Molecular Typing of Penicillium marneffei Isolates from Thailand by Not I Macrorestriction and Pulsed-Field Gel Electrophoresis

Abstract

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.

Analysis of Polymorphic Microsatellite Markers for Typing Penicillium marneffei Isolates

“…In contrast, Vanittanakom et al (47) detected only two HaeIII types among 46 isolates of P. marneffei. More recently, pulsed-field gel electrophoresis of NotI restriction endonuclease fragments was shown to be reproducible and highly discriminatory between isolates despite laborious sample preparation (46). The results of typing by PMM analysis in the present study of isolates ATCC 64102, 24100, 18224, and 64101 was consistent with the typing results obtained by NotI analysis of the same isolates by Vanittanakom et al (47); both methods showed these isolates to be unrelated.…”

“…However, RAPD profiles have been shown to be unstable in Aspergillus fumigatus or too sensitive to reaction conditions (4,27). Macrorestriction digestion profiles of NotI restriction fragments resolved by pulsed-field gel electrophoresis for 69 P. marneffei isolates obtained from AIDS patients in Thailand detected 54 patterns (46). Although it provided good discriminatory power, NotI restriction fragment length polymorphism analysis was reported to lack concordance with HaeIII analysis.…”

“…Although it provided good discriminatory power, NotI restriction fragment length polymorphism analysis was reported to lack concordance with HaeIII analysis. The method requires specialized equipment not available in many laboratories and tedious sample preparation (46).…”

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

“…Two HaeIII types for 46 isolates were reported (53). Randomly amplified polymorphic DNA and macrorestriction profiles have been utilized for typing, but these methods are difficult to reproduce or it is difficult to compare results between laboratories (23,26,51). Recently, microsatellite markers have been shown to provide high degree of discriminatory power and reproducibility for typing P. marneffei isolates (15,16,17,21,30).…”

MP1 Homologue-Based Multilocus Sequence System for Typing the Pathogenic Fungus Penicillium marneffei : a Novel Approach Using Lineage-Specific Genes

“…Trewatcharegon et al have used pulsed-field gel electrophoresis (PFGE) and Fisher et al have used multilocus microsatellite typing for typing P. marneffei (13,14,36). However, due to experimental variations, PFGE patterns are difficult to compare among different laboratories.…”