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Journal of Clinical Microbiology, March 2006, p. 1175-1177, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.1175-1177.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

CASE REPORT

Use of DNA Sequencing Analysis To Confirm Fungemia Due to Trichosporon dermatis in a Pediatric Patient

Shelly R. Gunn,^1* Xavier T. Reveles,² Jeanette D. Hamlington,² Lee C. Sadkowski,³ Teresa L. Johnson-Pais,² and James H. Jorgensen¹

Department of Pathology,¹ Department of Cellular and Structural Biology, University of Texas Health Science Center,² VA Mycology Reference Laboratory, Audie Murphy Memorial Veterans Hospital, San Antonio, Texas³

Received 7 October 2005/ Returned for modification 8 November 2005/ Accepted 30 November 2005

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This is the first reported case of human disease caused by Tricosporon dermatis, an organism recently transferred to the genus Trichosporon from Cryptococcus and now confirmed to be a human pathogen.

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A 13-month-old male with a history of autoimmune enteropathy developed a fever of unknown origin (FUO) and malaise. The patient was the product of a full-term uncomplicated pregnancy and delivery. At 9 days of life he had a rotavirus infection and subsequently developed a malabsorption-dysmotility syndrome. The etiology of his enteropathy was unknown, but it was suspected to be autoimmune. He had been on immunosuppressant therapy, including prednisolone and tacrolimus; and his caloric needs had been maintained with total parenteral nutrition through a central venous catheter (CVC) since his first month of life. Numerous bacterial line infections had required catheter replacement, and at the time of this presentation he had a Broviac catheter in place in his right internal jugular vein. His physical examination was notable for rhinitis with a temperature of 101.0°F, and the differential diagnosis for his FUO included viral illness versus bacterial line infection. The patient had a normal total white blood cell count of 5.01 x 10⁶ per liter, with relative lymphopenia (5%) and neutrophilia (73%). Following the collection of blood samples for microbiologic analysis, the patient was started on intravenous vancomycin therapy.

The blood specimens were taken from both the central line and a peripheral vein and were inoculated into BACTEC Plus Aerobic/F culture vials and processed in a blood culture system (BACTEC 9240 fluorescent series instruments; BD Diagnostic Systems, Sparks, MD). Growth was detected in the blood cultures after 48 h, with gram-positive organisms and yeast observed by microscopic examination. Subcultures were performed onto Trypticase soy sheep blood agar, which grew a coagulase-negative Staphylococcus, and CHROMagar, which produced mucoid mauve-colored colonies at 30°C after 24 h of incubation. Microscopic examination of subcultures grown on cornmeal agar revealed hyphae with the formation of arthroconidia and lateral and terminal blastoconidia (Fig. 1). The organism was found to be urease positive on Christensen's urea and displayed robust growth on Mycosel agar containing 0.01% cycloheximide. Urease positivity and cycloheximide resistance combined with the macroscopic and microscopic morphologies of the organism were consistent with the characteristics of Trichosporon spp. Further biochemical analysis with the VITEK 1 and 2 systems (bioMerieux, Inc., Durham, NC) identified Trichosporon mucoides with excellent confidence levels and t indices of 1.0 and 96.55, respectively. A second blood sample obtained 3 days after the initial sample was obtained, as well as a third sample obtained 8 days later, were again positive for T. mucoides. Based upon the initial colony morphology, which resembled a Candida sp. (especially Candida glabrata), a fluconazole susceptibility test was performed by the Clinical and Laboratory Standards Institute (Wayne, PA) procedure. On the following day, the zone diameter of 26 mm easily exceeded the 19-mm breakpoint for fluconazole susceptibility. The clinicians elected to remove the patient's catheter and treat the infection with voriconazole. After he remained afebrile for several days, the patient underwent catheter replacement and was discharged to home. The final report listed coagulase-negative Staphylococcus and Trichosporon mucoides as the organisms isolated from the patient's blood.

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FIG. 1. Wet mount of T. dermatis after 48 h of growth on cornmeal agar at 37°C demonstrating hyphae with the formation of arthroconidia.

Although the organism was positively identified as T. mucoides by the VITEK 1 and 2 systems, retrospective genetic analysis was performed to confirm the physiologic and biochemical findings by using a well-described PCR-based method for the rapid and specific identification of the medically relevant Trichosporon species. This technique incorporates PCR primers designed from DNA sequences within the conserved regions of the rRNA exons. These primers amplify variable sequences found in the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the tandemly repeated 18S, 5.8S, and 26S fungal rRNA gene loci. The species-specific sequences contained within the ITS1 region for the medically relevant Trichosporon spp. are 13 bp in length and in some cases differ by only a single nucleotide (11). The IGS1 sequences range in length from 195 to 719 bp (9), and thus, both the length of the amplified region and the exact sequence of the DNA can be used to advantage for the identification of Trichosporon spp. The IGS1 sequences have recently been shown to identify unambiguously all Trichosporon isolates to the species level (7).

In preparation for the sequencing studies, the blood isolate was grown to a stable state at 37°C in RPMI 1640 with morpholinepropanesulfonic acid buffer (Hardy Diagnostics). Briefly, yeast cells were collected by centrifugation; resuspended in distilled H₂O (dH₂O); combined with a mixture of 2% Triton X-100, 1% sodium dodecyl sulfate, 100 mM NaCl, 10 mM Tris-Cl, and 1 mM EDTA; and then combined with phenol-chloroform-isoamyl alcohol (25:24:1) and 0.3 g acid-washed glass beads (Sigma-G 8772). DNA was precipitated with 100% ethanol, spun in a microcentrifuge, and washed with 70% ethanol. Following air drying, the pellet was resuspended in 50 µl of dH₂O. The ITS and IGS regions were directly sequenced from the PCR products obtained with primer pair pITS-F (GTC GTA ACA AGG TTA ACC TGC GG) and pITS-R (TCC TCC GCT TAT TGA TAT GC) and with primer pair 26SF (5'-ATC CTT TGC AGA CGA CTT GA-3') and 5SR (5'-AGC TTG ACT TCG CAG ATC GG-3'), respectively (9, 11). PCR was performed in an MJ Research thermocycler (Bio-Rad) with an initial cycle of 10 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 56°C, and 1 min at 72°C, with a final extension of 10 min at 72°C. PCR products were confirmed by agarose gel electrophoresis and cleaned by the use of exonuclease 1-shrimp alkaline phosphatase. The sequencing reaction with the primers listed above was prepared according to the instructions of the manufacturer of the BigDye Terminator cycle sequencing kit (Applied Biosystems). Cycle sequencing was performed in an MJ Research thermocycler (Bio-Rad), and sequencing was performed with an ABI 3100 Avant genetic analyzer (Applied Biosystems).

Following amplification and sequencing of the ITS region, the species-specific sequence contained within the ITS1 region was compared to the published 13-bp sequence of Trichosporon mucoides. A 1-bp difference (G to A) at position 7 was identified for the clinical isolate (GenBank accession number AB018030). The species-specific ITS1 sequence of an environmental isolate of T. mucoides (GenBank accession number AB018031) was then compared to that of the isolate from the patient and was found to be identical (Table 1). In order to confirm the ITS identification of T. mucoides, IGS1 sequence alignment was performed by using the online BLAST 2 program (National Center for Biotechnology Information, Bethesda, MD), and the sequence obtained from the amplification of the IGS1 region of the isolate was compared to the nucleotide sequence in GenBank for the IGS1 region of Trichosporon mucoides (GenBank accession number AB066433) (Table 2). The length (357 bp) obtained for the amplified region was consistent with that for Trichosporon mucoides. However, the amplicon sequence showed a significant divergence from the published sequence, including five gap regions; and in the area where there was sufficient homology for comparison, only 196 of 224 (87%) bases matched. At this point it was observed that the IGS1 region for Trichosporon dermatis, a species recently added to the genus Trichosporon (10), is also 357 bp in length. BLAST comparison of the amplicon with the two identical published IGS1 sequences for Trichosporon dermatis (GenBank accession numbers AB066412 and AB072613) showed 99% homology with a difference of only 2 bp (C to T at position 243 and T to C at position 323). The 13-bp species-specific sequence contained within the ITS1 region for Trichosporon dermatis (GenBank accession number AB035581) was also compared to that of the amplicon and was found to be identical (Table 1). Further analysis showed that the carbohydrate assimilation profiles generated by the VITEK system and the ID32C yeast identification system (bioMerieux) for the isolate were consistent with the profile published for Trichosporon dermatis (10). The isolate has been deposited in the American Type Culture Collection (Manassas, VA) under catalog number MYA-3671.

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TABLE 1. Comparison of the ITS1 species-specific sequences from T. mucoides and T. dermatis with the sequence of the blood isolate

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TABLE 2. Comparison of IGS1 regions from T. mucoides and the T. dermatis isolatea

Over the past decade, increasing numbers of systemic human infections by Trichosporon spp. have been reported, particularly in susceptible immunocompromised patients (7). In this setting virtually any one of the Trichosporon spp. could potentially cause fungemia, but due to the limitations of physiologic and biochemical analysis, only six species of Trichosporon have previously been considered to be medically relevant (3). Most reported cases of Trichosporon fungemia are thought to be caused by varieties of Trichosporon asahii, but all other members of the six species identified as human pathogens (with the exception of Trichosporon ovoides) have been reported to cause opportunistic fungemia (1). Rare cases of disseminated trichosporonosis have also been caused by the presumably nonpathogenic species Trichosporon loubieri (5, 6).

The present case describes fungemia in an immunosuppressed pediatric patient, initially reported as Trichosporon mucoides by morphological and biochemical analysis and retrospectively identified by genetic analysis as Trichosporon dermatis. Although there are a number of case reports in the literature of disseminated infections caused by Trichosporon mucoides, there are to date no case reports of fungemia due to Trichosporon dermatis. These two pathogens share morphological and biochemical characteristics and have virtually identical carbohydrate assimilation profiles (10). Thus, positive identification at the species level can practically be achieved only by analyzing the interspecies divergence revealed at the DNA level. The current classification system makes species-specific identification of Trichosporon feasible, and it has been recommended that species identification be attempted for all relevant clinical isolates (5), as Trichosporon spp. with various antifungal susceptibilities are isolated from increasing numbers of patients (2).

The patient described in this report was treated with voriconazole, to which he had an excellent clinical response. This may be attributed to the efficacy of this drug, the timely removal of the CVC source of infection, and the patient's normal granulocytic response to fungemia. His tacrolimus level was therapeutic at the time of presentation, and although the patient had a relative lymphopenia, the relative neutrophilia revealed by the leukocyte differential was appropriate for the infection. Tacrolimus inhibits T-lymphocyte activation through interleukin-2 (4) and probably has little direct effect on the phagocytic oxidative burst or bone marrow production of granulocytes.

Despite a steady increase in the number of cases, Trichosporon sepsis is still relatively rare and is often not suspected as the causative agent in a blood culture positive for yeast. Even when trichosporonosis or another emerging mycosis is suspected, positive identification of the causative agent presents a diagnostic challenge because these organisms share morphological and biochemical features with many of the more commonly isolated pathogens. Techniques derived from molecular biology, such as DNA sequencing, can thus complement more traditional laboratory methods for the identification of pathogens (8). In this case report, biochemical and physiologic analysis as well as molecular analysis of the ITS1 sequence incorrectly identified the isolate as T. mucoides. However, sequencing of the IGS1 region correctly identified T. dermatis, supporting the findings of the recent study by Rodriguez-Tudela et al., which demonstrates that sequencing of the IGS1 region is the superior method for the molecular identification of Trichosporon isolates at the species level (7). Although IGS1 sequencing is not always available to the clinical microbiology laboratory, microscopy, susceptibility testing, and traditional biochemical identification methods are adequate for timely diagnosis and management in the majority of cases. However, when it is feasible, identification of the organism at the species level can positively influence patient care, particularly in the future choice of antifungal therapy for specific Trichosporon spp. with demonstrable resistance to certain antifungal drugs.

Nucleotide sequence accession numbers. The rRNA gene sequences of the IGS and ITS1 regions of the clinical isolate described here (isolate UH4277) have been deposited in GenBank under accession numbers DQ228143 and DQ228144, respectively.

 
We express our gratitude to Robin Leach for assistance with the DNA analysis.

This research was supported in part by a San Antonio Cancer Institute Cancer Center support grant (grant P30 CA54174).

 
* Corresponding author. Mailing address: UTHSCSA, Pathology, 7703 Floyd Curl Drive, San Antonio, TX 78229. Phone: (210) 567-3913. Fax: (210) 567-3803. E-mail: gunn{at}uthscsa.edu.

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Journal of Clinical Microbiology, March 2006, p. 1175-1177, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.1175-1177.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gunn, S. R. Articles by Jorgensen, J. H. Search for Related Content PubMed PubMed Citation Articles by Gunn, S. R. Articles by Jorgensen, J. H.