Mycopathologia 157: 43–47, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Correlation of in vitro itraconazole and fluconazole susceptibility with clinical outcome for patients with vulvovaginal candidiasis Milce Costa1 , Xisto Sena Passos1, Andr´e Thiago Borges Miranda1 , Rosane Silva Carneiro de Ara´ujo2 , Claudete Rodrigues Paula3 & Maria do Ros´ario Rodrigues Silva1 1 Instituto de Patologia Tropical e Sa´ ude P´ublica da Universidade Federal de Goi´as; 2
Ambulat´orio de Ginecologia e Obstetr´ıcia do Hospital das Cl´ınicas da Universidade Federal de Goi´as, Brasil; 3 Departamento de Microbiologia do Instituto de Ciências Biom´edicas da Universidade de São Paulo
Received 15 December 2002; accepted 28 February 2003
Abstract Between April 2001 and April 2002 were studied 106 women with a clinical diagnosis of vaginal candidiasis seen at the Gynecology and Obstetrics Ambulatory of the Hospital das Clínicas da Universidade Federal de Goiás. The patients were assessed on two occasions, before starting treatment with itraconazole or fluconazole (initial visit) and 14 days after treatment (return). At two visits the signs and symptoms were recorded and vaginal secretion was collected. According to the clinical evaluation, itraconazole was effective in 64.3%, while fluconazole was effective in 71.0% of the patients. The mycological cure rates (negative culture) in the return were 64.3% for the patients treated with itraconazole and 78.9% for the patients treated with fluconazole. The MICs of itraconazole and fluconazole for 80 Candida isolates were determined by Etest method. We investigated the correlation between in vitro susceptibility (Susceptible, Susceptibility Depending Dose and Resistant) to itraconazole and fluconazole with clinical outcome of the patients. The success rates were 63.9% for itraconazole and 90.6% for fluconazole in the susceptible category, 100.0% for both drugs in the susceptible dose dependent category, and 0.0% for both drugs in the resistant category. Our results showed there were a positive correlation between in vitro susceptibility test results with clinical outcome in vaginal Candida infections and that both drugs might be one choice in the treatment of vaginal candidiasis. Key-words: Fluconazole, in vitro susceptibility, itraconazole, vaginal candidiasis
Introduction Candida infection is a common disease of the lower genital tract in women. It is estimated that 75% of all women will experience at least one episode of acute vulvovaginal candidiasis during their child-bearing period [1]. Effective management of vaginitis depends on accurate diagnosis, selection and administration of effective specific therapy and good compliance of the patient [2]. Methods for testing the antifungal susceptibility could become important tools in the selection of an appropriate antifungal drug for the treatment and prophylaxis of fungal infections. At present, the Etest method, which is a new concept for quantitative sens-
itivity tests is used in order to assess the in vitro susceptibility of the yeast to antifungal agents [3]. Determination of minimum inhibitory concentration (MIC) for several antifungal agents using Etest has been shown to compare favorably with reference broth dilution of National Committee for Clinical Laboratory (NCCLS) testing against Candida and other yeast species [4–10]. To establish the relationship between in vitro results and clinical outcome of candidal infection has been a complicated issue, since the antifungal susceptibility pattern is not the only factor influencing clinical outcome [11]. Pharmacokinetics properties of the antifungal drugs as well as several host factors, such as underlying disease and immune status also determine
44 the fate of the infection. Thus, an infection caused by a susceptible organism may fail to respond to antifungal therapy; conversely, an infection due to a resistant strain usually, but not always, results in clinical failure [12]. We evaluated patients with vulvovaginal candidiasis treated with itraconazole or fluconazole and correlated the clinical outcomes with the itraconazole or fluconazole susceptibility test results according to Etest method.
Clinical and mycological evaluation. Interpretation of the clinical and mycological responses was done according to the following criteria: Clinical cure was indicated by resolution of clinical signs and symptoms of infection within 14 days after treatment with itraconazole or fluconazole. Clinical failure was indicated by persistence of signs and symptoms 14 days after therapy with itraconazole or fluconazole. Mycological cure was verified by negative culture 14 days after treatment and mycological failure was indicated by positive culture 14 days after treatment.
Patients and methods
Etest method. The Etest was performed according to the manufacturer’s instructions (AB Biodisk, Solna, Sweden). RPMI 1640 medium with L-glutamine and without sodium bicarbonate with 2% glucose buffered with potassium phosphate at pH 7.0 and 1.5% Bacto agar (Difco Laboratories, USA) were used to prepare Etest RPMI-agar plates. Prior to testing, each isolate was grown on Sabouraud dextrose agar for 24 h at 35 ◦ C. Suspensions were prepared from individual colonies in 3 mL of sterile 0.85% saline to density of a 0.5 Mac Farland standard. Each suspension was inoculated onto 150 mm Petri plates containing RPMI agar by using a sterile swab, streaking it on the entire surface of the agar. The plates were allowed to dry for 15 min. Following this, the Etest strips of itraconazole and fluconazole were placed onto the inoculated agar. After 24 h incubation at 35 ◦ C, the minimum inhibitory concentration (MIC) of azoles compounds was determined as the lowest drug concentration at which the border of the elliptical inhibitory zone intercepted the scale on the antifungal strip. Quality control strains of C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included on each day of testing to check the reproducibility of the results as recommended by NCCLS [14]. MICs breakpoints of antifungal agents. The interpretation of antifungal resistant-susceptible category among the yeast isolates was based on the NCCLS M27-A criteria of MIC breakpoints published for itraconazole and fluconazole. The MICs (µg/mL) breakpoints categories for itraconazole of susceptible (S), susceptibility depending upon dose (S-DD) and resistant (R) are ≤0.125 µg/mL, 0.25–0.5 µg/mL and ≥1 µg/mL respectively and for fluconazole categories of S, S-DD and R are ≤8 µg/mL, 16–32 µg/mL and ≥64 µg/mL respectively [14].
Patients. The study was conducted in 106 patients with a clinical diagnosis of vaginal candidiasis, consulted at the Department of Obstetrics and Gynecology of the Hospital das Clínicas da Universidade Federal de Goiás, Brazil, between April 2001 and April 2002. All patients agreed to participate in this study, which was approved by the local Ethics Committee. Women were excluded if they were pregnant or in lactation period, had a history of immunosuppressive disease, presented trichomoniasis and poorly-controlled diabetes. Women treated with steroids or immunosuppressive drugs were excluded as well. None of the women included in this study had received topical or systemic antifungals within 1 month before enrollment. Women were assessed on two occasions, before starting the treatment (itraconazole or fluconazole) and 14 days after treatment. In the two visits the clinical signs and symptoms as vaginal discharge, itching, burning, dysuria and dyspareunia were recorded. Vaginal secretion was collected with the aid of an unlubricated speculum from ectocervix region and from vaginal fundus of each patient using sterile swabs. Yeast isolation and identification. The yeasts were isolated in Sabouraud dextrose agar medium (Difco) and their identification were performed by standard microbiologic procedures [13] and by API-20C system. Treatment regimens. Each patient received medication at the first visit and was randomly allocated to either of the two treatments below: (a) Itraconazole: Single day dose with 400 mg. Two capsules after lunch and two capsules after dinner (Janssen-Cilag Pharmaceuticals). (b) Fluconazole: Single day dose with 150 mg (Pfizer Pharmaceuticals).
45 Table 1. Clinical signs and symptoms of vaginal candidiasis after treatment with itraconazole and fluconazole Signs and symptoms
Itraconazole Cure Failure
Fluconazole Cure Failure
Vaginal itching Vaginal burning Discharge Dysuria Dyspareunia
38 34 35 19 19
34 26 30 9 11
04 03 05 01 02
01 01 04 02 03
Statistical test – there was no significant difference between itraconazole and fluconazole (p < 0.05).
Results Of the 106 patients initially recruited, a total of 80 (75.5%) patients had vulvovaginal candidiasis. The identification of species showed C. albicans in 88.7% (71/80), C. parapsilosis in 6.2% (05/80) and C. krusei in 5% (04/80) of the cases. In the second visit, after treatment, 23 women already presented episode of vaginitis verified by mycological failure. The isolated strains were identified as C. albicans in 69.6% (16/23), C. krusei in 17.4% (04/23) and C. parapsilosis in 13% (03/23) of the cases. The majority of the patients treated with itraconazole (n = 42) or fluconazole (n = 38) had reported alleviation of symptoms after treatment. The signs and symptoms for vulvovaginal candidiasis treated with these drugs are listed in Table 1. A mycological and clinical effectiveness of antifungal agents regimens against vaginal candidiasis is shown in Table 2. According to the clinical evaluation itraconazole was effective in 64.3% of the cases, while fluconazole was in 71.0%. The mycological cure rates (negative culture) were 64.3% for the treatment with itraconazole and 78.9% for the treatment with fluconazole. In the clinical and mycological evaluation the responses were statistically significant at the end of treatment for both regimens. There was a reduction in the symptoms of vaginitis and a reduction of Candida in vaginal swabs between first visit and second visit (Chi square test, p < 0.05). There was no significant difference between the different regimens (p < 0.05). Itraconazole and fluconazole MICs by Etest method showed a broad range for C. albicans, C. parapsilosis and C. krusei. The highest MICs were observed for C. krusei. For itraconazole the breakpoints of Candida isolates showed that there were 36 isolates considered susceptible, 4 considered susceptibility depending dose
Table 2. Comparison of mycological eradication and clinical cure after treatment with itraconazole (400 mg) or fluconazole (150 mg) against Candida species Treatment
Negative culture and clinical curea
Itraconazole 23 n = 42 Fluconazole 27 n = 38
Negative culture and clinical failureb
Positive culture and clinical cure
Positive culture and clinical failure
04
04
11
03
00
08
There was no significant difference between the two regimens – (X2 p < 0.05). a Resolution of clinical signs and symptoms and mycological eradication. b Persistence of clinical signs and symptoms and mycological persistence. Table 3. In vitro and in vivo correlation for itraconazole (n = 42) and fluconazole (n = 35) in vaginal candidiasis obtained by mycological data. Antifungals
S Curea Failureb
Itraconazole 23 (n = 42) Fluconazole 29 (n = 35)
SDD Cure Failure
R Cure Failure
13
04
00
00
02
03
01
00
00
02
S = susceptible; S-DD = susceptibility depending dose; R = resistant. a Negative culture post therapy (return). b Positive culture post therapy (return).
and 2 resistant. Thirteen isolates considered S and all the resistant isolates had positive culture post therapy. The success rates were 63.9% for S, 100.0% for SDD and 0.0% for R. For fluconazole the breakpoints of Candida isolates showed that there were 32 isolates considered susceptible, 1 considered susceptibility depending dose and 2 resistant. Three isolates considered S and 2 resistant isolates had positive culture post therapy. The success rates were 90.6% for S, 100.0% for SDD and 0.0% for R. For 3 C. krusei isolates the fluconazole breakpoints weren’t applicable because this specie is intrinsically resistant to this drug (Table 3).
Discussion This study compares the effectiveness of two treatment regimens: Itraconazole, a single day oral dose with 400mg and Fluconazole, a single oral dose with
46 150mg for vulvovaginal candidiasis. The medications were well tolerated and there was a reduction of clinical signs and symptoms of vaginitis in the end of 14 days of treatment (return). Each regimen proved to be equally effective on the treatment of vaginal candidiasis. There wasn’t a significant difference between different therapies. In previous studies, Mikamo et al [15] have find similar results. These authors showed that the treatment with a daily 200mg oral dose of itraconazole for 3 days and a single 150mg oral dose of fluconazole proved be effective on the treatment of vaginal candidiasis, too. Our study suggests that the treatment of vaginal candidiasis with oral itraconazole or oral fluconazole would be effective and might represent one choice in the treatment of vaginal candidiasis. Antifungal susceptibility tests may be an important aid in the decisions of starting antifungal treatment and in monitoring the outcome. In this study, the evaluation of the in vitro activity of fluconazole and itraconazole against Candida species showed that both drugs are highly active. C. krusei isolates were resistant to fluconazole, because this specie is intrinsically resistant to this drug [12]. To predict the clinical outcome of antifungal treatment by determination of MICs against the infecting yeasts has been troublesome so far. As indicated in Table 3, some patients (16/77) treated with itraconazole and fluconazole for which the MICs breakpoints were considered susceptible, were associated with mycological failure. Explain to this point is very difficult. These results could be explained, because even though MIC can provide an estimate of drug activity, the MIC is only a part of the picture and many other factors can intervene to produce failure. Relevant factors as inherent variability of all in vivo systems (e.g., parameters immune such as leukocyte number) and poor absorption of the drug can potentially have more influence on clinical outcome than intrinsic drug susceptibility [11, 12, 16, 17]. Treatment with fluconazole or other azole antifungal drugs has become an important clinical problem in the management of vaginal candididiasis in these patients. Azole antifungal agents prevent the synthesis of ergosterol, a major component of fungal plasma membrane by inhibiting ergosterol biosynthesis, whereas, caspofungin belong to a class of antifungals known as the echinochandins exert antifungal activity via inhibition of 1,3-β-D glucan biosynthesis differing from the mechanism of action of azole and antagonism might not be expected. The use of combination antifungal therapy
may be considered if the combination results to increase the efficacy. The combination therapy of azole and caspofungin seems to be highly active against azole-susceptible and resistant isolates. In conclusion, it may constitute an effective therapeutic option for the treatment of Candida infections [18, 19]. However, all isolates for which the MICs breakpoints were considered resistant prior treatment showed mycological failure after treatment. These results reveal clinical correlation of MICs, when the susceptibility test showed resistance. Considering our results we would like to point out that the correlation of the MIC values of these drugs with clinical outcome is not a totally clarified issue and requires complex evaluation of several factors.
Acknowledgments This work was supported by Janssen-Cilag Pharmaceuticals and Pfizer Pharmaceuticals.
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