Abstract
The first outbreak of Candida haemulonii fungemia is described. The seven isolates from the blood of four neonates were identified by DNA sequencing of the ribosomal DNA. They were all resistant to amphotericin B, fluconazole, and itraconazole. This report highlights the emergence of C. haemulonii as an opportunistic pathogen in immunocompromised patients.
Candida haemulonii (van Uden & Kolipinski) S. A. Meyer & D. Yarrow (14) (syn. Torulopsis haemulonii) was originally described from the gut of a blue-striped grunt (Haemulon scirus) in 1962 (13). Subsequently, the species was isolated from the skin of dolphins and seawater off the coast of Portugal (13). Recently, it was associated with an epidemic in a laboratory-maintained colony of Ornithodoros moubata (an arthropod vector) in the Czech Republic (8). Lavarde et al. (6) reported the first isolation of this yeast from humans from the blood of a patient who died of renal failure. It has also been isolated from toe ulcers and the nails of diabetic patients (3). Identification of C. haemulonii is difficult because it is phenotypically very similar to Candida famata (teleomorph, Debaryomyces hansenii) and Candida guilliermondii (teleomorph, Pichia guilliermondii) (7). Lehmann et al. (7) studied 25 clinical isolates of C. haemulonii and described two genetically distinct groups within the species (group I and group II) on the basis of isoenzyme profiles, DNA reassociations, and physiological characteristics. Recently, Sugita et al. (12) described a new species, Candida pseudohaemulonii, which was isolated from the blood of a patient in Thailand that was resistant to amphotericin B and azoles. Here, we describe an outbreak of candidemia that was caused by C. haemulonii isolates that were resistant to amphotericin B and azoles and that involved four neonates in the neonatal intensive care unit (NICU) of a maternity hospital in Kuwait.
All the isolates were recovered in BACTEC Peds Plus/F culture vials after 2 to 3 days of incubation at 37°C. On Sabouraud dextrose agar, the colonies were off-white to cream colored and smooth. Microscopic examination showed ovoid to globose budding yeast-like cells. No chlamydospores or pseudohyphae were formed on cornmeal-Tween 80 medium (Difco Laboratories, Detroit, MI). The isolates were negative by the germ tube test. The Vitek2 yeast ID system (bioMerieux, Marcy l'Etoile, France) identified the isolates as Rhodotorula glutinis (low to good discrimination). Since the phenotypic characteristics of the isolates were not helpful, molecular methods were used for species-specific identification. The amplification of species-specific the internally transcribed spacer 1 (ITS-1) and ITS-2 regions of ribosomal DNA and the D1 and D2 regions of 26S rRNA was performed with genomic DNA extracted from the isolates by using panfungal primers (primers ITS1 and ITS4 and primers NL-1 and NL-4, respectively), and the amplicons were sequenced as described earlier (1). Based on the ITS-2 region sequence from C. haemulonii, a specific PCR-based method was developed for the direct detection of C. haemulonii DNA by using a C. haemulonii-specific primer (CHSP [5′-TCTAAATATCATGCCACAGTGAA-3′]) together with primer ITS4 (2).
The in vitro susceptibilities of the isolates to antifungal agents were determined by Etest (AB Biodisk, Solna, Sweden) on RPMI agar supplemented with 2% glucose. The plates were incubated at 35°C and were read after 40 h. The isolates were considered resistant if they exhibited the following MICs: fluconazole, ≥64 μg/ml; itraconazole, ≥1 μg/ml; flucytosine, ≥32 μg/ml; voriconazole, ≥4 μg/ml; amphotericin B, ≥1.0 μg/ml; and caspofungin, ≥2 μg/ml.
The characteristics of the four neonates whose blood yielded C. haemulonii in culture are presented in Table 1. The first isolation was made on 15 November 2005 from the blood of a neonate who was born full term but with a congenital diaphragmatic hernia (case 1). He was in the NICU for 42 days before he developed fungemia due to C. haemulonii. Despite aggressive treatment with liposomal amphotericin B (AmBisome), caspofungin, and/or fluconazole for more than 6 weeks, he succumbed to the infection. The other three cases (cases 2 to 4; Table 1) occurred within a period of 10 days after the patients had been in the NICU for 76, 45, and 30 days, respectively. All three were born preterm, and they were ventilated and had received courses of broad-spectrum antibiotics. While case 2 and case 3 survived the infection, case 4 died, despite treatment with amphotericin B. All four cases received total parenteral nutrition through central lines for various periods.
TABLE 1.
Characteristics of neonates with Candida haemulonii fungemiaa
| Characteristic | Case 1 | Case 2 | Case 3 | Case 4 |
|---|---|---|---|---|
| Birth date (day/mo/yr) | 3/10/05 | 7/11/05 | 29/11/05 | 21/12/05 |
| Gestation age (wk) | 35b | 26 | 25 | 31 |
| Sex | Male | Male | Female | Female |
| Birth wt (g) | 2,570 | 710 | 800 | 1,035 |
| Time on ventilation (days) | 115 | 60 | 34 | 4 |
| Prior antibiotic therapy | AMK, AMP, TZB | AMK, AMP, VAN | AMK, AMP, VAN, CX, PIP-TZB | AMK, AMP, MEM |
| Date blood culture first positive (day/mo/yr) | 15/11/05 | 23/1/06 | 14/1/06 | 20/1/06 |
| C. haemulonii isolation | + (once) | + (once) | + (twice) | + (thrice) |
| Antifungal therapy | AP (L) at 5 mg/kg of body weight for 30 days, CS at 1 to 2 mg/kg for 26 days, and FL at 6 mg/kg for 24 days | FL at 6 mg/kg for 21 days | AP at 1 mg/kg for 21 days, CS at 1 to 2 mg/kg for 15 days | AP (L) at 1.5/kg for 14 days |
| Outcome | Death | Survival | Survival | Death |
Abbreviations: FL, fluconazole; AP, amphotericin B; AP (L), liposomal amphotericin B; CS, caspofungin; AMK, amikacin; AMP, ampicillin; TZB, tazobactam; CX, cloxacillin; VAN, vancomycin; MEM, meropenem; PIP, piperacillin.
Born with a diaphragmatic hernia.
The carbon assimilation profile obtained with the API 20 C AUX yeast identification system yielded a code of 6102170 after 48 h of incubation at 30°C, whereas the ID 32C system yielded codes of 5163140215 and 7163170215 at 48 h and 120 h of incubation at 30°C, respectively. However, neither of these codes was included in the database for the identification of C. haemulonii. By Wickerham carbon assimilation tests, which were monitored for up to 8 weeks, the isolates assimilated trehalose, l-rhamnose, and glycerol but not l-sorbose or d-arabinose. The assimilation of melizitose and raffinose was delayed, becoming positive on days 12 and 14, respectively. The ITS and D1/D2 region sequences of all seven isolates matched nearly completely (1 and 0 nucleotide differences in the ITS and D1/D2 regions, respectively) with the corresponding sequences from reference strain C. haemulonii CBS 5149 (GenBank accession no. AY500375), thus confirming their identification as C. haemulonii. PCR amplification of genomic DNA from all the seven isolates with primers CHSP and ITS4 yielded the expected 106-bp product, while no amplicons were obtained from C. albicans or six other commonly encountered Candida species (data not shown). The DNA sequences have been deposited in the European Molecular Biology Laboratory (EMBL) nucleotide sequence database.
The data on the antifungal susceptibilities of the isolates are presented in Table 2. All the isolates were resistant to amphotericin B, fluconazole, and itraconazole and susceptible to voriconazole, flucytosine, and caspofungin. None of the neonates were treated with flucytosine or voriconazole, to which the yeast was susceptible. As part of the patient management strategy, the central venous catheters of these neonates were changed during their hospitalization. Unfortunately, the catheter tips were not cultured for the isolation of C. haemulonii. It is therefore not clear how much the removal of the catheters contributed to the infection, particularly in case 2, who responded to fluconazole treatment, even though the yeast was resistant to this drug.
TABLE 2.
Antifungal susceptibility data for C. haemulonii isolates on RPMI medium determined by Etest
| Case no. | Isolate no. | Date isolated (day/mo/yr) | MIC (μg/ml)a
|
|||||
|---|---|---|---|---|---|---|---|---|
| AP | FL | IT | VO | CS | FC | |||
| 1 | 2062/05 | 15/11/05 | 4 | 96 | 2 | 0.047 | 0.5 | 0.047 |
| 2 | 133/06 | 23/1/06 | 6 | >256 | 3 | 0.125 | 0.5 | 0.064 |
| 3 | 68/06 | 14/1/06 | 2 | >256 | 2 | 0.125 | 0.023 | 0.012 |
| 3 | 208/06 | 5/2/06 | 4 | >256 | 2 | 0.125 | 0.125 | 0.032 |
| 4 | 146/06 | 20/1/06 | 4 | >256 | 4 | 0.125 | 0.125 | 0.008 |
| 4 | 154/06 | 25/1/06 | 8 | >256 | 2 | 0.125 | 0.125 | 0.023 |
| 4 | 185/06 | 30/1/06 | 6 | >256 | 3 | 0.38 | 0.19 | 0.047 |
Abbreviations: AP, amphotericin B; FL, fluconazole; IT, itraconazole; VO, voriconazole; CS, caspofungin; FC, flucytosine.
This report is noteworthy in that it describes the emergence of C. haemulonii as an opportunistic fungal pathogen that is capable of causing an outbreak of fungemia. Although it is rarely isolated in clinical microbiology laboratories, C. haemulonii has been reported to be intrinsically resistant to amphotericin B and azoles (4, 11), thus posing diagnostic and therapeutic challenges. With the exception of some Candida lusitaniae strains, the occurrence of amphotericin B resistance among Candida species is rarely reported. In this context, it may be pointed out that Etest has a greater sensitivity than Clinical and Laboratory Standards Institute procedure M27-A2 (10) for the detection of Candida isolates exhibiting reduced susceptibility to amphotericin B. So far, there have been only a few reports on the isolation of C. haemulonii from blood (4, 6, 9, 11). The first report on the isolation of C. haemulonii from blood was for a patient who was on peritoneal dialysis due to renal failure and who died, despite therapy with amphotericin B and flucytosine (6). Subsequently, two additional cases of C. haemulonii fungemia were reported from patients with cancer and megaloblastic anemia (9, 11). Recently, Giusiano et al. (4) reported on the isolation of C. haemulonii from the blood and a catheter of a pediatric cancer patient. The isolates showed increased resistance to fluconazole, itraconazole, and amphotericin B. Scant information on the occurrence and distribution of C. haemulonii in clinical specimens is available. Recently, Goswami et al. (5) reported on the isolation of C. haemulonii from 9% of diabetic women and 30% of nondiabetic women with vaginal candidiasis. However, those authors did not describe the carbohydrate assimilation profiles of the isolates. Such a high frequency of C. haemulonii isolation from patients with vaginal candidiasis has not been reported previously.
This organism cannot be identified by using the currently available yeast identification systems, as it is not included in the systems' databases (3, 7). Most of the strains of C. haemulonii that were included in group I by Lehman et al. (7) yielded an assimilation code of 6102170 in the API 20C system, which is consistent with the codes for our isolates. However, with the ID 32C yeast identification kit, our isolates yielded two assimilation codes, 5163140215 at 48 h and 7163170215 at 120 h. Both of these assimilation codes were different from the ID 32C codes described by Sugita et al. (12) for C. haemulonii type I or type II strains. However, the DNA sequences of our isolates that were sequenced (information provided with the sequencing data) exhibited 99.6% and 100% identities with the corresponding ITS and D1/D2 sequences, respectively, from the reference strain C. haemulonii CBS 5149 (GenBank accession no. AY500375), thus confirming their identification as C. haemulonii. Since all the isolates were recovered over a short period of time and exhibited nearly identical phenotypic and molecular characteristics, we believe that they were part of an outbreak of fungemia due to this rare yeast species that probably originated from a common source. Although no specific containment measures were taken, the outbreak subsided by strict enforcement of infection control measures. Unfortunately, we could not undertake epidemiological studies to trace the source of C. haemulonii in the NICU. To the best of our knowledge, this is the first report describing an outbreak of C. haemulonii fungemia.
Nucleotide sequence accession numbers.
The DNA sequences have been deposited in EMBL under accession nos. AM231721 to AM231725, AM412999, and AM413000.
Acknowledgments
This study was supported by Kuwait University grant MI04/05; the Department of Microbiology, Faculty of Medicine, Kuwait University; and the Ministry of Health, Kuwait.
The technical support of Daad Farhat is acknowledged.
Footnotes
Published ahead of print on 11 April 2007.
REFERENCES
- 1.Ahmad, S., Z. Khan, E. Mokaddas, and Z. U. Khan. 2004. Isolation and molecular identification of Candida dubliniensis from non-human immunodeficiency virus-infected patients in Kuwait. J. Med. Microbiol. 53:633-637. [DOI] [PubMed] [Google Scholar]
- 2.Ahmad, S., Z. Khan, A. S. Mustafa, and Z. U. Khan. 2002. Seminested PCR for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification. J. Clin. Microbiol. 40:2483-2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Gargeya, I. B., W. R. Pruitt, S. A. Meyer, and D. G. Ahearn. 1991. Candida haemulonii from clinical specimens in the USA. J. Med. Vet. Mycol. 29:335-338. [PubMed] [Google Scholar]
- 4.Giusiano, G., M. Mangiaterra, V. Garcia Saito, F. Rojas, V. Gomez, and M. C. Diaz. 2006. Fluconazole and itraconazole resistance of yeasts isolated from the bloodstream and catheters of hospitalized pediatric patients. Chemotherapy 52:254-259. [DOI] [PubMed] [Google Scholar]
- 5.Goswami, R., V. Dadhwal, S. Tejaswi, K. Datta, H. Paul, R. N. Haricharan, U. Banerjee, and N. P. Kochupillai. 2000. Species-specific prevalence of vaginal candidiasis among patients with diabetes mellitus and its relation to their glycaemic status. J. Infect. 41:162-166. [DOI] [PubMed] [Google Scholar]
- 6.Lavarde, V., F. Daniel, H. Saez, M. Arnold, and B. Faguer. 1984. Peritonite mycosique a Torulopsis haemulonii. Bull. Soc. Fr. Mycol. Med. 13:173-176. [Google Scholar]
- 7.Lehmann, P. F., L. C. Wu, W. R. Pruitt, S. A. Meyer, and D. G. Ahearn. 1993. Unrelatedness of groups of yeasts within the Candida haemulonii complex. J. Clin. Microbiol. 31:1683-1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Loosova, G., P. Jindrak, and L. P. Kopacek. 2001. Mortality caused by experimental infection with the yeast Candida haemulonii in the adults of Ornithodoros moubata (Acarina: Argasidae). Folia Parasitol. (Praha) 48:149-153. [PubMed] [Google Scholar]
- 9.Marjolet, M. 1986. Torulopsis ernobii, Torulopsis haemulonii: levures opportunistes chez I'immunodéprimé? Bull. Soc. Fr. Mycol. Med. 15:143-146. [Google Scholar]
- 10.Peyron, F., A. Favel, A. Michel-Nguyen, M. Gilly, P. Regli, and A. Bolmstrom. 2001. Improved detection of amphotericin B-resistant isolates of Candida lusitaniae by Etest. J. Clin. Microbiol. 39:339-342. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Rodero, L., M. Cuenca-Estrella, S. Cordoba, P. Cahn, G. Davel, S. Kaufman, L. Guelfand, and J. L. Rodriguez-Tudela. 2002. Transient fungemia caused by an amphotericin B-resistant isolate of Candida haemulonii. J. Clin. Microbiol. 40:2266-2269. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sugita, T., M. Takashima, N. Poonwan, and N. Mekha. 2006. Candida pseudohaemulonii sp. nov., an amphotericin B- and azole-resistant yeast species, isolated from the blood of a patient from Thailand. Microbiol. Immunol. 50:469-473. [DOI] [PubMed] [Google Scholar]
- 13.van Uden, K., and M. C. Kolipinski. 1962. Torulopsis haemulonii nov. spec. a yeast from the Atlantic Ocean. Antonie Leeuwenhoek 28:78-80. [DOI] [PubMed] [Google Scholar]
- 14.Yarrow, D., and S. A. Meyer. 1978. Proposal for amendment of the diagnosis of the genus Candida Berkhout nom. cons. Int. J. Syst. Bacteriol. 28:611-615. [Google Scholar]