Invest Clin 60(4): 275 - 282, 2019 https://doi.org/10.22209/IC.v60n4a02

Fluconazole and voriconazole susceptibility in oral colonization isolates of Candida spp. in HIV patients.

Daniela Brito1, Johelia Fernández1, María Andrea Castillo1, Sandra Azuero1, Rosaura Hernández-Valles2, Yotsabeth Saúl-García2, Nereida Valero3,

Rafael Villalobos4, Neomar Semprún-Hernández1 and Dilia Martínez-Méndez1,5

1. Immunology Lab. Facultad Experimental de Ciencias. Universidad del Zulia. Maracaibo, Venezuela.

2. Micology Lab. Medical School. Universidad Nacional "Francisco de Miranda" (UNEFM). Coro, Venezuela.

3. Instituto de Investigaciones Clínicas “Dr. Américo Negrette”. Universidad del Zulia. Maracaibo, Venezuela.

4 HIV&AIDS Program. Servicio Autónomo Hospital Universitario de Maracaibo (SAHUM). Maracaibo, Venezuela.

5 GP Supervisor.International SOS. Malongo, Angola.

Key words: candidiasis; HIV; antifungal susceptibility; non-albicans species.

Abstract. The identification of Candida species and their antifungal sus- ceptibility is important for the treatment of infected patients. The aim was to determine the susceptibility to fluconazole and voriconazole in isolates from oral colonization of Candida spp. in HIV patients. From the 135 patients stud- ied, 33.3% were females and 66.7% males, with a mean age of 36.6 years and 83.7% of them were under treatment. The identification of the specie was per- formed by the API20CAUX® test and the antifungal susceptibility was deter- mined by the disc diffusion test. Strains of C. parapsilosis and C. krusei were used as quality controls. The Candida species identificated were: C. parapsi- losis complex 52%, C. albicans complex 36% and C. famata 12%. 60% of the isolates were susceptible to fluconazole and 40% were dose-dependent. All were susceptible to voriconazole. In this study, none of the patients had oropharyn- geal candidiasis however, 18.5% had Candida spp. colonization, this percentage being below other studies in HIV carriers. We found a high proportion of non- albicans species but no difference in the CD4+ counts between patients.

Corresponding author: Dilia Martínez-Méndez . GP Supervisor.International SOS. Malongo, Angola. Email: dkmartinez.mw@gmail.com

Susceptibilidad a fluconazol y voriconazol en aislados orales de Candida spp. en pacientes con VIH.

Invest Clin 2019; 60 (4): 275-282

Palabras clave: candidiasis; VIH; susceptibilidad antifúngica; especies no-albicans.

Resumen. Identificar las especies de Candida y la susceptibilidad a los antifúngicos es importante para el tratamiento adecuado de pacientes infec- tados. El objetivo fue determinar la susceptibilidad a fluconazol y voriconazol de Candida spp. en aislados de colonizaciones orales en pacientes con VIH. La identificación se realizó mediante la prueba API20CAUX® y la prueba de difusión de disco para la sensibilidad a los antifúngicos. Se utilizaron cepas de C. parapsilosis y C. krusei como control de calidad. De los 135 pacientes, el 33,3% eran mujeres y el 66,7% hombres con una edad media de 36,6 años y el 83,7% recibían tratamiento. Las especies de Candida identificadas fueron: complejo C. parapsilosis 52%, complejo C. albicans 36% y C. famata 12%. La susceptibilidad al fluconazol 60% eran susceptibles y 40% dependía de la dosis. Todas eran susceptibles al voriconazol. En este estudio, ninguno tenía candi- diasis orofaríngea, sin embargo, el 18.5% tenía colonización por Candida spp., aunque el porcentaje está por debajo de otros estudios en portadores de VIH. Encontramos una alta proporción de especies no-albicans. No hubo diferencias en el recuento de CD4+ entre los pacientes.

Received: 05-11-2018 Accepted:09-10-2109

INTRODUCTION

In HIV patients under antiretroviral therapy (ART) a reduction of oral candidia- sis by C. albicans have been observed; how- ever, the emergence of non-albicans species with resistance to antifungal agents has also been observed and the presence of oral Candida spp. in these patients can predict the development of oral candidiasis (1-3). To identify the Candida species involved and the study of antifungal susceptibility is important for the epidemiological mapping of the species and to improve the treatment of these patients(4). Fluconazole is used in the treatment of HIV/AIDS associated op- portunistic yeast infections and prolonged exposure is associated with the increase of resistance (1,4,5). The aim of this work was

to determine the susceptibility to flucon- azole (FCZ) and voriconazole (VCZ) in the isolates of the oral colonization of Candida spp. in HIV/AIDS patients.

METHODS

A cross-sectional study was made in 135 seropositive HIV patients previously di- agnosed by ELISA and confirmed by Western blot at the University Hospital (SAHUM) HIV/AIDS program, Maracaibo - Venezuela. None had clinical evidence of oral candidia- sis. Patient’s age, CD4+ T-lymphocyte count, World Health Organization (WHO) immu- nological classification for established HIV infection (5) and ART were obtained from their medical records.

Ethical clearance was obtained by the Medical board in accordance with the eth- ics committee of SAHUM and participants signed informed consent. Identification and susceptibility were reported to each subject and the attending physician.

Samples: A swab was collected from the oral mucosa, floor of the mouth and dor- sal tongue. Samples were cultured in Sab- ouraud-dextrose agar (SDA - BD Difco™ MD, USA) with ampicillin (500mg/L) for 24h at 35ºC. Blastoconidies were observed.

Identification: Pure colonies were plated in CHROMAgar® Candida (Biomedics®) for 48h at 35°C following manufacturer’s in- structions. Morphology and color on CHRO- MAgar® Candida colonies were noted. Germ tube test was achieved inoculating 0.5 ml of sheep sterile serum with a scoop (5 µl) of yeast, incubated for 3h at 37°C. Isolates with germ tube-positive were presumed to be C. al- bicans complex. Chlamydospores production was assessed by culturing on cornmeal agar supplemented with 1% Tween-80 (SIGMA-AL- DRICH®) at 25°C for 72h followed by micro- scopic observation. The isolates were identi- fied by API20CAUX® test (bioMérieux®SA, Marcy-l’Etoile, France) in accordance with manufacturer’s instructions.

Antifungal susceptibility of identified isolates as outlined in NCCLS M44-A2 docu- ment for disc diffusion susceptibility testing in yeasts using Mueller Hinton + 2% glucose

+ 0.5 g/mL methylene blue dye (GMB) agar and FCZ (25µg) and VCZ discs (1µg) (Bio- analyse®). After 24h at 37°C of incubation, the zones of inhibition were measured. FCZ breakpoint for C. albicans, C. parapsilo- sis and C. tropicalis was: sensible (S) ≥17; dose-dependent sensibility (DDS) 14-16 and resistance (R) ≤13. For C. glabrata (S) ≥17; SDD ≥15 y R ≤14. VCZ breakpoint was S

≥17; DDS 15- 16 and R ≤14 to C. albicans,

C. parapsilosis, C. tropicalis and S ≥15; DDS 13-14; R ≤12 to C. krusei (6,7). Mini- mal inhibitory concentration (CMI) results were interpreted according to CLSI interpre-

tive criteria (6,7). As quality control, ATCC strains of C. parapsilosis 22019 and C. kru- sei 6258 were used. For statistical analysis, the chi-square test and the Fisher’s exact test using the Graph Pad Prism 5.0 program were used.

RESULTS

A total of 135 patients with a con- firmed diagnosis of HIV were enrolled on this study, 33.3% (n=45) females and 66.7% (n=90) males with a mean age of

36.6 years (SD±11.28) ranging from 21 to 60 years (p=0.21). According to WHO

(5) immunological classification for estab- lished HIV infection (mm3/CD4+): 27.4% (n=37) of the patients had not significant, immunosuppression; 22.2% (n=30) mild; 16.3% (n=22) advanced and 17.8% (n=24) severe with less than 200cel/mm3. 16.3% (n=22) had no information on the medical chart because they are new incomings pa- tients to the HIV program at the day that sample was collected (p=0.48). 83.71% (n=113) patients were on ART and 16.9% (n=22) were not because they were the new incoming patients (p=0.96). The most fre- quent treatment regimen was the combina- tion with nucleoside reverse transcriptase inhibitor (NRTI) plus a protease inhibitor (PI) followed by a non-nucleoside transcrip- tase inhibitor (NNRTI) and a nucleotide reverse transcriptase inhibitor (NtRTI) or PI. Only one patient used a fusion inhibi- tor (FI) and none used integrase inhibitor

(II) (p=0.96). Of all 135 patients 67.4% (n=91) were not under antimicrobial ther- apy, but 11.9% (n=16) reported use of any antifungal (fluconazole, ketoconazole or amphotericin B), 20% (n=27) antibacterial (cotrimoxazole, amoxicillin, azithromycin or clarithromycin) and less than 1% (n=1) antiparasitic (metronidazole) (p=0.60). Non-statistical significance was observed between age, gender, CD4+ and use of the ART or antimicrobial (p>0.05) (Table I).

TABLE I

GENDER, CD4+ CEL/MM3, TREATMENT AND Candida spp. CULTURE

* New patients without information in the medical chart at the sample collection day.

All 135 patients had no clinical evi- dence of oral candidiasis (angular cheilitis, leukoplakia or oral ulcerations), however, 18.5% (n=25) were colonized by Candida spp. Of these, 52% (n=13) were identified as C. parapsilosis complex (C. parapsilo- sis sensu stricto, C. orthopsilosis and C. metapsilosis); 36% (n=9) C. albicans com- plex (C. dubliniensis, C. stellatoidea and C. africana) and 12% (n=3) C. famata. The fluconazole MIC at 48 hours showed that 60% (n=15/25) were susceptible, of these, 24% (n=6/25) C. parapsilosis complex, 24% (n=6/25) C. albicans complex and 12% (n=3/25) C. famata and 40% (n=10/25) had doses dependent susceptibility, 28%

(n=7/25) C. parapsilosis complex and 12% (n=3/25) C. albicans complex. None were resistant (p=0.75). All species of Candida identificated were susceptible to voricon- azole MIC (p=0.43). Four patients with Can- dida spp. colonization were new incomings patients to the HIV/AIDS program without ART. One of these was admitted to the hospi- tal with complicate tuberculosis plus oral C. parapsilosis and other with a neurocrypto- cocosis plus oral C. famata. Also, two of the seven C. parapsilosis with doses dependent susceptibility was using FCZ daily for more than a month (p=0.47). Non-statistical sig- nificance was observed (p>0.05) (Table II).

TABLE II

CANDIDA SPECIES IDENTIFICATION BY GENDER, ANTIFUNGAL SUSCEPTIBILITY, CD4+ CEL/MM3 AND TREATMENT.

* New patients without information in the medical record at the sample collection day. “ New patient with tuberculosis

^ Both with doses dependent susceptibility to fluconazole

º New patient with neurocryptococosis

DISCUSSION

The mean age of the patients was 36 years showing that HIV still is an infection among the young population as reported in other countries (5, 8-10). Within the 135 patients involved in this study, 90 were men and 45 were women, resulting in the ratio of two cases in men for every one case in women. Against what happens in some Af- rican countries with a female prevalence of 4.6% vs. 2.7% male prevalence and could be explained by the cultural, social, and eco- nomic vulnerability of women (8,9,11). The last HIV database from Venezuela show that more than 100.000 men were seropositive and approx. 38.000 women, so even with the non-statistical significance observed in our study, data shows that the incidence of HIV in men remains high, with a slow increase in women and both are worrying (12).

Candida spp. oral colonization among HIV patients may predict the development of oropharyngeal candidiasis (OPC) as the most common opportunistic infection in these patients and the progression is faster and more severe as a progressive depletion of CD4+ T cells and/or failure to ART (3,5,8). No patient from this study had oropharyn- geal candidiasis, however, 18.5% had Can- dida spp. colonization, being lower than the studies in HIV carriers in Argentina (72%), Brazil (62%), Cote d´Ívore (79.4%), India

(68%) and Thailand (61%) (9,13-17). We found no difference in the CD4+ counts be- tween patients colonized by Candida spp. as other studies had found (11). Suggesting, as other authors also observed, a protective ef- fect of ART regimens against OPC (1,5,18).

The patients without ART therapy one with complicated TBC and other with neu- rocryptococosis, both were considerate as severe immunodepression, using the WHO clinical staging as a guide for decision-mak- ing regardless of age or CD4+ testing, par- ticularly when the CD4+ count is not avail- able (5,18).

The susceptibility to an antifungal can be different between species and populations and the emergence of antifungal resistance made the identification of Candida spp. an essential to guide the therapeutic choice and clinical treatment (8,19-22). We have found no resistance against FCZ or VCZ, but 40% (n=10/25) of the species had DDS to FCZ: 12% (n=3/25) C. albicans complex and 28% (n=7/25) C. parapsilosis complex. Two of that seven were taking fluconazole, sug- gesting the exposure during ART provided a positive selection pressure for yeasts be- ing less sensitive (3,4,20,22), or could be because C. parapsilosis sensu extricto show more resistance to FCZ (23-26).

This study addresses the importance on identification and antifungal susceptibility as guide to proper treatment in any myco- sis. We find a high proportion of non-albicans species, against most of the studies in HIV carriers where C. albicans complex remains as the most common species (3,11,17). How- ever, our data support Venezuelan´s studies, where C. parapsilosis is the most frequent species isolated (21,25,27-29). The epidemi- ology of Candida infections is changing: new species, better adapted virulence factors, fur- ther antifungal resistance and worse clinical presentations.

ACKNOWLEDGMENT

To all SAHUM HIV/AIDS program per- sonnel and Ms. Zaida Bracho, UNEFM Mycol- ogy Lab.

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