Focus on limbal stem cell deficiency and limbal cell transplantation

Limbal stem cell deficiency(LSCD)causes severe vision impairment and can lead to blindness,representing one of the most challenging ocular surface disorders.Stem cell deficiency can be congenital or,more often,acquired.The categorization of ocular surface transplantation techniques is crucial to achieving treatment homogeneity and quality of care,according to the anatomic source of the tissue being transplanted,genetic source,autologous or allogenic transplantation(to reflect histocompatibility in the latter group),and cell culture and tissue engineering techniques.The aim of this minireview is to provide a summary of the management of LSCD,from clinical characteristics and therapeutic outcomes to the development of novel therapeutic approaches.The manuscript also briefly summarizes recent findings in the current literature and outlines the future challenges to overcome in the management of the major types of ocular surface failure.

Quantification of corneal neovascularization after ex vivo limbal epithelial stem cell therapy

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animalfree culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization,vessel caliber(VC), and invasive area(IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity(BCVA),epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction(improvement) of all three parameters of corneal neovascularization [neovascular area(NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31%(P =0.035), invasion area29.37%(P =0.018) and VC 14.29%(P =0.072). BCVA improved in all eyes(mean follow-up, 76 ±21mo).Epithelial transparency improved significantly from 2.00 ±0.93 to 0.88±1.25(P =0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing andmonitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method,successful cases could be differentiated from failed cases.

Midterm outcomes of penetrating keratoplasty following allogeneic cultivated limbal epithelial transplantation in patients with bilateral limbal stem cell deficiency

AIM:To evaluate the midterm outcomes of penetrating keratoplasty(PK)following allogeneic cultivated limbal epithelial transplantation(CLET)for bilateral total limbal stem cell deficiency(LSCD).METHODS:Ten patients(10 eyes)with bilateral LSCD were enrolled in this prospective noncomparative case series study.Each participant underwent PK approximately 6 mo after a CLET.Topical tacrolimus,topical and systemic steroids,and oral ciclosporin were administered postoperatively.Best-corrected visual acuity(BCVA),intraocular pressure(IOP),ocular surface grading scores(OSS),corneal graft epithelial rehabilitation,persistent epithelial defect(PED),immunological rejection,and graft survival rate were assessed.RESULTS:The time interval between PK and allogeneic CLET was 6.90±1.29(6-10)mo.BCVA improved from 2.46±0.32 log MAR preoperatively to 0.77±0.55 log MAR post-PK(P<0.001).Kaplan-Meier analysis of mean graft survival revealed graft survival rates of 100%at 12 and 24 mo and 80.0%at 36 mo.PEDs appeared in 5 eyes at different periods post-PK,and graft rejection occurred in 4 eyes.The total OSS decreased from 12.4±4.4 before allogeneic CLET to 1.4±1.51 after PK.CONCLUSION:A sequential therapy design of PK following allogeneic CLET can maintain a stable ocular surface with improved BCVA despite the relatively high graft rejection rate.

Cell-based therapies for limbal stem cell deficiency: a literature review

Background and Objective:Limbal stem cell deficiency(LSCD)is characterized by the insufficiency of limbal stem cells to maintain the corneal epithelium.Severe cases of LSCD may be treated with limbal transplantation from healthy autologous or allogeneic limbal tissue.Multiple cell-based therapies have been studied as alternative treatments to improve success rates and minimize immunosuppressive regimens after allogeneic transplants.In this review,we describe the success rates,and complications of different cell-based therapies for LSCD.We also discuss each therapy’s relative strengths and weaknesses,their history in animal and human studies,and their effectiveness compared to traditional transplants.Methods:PubMed was searched for publications using the terms LSCD,cell-based therapy,cultivated limbal epithelial transplantation(CLET),cultivated oral mucosal epithelial transplantation(COMET),and mesenchymal stem cells from 1989 to August 2022.Inclusion criteria were English language articles.Exclusion criteria were non-English language articles.Key Content and Findings:current cell-based therapies for LSCD are CLET and non-limbal epithelial cells.Non-limbal epithelial cell methods include COMET,conjunctival epithelial autografts,and mesenchymal stem/stromal cells(MSCs).Moreover,several alternative potential sources of non-limbal cells have described,including induced pluripotent stem cells(iPSCs),human embryonic stem cells(hESCs),human dental pulp stem cells,hair follicle bulge-derived epithelial stem cells,amniotic membrane epithelial cells,and human umbilical cord lining epithelial cells.Conclusions:Cell-based therapies are a promising treatment modality for LSCD.While CLET is currently the only approved cell-based therapy and is only approved in the European Union,more novel methods have also been shown to be effective in human or animal studies thus far.Non-limbal epithelial cells such as COMET are also an alternative treatment to allogeneic transplants especially as a surface stabilizing procedure.iPSCs are currently being studied in early phase trials and have the potential to revolutionize the way LSCD is treated.Lastly,cell-based therapies for restoring the limbal niche such as mesenchymal stem cells have also shown promising results in the first human proof-of-concept study.Several potential sources of non-limbal cells are under investigation.

Limbal epithelial stem cells in corneal surface reconstruction

Cornea serves as the partial front barrier and major light reflection organ of the eye.The integrity of corneal surface is essential for ocular function.Injuries or congenital diseases could significantly destruct the homeostasis of the ocular surface,especially the microenvironment of limbal epithelial stem cells(LESCs),and will eventually cause dysfunction of corneal regeneration and diminish of LESCs.The loss of LESCs by different reasons are named limbal stem cell deficiency(LSCD),which is one of the leading cause of vision loss worldwide.To restore the corneal surface,LESC transplantation in the form of tissue or cell cultures is currently a viable and promising method to treat LSCD.In this review,we aim to introduce the characters and niche of LESCs,and discuss different aspects of its application in cornea surface reconstruction.

Limbal stem cells: Central concepts of corneal epithelial homeostasis

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

Simple keratectomy and corneal tattooing for limbal dermoids:results of a 3-year study

AIM:To evaluate and report the efficacy of combined surgical excision and corneal tattooing in patients with limbal dermoids.·METHODS:In a retrospective interventional case series,9 eyes of 8 patients were treated with combined surgery of simple keratectomy and corneal tattooing for limbal dermoids.Medical records,including best-corrected visual acuity,anterior segment photography,demographic,clinical data,and follow-up information were reviewed.·RESULTS:The mean follow up period in this study was 50±15(range 36-77) months.There was no evidence of infection or recurrent limbal dermoids in any of the eyes during the follow-up period.All patients achieved good cosmetic outcomes with no complications.·CONCLUSION:Simple keratectomy and corneal tattooing of limbal dermoids could be an alternative option for surgery,especially when a donor cornea is not available.

Therapeutic effect of secretome from TNF-αstimulated mesenchymal stem cells in an experimental model of corneal limbal stem cell deficiency

AIM:To explore the secretome efficacy in tumor necrosis factor(TNF)-αstimulated mouse mesenchymal stem cells(MSCs)in a murine model of corneal limbal alkali injury.METHODS:Corneal limbal stem cell deficiency(LSCD)was created in the eyes of male C57 mice.Concentrated conditioned medium from TNF-αstimulated MSCs(MSC-CMT)was applied topically for 4 wk,with basal medium and conditioned medium from MSCs as controls.Corneal opacification,corneal inflammatory response,and corneal neovascularization(NV)were evaluated.Corneal epithelial cell apoptosis,corneal conjunctivation,and inflammatory cell infiltration were assessed with TUNEL staining,CK3 and Muc-5 AC immunostaining,and CD11 b immunofluorescence staining,respectively.The effect of TSG-6 was further evaluated by knockdown with short hairpin RNA(sh RNA).RESULTS:Compared to the controls,topical administration of MSC-CMT significantly ameliorated the clinical symptoms of alkali-induced LSCD,with restrained corneal NV,reduced corneal epithelial cell apoptosis,and inhibition of corneal conjunctivation.In addition,MSC-CMT treatment significantly reduced CD11 b+inflammatory cell infiltration,and inhibited the expression of pro-inflammatory cytokines(IL-1β,TNF-αand IL-6).Furthermore,the promotion of corneal epithelial reconstruction by MSC-CMT was largely abolished by TSG-6 knockdown.CONCLUSION:Our study provides evidence that MSCCMT enhances the alleviation of corneal alkali injuries,partially through TSG-6-mediated anti-inflammatory protective mechanisms.MSC-CMT may serve as a potential strategy for treating corneal disorders.

Effects of preservation time on proliferative potential of human limbal stem/progenitor cells

AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the expression of stem cell markers. METHODS: Thirty limbal rims were split into 4 parts and stored in corneal preservation medium at 4 degrees C for 0, 1, 4, or 7 days. The limbal stem cell and mitotic markers P63, CK19, proliferating cell nuclear antigen (PCNA), and Ki67 were determined by immunohistochemical staining. The proliferative potential of limbal epithelial cells was assessed by cell viability, the ability of generating stratified epithelium, and colony forming assay. RESULTS: The stored tissues maintained limbal stratified structure to 7 days and exhibited comparable expression level of stem cell and mitotic markers. The proportion of viable cells decreased with the prolonged preservation time, while colony forming efficiency decreased from the 1st day and disappeared at the 4th day. When inoculated on amniotic membrane, the cells preserved for 1 day formed a stratified epithelium, while the cells from 4 days' preservation formed a discontinuous layer. CONCLUSION: The colony forming efficiency of limbal epithelial stem/progenitor cells decreased rapidly with the increasing preservation time, while the expression level of markers and capacity of forming epithelial monolayer on amniotic membrane decreased gradually. The limbal epithelial stem cells lost their function earlier than the lost expression level of stem cell markers. This may help us to better choose the appropriate preservation grafts for future limbal stem cell transplantation.

Preoperative evaluation and outcome of corneal transplantation for limbal dermoids：aten-yearfollow-up study

AIM： To summarize preoperative evaluation and outcome of corneal transplantation for limbal dermoids for ten years.METHODS： Eighty-five patients diagnosed with limbal dermoids and treated with corneal transplantation were analyzed retrospectively. All patients were further divided into two groups according to absence or presence of neovascularization surrounding the dermoids in the corneal stroma. Eighty-two eyes were treated with tumor excision combined with partial lamellar sclerokeratoplasty, and the other three eyes were performed by penetrating keratoplasty. The size and location of the tumor, the associated ocular and systemic anomalies, the depth of the corneal penetration of tumor tissues, the preoperative and postoperative best-corrected visual acuity （BCVA）, graft survival and cosmetic outcome, and surgical complications were recorded respectively.RESULTS： The average age at surgery was 5.3y （range, 3mo-36y）. The mean size of dermoids was 6.1±1.6 mm. The 43.5% of eyes （37/85） were present with hair at the surface of the dermoid and 72.9% of dermoids were located inferotemporal of the eye. Amplyopia was present in 34.1% of patients （29/85） and 9.4% of patients （8/85） had lipodermoids. Eighteen patients suffered from Goldenhar’s syndrome with an accessory ear. The 75% of patients in group 1 had involvement of the corneal deep stroma down to Descemet’s membrane without involving it, but 71.4% of patients had Descemet’s membrane involvement in group 2. Preoperative BCVA ranged from counting fingers to 20/20. Postoperatively 81.1% had a BCVA of 20/800 or better. There was no significant difference between the post-surgical BCVA of the two groups （t=1.584, P〉0.05）. The grafts of 70.5% patients were present as 1＋ opacity, 21.1% as 2＋ opacity, 8.2% as 3＋ opacity and none as 4＋ opacity. Surgical complications included graft rejection, microperforation, prolonged reepithelialization, steroid glaucoma, interface neovascularization, and interface hemorrhage.CONCLUSION： The dermoids with neovascularization surrounding them in the corneal stroma invaded deeper tissues in the cornea than those with no neovascularization surrounding them in the corneal stroma. Therefore, surgeons should take care to avoid corneal perforation during the corneal transplantation operation. The majority of patients markedly improved their cosmetic appearance after surgery.

Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

AIM： To explore the possibility of human umbilical cord mesenchymal stem cells（h UCMSCs）, human umbilical vein endothelial cells（h UVECs）, human dental pulp stem cells（h DPSCs） and human periodontal ligament stem cells（h PDLSCs） serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS： Different feeder layers were cultured in Dulbecco＇s modified Eagle＇s medium（DMEM）/F12 and were treated with mitomycin C. Rabbits limbal stem cells（LSCs） were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency（CFE） assay and immunofluorescence（IPO13,CK3/12）.RESULTS： The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION： hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.

Chronic ocular GVHD:limbal and conjunctival stem cell allografts from the same hematopoietic stem cell donor

Dear Editor,I am Dr.Elias F Jarade from the Beirut Eye and ENT Specialist Hospital, Beirut, Lebanon. I write to describe a novel surgical technique in the management of chronic ocular graft-versus-host disease（GVHD）.

Single-cell RNA sequencing in cornea research:Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.

The Effect of Limbal Autograft in Recurrence of Pterygium

Background: Assessing the effect of limbal autograft shifting in recurrence of pterygium. Methods: This single-center study was carried out in a tertiary health facility. A review of data on consecutive patients who underwent pure limbal autografts shifting after pterygium resection was done. In all the cases, the pterygia extended at least 3 mm beyond the limbus. The resected each limbal grafts included a width of 1.5 mm and a length of 2 or 3 mm of limbus and a depth of 250 μm. Schmer test was performed at the eighth month postoperatively. Pterygium recurrence was accepted endpoint of the study. One patient had recurrent pterygium, whereas the others had primary pterygium. Patients with other ocular surface diseases or ocular pathology and, patients who discontinued follow-up visits were excluded from the study. Results: The study included 10 patients, with 5 males and 5 females. Median age of the patients was 40 (25 - 70). Follow-up was conducted for a minimum of 8 months for patients with recurrence and at least for 16 months for non-recurrent cases. Recurrence was observed in 6 patients out of 10, in one patient, atypia was reported and excluded from the study. Four recurrent patients experienced decreased levels of tears. The rest one patient with recurrence had not any tear abnormality. The remaining 4 patients responded well to the surgery. Because of the high recurrence rate, it was decided to terminate the study. Conclusions: Limbal autografts shifting alone is not an appropriate treatment for primary pterygium because of the high recurrence rate.

Cryopreserved limbal lamellar keratoplasty for peripheral corneal and limbal reconstruction

This study aimed to evaluate the outcomes and described the recovery process of cryopreserved limbal lamellar keratoplasty（CLLK） for peripheral corneal and limbal diseases. Thirteen eyes of 12 patients with a mean age of 41±23.9 y were included. The average follow-up was 12.1±5.6 mo. Stable ocular surface was achieved in all eyes at last follow-up. Epithelialization originated from both recipient and graft in 9 eyes. We conclude that CLLK compensates for the shortage of donor corneas and cryopreserved limbal grafts provide epithelialization sources in ocular surface reconstruction.

The protective effect of zeaxanthin on human limbal and conjunctival epithelial cells against UV-induced cell death and oxidative stress

AIM: To explore the protective effect of zeaxanthin on human limbal and conjunctival epithelial cells against UVradiation and excessive oxidative stress.METHODS: Human limbal and conjunctival epithelial cells were isolated from cadaver and cultured in vitro. They were challenged with UVB radiation and H2 O2 with and without zeaxanthin pretreatment. Cell viability, p38 and c-JUN NH(2)-terminal kinase(JNK) phosphorylation, IL-6, IL-8 and MCP-1 secretion and malondialdehyde(MDA) content were measured.RESULTS: Zeaxanthin had no measurable cytotoxicity on limbal or conjunctival epithelial cells when used at concentrations of 5 μg/mL and below. At 30 mJ/cm2 UVB, the pretreatment of zeaxanthin increased the percentage of live cells from 50% to 69%(P=0.01) and from 66% to 75%(P=0.05) for limbal and conjunctival epithelial cells, respectively. The concentrations of IL-6, IL-8 and MCP-1 in the culture medium reduced to 66%(for IL-6 and MCP-1)and 56%(for IL-8) of the levels without zeaxanthin. This was accompanied by reduced p38 and JNK protein phosphorylation. Pretreatment of zeaxanthin also reduced intracellular MDA content caused by H2 O2 stimulation from 0.86 μmol/L to 0.52 μmol/L(P=0.02) in limbal epithelial cells and from 0.96 μmol/L to 0.56 μmol/L in conjunctival epithelial cells(P=0.03). However, zeaxanthin did nothave significant effect on H2 O2-induced cell death in limbal or conjunctival epithelial cells.CONCLUSION: Zeaxanthin is an effective reagent in reducing the detrimental effect of UV-radiation and oxidative stress on ocular surface epithelial cells.

Investigation of immunogenicity of cryopreserved limbal stem cells

AIM: To investigate changes in immunogenicity of cryopreserved limbal stem cells. METHODS: Cryopreserved limbal stem cells, fresh primary limbal stem cells and blank controls were inoculated subcutaneously in C57BL-6 mice and the percentage of CD25 cells in limbal explants was determined by flow cytometry at day 21 post inoculation. Morphological studies were performed by light and electron microscopy of limbal explant sections. RESULTS: The number of regional and systemic lymphocytes derived from cryopreserved limbal stem cells was lower than that from fresh primary limbal stem cells. CONCLUSION: Lymphocytes derived from cryopreserved limbal stem cells showed changes in immunogenicity, but the significance is unknown. The cryopreservation and thawing methods await further study.

Lamellar keratoplasty with corneoscleral graft for limbal dermoids

To assess the postoperative outcomes of limbal dermoid excision with corneoscleral graft transplantation. The charts of 8 consecutive patients （mean age： 13.0y） who had undergone limbal dermoid excision with lamellar corneosclerai graft transplantation by a single surgeon were retrospectively reviewed. Mean dermoid size was 7.75 mm （6.0-12.0 mm）. Mean visual acuities （in IogMAR units） before and after surgery were 1.8 and 1.7, respectively （P=0.29）. Spherical equivalents were 1.3 diopter （D） before surgery and 0.7 D after surgery （P=0.40）. The mean astigmatism measurements before and after surgery were 2.4 D and 1.5 D, respectively （P=0.17）. Vector analysis revealed a mild change in astigmatism with a mean ＂d＂ of 3,2 （0,56-6.89）, No intra- or post-operative complications occurred. Lamellar keratoplasty for limbal dermoids is safe and offers good cosmesis and tectonic stability. A significant decrease in the amount of astigmatism is not expected following surgery.

Limbal leproma in lepromatous leprosy

Dear Sir, W e are writing to you to present an unusual case of leproma growing at the limbus of a lepromatous leprosy patient. This the first report of ocular leprosy in Brunei, where leprosy is extremely rare. Leprosy, otherwise known as Hansen’s disease, is a chronic granulomatous communicable infection caused by Mycobacterium leprae and its genetic variant (Mycobacterium lepromatosis) [1]. A leproma is a superficial, ci