High prevalence of Candida albicans genotype C in HIV-positive patients [627842]

Romanian Review of Laboratory Medicine
High prevalence of Candida albicans genotype C in HIV-positive patients
–Manuscript Draft–

Manuscript Number: RRLM-D-19-00005R1
Full Title: High prevalence of Candida albicans genotype C in HIV-positive patients
Article Type: Short Communication
Section/Category: Medicine
Keywords: Candida albicans; prevalence; genotype C; HIV-patients; Romania
Abstract: Background
Fungal infections are a health issue paradoxically fuelled by the developments in
medical care.
Objectives
Our study is an investigation on the correlation between the prevalence of the
genotypes of Candida albicans strains isolated from various infection sites of
Romanian patients.
Methods
There have been investigated 301 isolates collected from patients in terms of genotype
determination (G).
Results and conclusions
The isolates were clustered in three groups: 57.47% from superficial infections, 20.59%
from blood culture and 21.92% from deep-seated infections. No significant correlation
was found between the genotype and the infection site, but a significant correlation
was found between genotype C and isolates from HIV patients proving that C. albicans
pathogenicity probably relies on factors related to the host.
Manuscript Classifications: 20: MEDICINE; 20.100: Laboratory Medicine; 20.500: Infectious Diseases
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High prevalence of Candida albicans genotype C in HIV -positive patients

Abstract
Background: Fungal infections are a health issue paradoxically fuelled by the
developments in medical care. Objectives: Our study is an investigation on the correl ation
between the prevalence of the genotype s of Candida albicans strains isolated from various
infection sites of Romanian patients. Methods: There have been investigated 301 isolates
collected from patients in terms of genotype determination (G) . Results and conclusions :
The isolates were clustered in three groups: 57.47% from superficial infections, 20.59% from
blood culture and 21.92% from deep -seated infections. No significant correlation was found
between the genotype and the infection site , but a sig nificant correlation was found between
genotype C and isolat es from HIV patients proving that C. albicans pathogenicity probably
relies on factors related to the host.
Key words: C. albicans , prevalence, genotype C, HIV-patients, Romania. Manuscript without authors and affiliations
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Introduction. Fungal infections are a major health issue counterintuitive fuelled by
the development s in medical care. The great majority of clinically relevant fungi are
opportunistic pathogens for patients that receive immunosuppressive therapy, broad spectrum
antibio tics, chemotherapy, patients with diabetes or HIV infection, etc. [1]. Candida yeasts
are the most frequent fungal pathogen in humans, being t he fourth cause of bloodstream
infections [2], and between these C. albicans is the dominant species. Although much
progress has been made in understanding the phenotypic and genotypic C. albicans profile,
still less is known regarding their interaction with the host [3]. This study is an investigation
providing data on the genotypic profiles of C. albican s strains isolated from Romanian
patients in terms of their prevalence.
Methods. The investigation was performed on 301 clinical y east isolates collected in
four tertiary hospitals from different regions of Romania during 2010 -2011 (i.e. Iași,
Bucureș ti, Cluj-Napoca, Timișoara and Tîrgu Mureș) as follows: 62 were recovered from
confirmed bloodstream infections (BSI), 66 from deep -seated mycoses (DEEP) and 173 from
superficial mycoses (SUP) . DEEP mycoses were represented by the following samples: upper
urinary tract in non -catheterized patients (U) , urinary tract of HIV infected patients (U -HIV),
low respiratory tract (RT), cerebrospinal fluid (CS), bronchial aspirate (BA), bronchial
aspirate from tuberculosis patients (BA -TB), sputum (S), sputum from tu berculosis patients
(S-TB), and drain tube (DT) . SUP infections samples were divided into: female genital tract –
vaginal discharge (VD) , vaginal discharge from pregnant women (VD-P), scraped lingual
(SL), pharyngeal exudate (PE), onycomycosis (O), gastroi ntestinal tract (GT), oral sampling
(OS), oral sampling from patients with diabetes (OS -D), oral sampling from pregnant patients
(OS-P), oral sampling from HIV patients (OS -HIV), oral sampling from patients with denture
(OS-D), oral sampling from patients with tuberculosis (OS -TB), nail sample (NS), samples
from patients with balanitis (B), samples from pa tients with periodontitis (P), faeces samples
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(F), faces from HIV infected patients (F-HIV). The isolates were collected from patients that
satisfy simultaneously at least two of the following conditions : low birth weight (<1500 g),
old age (>65 years), insulin -dependent diabetes mellitus, recent major surgery, broad –
spectrum ant ibiotic therapy, central venous catheter, organ transplantation, prolong ed ICU
stay (>48h), immunosuppression (HIV infection or other predisposing conditions), total
parenteral nutrition or mechanical ventilation.
The isolates were submitted to the Laboratory of Antimicrobial C hemotherapy from
the Department of Public Health o f “Ion Ionescu de la Brad” University, Iasi, Romania. All
isolates were checked for purity and stored in 10% glycerol at -70°C. The final identification
was performed using ID32C strips (bioMérieux, France). For DNA extraction , 4-5 individual
colonies of C. albican s were picked up from Sabouraud Dextrose Agar (SDA ) plates and
suspended in 200 μL distilled wate r in a sterile Eppendorf tube. DNA was obtained by lysing
the yeast cells at 95°C for 5 min followed by immersing in ice and centrifugation. PCR to
detect the genotypes was performed according to the method p reviously described [4-5] using
the primers
CA-NT-L (5’ -ATAAGGGAAGTCGGCAAAATAGATCCGTAA -3’) and CA -NT-R
(5’-CCTTGGCTGTGGTTTCGCTAGATAGTAGAT -3’). All PCR amplification reactions
were performed in 50 μL of distilled water containing 2.0 μL of each primer, 2.0 μL of
genomic DNA (5 μg/mL) and one PCR bead (Ready -to-Go PCR beads; Amersham Pharmacia
Biotech, Piscataway, NJ, USA). The PCR conditions were: denaturation for 5 min a t 93°C , 40
cycles of 93°C fo r 30 s, 55°C for 45 s, and 72 °C for 4 5 s and a final extension at 72 °C for 10
min. The products were analysed by using DNA microfluidic chips of Experion Automated
Electrophoresis System (Bio -Rad, US) . The genotypes of C. albicans can be divided into 5
groups by the size of DNA amplified fragments (450 bp for group A, 840 bp for group B, 450
and 840 bp for group C, 1080 bp for g roup D and 1400 bp for group E) [5-7]. The results
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were submitted to a preliminary multiple correspondence analysis (MCA) which had been
assessed by using XlStat -Ecology 2018.7 version .
Results. According to prevalence 57.47% of the isolates were from superficial
infections, 20.59 % from blod culture and 21.92% had deep -seated systemic infections origin
(see Table 1). C. albicans strains were dived into three different groups, based on the length
of polymerase chain reaction (PCR) amplification product, namely genotype A with 52.92%,
B with 17 .85%, respectively C, with 29.23 % (Table 1). Genotypes D and E were not detected.
Table No 1 insertion: Table 1. Genotype comparisons in C. albicans infections.
From the superficial mycoses (SUP), most of the samples were from vaginal discharge
(VD) (2 0.54%) and oral samples (OS) ( 17.12%), and from deep seated mycoses (DEEP), the
majority of th e samples were from sputum (S) (39.39%) and bronchial aspirate from patients
with tuberculosis (BA-TB) (22.72%) ( see Table 1) .
In many samples, genotype A was the predominant genotype , like those from vaginal
discharge (48.64 %), oral sampling (62.5% ) or faeces (72.72%). Genotype C was found mostly
in oral sampli ng from patients with HIV (45.16 %) (Table 1, Figure 1). Genotype B was
mostly found in samples from urine and respiratory tract (33.33%) and also from the
bronchi al aspirate of patients with TB (28.58%). It can be noticed also that the oral samples
from patients with HIV had genotype C (45.16%), instead of genotype A , which was found in
normal patients (up to 100%) .
Figure No 1 insertion: Figure 1. Multiple correspondence analysis diagrams of the associations among
genotype and prevalence of C. albicans. DEEP (deep seated mycoses) : U (upper urinary tract in non –
catheterized patients); U-HIV (urinary tract of HIV patients), RT (low respiratory tract), CS (cerebrospinal fluid),
BA (bronchial aspi rate), BA-TB (bronchial aspirate from tuberculosis patients), S (sputum), S-TB (sputum from
tuberculosis patients), and DT (drain tube). BSI (blood stream infections) : BC (blood culture). Superficial
infections (SUP) : VD (female genital tract – vaginal discharge), VD-P (vaginal discharge from pregnant patients),
SL (scraped lingual), PE (pharyngeal exudate), O (onycomycosis), GT (gastrointestinal tract), OS (oral
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sampling), OS-D (oral sampling from patients with diabete s), OS-P (oral sampling from pregnant patients), OS-
HIV (oral sampling from HIV patients), OS-D (oral sampling from patients with denture), OS-TB (oral sampling
from patients with tuberculosis), NS (nail samples), B (samples from patients with balanitis) , P (samples from
patients with periodontitis), F (faeces samples), F-HIV (faces from patients with HIV).
According to the correspondence analysis there was no significant correlation between
the prevalence of the strains and their genotype (p = 0.449) . It was noticed a significant
correlation between HIV patients and prevalence (p<0.0001) for the faeces, urine and oral
samples. This correlation was not found for the non -HIV samples w ith the same prevalence ( p
= 0.439).
The analysed data were clustered in to three classes using multivariate statistical
analysis (Figure 1). The first group was described by the superficial mycoses (SUP) which are
usually presenting genotype A or C. The second group was made up by the blood stream
infections (BSI), which are c haracterized mostly by genotype A and C. The third group was
formed by the deep seated mycoses (DEEP), which are related to all three types of genotypes.
The relative proportion of each group is presented in Figure 1.
Discu ssions . Whether the virulence of C. albicans is related to ge notypes remains a
continuous debate . One study provides that genotype A was more prevalent among invasive
strains and genotype B and C were more prevale nt among none -invasive strains [8]. Al-
Kara awi et al . reported that genotype A is the most predominant type in patients with oral
Candida infections [9]. Our study proves that genotype A is not only dominant in patients
with oral candidiasis, but also in patients with other superficial mycoses, deep seated my coses
and also with blood steam infections. Genotype determination of C. albicans strains tend to be
genetically similar to each other, when originated from similar population groups, in relation
to the immune condition, anatomic s ite or to geographical lo cation [9]. Genotypes A, B and C
were also detected in 73 strains sampled from dental biofilms o f severe early childhood caries
[5]. In another study genotypes were detected by analysing 151 strains of C. albicans (71
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samples from infant patients with cutaneous candidiasis and 61 samples from females with
vaginal candidiasis), and no distinctive association was found between genotype and the site
of cutaneous infection [7]. Barros et al ., found sixteen genotypes among 56 samples of C.
albicans isolated from the oral cavity [4]. A comparison between this study and the previously
ones [4-5,7] in terms of genotype comparisons is difficult due to the different number of
strains and also to their prevalence in various clinical conditions, body sites or geographical
conditions . Genotype A was predominant in all the examined groups and s urprisingly,
genotype C was identified more frequently in isolates sampled from patients with HIV (see
Table 1). Genotype C is considered a hybrid of genotype A and B , containing the intron in
only one allele [10-11].
Conclusions. A significant correlation was not found between the infection site and a
particular genotype. C. albicans is a diploid organism and its pathogenicity is linked to a
series of inherent and environmental factors and it’s mostly related to the host status .
However, in order to prove the above statements, it was found a significant correlation
between prevalenc e and genotype in HIV patients.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research,
authorship, and/or publication of this article.
Acknowledgments The authors are grateful for the financial support from the H2020
ERA Chairs Project no. 667387: SupraChem Lab laboratory of Supramolecular Chemistry for
Adaptative Delivery Systems ERA Chair initiative .
Ethical approval . All procedures performed in studi es involving human participants
were in accordance with the ethical standards of the institutional and/or national research
committee and with the 1964 Helsinki declaration and its later amendments o r comparable
ethical standards.
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References
[1] Pierce CG, Lopez -Ribot JL . Candidiasis drug discovery and development: new approaches
targeting virulence for discovering and identifying new drugs. Expert Opin Drug Discov.
2013 Sep;8(9):1117 -26.
[2] Rosca I, Bostanaru AC, Minea B, Nastasa V, Gherghel I, Panzaru CV, et al. Phenotypic and
genotypic variations in Candida albicans isolates from Romanian patients. Rev Romana
Med Lab. 2018;26(4):405 -13.
[3]Wong SSW, Samaranayake LP, Senevirtane CJ . In pursuit of the ideal antifungal agent for
Candida infections: high -throughput screening of small molecules. Drug Discov Today.
2014 Nov;19(11):1721 -1730 .
[4] Barros LM, Boriollo MFG, Alves AC, Klein MI, Gonclaves RB, Hofling JF. Genetic
diversity and exoenzyme activities of Candida albicans and Candida dubliniensis isola ted
from the oral cavity of Brazilian periodontal patients. Arch Or al Biol. 2008
Dec;53(12):1172 -8.
[5] Li W, Yu D, Gao S, Liu J, Chen Z, Zhao W. Ro le of Candida albicans secreted aspartyl
proteinases (Saps) in severe early childhood caries. Int J Mol Sci. 2014 Jun
13;15 (6):10766 -79.
[6] Tamura M, Watanabe K, Mikami J, Yazawa K, Nishimura K . Molecular characterization
of new clinical isolates of Candida albicans and C. dubliniensis in Japan: analysis reveals
a new genotype of C. albicans with group I intron . J Clin Mic robiol. 2001
Dec;39(12):4309 -15.
[7] She XD, Wang XJ, Fu MH, Shen YN, Liu WD. Genotype comparisons of strains of
Candida albicans from patients with cutaneous candidiasis and vaginal candidiasis. Chin
Med J (Engl). 2008 Aug 5;121(15):1450 -5.
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[8] Schaller M, Bein M, Korting HC, Baur S, Hamm G, Monod M, et al. The secreted aspartyl
proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis
based on reconstituted human vaginal epithelium. Infec t Immun. 2003 Jun;71(6):3227 -34.
[9] Al-Karaawi ZM, Manfredi M, Waugh ACW, McCullough MJ, Jorge J, Scully C, et al.
Characterization of Candida spp. isolated from the oral cavities of patients from diverse
clinical settings. Oral Microbi ol Immunol. 2002 Feb ;17(1):44 -9.
[10] McCullough MJ, Clemons KV, Stevens DA. Molecular and phenotypic characterization
of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and
Candida stellatoidea . J Clin M icrobiol. 1999 Feb;37(2):417 -21.
[11] Abdulrahim MH, McManus, BA, Fliut SR, Coleman DC. 2016. Genotyping Candida
albicans from Candida leukoplakia and non -Candida leukoplakia shows no enrichment of
multilocus sequence typing clades but enrichment of ABC genotype C in Candida
leukoplakia . PLoS One. 2013 Sep 18;8(9):e73738 .

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Table 1. Genotype comparisons in C. albicans infections.
Sample type Genotype
n %A %B %C
Superficial mycoses (SUP) 173
Vaginal discharge (VD) 39 48.64 27.02 24.34
Vaginal discharge in pregnant (VD -P) 28 62.96 7.40 29.64
Scraped lingual (SL) 5 40 40 20
Pharyngeal exudate (PE) 4 50 50 0
Onycomycosis (O) 2 50 0 50
Gastrointestinal tract (GT) 2 100 0 0
Oral sampling (OS) 32 62.5 6.25 31.25
Oral sampling diabetes (OS -D) 6 100 0 0
Oral sampling pregnant (OS -P) 4 100 0 0
Oral sampling HIV (OS – HIV) 26 38.46 16.38 45.16
Oral sampling denture (OS -D) 3 66.66 0 33.34
Oral sampling TB (OS – TB) 1 0 0 100
Nail sample (NS) 1 100 0 0
Balanitis (B) 1 100 0 0
Periodontitis (P) 1 0 0 100
Faeces (F) 11 72.72 9.09 18.19
Faeces HIV (F -HIV) 6 55.55 11.11 33.33
Colecistitis (Co) 1 0 0 100

Blood stream infections (BSI) 62
Blood culture (BC) 62 50 14,51 35,49

Deep seated mycoses (DEEP) 66
Urine (U) 3 33.33 33.33 33.33
Urine HIV (U -HIV) 1 0 0 100
Respiratory tract (RT) 3 66.66 33.33 0
Cerebrospinal fluid (CS) 1 0 0 100
Bronchial aspirate (BA) 2 50 0 50
Bronchial aspirate TB (BA -TB) 15 50 28.58 21.42
Sputum (S) 26 52 24 24
Sputum TB (S -TB) 8 50 50 0
Drain tube (DT) 7 50 25 25

Table
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Figure
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Dear Editor -in-Chief,

According to your recommendation, we made the required changes in the text of
the manuscript RRML -D-19-00005 as follows:
– Removal the authors and their affiliations
– Modification of the text in Materials and Methods Section
– Modification of Figure 1

Sincerely yours,
Mihai MARES
Corresponding author

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