Identification of Strains in Culture

By using several molecular targets (Table 7.2) and by increasing the number of available DNA sequences in international databases (e.g. GenBank), several studies have shown that accurate molecular identification to the species level of the zygomycetes pathogenic for humans is feasible (Machouart et al., 2006; Schwarz

Table 7.2 Molecular methods used for identification of zygomycetes strains in culture


Target rDNA "region



Several species 28S

Several species ITS

Several species 18S

Several species ITS

Several speciesb 28S

Rhizopus species ITS

Apophysomyces ITS


Rhizopus microsporus ITS (Case report)

Rhizomucor pusillus ITS

(Case report) Rhizopus microsporus 18S (Case report)

PCR (specific primers for

13 taxons) PCR + Sequencing PCR + RFLP

PCR + Sequencing MicroSeq D2 large-subunit ribosomal DNA sequencing kit Multiplex PCR PCR + RFLP

PCR + hybridization with specific probes on DNA microarray PCR + Sequencing


Voigt et al.(1999)

Schwarz et al. (2006) Machouart et al. (2006)

Nagao et al. (2005) Chakrabarti et al.

a ribosomal DNA.

b Inaccurate identification in almost 50% of the cases.

et al., 2006; Voigt et al., 1999). In a large study, Voigt et al. (1999) have PCR amplified and sequenced the nuclear small subunit (18S) ribosomal DNA and the variable domains of the nuclear large subunit (28S) ribosomal DNA from 42 zygomycetes isolates including all the species known to be pathogenic for human and animals. They have shown that the 18S sequences are highly conserved but that sequences of the D1 and D2 domains of the 28S rDNA were more variable. This variability was used to design specific primers for 13 different species that could be used for identification. Analysis of intra and interspecies variability of ITS sequences obtained from 54 zygomycetes isolates belonging to 16 different species has also been evaluated recently (Schwarz et al., 2006). The whole ITS1-5.8S-ITS2 region was PCR amplified with universal fungal primers, and sequenced. Overall, sequences comparison showed that for a given species, the variability between isolates was very low. In contrast, sequences were very different between species (Table 7.3). These results showed that sequencing of ITS region is a reliable method for an accurate identification of zygomycetes to the species level. A different approach combining PCR and RFLP has been used in another study (Machouart et al., 2006). By amplification of a part of the 18S r DNA with a mix of 4 specific sense primers and a degenerated antisense primer followed by restriction enzyme digestion, it has been shown that specific patterns were obtained, allowing for precise species identification. Both ITS sequencing and PCR amplification of 18S combined with RFLP have been successfully employed for strain identification in

Table 7.3 Intraspecies and interspecies variability of ITS1-5.8S-ITS2 sequences for five zygomycetes pathogenic for humans


Percentage of similarity

Within species

Between species

Rhizopus oryzae Rhizopus microsporus Rhizomucor pusillus Absidia corymbifera Mucor circinelloides

99-100 99-100 100

99-100 99

36-61 33-61




individual case reports (Larche et al., 2005; Iwen et al., 2005). Alternative techniques have also been used. In one case report of R. microporus peritonitis, identification of the causative organism was achieved by hybridization of the panfungal PCR product against a set of specific probes on a DNA microarray (Monecke et al., 2006). More specific studies (Table 7.2) have also demonstrated the usefulness of molecular-based technique for identification of Apophysomyces elegans (Chakrabarti et al., 2003) and Rhizopus species (Nagao et al., 2005).

The ITS sequence-based method has been used in a recent prospective surveillance study of 27 patients with zygomycosis in a large cancer center (Kontoyiannis et al., 2005). Interestingly, molecular identification of strains showed that morphological-based identification was erroneous in >20% of the cases. These results highlighted the difficulty of identification of zygomycetes to the species level by mycological standard procedures for a nonspecialized laboratory. Although these different recent studies (Kontoyiannis et al., 2005; Schwarz et al., 2006; Voigt et al., 1999; Machouart et al., 2006) demonstrated that molecular approaches are useful for species identification, it should be noted that these techniques need reliable methodologies and accurate as well as comprehensive sequence databases for comparison purposes. Indeed, as previously discussed (Greenberg et al., 2004) the use of the MicroSeq D2 large-subunit DNA sequencing kit, a commercial kit for molecular identification of filamentous fungi, led to misidentification of the zygo-mycetes isolates (including the most frequently pathogenic species) in almost 50% of the cases (Hall et al., 2004). These misidentifications could be related, at least in part, to the incomplete sequences library associated to the kit.

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