Molecular Typing of Australian Scedosporium Isolates Showing Genetic Variability and Numerous S. aurantiacum

Molecular typing showed genetic diversity, dismissed 2 suspected outbreaks of scedosporiosis, and identified multiple strains of the newly described species S. aurantiacum.

One hundred clinical isolates from a prospective nationwide study of scedosporiosis in Australia (2003)(2004)(2005) and 46 additional isolates were genotyped by internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, ITS sequencing, and M13 PCR fi ngerprinting. ITS-RFLP and PCR fi ngerprinting identifi ed 3 distinct genetic groups. The fi rst group corresponded to Scedosporium prolifi cans (n = 83), and the other 2 comprised isolates previously identifi ed as S. apiospermum: one of these corresponded to S. apiospermum (n = 33) and the other to the newly described species S. aurantiacum (n = 30). Intraspecies variation was highest for S. apiospermum (58%), followed by S. prolifi cans (45%) and S. aurantiacum (28%) as determined by PCR fi ngerprinting. ITS sequence variation of 2.2% was observed among S. apiospermum isolates. No correlation was found between genotype of strains and their geographic origin, body site from which they were cultured, or colonization versus invasive disease. Twelve S. prolifi cans isolates from 2 suspected case clusters were examined by amplifi ed fragment length polymorphism analysis. No specifi c clusters were confi rmed.
Since scedosporiosis, in particular that caused by S. prolifi cans, is often refractory to treatment (3,5), preventive strategies are of paramount importance. However, the epidemiology and mode of transmission of infection are not well understood. Furthermore, the environmental reservoir of S. prolifi cans is unknown. Molecular typing techniques now provide the means to elucidate the epidemiology of Scedosporium infections and to investigate potential case clusters (16,18,19). Strains recovered from patients with cystic fi brosis have demonstrated a high degree of genetic variability (10,20), although a single genetic profi le predominated in 1 study (8). The degree of genetic variation within S. prolifi cans is more controversial. Two studies have reported low to no intraspecies genetic heterogeneity (16,21), while a third noted substantial genetic diversity (19). The results of these studies may be biased because they included only small numbers of isolates from specifi c patient populations. Genetic variability among S. aurantiacum has not yet been studied.
In this study, we used 4 molecular tools to examine genetic variation among a large number of Australian clinical Scedosporium isolates: 1) internal transcribed spacer (ITS)-based restriction fragment length polymorphism (ITS-RFLP) analysis; 2) DNA sequence analysis of the ITS region (selected isolates); 3) PCR fi ngerprinting using the microsatellite specifi c core sequence of phage M13; and 4) amplifi ed fragment length polymorphism (AFLP) analysis (isolates from suspected case clusters). We also searched for the newly described species, S. aurantiacum and for genetic clustering of strains according to their geographic origin, body site from which they were cultured, and ability to cause invasive disease.

Scedosporium Isolates and Data Collection
A total of 146 Scedosporium isolates were studied (online Technical Appendix, available from www.cdc. gov/EID/content/14/2/282-Techapp.pdf). Forty-six were from the culture collection at the Clinical Mycology Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Sydney, Australia. For these isolates, the following data were captured: demographic information, patient coexisting conditions and risk factors (summarized in the online Technical Appendix). The remaining 100 isolates were obtained through a national, prospective, laboratory-based surveillance for scedosporiosis in Australia (the Australian Scedosporium [AUSCEDO] Study) from January 2003 to December 2005. The following data were collected: clinical status, risk factor (defi ned according to published risk factors for scedosporiosis [4,[12][13][14][15]), major comorbidity (based on the International Classifi cation of Diseases, 10th revision, Australian Modifi cation [ICD-10 AM] diagnostic classifi cation system [22]), isolated species, treatment and outcome. Scedosporium strains obtained from a single colony from the primary isolation plate from all patients were forwarded to the Molecular Mycology Research Laboratory, Westmead Hospital, for genotyping. Isolates were identifi ed as S. prolifi cans or S. apiospermum by standard phenotypic methods (23). Species were confi rmed as S. prolifi cans or S. apiospermum, and S. aurantiacum was identifi ed (11) by ITS-RFLP analysis.

Defi nitions
An episode of scedosporiosis was defi ned as the incident isolation of Scedosporium spp. from any body site. Two or more episodes, fulfi lling the case defi nition and occurring in different patients that were epidemiologically linked were defi ned as a potential case cluster. Invasive disease was defi ned according to the European Organization for Treatment of Cancer/Mycoses Study Group criteria for "defi nite" or "probable" infection (24). All other patients not fulfi lling these criteria, including those with "possible" infection were considered colonized. Coincident hospital renovations or construction was considered to be a potential risk factor if major work was undertaken within 3 months before the isolation of Scedosporium spp. from a patient.

Description of 2 Potential Case Clusters
The fi rst potential case cluster involved 8 patients located in the same hematology/hemopoietic stem cell transplant (HSCT) unit at the Alfred Hospital, a large university hospital in Melbourne (September 2000-October 2001; [15]). The second consisted of 3 patients located in the same hematology/HSCT ward at Westmead Hospital a major university hospital in Sydney (September 2003-January 2004; unpub. data). Details of the patients involved in these suspected case clusters are summarized in the online Technical Appendix). On each occasion, patient isolates were submitted for genetic analyses to inform infection control responses (see Results).
Genomic DNA was isolated as described previously (18). The ITS1, 5.8S, and ITS2 regions of the rDNA gene cluster were amplifi ed with the primers SR6R and LR1 (Table 1) as described previously (25). Amplicons were double digested with the restriction endonucleases Sau96I and HhaI (New England BioLabs, Ipswich, MA, USA) in accordance with the manufacturer's recommendations. Digested products were separated by electrophoresis in 3% agarose gels at 100 V for 3-4 h. Banding patterns were analyzed visually.

ITS Sequencing
Eleven isolates, representative of each of 3 ITS-RFLP patterns obtained, were selected for ITS sequencing: ITS-RFLP profi le A (S. . The ITS region was amplifi ed as described above and commercially sequenced in both directions by using SR6R or LR1 (Table 1) as forward and reverse primers.

PCR Fingerprinting
The minisatellite-specifi c core sequence of the wildtype phage M13 was used as a single primer for PCR fi ngerprinting (Table 1). Amplifi cation reactions were performed as previously described (18). Blank control tubes containing all reagents except template DNA were included for each run; each sample was analyzed at least twice. PCR products were separated by electrophoresis on 1.4% agarose gels at 60 V for 14 cm. Strains were defi ned to be identical if their PCR fi ngerprinting profi les had a similarity of >97% ( = 1 band difference). Reproducibility of the PCR fi ngerprinting technique was accessed by re-amplifying 1 strain of each of the 3 Scedosporium spp. with all PCR amplifi cations carried out and re-running those on each gel.

AFLP Analysis
AFLP analysis was performed as described previously by using either EcoRI-GT 6-FAM-labeled and MseI-GT or EcoRI-TC 6-FAM-labeled and MseI-CA as selective primer pairs (QIAGEN, Valencia, CA, USA; Table 1) (26). All samples were analyzed by using the ABI Prism 3730 system (Applied Biosystems, Foster City, CA, USA). Data collation, fragment sizing, and pattern analyses were performed with GeneMapper software version 3.5 (Applied Biosystems). Only electrophoregram peaks above 1,000 fl uorescent units were scored for the presence or absence of bands of the same size (range 50-500 bp) relative to the GeneScan 500 LIZ DNA size standard (Applied Biosystems). Only bands detected in duplicate AFLP experiments were included in the analysis.

Clinical Data
Statistical analysis was performed by using SPSS version 10.0.07 (SPSS, Chicago, IL, USA) and EpiInfo version 6.0 (Centers for Disease Control and Prevention, Atlanta, GA, USA). Proportions were compared by using the χ 2 or Fisher exact test. A p value <0.05 was statistically signifi cant.

ITS Sequences
ITS sequences obtained from 11 isolates (see above) were aligned with the ITS sequences of the following reference strains obtained from GenBank: S. apiospermum

PCR Fingerprinting Patterns and AFLP Fragments
PCR fi ngerprinting patterns were analyzed by using the 1D gel analysis module (BioGalaxy [BioAware, Hannut, Belgium]) in BioloMICS version 7.5.30 (BioAware). Images were normalized for lane to-lane differences in mobility by the alignment of patterns obtained on multiple loadings of the 1kb DNA size marker (GIBCO-BRL, Gaithersburg, MD, USA). The unweighted-pair group method by using arithmetic averages and the procedures of Nei and Li (28), both implemented in BioloMICS, were used to generate dendograms based on the coeffi cient of similarity (29) between the isolates. In addition, principal coordinate analysis (PcoA; BioloMICS) was conducted to give an overall representation of the observed strain variation. AFLP fragments were analyzed with BioloMICS.

Results
A total of 146 Scedosporium isolates from 120 episodes (119 patients) were studied (online Technical Appendix). Demographic data were available for 108 (90%) episodes and coexisting conditions and risk factor data for 115 (95.8%). Most episodes were reported from New South Wales (64.2%), followed by Victoria (19.2%) and Western Australia (9.2%). The male: female ratio was 1.3: 1. The major patient coexisting conditions and known risk factors for scedosporiosis are summarized in the online Technical Appendix. Thirty-nine patients (32.7%) had no underlying medical condition. Coincident building construction was noted in 27 cases (22.5%). Scedosporium isolates were associated with invasive disease in 46 (38.3%) instances; the remaining 74 (61.7%) were isolated from patients who were colonized (Table 2).

Molecular Typing of Scedosporium Isolates
All 146 isolates were examined by ITS-RFLP analysis and PCR fi ngerprinting. ITS sequencing was performed on 11 strains as described above. AFLP analysis was performed only for selected S. prolifi cans isolates, including the isolates of the suspected case clusters and isolates representative of the S. prolifi cans branches identifi ed by PCR fi ngerprinting (online Appendix Figure 1, available from www.cdc.gov/EID/content/14/2/282-appG1.htm).

ITS-RFLP Analysis
RFLP analysis found 1 RFLP profi le specifi c for S. prolifi cans isolates (ITS-RFLP profi le A) and 2 profi les (ITS-RFLP profi les B and C) for isolates previously phenotypically identifi ed as S. apiospermum (Figure 1, panel  A). ITS-RFLP profi le B corresponded to S. apiospermum and ITS-RFLP profi le C to the newly described species, S. aurantiacum.

ITS Sequencing
Sequencing of the ITS 1, 5.8S, and ITS2 regions of the 11 strains, representative of each of the 3 ITS-RFLP profi les found the following results: BLAST  Phylogenetic analysis of the sequences demonstrated 3 distinct clades, the fi rst corresponding to S. prolifi cans as the basal clade. The other 2 corresponded to the 2 more closely related but clearly distinct clades, S. apiospermum, and S. aurantiacum (Figure 2). S. apiospermum showed intraspecies sequence variation of 2.2% compared to S. aurantiacum and S. prolifi cans, which displayed no variation. PCR fi ngerprinting delineated 3 major clusters concordant with S. apiospermum, S. aurantiacum, and S. prolificans (online Appendix Figure 1; Figure 1, panel B; Figure  3). Clusters corresponding to S. aurantiacum and S. prolificans were substantially more densely grouped than the S. apiospermum cluster (Figure 3). PCR fi ngerprinting profi les showed polymorphisms within each of the 3 species, allowing for a clear differentiation, by using a "cut-off point" of >97% similarity. Multiple isolates from the same patient obtained from different anatomic sites (online Technical Appendix) had identical or >97% similarity between their PCR fi ngerprints, except for 1 patient (patient 118). In 8 instances, PCR fi ngerprinting showed that patients were infected with 2 different strains: (patients 1, 10, 27, 57, 83 99, 118 [online Appendix Figure 1, online Technical Appendix]). For all species, genetic profi les were independent of geographic origin, body site of isolation or whether the patient was infected or colonized (online Appendix Figure 1). Profi les were also independent of patient comorbidityity and risk factors for scedosporiosis (data not shown). Intraspecies PCR fi ngerprinting variation was highest for S. apiospermum (58%) followed by S. prolifi cans (45%) and S. aurantiacum (28%) (online Appendix Figure 1).  Figure 1) were further investigated by AFLP typing. S. prolifi cans was not isolated from the environment in either setting despite extensive sampling. The AFLP bands were found to be 50-493 bp by using the primers EcoRI-GT and MseI-GT (data not shown), and from 52-468 bp by using the primers EcoRI-TG and MseI-CA (online Appendix Figure 2, available from www.cdc.gov/EID/content/14/2/ 282-appG2.htm). These 35 isolates exhibited 32 different AFLP profi les, with isolates from the same patient (patients 1, 73, and 119) showing identical profi les (online Appendix Figure 2), confi rming the PCR fi ngerprinting results (online Appendix Figure 1). PcoA of the combined AFLP and PCR fi ngerprinting data demonstrated no clustering of these isolates (Figure 4), which ruled out the possibility of nosocomial transmission.

Discussion
We examined genetic variation among a large number of population-derived Scedosporium isolates across the Australian continent. In line with previously reported genetic variability in the S. apiospermum/P. boydii species complex (30-32), we observed 2 distinct ITS-RFLP patterns among S. apiospermum isolates, showing the presence of the newly described species S. aurantiacum (11). Notably, we have identifi ed by ITS sequencing that S. aurantiacum comprised 45% of the current collection of Australian "S. apiospermum" isolates and documents genetic variability within S. aurantiacum.
Epidemiologic investigation of Scedosporium infection requires accurate identifi cation and typing. S. apiospermum, S. aurantiacum, and S. prolifi cans were clearly distinguished from each other by PCR fi ngerprinting and ITS-RFLP analysis. This is consistent with previous rDNA sequence-based studies (30,33,34). The observation of 2 distinct genetic groups, corresponding to S. aurantiacum and S. apiospermum, supports the proposal that S. aurantiacum be designated a separate species (11). This proposal is also supported by the 5%-10% ITS sequence variation found between S. aurantiacum and S. apiospermum compared to an absence of intraspecies variation in S. aurantiacum and S. prolifi cans and a 2.2% variation in S. apiospermum (30,32; current study).
The intraspecies PCR fi ngerprint variation in S. prolifi cans (45%) was greater than that in S. aurantiacum but less than that in S. apiospermum. Given that S. aurantiacum is phylogenetically more closely related to S. apiospermum than to S. prolifi cans (11,33; current study), this result was unexpected. It may be due to different evolutionary pressures acting on the 3 different species or the relatively small numbers of S. aurantiacum isolates studied to date. The moderate genetic diversity among S. prolificans confi rms previous fi ndings (19). Despite the observed polymorphisms, PcoA of PCR fi ngerprint profi les showed dense clustering for S. prolifi cans (Figure 3), which is consistent with the low to absent intraspecies variability in S. prolifi cans found by others (20,21,33). These apparently contradictory fi ndings emphasize the importance of choosing the optimum molecular typing tool with the most appropriate discriminatory power for the organism or species being studied. The high degree of intraspecies variation detected by PCR fi ngerprinting and AFLP analysis supports the use of these methods to establish genetic relatedness between isolates recovered from different patients or multiple isolates from the same patient. In comparison, the variation detected by ITS-RFLP analysis and ITS sequencing corresponded to interspecies variation, which makes those techniques ideal for identifi cation of any given isolate to the species level. Individual patients are most likely infected or colonized with genetically distinct strains (19-21; this study). Identical PCR fi ngerprint or AFLP profi les were noted in multiple isolates recovered simultaneously from different anatomic sites in the same patient (21; current study). However, 8 patients were infected or colonized by at least 2 strains as refl ected by their different genetic profi les (online Technical Appendix). Possible explanations include concomitant infection by multiple strains from which only a restricted number were recovered, or colonization by 1 strain followed by infection or colonization with a second strain of a different genotype. Longitudinal genotyping studies are required to determine the likelihood that persistence of >1 genotypes later leads to clinically important infection or whether the disease is more likely to be caused by an unrelated genotype. In this context, the development of a multilocus sequence typing scheme for Scedosporium, as has been developed for Candida spp. (35), would be of great advantage to overcome interlaboratory reproducibility problems, which are known to be associated with PCR fi ngerprinting or AFLP data. However, developing such a scheme remains cumbersome due to the current lack of genomic data of Scedosporium spp.
For all 3 Scedosporium spp., there was no clustering of strains according to their geographic or body site of origin or by their ability to cause invasive disease, which is in agreement with previous fi ndings for S. apiospermum (20,30) and S. prolifi cans (16,17,21). Of note, no specifi c genotypes were associated with underlying medical conditions or risk factors. Compared with S. apiospermum and S. aurantiacum S. prolifi cans was more frequently associated with coincident hospital renovation, and invasive disease, had a greater predilection to cause disseminated infection and was the predominant species isolated from blood and other sterile sites (12)(13)(14)(15)(16)36; current study). Our preliminary observations indicate that the epidemiology and clinical relevance of recovering S. aurantiacum may be similar to that of S. apiospermum. S. aurantiacum has been reported to colonize the respiratory tract of at-risk patients (8). 288 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 2, February 2008   In addition to PCR fi ngerprinting, we applied AFLP analysis to investigate the possibility of 2 case clusters caused by S. prolifi cans. AFLP analysis was chosen as an independent technique using 2 combinations of selective primers (Table 1), which have been previously shown to have good discriminatory power for fungal strain differentiation (26). Both techniques, previously used to identify outbreak strain clusters in the recent cryptococcosis outbreak on Vancouver Island (37), generated in the current situation distinct patterns from all S. prolifi cans isolates except serial isolates obtained from the same patient (online Appendix Figures 1, 2). These fi ndings exclude the occurrence of nosocomial outbreaks or any close relationship with the nonoutbreak isolates, a result similar to those obtained previously (38). Overall nosocomial acquisition of infection has been demonstrated in only 2 instances (16,17). Scedosporium spp. have rarely been isolated from hospital air or from indoor or outdoor surface samples (13,39,40, current study), which raises questions about the mode of acquisition by patients and the mechanisms of the selection of this specifi c fungus as an infectious agent from among the high biodiversity of environmental molds.
In conclusion, ITS-RFLP analysis is a powerful tool for distinguishing between isolates of the new species S. aurantiacum and S. apiospermum. PCR fi ngerprinting and AFLP analysis are useful techniques for determining genetic relatedness between Scedosporium isolates and for investigating potential case clusters.