Oral Candida isolates and fluconazole susceptibility patterns in older Mexican women

Accepted Manuscript Title: Oral Candida isolates and fluconazole susceptibility patterns in older Mexicans women Author: Beatriz Benito-Cruz Saray Aranda-Romo Francisco Javier L´opez-Esqueda Estela de la Rosa-Garc´ıa Rebeca Rosas-Hern´andez Luis Octavio S´anchez-Vargas PII: DOI: Reference:

S0167-4943(16)30064-4 http://dx.doi.org/doi:10.1016/j.archger.2016.04.001 AGG 3310

To appear in:

Archives of Gerontology and Geriatrics

Received date: Revised date: Accepted date:

8-7-2015 9-3-2016 3-4-2016

Please cite this article as: Benito-Cruz, Beatriz, Aranda-Romo, Saray, L´opezEsqueda, Francisco Javier, de la Rosa-Garc´ia, Estela, Rosas-Hern´andez, Rebeca, S´anchez-Vargas, Luis Octavio, Oral Candida isolates and fluconazole susceptibility patterns in older Mexicans women.Archives of Gerontology and Geriatrics http://dx.doi.org/10.1016/j.archger.2016.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

TITLE: Oral Candida isolates and fluconazole susceptibility patterns in older Mexicans women

AUTHORS: Beatriz Benito-Cruz1; Saray Aranda-Romo 2, Francisco Javier López-Esqueda 3, de la Rosa-García Estela4, Rebeca Rosas-Hernández5 y Luis Octavio SánchezVargas2*.

1

Master in Biomedical Science. Faculty of Medicine, University Autonomous of San

Luis Potosí, México 2

Oral Microbiology, Pathology and Biochemical laboratory. Faculty of Stomatology,

University Autonomous of San Luis Potosí, México. 3

Geriatric Unit. Central Hospital “Ignacio Morones Prieto”. University Autonomous of

San Luis Potosí, México 4

Department of Attention to Health. Metropolitan Autonomous University,

Xochimilco, México City. 5

Molecular Biophysics laboratory. Physics Institute "Manuel Sandoval Vallarta".

University Autonomous of San Luis Potosí, México. SHORT TITLE: Candida colonization and infection in older adults CORRESPONDING AUTHOR: 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., México. e-mail: [email protected] KEY WORDS: Candida, Diabetes, elderly, infection, epidemiology.

Abstract 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-oldgroup. About fluconazole susceptibility: for C. albicans, 20.3%, about Candida nonalbicans species 15.3% were dose dependently susceptible (DDS) and 17.9% were resistant. Conclusions: After 80 years of age, there is a considerable increase in Candida nonalbicans species and a reduced susceptibility to fluconazole.

Introduction 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

11

. When Candida penetrates the epithelium and invades the host tissues,

septicemia and systemic infections may result

12

. 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

10

. Given the reports of therapeutic

failure

17,18

, 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 counterclockwise 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 funginegative. 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 Lglutamine

(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 SPSS Inc; USA).

Results Study population This study involved 120 older Mexicans; with an average age of 72.1 years (range 65102). 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 4.

Discussion 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. Candida

21

progressively colonizes and causes infections ranging from superficial

disseminated

23

22

to

. 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 years)24. 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

and

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

36

; 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.

38

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, et.al., 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

21,26

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

40

, 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

30

, 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%

7,8

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.

41

.

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

31

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

42

. 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

43,44

. The Infectious Disease Society of America (IDSA) guidelines suggest

using itraconazole solution if fluconazole failure occurs

45

. 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

26,46

of antifungals in local, systemic and

hospital use.

Conclusions 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.

Acknowledgements This work was supported by the project PROMEP/103.5/13/6575, folio UASLP-PTC464. 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

Women Men

60-69 years N=53 N % 42 79.2 11 20.8

Age group 70-79 years N=43 N % 39 90.7 4 9.3

≥80 years N=24 N % 21 87.5 3 12.5

Hospitalization

19

35.8

19

44.2

22

91.7

0.001

Partially of completely edentulous

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 Protetic estomatitis

7 2

13.2 3.8

9 3

20.9 7

8 1

33.3 4.2

NS

Characteristics

P NS

Eritematous Pseudomembranosa Queilitis Angular ≥ 2 types Yeast Colonization/infection Multi-species NS=Not Significant

2 1 2 1

3.8 1.9 3.8 1.9

1 2 1 3

2.3 4.7 2.3 7

2 0 3 2

8.3 0 12.5 8.3

35

66

29

67.4

15

62.5

NS

10

18.9

9

20.9

7

29.2

NS

NS

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

Yeast species isolates C. albicans Candida no albicans C. glabrata C. tropicalis C. krusei C. sphaerica C. dubliniensis C. sake C. pulcherrima C. kefyr

60-69 years N=53 N % 47 100 29 61.7 16 34.0 10 21.3 3 6.4 0 0 1 2.1 0 0 1 2.1 1 2.1 0 0

Age group 70-79 years N=43 N % 42 100 26 61.9 15 35.7 10 23.8 4 9.5 0 0 0 0 0 0 0 0 0 0 1 2.4

N 24 9 14 7 4 1 0 1 1 0 0

Sacharomyces cerevisiae

2

1

1

Characteristics

4.3

2.4

≥80 years N=24 % 100 37.5 58.3 29.1 16.6 4.2 0 4.2 4.2 0 0

P

0.001

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 species identify

60-69 years N=53 n (%) a b c R SSD S 3 (10) 26 (90)

C. albicans (n=64) C. glabrata (n=27) C. tropicalis (n=11) C. krusei (n= 1)

1 (10) 2 (67)

1 (10)

Age group 70-79 years N=43 n (%) R SSD S 6 (23) 20 (77)

8 (80) 1 (33)

1 (25)

2 (20)

8 (80)

2 (50)

1 (25)

R 4 (44)

1 (14) 2 (50) 1 (100)

a

Resistent

≥80 years N=24 n (%) SSD

P S 5 (56)

0.74

6 (86)

NS

2 (50)

NS NA

b

Dose-dependent susceptibility

c

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 Susceptibility C. sphaerica C. dubliniensis C. sake C. pulcherrima C. kefyr Sacharomyces cerevisiae

60-69 years MIC50 µg/ml 0.125

a

70-79 years MIC50 µg/ml

0.125 0.250

8 1 2-8

≥80 years MIC50 µg/ml

32 2

2