Genetic identification of the main opportunistic Mucorales by PCR-restriction fragment length polymorphism

Abstract

Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis.

FIG.1.

Alignment of partial 18S rRNA gene sequences for Mucorales, other filamentous fungi, yeasts, and human. Crosses and dots indicate sequence homologies or polymorphisms in this domain, respectively. The binding regions of the sense and antisense primers are boxed. The sequence polymorphisms between the four main pathogenic Mucorales (in boldface) or the human sequence are highlighted in gray. Sequences were obtained from GenBank and represent parts of the sequences given under accession numbers listed in Table 1.

FIG. 2.

Electrophoresis patterns of amplicons obtained from fungal and human DNA with the panfungal primers Lap and Rap. Lanes M, 100-bp ladder; lanes T−, negative control (sterile water); lane 1, Mucor sp.; lane 2, Acremonium sp.; lane 3, Aspergillus fumigatus; lane 4, Candida albicans; lane 5, Fusarium sp.; lane 6, Geotrichum sp.; lane 7, Alternaria sp.; lane 8, Scopulariopsis brevicaulis; lane 9, Scedosporium sp.; lane 10, Penicillium sp.; lane 11, DNA from Absidia corymbifera; lane 12, human DNA.

FIG. 3.

Amplification and restriction patterns obtained from PCR-RFLP on fungal cultures (A) and on clinical samples (B and C). Lanes M, 100-bp ladder; lane 1, nondigested PCR product (Mucor sp.); lane 2, digestion with AclI (Absidia corymbifera); lane 3, digestion with AflII (Mucor sp.); lane 4, digestion with BmgBI (Rhizopus sp.); lane 5, digestion with PpuMI (Rhizomucor sp.); lanes 6 to 8, PCR-RFLP on a cutaneous biopsy specimen of a back lesion. The first 100-bp band of the ladder is not distinguishable on this photograph. Lanes 9 and 10, PCR-RFLP on a biopsy specimen from pulmonary abscess; lanes 6 and 9, nondigested amplicons; lane 7, digestion with BmgBI (Rhizopus sp.); lane 8, digestion with AseI (Rhizopus microsporus or Rhizopus azygosporus); lane 10, digestion with AclI (Absidia corymbifera).