinical samples, one Acanthamoeba-positive sample (proved with pathology) was falsely negative according to the PDH assay. The sensitivity and specificity of the PDH assay for diagnosing Acanthamoeba keratitis were 87.5% and 100%, respectively, while the sensitivity and specificity for diagnosing microsporidia keratitis were 100%. The infectious agent of all clinical samples of microsporidia keratitis was identified as Vittaforma corneae with DNA sequencing, while those of Acanthamoeba keratitis were caused by four species of Acanthamoeba, with Acanthamoeba castellanii found in four samples (50%, 4/8). CONCLUSIONS: The PDH model has the potential to be a molecular assay for diagnosing Acanthamoeba and microsporidia keratitis. However, a prospective clinical study might be needed before the model is adopted in routine clinical practice.

Characterizing clinical isolates of Acanthamoeba castellanii with high resistance to polyhexamethylene biguanide in Taiwan.

BACKGROUND/PURPOSE: Acanthamoeba keratitis (AK), a painful infectious corneal disease, is caused by the free-living pathogenic species Acanthamoeba. The symptoms include corneal infiltrate, epithelial, and stromal destruction, and loss of vision. Current treatment generally involves an hourly application of polyhexamethylene biguanide (PHMB) over a period of several days; however, even this is not entirely effective against all strains/isolates. The aims of this study were to confirm the existence of pathogenic strains in Taiwan which are highly resistant to drugs and to characterize the behavior of these strains. METHODS: An in vitro Acanthamoeba species culture platform was established to observe the effectiveness of treatment and chart the morphological changes that occur under the effects of drugs using a light microscope and time-lapse recording. Changes in gene expression were examined using reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS: Over 90% of the standard strain cells (ATCC 30010) were lysed after being treated with PHMB for 1 hour; however, clinical isolates of Acanthamoeba castellanii that differed in their susceptibility to the treatment drug were only partly lysed. Following treatment with PHMB, National Cheng Kung University Hospital isolation B (NCKH_B) transformed into a pseudocyst under the effects of drug stress; however, National Cheng Kung University Hospital isolation D (NCKH_D), an isolate with higher tolerance for PHMB, did not transform. CONCLUSION: Our results confirm the existence of clinical isolates of A. castellanii with high resistance to PHMB in Taiwan and present the alternative drug tolerance of A. castellanii in addition to the transformation of pseudocyst/cyst.