- Email: [email protected]
Superficial candidosis by Candida duobushaemulonii: An emerging microorganism
Accepted Manuscript Superficial candidosis by Candida duobushaemulonii: An emerging microorganism
María Guadalupe Frías-De-León, Erick Martínez-Herrera, Gustavo Acosta-Altamirano, Roberto Arenas, Carmen RodríguezCerdeira PII: DOI: Article Number: Reference:
S1567-1348(19)30179-0 https://doi.org/10.1016/j.meegid.2019.103960 103960 MEEGID 103960
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
11 March 2019 5 July 2019 9 July 2019
Please cite this article as: M.G. Frías-De-León, E. Martínez-Herrera, G. AcostaAltamirano, et al., Superficial candidosis by Candida duobushaemulonii: An emerging microorganism, Infection, Genetics and Evolution, https://doi.org/10.1016/ j.meegid.2019.103960
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT Superficial candidosis by Candida duobushaemulonii: an emerging microorganism
María Guadalupe Frías-De-León1,5,6*, Erick Martínez-Herrera1,5,6*, Gustavo Acosta-
T
Altamirano1,6, Roberto Arenas2,3,5,6, Carmen Rodríguez-Cerdeira3,4,5,6**
Research Unit, High Specialty Regional Hospital of Ixtapaluca. Ixtapaluca, Edo. Mexico
2
Mycoloy Service, Hospital Manuel Gea González, Mexico City, Mexico
3
Efficiency, quality and costs in Health Services Research Group (EFISALUD), Health
CR
IP
1
US
Research Institute. SERGAS-UVIGO.
Dermatology Department, Hospital do Meixoeiro and University of Vigo, Vigo, Spain
5
European Women’s Dermatologic and Venereologic Society (EWDVS), Vigo, Spain
6
Psychodermatology task force of the Ibero-Latin American College of Dermatology
M
AN
4
CE
PT
ED
(CILAD)
AC
*Correspondence should be addressed to: Carmen Rodriguez-Cerdeira Meixoeiro Hospital,CHUVI, C/Meixoeiro S/N 36200, Vigo, Spain Tel: 0034986814517/0034600536114 Fax: 004986276416 E-mail: [email protected]
ACCEPTED MANUSCRIPT Introduction Species of the genus Candida are the most common causative agents of fungal infections worldwide. They can cause superficial (cutaneous, mucocutaneous, onychomycosis) and systemic (meningitis, sepsis, candidemia, others) infections that can be disseminated or deep (chronic mucocutaneous candidosis, candida granuloma) (1). Among these, the most common fungal species is C. albicans; however, currently non-C. albicans species (NCA)
T
are emerging as pathogens in immunocompetent and immunosuppressed individuals.
IP
Several NCAs have been isolated from superficial infections, including C. parapsilosis, C.
CR
glabrata, C. orthopsilosis, C. metapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. intermedia, and C. sake (2-6). Other rarely found species, such as those of the
C.
US
haemulonii complex, exhibit patterns of variable susceptibility to amphotericin B and azole derivatives (7, 8). The C. haemulonii complex comprises C. haemulonii, C. haemulonii var.
AN
vulnera, and C. duobushaemulonii (9), which have been isolated from infections in humans (10). However, information regarding the occurrence and distribution of C. haemulonii
M
complex species in clinical samples is limited because the methods, such as the VITEK 2 system, API 20C, and CHROMagar Candida, commonly used for the identification of these
ED
fungi, cannot distinctly identify these organisms (11). While methods exist that do permit distinction among closely related species, including MALDI-TOF and sequencing of the
PT
internal transcribed spacer region (ITS) of ribosomal DNA (12), these methods are not widely available in hospital laboratories. Due to the emergence of the C. haemulonii
CE
complex and other phylogenetically related species as pathogens resistant to azole antifungals, Kathuria et al. (13) recommended the establishment of a reliable method for
AC
the identification and testing of antifungal susceptibility of rare yeasts, as accurate identification and appropriate treatment are of particular importance in clinical settings. Similar studies were performed by Cendejas-Bueno et al. (9), where they observed that the C. haemulonii complex was resistant to amphotericin B and exhibited cross-resistance to azole compounds. Ramos et al. (12) observed two samples of C. duobushaemulonii that were susceptible to itraconazole, and the remaining experimental samples (75%) were resistant to this antifungal. Out of the 12 samples, eight (66.7%) were resistant to voriconazole; however, all isolates were susceptible to caspofungin (MIC = 0.5 mg / L).
ACCEPTED MANUSCRIPT Here, we report two clinical isolates obtained from a patient with total dystrophic onychomycosis and another with mucocutaneous candidosis. These isolates were originally identified as C. albicans by CHROMagar Candida (Becton-Dickinson, New Jersey, USA) and Trichosporon spp. by isolation in Sabouraud agar and subsequent microscopic morphological identification, and as C. haemulonii by the VITEK® 2 system (BioMérieux®). Our molecular analysis of these isolates resulted in their re-identification
IP
T
as C. duobushaemulonii.
CR
Material and methods
Two yeast-like isolates (77-18 and 553) were analysed, and these isolates were obtained
US
from two male patients (aged 33 and 75 years), who attended a dermatological consultation due to a perianal lesion present for 4 months and due to a 2-year infection of all toenails,
AN
respectively (Fig. 1).
Originally, the isolates were identified as C. albicans by CHROMagar Candida (Becton-
M
Dickinson, New Jersey, USA), as Trichosporon spp. following isolation on Sabouraud agar and subsequent microscopic morphological identification, and as C. haemulonii by the
ED
VITEK® 2 system (BioMérieux®). The patient with onychomycosis was treated with 200 mg of itraconazole daily for three months. However, this patient did not return to
PT
consultation, so we were unable to complete his follow-up. The patient with mucocutaneous candidosis was administered itraconazole at 100 mg / day
CE
and exhibited significant improvement at 15 days. After additional 15 days of treatment, the
AC
pathology observed in this patient was satisfactorily resolved.
Molecular identification DNA extraction: Isolate DNA was extracted using the Yeast DNA Preparation kit (Jena Bioscience, GE) according to manufacturer instructions. Amplification of the D1 / D2 region of the 26S rDNA: The DNA of the two isolates was analysed
by
PCR
using
GCATATCAATAAGCGGAGGAAAAG-3') GGTCCGTGTTTCAAGACGG-3') (14).
the
oligonucleotides and
NL1 NL4
(5'(5'-
ACCEPTED MANUSCRIPT The reactions were performed in a reaction volume of 25 μL that included 2.5 mM MgCl2, 0.2 mM dATP, dCTP, dGTP, and dTTP (Jena Bioscience), 0.1 μM of each NL1 and NL4 oligonucleotide (Sigma-Aldrich, USA), 1 U of Taq polymerase (Jena Bioscience), and 20 ng of DNA. Amplifications were performed in a T100 ™ thermocycler (Bio-Rad, Laboratories, Inc., USA) with the following parameters: 95ºC for 7 min, followed by 40 cycles at 95ºC for 1 min, 53ºC for 2 min, 72ºC for 1 min, and a final extension at 72ºC for
T
10 min. Deionised water was used as a negative control. The amplification products were
IP
separated by electrophoresis on 1.5% agarose gels in 0.5X TBE buffer (45 mM Tris-Base,
CR
45 mM boric acid, 1 mM EDTA and pH 8.3) at 90 v for 60 min, and they were visualized by staining with GelRed 30X (Biotium, USA). The 100 bp DNA ladder (Jena Bioscience)
US
was used as a molecular size marker. Gel images were captured in a Molecular Imager® Gel Doc ™ XR photodocument, (Bio-Rad Laboratories).
AN
Sequencing of amplicons: The PCR products were purified using the PCR Purification Kit (Jena Bioscience) according to manufacturer instructions. The purified amplicons were
M
sequenced at the National Laboratory of Genomics for Biodiversity, Langebio - Cinvestav
ED
Irapuato, Gto., Mexico. The sequences were deposited into the GenBank database.
Identification by sequence similarity: Sequence similarity was analysed by the BLASTN of
the
National
Center
for
Biotechnology
Information
(NCBI)
PT
program
(ncbi.nlm.nih.gov/BLAST/). The identity of each isolate was determined according to the
CE
similarity of the experimental sequences to the sequences deposited in the GenBank
Results
AC
database. The parameters used included similarity >97% (15).
Both isolates (77-18 and 553) amplified a fragment of approximately 620 bp. BLAST analysis revealed 100% identity and 100% coverage (similarity), and this analysis also indicated an "e-value" close to zero (4e-83) among the sequences of each isolate and the sequences of the D1 region / D2 of the 26S rDNA of C. duobushaemulonii. The sequences derived from both isolates were deposited into GenBank (Access No.: xx and zz).
Discussion
ACCEPTED MANUSCRIPT This study highlights the importance of implementing more specific microbiological identification methods, as the number of recognized pathogenic yeasts is steadily increasing. This increase is associated with the description of new species that are closely related to currently identified species, and in certain cases these new species have been found to exist in complexes, such as those that comprise the C. haemoulonii complex (12). Within the C. haemoulonii complex, all species have been shown to cause infections in
T
humans. C. duobushaemulonii has been reported by Fang et al. (16) to be present in deep
IP
cutaneous infections. Specifically, this infection was observed in a 68-year-old male patient
CR
who presented with ulcerative crusting and confluent lesions located on the right forearm and arm. The patient was treated with amphotericin B and then switched to intravenous
revealed a complete resolution of the problem.
US
fluconazole 200 mg / once daily for 7 days. Follow-up after 6 months post-treatment
AN
Infection of chronic wounds in lower extremities with the diagnosis of candidemia was published by Almeida et al. (10). In Brazil between January 2010 and March 2015, the
M
authors found 9 samples positive for C. duobushaemulonii. Majority of the patients were diabetic, and another group of patients were organ transplant recipients.
ED
In another study conducted by Boatto et al. (17), 13 women diagnosed with RVVC and their partners were selected investigation. Isolates were obtained using CHROMagar
PT
Candida medium, and species identification was determined by phenotypic and molecular methods. Susceptibility to fluconazole was evaluated by E-test. Out of the 26 strains, six
CE
were identified as C. duobushaemulonii. These patients exhibited a very low sensitivity to fluconazole (17). Majority of authors agree that patients diagnosed with diabetes mellitus
AC
are more likely to be infected with this fungus (10, 16). Information regarding the occurrence and distribution of species of the C. haemulonii complex in clinical samples is scarce, and this is likely due to the fact that phenotypic identification methods are used in many countries, and these do not allow for discrimination among closely related species. In this study, we verified that both the CHROMagar and the VITEK 2 systems are limited for the identification of C. duobushaemulonii, despite the known differences between these methods. For CHROMagar, C. albicans was identified, while the automated system VITEK 2 indicated C. haemoulonii with a high percentage of probability; however, only the C. haemoulonii complex was determined. This type of erroneous microbiological
ACCEPTED MANUSCRIPT identification results in poor therapeutic management of patients, as the fungi comprising the C. haemoulonii complex typically exhibit resistance to amphotericin B and azole derivatives (7, 8). Given this, it is important to reiterate the need to implement molecular methods to achieve a specific diagnosis and adequate treatment of patients who are affected by emerging pathogens.
T
Conclusion
IP
In summary, we show that C. duobushaemulonii from the species complex of C.
CR
haemulonii is an emerging pathogen, and thus its possible presence must be taken into account when diagnosing several cutaneous mycoses in patients, especially diabetic
US
patients or solid transplant recipients. In addition, this pathogen can be isolated from chronic wounds of the lower extremities, superficial lesions, oral and vaginal mucous
AN
membranes, and nails. It may also be present in the central venous catheter of hospitalized patients, and therefore the device should be immediately removed.
M
We believe that in all cases of mycosis caused by the C. haemulonii spp. complex, especially by C. duobushaemulonii, the empirical use of antifungals, such as amphotericin
ED
B or azole derivatives should be avoided, and antifungal sensitivity tests must be performed to prescribe the appropriate antifungal therapy.
PT
Finally, it should be noted that to the best of our knowledge, this is the first study to report
AC
Funding
CE
superficial infection by C. duobushaemulonii in Mexico
No funding was received.
Availability of data and materials The datasets used and/or analysed during the present study are available from the corresponding author on reasonable request.
ACCEPTED MANUSCRIPT
Ethics approval and consent to participate The authors declare that the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and
IP
T
national) and with the Helsinki Declaration of 1975, as revised in 2000.
CR
Patient consent for publication
AN
case details and any accompanying images.
US
Written informed consent was obtained from the patient regarding the publication of the
Authors’ contributions
M
CRC, GF-d-L, BV and GA-A followed up the patient, wrote the manuscript and designed
ED
the study. Moreover, they contributed to addressing all questions related to the accuracy and integrity of this study. RA and EM-H provided the analyses of the isolates and the
PT
molecular identification results. All the authors have read and approved the final version of
CE
the manuscript.
Competing interests
AC
The authors declare no potential competing interests with respect to the research, authorship and/or publication of this article.
References
1. Guarana, M., Nucci, M., 2018. Acute disseminated candidiasis with skin lesions: a systematic review. Clin. Microbiol. Infect. 24, 246-250. https:// doi: 10.1016/j.cmi.2017.08.016
ACCEPTED MANUSCRIPT 2. Reyes-Montes,
M.R.,
Duarte-Escalante
E.,
Martínez-Herrera,
E.,
Acosta-
Altamirano, Frías-De León, M.G., 2017. Current status of the etiology of candidiasis in Mexico. Rev. Iberoam. Micol. 34: 203–210. 3. Sadeghi, G., Ebrahimi-Rad, M., Mousavi, S.F., Shams-Ghahfarokhi, M., RazzaghiAbyaneh, M., 2018. Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile. J. Mycol. Med. 28, 51-58. https://
T
doi: 10.1016/j.mycmed.2017.12.008.
IP
4. Saúl-García, Y., Humbría-García, L., Hernández-Valle, R., 2015. Species
CR
distribution and antifungal susceptibility of Candida spp. causing superficial mycosis. Coro, Falcon state, Venezuela. Invest Clin. 56, 276-283.
US
5. Razzaghi-Abyaneh, M., Sadeghi, G., Zeinal, et al., 2014. Species distribution and antifungal susceptibility of Candida spp. isolated from superficial candidiasis in in
Iran.
J.
Mycol.
10.1016/j.mycmed.2014.01.004.
Med.
24,
e43-50.
https://
doi:
AN
outpatients
M
6. Feng, X., Ling, B., Yang, G., Yu, X., Ren, D., Yao, Z., 2012. Prevalence and distribution profiles of Candida parapsilosis, Candida orthopsilosis and Candida
ED
metapsilosis responsible for superficial candidiasis in a Chinese university hospital. Mycopathologia. 173, 229-34. https://. doi: 10.1007/s11046-011-9496-5.
species,
PT
7. Colombo, A.L., Júnior, J.N.A., Guinea J. Emerging multidrug-resistant Candida 2017.
Curr.
Opin.
Infect.
Dis.
30,
528-538.
https://
doi:
CE
10.1097/QCO.0000000000000411. 8. Kim, M.N., Shin, J.H., Sung, H., et al., 2009. Candida haemulonii and closely
AC
related species at 5 university hospitals in Korea: identification, antifungal susceptibility, and clinical features. Clin. Infect. Dis. 48, e57-61. https:// doi: 10.1086/597108. 9. Cendejas-Bueno, E., Kolecka, A., Alastruey-Izquierdo, A., et al. Reclassification of the Candida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant human pathogenic yeasts. J. Clin. Microbiol. 50, 3641-3651.
ACCEPTED MANUSCRIPT 10. de Almeida, J.N. Jr., Assy, J.G., Levin, A.S, et al., 2016. Candida haemulonii Complex Species, Brazil, January 2010-March 2015. Emerg. Infect. Dis. 22, 561563. https:// doi: 10.3201/eid2203.151610. 11. Hou, X., Xiao, M., Chen, S.C., et al., 2016. Identification and antifungal susceptibility profiles of Candida haemulonii species complex clinical isolates from a multicenter study in China. J. Clin. Microbiol. 54, 2676-2680.
T
12. Ramos, L. S., Figueiredo-Carvalho, M. H. G., Barbedo, L. S., et al., 2015. Candida
IP
haemulonii complex: species identification and antifungal susceptibility profiles of
CR
clinical isolates from Brazil. J.. Antimicrob. Chemother. 70, 111–115. https:// doi 10.1093/jac/dku321
US
13. Kathuria, S., Singh, P.K., Sharma, Ch., et al., 2015. Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted
AN
Laser Desorption Ionization–Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI
doi: 10.1128/JCM.00367-15.
M
Broth Microdilution, and Etest Method. J. Clin Microbiol. 53, 1823–1830. https://
ED
14. Kurtzman, C.P., Robnett, C, J., 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-
PT
subunit (26S) ribosomal DNA gene. J. Clin. Microbiol. 35, 1216-1223 15. Nilsson, R.H., Kristiansson, E., Ryberg, M., Hallenberg, N., Larsson, K.H. 2008.
CE
Intraspecific ITS variability in the kingdom fungi as expressed in the international sequence databases and its implications for molecular species identification. Evol
AC
Bioinform Online. 4, 193–201. 16. Fang SY, Wei KC, Chen WC, et al., 2016. Primary deep cutaneous candidiasis caused by Candida duobushaemulonii in a 68-year-old man: the first case report and literature review. Mycoses. 59: 818–821. https:// doi:10.1111/myc.12540 17. Boatto, H.F., Cavalcanti, S.D., Del Negro, G.M., et al., 2016. Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil. Mem Inst Oswaldo Cruz. 111, 407-410. https:// doi: 10.1590/0074-02760160166.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Legends Figure. 1. PCR electrophoresis for the molecular identification of experimental isolates (77-18 and 553) using the oligonucleotides NL-1 and NL-4 as described in Materials and
AC
CE
PT
ED
M
AN
US
CR
IP
T
Methods. M: 100 bp molecular size marker; C-: Negative control (without DNA).
ACCEPTED MANUSCRIPT Table 1. Antifungal susceptibility of the isolates studied by the broth microdilution method
Isolate 77-18 553
Minimum Inhibitory Concentration (mg/L) AMB 5-FC FLC KTC ITC VRC MCF ANF 2 0.25 16 0.25 0.06 0.25 0.06 0.03 2 0.12 16 0.25 0.06 0.12 0.25 0.06
AC
CE
PT
ED
M
AN
US
CR
IP
T
AMB: amphotericin B; 5-FC: flucytosine; FLC: fluconazole; KTC: ketoconazole; ITC: itraconazole; VRC: voriconazole; MCF: micafungin; ANF: anidulagungin.
ACCEPTED MANUSCRIPT Candida duobushaemulonii (CD) causes superficial and invasive infections CD cause infections in rare cases and is often misidentified Isolates was previously identified C. haemulonii by the VITEK® 2 system Expanded laboratory surveillance using PCR electrophoresis is an important step
AC
CE
PT
ED
M
AN
US
CR
IP
T
Detection and control of such emerging pathogens is currently a priority
María Guadalupe Frías-De-León, Erick Martínez-Herrera, Gustavo Acosta-Altamirano, Roberto Arenas, Carmen RodríguezCerdeira PII: DOI: Article Number: Reference:
S1567-1348(19)30179-0 https://doi.org/10.1016/j.meegid.2019.103960 103960 MEEGID 103960
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
11 March 2019 5 July 2019 9 July 2019
Please cite this article as: M.G. Frías-De-León, E. Martínez-Herrera, G. AcostaAltamirano, et al., Superficial candidosis by Candida duobushaemulonii: An emerging microorganism, Infection, Genetics and Evolution, https://doi.org/10.1016/ j.meegid.2019.103960
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT Superficial candidosis by Candida duobushaemulonii: an emerging microorganism
María Guadalupe Frías-De-León1,5,6*, Erick Martínez-Herrera1,5,6*, Gustavo Acosta-
T
Altamirano1,6, Roberto Arenas2,3,5,6, Carmen Rodríguez-Cerdeira3,4,5,6**
Research Unit, High Specialty Regional Hospital of Ixtapaluca. Ixtapaluca, Edo. Mexico
2
Mycoloy Service, Hospital Manuel Gea González, Mexico City, Mexico
3
Efficiency, quality and costs in Health Services Research Group (EFISALUD), Health
CR
IP
1
US
Research Institute. SERGAS-UVIGO.
Dermatology Department, Hospital do Meixoeiro and University of Vigo, Vigo, Spain
5
European Women’s Dermatologic and Venereologic Society (EWDVS), Vigo, Spain
6
Psychodermatology task force of the Ibero-Latin American College of Dermatology
M
AN
4
CE
PT
ED
(CILAD)
AC
*Correspondence should be addressed to: Carmen Rodriguez-Cerdeira Meixoeiro Hospital,CHUVI, C/Meixoeiro S/N 36200, Vigo, Spain Tel: 0034986814517/0034600536114 Fax: 004986276416 E-mail: [email protected]
ACCEPTED MANUSCRIPT Introduction Species of the genus Candida are the most common causative agents of fungal infections worldwide. They can cause superficial (cutaneous, mucocutaneous, onychomycosis) and systemic (meningitis, sepsis, candidemia, others) infections that can be disseminated or deep (chronic mucocutaneous candidosis, candida granuloma) (1). Among these, the most common fungal species is C. albicans; however, currently non-C. albicans species (NCA)
T
are emerging as pathogens in immunocompetent and immunosuppressed individuals.
IP
Several NCAs have been isolated from superficial infections, including C. parapsilosis, C.
CR
glabrata, C. orthopsilosis, C. metapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. intermedia, and C. sake (2-6). Other rarely found species, such as those of the
C.
US
haemulonii complex, exhibit patterns of variable susceptibility to amphotericin B and azole derivatives (7, 8). The C. haemulonii complex comprises C. haemulonii, C. haemulonii var.
AN
vulnera, and C. duobushaemulonii (9), which have been isolated from infections in humans (10). However, information regarding the occurrence and distribution of C. haemulonii
M
complex species in clinical samples is limited because the methods, such as the VITEK 2 system, API 20C, and CHROMagar Candida, commonly used for the identification of these
ED
fungi, cannot distinctly identify these organisms (11). While methods exist that do permit distinction among closely related species, including MALDI-TOF and sequencing of the
PT
internal transcribed spacer region (ITS) of ribosomal DNA (12), these methods are not widely available in hospital laboratories. Due to the emergence of the C. haemulonii
CE
complex and other phylogenetically related species as pathogens resistant to azole antifungals, Kathuria et al. (13) recommended the establishment of a reliable method for
AC
the identification and testing of antifungal susceptibility of rare yeasts, as accurate identification and appropriate treatment are of particular importance in clinical settings. Similar studies were performed by Cendejas-Bueno et al. (9), where they observed that the C. haemulonii complex was resistant to amphotericin B and exhibited cross-resistance to azole compounds. Ramos et al. (12) observed two samples of C. duobushaemulonii that were susceptible to itraconazole, and the remaining experimental samples (75%) were resistant to this antifungal. Out of the 12 samples, eight (66.7%) were resistant to voriconazole; however, all isolates were susceptible to caspofungin (MIC = 0.5 mg / L).
ACCEPTED MANUSCRIPT Here, we report two clinical isolates obtained from a patient with total dystrophic onychomycosis and another with mucocutaneous candidosis. These isolates were originally identified as C. albicans by CHROMagar Candida (Becton-Dickinson, New Jersey, USA) and Trichosporon spp. by isolation in Sabouraud agar and subsequent microscopic morphological identification, and as C. haemulonii by the VITEK® 2 system (BioMérieux®). Our molecular analysis of these isolates resulted in their re-identification
IP
T
as C. duobushaemulonii.
CR
Material and methods
Two yeast-like isolates (77-18 and 553) were analysed, and these isolates were obtained
US
from two male patients (aged 33 and 75 years), who attended a dermatological consultation due to a perianal lesion present for 4 months and due to a 2-year infection of all toenails,
AN
respectively (Fig. 1).
Originally, the isolates were identified as C. albicans by CHROMagar Candida (Becton-
M
Dickinson, New Jersey, USA), as Trichosporon spp. following isolation on Sabouraud agar and subsequent microscopic morphological identification, and as C. haemulonii by the
ED
VITEK® 2 system (BioMérieux®). The patient with onychomycosis was treated with 200 mg of itraconazole daily for three months. However, this patient did not return to
PT
consultation, so we were unable to complete his follow-up. The patient with mucocutaneous candidosis was administered itraconazole at 100 mg / day
CE
and exhibited significant improvement at 15 days. After additional 15 days of treatment, the
AC
pathology observed in this patient was satisfactorily resolved.
Molecular identification DNA extraction: Isolate DNA was extracted using the Yeast DNA Preparation kit (Jena Bioscience, GE) according to manufacturer instructions. Amplification of the D1 / D2 region of the 26S rDNA: The DNA of the two isolates was analysed
by
PCR
using
GCATATCAATAAGCGGAGGAAAAG-3') GGTCCGTGTTTCAAGACGG-3') (14).
the
oligonucleotides and
NL1 NL4
(5'(5'-
ACCEPTED MANUSCRIPT The reactions were performed in a reaction volume of 25 μL that included 2.5 mM MgCl2, 0.2 mM dATP, dCTP, dGTP, and dTTP (Jena Bioscience), 0.1 μM of each NL1 and NL4 oligonucleotide (Sigma-Aldrich, USA), 1 U of Taq polymerase (Jena Bioscience), and 20 ng of DNA. Amplifications were performed in a T100 ™ thermocycler (Bio-Rad, Laboratories, Inc., USA) with the following parameters: 95ºC for 7 min, followed by 40 cycles at 95ºC for 1 min, 53ºC for 2 min, 72ºC for 1 min, and a final extension at 72ºC for
T
10 min. Deionised water was used as a negative control. The amplification products were
IP
separated by electrophoresis on 1.5% agarose gels in 0.5X TBE buffer (45 mM Tris-Base,
CR
45 mM boric acid, 1 mM EDTA and pH 8.3) at 90 v for 60 min, and they were visualized by staining with GelRed 30X (Biotium, USA). The 100 bp DNA ladder (Jena Bioscience)
US
was used as a molecular size marker. Gel images were captured in a Molecular Imager® Gel Doc ™ XR photodocument, (Bio-Rad Laboratories).
AN
Sequencing of amplicons: The PCR products were purified using the PCR Purification Kit (Jena Bioscience) according to manufacturer instructions. The purified amplicons were
M
sequenced at the National Laboratory of Genomics for Biodiversity, Langebio - Cinvestav
ED
Irapuato, Gto., Mexico. The sequences were deposited into the GenBank database.
Identification by sequence similarity: Sequence similarity was analysed by the BLASTN of
the
National
Center
for
Biotechnology
Information
(NCBI)
PT
program
(ncbi.nlm.nih.gov/BLAST/). The identity of each isolate was determined according to the
CE
similarity of the experimental sequences to the sequences deposited in the GenBank
Results
AC
database. The parameters used included similarity >97% (15).
Both isolates (77-18 and 553) amplified a fragment of approximately 620 bp. BLAST analysis revealed 100% identity and 100% coverage (similarity), and this analysis also indicated an "e-value" close to zero (4e-83) among the sequences of each isolate and the sequences of the D1 region / D2 of the 26S rDNA of C. duobushaemulonii. The sequences derived from both isolates were deposited into GenBank (Access No.: xx and zz).
Discussion
ACCEPTED MANUSCRIPT This study highlights the importance of implementing more specific microbiological identification methods, as the number of recognized pathogenic yeasts is steadily increasing. This increase is associated with the description of new species that are closely related to currently identified species, and in certain cases these new species have been found to exist in complexes, such as those that comprise the C. haemoulonii complex (12). Within the C. haemoulonii complex, all species have been shown to cause infections in
T
humans. C. duobushaemulonii has been reported by Fang et al. (16) to be present in deep
IP
cutaneous infections. Specifically, this infection was observed in a 68-year-old male patient
CR
who presented with ulcerative crusting and confluent lesions located on the right forearm and arm. The patient was treated with amphotericin B and then switched to intravenous
revealed a complete resolution of the problem.
US
fluconazole 200 mg / once daily for 7 days. Follow-up after 6 months post-treatment
AN
Infection of chronic wounds in lower extremities with the diagnosis of candidemia was published by Almeida et al. (10). In Brazil between January 2010 and March 2015, the
M
authors found 9 samples positive for C. duobushaemulonii. Majority of the patients were diabetic, and another group of patients were organ transplant recipients.
ED
In another study conducted by Boatto et al. (17), 13 women diagnosed with RVVC and their partners were selected investigation. Isolates were obtained using CHROMagar
PT
Candida medium, and species identification was determined by phenotypic and molecular methods. Susceptibility to fluconazole was evaluated by E-test. Out of the 26 strains, six
CE
were identified as C. duobushaemulonii. These patients exhibited a very low sensitivity to fluconazole (17). Majority of authors agree that patients diagnosed with diabetes mellitus
AC
are more likely to be infected with this fungus (10, 16). Information regarding the occurrence and distribution of species of the C. haemulonii complex in clinical samples is scarce, and this is likely due to the fact that phenotypic identification methods are used in many countries, and these do not allow for discrimination among closely related species. In this study, we verified that both the CHROMagar and the VITEK 2 systems are limited for the identification of C. duobushaemulonii, despite the known differences between these methods. For CHROMagar, C. albicans was identified, while the automated system VITEK 2 indicated C. haemoulonii with a high percentage of probability; however, only the C. haemoulonii complex was determined. This type of erroneous microbiological
ACCEPTED MANUSCRIPT identification results in poor therapeutic management of patients, as the fungi comprising the C. haemoulonii complex typically exhibit resistance to amphotericin B and azole derivatives (7, 8). Given this, it is important to reiterate the need to implement molecular methods to achieve a specific diagnosis and adequate treatment of patients who are affected by emerging pathogens.
T
Conclusion
IP
In summary, we show that C. duobushaemulonii from the species complex of C.
CR
haemulonii is an emerging pathogen, and thus its possible presence must be taken into account when diagnosing several cutaneous mycoses in patients, especially diabetic
US
patients or solid transplant recipients. In addition, this pathogen can be isolated from chronic wounds of the lower extremities, superficial lesions, oral and vaginal mucous
AN
membranes, and nails. It may also be present in the central venous catheter of hospitalized patients, and therefore the device should be immediately removed.
M
We believe that in all cases of mycosis caused by the C. haemulonii spp. complex, especially by C. duobushaemulonii, the empirical use of antifungals, such as amphotericin
ED
B or azole derivatives should be avoided, and antifungal sensitivity tests must be performed to prescribe the appropriate antifungal therapy.
PT
Finally, it should be noted that to the best of our knowledge, this is the first study to report
AC
Funding
CE
superficial infection by C. duobushaemulonii in Mexico
No funding was received.
Availability of data and materials The datasets used and/or analysed during the present study are available from the corresponding author on reasonable request.
ACCEPTED MANUSCRIPT
Ethics approval and consent to participate The authors declare that the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and
IP
T
national) and with the Helsinki Declaration of 1975, as revised in 2000.
CR
Patient consent for publication
AN
case details and any accompanying images.
US
Written informed consent was obtained from the patient regarding the publication of the
Authors’ contributions
M
CRC, GF-d-L, BV and GA-A followed up the patient, wrote the manuscript and designed
ED
the study. Moreover, they contributed to addressing all questions related to the accuracy and integrity of this study. RA and EM-H provided the analyses of the isolates and the
PT
molecular identification results. All the authors have read and approved the final version of
CE
the manuscript.
Competing interests
AC
The authors declare no potential competing interests with respect to the research, authorship and/or publication of this article.
References
1. Guarana, M., Nucci, M., 2018. Acute disseminated candidiasis with skin lesions: a systematic review. Clin. Microbiol. Infect. 24, 246-250. https:// doi: 10.1016/j.cmi.2017.08.016
ACCEPTED MANUSCRIPT 2. Reyes-Montes,
M.R.,
Duarte-Escalante
E.,
Martínez-Herrera,
E.,
Acosta-
Altamirano, Frías-De León, M.G., 2017. Current status of the etiology of candidiasis in Mexico. Rev. Iberoam. Micol. 34: 203–210. 3. Sadeghi, G., Ebrahimi-Rad, M., Mousavi, S.F., Shams-Ghahfarokhi, M., RazzaghiAbyaneh, M., 2018. Emergence of non-Candida albicans species: Epidemiology, phylogeny and fluconazole susceptibility profile. J. Mycol. Med. 28, 51-58. https://
T
doi: 10.1016/j.mycmed.2017.12.008.
IP
4. Saúl-García, Y., Humbría-García, L., Hernández-Valle, R., 2015. Species
CR
distribution and antifungal susceptibility of Candida spp. causing superficial mycosis. Coro, Falcon state, Venezuela. Invest Clin. 56, 276-283.
US
5. Razzaghi-Abyaneh, M., Sadeghi, G., Zeinal, et al., 2014. Species distribution and antifungal susceptibility of Candida spp. isolated from superficial candidiasis in in
Iran.
J.
Mycol.
10.1016/j.mycmed.2014.01.004.
Med.
24,
e43-50.
https://
doi:
AN
outpatients
M
6. Feng, X., Ling, B., Yang, G., Yu, X., Ren, D., Yao, Z., 2012. Prevalence and distribution profiles of Candida parapsilosis, Candida orthopsilosis and Candida
ED
metapsilosis responsible for superficial candidiasis in a Chinese university hospital. Mycopathologia. 173, 229-34. https://. doi: 10.1007/s11046-011-9496-5.
species,
PT
7. Colombo, A.L., Júnior, J.N.A., Guinea J. Emerging multidrug-resistant Candida 2017.
Curr.
Opin.
Infect.
Dis.
30,
528-538.
https://
doi:
CE
10.1097/QCO.0000000000000411. 8. Kim, M.N., Shin, J.H., Sung, H., et al., 2009. Candida haemulonii and closely
AC
related species at 5 university hospitals in Korea: identification, antifungal susceptibility, and clinical features. Clin. Infect. Dis. 48, e57-61. https:// doi: 10.1086/597108. 9. Cendejas-Bueno, E., Kolecka, A., Alastruey-Izquierdo, A., et al. Reclassification of the Candida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant human pathogenic yeasts. J. Clin. Microbiol. 50, 3641-3651.
ACCEPTED MANUSCRIPT 10. de Almeida, J.N. Jr., Assy, J.G., Levin, A.S, et al., 2016. Candida haemulonii Complex Species, Brazil, January 2010-March 2015. Emerg. Infect. Dis. 22, 561563. https:// doi: 10.3201/eid2203.151610. 11. Hou, X., Xiao, M., Chen, S.C., et al., 2016. Identification and antifungal susceptibility profiles of Candida haemulonii species complex clinical isolates from a multicenter study in China. J. Clin. Microbiol. 54, 2676-2680.
T
12. Ramos, L. S., Figueiredo-Carvalho, M. H. G., Barbedo, L. S., et al., 2015. Candida
IP
haemulonii complex: species identification and antifungal susceptibility profiles of
CR
clinical isolates from Brazil. J.. Antimicrob. Chemother. 70, 111–115. https:// doi 10.1093/jac/dku321
US
13. Kathuria, S., Singh, P.K., Sharma, Ch., et al., 2015. Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted
AN
Laser Desorption Ionization–Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI
doi: 10.1128/JCM.00367-15.
M
Broth Microdilution, and Etest Method. J. Clin Microbiol. 53, 1823–1830. https://
ED
14. Kurtzman, C.P., Robnett, C, J., 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-
PT
subunit (26S) ribosomal DNA gene. J. Clin. Microbiol. 35, 1216-1223 15. Nilsson, R.H., Kristiansson, E., Ryberg, M., Hallenberg, N., Larsson, K.H. 2008.
CE
Intraspecific ITS variability in the kingdom fungi as expressed in the international sequence databases and its implications for molecular species identification. Evol
AC
Bioinform Online. 4, 193–201. 16. Fang SY, Wei KC, Chen WC, et al., 2016. Primary deep cutaneous candidiasis caused by Candida duobushaemulonii in a 68-year-old man: the first case report and literature review. Mycoses. 59: 818–821. https:// doi:10.1111/myc.12540 17. Boatto, H.F., Cavalcanti, S.D., Del Negro, G.M., et al., 2016. Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil. Mem Inst Oswaldo Cruz. 111, 407-410. https:// doi: 10.1590/0074-02760160166.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Legends Figure. 1. PCR electrophoresis for the molecular identification of experimental isolates (77-18 and 553) using the oligonucleotides NL-1 and NL-4 as described in Materials and
AC
CE
PT
ED
M
AN
US
CR
IP
T
Methods. M: 100 bp molecular size marker; C-: Negative control (without DNA).
ACCEPTED MANUSCRIPT Table 1. Antifungal susceptibility of the isolates studied by the broth microdilution method
Isolate 77-18 553
Minimum Inhibitory Concentration (mg/L) AMB 5-FC FLC KTC ITC VRC MCF ANF 2 0.25 16 0.25 0.06 0.25 0.06 0.03 2 0.12 16 0.25 0.06 0.12 0.25 0.06
AC
CE
PT
ED
M
AN
US
CR
IP
T
AMB: amphotericin B; 5-FC: flucytosine; FLC: fluconazole; KTC: ketoconazole; ITC: itraconazole; VRC: voriconazole; MCF: micafungin; ANF: anidulagungin.
ACCEPTED MANUSCRIPT Candida duobushaemulonii (CD) causes superficial and invasive infections CD cause infections in rare cases and is often misidentified Isolates was previously identified C. haemulonii by the VITEK® 2 system Expanded laboratory surveillance using PCR electrophoresis is an important step
AC
CE
PT
ED
M
AN
US
CR
IP
T
Detection and control of such emerging pathogens is currently a priority