Nucleotide Sequence-Based Analysis for Determining the Molecular Epidemiology of Penicillium marneffei

摘要

The dimorphic fungus, Penicillium marneffei, is an emerging opportunistic pathogen endemic in Southeast Asia, especially for those with impaired cellular immunity such as human immunodeficiency virus-infected persons. A discriminatory and reproducible method based on the analysis of nucleotide sequences would facilitate epidemiologic investigations of this fungus. Twenty-four clinical or environmental isolates of P. marneffei obtained from China, Thailand, and Vietnam were analyzed by nucleotide sequence analysis. A total of 3,803 bp, consisting of eight nuclear gene fragments (transcription factor [AbaA], catalase [CpeA]], homodomain transcription factor [StlA], isocitrate lyase [Icl1], polyaromatic amino acid biosynthesis [PAA], NADH-dependent glutamate synthase [NGS], lovastatin nonaketide synthase [LNS], a cell wall mannoprotein [MP1], and a gene fragment of the cytochrome oxidase subunit 1 gene [COX1] of the P. marneffei mitochondrial genome) were amplified by PCR and then sequenced. No polymorphic sites within the Cox1 gene fragment were observed. Likewise, no nucleotide sequence polymorphisms were observed for three gene fragments: StlA, AbaA, and NGS. Seven single-nucleotide polymorphisms were observed for three gene fragments, Icl1, CpeA, and PAA, providing only a low degree of discriminatory power (D = 0.747). In contrast, the gene fragment for an antigenic cell wall glycoprotein, MP1, a useful immunologic marker for infection, was observed to be highly polymorphic with 12 different MP1 types (D = 0.887). Single-nucleotide polymorphisms were observed at 21 different locations in the MP1 gene fragment. Indels of 3, 21, 24, and 42 bp were observed and were in frame for protein translation. The relatively high degree of MP1 polymorphisms suggests the sequence is rapidly evolving in order to evade host immune responses. After all polymorphic gene sequences were combined, a high degree of genetic variation was observed (D = 0.949) for a total of 16 different haploid sequence types with 11 genotypes represented by single isolates. Phylogenetic analysis detected clusters composed of isolates obtained only from China or Thailand, as well as clusters with a combination of isolates from these two countries, indicating some mixing or common descent. Identical sequences were observed for isolates passed in vitro for 8 weeks, suggesting good reproducibility. The low degree of nucleotide diversity in housekeeping and regulatory genes suggests the recent emergence and spread as a species or an evolutionary bottleneck. In summary, multilocus sequence typing demonstrated a high degree of discriminatory power and reproducibility and may provide a robust and reliable adjunct method for genotyping isolates of P. marneffei and facilitating interlaboratory comparisons.