Random amplified polymorphic DNA typing and phylogeny of Pythium insidiosum clinical isolates in Thailand.

Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.

Malassezia furfur in infantile seborrheic dermatitis.

Our objective was to study both incidence and various strains of Malassezia in infantile seborrheic dermatitis (ISD). Sixty infants between 2 weeks and 2 years old with clinical diagnosis of ISD at the Department of Pediatrics, King Chulalongkorn Memorial Hospital from May 2002 to April 2003 were recruited. Malassezia spp. were isolated from cultured skin samples of the patients, genomic DNA was extracted and the ITS1 rDNA region was amplified. The PCR product was examined by agarose gel electrophoresis and DNA sequences were determined. The ITS1 sequences were also subjected to phylogenetic analysis and species identification. ISD is most commonly found in infants below the age of 2 months (64%), followed by those between 2 and 4 months (28%) old. Cultures yielded yeast-like colonies in 15 specimens. PCR yielded 200-bp products (Candida) in 3 patients and 300-bp products (Malassezia furfur) in 12 patients (18%). Sugar fermentation using API 20C aux performed on the three 200-bp PCR products yielded Candida species. M. furfur was the only Malassezia recovered from skin scrapings of children with ISD.

Intratypic variations among Thai herpes simplex virus (HSV) isolates determined by restriction fragment length polymorphism (RFLP) analysis.

Whole genomic polymorphisms for 20 HSV-1 and 20 HSV-2 isolates from Thai patients were analyzed by means of Restriction Fragment Length Polymorphism (RFLP) analysis using 4 restriction endonucleases: BamHI, Kpnl, HindIII, and EcoRI. Variations in cleavage sites among the HSV-1 and HSV-2 isolates were compared to the cleavage patterns of standard HSV-1 strain KOS and HSV-2 strain Baylor 186. Although 70% of HSV-1 isolates with BamHI digestion, 50% with Kpnl, 75% with HindIII and 70% with EcoRI digestion were found to be similar to the standard HSV-1 (KOS) pattern, new BamHI restriction sites were detected in some HSV-1 isolates. For HSV-2 isolates, 85% had the same pattern as the standard HSV-2 (Baylor 186) after digestion with BamHI, HindIII, and EcoRI. No difference was observed with Kpnl digestion. When the patterns from the 4 enzymes were combined, HSV-1 isolates showed more divergence than the HSV-2 isolates. HSV-1 isolates found in both non-genital and genital lesions had more variety than the HSV-2 isolates. This suggests that intratypic variations in HSV-2 are fewer than in HSV-1.

Antifungal susceptibility and molecular typing of candida albicans isolated from AIDS and non-AIDS Thai patients.

The antifungal susceptibility of Candida albicans isolated from 2 groups of Thai patiens; AIDS patients and non-AIDS patients was investigated. Two hundread and seventeen C. albicans were isolated from the specimens from 54 AIDS patients and 163 non AIDS patients. All isolate were included in the antifungal susceptibility test against amphotericin B, fluconazole, ketoconazole and nystatin. A hundred isolates were randomly selected from both groups for the electrophoretic karyotypes determination. There was not much difference in the value of Mic, MIC50 and MIC90 of all antifungal agent for C. albicans isolates between AIDS and non-AIDS patients. The amphoter B MIC for 61.0% if AIDS isolates and 71.0% of non-AIDS isolates were >0.5 mg/l, while ketoconazole MIC for 94.4% of AIDS and 74.9% of non-AIDS isolates were >0.125 mg/l and fluconazole MIC of 100% of AIDS and non-AIDS isolates were 2.0 mg/l. For nystatin, the MIC for more than 90% of both isolates was <8 mg/l. The MIC50 and MIC90 of all antifungal agents for the two groups of isolates were almost at the same concentration except for fluconazole which showed a two-fold difference of MIC50. The chromosomal DNA karyotypic of C. albicans indicated genetic diversity among all isolates. Twenty-three distinct pulsed-field gel electrophoresis karyotypes and molecular sizes ranging from 3.5 to 0.5 megabases were identified. Most isolates (61%) from both AIDS and non-AIDS isolates belong to type 1 and type 3. Among the AIDS isolates, 38.3% and 29.8% were type 1 and 3, respectivety, and non-AIDS isolates, 20.8% and 33.9% were type 1 and 3, respectively. C. albicans isolates were show to have 8 to 10 chromosomal DNA brands. Most of the isolates (78%) along with C. albicans FC18 control strain had 8 band patterns. The recovery of the common karyotypes in AIDS patients, as well as in non-AIDS patients, suggests that C. albicans infection may develop from a common source by the cross contamination between both groups of patients.