Napaporn Tananuvat

Clinical and Epidemiological Analyses of Human Pythiosis in Thailand

BACKGROUND Pythiosis is an emerging and life-threatening infectious disease in humans and animals that is caused by the pathogenic oomycete Pythium insidiosum. Human pythiosis is found mostly in Thailand, although disease in animals has been increasingly reported worldwide. Clinical information on human pythiosis is limited, and health care professionals are unfamiliar with the disease, leading to underdiagnosis, delayed treatment, and poor prognosis. METHODS To retrospectively study the clinical and epidemiological features of human pythiosis, we analyzed clinical data from patients with pythiosis diagnosed during the period of January 1985 through June 2003 at 9 tertiary care hospitals throughout Thailand. RESULTS A total of 102 cases of human pythiosis were documented nationwide. A substantial proportion (40%) of cases occurred in the last 4 years of the 18-year study interval. Clinical presentations fell into 4 groups: cutaneous/subcutaneous cases (5% of cases), vascular cases (59%), ocular cases (33%), and disseminated cases (3%). Almost all patients with cutaneous/subcutaneous, vascular, and disseminated pythiosis (85%) had underlying thalassemia-hemoglobinopathy syndrome. Most ocular cases (84%) were associated with no underlying disease. A majority of the patients were male (71%), were aged 20-60 years (86%), and reported an agricultural occupation (75%). Regarding treatment outcomes, all patients with disseminated infection died; 78% of patients with vascular disease required limb amputation, and 40% of these patients died; and 79% of patients with ocular pythiosis required enucleation/evisceration. CONCLUSIONS Here, we report, to our knowledge, the largest case study of human pythiosis. The disease has high rates of morbidity and mortality. Early diagnosis and effective treatment are urgently needed to improve clinical outcomes. Because P. insidiosum is distributed worldwide and can infect healthy individuals, an awareness of human pythiosis should be promoted in Thailand and in other countries.

Identification of Emerging Human-Pathogenic Pythium insidiosum by Serological and Molecular Assay-Based Methods

ABSTRACT Pythium insidiosum is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of P. insidiosum. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the P. insidiosum isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described P. insidiosum DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for P. insidiosum was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.

Clinical Manifestations of 17 Patients Affected With Mucopolysaccharidosis Type VI and Eight Novel ARSB Mutations

Mucopolysaccharidosis (MPS) type VI or Maroteaux‐Lamy syndrome is a very rare autosomal recessive lysosomal storage disease, caused by a deficiency of the enzyme N‐acetylgalactosamine‐4‐sulfatase (Arylsulfatase B, ARSB). Clinical examination, biochemical studies, and molecular genetic analyses have been performed in 17 patients affected with MPS VI from 15 unrelated families from Thailand, India, and Turkey. Large ear lobule appears to be a newly recognized finding of this syndrome. Mutation analysis of the ARSB gene revealed seven missense and three frameshift mutations of which eight were novel. Novel missense mutations were p.Asp53Asn, p.Val376Glu, p.Glu390Lys, p.Pro445Leu, and p.Trp450Cys, while an Indian patient was homozygous for two novel missense mutations (p.Pro445Leu and p.Trp450Cys). Three novel frameshift mutations were p.Pro70fsX123, p.Ser403fs, and p.Thr526fs. Two previously reported mutations, p.Arg160Gln and p.Leu321Pro, were also observed in our cohort. The amino acid Arg160 appears to be the mutational hot spot for the ARSB gene. Five patients homozygous for p.Leu321Pro mutation had early onset of the disease, and haplotype analysis showed that the mutation is a founder mutation in Turkish population

Cultivation and Phenotypic Characterization of Rabbit Epithelial Cells Expanded Ex Vivo from Fresh and Cryopreserved Limbal and Oral Mucosal Explants

Abstract Purpose: To compare the morphology of cultured rabbit epithelial sheets and the expression of stem cells with differentiated cell markers of cultivated epithelial cells from fresh and cryopreserved limbal and oral mucosal biopsies. Materials and methods: Six New Zealand white rabbits were divided into two groups of three, from which limbal and oral mucosal biopsies were taken. Harvested tissues from each rabbit were brought to immediate cultivation, while another set of tissues was cryopreserved. Cultivation was performed by the explant culture technique using human amniotic membrane as a culture substrate, co-culturing with 3T3 fibroblasts and using the air-lifting method. Cells were cultured for three weeks; then cultured epithelial sheets were stained with hematoxylin–eosin and examined for expression patterns of p63, keratin 3 (K3) and connexin 43 (Cx43). Cryopreservation was carried out using the vitrification method. Tissues were preserved in liquid nitrogen using 25% dimethyl sulfoxide combined with 25% propylene glycol in Dulbecco’s Modified Eagle’s Medium containing 20% fetal bovine serum. After two months, the tissues were warmed, cultured and stained using the same processes as for fresh tissue cultures. Results: Cultivation of fresh limbal and fresh oral mucosal tissues showed epithelial stratification, with two to five cell layers. Immunohistochemical staining showed p63-positive cells in basal and intermediate cell layers. K3 staining was observed in cells in the suprabasal layer, while expression of Cx43 was scattered throughout all layers of the epithelia. All culture sheets expressed p63, K3 and Cx43 with the exception of one sheet from the oral mucosal culture that was p63-negative. Cultured epithelial sheets from cryopreserved tissues showed results similar to those from fresh tissue culture. Conclusions: This study found that cells in cultivated fresh limbal and oral mucosal tissues had similar morphology to cells in cultivated cryopreserved limbal and oral mucosal tissues, both containing a heterogeneous population of cells including stem cells and differentiated cells.

Root dentin anomaly and a PLG mutation

We report a Thai girl affected with plasminogen deficiency, Type I. Ligneous conjunctivitis was first observed when she was one-month-old. The newly recognized findings include tapered incisor roots as a result of thin root dentin, generalized short tooth roots, and mandibular prognathism. Mutation analysis of PLG demonstrated homozygous c.1193G>A missense mutation. The parents were heterozygous for c.1193G>A mutation. The c.1193G>A mutation is novel and predicted to cause amino acid substitution p.Cys398Tyr. Thin root dentin in the patient who was affected with PLG mutation and immunolocalization of Plg during early root development in mice imply the role of plasminogen in root dentin formation.

Superior keratoconus with hydrops.

Superior keratoconus is a rare form of keratoconus, in which the corneal apex is atypically superior. We report a 32-year-old man who presented with corneal hydrops in his right eye. Ophthalmic examination revealed corneal edema with Descemet’s break involving the superior part of his right cornea. Superior steepening in the left cornea demonstrated by corneal topography was consistent with subclinical keratoconus

Limbal stem cell and oral mucosal epithelial transplantation from ex vivo cultivation in LSCD-induced rabbits: histology and immunologic study of the transplant epithelial sheet.

PurposeTo evaluate the results of cultivated limbal epithelial and oral mucosal epithelial transplantation (CLET and COMET) in limbal stem cell deficiency (LSCD)-induced rabbit model.Materials and methodsSix New Zealand white rabbits were divided into two groups of three rabbits each. Limbal tissue was harvested from the first group, and oral mucosal biopsy was obtained from the second group. The tissues were cultured using an explant technique with amniotic membrane as a substrate and co-culture with the 3T3 fibroblast and air-lifting method. The right eye of each rabbit was induced to have LSCD using alkali burns. After three weeks, the LSCD-induced rabbit eyes were transplanted with the cultivated limbal and oral mucosal epithelial sheet in the first and second group, respectively. The transplanted eye was evaluated weekly post-operation. After 2 months, all transplanted eyes were enucleated and the epithelial morphology and phenotype of ocular surfaces were studied and compared with normal corneal and oral mucosal tissue.ResultsAt 2-month post-transplantation, the eyes of four animals recovered with corneal transparency, one partially recovered, and one failed. The histology of the majority of transplanted eyes was stratified layers of corneal epithelia similar to normal rabbit cornea with some different findings such as goblet cells in the limbal region. Corneal epithelial thickening and stromal vascularization in two animals were observed. Phenotypic characterization of transplanted eyes showed a similar pattern of marker expression with the absence of p63 expression in the limbal or corneal epithelium in the COMET group.ConclusionsThe histology and phenotype of transplanted eyes after CLET and COMET were most likely to have similar characteristics as a normal healthy rabbit eye even though the COMET eyes have some inferior characteristics to the CLET eyes.

Outcomes of LASIK for Myopia or Myopic Astigmatism Correction with the FS200 Femtosecond Laser and EX500 Excimer Laser Platform

Purpose: To evaluate the efficacy, predictability, stability and safety of laser in situ keratomileusis (LASIK) using the FS200 femtosecond laser and EX500 excimer laser platform. Methods: The outcomes of 254 eyes of 129 consecutive patients with myopia or myopic astigmatism who underwent full correction femtosecond laser-assisted LASIK at CMU LASIK Center were assessed. Pre-operative and post-operative parameters including manifest refraction, Uncorrected Distance Visual Acuity (UDVA), Best Corrected Distance Visual Acuity (BDVA), corneal topography and tomography were analyzed. The results between low to moderate myopia and high myopia were compared up to 12 months. Results: Mean pre-operative Spherical Equivalent (SE) was -5.15±2.41 Diopters (D) (range -0.50 to -11.50 D) and -0.13±0.28 D, -0.13±0.27 D, -0.13±0.28 D and -0.14±0.30 D at 1, 3, 6, and 12 months, post-operatively. At 12 months, the propor¬tion of eyes achieving UDVA ≥ 20/20 was 90.0% and ≥20/40 was 98.8%. The proportion of eyes achieving post-operative mean SE ±0.5 D, and ±1 D was 91.3%, and 98.5%. No eyes lost more than two lines of BDVA. The low to moderate myopic group had a statistically significant better UDVA at one (p=0.017) and three months (p=0.014) but no difference at six (p=0.061) and 12 months (p=0.091). The mean post-operative SE was better in low to moderate myopic group at every follow-up visit (p=0.001, 0.007, <0.001 and <0.001). Conclusion: One-year clinical results of LASIK with the FS200 femtosecond laser and EX500 excimer laser showed high efficacy, predictability, stability and safety.

Corneal perforation after noncontact tonometry in patients with active recurrent herpes simplex keratitis: case report

PurposeTo report iatrogenic complications and to review the potential complications caused by noncontact tonometry (NCT) in related literature.MethodsThis case report describes two cases of active recurrent herpes simplex keratitis (HSK) on top of a thin corneal scar. The cornea was perforated by the air pulse from the NCT, resulting in an air bubble in the anterior chamber.ResultsBoth patients were diagnosed with active recurrent necrotizing stromal HSK on top of a thinned corneal scar after previous therapeutic treatment involving tissue adhesive glue with a bandage contact lens (BCL) to treat a perforated cornea. During a follow-up visit, both patients reported similar symptoms of acute pain and fluid exuding from their eyes immediately after undergoing NCT. Slit-lamp examination revealed a perforated cornea with an intracameral air bubble. Treatment involved use of tissue adhesive glue and BCL in both cases.ConclusionNCT may not be sufficiently safe in patients with active infectious keratitis, particularly in cases where the cornea is relatively thin and necrotic.

Ocular Basidiobolomycosis: A Case Report

Background: Ocular basidiobolomycosis is an unusual infection caused by fungus of the order Entomophthorales. This fungus has been previously reported as a common cause of skin, subcutaneous, and gastrointestinal tract infection. The fungus isolation and its typical characteristics are clues for diagnosis of this uncommon pathogen. Case Report: A 47-year-old male patient with nodular scleritis in the left eye after an eye injury from sawdust was treated as bacterial scleritis. The lesion improved with early surgical drainage and antibacterial therapy; then, he was discharged from the hospital. Thereafter, the patient was re-admitted due to progression of infectious scleritis with keratitis and orbital cellulitis. Surgical abscess drainage was performed again. The microbiological study demonstrated Basidiobolus ranarum. The patient was treated with topical ketoconazole, subconjunctival fluconazole injection, and oral itraconazole with partial response to the treatment. However, the patient eventually denied any further treatment and did not return for follow-up. Conclusions: B. ranarum is a rare pathogen of ocular infection in which a definite diagnosis requires isolation of the causative organism. Delay in diagnosis and appropriate treatment can lead to extension of the infection and poor outcomes.