SEARCH

SEARCH BY CITATION

Keywords:

  • Pseudozyma aphidis ;
  • fungaemia;
  • neonate;
  • antifungal susceptibility;
  • India

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

The Ustilaginomycetous basidiomycete yeast, Pseudozyma aphidis has recently been implicated in potentially fatal disorders ranging from subcutaneous mycoses to disseminated infections. Till date a solitary case of P. aphidis fungaemia in a paediatric patient has been reported. We present a case of fungaemia due to P. aphidis in a rhesus factor-isoimmunised, low-birth-weight neonate. The isolate was identified by sequencing the D1/D2 domain of the LSU region. Antifungal susceptibility of the isolate revealed susceptibility to amphotericin B, voriconazole, itraconazole, isavuconazole and posaconazole. It had high minimum inhibitory concentrations of fluconazole and was resistant to flucytosine and echinocandins. Consequently, the patient was successfully treated with intravenous amphotericin B. Although the source of infection could not be traced, as the neonate developed fungaemia on the first day of life, it could possibly be from the maternal urogenital tract or intrahospital transmission. A review of previously published cases revealed that risk factors for invasive Pseudozyma spp. infections were similar to those previously reported for non-albicans Candida spp. Pseudozyma species are underreported due to the difficulty of identifying this rare yeast pathogen by commercial identification systems. Considering that Pseudozyma spp. cause invasive fungal infections globally and are resistant to flucytosine, fluconazole and echinocandins, this pathogen assumes a greater clinical significance.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

Pseudozyma species are yeast-like fungi which have been rarely incriminated in human mycoses. They belong to the phylum Basidiomycota, subphylum Ustilaginomycotina, class Ustilaginomycetes and order Ustilaginales.[1] Pseudozyma species were not known as human pathogens until 2003, when Sugita et al. [2] isolated three Pseudozyma species; P. antarctica, P. parantarctica and P. thailandica from the blood of three Thai patients. So far, a solitary case of fungaemia due to P. aphidis has been reported from the USA in 2008.[3] Herein, we report the first case of fungaemia in a neonate due to P. aphidis from India and present an update of the cases reported so far.

Case report

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

A low-birth-weight, full-term, male baby was born to a rhesus factor (Rh)-negative mother by normal vaginal delivery on 20 October, 2012 at a private hospital in Agra, Uttar Pradesh, India. The same day, he developed lethargy and poor feeding associated with early neonatal jaundice and was referred to a tertiary care hospital in Delhi, on 22 October, 2012 where he was immediately admitted to the neonatal intensive care unit with suspected neonatal sepsis. Laboratory investigations showed haemoglobin of 18.5 g dl−1, total bilirubin −25 mg dl−1, blood group – B (Rh-positive) and a positive direct Coomb's test suggestive of Rh-isoimmunisation. Other causes of neonatal jaundice such as glucose-6-phosphate dehydrogenase deficiency and intrauterine infections were ruled out. The baby received intensive phototherapy and was treated with intravenous piperacillin and tazobactam combination for suspected sepsis. The blood sample was collected aseptically on day 1 of admission and processed for bacterial and fungal pathogens. Also, double volume exchange transfusion and intravenous immunoglobulin were commenced. He developed thrombocytopenia and was infused platelet concentrates. Postexchange transfusion, total bilirubin level, dropped to 11.9 mg dl−1 on day 2 after which phototherapy was stopped. On day 3 of admission, the blood cultures showed growth of yeast-like colonies, however, culture was negative for bacteria. Therefore, a presumptive diagnosis of fungaemia was considered and the baby was administered intravenous amphotericin B (0.6 mg kg−1 day−1) for 1 week. A repeat blood culture on day 6 of admission showed clearance of fungaemia. The subsequent stay of the baby was uneventful and repeated blood cultures done twice were sterile. He was discharged on day 20 of admission with oral voriconazole (4 mg kg−1 per dose twice a day) as domiciliary treatment for 7 days. Currently, the baby continues to be healthy.

Mycological investigations

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

The isolate was assigned an accession number VPCI 1049/P/12 and showed moist, yeast-like, tan-yellow and wrinkled colonies on Sabouraud's glucose agar after 4 days of incubation at 37 °C (Fig. 1a). On microscopic examination, lactophenol cotton blue mount showed fusiform spindle-shaped elongated blastoconidia and presence of hyphae (Fig. 1b). On CHROMagar Candida medium (Difco, Becton Dickinson, Baltimore, MD, USA) the isolate formed rough green colonies after 48 h of incubation at 37 °C. However, germ tube test and chlamydospore formation were negative. The isolate showed a positive test for diazonium blue B (DBB), hydrolysed urea and was inhibited on 0.1% cycloheximide-containing medium. API ID 32C and VITEK2 compact (bioMérieux, Marcy I'Etoile, France) gave inconclusive profiles. The isolate assimilated sucrose, raffinose, soluble starch, trehalose, lactose, maltose and nitrate. Furthermore, molecular identification was done by the amplification and sequencing of the D1/D2 domain of the LSU region.[4] GenBank BLAST searches were performed for species identification. The sequence exhibited 99% identity with P. aphidis (GenBank accession no. HQ676615). The LSU sequence of the isolate was submitted to GenBank under the accession number KC812275. The isolate, VPCI 1049/P/12 has been deposited in the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands under the accession number CBS 12818.

image

Figure 1. (a) Multiple yeast-like, moist, tan-yellow and wrinkled colonies of Pseudozyma aphidis VPCI 1049/P/12 on Sabouraud glucose agar plate as seen after 4 days of incubation at 37 °C. (b) Lactophenol cotton blue mount of the same P. aphidis isolate showing fusiform spindle-shaped blastoconidia (arrow upwards) and hyphae (arrow downwards) ×1000.

Download figure to PowerPoint

In vitro antifungal susceptibility testing

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

Antifungal susceptibility testing of the isolate was determined using the Clinical and Laboratory Standards Institute (CLSI) microbroth dilution method, following the M27-A3 guidelines.[5] The antifungals tested were amphotericin B (Sigma, St. Louis, MO, USA), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Lee Pharma, Hyderabad, India), voriconazole (Pfizer), posaconazole (Schering-Plough, Kenilworth, NJ, USA, now Merck), isavuconazole (Basilea Pharmaceutica, Basel, Switzerland, now Astellas), flucytosine (Sigma), caspofungin (Merck, Whitehouse Station, NJ, USA), micafungin (Astellas, Toyama, Japan) and anidulafungin (Pfizer). CLSI-recommended quality control strains Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were used. The minimum inhibitory concentration (MIC) end points were defined as the lowest drug concentration that caused a prominent decrease in growth (50%) vis-à-vis the controls and read visually after 48 h for fluconazole, voriconazole, itraconazole, isavuconazole, posaconazole and flucytosine and after 24 h for echinocandins. For amphotericin B, the MIC was defined as the lowest concentration at which there was 100% inhibition of growth compared with the drug-free control wells. The isolate was susceptible to amphotericin B (MIC, 0.03 μg ml−1), itraconazole (MIC, 0.03 μg ml−1), posaconazole (MIC, 0.03 μg ml−1), voriconazole (MIC, 0.06 μg ml−1) and isavuconazole (MIC, 0.25 μg ml−1). However, it had high MICs of fluconazole (MIC, 8 μg ml−1), and was resistant to anidulafungin (MIC, 8 μg ml−1), caspofungin (MIC, 8 μg ml−1), micafungin (MIC, >8 μg ml−1) and flucytosine (MIC, >64 μg ml−1).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

The genus Pseudozyma contains 18 described species which are phylogenetically related to Ustilago maydis and other smut fungi.[1, 6-9] Pseudozyma aphidis is either epiphytic or saprophytic and is known from secretions of insects (family: Aphididae) on leaves.[1] It has been reported from leaves of apple, cherry, apricot and grasses.[10, 11] Of the 18 species only four are reported as human pathogens till date and little is known about their pathogenicity.[2, 3, 12-14] The analysis of the global distribution of eight cases of human infection due to Pseudozyma species including the present case is shown in Table 1.[2, 3, 12-14] It is pertinent to mention that barring a solitary case of mycetoma all other infections due to this pathogen are invasive. The present case represents the first case of fungaemia due to P. aphidis in a neonate reported so far. In another case of fungaemia in a 7-year-old paediatric patient due to P. aphidis, the patient had received parenteral nutrition through a long-term indwelling central venous catheter (CVC) due to her short bowel syndrome.[3] Her CVC had been replaced three times since birth due to line infections and the possible entry of P. aphidis through CVC was considered.[3] Another case of pulmonary mycosis reported by Parahym et al. [14] occurred in a 17-year-old male under treatment for Burkitt's lymphoma who presented with febrile neutropenia. The pleural fluid culture yielded P. aphidis, sensitive to amphotericin B and azoles but resistant to caspofungin. It may be noted with regard to the above two cases that the patients had one or more predisposing factors for fungaemia such as impaired mucosal defences, prolonged indwelling venous catheters, total parenteral nutrition, haematological malignancy and chemotherapy.[3, 14] In the other reports of brain abscess and mycetoma, P. aphidis was isolated along with primary bacterial pathogens.[12, 13] In both cases, P. aphidis was isolated from deep seated, usually sterile tissue which underscores its potential pathogenicity. In the present case, as the newborn developed fungaemia on the first day of his life, vaginal or nosocomial transmission of this species might have occurred. Since a vaginal swab of the mother or hand swabs of health care personnel were not investigated, the source remains enigmatic. Notably, risk factors associated with invasive P. aphidis infections including the present case of fungaemia are similar to those previously reported for non-albicans Candida spp viz. age <65 years, cancer chemotherapy, neutropenia (<3000 cells μl−1) and severe thrombocytopenia.[15] In three cases of fungaemia due to Pseudozyma species reported by Sugita et al. [2], clinical features of the patients and the clinical impact of the organisms have not been presented.

Table 1. Global literature review of eight human cases reported so far due to Pseudozyma species and their in vitro antifungal susceptibility profile
Pseudozyma spp.Country (no. of isolates)Clinical specimenUnderlying diseaseTreatment and outcomeMICs (μg ml−1) of antifungalsReference
  1. AMB, amphotericin B; FC, flucytosine; FLC, fluconazole; ITC, itraconazole; MCZ, miconazole; VRC, voriconazole; POS, posaconazole; ISA, isavuconazole; CAS, caspofungin; MFG, micafungin; AFG, anidulafungin.

  2. a

    Coexisting with Methicillin-resistant Staphylococcus aureus.

  3. b

    Coexisting with Nocardia otitidiscaviarum.

P. antarctica Thailand (1)BloodPneumothoraxNot reportedAMB, 0.125; FC, >64; FLC, 0.5; ITC, 0.06; MCZ, 0.125Sugita et al. [2]
P. parantarctica Thailand (1)BloodLeptospirosis and aseptic meningitisNot reportedAMB, 0.125; FC, >64; FLC, 2; ITC, 0.25; MCZ, 1Sugita et al. [2]
P. thailandica Thailand (1)BloodAcute asthmatic attack and respiratory failureNot reportedAMB, 0.125; FC, >64; FLC, >64; ITC, >8; MCZ, 4Sugita et al. [2]
Pseudozyma spp.aKorea (1)Pus from brain abscessAstrocytomaAntifungal therapy not administered, expired due to multiorgan failureNot doneHwang et al. [12]
P. aphidis USA (1)BloodShort bowel syndromeI.V. FLC (loading dose of 10 mg/kg followed 24 h later by a 5 mg kg−1 day−1 maintenance dose), patient improvedAMB, 0.25; FLC, 4; ITC, 0.125Lin et al. [3]
China (1)Mycetoma sinus dischargebMycetoma of legITC 100 mg orally twice daily for 7 days along with repeated debridement, patient improvedNot doneChen et al. [13]
Brazil (1)Pleural fluidBurkitt lymphoma with respiratory insufficiency due to a probable pulmonary infection10-day course of i.v. AMB (5 mg kg−1 day−1) and, afterwards, with a 15-day course of oral VRC (400 mg day−1), patient improvedAMB, 0.25; FLC, 4; ITC, 0.25; VRC, 0.03; CAS, 4; AFG, 4Parahym et al. [14]
India (1)BloodHaemolytic jaundice due to Rh-isoimmunisationI.V. AMB (0.6 mg kg−1 day−1), patient improvedAMB, 0.03; FC, >64; FLC, 8; ITC, 0.03; VRC, 0.06; ISA, 0.25; POS, 0.03; CAS, >8; AFG, 8; MFG, >8Present report

This species cannot be identified by commercial systems available in routine diagnostic laboratories. Therefore, isolation of yeast, showing fusiform blastoconida that hydrolyze urea, are DBB positive and assimilate myo-inositol and d-glucuronate may represent rare basidiomycetes. Such isolates should be confirmed by sequencing. Due to the rare isolation of P. aphidis in human infections, there is paucity of antifungal susceptibility data. Sugita et al. [2] have reported all the three species of Pseudozyma resistant to flucytosine and P. thailandica additionally resistant to both fluconazole and itraconazole. In contrast, Lin et al. [3] and Parahym et al. [14] have reported low MICs of fluconazole and itraconazole for P. aphidis. Our P. aphidis isolate was susceptible to amphotericin B, voriconazole, itraconazole, isavuconazole and posaconazole, whereas it showed high MICs of fluconazole and was resistant to flucytosine and echinocandins. The neonate was treated successfully with amphotericin B and voriconazole. P. aphidis has been prevalent globally and so far infections have been reported from Brazil, China, Korea, Thailand and the USA.[2, 3, 12-14] In conclusion, Pseudozyma species are underreported due to the difficulty in identifying this rare yeast pathogen by conventional and commercial identification systems. Considering that Pseudozyma species cause invasive fungal infections and are resistant to flucytosine and fluconazole, the pathogens assume a greater clinical significance.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

This work was carried out, in part, with financial assistance from the Department of Biotechnology (BT/39/NE/TBP/2010), Government of India, New Delhi, India. J.F.M has been supported by Qatar National Research Fund (Grant NPRP 5-298-3-086).

Conflict of interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References

J.F.M received grants from Astellas, Merck, Pfizer, Schering-Plough, Gilead and Janssen Pharmaceuticals. He has been a consultant to Basilea and Merck and received speaker's fees from Merck, Pfizer, Schering-Plough, Gilead and Janssen Pharmaceutica. All other authors: no potential conflicts of interest. The authors alone are responsible for the content and writing of the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Case report
  5. Mycological investigations
  6. In vitro antifungal susceptibility testing
  7. Discussion
  8. Acknowledgments
  9. Conflict of interest
  10. References
  • 1
    Kurtzman CP, Fell JW, Boekhout T. The Yeasts: A Taxonomic Study, 5th edn. London: Elsevier, 2011.
  • 2
    Sugita T, Takashima M, Poonwan N et al. The first isolation of ustilaginomycetous anamorphic yeasts, Pseudozyma species, from patients' blood and a description of two new species: P. parantarctica and P. thailandica. Microbiol Immunol 2003; 47: 18390.
  • 3
    Lin SS, Pranikoff T, Smith SF et al. Central venous catheter infection associated with Pseudozyma aphidis in a child with short gut syndrome. J Med Microbiol 2008; 57: 5168.
  • 4
    White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: A Guide to Methods and Applications. San Diego, CA: Academic Press, 1990: 31522.
  • 5
    Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, 3rd edn. Approved standard, M27-A3. Wayne, PA: CLSI, 2008.
  • 6
    Seo HS, Um HJ, Min J et al. Pseudozyma jejuensis sp. nov., a novel cutinolytic ustilaginomycetous yeast species that is able to degrade plastic waste. FEMS Yeast Res 2007; 7: 103545.
  • 7
    Fukuoka T, Kawamura M, Morita T et al. A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants. Carbohydr Res 2008; 343: 294755.
  • 8
    Morita T, Konishi M, Fukuoka T, Imura T, Kitamoto D. Production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma siamensis CBS 9960 and their interfacial properties. J Biosci Bioeng 2008; 105: 493502.
  • 9
    Morita T, Ogura Y, Takashima M et al. Isolation of Pseudozyma churashimaensis sp. nov., a novel ustilaginomycetous yeast species as a producer of glycolipid biosurfactants, mannosylerythritol lipids. J Biosci Bioeng 2011; 112: 13744.
  • 10
    Sláviková E, Vadkertiová R, Vránová D. Yeasts colonizing the leaves of fruit trees. Ann Microbiol 2009; 59: 41924.
  • 11
    Allen TW, Quayyum HA, Burpee LL, Buck JW. Effect of foliar disease on the epiphytic yeast communities of creeping bentgrass and tall fescue. Can J Microbiol 2004; 50: 85360.
  • 12
    Hwang S, Kim J, Yoon S et al. First report of brain abscess associated with Pseudozyma species in a patient with astrocytoma. Korean J Lab Med 2010; 30: 2848.
  • 13
    Chen B, Zhu LY, Xuan X et al. Isolation of both Pseudozyma aphidis and Nocardia otitidiscaviarum from a mycetoma on the leg. Int J Dermatol 2011; 50: 7149.
  • 14
    Parahym AM, da Silva CM, Domingos Ide F et al. Pulmonary infection due to Pseudozyma aphidis in a patient with Burkitt lymphoma: first case report. Diagn Microbiol Infect Dis 2013; 75: 1046.
  • 15
    Cheng MF, Yang YL, Yao TJ et al. Risk factors for fatal candidemia caused by Candida albicans and non-albicans Candida species. BMC Infect Dis 2005; 5: 22.