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Accepted Manuscript

Title: Oral Candida isolates and fluconazole susceptibility

patterns in older Mexicans women

Author: Beatriz Benito-Cruz Saray Aranda-Romo Francisco

Javier López-Esqueda Estela de la Rosa-Garcı́a Rebeca
Rosas-Hernández Luis Octavio Sánchez-Vargas

PII: S0167-4943(16)30064-4
Reference: AGG 3310

To appear in: Archives of Gerontology and Geriatrics

Received date: 8-7-2015

Revised date: 9-3-2016
Accepted date: 3-4-2016

Please cite this article as: Benito-Cruz, Beatriz, Aranda-Romo, Saray, López-
Esqueda, Francisco Javier, de la Rosa-García, Estela, Rosas-Hernández, Rebeca,
Sánchez-Vargas, Luis Octavio, Oral Candida isolates and fluconazole susceptibility
patterns in older Mexicans women.Archives of Gerontology and Geriatrics

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Oral Candida isolates and fluconazole susceptibility patterns in older Mexicans

Beatriz Benito-Cruz1; Saray Aranda-Romo 2, Francisco Javier López-Esqueda 3,
de la Rosa-García Estela4, Rebeca Rosas-Hernández5 y Luis Octavio Sánchez-

Master in Biomedical Science. Faculty of Medicine, University Autonomous of San
Luis Potosí, México
Oral Microbiology, Pathology and Biochemical laboratory. Faculty of Stomatology,
University Autonomous of San Luis Potosí, México.
Geriatric Unit. Central Hospital “Ignacio Morones Prieto”. University Autonomous of
San Luis Potosí, México
Department of Attention to Health. Metropolitan Autonomous University,
Xochimilco, México City.
Molecular Biophysics laboratory. Physics Institute "Manuel Sandoval Vallarta".
University Autonomous of San Luis Potosí, México.


Candida colonization and infection in older adults

Dr. Luis Octavio Sánchez Vargas. Laboratory of Biochemistry, Microbiology and
Pathology. Facultad de Estomatología. Universidad Autónoma de San Luis Potosí. Av.
Dr. Manuel Nava No. 2. Zona Universitaria. C.P. 78290. San Luis Potosí. S.L.P.,



Candida, Diabetes, elderly, infection, epidemiology.

Objectives: To assess the epidemiologic and microbiologic profile and in vitro
fluconazole susceptibility of yeasts isolated from the oral mucosa colonization/infection
of elderly patients.

Background: It has been reported that in older adults increases the oral colonization by
Candida particularly C. non-albicans, showing a decreased response to fluconazole,
which increases the risk of recalcitrant local and disseminated candidiasis.

Materials and Methods: This was a prospective cross-sectional study conducted in 120
elderly patients. Oral samples were obtained of mucosal Candida colonization or
infection by swabbing. Each sample was plated on CHROMagar®Candida and
incubated (36±1.5°C) for two days. The yeast species were identified using the
API®ID32-C-AUX. Fluconazole susceptibility was tested using a broth microdilution
assay according to the CLSI methods.

Results: The yeast colonization/infection frequency in the total population was 65.8%.
The frequency of the highest Candida carriers was 67.4% in the 70-79-year-old-group.
Oral candidiasis was present in 20%, with a tendency to increase with age (33.3% of
adults aged>80 years), it was determined that the use of prosthesis is associated with a
higher colonization rate (Chi2, p=0.011). The frequency of colonization/infection cases
with more than one species showed a tendency to increase with age; 18.9% in the 60-69
year-old-group, 20.9% in the 70-79-year-old-group and 29.2% in the ≥80 year-old-
group. About fluconazole susceptibility: for C. albicans, 20.3%, about Candida non-
albicans species 15.3% were dose dependently susceptible (DDS) and 17.9% were

Conclusions: After 80 years of age, there is a considerable increase in Candida non-

albicans species and a reduced susceptibility to fluconazole.


Healthcare services will face the challenge of meeting the needs of an increasingly
aging population over the next decades. The world population of over 65-year-old
individuals is predicted to reach 400 million by the year 2050 1. In Mexico, this
population is 9% of the total population 2 and is highly affected by chronic degenerative
diseases including diabetes and hypertension 3.
An aging population is at risk of developing diseases due to a number of factors,
including systemic diseases and oral lesions. There are inherent factors, such as tissular
senescence, mucosal fragility, alteration of function due to an impaired immune
response, and reduction of the protective effect of the saliva because of low salivary
flow. Second, there are extrinsic factors, such as poly-pathologies, including chronic
degenerative diseases and malignancies, poly-medications and malnutrition.4 These
factors, which contribute to changes during aging, may also disturb the balance in the
oral microbial ecosystem. A disturbance in oral homeostasis between bacteria and fungi
may cause oral infectious diseases. Candida species are a part of the commensal oral
microbiota of healthy individuals at all ages with a reported cultivable prevalence
between 15–75% 5,6 and up to 80% in elders, especially denture wearers 7,8
. The use of
antibiotics and corticosteroids 9, chemotherapy, malnutrition, premature birth and old
age are among the most common predisposing factors for opportunistic mycoses.10
Candida in the oral cavity serves as a reservoir for inoculation and infections elsewhere
in the body . When Candida penetrates the epithelium and invades the host tissues,
septicemia and systemic infections may result . These infections are difficult to treat
with antifungals, particularly in older adults, and therefore have a high reported
mortality (40%)13. The most common Candida albicans systemic infections, in addition
to septicemia, are catheter-related, intra-abdominal and urinary tract infections;
additionally, some groups have reported other Candida non-albicans species, which are
particularly resistant to azoles such as fluconazole and other antifungals 13. Candidemia
is a leading cause of morbidity and mortality in both immunocompetent and
immunocompromised critically ill patients14,15. The incidence of nosocomial yeast
infections has increased markedly in recent decades, especially among older people. On
the other hand, older adults are especially predisposed to oral infectious diseases such as
candidiasis, which is associated with diabetes and prosthetic dentures. Wearing
complete dentures is also a risk factor because they can promote Candida colonization,
Candidal biofilm formation and oral candidiasis16.
Furthermore, the cases of invasive mycosis have been increasing; these are generated
both in the intensive care unit and the outpatient setting, where most types of mycosis
represent endogenous infection in which the normally commensal host microbiota take
advantage of the “opportunity” to cause infection . Given the reports of therapeutic
failure , it is very important to determine the profile of the antifungal susceptibility
of oral Candida isolates and whether they are infectious or commensal. The aim of this
study was to assess the epidemiologic and microbiologic profile and the in vitro
susceptibility to fluconazole of yeasts isolated from the colonization or infection of the
oral mucosa of elderly patients.

Materials and Methods

Study design and clinical isolates.

Following approval from the Ethical Committees of the participant institutions, we

conducted a prospective cross-sectional study, conducted over a 6 month period in 120
older people participants (over the age of 65 during the period of study); half of these
were sampled during a stay not exceeding three days in a second level hospital wing
from the San Luis Potosí Central Hospital “Ignacio Morones Prieto”. The second half of
the population was sampled in recreation centers for older people [CEPITE –Centro
Potosino de la Tercera Edad/Potosino Senior Center] under strict systemic,
pharmacological and metabolic disease control. Characteristics considered to be risk
factors for Candida infection were recorded. We analyzed the following factors: age,
the presence of a complete and/or a partial dental prosthesis, natural dentition and oral
hygiene. Underlying diseases including diabetes and arterial hypertension were noted.
Other factors were also recorded, including systemic antibiotic therapy and all
medications used. Older patients in intensive care and pre and post-surgical patients
were excluded.

The participants were informed of the methods for sample collection and signed an
informed consent form. Samples were taken following the same order in each patient.
Briefly each sample was collected by passing a sterile cotton swab 10 times counter-
clockwise across the dorsal surface of the tongue, soft and hard palate from incisive
papila to uvula and buccal mucosa beginning from posterior right check and ending
with the left. Each swab was placed in its sterile container with Stuart transportation
medium (Copan, Italy) and taken to the laboratory within two hours to maintain the
viability of Candida. Oral specimens were coded according to the clinical group and
patient. Infection (candidiasis) was confirmed via cytology of the lesion: observing
gemmation and Candida pseudohyphae and hyphae formation, using smears stained
with periodic acid Schiff stain (Hycel, México).
Microbiological assessment and identification of isolates

Yeasts were identified to the species level using standard methods, such as the germ
tube test, filamentation strains on Corn meal agar (BD, France) - Tween 80 (Hycel,
México) plates (blastoconidia, pseudohyphae and true hyphae formation, as well as
chlamydoconidia production) were identified by observing the plates at 10X
magnification using a stereoscopic microscope (Leica® EZ4HD Microsystems,
Singapore) also each colony smear was stained with Trypan blue (Sigma, USA) and
observed by optical microscopy (100X) and carbohydrate assimilation patterns using
the API ID 32 C system (BioMerieux, France)

Each sample of oral mucosa collected was plated on differential and selective
CHROMagar® Candida medium (CHROMAgar, París, France) and incubated
aerobically at 36°C for two days for Candida yeast growth; if fungi did not grow, they
remained incubated for an additional period of seven days at a temperature of 30 ± 1ºC
(ideal temperature for growth of fungi other than Candida) prior to evaluation as fungi-
negative. With this chromogenic culture, a presumptive identification of Candida
species was made. According to the colorimetric characteristics given by the
manufacturer for each species, the use of this medium allowed us to separate two or
more strains in overgrowth of different species from the same sample. Cultures that
were positive for one or more species and purified cultures were reseeded and purified
in plates of Sabouraud glucose agar (BD, USA) by incubating them at 36°C (± 1°C) for
two days; the identifications were confirmed using an optical microscope (100X).
Subsequently, the yeast species were identified through the API ID32 C-AUX
(BioMerieux, France) carbohydrate assimilation system and Apiweb database
(BioMerieux, France). In our tests, reference Candida strains (C. albicans ATCC
90028, C. krusei ATCC 6258, C. glabrata ATCC 2001 and C. tropicalis ATCC 0750)
were used as controls in each experiment. Previously phenotyped isolates were
preserved in glycerol/YPD (Yeast extract, Peptone, Dextrose) broth 50:50 vol by
freezing (-70°C) for later use.

Fluconazole susceptibility testing

Prior to susceptibility testing, each isolate was sub-cultured on Sabouraud dextrose agar
and CHROMagar Candida (CHROMagar, Paris, France) to ensure purity and viability.
Susceptibility to fluconazole was tested using a broth microdilution assay, according to
the methods (document M27-A3) of the Clinical and Laboratory Standards Institute
(CLSI)19 using fluconazole (Pfizer, Inc., New York, NY, USA).
For susceptibility test, a RPMI-1640 medium without bicarbonate was prepared with L-
glutamine (SIGMA, USA) and buffered at pH 7.0 with 0.165 M of
morpholinopropanesulfonic acid [MOPS] (SIGMA, USA). This enriched medium used
for eukaryotic cell culture is recommended by the CLSI. Each yeast inoculum
suspension for the susceptibility test was prepared using a spectrophotometer UV-VIS
(Thermo Scientific. Madison, WI, USA) to obtain a final concentration of 0.5-2.5 × 103
cells/mL. The trays were incubated in air at 35°C, and the minimal inhibitory
concentration (MIC) endpoints were read visually after 48 h. To eliminate the effects of
trailing growth, spectrophotometric end points for fluconazole were also determined
after 48 h. Visual and spectrophotometric end points were defined as the lowest drug
concentrations that resulted in a prominent decrease in growth and a 50% reduction in
optical density, respectively, compared to the data from the drug-free growth control
well. Quality control was performed by testing the Candida strains recommended by
CLSI, which were C. parapsilosis ATCC 22019 and the C. krusei ATCC 6258.
The interpretive susceptibility criteria (breakpoints) used for fluconazole were the same
as those specified by the CLSI in document M27-S4 20, considering three categories: S=
susceptible; SDD= susceptible dependent upon dose; and R=Resistant. Isolates of C.
krusei are considered resistant to fluconazole irrespective of the MIC. Under infrequent
species are not described breakpoints for fluconazole, so the MCI is expressed

Statistical analysis
A descriptive analysis was performed, categorizing the study population into three age
groups: I) 60 to 69 years old; II) 70 to 79 years old and III) ≥ 80 years old. Differences
between categorical variables were evaluated using a chi-square test; comparisons of
species distribution and MIC distribution were determined using the chi-square test for
categorical variables. Logistic regression analysis was performed to identify risk factors,
using colonization / infection by Candida as the dependent variable and using age, the
use of oral prostheses, and / or hospitalization as the independent variables. P < 0.05
were considered statistically significant. For statistical analysis, we used the software
Statistical Package for the Social Sciences (SPSS) version 15.1 for Windows (IBM
Study population
This study involved 120 older Mexicans; with an average age of 72.1 years (range 65-
102). For analysis, the participants were classified according to three age ranges: Group
I: 60-69 years old; Group II: 70-79 years old; Group III: ≥80 years old. The
demographics and backgrounds of the participants by age group are shown in Table 1.
In all groups, the majority of participants were female, 79.2, 90.7 and 87.5 %, and
smoking status was not significantly different among groups.
Group I: 60-69 years old
In this group, thirty percent were alcoholic, 94.3% were partially or completely
edentulous, and only 30% used prosthesis. Type 2 diabetes was observed in 37.7% of
the participants, and hypertension was observed in 41.5% of the participants. The use of
hypoglycemic drugs was observed with a frequency of 39.6%. Sixty-six percent of the
participants were carriers of Candida, either colonizing or infecting, and 13.2% of the
participants presented candidiasis in some clinical form.
Group II: 70-79 years old
In this group, alcoholism increased to 34.9%; 95.3% of the participants were partially or
completely edentulous, and 46.5% of the participants used prostheses. Type 2 diabetes
was observed in 25.6% of the participants, and hypertension increased to 62.8%. The
use of multiple anti-hypertensive drugs (1 to 3 different drugs) was observed in 55.8%
of the participants. The use of hypoglycemic drugs was observed in 25.6% of the
participants. Sixty-seven percent of the participants were carriers of Candida, either
colonizing or infecting, with 20.9% of the participants presenting candidiasis in some
clinical form.
Group III: ≥80 years old
In this group, the habit of alcoholism was significantly lower (Chi2, p = 0.018) with a
decrease in alcoholism to 4.2%. Most of the participants (91.7%) of this group were
sampled during a short stay in a hospital. The lack of oral hygiene had a tendency to
increase with age, and 45.8% of the participants in this group did not practice any daily
oral hygiene (Chi2, p = 0.014).
Pharmacologic therapies in the elder population studied showed some differences by
age group. The use of multiple anti-hypertensive drugs was observed in 37.5% of the
participants, and 75% of the participants reported chronic consumption of two or more
nonsteroidal analgesics. Considering that this group consumed a larger quantity of
analgesics (Chi2, p = 0.002), the use of hypoglycemic drugs presented a downward
trend as the age of the participants increased, with a frequency of consumption of 16.6%
in this group (Chi2, p=0.015).
Hospitalized and non-hospitalized
An analysis of the study variables among hospitalized and non-hospitalized participants
was conducted; no significant differences were found in the demographic or clinical
conditions of the geriatric population, regardless of whether they were sampled in the
hospital or in the recreation center. From a total of 20 edentulous participants, 16 were
hospitalized and only 4 were non-hospitalized (p ≥ 0.01). Of the hospitalized
participants, 27 reported that they did not practice any oral hygiene procedures
regularly, whereas 58 of the non-hospitalized participants reported they practice some
form of oral hygiene procedure daily (p ≥ 0.01). The use of antihypertensive and
hypoglycemic drugs for the control of hypertension and diabetes, respectively, was
significantly higher (p ≥ 0.003 and p ≥ 0.01) in the non-hospitalized participants.
Analgesics and antibiotics, used together as dual therapy, were the most commonly used
drugs by hospital participants, with differences between the groups for analgesics (p ≥
0.001). No drug therapy was statistically associated with a higher frequency of
colonization / infection by Candida.
Yeast colonization / infection.
The frequency of yeast colonization / infection in the total study population of older
adults was 65.8%. The frequency of Candida carriers, either colonizing or infecting, was
the highest (67.4%) in age group II. No significant differences were observed in the
frequency of Candida among groups.
Oral candidiasis was present in 20% of the total population, and a clear tendency for
oral candidiasis to increase with age was observed; the highest frequency of oral
candidiasis (33.3%) was in group III. (Table 1)
According to a logistic regression analysis to determine which factors could influence
colonization / infection with Candida in the elders, it was determined that the use of a
prosthesis was associated with a higher colonization rate than for those who do not use a
prosthesis (Chi2, p = 0.011), with an odds ratio of 3.59 (C.I. 95%. 1.47-8.77, p = 0.003).
For the development of oral candidiasis, the associated factor was short term
hospitalization, with an odds ratio of 1.8 (C.I. 95%. 0.90-2.18, p = 0.05).
In group I, from 35 colonized / infected patients, 47 isolates were obtained, and 61.6%
of these were identified to be C. albicans, and 34% were Candida non-albicans. C.
glabrata was the most frequent non-albicans species, followed by C. tropicalis and
other Candida species, as well as a 4.3% (2 cases) of S. cerevisiae. In age group II, the
species distribution was very similar to that of the previous group. Of 29 patients
colonized or infected, 42 yeast isolates were obtained; of these, 61.9% were identified to
be C. albicans, and 35.7% were Candida non-albicans. C. glabrata was the most
frequent, followed by C. tropicalis, along with one case of C. kefyr and one case of S.
cerevisiae. In age group III, a different distribution of yeast species was observed (Chi2,
p = 0.001). Of 15 participants colonized or infected, 25 isolates were obtained. In this
group, most cases presented as multispecies infections. Of these, 36% were identified to
be C. albicans, and 60% were Candida non-albicans. Among the non-albicans, C.
glabrata was the most frequent, followed by C. tropicalis, and then four cases of other
Candida species and a case of S. cerevisiae.
The frequency of cases of colonization or infection with more than one species showed
a tendency to increase with age, with 18.9% of the cases in group I, 20.9% of the cases
in group II, and 29.2% of the cases in group III. No significant differences were
observed in the frequency of multi-species cases by age group. A description of the
frequencies by species is presented in Table 2.
Fluconazole susceptibility
With respect to susceptibility to fluconazole (Table 3), it was determined that for C.
albicans, 52 of 64 (81.2%) were susceptible and 13 of 64 (20.3%) resistant. For C.
glabrata, 22 of 27 (81.5%) were susceptible, 4 of 27 (14.8%) were dose dependent
susceptible and 1 of 27 (3.7%) was resistant. For C. tropicalis, 4 of 11 (36.3%) were
susceptible, 2 of 11 (18.1%) were dose dependent susceptible, and 5 of 11 (45.4%) were
resistant. For the more infrequent species of Candida, the MICs are presented in Table
During 2012 in Mexico, 43.2% of older people adults were classified as being in
multidimensional poverty2 according to the National Assessment of Social
Development Policy of the Federal Public Administration (CONEVAL-Consejo
Nacional de Evaluación de la Política de Desarrollo Social). The elder are a vulnerable
and growing population, and pharmacological and systemic control of their diseases is
fundamental to a better quality of life.
In our population, similar to those of other countries, chronic degenerative diseases are
the leading causes of morbidity and mortality 1,3. In this study, we observed that type 2
diabetes is a highly prevalent condition in the population of patients older than 60 years;
the complications associated with this disease include significant changes in the
conformation of the normal microbiota, increasing the frequency of opportunistic fungi.
21 22
Candida progressively colonizes and causes infections ranging from superficial to
disseminated . In all of these studies, C. albicans has been the yeast most commonly
isolated from the oral cavity.
In our results, we did not observe differences in oral colonization among diabetics and
non-diabetics, despite their pharmacological control or duration of disease. However, its
frequency decreased after 80 years of age, most likely due to the high mortality
attributable to old age (relative mortality rate: 638.47 per 100 thousand inhabitants > 80
Oral Candida colonization by different species has been studied in several populations,
including HIV patients 25, patients with diabetes 26, neonatal patients 27, children 28
older adults 29–33.
The elder have several metabolic disorders, frequently a decreased hepatic and renal
function34, use multiple drugs, have poor nutrition35, and several local factors associated
with the colonization and infection with Candida ; which may explain the high
frequency observed by us; noting the difficulty to explain the differences between
colonization, heavy carriage29 and infection of yeast37. In our study population, oral
candidiasis was present in 20% of the total population, and a clear tendency for oral
candidiasis to increase with age was observed; candidiasis appeared in 13.2%, 20.9%
and 33.3% of the adults aged 60-69, 70-79 and > 80 years, respectively; probably by
increasing of the metabolic impairment associated with increased age range.
Denture stomatitis was the most common type in the 70-79-year-old group, and
erythematous candidiasis or the presence of ≥ 2 clinical types was the most frequent
condition for the population of adults aged > 80 years. This trend may be because
increasing age modifies physiological factors and oral local factors and because the
immune system is diminished. The increase of these lesions may be due to changes in
physiological and local factors related to decreased immune response in older people.
An age-related increase in Candida non-albicans frequency has been reported,
particularly of C. glabrata 38, 39. According to this, we found that Candida non-albicans
species colonizing the oral cavity increased with age, with a higher frequency of these
species than C. albicans, independently of denture use or overall health. In the age
groups we used for the analysis, gender and other conditions were also not associated
with colonization. However, oral colonization was high in the >60-year-old population
compared with the frequencies reported in those of <60 years of age.
A very interesting finding in our study is the high frequency of oral colonization by
multi-species of Candida. Malani, et al. reported a 25.1% frequency of multi-species
colonization, most frequently by C. glabrata and C. albicans. We observed the
frequency of cases of multi-species colonization increasing with age from 18.9 to
29.2%, and also most frequently by C. glabrata and C. albicans. We agree whit Malani,, that local factors such as the use of dentures and poor oral hygiene were factors
that favored colonization by multiple species of Candida.
The high frequency of colonization and infection by oral Candida observed in 67% of
our population is of great importance in the elder population for both diagnostic and
therapeutic evaluation. This is because the decrease in immune response allows
Candida to colonize unusual sites, penetrate deeply into tissues and spread easily, with
serious consequences in debilitated patients in whom changes in microbial
ecosystems are favorable for the establishment of pathogens for long periods or
permanently. Also, a history of use of broad-spectrum antibiotics, antifungals, and other
drugs favors the selection of resistant strains in microbial ecosystems , promotes the
generation of microbial resistance and allows for the establishment of emerging strains
with intrinsic resistance to these antifungals.
The mouth plays a vital role in the quality of life of the older adults 31; good oral health
significantly increases quality of life , by facilitating good nutrition, communication
and the maintenance of emotional and social stability. Candida is a microorganism that
normally colonizes the oral tissues. The prevalence has been reported to range from
80% to 97%37. Our results found an oral Candida frequency of 66% in persons <70
years of age, 67.4% in persons 70-79 years of age, and interestingly, a decrease in the
frequency in persons > 80 years of age. The difference in frequency between our study
and the study of Kraneveld could be due to the lower detection limit and higher
selectivity of the culturing technique compared with specific qPCR on the Candida
internal transcriber spacer (ITS) region, as used by Kraneveld EA. However, the study
of the frequency of oral colonizing isolates and virulence determinants could possibly
explain the increase in disseminated infections by this yeast. In this sense, it becomes
important to study colonization, particularly by multiple species of Candida because it
is more like that species such as C. glabrata and C. tropicalis, which we observed in
meaningful frequencies, which we observed in meaningful frequencies, will be detected
and that have increased virulence, resistance and worse prognosis, than when was
colonized by C. albicans. We agree with the results of the study by Lockhart, et al. .
who demonstrated that the frequency of carriage, the intensity of carriage, and the
presence of multispecies all increase as a function of age and differ according to gender,
independently of denture use. This suggested that the natural suppression of yeast
carriage in the oral cavity breaks down in older people 41. They added that C. glabrata
colonized the oral cavities of elderly individuals without dentures only after 80 years of
age, suggesting that there are age-related compromising conditions other than denture
use in this age group 38,41.
The role of Candida in oral microbiological changes in old age is still not clear.
Nevertheless, it has been observed that physiological changes in the oral mucosa are
associated with age, such as the thinning of the mucosa, changes in the oral biochemical
microenvironment and decreases in salivary flow and that these changes may contribute
to Candida colonization 4,39.
The current findings strongly suggest that acidification of the oral environment
coincides with a high load of Candida and is the major ecological factor that perturbs
the oral commensal microbiome, leading to the shift toward an increase of aciduric
microbiota and a reduction of natural diversity in the bacterial microbiome. The actual
sequential order of the events (i.e., whether Candida outgrowth is followed by
acidification or whether primary acidification of the oral ecosystem due to other factors
such as dry mouth or high carbohydrate intake leads to Candida outgrowth) can be
elucidated in only well-planned longitudinal studies37
Oral fluconazole is a first-line systemic therapy for oropharyngeal candidiasis.
Treatment with oral fluconazole using 100 or 200 mg daily is easy and highly
efficacious. A recent trial also suggests that a single 750 mg dose of oral fluconazole
has equal efficacy and relapse rates to those of daily treatment . Itraconazole,
voriconazole, and posaconazole also have efficacy in the treatment of oropharyngeal
candidiasis. However, these agents would typically be used only as salvage therapies in
the setting of resistance or oropharyngeal candidiasis with Candida non-albicans
species . The Infectious Disease Society of America (IDSA) guidelines suggest
using itraconazole solution if fluconazole failure occurs . The results of our study
show that oral clinical isolates of colonizing or infecting Candida in the elder
population present patterns of reduced susceptibility with a considerable frequency.
This frequency increases with age because susceptibility to fluconazole is an important
marker of pathogenicity; therefore it allows us to establish better clinical and therapeutic
forecasts and more adequate management of antifungals in local, systemic and
hospital use.

Based on the results of this study, the oral cavity of elderly patients appears to be
colonized more frequently by C. albicans in patients under 80 years of age. After 80
years of age, there is a change in the pattern of Candida oral colonization by different
species, with a considerable increase in Candida non-albicans species, and the presence
of multispecies. This colonization depends on several factors, including the use of
partial or complete dentures and short-term hospitalization.

In addition, the oral isolates showed a reduced susceptibility to fluconazole (SSD and
R) in the ≥80 age group, which was higher than that observed in the <70 years
population. These cases of resistance and SSD in oral isolates of elderly patients are an
important parameter to consider in recalcitrant candidiasis, which may explain the
increase in therapeutic failure of azole antifungals in local and disseminated candidiasis.

This work was supported by the project PROMEP/103.5/13/6575, folio UASLP-PTC-

We appreciate the technical support and academic collaboration of QFB Virginia Flores
Gutierrez and MenC. Araceli Gómez Hernández. We also appreciate the facilities
provided by the staff of CEPITE (Centro Potosíno de la Tercera Edad).

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Table 1. Differences between personal, medical and fungal characteristics of the participants in
three age groups

Age group
Characteristics 60-69 years 70-79 years ≥80 years
N=53 N=43 N=24
N % N % N %
Women 42 79.2 39 90.7 21 87.5 NS
Men 11 20.8 4 9.3 3 12.5

Hospitalization 19 35.8 19 44.2 22 91.7 0.001

Partially of completely 50 94.3 41 95.3 24 100 NS


Natural teeth (28) 3 5.7 2 4.7 0 0 NS

Prosthesis used 16 30.2 20 46.5 11 45.8 NS

Oral hygienic 8 15.1 10 23.3 11 45.8 0.014

Alcoholism 16 30.2 15 34.9 1 4.2 0.018

Smoking 7 13.2 3 7 2 8.3 NS

Diabetes Mellitus 2 20 37.7 11 25.6 4 16.7 NS

Hypertension 22 41.5 27 62.8 11 45.8 NS

Oral candidiasis 7 13.2 9 20.9 8 33.3 NS

Protetic estomatitis 2 3.8 3 7 1 4.2
Eritematous 2 3.8 1 2.3 2 8.3
Pseudomembranosa 1 1.9 2 4.7 0 0 NS
Queilitis Angular 2 3.8 1 2.3 3 12.5
≥ 2 types 1 1.9 3 7 2 8.3
35 66 29 67.4 15 62.5 NS
Multi-species 10 18.9 9 20.9 7 29.2 NS
NS=Not Significant

Table 2 Differences between oral clinical yeast species identified of the participants in three
age groups.

Age group
Characteristics 60-69 years 70-79 years ≥80 years
N=53 N=43 N=24
N % N % N %
Yeast species isolates 47 100 42 100 24 100
C. albicans 29 61.7 26 61.9 9 37.5
Candida no albicans 16 34.0 15 35.7 14 58.3
C. glabrata 10 21.3 10 23.8 7 29.1
C. tropicalis 3 6.4 4 9.5 4 16.6
C. krusei 0 0 0 0 1 4.2
C. sphaerica 1 2.1 0 0 0 0
C. dubliniensis 0 0 0 0 1 4.2
C. sake 1 2.1 0 0 1 4.2
C. pulcherrima 1 2.1 0 0 0 0
C. kefyr 0 0 1 2.4 0 0

Sacharomyces cerevisiae 2 4.3 1 2.4 1 4.2

Table 3. Differences between fluconazole susceptibility of oral by Candida species

isolates from the participants in three age groups (brackets contain percentages)

Candida Age group

species 60-69 years 70-79 years ≥80 years
identify N=53 N=43 N=24
n (%) n (%) n (%)
a b c
C. albicans 3 (10) 26 (90) 6 (23) 20 (77) 4 (44) 5 (56) 0.74
C. glabrata 1 (10) 1 (10) 8 (80) 2 (20) 8 (80) 1 (14) 6 (86) NS
C. tropicalis 2 (67) 1 (33) 1 (25) 2 (50) 1 (25) 2 (50) 2 (50) NS
C. krusei 1 (100) NA
(n= 1)
Dose-dependent susceptibility

NS=Not Significant

NA=Not Applicable

Table 4. Fluconazole minimal inhibitory concentration (MIC50) of oral infrequent Candida

species isolates from the participants in three age groups.

Fluconazole 60-69 years 70-79 years ≥80 years

Susceptibility MIC50 µg/ml MIC50 µg/ml MIC50 µg/ml
C. sphaerica 0.125
C. dubliniensis 0.125
C. sake 8 0.250
C. pulcherrima 1
C. kefyr 32
Sacharomyces cerevisiae 2-8 2 2