- Email: [email protected]
Candida blankii: an emerging yeast in an outbreak of fungemia in neonates in Delhi, India
Journal Pre-proof Candida blankii: an emerging yeast in an outbreak of fungemia in neonates in Delhi, India Anuradha Chowdhary, J. Benjamin Stielow, Gargi Upadhaya, Pradeep K. Singh, Ashutosh Singh, Jacques F. Meis PII:
S1198-743X(20)30002-1
DOI:
https://doi.org/10.1016/j.cmi.2020.01.001
Reference:
CMI 1887
To appear in:
Clinical Microbiology and Infection
Received Date: 3 October 2019 Revised Date:
11 December 2019
Accepted Date: 1 January 2020
Please cite this article as: Chowdhary A, Stielow JB, Upadhaya G, Singh PK, Singh A, Meis JF, Candida blankii: an emerging yeast in an outbreak of fungemia in neonates in Delhi, India, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2020.01.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.
1
Candida blankii: an emerging yeast in an outbreak of fungemia in neonates in Delhi, India
2 3
Anuradha Chowdhary1#, J. Benjamin Stielow2,3, Gargi Upadhaya1, Pradeep K Singh1, Ashutosh
4
Singh1, Jacques F Meis3,4
5 6 7
1
Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi;
8
2
Thermo Fisher Diagnostics, Specialty Diagnostics Group, Landsmeer, The Netherlands; 3Centre
9
of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands; 4Department of
10
Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ),
11
Nijmegen, The Netherlands.
12 13 14 15 16
Keywords: Candida blankii, Outbreak, Whole genome sequencing, Neonates, India
17
Running Title: Candida blankii outbreak in NICU, India
18 19 20 21 22 23 24 25 26
#Corresponding author Prof. Anuradha Chowdhary Department of Medical Mycology V. P. Chest Institute, University of Delhi Delhi-110007, India Email: [email protected]
27 1
28
Abstract
29
Objective: Outbreaks of fungal sepsis due to emerging and rare multidrug resistant Candida
30
species are increasingly reported in healthcare settings. We report an outbreak of fungemia due
31
to the rare multidrug-resistant yeast Candida blankii in an Indian neonatal unit.
32
Material and Methods: Blood cultures grew C. blankii in nine neonates in the NICU of a
33
multispecialty hospital, Delhi during a period of seven months. Isolates were identified by ITS
34
and D1/D2 region sequencing. Antifungal susceptibility testing was performed by M27-A3 CLSI
35
broth microdilution. To determine genetic relatedness among C. blankii isolates we undertook
36
amplified fragment length polymorphism analysis and DNA sequencing using the Illumina
37
NextSeq500 platform.
38
Results: C. blankii fungemia occurred at 2-3 postnatal days in nine LBW/VLBW neonates. All
39
neonates were treated with fluconazole and four of nine patients died, resulting in a case fatality
40
rate of 45%. C. blankii was misidentified or not identified by automated identification systems.
41
Fluconazole had higher MICs (GM-MIC, 8 µg/ml) than the other azoles. Also, anidulafungin
42
(GM-MIC, 2 µg/ml) had high MICs. Genome sequencing confirmed transmission of genetically
43
mostly indistinguishable strains. The C. blankii genome showed an altered 1,3-beta-D-glucan
44
synthase protein and several larger deletions in the echinocandin target FKS1 gene suggesting
45
potential for development of antifungal resistance.
46
Conclusions: The study emphasizes the emergence of a rare and uncommon yeast C. blankii,
47
with reduced susceptibility to one or more antifungal agents, in nosocomial fungemia. Genomic
48
insights of this rare yeast are reported using whole genome sequencing typing.
49
2
50
Introduction
51
Healthcare-associated fungal outbreaks due to uncommon and new species of fungi have been
52
frequently described in recent years [1]. Candida species are the third most common cause of
53
bloodstream infections in a recent point prevalence survey among hospitalized adults and
54
children in the United States [2]. In the last decade outbreaks of fungal sepsis due to emerging
55
and rare Candida species, particularly multidrug-resistant Candida auris have gained ample
56
attention among healthcare personnel [1,3]. All outbreaks emphasized the importance of
57
heightened awareness, rapid diagnostic methods and molecular typing tools such as Whole
58
Genome Sequencing (WGS), to promptly investigate and implement aggressive interventions in
59
controlling and preventing these infections in healthcare settings [3]. We report a cluster of
60
nosocomial fungemia in a neonatal intensive care unit (NICU) of a multispecialty hospital due to
61
the hitherto rare pathogen C. blankii over a period of eight months.
62
Materials and Methods
63
During June 2016 to January 2017, C. blankii fungemia occurred in nine neonates in the NICU
64
of a multispecialty hospital in North Delhi, India.
65
identified as C. blankii by sequencing of ITS and D1/D2 regions. MALDI-TOF MS Biotyper
66
(Bruker) yielded no identification of yeasts and therefore an in-house data base was created using
67
the first two molecularly identified strains as described previously [4]. The methods for
68
identification, antifungal susceptibility testing (AFST), amplified fragment length polymorphism
69
(AFLP) and WGS are detailed in Supplementary tables S1-S4 [5-7]. No ethical approval was
70
required given the retrospective nature of the study and the focus on the pathogen.
71
Results
Blood samples grew yeasts that were
3
72
The first case of C. blankii fungemia occurred in a very low birth weight (VLBW), neonate with
73
severe asphyxia. The patient developed symptoms of sepsis on day 2 after the birth and the blood
74
culture was collected which grew C. blankii two days after collection. As fluconazole (FLU)
75
treatment was initiated with a loading dose of 12mg/kg body weight followed by 6mg/kg body
76
weight, however, he expired on day 10. The time span between the first and second patient was 6
77
weeks. The second case also occurred in a low birth weight (LBW), preterm neonate whose
78
blood culture was collected at day 2 after birth and was positive for C. blankii on day 4 postnatal.
79
An epidemiological investigation was conducted after the third patient was identified. Overall,
80
nine neonates had C. blankii fungemia within a period of seven months (table 1). All neonates
81
had LBW and age at onset of fungemia ranged from 2 to 3 postnatal days. Of nine neonates, five
82
had a vaginal delivery and four were born with caesarean section. They had one or more
83
common risk factors i.e., preterm delivery, LBW/VLBW, and six had a central venous line and
84
required ventilator support. All were treated with FLU and four of nine patients died, resulting in
85
a case fatality rate of 45%. After the second case of fungemia was detected, enhanced infection
86
control measures including strict compliance with standard precautions in particular hand
87
hygiene, and contact isolation were instituted.
88
personnel in contact with neonates in pediatric and maternity units were obtained. The sampling
89
included screening for hand carriage and sampling of fomites, equipment, disinfectants and vials.
90
Considering that all patients developed early-onset sepsis, maternal high vaginal swabs (HVS)
91
were also cultured. However, all environmental and HVS cultures were negative for C. blankii.
92
C. blankii showed elongated to oval budding yeast cells and grew at 37°C and 45°C as white to
93
cream-colored colonies on SDA. VITEK-2 (bioMerieux) misidentified five C. blankii isolates as
94
C. cifferi and another five as Trichosporon asahii. An in-house C. blankii database (n=2) in
Surveillance cultures from all health care
4
95
MALDI Biotyper correctly identified the remaining 8 isolates with score values of 2-2.2. AFST
96
showed that FLU had higher MICs (GM MIC, 8 µg/ml) than the other azoles i.e., isavuconazole
97
(GM MIC, 0.07 µg/ml), posaconazole (GM MIC, 0.13 µg/ml), itraconazole (GM MIC, 0.18
98
µg/ml), and voriconazole (GM MIC, 0.25 µg/ml). Anidulafungin (GM MIC, 2 µg/ml) had high
99
MICs whereas micafungin (GM MIC, 0.06 µg/ml) exhibited potent activity (table 2).
100
The average draft genome sequence size of C. blankii is 13.65 Mb with an average G+C content
101
of 54.3%. The six assemblies ranged between 1706 and 11176 contigs and encoded an average
102
of 4370 protein coding genes. The qualitatively best genome contained 4626 gene models, which
103
appears to be a reasonable prediction when compared to the closest available sister species,
104
Tortispora caseinolytica encoding 4657 protein coding gene models. Overall, 47 gene models
105
from other Candida species were used to determine particular genes involved in antifungal
106
resistance. Such genes showed partially expected homologies (ERG11) to other Candida species
107
but were also contrasted by relatedness (FKS1) to unrelated black yeasts (Cladophialophora and
108
Fonsecaea). Clonality of outbreak isolates was determined by read mapping against the
109
qualitatively best reference genomes assembled within this study which yielded a maximum total
110
distance between genomes of 1.17% and a minimum distance of 0.34% respectively (see
111
supplementary material table S4). A single nucleotide polymorphism analysis indicated high
112
clonality of the investigated strain set, except for one isolate (VPCI_963/P/16) differing in
113
approximately 69.000 total polymorphic variants while strain to strain variation between all
114
remaining isolates was between 50 and 277 variants in total. The next generation sequencing
115
data together with the AFLP analysis of Indian C. blankii isolated clearly showed a clonal origin
116
for most isolates which was genotypically distinct from the type strain (AFLP pattern; figure 1)
5
117
Discussion
118
The study emphasizes the emergence of C. blankii in nosocomial fungemia and reports for the
119
first time the genomic insights of this rare yeast using WGS typing in an outbreak setting.
120
Candida blankii was described for the first time in 1968 from blood of a mink in Canada [8].
121
This yeast has been rarely isolated from human samples before this outbreak [9-13]. In a
122
prospective candidemia study in Norway a single isolate of C. blankii was identified [9]. In 2000,
123
C. blankii was isolated from the hands of a 2-month-old infant with atopic dermatitis in Russia
124
[10]. Subsequently it was isolated from respiratory samples of a 14-year-old cystic fibrosis (CF)
125
patient in Argentina [11]. So far only two cases of candidemia with clinical details have been
126
recorded in a 16-year-old female with CF in Brazil who underwent lung transplantation and in a
127
27-week-old preterm neonate in Kuwait [13]. Notable findings in both cases indicated that C.
128
blankii has reduced susceptibility to azoles and echinocandins. Despite that all isolates recovered
129
during this outbreak exhibited high MICs of FLU, five neonates treated with high doses of FLU
130
for 10-21 days survived and had cleared the fungemia. The lack of susceptibility breakpoints for
131
C. blankii and limited therapeutic data precludes the use of any specific antifungals in neonatal
132
sepsis.
133
It is not known if C. blankii exist on human skin or mucosa but its isolation from the skin of an
134
atopic dermatitis patient suggest it may colonize damaged skin [10]. Like C. auris, this yeast
135
grows at elevated temperature and is tolerant to high salt concentrations and thus has appropriate
136
attributes to be a human colonizer and pathogen. Killing assays in a Galleria mellonella model
137
has previously shown variable strain pathogenicity of C. blankii [12]. Remarkably, a strongly
138
altered 1,3-beta-D-glucan synthase protein sequence was inferred from the C. blankii genome. A
139
large deletion through almost the entire FKS1 region was identified together with multiple larger 6
140
deletions in the glucan synthase subunit. Interestingly, the evolutionary relation with respect to
141
FKS1, the cytochrome P450 superfamily was affiliated to black yeasts in the order
142
Chaetothyriales that are often associated with hydrocarbon-rich environments and are pathogenic
143
to humans (e.g. C. bantiana) [14]. Notably, C. blankii and its sister species such as C.
144
digboiensis and C. bituminiphila occur in similar hydrocarbon-rich environments, suggesting a
145
probable link between two unrelated higher-level taxonomic entities. Earlier, in India, C. blankii
146
was identified as the most frequently isolated species (4.7%) from nectary gland samples of
147
flowers [15]. In the present outbreak all isolates, except for one were genotypically
148
indistinguishable with very high genome to genome identity suggesting at least a single, yet
149
undetermined origin and route of transmission. The study is limited by the fact that in spite of
150
thorough investigations the source of C. blankii could not be traced. This report alerts to the
151
importance of the rare and uncommon yeast C. blankii that exhibit reduced susceptibility to one
152
or more antifungal agents and has the potential of causing serious invasive infections warranting
153
thorough understanding of its epidemiology and transmission.
154
7
155
Financial support
156
This work was supported in part by a research grant from India Council of Medical Research [F.
157
No. OMI/13/2014-ECD-I to A. C.], Government of India, New Delhi, India.
158
159
Potential conflict of interest
160
J. F. M. has received grants from F2G and Pulmozyme; has been a consultant to Scynexis; and
161
has received speaker’s fees from United Medical, TEVA and Gilead. J. B. S. is employee of
162
Thermo Fisher Scientific, Landsmeer, the Netherlands. All other authors: none to declare.
163 164
Acknowledgment
165
We thank Mr. Pradip Roy, Department of Medical Mycology, Vallabhbhai Patel Chest Institute,
166
University of Delhi for technical assistance.
167
8
168
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169
1. Benedict K, Richardson M, Vallabhaneni S, Jackson BR, Chiller T. Emerging issues,
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4. De Carolis E, Posteraro B, Lass-Flörl C et al. Species identification of Aspergillus,
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http://dx.doi .org/10.1111/j.1469-0691.2011.03599.x.
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5. Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution
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Antifungal Susceptibility Testing of Yeasts; Approved Standard—Third Edition: CLSI
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Document M27-A3. CLSI, Wayne, PA, USA, 2008.
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6. de Valk HA, Meis JF, de Pauw BE, Donnelly PJ, Klaassen CH. Comparison of two
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highly discriminatory molecular fingerprinting assays for analysis of multiple Aspergillus
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fumigatus isolates from patients with invasive aspergillosis. J Clin Microbiol. 2007; 45:
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1415–1419.
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7. Chatterjee S, Alampalli SV, Nageshan RK et al. Draft genome of a commonly
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misdiagnosed multidrug resistant pathogen Candida auris. BMC Genomics. 2015; 16:
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686. doi: 10.1186/s12864-015-1863-z.
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8. Buckley HR, van Uden N. Five new Candida species. Mycopathol Mycol Appl. 1968; 36: 257-266. 9. Sandven P, Bevanger L, Digranes A et al. Candidemia in Norway (1991 to 2003): results from a nationwide study. J Clin Microbiol. 2006; 44: 1977-1981. 10. Arzumanyan
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allergic diseases: species variety and sensitivity to antifungal drugs. Bull Exp Biol
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Med. 2000; 129: 601-604.
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11. Zaragoza S, Galanternik L, Vazquez M, Teper A, Córdoba S, Finquelievich J. Candida
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blankii: New agent in cystic fibrosis airways? J Cyst Fibros. 2015; 14:S140.
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doi: https://doi.org/10.1016/S1569-1993(15)30492-6.
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12. Nobrega de Almeida J Jr, Campos SV, Thomaz DY et al. Candida blankii: an emergent
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opportunistic yeast with reduced susceptibility to antifungals.
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Infect. 2018; 7: 24. doi: 10.1038/s41426-017-0015-8.
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13. Al-Haqqan A, Al-Sweih N, Ahmad S et al. Azole-resistant Candida blankii as a newly
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recognized cause of bloodstream infection. New Microb New Infect. 2018; 26: 25-29.
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14. Teixeira MM, Moreno LF, Stielow BJ et al. Exploring the genomic diversity of black
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15. Sandhu DK, Waraich MK. Yeasts associated with pollinating bees and flower nectar. Microb Ecol. 1985; 11: 51-58. doi: 10.1007/BF02015108.
213
11
214
Legend
215
Figure 1: Amplified fragment length polymorphism analysis showing all Indian C. blankii strains
216
had a clonal origin and was genotypically distinct from the C. blankii type strain from Canada
217
(CBS1898). The dendrogram was constructed by using UPGMA (unweighted pair group method
218
with averages) in combination with the Pearson correlation coefficient and was restricted to
219
fragments of 60–400 bp. Scale bar indicates the percentage similarity.
220 221 222
12
Table 1: Clinical details of 9 neonates with Candida blankii fungemia. Patient
DOB/DOA (day/month/year)
Isolate ID
GA (weeks)/ sex/mode of delivery
BW (gm)
Risk factors
Antifungal therapy
NICU stay/ Outcome
FLU for 10 days (12 mg/ kg BW loading dose followed by 6 mg/kg); Van & Mero for 7 days
10 days/ Death
01
VPCI 936/P/16#
05/06/2016
41/M/VD
1200
VLBW, IUGR, thrombocytopenia, CVC, severe asphyxia, sepsis, mechanical ventilation
02
VPCI 1130/P/16#
19/07/2016
37/M/VD
1900
LBW, IUGR, sepsis, thrombocytopenia
FLU for 14 days Van & Mero for 10 days
15 days/ Survived
03
VPCI 1168/P16#
02/08/2016
35/F/VD
1780
PT,LBW, sepsis, thrombocytopenia
FLU 14 days Van & Mero 14 days
23 days/ Survived
04
#
1500
PT, LBW, IUGR, thrombocytopenia, Maternal history of Pre-eclampsia, anti partum hemorrhage,
FLU for 12 days Ami & Cip for 10 days
14 days/ Survived
FLU for 6 days Van & Mero for 6 days
6 days/ Death
VPCI 1175/P/16
06/08/2016
36/F/LSCS
05
VPCI 2237/P/16
04/09/2016
38/F/LSCS
1100
VLBW, Persistent hypoglycemia, severe asphyxia, sepsis,CVC mechanical ventilation
06
VPCI 2251/P/16
05/12/2016
24/M/VD
855
Extremely PT, ELBW, Severe asphyxia, CVC, sepsis, mechanical ventilation
FLU for 10 days
12 days/ Death
07*
VPCI 3415/P/16# & VPCI 54/P/17
21/12/2016
24/M/LSCS
1035
Extremely PT, VLBW, sepsis, persistent hypoglycemia, sever asphyxia, CVC, mechanical ventilation
FLU for 10 days Van & Mero for 7 days
18 days/ Survived
08
VPCI 3429/P/16
26/12/2016
40/M/LSCS
3440
Severe asphyxia, CVC, thrombocytopenia, persistent hypoglycemia, mechanical ventilation
FLU for 5 days Van & Mero for 5 days
5 days/ Death
09
VPCI 45/P/17#
09/01/2017
28/M/LSCS
855
Very preterm, ELBW, Severe asphyxia, thrombocytopenia, sepsis, CVC, mechanical ventilation
FLU for 21 days Van & Mero for 14 days
25 days/ Survived
*Two isolates from two blood cultures were obtained from the same patient. # Isolates selected for whole genome sequencing; DOB, date of birth; DOA, date of admission; GA, gestational age; M, male; VD, vaginal delivery; F, female; LSCS, lower segment cesarean section; BW, birth weight; VLBW, very low birth
weight; IUGR, intrauterine growth restriction, CVC, central venous catheter; PT, Preterm; LBW, low birth weight; ELBW, extremely low birth weight; FLU, fluconazole; Van, Vancomycin; Mero, Meropenem; Ami, Amikacin; Cip, ciprofloxacin. All neonates were given FLU 12 mg/ kg BW (loading dose) followed by 6 mg/kg body weight.
Table 2: Antifungal susceptibility profile of Candida blankii isolates (n=10) Isolate no. VPCI 936/P/16 VPCI 1130/P/16 VPCI 1168/P/16 VPCI 1175/P/16 VPCI 2237/P/16 VPCI 2251/P/16 VPCI 3415/P/16 VPCI 3429/P/16 VPCI 45/P/17 VPCI 54/P/17 Range MIC50 MIC90 GM
FLU 8 8 8 8 8 8 8 8 8 8 8 8 8 8
ITC 0.25 0.125 0.25 0.25 0.125 0.25 0.125 0.125 0.25 0.25 0.125-0.25 0.25 0.25 0.18
VRC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
Drugs* ISA PSC AMB 0.06 0.25 0.5 0.125 0.125 0.25 0.06 0.06 0.25 0.125 0.25 0.25 0.06 0.125 0.25 0.03 0.06 0.25 0.125 0.125 0.25 0.03 0.125 0.25 0.125 0.125 0.25 0.06 0.25 0.5 0.03-0.125 0.06-0.25 0.25-0.5 0.06 0.125 0.25 0.125 0.25 0.5 0.07 0.13 0.28
CAS 1 1 1 1 1 2 2 1 2 2 1-2 1 2 1.31
MFG 0.125 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.125 0.06-0.125 0.06 0.125 0.06
AFG 2 2 2 2 2 2 2 2 2 2 2 2 2 2
FC 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.12
* FLU, fluconazole; ITC, itraconazole; VRC, voriconazole; ISA, isavuconazole; PSC, posaconazole; AMB, ampotericine B; CAS, caspofungin; MFG, micafungin; AFG, anidulafungin; FC, 5-flucytocine
S1198-743X(20)30002-1
DOI:
https://doi.org/10.1016/j.cmi.2020.01.001
Reference:
CMI 1887
To appear in:
Clinical Microbiology and Infection
Received Date: 3 October 2019 Revised Date:
11 December 2019
Accepted Date: 1 January 2020
Please cite this article as: Chowdhary A, Stielow JB, Upadhaya G, Singh PK, Singh A, Meis JF, Candida blankii: an emerging yeast in an outbreak of fungemia in neonates in Delhi, India, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2020.01.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.
1
Candida blankii: an emerging yeast in an outbreak of fungemia in neonates in Delhi, India
2 3
Anuradha Chowdhary1#, J. Benjamin Stielow2,3, Gargi Upadhaya1, Pradeep K Singh1, Ashutosh
4
Singh1, Jacques F Meis3,4
5 6 7
1
Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi;
8
2
Thermo Fisher Diagnostics, Specialty Diagnostics Group, Landsmeer, The Netherlands; 3Centre
9
of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands; 4Department of
10
Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ),
11
Nijmegen, The Netherlands.
12 13 14 15 16
Keywords: Candida blankii, Outbreak, Whole genome sequencing, Neonates, India
17
Running Title: Candida blankii outbreak in NICU, India
18 19 20 21 22 23 24 25 26
#Corresponding author Prof. Anuradha Chowdhary Department of Medical Mycology V. P. Chest Institute, University of Delhi Delhi-110007, India Email: [email protected]
27 1
28
Abstract
29
Objective: Outbreaks of fungal sepsis due to emerging and rare multidrug resistant Candida
30
species are increasingly reported in healthcare settings. We report an outbreak of fungemia due
31
to the rare multidrug-resistant yeast Candida blankii in an Indian neonatal unit.
32
Material and Methods: Blood cultures grew C. blankii in nine neonates in the NICU of a
33
multispecialty hospital, Delhi during a period of seven months. Isolates were identified by ITS
34
and D1/D2 region sequencing. Antifungal susceptibility testing was performed by M27-A3 CLSI
35
broth microdilution. To determine genetic relatedness among C. blankii isolates we undertook
36
amplified fragment length polymorphism analysis and DNA sequencing using the Illumina
37
NextSeq500 platform.
38
Results: C. blankii fungemia occurred at 2-3 postnatal days in nine LBW/VLBW neonates. All
39
neonates were treated with fluconazole and four of nine patients died, resulting in a case fatality
40
rate of 45%. C. blankii was misidentified or not identified by automated identification systems.
41
Fluconazole had higher MICs (GM-MIC, 8 µg/ml) than the other azoles. Also, anidulafungin
42
(GM-MIC, 2 µg/ml) had high MICs. Genome sequencing confirmed transmission of genetically
43
mostly indistinguishable strains. The C. blankii genome showed an altered 1,3-beta-D-glucan
44
synthase protein and several larger deletions in the echinocandin target FKS1 gene suggesting
45
potential for development of antifungal resistance.
46
Conclusions: The study emphasizes the emergence of a rare and uncommon yeast C. blankii,
47
with reduced susceptibility to one or more antifungal agents, in nosocomial fungemia. Genomic
48
insights of this rare yeast are reported using whole genome sequencing typing.
49
2
50
Introduction
51
Healthcare-associated fungal outbreaks due to uncommon and new species of fungi have been
52
frequently described in recent years [1]. Candida species are the third most common cause of
53
bloodstream infections in a recent point prevalence survey among hospitalized adults and
54
children in the United States [2]. In the last decade outbreaks of fungal sepsis due to emerging
55
and rare Candida species, particularly multidrug-resistant Candida auris have gained ample
56
attention among healthcare personnel [1,3]. All outbreaks emphasized the importance of
57
heightened awareness, rapid diagnostic methods and molecular typing tools such as Whole
58
Genome Sequencing (WGS), to promptly investigate and implement aggressive interventions in
59
controlling and preventing these infections in healthcare settings [3]. We report a cluster of
60
nosocomial fungemia in a neonatal intensive care unit (NICU) of a multispecialty hospital due to
61
the hitherto rare pathogen C. blankii over a period of eight months.
62
Materials and Methods
63
During June 2016 to January 2017, C. blankii fungemia occurred in nine neonates in the NICU
64
of a multispecialty hospital in North Delhi, India.
65
identified as C. blankii by sequencing of ITS and D1/D2 regions. MALDI-TOF MS Biotyper
66
(Bruker) yielded no identification of yeasts and therefore an in-house data base was created using
67
the first two molecularly identified strains as described previously [4]. The methods for
68
identification, antifungal susceptibility testing (AFST), amplified fragment length polymorphism
69
(AFLP) and WGS are detailed in Supplementary tables S1-S4 [5-7]. No ethical approval was
70
required given the retrospective nature of the study and the focus on the pathogen.
71
Results
Blood samples grew yeasts that were
3
72
The first case of C. blankii fungemia occurred in a very low birth weight (VLBW), neonate with
73
severe asphyxia. The patient developed symptoms of sepsis on day 2 after the birth and the blood
74
culture was collected which grew C. blankii two days after collection. As fluconazole (FLU)
75
treatment was initiated with a loading dose of 12mg/kg body weight followed by 6mg/kg body
76
weight, however, he expired on day 10. The time span between the first and second patient was 6
77
weeks. The second case also occurred in a low birth weight (LBW), preterm neonate whose
78
blood culture was collected at day 2 after birth and was positive for C. blankii on day 4 postnatal.
79
An epidemiological investigation was conducted after the third patient was identified. Overall,
80
nine neonates had C. blankii fungemia within a period of seven months (table 1). All neonates
81
had LBW and age at onset of fungemia ranged from 2 to 3 postnatal days. Of nine neonates, five
82
had a vaginal delivery and four were born with caesarean section. They had one or more
83
common risk factors i.e., preterm delivery, LBW/VLBW, and six had a central venous line and
84
required ventilator support. All were treated with FLU and four of nine patients died, resulting in
85
a case fatality rate of 45%. After the second case of fungemia was detected, enhanced infection
86
control measures including strict compliance with standard precautions in particular hand
87
hygiene, and contact isolation were instituted.
88
personnel in contact with neonates in pediatric and maternity units were obtained. The sampling
89
included screening for hand carriage and sampling of fomites, equipment, disinfectants and vials.
90
Considering that all patients developed early-onset sepsis, maternal high vaginal swabs (HVS)
91
were also cultured. However, all environmental and HVS cultures were negative for C. blankii.
92
C. blankii showed elongated to oval budding yeast cells and grew at 37°C and 45°C as white to
93
cream-colored colonies on SDA. VITEK-2 (bioMerieux) misidentified five C. blankii isolates as
94
C. cifferi and another five as Trichosporon asahii. An in-house C. blankii database (n=2) in
Surveillance cultures from all health care
4
95
MALDI Biotyper correctly identified the remaining 8 isolates with score values of 2-2.2. AFST
96
showed that FLU had higher MICs (GM MIC, 8 µg/ml) than the other azoles i.e., isavuconazole
97
(GM MIC, 0.07 µg/ml), posaconazole (GM MIC, 0.13 µg/ml), itraconazole (GM MIC, 0.18
98
µg/ml), and voriconazole (GM MIC, 0.25 µg/ml). Anidulafungin (GM MIC, 2 µg/ml) had high
99
MICs whereas micafungin (GM MIC, 0.06 µg/ml) exhibited potent activity (table 2).
100
The average draft genome sequence size of C. blankii is 13.65 Mb with an average G+C content
101
of 54.3%. The six assemblies ranged between 1706 and 11176 contigs and encoded an average
102
of 4370 protein coding genes. The qualitatively best genome contained 4626 gene models, which
103
appears to be a reasonable prediction when compared to the closest available sister species,
104
Tortispora caseinolytica encoding 4657 protein coding gene models. Overall, 47 gene models
105
from other Candida species were used to determine particular genes involved in antifungal
106
resistance. Such genes showed partially expected homologies (ERG11) to other Candida species
107
but were also contrasted by relatedness (FKS1) to unrelated black yeasts (Cladophialophora and
108
Fonsecaea). Clonality of outbreak isolates was determined by read mapping against the
109
qualitatively best reference genomes assembled within this study which yielded a maximum total
110
distance between genomes of 1.17% and a minimum distance of 0.34% respectively (see
111
supplementary material table S4). A single nucleotide polymorphism analysis indicated high
112
clonality of the investigated strain set, except for one isolate (VPCI_963/P/16) differing in
113
approximately 69.000 total polymorphic variants while strain to strain variation between all
114
remaining isolates was between 50 and 277 variants in total. The next generation sequencing
115
data together with the AFLP analysis of Indian C. blankii isolated clearly showed a clonal origin
116
for most isolates which was genotypically distinct from the type strain (AFLP pattern; figure 1)
5
117
Discussion
118
The study emphasizes the emergence of C. blankii in nosocomial fungemia and reports for the
119
first time the genomic insights of this rare yeast using WGS typing in an outbreak setting.
120
Candida blankii was described for the first time in 1968 from blood of a mink in Canada [8].
121
This yeast has been rarely isolated from human samples before this outbreak [9-13]. In a
122
prospective candidemia study in Norway a single isolate of C. blankii was identified [9]. In 2000,
123
C. blankii was isolated from the hands of a 2-month-old infant with atopic dermatitis in Russia
124
[10]. Subsequently it was isolated from respiratory samples of a 14-year-old cystic fibrosis (CF)
125
patient in Argentina [11]. So far only two cases of candidemia with clinical details have been
126
recorded in a 16-year-old female with CF in Brazil who underwent lung transplantation and in a
127
27-week-old preterm neonate in Kuwait [13]. Notable findings in both cases indicated that C.
128
blankii has reduced susceptibility to azoles and echinocandins. Despite that all isolates recovered
129
during this outbreak exhibited high MICs of FLU, five neonates treated with high doses of FLU
130
for 10-21 days survived and had cleared the fungemia. The lack of susceptibility breakpoints for
131
C. blankii and limited therapeutic data precludes the use of any specific antifungals in neonatal
132
sepsis.
133
It is not known if C. blankii exist on human skin or mucosa but its isolation from the skin of an
134
atopic dermatitis patient suggest it may colonize damaged skin [10]. Like C. auris, this yeast
135
grows at elevated temperature and is tolerant to high salt concentrations and thus has appropriate
136
attributes to be a human colonizer and pathogen. Killing assays in a Galleria mellonella model
137
has previously shown variable strain pathogenicity of C. blankii [12]. Remarkably, a strongly
138
altered 1,3-beta-D-glucan synthase protein sequence was inferred from the C. blankii genome. A
139
large deletion through almost the entire FKS1 region was identified together with multiple larger 6
140
deletions in the glucan synthase subunit. Interestingly, the evolutionary relation with respect to
141
FKS1, the cytochrome P450 superfamily was affiliated to black yeasts in the order
142
Chaetothyriales that are often associated with hydrocarbon-rich environments and are pathogenic
143
to humans (e.g. C. bantiana) [14]. Notably, C. blankii and its sister species such as C.
144
digboiensis and C. bituminiphila occur in similar hydrocarbon-rich environments, suggesting a
145
probable link between two unrelated higher-level taxonomic entities. Earlier, in India, C. blankii
146
was identified as the most frequently isolated species (4.7%) from nectary gland samples of
147
flowers [15]. In the present outbreak all isolates, except for one were genotypically
148
indistinguishable with very high genome to genome identity suggesting at least a single, yet
149
undetermined origin and route of transmission. The study is limited by the fact that in spite of
150
thorough investigations the source of C. blankii could not be traced. This report alerts to the
151
importance of the rare and uncommon yeast C. blankii that exhibit reduced susceptibility to one
152
or more antifungal agents and has the potential of causing serious invasive infections warranting
153
thorough understanding of its epidemiology and transmission.
154
7
155
Financial support
156
This work was supported in part by a research grant from India Council of Medical Research [F.
157
No. OMI/13/2014-ECD-I to A. C.], Government of India, New Delhi, India.
158
159
Potential conflict of interest
160
J. F. M. has received grants from F2G and Pulmozyme; has been a consultant to Scynexis; and
161
has received speaker’s fees from United Medical, TEVA and Gilead. J. B. S. is employee of
162
Thermo Fisher Scientific, Landsmeer, the Netherlands. All other authors: none to declare.
163 164
Acknowledgment
165
We thank Mr. Pradip Roy, Department of Medical Mycology, Vallabhbhai Patel Chest Institute,
166
University of Delhi for technical assistance.
167
8
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11
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Legend
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Figure 1: Amplified fragment length polymorphism analysis showing all Indian C. blankii strains
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had a clonal origin and was genotypically distinct from the C. blankii type strain from Canada
217
(CBS1898). The dendrogram was constructed by using UPGMA (unweighted pair group method
218
with averages) in combination with the Pearson correlation coefficient and was restricted to
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fragments of 60–400 bp. Scale bar indicates the percentage similarity.
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12
Table 1: Clinical details of 9 neonates with Candida blankii fungemia. Patient
DOB/DOA (day/month/year)
Isolate ID
GA (weeks)/ sex/mode of delivery
BW (gm)
Risk factors
Antifungal therapy
NICU stay/ Outcome
FLU for 10 days (12 mg/ kg BW loading dose followed by 6 mg/kg); Van & Mero for 7 days
10 days/ Death
01
VPCI 936/P/16#
05/06/2016
41/M/VD
1200
VLBW, IUGR, thrombocytopenia, CVC, severe asphyxia, sepsis, mechanical ventilation
02
VPCI 1130/P/16#
19/07/2016
37/M/VD
1900
LBW, IUGR, sepsis, thrombocytopenia
FLU for 14 days Van & Mero for 10 days
15 days/ Survived
03
VPCI 1168/P16#
02/08/2016
35/F/VD
1780
PT,LBW, sepsis, thrombocytopenia
FLU 14 days Van & Mero 14 days
23 days/ Survived
04
#
1500
PT, LBW, IUGR, thrombocytopenia, Maternal history of Pre-eclampsia, anti partum hemorrhage,
FLU for 12 days Ami & Cip for 10 days
14 days/ Survived
FLU for 6 days Van & Mero for 6 days
6 days/ Death
VPCI 1175/P/16
06/08/2016
36/F/LSCS
05
VPCI 2237/P/16
04/09/2016
38/F/LSCS
1100
VLBW, Persistent hypoglycemia, severe asphyxia, sepsis,CVC mechanical ventilation
06
VPCI 2251/P/16
05/12/2016
24/M/VD
855
Extremely PT, ELBW, Severe asphyxia, CVC, sepsis, mechanical ventilation
FLU for 10 days
12 days/ Death
07*
VPCI 3415/P/16# & VPCI 54/P/17
21/12/2016
24/M/LSCS
1035
Extremely PT, VLBW, sepsis, persistent hypoglycemia, sever asphyxia, CVC, mechanical ventilation
FLU for 10 days Van & Mero for 7 days
18 days/ Survived
08
VPCI 3429/P/16
26/12/2016
40/M/LSCS
3440
Severe asphyxia, CVC, thrombocytopenia, persistent hypoglycemia, mechanical ventilation
FLU for 5 days Van & Mero for 5 days
5 days/ Death
09
VPCI 45/P/17#
09/01/2017
28/M/LSCS
855
Very preterm, ELBW, Severe asphyxia, thrombocytopenia, sepsis, CVC, mechanical ventilation
FLU for 21 days Van & Mero for 14 days
25 days/ Survived
*Two isolates from two blood cultures were obtained from the same patient. # Isolates selected for whole genome sequencing; DOB, date of birth; DOA, date of admission; GA, gestational age; M, male; VD, vaginal delivery; F, female; LSCS, lower segment cesarean section; BW, birth weight; VLBW, very low birth
weight; IUGR, intrauterine growth restriction, CVC, central venous catheter; PT, Preterm; LBW, low birth weight; ELBW, extremely low birth weight; FLU, fluconazole; Van, Vancomycin; Mero, Meropenem; Ami, Amikacin; Cip, ciprofloxacin. All neonates were given FLU 12 mg/ kg BW (loading dose) followed by 6 mg/kg body weight.
Table 2: Antifungal susceptibility profile of Candida blankii isolates (n=10) Isolate no. VPCI 936/P/16 VPCI 1130/P/16 VPCI 1168/P/16 VPCI 1175/P/16 VPCI 2237/P/16 VPCI 2251/P/16 VPCI 3415/P/16 VPCI 3429/P/16 VPCI 45/P/17 VPCI 54/P/17 Range MIC50 MIC90 GM
FLU 8 8 8 8 8 8 8 8 8 8 8 8 8 8
ITC 0.25 0.125 0.25 0.25 0.125 0.25 0.125 0.125 0.25 0.25 0.125-0.25 0.25 0.25 0.18
VRC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
Drugs* ISA PSC AMB 0.06 0.25 0.5 0.125 0.125 0.25 0.06 0.06 0.25 0.125 0.25 0.25 0.06 0.125 0.25 0.03 0.06 0.25 0.125 0.125 0.25 0.03 0.125 0.25 0.125 0.125 0.25 0.06 0.25 0.5 0.03-0.125 0.06-0.25 0.25-0.5 0.06 0.125 0.25 0.125 0.25 0.5 0.07 0.13 0.28
CAS 1 1 1 1 1 2 2 1 2 2 1-2 1 2 1.31
MFG 0.125 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.125 0.06-0.125 0.06 0.125 0.06
AFG 2 2 2 2 2 2 2 2 2 2 2 2 2 2
FC 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.12
* FLU, fluconazole; ITC, itraconazole; VRC, voriconazole; ISA, isavuconazole; PSC, posaconazole; AMB, ampotericine B; CAS, caspofungin; MFG, micafungin; AFG, anidulafungin; FC, 5-flucytocine