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Penicilliosis marneffei is a disemminated and progressive infection caused by a dimorphic fungus, Penicillium marneffei (6). P. marneffei infections are increasingly common in Southeast Asia, particularly Thailand (3, 12), southern China (4, 8), and Hong Kong (14). With the spread of human immunodeficiency virus (HIV) into northern Thailand, approximately 1,115 cases of penicilliosis marneffei associated with HIV infection were diagnosed at Chiang Mai University Hospital in Chiang Mai, Thailand, over a 7-year period (10). There, the disease was the third most common opportunistic infection after tuberculosis and cryptococcosis (13). The routes and mechanism of infection by P. marneffei are still poorly understood, but the original infection was possibly through inhalation of conidia. In addition to the infection in humans, P. marneffei was also reported to be isolated from the internal organs of the bamboo rat. In Thailand, the first survey was carried out in the central plains from June to September 1987 (1). Thirty-one small bamboo rats (Cannomys badius) and eight hoary bamboo rats (Rhizomys pruinosus) were trapped. P. marneffei was isolated from the internal organs of 6 (19%) C. badius and 6 (75%) R. pruinosus rats. Notably, the rats did not show any signs or symptoms of the disease like those found in humans, but they might serve as natural reservoirs for P. marneffei.

A recent molecular biology-based study based on HaeIII restriction endonuclease analysis has led to the division of P. marneffei from northern Thailand into two DNA types (15). Evaluation of 22 human isolates revealed that 16 (72.7%) were of type I and 6 (27.3%) were of type II. Since only two genotypes accounted for the majority of isolates, further analysis was necessary to differentiate strains of the same DNA type more effectively. In the present study, we used pulsed-field gel electrophoresis (PFGE) in an attempt to characterize P. marneffei isolates recovered from AIDS patients from several regions in Thailand. PFGE is based on the digestion of chromosomal DNA with a restriction endonuclease that cleaves infrequently and that produces only a few high-molecular-weight fragments that can be separated under special conditions of electrophoresis. PFGE has been demonstrated to have advantages in discriminatory power, typeability, and reproducibility (2, 9) and has been widely used to investigate epidemics of several fungi (11). We also assessed the performance of the PFGE technique by comparing the results of PFGE with those of restriction endonuclease typing of P. marneffei previously reported by Vanittanakom et al. (15).

Fungal isolates. Sixty-four clinical isolates of P. marneffei from 62 patients with sporadic cases of infection were used for the genotypic study. Among these isolates, 14 were obtained from Lanna Medical Laboratory in the city of Chiang Mai, which is in northern Thailand. These isolates were recovered from patients who lived in Muang District, Maerim District, and Hang Dong District, which are in Chiang Mai Province. Twenty-three isolates were from Microbiology Laboratory, Prince of Songkla Hospital, which is in the city of Songkla, in southern Thailand, and the remaining 27 isolates were from several hospitals located in the central region of the country. Most patients who were admitted to Prince of Songkla Hospital were from Muang District, Haad Yai District, and Ranode District, which are in Songkla Province. Four isolates were purchased from the American Type Culture Collection (ATCC). Two isolates (isolates ATCC 64101 and ATCC 24100) were clinical isolates, and the others (isolates ATCC 64102 and ATCC 18224) were from bamboo rats. Isolate ATCC 18244 was obtained from the liver of a Rhizomys sinensis rat captured in Vietnam in 1956, whereas isolate ATCC 64102 was obtained from the lung of an R. pruinosus rat captured in China in 1986. The details about each isolate obtained from the different geographical regions of Thailand are summarized in Table 1. Three clinical isolates (isolates 13, 14, and 15) were obtained from the skin, blood, and liver, respectively, of a patient from southern Thailand. Other bamboo rat isolates were gifts from Samaniya Sukroongreung, Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand. These isolates were obtained in 1987 (1). The isolates were recovered from different internal organs of one bamboo rat and displayed the same macrorestriction pattern. As a result, only one bamboo rat isolate was chosen as a representative isolate. Every isolate was cultured on Sabouraud dextrose agar and was incubated at 25°C for 1 to 2 weeks. To collect spores, 2 ml of 0.01% Tween 80 (Sigma-Aldrich, St. Louis, Mo.) in sterile distilled water was added to each fully grown culture. Approximately 10⁶ cells were inoculated into a 500-ml flat-bottom bottle containing Sabouraud dextrose broth. The cultures were incubated at 25°C in a gyratory shaker (New Brunswick Scientific Co., Inc.) set at 160 rpm for 2 days.

Preparation of DNA and PFGE. Fungal mycelia (40 mg) were washed three times with 50 mM EDTA and suspended in 1 ml of cell wall lysis buffer containing 125 M EDTA, 50 mM sodium citrate, 25 µg of chitinase (Sigma-Aldrich) per ml, and 200 U of lyticase (Sigma) per ml. The mycelial suspension was mixed with low-melting-point agarose (SeaPlaque; FMC Bioproducts, Rockland, Maine) to a final concentration of 1.0%. The mixture was cast into molds (Bio-Rad, Richmond, Calif.) and was allowed to solidify for 10 min at 4°C. The plugs were incubated in a buffer containing 0.4 M EDTA and 50 mM sodium citrate supplemented with 1% 2-mercaptoethanol for 24 h at 37°C, followed by three washings with 50 mM EDTA. The protoplasts in the plugs were disintegrated, and the proteins were degraded with lysis buffer (0.5 M EDTA, 10 mM Tris-HCl, 1% N-laurylsarcosine, 2 mg of proteinase K per ml) for 24 h at 50°C. The plugs were finally washed three times with 50 mM EDTA and were stored at 4°C until use.

Data analysis. The DNA banding patterns obtained were photographed with a digital camera (Vilber Lourmat, Mame Lavallec, France) and saved as TIFF files for use with BIO-PROFIL (Vilber Lourmat). Normalization was done according to the molecular weight standards on each gel, with one molecular weight standard being used for every six samples. Construction of similarity matrices was carried out by comparison of Dice coefficients (5). In all cases, the unweighted pair group method with average linkages was used to cluster the patterns.

HaeIII restriction endonuclease (RE) analysis. Restriction fragment length polymorphism (RFLP) analysis of P. marneffei with restriction enzyme HaeIII was performed by the method previously described by Vanittanakom et al. (15), with some modification. In brief, protoplasts from 40 mg of fungal mycelia were suspended in 1 ml of lysis buffer (50 mM Tris-HCl, 100 mM EDTA, 0.5% sodium dodecyl sulfate, 0.3 M sodium acetate). After incubation at 65°C for 30 min, an equal volume of phenol-chloroform-isoamyl alcohol was added and the upper aqueous phase was separated by centrifugation at 1,600 × g for 10 min. DNA was precipitated from the aqueous phase with isopropanol and was finally suspended in TE buffer containing 50 µg of RNase A per ml. A digestion reaction was performed with the HaeIII restriction enzyme. After 1 h of incubation, electrophoresis was carried out for 3 h at 3 V/cm in a 1% agarose gel.

Results and discussion. The experimental variation between duplicate experiments was determined for four replicate experiments with four P. marneffei strains. The reproducibility of PFGE was 97%, and only one pattern for each isolate was imported into BIO-PROFIL.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   PFGE pattern representations. (a) Lanes 1 to 8 (unnumbered, from left to right, respectively), P. marneffei isolates classified as MPI by cluster analysis; (b) lanes 1 to 8 (unnumbered, from left to right, respectively), isolates classified as MPII. Bacteriophage concatemer ladders (in kilobase pairs) are indicated to the left of the panel.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Dendrogram of PFGE patterns with designated bands. Cluster analysis was performed as described in the text. Isolates which were untypeable are not shown.