Atencia Carrera

PLOS ONE
RESEARCH ARTICLE
Prevalence of biofilms in Candida spp.

bloodstream infections: A meta-analysis
Marı́a Belén Atiencia-Carrera ID1☯, Fausto Sebastián Cabezas-Mera ID1☯, Eduardo Tejera2*,
António Machado ID1*
1 Instituto de Microbiologı́a, Colegio de Ciencias Biológicas y Ambientales (COCIBA), Universidad San
Francisco de Quito (USFQ), Diego de Robles y Vı́a Interoceánica, Campus Cumbayá, Quito, Pichincha,
Ecuador, 2 Facultad de Ingenierı́a y Ciencias Agropecuarias Aplicadas, Grupo de Bioquimioinformática,
Universidad de Las Américas, Quito, Pichincha, Ecuador
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☯ These authors contributed equally to this work.
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* eduardo.tejera@udla.edu.ec (ET); amachado@usfq.edu.ec (AM)
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Abstract
Context
OPEN ACCESS
Candida-related infections are nowadays a serious Public Health Problem emerging multi-
Citation: Atiencia-Carrera MB, Cabezas-Mera FS,
Tejera E, Machado A (2022) Prevalence of biofilms drug-resistant strains. Candida biofilm also leads bloodstream infections to invasive sys-
in Candida spp. bloodstream infections: A meta- temic infections.
analysis. PLoS ONE 17(2): e0263522. https://doi.
org/10.1371/journal.pone.0263522
Objective
Editor: Surasak Saokaew, University of Phayao,
THAILAND The present meta-analysis aimed to analyze Candida biofilm rate, type, and antifungal resis-
Received: July 28, 2021 tance among hospitalized patients between 1995 and 2020.
Accepted: January 20, 2022
Published: February 3, 2022

Data sources
Web of Science, Scopus, PubMed, and Google Scholar databases were searched for
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review English papers using the following medical subject heading terms (MESH): “invasive candi-
process; therefore, we enable the publication of diasis”; “bloodstream infections”; “biofilm formation”; “biofilm-related infections”; “mortality”;
all of the content of peer review and author and “prevalence”.
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0263522 Study selection
Copyright: © 2022 Atiencia-Carrera et al. This is an The major inclusion criteria included reporting the rate of biofilm formation and the preva-
open access article distributed under the terms of
lence of biofilm-related to Candida species, including observational studies (more exactly,
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and cohort, retrospective, and case-control studies). Furthermore, data regarding the mortality
reproduction in any medium, provided the original rate, the geographical location of the study set, and the use of anti-fungal agents in clinical
author and source are credited. isolates were also extracted from the studies.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Data extraction
Funding: This work was supported by COCIBA Independent extraction of articles by 2 authors using predefined data fields, including study
Research Grant 2018-2019 through project ID: quality indicators.
PLOS ONE | https://doi.org/10.1371/journal.pone.0263522 February 3, 2022 1 / 23

PLOS ONE Prevalence of biofilms in candidiasis: A meta-analysis
12260 entitled “Adhesión inicial y resistencia Data synthesis

antimicrobiana de Candida sp. aisladas de la
microbiota humana”, under regulations of the A total of 31 studies from publicly available databases met our inclusion criteria. The biofilm
Ministry of Health of Ecuador (Contrato Marco de formation in the data set varied greatly from 16 to 100% in blood samples. Most of the stud-
Acceso a los Recursos Genéticos No. MAE-DNB- ies belonged to Europe (17/31) and Asia (9/31). Forest plot showed a pooled rate of biofilm
CM-2016-0046).
formation of 80.0% (CI: 67–90), with high heterogeneity (Q = 2567.45, I2 = 98.83, τ2 =
Competing interests: The authors have declared 0.150) in random effects model (p < 0.001). The funnel plot and Egger’s linear regression
that no competing interests exist.
test failed to find publication bias (p = 0.896). The mortality rate in Candida-related blood-
stream infections was 37.9% of which 70.0% were from biofilm-associated infections. Fur-
thermore, Candida isolates were also characterized in low, intermediate, or high biofilm
formers through their level of biofilm mass (crystal violet staining or XTT assays) after a 24h
growth. When comparing between countries, statistical differences were obtained (p =
0.0074), showing the lower and higher biofilm prevalence values in Italy and Spain, respec-
tively. The prevalence of low, intermediate, and high biofilms were 36.2, 18.9, and 35.0% (p
< 0.0001), respectively. C. tropicalis was the prevalent species in high biofilm formation
(67.5%) showing statistically significant differences when compared to other Candida spe-
cies, except for C. krusei and C. glabrata. Finally, the rates of antifungal resistance to flucon-
azole, voriconazole, and caspofungin related to biofilm were 70.5, 67.9 and 72.8% (p <
0.001), respectively.
Conclusions
Early detection of biofilms and a better characterization of Candida spp. bloodstream infec-
tions should be considered, which eventually will help preserve public health resources and
ultimately diminish mortality among patients.
Introduction
Invasive candidiasis is a systemic mycosis caused by Candida species, being commonly
described as an opportunistic infection. The population group more vulnerable for invasive
candidiasis includes patients with critical illness or immunosuppression (such as hematologi-
cal and solid organ malignancy, hematopoietic cell and solid organ transplantation, recent
abdominal surgery, and hemodialysis), or even people with a central venous catheter, paren-
teral nutrition. In addition, people that received broad-spectrum antibiotics or with drug hab-
its are also susceptible to invasive candidiasis, as well as premature newborns [1]. All these
plausible scenarios lead this systemic infection to be nowadays the 4th leading nosocomial
infection in the United States, demonstrating mortality of up to 40% [2]. In Europe, Bassetti
and colleagues realized a multinational and multicenter study in 2019 reporting 7.07 episodes
per 1000 in European intensive care units (ICUs) with a 30-day mortality of 42% [3]. While, in
the Asia-Pacific region, Hsueh and colleagues reported a candidemia incidence in ICUs of 5-
to 10-fold higher than in the entire hospital and a mortality rate of patients between 35% and
60% [4]. In Latin America, Nucci and colleagues realized a laboratory-based survey between
November 2008 and October 2010 among 20 tertiary care hospitals in seven Latin American
countries, reporting an overall incidence of 1.18 cases per 1,000 in general admissions [5]. The
mortality associated with invasive candidiasis is similar or even higher in other worldwide
countries [6].

To understand the dimension of this infection and its virulence, we must define the term
invasive candidiasis as both forms of candidemia detected in the blood and tissues or deep
organs under the mucosal surfaces (also known as deep candidiasis). Deep candidiasis can
remain localized or spread causing a secondary infection [7]. Patients with a systemic infection
induced by Candida spp. can be subdivided into three groups: (1) those who present with
bloodstream infection (candidemia); (2) those who develop deep-seated candidiasis (most fre-
quently intra-abdominal candidiasis); and, (3) those who develop a combination of these two
groups [8].
The gold standard for the diagnosis of invasive candidiasis is the growth culture, being
blood culture commonly used to diagnose candidemia while culture media is applied to
diagnose deep candidiasis from tissue biopsies [9]. In this meta-analysis, we only evaluated
studies using positive blood cultures to evaluate the biofilm formation and other related fac-
tors in candidiasis virulence. More exactly, the selected studies performed an in vitro bio-
film assay using Candida isolates from blood samples of the patients with catheter-related
candidemia (CRC) and non-CRC. In cases of patients with CRC, the standard procedure
was blood cultures from obtained the catheter and peripheral veins, whereas non-CRC was
indicated by the recovery of Candida spp. from only blood samples, as previously described
by Guembe and colleagues [10].
Nosocomial infections are closely associated with biofilms growing attached to medical
devices or host tissues [11]. Biofilms are the predominant growth state of many microorgan-
isms, being a community of irreversible adherent cells with different phenotypic and structural
properties when compared to free-floating (planktonic) cells. National Institutes of Health esti-
mated that biofilms are responsible, in one way or another, for more than 80% of all microbial
infections in the United States [12]. Candida species can produce well-structured biofilms
composed of multiple types of cell and even microbial species, leading to an intrinsic resistance
against a wide variety of stress factors, such as various antifungal drugs and immune defense
mechanisms [13]. Although the dynamics biofilm-host is not yet fully understood, it is well-
known that Candida biofilms inhibit the innate immune system of the host [14]. Therefore,
our main goal was to analyze the relationship between biofilms and mortality in Candida spp.
related infections, showing a severe menace to the Public Health System with serious
outcomes.
Results
Study inclusion criteria and characteristics of the eligible studies
A total of 214 studies were retrieved and 70 full texts were reviewed from publicly available
databases (Web of Science, Scopus, PubMed, and Google Scholar). Thirty-one studies met our
inclusion criteria (Fig 1). The final data set included studies covering different global regions
(most of them in Europe). All available and relevant data were extracted of each study, more
exactly, biofilm rate, biofilm type, underlying disease of the patients, Candida species reported,
and antifungal resistance. The data was then used to create other databases, collecting informa-
tion of at least five or more papers, and consequently, each paper was cited more than once.
These additional databases were chosen to realize subgroup analysis using a random-effect
model and to answer relevant questions about Candida-related biofilms, such as the mortality
rate related to biofilms, the geographical distribution of biofilms, the characterization of bio-
film production among Candida species, and the correlation between biofilm formation and
antifungal resistance (S1 and S2 Files).
As shown in Fig 1, a total data set of 31 studies was achieved for the present meta-analysis
following the eligibility criteria, screening process, and quality assessment.

Fig 1. Prisma flow chart of included and excluded studies of the selection process.
https://doi.org/10.1371/journal.pone.0263522.g001
Overall effects of Candida biofilms

The data set reported biofilm rates of Candida-related infections among hospitalized patients
between 1995 and 2020 in several countries worldwide. As shown in Table 1, the biofilm for-
mation by Candida spp. isolates in the data set varied greatly from 16% to 100% in blood sam-
ples from hospitalized patients. Most of the data set belonged to studies realized in Europe (17/
31), followed by Asia (9/31), South America (3/31), and North America (2/31).
Although the methodologies to quantify biofilm biomass varied between studies, these
methodologies are based on the optical density (OD) obtained by the combination of a certain
colorimetric compound or a simple dissolution in a buffer or water with the growth of the iso-
lated Candida sp. and then it’s compared with reference Candida strains in the same growth
conditions. The main methodologies in our study set were crystal violet (CV) assays using
microplate reader (51.6%; 16/31), assays with tetrazolium dye (2,3-bis-(2-methoxy-4-nitro-
5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide, XTT) using micro plate reader (35.5%; 11/
31), and Branchini’s method (9.7%; 3/31). The Branchini’s method, also called slime produc-
tion method, is based on the production of a viscid slime layer by the growth of the Candida
isolate in a tube containing Sabouraud broth [15].
Regardless of the applied methodology in the studies, all these authors were able to evaluate
biofilm formation among Candida isolates. However, only 18 of 31 studies were able to catego-
rize the biofilm formation, and so just 5 studies were able to evaluate a positive correlation
between biofilm presence and increment of antifungal resistance in the treatment. Finally, the

Table 1. General information extracted from the data set selected for the present meta-analysis.
First author Publication Region Country Methodology to Biofilm Biofilm formation, n (%) Correlation between Attributable
(year) measure biofilm rate, n (%) biofilm and mortality, n
resistance (%)
High Medium Low
Atalay 2015 Asia Turkey CV (450 nm) 8/50 (16) No
Tumbarello 2007 Europe Italy PBS (405 nm) & XTT 80/294 No 56 (70.0)
(490 nm) (27.2)
Tortonaro 2013 Europe Italy XTT (490 nm) 160/451 116 44 No 11 (6.9)
(35.4) (72.5) (27.5)
Banerjee 2015 Asia India Branchini’s method 31/80 No 5 (16.1)
(38.8)
Tumbarello 2012 Europe Italy PBS (405 nm) & XTT 84/207 No 43 (51.2)
(490 nm) (40.6)
Pongracz 2016 Europe Hungary CV (570 nm) & XTT 43/93 12 31 Yes 23 (53.49)
(490 nm) (46.2) (27.9) (72.1)
Sida 2015 Asia India Branchini’s method 2/4 (50) No
Rodrigues 2019 South Brazil Christensen’s method 15/28 No 6 (40.0)
America (53.8)
Gangneux 2018 Europe France BioFilm Ring Test 181/319 132 49 No 55 (30.4)
(56.7) (72.9) (27.1)
Shin 2002 Asia Korea DW (405 nm) 58/101 No
(57.4)
Pannanusorn 2012 Europe Sweden XTT (590 nm) 231/393 101 130 No
(58.7) (43.7) (56.3)
Tascini 2018 Europe Italy XTT (490 nm) 57/89 No 25 (43.9)
(64.0)
Tobudic 2011 Europe Austria CV (630 nm), PBS 34/47 No 18 (52.9)
(405 nm) & XTT (620 (72.3)
nm)
Tulasidas 2018 Asia India CV (570 nm) 55/74 No
(74.3)
Pfaller 1995 North USA Branchini’s method 13/17 3 6(46.1) 4 No
America (76.5) (23.1) (30.8)
Pham 2019 Asia Thailand XTT (490 nm) 38/46 25 13 No 13 (34.2)
(76.4) (65.8) (34.2)
Guembe 2014 Europe Spain CV (550 nm) 45/54 No
(76.4)
Kumar 2006 Asia India UPW (405 nm) 30/36 No
(83.3)
Rajendran 2016 Europe Scotland CV (570 nm) 245/280 56 44 (17.9) 144 Yes
(87.7) (22.9) (58.9)
Stojanovic 2015 Europe Serbia CV (595 nm) 7/8 (87.5) 2 3 (42.8) 2 Yes
(28.6) (28.6)
Turan 2018 Asia Turkey CV (540 nm) 145/162 37 61 (42.1) 47 Yes
(89.5) (25.5) (32.4)
Tulyaprawat 2020 Asia India XTT (490 nm) 45/48 26 19 No
(93.8) (57.8) (42.2)
Muñoz 2018 Europe Spain CV (540 nm) 280/280 90 190 No 95 (33.9)
(100.0) (32.1) (67.9)
Soldini 2017 Europe Italy CV (540 nm) 190/190 84 38 (20.0) 68 No 89 (46.8)
(100.0) (44.2) (35.8)
Vitális 2020 Europe Hungary CV (550 nm) 127/127 28 69 (54.4) 30 No 70 (55.1)
(100.0) (22.0) (23.6)
Prigitano 2013 Europe Italy XTT (490 nm) 297/297 96 141 60 No 65 (21.9)
(100.0) (32.3) (47.5) (20.2)
(Continued )

Table 1. (Continued)
First author Publication Region Country Methodology to Biofilm Biofilm formation, n (%) Correlation between Attributable
(year) measure biofilm rate, n (%) biofilm and mortality, n
resistance (%)
High Medium Low
Treviño- 2018 North México CV (595 nm) 89/89 No 32 (35.9)
Rangel America (100.0)
Marcos- 2017 Europe Spain CV (540 nm) 22/22 13 (59.1) 9 Yes 3 (13.6)
Zambrano (100.0) (40.9)
Marcos- 2014 Europe Spain CV (540 nm) 564/564 194 187 181 No
Zambrano (100.0) (34.4) (33.1) (32.1)
Thomaz 2019 South Brazil CV (595 nm) & XTT 38/38 3 (7.9) 35 No
America (490 nm) (100.0) (92.1)
Herek 2019 South Brazil CV (570 nm) 13/13 3 7 (53.8) 3 No
America (100.0) (23.1) (23.1)
The prevalence of biofilm formation was calculated with 95% CI through random-model and significance level �0.05 (p-value). The sample size and prevalence were
used to calculate the combined biofilm produced. Attribute mortality was calculated by the number of deaths among patients with biofilm in blood samples. The
information summarized in the table did not show information on the patients’ underlying diseases and resistance. The methodologies used to measure biofilm in the
studies were based in the optical density (nm, i.e., wavelength in the assay) of the biomass from growth culture, more exactly: XTT—using micro plate reader with
yellow tetrazolium salt; CV—using micro plate reader with crystal violet staining; UPW—using micro plate reader with ultra-pure water; DW—using microplate reader
with distilled water; Branchini’s method—evaluating the adherent growth of the biofilm’s slime production; BioFilm Ring Test—using micro plate reader with a BioFilm
Index (BFI) software; and, Christensen’s method—evaluating the adherent growth of the biofilm in Falcon tube with safranin or trypan blue staining.
https://doi.org/10.1371/journal.pone.0263522.t001
incidence of mortality among patients varied considerably among studies, reporting the values
of attributable mortality between 6.9 and 70%. All the information extracted is available in the
supplementary section.
Analysis of the forest plot was then realized with data set, showing a pooled rate of biofilm
formation of 80.0% (CI: 67–90), as shown in Fig 2. The heterogeneity indices obtained using
random effects model (p < 0.001) were Q = 2567.45 (p < 0.001), I2 = 98.83, and τ 2 = 0.150.
Fig 2. Forest plot of the meta-analysis of the prevalence of biofilm formation in Candida spp. isolated from blood
clinical samples.

The pooled rate of biofilm formation obtained needs to be carefully analyzed given the high
value of heterogeneity. This will be addressed in our discussion.
A funnel plot was realized to evaluate the existence of publication bias in the final data set
(Fig 3). Furthermore, Egger’s linear regression test was also used to reveal any publication bias
and possible asymmetric data distribution in the funnel plot.
No publication bias was identified by the Egger’s linear regression test (p = 0.896). How-
ever, as we will discuss in the next section the qualitative analysis of the funnel clearly suggests
some biases from the departure of the geometry from the expected triangular form. The funnel
plot of this study illustrates the effect size (biofilm prevalence) on the x-axis and the standard
error (SE) on the y-axis. In case of no publication bias in the data set, the studies are distributed
evenly around the pooled effect size. The smaller studies should appear near the bottom due to
their higher variance when compared to the larger studies, which should be placed at the top
of the plot. The diagonal lines show the expected 95% confidence intervals around the sum-
mary estimate. In the absence of heterogeneity, the studies of the data set should lie within the
funnel defined by these diagonal lines. However, heterogeneity and some asymmetries among
the studies of the data set were illustrated by the funnel plot. In our case, we found studies with
low errors (similar sizes) but with drastic differences in the biofilm prevalence. This type of
pattern probably indicates the presence of confounding variables (sub-groups undelaying
structures) which are not included in the global analysis.
Fig 3. Funnel plot of the meta-analysis on the biofilm formation rate in Candida spp. isolated from blood clinical samples. Studies are represented by a
point. The X-axis represents the effect size (biofilm prevalence), and the Y-axis shows the standard error. Despite some asymmetry revealed by the funnel plot in
the data set, Egger’s test failed to show publication bias (p = 0.896).

Although an obvious biofilm prevalence was found in the data set, the selected studies
poorly described the underlying conditions of the patient with biofilm production. The analy-
sis of these conditions among the patients was merely descriptive, as shown in Table 2.
The lack of a detail description of the clinical background and host factors in the patients
among the studies represents a main drawback of the present meta-analysis precluding the
evaluation of clinical or patient factors and the ability of Candida isolates to establish biofilm.
Nonetheless, the ability to establish biofilm is a virulence factor by itself and should be evaluate
as risk factor in the treatment of patients with Candida-related blood infections. As summa-
rized in Table 2, only 16 of 31 studies reported some sort of clinical background of the patients
with Candida-related bloodstream infections. From this subset of studies, patients evidenced
mainly the following clinical conditions: hematological or solid cancer (68.8%, 11/16), surgery
interventions (62.5%, 10/16); patients with central venous catheter (56.3%, 9/16); adults under
total parenteral nutrition (50.0%, 8/16); patients with human immunodeficiency virus (HIV;
50.0%, and 8/16); patients with diabetes (43.8%; 7/16); patients in the intensive care unit (ICU;
37.5%, and 6/16); patients with immunosuppressive therapy (37.5%, 6/16) and, the remaining
clinical backgrounds were only described in 25% or less of the studies in this subset, such as
neutropenia (4/16), cardiovascular diseases (3/16), pulmonary diseases (3/16), urinary catheter
(3/16), chemotherapy (2/16), and renal insufficiency (2/16). The heterogeneity of the clinical
background of the patients and the gap of the host epidemiological factors in these studies
excluded further analysis between Candida-related biofilm isolates and clinical history.
Mortality among patients with Candida biofilm

Further subgroup analysis using a random-effect model was realized to differentiate the Can-
dida-related mortality rates between bloodstream infections with planktonic cells and biofilm
formation. From the initial data set, only 15 studies evaluated the mortality among patients
with Candida-related bloodstream infections. As shown in Table 3, the pooled mortality rate
due to Candida-related bloodstream infections was 37.9% (95% CI: 26.2–50.2) of which the
mortality associated with biofilm-forming infections was 70.0% (95% CI: 52.8–84.8).
In both scenarios, the mortality rate was statistically incremented among hospitalized
patients (p < 0.0001). However, biofilm-related infections evidenced almost the double value
of mortality rate in patients, when compared to all Candida-related bloodstream infections.
Geographical distribution of biofilm-forming Candida spp. isolates

The prevalence rate of biofilm-related infections significantly varied among studies of different
countries and regions. Therefore, a subgroup analysis was realized between the biofilm forma-
tion rates and the geographical region to evaluate possible statistically significant differences
(Table 4). Subgroup analysis evaluated the biofilm prevalence between regions and countries
with a minimum of published studies, at least two and three studies per region and country,
respectively. However, Egger’s test was not applied due to the low number of studies in this
analysis.
Although the biofilm prevalence varied among regions, there were no statistically signifi-
cant differences (p = 0.4049). Europe reported a greater number of studies and showed an
intermediate biofilm prevalence among Candida spp. infections. Meanwhile, when comparing
prevalence rates between countries, a statistically significant value was obtained (p = 0.0074).
In the pairwise comparison analyses, Spain was significantly superior to Brazil (p < 0.0001),
Italy (p = 0.0263), and India (p = 0.0030).

Table 2. The reported clinical background of the patients with Candida-related bloodstream infections in the study set.
Study set Total Biofilm Mortality Mortality- Adult clinical conditions Pediatric clinical conditions
related CA IT MV CD Neu ND CO PD GI QMT DI AL CRF UC CVC RI NGT TPN GAD HIV ANF ANT SC ICU PCVC PVC PB LWB
biofilm
Stojanovic 8 7 0 0 4 4 NR NR NR NR NR NR NR NR 5 2 3 NR 6 NR 4 5 NR NR NR 6 4 6 NR NR NR NR
PLOS ONE
et al., 2015
Banerjee et al., 80 31 16 5 11 NR 9 5 6 6 7 11 19 NR 17 16 13 27 58 28 NR NR NR 1 NR 42 9 NR 0 19 14 13
2015
Guembe et al., 54 45 0 0 16 NR NR 6 NR NR NR NR 6 NR NR NR NR NR 23 NR NR NR NR NR NR NR NR NR NR NR NR 10
2014
Pongracz et al., 93 43 43 23 25 19 NR NR NR NR NR NR NR NR 20 NR NR NR NR NR NR 22 NR 11 NR NR 51 NR NR NR NR NR
2016
Vitalis et al., 127 127 70 70 28 13 87 NR NR NR NR NR NR NR 41 NR NR NR NR NR NR 68 NR 13 162 91 8 100 NR NR NR NR
2020
Kumar et al., 36 30 0 0 35 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 1 NR NR NR NR NR NR NR NR
2006
Tumbarello 207 84 82 43 42 16 NR NR NR NR 29 17 9 NR NR NR 21 NR 56 NR 27 58 NR 1 NR 75 38 NR NR NR NR NR
et al., 2012
Tumbarello 294 80 154 56 88 82 NR NR 10 NR NR NR 16 NR NR NR NR 136 30 NR NR 72 NR NR NR NR 100 57 NR NR NR NR
et al., 2007
Marcos- 22 22 0 0 21 13 NR NR 4 NR NR NR 1 NR 76 NR 4 NR 19 NR NR 13 NR 1 7 NR 4 2 NR NR NR NR
Zambrano
et al., 2017
Tortonaro 451 160 13 11 136 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 219 158 NR NR 17 NR
et al., 2013
Muñoz et al., 280 280 0 95 151 22 50 91 18 70 78 59 NR 53 69 NR 61 NR 201 NR NR 152 NR 6 62 253 136 28 NR NR NR NR
2018
PLOS ONE | https://doi.org/10.1371/journal.pone.0263522 February 3, 2022

Soldini et al., 190 190 89 89 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 152 NR NR 132 NR NR NR 177 NR 28 NR NR NR NR
2017
Tascini et al., 89 57 42 25 NR NR NR NR NR NR NR NR NR NR NR NR NR 47 80 NR 25 62 NR NR 75 NR 35 NR NR NR NR NR
2018
Treviño-Rangel 89 89 32 32 NR NR 24 NR NR NR 13 NR NR 7 NR NR NR 37 50 1 NR 30 NR 13 30 53 38 NR NR NR 4 NR
et al., 2018
Shin et al., 2002 101 58 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 41 NR NR 35 NR NR NR NR NR NR NR NR NR NR
Atalay et al., 50 8 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR 18 NR NR NR NR NR NR NR NR NR NR NR NR NR
2015
Gangneux 319 181 105 55 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
et al., 2018
Herek et al., 13 13 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
2019
Marcos- 564 564 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
Zambrano
et al., 2014
Pannanusorn 393 231 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
et al., 2012
Pfaller et al., 17 13 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
1995
Pham et al., 46 38 23 13 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
2019
Prigitano et al., 297 297 130 65 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
2013
Rajendran 280 245 0 0 NR NR NR NR NR NR NR NR 121 30 153 128 NR NR NR 118 NR 123 133 NR 119 NR 40 128 NR NR NR NR
et al., 2016
(Continued )
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Prevalence of biofilms in candidiasis: A meta-analysis
Table 2. (Continued)
Study set Total Biofilm Mortality Mortality- Adult clinical conditions Pediatric clinical conditions
related
CA IT MV CD Neu ND CO PD GI QMT DI AL CRF UC CVC RI NGT TPN GAD HIV ANF ANT SC ICU PCVC PVC PB LWB
biofilm
Rodrigues 28 15 13 6 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
PLOS ONE
et al., 2019
Sida et al., 2015 4 2 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
Thomaz et al., 38 38 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
2019
Tobudic et al., 47 34 25 18 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
2011
Tulasidas et al., 74 55 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
2018
Tulyaprawat 48 45 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
et al., 2020
Turan et al., 162 145 0 0 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
2018
CA: malignancy; IT: Immunosuppressive Therapy; MV: Mechanical Ventilation; CD: Cardiovascular Disease; Neu: Neutropenia; ND: Neurological Disorders, CO: Corticoids; PD: Pulmonary
Disorders; GI: Gastro Intestinal and Hepatically Disease; QMT: Chemotherapy; DI: Diabetes; AL: Alcoholism; CRF: Chronic Renal Failure; UC; Urinary Catheter; CVC: Central Venous Catheter;
RI: Renal Insufficiency; NGT: Nasogastric Tube, TPN: Total Parenteral Nutrition; GAD: Genetic Autoimmune Disorders; HIV: Human Immunodeficiency Virus; ANF: Prior Antifungal Therapy;
ANT: Prior Antibacterial Therapy; SC: Surgical conditions; ICU: Intensive Care Unit; PCVC: Pediatric Central Venus Catheter; PVC: Peripheric Venus Catheter; PB: Preterm Bird; LBW: Low
Weight Bird; NR: Not Reported in the study.
PLOS ONE | https://doi.org/10.1371/journal.pone.0263522 February 3, 2022

10 / 23
Prevalence of biofilms in candidiasis: A meta-analysis
Table 3. Pooled mortality rates in bloodstream infections due to Candida spp.

k Mortality rate (95% CI) (%) Random model
Q I2 τ p
All Candida spp. bloodstream infections 15 37.9 (26.2–50.2) 493.82 97.2 0.237 < 0.0001
Biofilm-forming 15 70.0 (52.8–84.8) 345.47 95.9 0.331 < 0.0001
k, Number of studies; Q, I2 and τ, Heterogeneity indexes; p, Random effect model significance level. Mortality rates
were estimated within 30 days after diagnosis and confirmation of Candida spp. bloodstream infection. The studies
considered (k = 15) were those in which a sample corresponded to an individual and reported deaths related to
biofilm-formers strains.
Biofilm-forming capability in Candida spp. isolates

Candida spp. isolates vary in their ability to form biofilms, being usually categorized as low
(LBF), intermediate (IBF), and high biofilm formers (HBF) according to biomass production
(S1–S3 Figs). Briefly, biofilm forming capacity was assessed using the crystal violet or XTT
assays, measuring the biofilm mass. Candida isolates were cultured in 96-well plates at 37˚C
for 24 h and the biomass of each isolate was measured. Then, isolates were grouped based on
their level of biomass, more exactly: low biofilm formers (LBF) showed a biomass production
below the 1st quartile (Q1; Absisolate < 0.432), intermediate biofilm formers (IBF) evidenced a
biomass production in the 2nd quartile (Q2; 0.432 < Absisolate < 1.07), and high biofilm form-
ers (HBF) demonstrated a biomass production higher the 1st quartile 3rd quartile (Q3; Absisolate
> 1.07), as previously described by Monfredini et al. [16] and Vitális et al. [17]. Eighteen stud-
ies reported this biofilm classification and so a subgroup analysis was realized (Table 5).
Statistically significant differences were found among Candida isolates according to their
biofilm-forming capability (p < 0.0001), evidencing a low number of Candida isolates related
to intermediate biofilms. No publication bias was detected in both subgroups according to
Egger’s linear regression test.
Evaluation of biofilm formation between different Candida species

Although Candida spp. isolates vary in their ability to form biofilms, little is known about this
biofilm-forming ability among Candida species. Each category of biofilm was further evaluated
among Candida species to evaluate the most virulent Candida species (S1 Table). When
Table 4. Subgroup analysis for different geographical regions and countries.

Subgroups k Prevalence (95% CI) (%) Random model
Region Q I2 τ p�
Europe 17 81.0 (63.3–94.0) 2267.21 99.3 0.407 0.4049
Asia 9 67.9 (48.1–85.0) 171.49 95.3 0.283
South America 3 91.6 (50.7–100.0) 31.83 93.7 0.387
North America 2 94.0 (55.1–100.0) 12.94 92.3 0.319
Country (�3 studies)
Italy 6 69.1 (32.0–95.8) 1095.33 99.5 0.471 0.0074
India 5 72.3 (46.2–92.7) 55.54 92.8 0.267
Spain 4 98.9 (93.5–100.0) 33.85 91.1 0.126
Brazil 3 91.6 (50.7–100.0) 31.83 93.7 0.387
2
k, Number of studies; Q, I and τ, Heterogeneity indexes; p� , Significance level in subgroup analysis.

Table 5. Overall effects in subgroups based on biofilm-forming capability.

Biofilm-forming capability k Prevalence (95% CI) (%) Egger’s test Random model
p Q I2 τ p�
High (HBF) 18 35.0 (26.6–43.9) 0.768 313.94 94.58 0.177 < 0.0001
Intermediate (IBF) 18 18.9 (7.8–33.1) 0.457 1074.52 98.42 0.334 < 0.0001
Low (LBF) 18 36.2 (24.7–48.5) 0.370 623.25 97.27 0.253 < 0.0001
2 �
k, Number of studies; Q, I and τ, Heterogeneity indexes; p , Random effect model significance level in subgroup
analysis. The selected studies (k = 18) categorized the strains according to their biofilm-forming capability using only
methods based on biomass quantification through spectrophotometric measures.
analyzing HBF (Table 6), C. tropicalis was the most prevalent HBF overpassing C. albicans and
C. parapsilosis by a factor of 2. More precisely, the HBF prevalence of C. tropicalis was the high-
est showing statistically significant differences with the other Candida species, except for C.
krusei (p = 0.5477) and C. glabrata (p = 0.0896).
In order to comprehend how these two major factors: countries and Candida species could
actually explain the high heterogeneity showed in our data, we carried out a meta-regression
analysis. The inclusion of both variables as interacting variables in a multiplicative model (R2
= 59.13%, p < 0.0001) explained more than an additive model (R2 = 43.48%, p < 0.0001),
regarding the prevalence of biofilm formation.
Evaluation of antifungal resistance pattern among Candida isolates

Multiple antifungal resistance among candidiasis has become a serious public health issue,
leading to clinical complications and expensive costs. A subgroup analysis based on antifungal
resistance was also realized among our study set. Due to the different methodologies used to
test susceptibility, the number of studies not enough to analyze statistically antifungal resis-
tance rates between Candida species. As shown in Table 7, the rates of antifungal resistance to
fluconazole, voriconazole, and caspofungin related to biofilm-forming strains were 70.5, 67.9,
and 72.8%, respectively.
Table 6. Subgroup analysis between different Candida species.

Species k BF strains Prevalence of HBF % (95% Random model
(n) CI) Q I2 τ p�
C. albicans 22 1461 30.3 (20.5–41.0) 225.66 95.6 0.173 0.0454a
non-albicans Candida species 26 1868 43.6 (34.5–52.9) 306.69 87.6 0.230
C. albicans 22 1461 30.3 (20.5–41.0) 225.66 95.6 0.173
C. glabrata 17 387 37.6 (0.1–71.0) 95.0 95.8 0.325 < 0.0001b
C. tropicalis 17 331 67.5 (58.3–76.3) 11.71 31.7 0.069
C. parapsilosis 20 744 29.6 (20.3–39.9) 69.9 84.3 0.154
C. krusei 10 68 52.8 (0.1–94.9) 30.12 83.4 0.409
��
Other species 20 338 40.7 (26.5–55.6) 22.49 60.0 0.139
k, Number of studies; Q, I2 and τ, Heterogeneity indexes; p� , Random effect model significance level in subgroup
analysis.
a
Comparison between C. albicans and non-albicans Candida species.
b
Comparison between all Candida species.
��
Other species includes C. dublinensis (n = 12), C. quilliermondi (n = 25), C. lusitaniae (n = 10), C. haemulonii
(n = 4), C. lypolitica (n = 1), C. pelliculosa (n = 1) and unreported species (n = 285).

Table 7. Summary of subgroup analysis for antifungal resistance in Candida spp. isolates.
Studies k Antifungal resistance rate % (95% CI)
Fluconazole Voriconazole Caspofungin
Mixed/Planktonic cells 3 15.1 (0.7–41.2) 1.6 (0.1–4.4) 3.1 (0.0–20.76)
Biofilm-forming strains 2 70.5 (54.6–84.5) 67.9 (51.8–82.3) 72.8 (55.1–87.8)
�
Cochran’s Q 11.68 85.15 22.88
p-value�� 0.0006 < 0.0001 < 0.0001
Not reported/ Other methods 26 - - -
k, Number of studies; Q� , Test of heterogeneity between groups; p�� , Random effect model significance level in
subgroup analysis. Subgroup analysis based on antifungal resistance contains k = 5 studies. Egger’s test may lack the
statistical power to detect bias when the number of studies is small (i.e., k < 10).
When comparing to planktonic cells, all Candida-related biofilm isolates showed a statisti-
cal increment of resistance against the three antifungals evaluated in the study (p < 0.001).
Discussion
The present study evaluated a possible relationship between Candida-related biofilm forma-
tion, bloodstream infections, and mortality among hospitalized patients. Invasive mycoses are
responsible every year for more than two million infections worldwide and for, at least, as
many deaths as tuberculosis or malaria. Candidiasis, aspergillosis, cryptococcosis, and pneu-
mocystosis cause more than 90% of reported deaths associated with invasive mycoses [18].
Among them, the most frequent mycosis is invasive candidiasis causing high morbidity in crit-
ically ill patients [19].
Overall effects of Candida biofilms in infections and mortality

As previously referred, around 70.0% of candidemia reports were caused by biofilm-forming
strains. However, its biofilm formation was less than in isolates from urogenital infections
[20–23] and even respiratory tract infections [22, 23]. Still, the rate of candidemia-associated
biofilm infections was higher than oral-related biofilm infections [24] and more than invasive
infections [25]. These findings are in agreement with the Institute of Health in the United
States, which estimates that biofilms are responsible, in one way or another, for over 80% of all
microbial infections [12]. Yet, the reports of Candida-associated biofilm infections varied
greatly between published studies possibly due to the lack of differentiation between Candida
species, the experience of the researchers, the number of Candida isolates in the study set, and
the diversity of biofilm detection and quantification methodologies and its subsequent classifi-
cation within the study set, such as crystal violet assay, biomass measure, XTT reduction assay,
and microtiter plate method [8, 12].
Another issue concerns the lack of differentiation between planktonic and biofilm-related
Candida infections in the diagnosis of the clinical laboratories at public health system [19, 26].
The traditional clinical microbiology laboratories have focused on testing planktonically iso-
lated microorganisms and reporting the susceptibility to various antimicrobials under plank-
tonic growth conditions [27]. While the authors from the studies of this meta-analysis applied
a further analysis by evaluating the ability of biofilm production in Candida isolates through
an in vitro biofilm assay. In Candida biofilms, traditional techniques require device removal
followed by culture or microscopy of a catheter segment, while catheter-sparing diagnostic
tests include paired quantitative blood cultures. However, as previously indicated by Høiby
et al. (2015) and Bouza et al. (2013), the number of positive peripheral blood cultures also

seems to be a promising diagnostic tool to diagnose catheter-related candidemia without

directly removing the catheter [27, 28]. Therefore, an implementation of a new gold standard
methodology is vital to a better characterization of microbial-associated infections avoiding
unproductive treatments among hospitalized patients. The mortality rate caused by biofilm
formation in Candida-related infections was almost double when compared to planktonic
infections. Other studies already stated the burden of invasive candidiasis and its severe out-
comes [1, 29], indicating biofilm formation and antifungal resistance as main risk factors
among patients. Moreover, we report a pooled attributable mortality of 37.9% to Candida-
related bloodstream infection with planktonic cells, which is in agreement with previous
reports [1, 18, 30, 31]. These studies reported a mortality range between 25 and 40%, showing
a higher mortality incidence among ICU or burn patients, and immunocompromised patients
[32]. While the mortality associated with biofilm-forming strains was 70.0% in Candida-
related bloodstream infections. However, this correlation has been debated by several authors
[10, 16, 33, 34], reporting different mortality rates (25–70%).
It is also important to mention that the ability to quicky proliferate and to establish biofilm
is not exclusively dependent of the type of Candida species and even strains in a blood-related
infection, but it is also influenced by their interaction with host homeostasis and variations
(mucosal pH shifts or nutritional changes), previous use of antibiotics, and immune system
alterations (such as secondary effect of stress or immunosuppressant therapy) [35].
The I2 observed in the forest plot indicate a high heterogenic data. The I2 is a measure-
ment of the heterogeneity that is not caused by variations in the sample size considered in
each study. Therefore, this high value and also the geometry of the funnel plot indicates the
possibility of major sources of variation across the studies. Some of the sources of variations
can clearly be related with the differences previously described (i.e., methodology, Candida
species, etc.) and consequently the pooled effect around the 80% need to be considered with
caution. Several factors can be modulating this pooled effect leading to higher and/or lower
values. In this context, the present meta-analysis was unable to study any correlations
between clinical or epidemiological factors and mortality in patients with biofilm-related
blood infections. These heterogeneity and gaps on the selected studies constitute the main
drawback of our study. However, it is also well-known that the ability to establish biofilms
among Candida species is an important virulence factor contributing to a more severe
infection in patients [36] and it is worth to be studied. The observed heterogeneity was the
leading cause to consider the effect of several variables like geographical distribution and
Candida species. However, the missing information in the consulted scientific literature can
be an important source of unexplained variation.
Geographical distribution of Candida biofilm-related infections

World incidence of invasive candidiasis is difficult to estimate because the criteria used for diag-
nosing and categorizing invasive candidiasis are quite different [6, 8, 9]. Also, most studies
restricted many factors in their group set, such as the range age of patients and their health sta-
tus. The present meta-analysis recollected data from diverse study sets demonstrating the Can-
dida-related biofilm infections as a main nosocomial infection, but only 16 of 31 studies
partially reported the clinical background of the patients (Table 2), such as patients suffering
from immunodeficiency, receiving organ transplantations, under major surgery, or treated with
cancer chemotherapy and different primary hospitalizations, and no epidemiological factors
were available. Only a study realized in a tertiary care hospital of southern India reported the
clinical backgrounds in adult and pediatric patients [37], evidencing central venous catheter
and low weight at birth as the most prevalent risk factors in these population sets, respectively.

Generally, the number of patients in surveillance studies is very low and there are many
gaps in our knowledge on the true epidemiology of invasive candidiasis in many regions of the
world [19]. As expected, around 55% of our data set belonged to European studies (17/31),
where the rate of biofilm-related infections varied greatly among countries showing Spain
with statistical differences in the incidence of Candida-related biofilm infections in hospital-
ized patients in comparison with other countries. However, Cesta and colleagues recently
reported Italy as the one region with a higher number of deaths caused by antibiotic-resistant
bacteria and biofilm-related infections [38]. Due to European Centre for Disease Prevention
and Control (ECDC) reported a spread of multi-drug resistant strains (MDR) in Italy, in par-
ticular of the bacterial species of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acineto-
bacter baumannii [38], it is plausible that the Candida-related biofilm incidence among
hospitalized patients in Italy had been underrated. Likewise, only two and three studies in our
data set belong to North and South America, respectively. All three studies of South America
were indeed from Brazil, demonstrating one of the highest Candida-related biofilm incidences
among hospitalized patients (91.6%). However, no further information was available in the
remaining Latin-American countries with the criteria selection of the present meta-analysis.
We can notice in the meta-analysis that the values of I2, Q and other indicators also suggest
a high heterogeneity within each group. It is an indicator that other factors can be involved.
For example, if we consider only the articles from Italy, we can notice that the sample size in 5
of 6 studies do not considerably differ but the effect size is quite different (this will impact
directly in the funnel plot geometry as presented in Fig 3). In three studies, we found a low
prevalence of biofilm formation [33, 39, 40] while in other two articles we found a high preva-
lence of biofilm formation [41, 42]. This distribution suggests that factors quite beyond the
geography are possible causes of heterogeneity within groups.
Association between different Candida species in biofilm and infections

The number of Candida species with clinical importance in humans is relatively small, more
exactly, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and
Candida dubliniensis [43]. C. albicans is the most reported Candida species worldwide in dif-
ferent ethnic populations [34, 44–47], being responsible for the majority of oral and systemic
candidiasis cases. However, there has been an increase in the number of reports about non-
albicans Candida infection in the last years and even surpassing C. albicans in terms of inci-
dence and attributable mortality [25, 31, 34, 42, 48–51]. This new scenario could be attributed
to the implementation of better molecular techniques in the identification of Candida species
[21, 29, 52].
Our results demonstrated C. tropicalis as the most prevalent HBF evidencing statistical
dominance among Candida species. Although C. tropicalis is described as a species with nor-
mal to high biofilm-forming capacity [36], it is commonly related to infections in prosthetic
joints, endodontic issues, ulcerative colitis [53–55]. C. tropicalis biofilm is characterized by
chains of cells with thin, but large, amounts of extracellular matrix material with low sums of
carbohydrate and protein [36, 40]. Furthermore, Silva and colleagues showed that matrix
material extracted from biofilms of C. tropicalis and C. albicans contained carbohydrates, pro-
teins, hexosamine, phosphorus and uronic acid [55]. However, hexosamine was the major
component quantified in C. tropicalis biofilm (27%). C. tropicalis biofilms are described as a
dense network of yeast cells with evident different filamentous morphologies [36].
After C. tropicalis, the present meta-analysis showed C. krusei and C. glabrata as the second
and third most prevalent HBF among Candida species, more exactly, 52.8 and 37.6%, respec-
tively. C. krusei is characterized by a thick multilayered biofilm of pseudohyphal forms

embedded within the polymer matrix [56], being categorized with a high ability to establish
biofilm [36]. Several mucosal infections and pneumonia are caused by C. krusei [23, 56].
Although C. glabrata is known to develop less biofilm, it is characterized to produce high con-
tent of both protein and carbohydrate [40, 57]. C. glabrata is commonly associated with infec-
tions among patients with total parenteral nutrition, periodontal disease, ventilator-associated
and non-healing surgical wounds [58]. C. glabrata biofilms are structured on multilayers of
blastospores with high cohesion among them [55]. The elucidation of these biofilm-forming
abilities and properties among Candida species could provide a promising step toward the
improvement of treatments.
Until this point, we have showed that Candida species and geographical distribution can be
related with our data heterogeneity. The actual combination of both variables in a multiple
meta-regression model as interacting variables explained more than the 50% of the global vari-
ability. The lack of clinical information and many other discussed variables are probably
related, at least partially, with the remained variability. Unfortunately, as previously explained,
this information is not accessible for most of the studies and constitute by itself a recommen-
dation in further studies.
Antifungal resistance among Candida-related biofilm infections

Candida spp. infections had successfully become more difficult to treat in the last decade due
to the growth of immunogenic diseases, the disproportionate use of immunosuppressive
drugs, malnutrition, endocrine disorders, the widespread use of indwelling medical devices,
broad-spectrum antibiotics, aging, and an increase of the number of patients among the popu-
lation [36, 59]. Thus, the morbidity and mortality associated with candidiasis are still very
high, even using the actual antifungal drugs [59]. The main antifungal drugs applied to Can-
dida infections are azoles, polyenes, and echinocandins [60]. Briefly, azoles (such as flucona-
zole and voriconazole) block ergosterol synthesis by targeting the enzyme lanosterol 14α-
demethylase and leading to an accumulation of toxic sterol pathway intermediates. While echi-
nocandins (such as caspofungin) aim for the synthesis of 1,3-β-glucan (a cell wall component),
being the ideal antifungal drug of choice in severe cases of candidemia [61, 62]. As previously
referred, the rates of antifungal resistance to fluconazole, caspofungin, and voriconazole in
biofilm cells surpassed planktonic cells by a factor of 4.7, 23.5, and 42.4, respectively. Despite
the number of studies comparing resistance between planktonic and biofilm cells among Can-
dida species is still scarce, these results are in agreement with the literature postulations [36,
63]. Numerous reasons are attributed to this enormous resistance against antifungal drugs in
Candida-related biofilms, such as high cell density, growth rate reduction, nutrient limitation,
matrix extracellular production, presence of persister (dormant and non-dividing) cells, phe-
notypic shift, and high sterols content on membrane cell [36, 59, 63]. So, the treatment for
Candida-biofilm infections requires a comprehensive knowledge of the complex mechanisms
underlying the interaction between a biofilm and its host.
Although no efficient treatment for Candida biofilms has been found yet, several promising
strategies are being explored. New therapeutic targets, such as the genes involved in biofilm
development and the quorum-sensing systems, are considered an alternative treatment to the
currently antifungal drugs.
Conclusions
In summary, several studies on the prevalence of Candida biofilms in bloodstream infections
have been published across the world, allowing some conclusions on its mortality, species, and
virulence in different geographic regions. However, a lot of information is missing, such as the

lack of a thorough clinical background from the patients and the diversity of the primary infec-
tions from the patients. Further studies are needed to close gaps in our understanding of the
incidence of Candida biofilms and to monitor trends in antifungal resistance and species
shifts.
To the authors’ best knowledge, this meta-analysis is one of the few that explored the associ-
ation of biofilm production among different Candida species in bloodstream infections [64–
67], using data published worldwide and adhering to the Preferred Reporting Items for Sys-
tematic Reviews and Meta-Analyses guideline. Although the present meta-analysis was per-
formed methodically, there are some limitations of this study: (1) heterogeneity exists in some
subgroup and overall analyses; (2) relationship between mortality and each Candida-related
biofilm species could not be assessed; and, (3) a detailed analysis of antifungal resistance in
Candida biofilms was not possible. These limitations are due to a lack of sufficient published
data. Therefore, early detection of biofilms and a better characterization of Candida spp.
bloodstream infections should be considered, which eventually will help preserve public health
resources and ultimately diminish mortality among patients.
Materials and methods

Data selection, search strategy, and study guidelines
This study was conducted following Preferred Reporting Items for Systematic Reviews and
Meta-Analyses (PRISMA) strategies (S1 File) [68]. Web of Science, Scopus, PubMed, and Goo-
gle Scholar databases were searched for English papers using the following medical subject
heading terms (MESH): “invasive candidiasis”; “bloodstream infections”; “biofilm formation”;
“biofilm-related infections”; “mortality”; and, “prevalence”.
In each electronic database, a combination of MESH terms was used to conduct the search
applying the following strategy (in the MEDLINE for example): ‘‘(Candida) AND (biofilm
[Title/Abstract]) AND (mortality).” All studies published until 30th July of 2020 were
retrieved. The articles reporting the prevalence of bloodstream infections biofilm-related, the
mortality rates, and the species identification of Candida isolates were included. The references
of these articles were also checked for finding additional records. The data selection was lim-
ited to human clinical isolates and studies in English. All references were compiled into a data-
base Zotero Library and then managed using Excel.
Screening process
Duplicates were initially identified and eliminated in Zotero after entering all the recognized
studies into an Excel self-created database (S2 File). All articles were assessed by two reviewers
(MBA-C and FSC-M) by screening titles, abstracts, topics, and finally full texts. At each level,
the reviewers independently screened the articles and finally merged their conclusions. An
additional examination of the selected articles was realized by a third author (AM) focused on
the homogeneity of the eligibility criteria of previous reviewers in the initial data set. Discrep-
ancies were resolved by discussion before finalizing the records for the evaluation of eligibility
criteria. In case of disagreements, the third assessor (AM) was assigned to make a final
decision.
Eligibility criteria
The major inclusion criteria included reporting the rate of biofilm formation and the preva-
lence of biofilm-related to Candida species, including observational studies (more exactly,
cohort, retrospective, and case-control studies). Furthermore, data regarding the mortality

rate, the geographical location of the study set, and the use of anti-fungal agents in clinical iso-
lates were also extracted from the studies.
All studies without information about biofilm formation or clinical Candida isolates were
consequently excluded. The method to quantify biofilm biomass was not a criterion to include
or exclude any paper in this meta-analysis. Concerning antifungal resistance rate, only studies
that used the standard susceptibility tests according to the Clinical and Laboratory Standards
Institute (CLSI) or European Committee on Antimicrobial Susceptibility Testing EUCAST
were selected for the present study.
Reviews, editorials, congress or meeting abstracts, literature in languages other than
English, case reports, and letters to editors were excluded from the final data set. Finally, arti-
cles without full text available, duplicate reports on different databases, and studies with
unclear or missing data were also omitted.
Data extraction and quality assessment

Methodological quality assessment of the studies was performed using a checklist for neces-
sary items as outlined in the Critical Appraisal Skills Programmed checklists [69]. For each
article, a series of critical questions were asked. If the pertinent data were presented, the
question was scored ‘‘yes.” If there was any doubt or no information in the study, that ques-
tion was marked as ‘‘no”. A data extraction form was designed to extract the relevant char-
acteristics of each study (S1 and S2 Files). The extracted information included the first
authors’ names, time of the study, year of publication, location, sample size, biofilm forma-
tion rate, Candida species and its categorization (as C. albicans and non-albicans Candida
species), the correlation between biofilm formation and antifungal resistance, and the type
of biofilm. The type of biofilm was categorized as low biofilm formers (LBF), intermediate
biofilm formers (IBF), and high biofilm formers (HBF). The initial two authors (MBA-C
and FSC-M) extracted all data, further confirmation and final evaluation were realized by
the lead authors (AM and ET).
Data analysis and statistical methods

Meta-analysis was performed using several R packages ("meta" [70], "metafor" [71], "poibin"
[72], and "stringr" [73]) of R version 3.4.3 [74] and RStudio version 1.3.1073 [75] (S3 File). The
rate of biofilm formation was computed, and values were reported with confidence intervals
(CI) of 95%. The heterogeneity was assessed by the Cochrane Q and I2 tests. The I2 metric indi-
cates the amount of heterogeneity that is not related with sampling size variation. Moreover, it
is also independent of the number of studies included in the meta-analysis (in contrast to the
Cochrane Q metric). Considering the heterogeneity indices, the random-effects model was
then used for meta-analysis of the selected studies, and the Freeman-Tukey transformation
was also applied to calculate the pooled frequencies. To estimate the between-study variance in
a random-effects model we use tau-squared, and its square root is the estimated standard devi-
ation of underlying effects across studies. Subgroup analyses were conducted based on the type
of biofilm, biofilm-related species, geographical regions, and antifungal resistance rates. Outli-
ers’ analysis was done with the Baujat diagram, while quantitative Egger weighted regression
test and Funnel plot were used to evaluate the eventual existence of publication bias. In statisti-
cal analysis, p-values <0.05 were considered as significant statistical results. We used the mul-
tiple meta-regression analysis with the "metareg" function from "meta" to explore the
contribution to model heterogeneity of several variables. In this approach, the maximum-like-
lihood method was used.

Supporting information
S1 Fig. Forest plot of the meta-analysis of the prevalence of high biofilm producers in Can-
dida spp. isolates.
(TIF)
S2 Fig. Forest plot of the meta-analysis of the prevalence of intermediate biofilm producers
in Candida spp. isolates.
(TIF)
S3 Fig. Forest plot of the meta-analysis of the prevalence of low biofilm producers in Can-
dida spp. isolates.
(TIF)
S1 Table. Subgroup analysis between different Candida species and biofilm-forming capa-
bility.
(DOCX)
S1 File. The PRISMA statement for reporting meta-analysis of the present study.
(DOCX)
S2 File. The databases of the present study for metanalyses process.
(XLSX)
S3 File. The R-code used in the present meta-analysis.
(TXT)
Acknowledgments
A special recognition deserves all colleagues of the Microbiology Institute of USFQ and
COCIBA, as well as the Research Office of Universidad San Francisco de Quito.
Author Contributions
Conceptualization: Marı́a Belén Atiencia-Carrera, António Machado.
Data curation: Marı́a Belén Atiencia-Carrera, Fausto Sebastián Cabezas-Mera, António
Machado.
Formal analysis: Marı́a Belén Atiencia-Carrera, Fausto Sebastián Cabezas-Mera, António
Machado.
Funding acquisition: António Machado.
Investigation: Marı́a Belén Atiencia-Carrera, Fausto Sebastián Cabezas-Mera, António
Machado.
Methodology: Fausto Sebastián Cabezas-Mera, Eduardo Tejera, António Machado.
Project administration: António Machado.
Software: Fausto Sebastián Cabezas-Mera, Eduardo Tejera.
Supervision: António Machado.
Validation: Eduardo Tejera, António Machado.
Visualization: Fausto Sebastián Cabezas-Mera.

Writing – original draft: Marı́a Belén Atiencia-Carrera, Fausto Sebastián Cabezas-Mera,

António Machado.
Writing – review & editing: Eduardo Tejera, António Machado.
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PLOS ONE

Prevalence of biofilms in Candida spp.

Accepted: January 20, 2022

Published: February 3, 2022

PLOS ONE | https://doi.org/10.1371/journal.pone.0263522 February 3, 2022 1 / 23

12260 entitled “Adhesión inicial y resistencia Data synthesis

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Overall effects of Candida biofilms

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Mortality among patients with Candida biofilm

Geographical distribution of biofilm-forming Candida spp. isolates

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Table 3. Pooled mortality rates in bloodstream infections due to Candida spp.

Biofilm-forming capability in Candida spp. isolates

Evaluation of biofilm formation between different Candida species

Table 4. Subgroup analysis for different geographical regions and countries.

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Table 5. Overall effects in subgroups based on biofilm-forming capability.

Evaluation of antifungal resistance pattern among Candida isolates

Table 6. Subgroup analysis between different Candida species.

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Overall effects of Candida biofilms in infections and mortality

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seems to be a promising diagnostic tool to diagnose catheter-related candidemia without

Geographical distribution of Candida biofilm-related infections

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Association between different Candida species in biofilm and infections

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Antifungal resistance among Candida-related biofilm infections

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Materials and methods

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Data extraction and quality assessment

Data analysis and statistical methods

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Writing – original draft: Marı́a Belén Atiencia-Carrera, Fausto Sebastián Cabezas-Mera,

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