Emericella quadrilineata as Cause of Invasive Aspergillosis

This opportunistic fungus is frequently misidentified because of its morphologic similarity to E. nidulans.

T he genus Aspergillus includes >250 species; ≈20 have been reported to cause opportunistic infections in humans. The most important human pathogens in this genus are A. fumigatus, A. fl avus, A. niger, A. terreus, and Emericella nidulans (anamorph: A. nidulans) (1). A. fumigatus is the most common cause of invasive aspergillosis, a condition associated with substantial severity and mortality rates (2). Invasive infections caused by E. nidulans are uncommon in animals and humans (3)(4)(5); in humans they appear to occur predominantly in patients who have chronic granulomatous disease (CGD), a rare disorder of phagocytes in which the absence of both superoxide and hydrogen peroxide production in phagocytes predisposes patients to bacterial and fun-gal infections. Invasive E. nidulans infections in this patient group are associated with higher mortality rates than those caused by A. fumigatus (6,7). The most common site of infection is the lungs; other manifestations are subcutaneous abscesses or liver abscesses, suppurative adenitis, osteomyelitis, fungemia, cellulitis, and meningitis (7,8). Within the genus Emericella, other species have only rarely been identifi ed as agents of human or animal infections.
The identifi cation of E. nidulans in clinical microbiology laboratories is commonly based on the characteristic microscopic morphology, the production of hülle cells, or the production of ascospores. A. fumigatus is identifi ed by its heat tolerance; other species fail to grow when incubated at high temperature, typically 48°C.
We recently noted a cluster of infection or colonization by E. quadrilineata, a species closely related to E. nidulans. Within a 3-month period, 4 cases were identifi ed at the Radboud University Nijmegen Medical Center. No apparent epidemiologic link between the cases was found because each patient was cared for in a different ward, and 2 patients with invasive aspergillosis were admitted directly from home. One of the latter 2 patients was a 10-year-old boy with X-linked CGD and a probable diagnosis of invasive pulmonary aspergillosis (9); the other patient was a 60-year-old man who had chronic lymphocytic leukemia and in whom cerebral aspergillosis later developed and was confi rmed by biopsy. From the other 2 patients, who had no signs and symptoms of invasive fungal disease, E. quadrilineata was cultured from respiratory specimens. No laboratory contamination was evident during the period in which the cluster occurred. No subsequent cases were identifi ed. Morphologic species identifi cation was diffi cult, and we had to rely on sequence-based identifi cation, which prompted this investigation of the role of E. quadrilineata as a causative agent of invasive aspergillosis.

Data and Strain Collection
We searched the PubMed literature for cases of infections caused by E. quadrilineata (anamorph: Aspergillus tetrazonus) or E. nidulans; search terms were Emericella, quadrilineata, tetrazonus, nidulans, and aspergillosis. For those articles that described infections by E. quadrilineata or E. nidulans, we asked the authors to send us their isolates for sequence analysis. We also approached colleagues who care for patients with CGD or might otherwise have a collection of E. nidulans isolates. We also searched our department's fungal culture collection for E. nidulans isolates. It is our policy to store all Aspergillus isolates cultured from clinical specimens sent to our laboratory, regardless of the clinical relevance of the isolate. Finally, we added E. nidulans and E. quadrilineata isolates deposited in the culture collection of the Centraal Bureau voor Schimmelcultures ([CBS], Utrecht, the Netherlands). The fi nal collection totaled 33 Emericella isolates, with 11 isolates from the CBS culture collection (type strains E. quadrilineata, E. nidulans, E. nidulans var. echinulata) and 1 isolate from the National Collection of Pathogenic Fungi. Ten isolates were from our own culture collection (including the 4 encountered in the cluster of cases), and 11 isolates were from 5 other medical centers; some of these isolates had been cultured as causes of infection and previously reported (online Appendix Table, available from www.cdc.gov/ EID/content/14/4/566-appT.htm) (8,(9)(10)(11)(12). Seven isolates had been cultured from patients with confi rmed invasive aspergillosis, and 2 were from patients with probable cases (online Appendix Table).

Morphologic Identifi cation
Aspergillus isolates are routinely identifi ed by their macroscopic colony morphology and the microscopic morphology of their anamorphic features. When teleomorph features were also used to identify an isolate, teleomorph nomenclature, such as Emericella spp., was used to report the strain. In addition, the isolates were incubated at 48ºC, which precludes the growth of most Aspergillus spp. except A. fumigatus.

Sequence-based Identifi cation
Sequence-based identifi cation in the routine clinical microbiology laboratory was carried out by sequencing of parts of the internal transcribed spacer (ITS) 1 and 2 regions. Total DNA of the Emericella cultures was extracted by using the MagNa Pure Total NA isolation kit (Roche Diagnostics Nederland BV, Almere, the Netherlands). Then the ITS 1 and 2 sequence was amplifi ed by PCR with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as described (13).
After purifi cation, the PCR product was sequenced with the BigDye Terminator v3 kit (Applied Biosystems, Foster City, CA, USA).
In addition to the above-mentioned sequenced-based identifi cation, parts of the β-tubulin and calmodulin genes were sequenced. The Emericella cultures were cultivated in 2 mL malt peptone broth by using 10% (vol/vol) of malt extract (Oxoid, Basingstoke, UK) and 0.1% (wt/vol) bacto peptone (Difco, Becton Dickinson, Le Pont de Claix, France). The cultures were incubated at 25°C for 7 days. DNA was extracted from the cells by using the Masterpure yeast DNA purifi cation kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer's instructions. Amplifi cation of part of the β-tubulin gene was performed by using the primers Bt2a and Bt2b (14,15). Amplifi cations of the partial calmodulin gene were set up as described (16). Sequence analysis was performed with the BigDye Terminator Cycle Sequencing Ready Reaction Kit for both strands. Sequences were aligned by using ClustalX software (17) and were improved manually.
Evolutionary distances between the sequences were calculated by using the Kimura formula (18) and DNA-DIST program of the PHYLIP program package (19). Phylogenetic trees were prepared by using the neighbor-joining method (20) and the NEIGHBOR program of the PHYLIP package. Bootstrap values were calculated from 1,000 replications of the bootstrap procedure by using programs SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE of the PHYLIP package (19,21). For parsimony analysis, PAUP* version 4.0 software was used (22). E. heterothallica was used as an outgroup in these experiments. The unique β-tubulin and calmodulin sequences were deposited in the GenBank nucleotide sequence database under accession nos. EF591677-EF591702.

Statistical Analysis
After being transformed logarithmically, MIC dilutions were compared by using the Mann-Whitney U test.
Data on growth at different antifungal drug concentrations were normalized by setting the corrected optical density of the growth control at 0% and the lowest optical density at 100%. Growth characteristics were analyzed by nonlinear regression analysis that used a 4-parameter logistic model and created a sigmoidal curve. Test runs determined deviation of the model, and goodness-of-fi t was tested by determining r 2 values. In addition to comparing MICs, we determined the drug concentration at which growth was 50% that of the control (50% maximal effective concentration [EC 50 ]) and calculated and fi tted the slope of the curve (GraphPad Prism, San Diego, CA, USA). For all drugs except caspofungin, the EC 50 values and slopes were compared for E. nidulans and E. quadrilineata.

Species Identifi cation
The 4 isolates from the cluster of cases grew on Sabouraud-dextrose agar as velvety, brownish-green colonies with a purplish reverse side. Conidiophores were light brown with hemispherical vesicles bearing metulae and biseriate philalides on the upper half. Conidia were spherical, smooth walled, subhyaline, fi nely roughened, and 3-4 μm in diameter. After ≈3 weeks of incubation, purple ascocarps formed, surrounded by characteristic hülle cells. Asci were spherical, 8 spored, 10-13 μm in diameter, and evanescent. The ascospores were reddish purple, lenticular, 5-6 × 3-4 μm, and smooth. The morphologic features were consistent with E. nidulans. As part of the diagnostic process, the isolates were incubated at 48ºC, and all isolates showed some growth, which was considered inconsistent with E. nidulans. Because of this discrepancy, sequence-based identifi cation was performed. However, all 33 isolates from the subgenus Nidulantes section that were analyzed in this study grew at 48°C, which indicates that incubation at this temperature does not fully distinguish between this section and A. fumigatus.

Sequence-based Analysis
The ITS sequence analysis of the 4 isolates was consistent with that of E. quadrilineata, although there were only 1 or 2 mismatches with the base-pair sequence of E. nidulans. The morphologic features of E. nidulans and E. quadrilineata are very similar; only the microscopic shape of the ascospores shows subtle differences. Ascospores of E. nidulans have 2 longitudinal crests, as opposed to E. quadrilineata, which has 4 short equatorial crests. The resolution of the ITS region was considered too low to unambiguously differentiate between E. nidulans and E. quadrilineata, and further sequence-based identifi cation was performed at CBS by using partial β-tubulin and calmodulin sequence data.
During analysis of part of partial β-tubulin gene sequences, we analyzed 367 bases of all 33 isolates. Among the polymorphic sites, 23 were phylogenetically informative. The neighbor-joining tree ( Figure 1) based on partial β-tubulin gene sequences had the same topologic features as 1 of the 2 maximum-parsimony trees constructed by the PAUP program (length 94 steps, consistency index 0.9787, retention index 0.9762). The calmodulin dataset included 489 bases, with 50 parsimony informative sites. The topologic features of the neighbor-joining tree ( Figure 2) and 1 of the 2 most parsimonious trees were the same (tree length 162, consistency index 0.9691, retention index 0.9854). Molecular data indicated that 12 of 33 isolates could be classifi ed as E. nidulans, all of which had previously been identifi ed as E. nidulans by microscopic examination of morphologic characteristics or other methods. For the 12 isolates classifi ed as E. quadrilineata, only 6 had previously been identifi ed accordingly. These 6 isolates included the 4 in our cluster, 1 from the CBS culture collection, and 1 previously reported as the cause of onychomycosis (12). The remaining 6 isolates had been previously identifi ed as E. nidulans (online Appendix Table). Of these, 1 belonged to the CBS culture collection, 1 was reported as the cause of cerebral aspergillosis (11), and 2 were from patients with CGD and confi rmed invasive aspergillosis.
A total of 4 isolates were classifi ed as E. rugulosa, 1 of which had been previously reported as E. nidulans (10). A total of 4 isolates were identifi ed as E. nidulans var. echinulata, 2 of which had caused invasive aspergillosis in patients with CGD and had been presumptively identi-  (Figure 3) (25).

In Vitro Susceptibility
The in vitro activity of antifungal agents against the Emericella isolates is shown in the online Appendix Table. Overall, terbinafi ne was the most active drug in vitro, followed by posaconazole, which was the most active azole. For statistical comparisons, we used 12 E. nidulans and 12 E. quadrilineata isolates. By comparing MICs, statistically signifi cant differences in drug activity were found for amphotericin B, voriconazole, and posaconazole (Table). These differences were also found when EC 50 values and slopes were compared for both species (data not shown). Comparison of minimum effective concentrations showed caspofungin to be signifi cantly more active against the E. nidulans isolates (p<0.05). Although only 4 E. nidulans var. echinulata isolates were analyzed, the susceptibility profi le of these isolates was similar to that of E. quadrilineata (and not of E. nidulans); for amphotericin B, MICs were low, and for caspofungin, MICs were high.

Literature Review
Three cases of infection due to E. quadrilineata have been documented. One patient was a 60-year-old man from northern India, who had a fi ngernail infection that affected all 5 nails of 1 hand. The strain was repeatedly cultured from 1 nail, and septate hyphal elements were seen in a portion of an excised nail. The patient was treated with itraconazole, but the response could not be evaluated (12). Invasive aspergillosis caused by E. quadrilineata has been described for 2 patients, both of whom had sinusitis. One of these, a 28-year-old woman, had acute nonlymphoblastic leukemia and had undergone allogeneic bone marrow transplantation. She developed sinusitis with orbital involvement 2 months after transplantation. The diagnosis was confi rmed by biopsy, and the patient was successfully treated with a combination of surgical debridement, granulocyte transfusions, and intravenous amphotericin B-cholesterol sulfate colloidal dispersion (26). The other patient was a 28-year-old man who had received an allogeneic bone marrow transplant for acute myeloid leukemia. Left orbital swelling, facial pain, and nasal congestion developed 68 days after transplantation. E. quadrilineata was cultured from a biopsy specimen; the patient was successfully treated with external ethmoidectomy, granulocyte transfusions, and topical and systemic therapy with a lipid formulation of amphotericin B. The fungal infection resolved (27).

Discussion
Until this report, 2 cases of invasive aspergillosis caused by E. quadrilineata had been described; each case had been reported as sinusitis in patients who had undergone bone marrow transplantation for hematologic malignancy. We add 1 case of central nervous system aspergillosis and 3 cases of invasive pulmonary aspergillosis in patients with CGD. The 3 cases may not be surprising because E. quadrilineata is very closely related to E. nidulans, a fungus known to cause infections in humans (3,5,(28)(29)(30), primarily in patients with CGD (6,7). Emericella spp. other than E. nidulans are less frequently reported as causative agents of infectious disease. In addition to the above-mentioned human infections, E. quadrilineata has been identifi ed as a causative agent of mycosis in animals (31). We have found only 1 report each of E. rugulosa (4) and E. nidulans var. echinulata (32) as the cause of human or animal infections.
Discriminating E. nidulans and E. quadrilineata by morphologic characteristics is virtually impossible. Only the ascospore ornamentation differs, and the subtle differences cannot be seen by using light microscopy. The most frequently used technique for their unambiguous identifi cation is scanning electron microscopic examination of the lining of the ascospores (25). However, fruiting bodies are usually formed after a rather long incubation period (1-2 weeks). And although E. nidulans was found to form cleistothecia in the human body (33), clinical isolates often lose their ability to form sexual reproductive structures and ascospores (34,35). Given these diffi culties, we anticipate that reliance on phenotypic characteristics alone would cause misidentifi cation of E. nidulans, rather than correct identifi cation of E. quadrilineata, as the cause of invasive aspergillosis. The sequence-based analysis showed that this was indeed the situation; 50% of the E. quadrilineata isolates had previously been identifi ed as E. nidulans. Among these was a case of cerebral aspergillosis, the second case observed in our cluster of cases. Despite the close morphologic and genetic relatedness between E. nidulans and E. quadrilineata, the activity of antifungal agents differed signifi cantly, which supports the conclusion that biological differences exist between these species. The triazoles were active in vitro; posaconazole showed the greatest activity, which is also observed for most Aspergillus spp. Although signifi cant differences were found for activity of voriconazole and posaconazole, these differences appear to be not clinically relevant, given the small differences in MIC values (Table). However, for amphotericin B and caspofungin, the observed differences in activity may be clinically important. Amphotericin B was less active against E. nidulans than against E. quadrilineata. In vitro resistance of E. nidulans against amphotericin B has been recognized (36), although the testing method has been shown to substantially affect the activity found (37). However, lack of activity of amphotericin B has also been reported in experimental models of infection and in cases reported in the literature (8,38). Caspofungin was less active against E. quadrilineata than against E. nidulans. Caspofungin was shown to be effective against E. nidulans in a murine mod-el of systemic infection (38), but no data are available for E. quadrilineata. Although the allylamine terbinafi ne is not used for treatment of patients with invasive aspergillosis, the drug is highly active against both E. nidulans and E. quadrilineata. The isolates were inhibited at concentrations as low as 0.015 mg/L. Identifi cation of molds primarily relies on morphologic criteria such as the macroscopic colony morphology and the microscopic morphology of the conidia and the structures bearing the conidia. Morphologic identifi cation underestimates differences among species and among members of the same species. This was recently shown for the section Fumigati, in which A. lentulus and A. udagawae were among isolates phenotypically identifi ed as A. fumigatus (34,35). We made a similar observation when 10 of 33 Emericella isolates were found to be misidentifi ed. Correct species demarcation is important from a taxonomic viewpoint but can also have clinical relevance. Within the Aspergillus section of Fumigati, the newly identifi ed species A. lentulus was shown to be more resistant than A. fumigatus to antifungal drugs (35). Therefore, correct species identifi cation will affect the choice of antifungal therapy. Differences in drug activity were also apparent in the Emericella spp. we examined. Another important reason for correct species identifi cation is the detection of outbreaks of infection, which could warrant interventions to prevent invasive fungal infection in immunocompromised persons or lead to epidemiologic surveys to identify sources of spread of fungal spores. However, the resolution of sequencing of the ITS region is too low to reliably dif-   ferentiate between E. nidulans and E. quadrilineata; therefore, in vitro susceptibility testing might be appropriate in those laboratories that do not have access to sequencing of β-tubulin and calmodulin genes. Molecular techniques in addition to morphologic identifi cation have identifi ed a role of E. quadrilineata as an opportunistic fungal pathogen, especially in patients with CGD and in those with hematologic malignancy. These molecular techniques will help identify and discriminate more accurately within the current fungal species and will give more insight into the pathogenesis of fungal infection.