Case Report

Itraconazole-resistant Candida auris with phospholipase, proteinase and

hemolysin activity from a case of vulvovaginitis

Dharmendra Kumar, Tuhina Banerjee, Chandra Bhan Pratap, Ragini Tilak

Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

Abstract
Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual
species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida
auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described.
Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors
including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any
invasive fungemia.
Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-
albicans candida species over C. albicans and the diversity of Candida spp causing infections.

Key words: C. auris; non-albicans; resistance; phospholipase.

J Infect Dev Ctries 2015; 9(4):435-437. doi:10.3855/jidc.4582

(Received 18 December 2013 – Accepted 15 April 2014)

Copyright © 2015 Kumar et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction culture analysis was requested. Two high vaginal

Since the first report on Candida auris from Japan swabswerecollected aseptically with the help of a
in 2009[1], infrequent reports of this species from speculum and a post vaginal wall retractor and sent
clinical specimens have been documented. Emergence immediately to the Microbiology department for
of non-albicans Candida spp as pathogens arebeing further analysis.
increasingly reported from different parts of the world A Gram stained smear was prepared and
[2], thus emphasizing the importance of identification examined, which showed the presence of ovoid
of Candida to a species levelin all laboratories. At the budding yeast cells. The sample was inoculated on
same time, these emerging species also exhibit varying Sabouraud’s dextrose agar (SDA) and incubated at
drug susceptibility profiles. Therefore, understanding 37°C for 5 days for fungal culture and blood agar and
drug susceptibility of these different species can help MacConkey agar for bacterial culture. Following
develop protocols for appropriate empirical treatment incubation, dull white to cream coloured, smooth
of these infections. In this study we report a case of colonies were seen on SDA, showing globose budding
vulvovaginal candidiasis caused by C. auris, identified yeast cells on microscopic examination (Figure 1A).
by molecular methods, along with the characterization Bacterial culture was negative. For further speciation,
of the drug resistance profile and virulence traitsof the germ tube and chlamydospore formation on Cornmeal
isolate. agar, sugar assimilation and fermentation tests and
growth on CHROM agar were performed [3].
Case Report Antifungal susceptibility testing was performedby disc
A 28-year-old woman with complaint of vaginal diffusion testing using fluconazole (25 µg),
discharge, burning itchy sensation along with itraconazole (10 µg), voriconazole (1 µg) and
dyspareunia and low back pain presented as outpatient amphotericin B (100 units/disc) (Hi Media Labs,
at the Department of Gynaecology, Sir Sunderlal Mumbai, India); determination of minimum inhibitory
Hospital, a tertiary care university hospital in concentration (MIC) against the sameantifungals was
Varanasi, north India. A clinical diagnosis of performed based on standard protocol [4].
vulvovaginitis was made. A bacterial and fungal
Kumar et al. – C. auris causing vulvovaginitis J Infect Dev Ctries 2015; 9(4):435-437.

The isolate showed no germ tube and Figure 1. A. Globose yeast cells in Gram’s staining; B. C.auris
chlamydospore formation, produced no characteristic C.auris growth on CHROMagar
colour on CHROMagar (Figure 1B). Species
identification by sugar assimilation and fermentation
tests were inconclusive. The isolate grew at 42°C but
failed to grow at 45°C. Due to unavailability of
automated methods and a known confirmatory
method, molecular identification methods by
amplifying a conserved portion of 18SrDNA region,
adjacent ITS1and a portion of the 28SrDNA were
performed,as described elsewhere [5]. On
visualization, a band of approximately 479 bp (which Figure 2. PCR amplification product of Candida spp
was slightly lower than that for C. glabrata) was seen
(Figure 2). Polymerase chain reaction products were
extracted and purified (extraction kit, Qiagen Inc.,
Valencia, USA) and subjected to DNA sequencing
(Genei, Bangalore, India) followed by sequence
analysis using BLAST suite programs and compared
with the gene bank databases of the national center for
biotechnology information
(www.ncbi.nl.nih.gov/entrez). The isolate showed
greater than 99% similarity to C. auris and was
identified as such.
Due to infrequent isolation of this species from treatment, without any event of breakthrough
clinical specimens and in order to reveal its pathogenic fungemia.
nature, the isolate was further studied for virulence
determinants as detailed elsewhere [6]. Briefly, Discussion
determination of phospholipase, proteinase and Candidal vulvovaginitis is a worldwide problem,
hemolysin activity was carried out. For phospholipase being the second most common cause of vaginitis,
detection, 5µl of standard yeast suspension (108 yeast affecting millions of women especially in the
cells/ml) was dropped over egg yolk agar media and reproductive age group [7]. Previous studies on
air-dried, followed by incubation at 37°C for 48 hours. vulvovaginitis cases due to Candida spp have already
Phospholipase activity was expressed as Pz value documented the increasing prevalence of non-albicans
which is the ratio of the diameter of the colony to the Candida spp over C. albicans. Indiscriminate use of
diameter of the colony plus the precipitation zone antifungal agents along with incorrect dosage
measured in mm. For proteinase activity, Prz value was schedules, prolonged hospitalization, poor immune
measured as the ratio of the diameter of the colony to status are some of the implicated factors behind the
that of the clear zone of proteolysis following emergence of unusual species [8].
inoculation on 1% bovine serum albumin (BSA) plate There have been a handful of reports on C. auris as
and staining and destaining after 5 days incubation. a pathogen causing fungemia [9,10] but in this case C.
Hemolysin activity was detected on SDA with 3% auris was found as the causative agent for
glucose and 7% sheep blood and expressed as vulvovaginitis. The increasing isolation of C. auris
hemolytic index (Hz).Candida albicans ATCC 90028 from various clinical specimens clearly indicates its
was used as positive control. ability to colonise, invade and cause disease with
The isolate was resistant to itraconazole (MIC≥ 2 varying severity. Though a limited number of studies
µg/ml) and sensitive to fluconazole (MIC ≤16µg/ml), have addressed this isolate, the majority of them have
voriconazole (MIC≤0.5µg/ml)and amphotericin B reported this species to be intrinsically resistant to
(MIC≤0.5µg/ml). The isolate showed phospholipase azoles and amphotericin B [11]. C. auris is
activity (Pz value 0.72), proteinase activity (Przvalue intrinsically less susceptible to fluconazole with
0.66) and hemolysin activity (Hz value 0.74). propensity to develop high-level resistance. In this
However, the patient responded to oral fluconazole report, we found the isolate to be resistant to
itraconazole only. However, it had been previously

436
Kumar et al. – C. auris causing vulvovaginitis J Infect Dev Ctries 2015; 9(4):435-437.

commented that despite invitro susceptibility to azoles References

and polyenes, these agents might not be that effective 1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K,
Yamaguchi H (2009) Candida auris sp. nov., a novel
in vivo. Other therapeutic options should be tested in ascomycetous yeast isolated from the external ear canal of an
this context. Moreover, because of this association of inpatient in a Japanese hospital. MicrobiolImmunol 53: 41–44
drug resistance with this species of Candida, accurate 2. MohantyS, Xess I, Hasan F, KapilA, Mittal S, Jorge E (2007)
species identification is very important not only for Prevalence & susceptibility to fluconazole of Candida species
epidemiological significance, but also to provide causing vulvovaginitis. Indian J Med Res126: 216-19.
3. Nadagir SD, Chunchanur SK, Halesh LH, Yasmeen K,
guidance to physicians for effective treatment. Chandrashakhar MR, Patil B (2008) Significance of Isolation
Elaboration of various virulence factors associated And Drug Susceptibility Testing Of Non-Candida albicans
with colonizing and pathogenic strains of Candida Species Causing Oropharyngeal Candidiasis In HIV Patients.
have revealed them to be bearers of several traits Southeast Asian J Trop Med Public Health39: 492-495
4. Clinical and Laboratory Standards Institute (CLSI) Reference
responsible for their ability to easily survive and method for broth dilution antifungal susceptibility testing of
persist ininfection sites. The presence of yeasts: approved standard M27-A2. 2nd ed. Wayne, PA:
phospholipase and proteinase activity was CLSI, 2002.
demonstrated in C. auris isolates in this study. 5. Mahmoudi Rad M, Zafarghandi ASh, Amel Zabihi M,
Extracellular hydrolytic enzymes like phospholipases Tavallaee M, Mirdamadi Y (2012) Identification of Candida
species associated with vulvovaginal candidiasis by multiplex
act as important virulence factors helping in adherence PCR. Infect Dis Obstet Gynecol 2012: 872169.
and invasion of host cells. Similarly, hemolysin 6. Tsang CSP, Chu FCS, Leung WK, Jin LJ, Samaranayake LP,
production is followed by acquisition of iron that helps Siu SC (2007) Phospholipase, proteinase and haemolytic
in hyphal extension and invasive disease thus leading activities of Candida albicans isolated from oral cavities of
patients with type 2 diabetes mellitus. J Med Microbiol 56:
to widespread infection [6]. 1393-1398
Even after initial reports of isolation of C. auris 7. Sobel JD (1997) Vaginitis. N Eng J Med 337: 1896-1903
from different body sites, its exact role in pathogenesis 8. Ferrer J (2000) Vaginal candidosis: epidemiological and
could not be ascertained in previous studies. Few etiological factors. Int J Gynecol Obstet 71: 21-7.
researchers have in fact proved its pathogenic nature 9. Sarma S, Kumar N, Sharma S, Govil D, Ali T, Mehta Y,
Rattan A (2013) Candidemia caused by amphotericin B and
by performing histopathological study [11]. Instead, fluconazole resistant Candida auris. Indian J Med Microbiol
we studied the virulence traits of this isolate to reveal 31: 90-101
its pathogenic character. 10. Kim MN, Shin JH, Sung H, Lee K, Kim EC, Ryoo N, Lee JS,
Candida spp, most commonly C. albicans is a Jung SI, Park KH, Kee SJ, Kim SH, Shin MG, Suh SP, Ryang
DW (2009) Candida haemulonii and closely related species at
commensal of the vaginal flora in 20%-50% of 5 university hospitals in Korea: identification,
women, but there has been an increasing trend in antifungalsusceptibility, and clinical features. Clin Infect
isolation of C. glabrata, C. krusei, C. parapsilosis Dis48: e57-61
from patients with symptomatic vulvovaginitis and 11. LeeWK,Shin JH,Kang MG,Kim SH,Park KH,Jang H
their increasing resistance to commonly used (2011)First three reported cases of nosocomial fungemia
caused by Candida auris. J Clin Microbiol 49: 3139–3142
antifungal drugs [5]. In this context, C. auris isolated
in this patient and the association with drug resistance Corresponding author
and virulence traits hints towards its increasing Ragini Tilak,
pathogenicity. Therefore, it should be realized that due Department of Microbiology, Institute of Medical Sciences,
to the diverse spectrum of pathogenic non-albicans Banaras Hindu University,Varanasi-221005, India
Candida spp widens, laboratories face a real challenge Phone: +91 9415396353
Email: [email protected]
in their prompt identification with available means.
Improved molecular assays should be tried whenever Conflict of interests: No conflict of interests is declared.
conventional methods become insufficient.

437