Recurrent Oropharyngeal Candidiasis Research Articles

<i>CDR1, CDR2, MDR1</i> and <i>ERG11</i> expression in azole resistant <i>Сandida albicans</i> isolated from HIV-infected patients in city of Moscow

Candida fungi are common opportunistic microorganisms capable of causing infections of various localization, as well as life-threatening conditions in immunocompromised patients, such as HIV-infected individuals, oncology patients, subjects undergoing HSCT, which number has been steadily increasing in recent years. In addition, resistance to anti-fungal drugs has been spreading as well. Naturally sensitive to azoles, C. albicans possess a variety of mechanisms of acquired resistance, including efflux transporters and target protein-encoding gene amplification. This study was conducted to assess a prevalence of such mechanisms in the isolates sample obtained from HIV-infected patients in the Moscow region of the Russian Federation, characterize a relationship between these mechanisms and patterns of developing drug resistance. 18 strains of C. albicans resistant to fluconazole and voriconazole were isolated from HIV-infected patients with recurrent oropharyngeal candidiasis in the Moscow region. The expression levels of the ERG11, MDR1, CDR1, CDR2 genes involved in the formation of acquired azole resistance were measured using quantitative PCR, the 2CT method with ACT and PMA genes as control genes and reference values of sensitive isolates. Expression levels exceeding the average values of sensitive isolates by more than 3 standard deviations were considered significantly elevated. In most of the isolates, elevated levels of CDR1 and CDR2 gene expression were found: 89% and 78%, respectively. The expression level of the MDR1 gene was increased only in 28% of cases. ERG11 expression levels were significantly elevated in 78% of the isolates. Expression levels of all resistance genes studied were significantly increased in 4 strains. In this sample of C. albicans isolates, acquired resistance is mainly associated with efflux vectors encoded by the CDR1 and CDR2 genes. Also, in most isolates, an increased expression level for the azole target protein gene ERG11 was detected. The expression level of the efflux transporter gene MDR1 was increased in the smallest number of samples. It is also impossible to exclude a potential role of other mechanisms in developing acquired resistance, such as mutations in the ERG11 gene. It can be assumed that the identified mechanisms of resistance result from long-term, widespread, and sometimes uncontrolled use of azoles, including those in treatment and prevention of candidiasis in HIV-infected patients.

Candida isolates causing refractory or recurrent oropharyngeal candidiasis in 11 hospitals in China.

Introduction: We studied the species distribution and antifungal susceptibilities of Candida isolates causing refractory or recurrent oropharyngeal candidiasis (OPC) in a multicenter study in China (2013–2016).Methods: Species identification was performed using the Bruker Biotyper (Bruker Daltonics, Germany) matrix-assisted laser desorption/ionization time of flight mass spectrometry system supplemented by internal transcribed spacer sequencing as required. Antifungal susceptibilities were determined by the Clinical and Laboratory Standards Institute document (CLSI) M27-A3 broth microdilution methodology.Results: A total of 558 non-duplicate Candida isolates comprising 10 species were obtained from 535 patients. Candida albicans was the most common species (89.6%), followed by C. glabrata (5.2%), C. tropicalis (2.9%), and C. parapsilosis (0.7%). Azoles were active against C. albicans with susceptibility rates of 96% and 95.8% for fluconazole and voriconazole, respectively. MIC50 values of C. albicans to fluconazole, voriconazole, itraconazole, and miconazole were 1, 0.03, 0.25 and 0.12 μg/mL, respectively, higher than those in previous studies of which OPC patients (corresponding MIC50 values of 0.25 , 0.015 , 0.06 , and 0.03 μg/mL). Except for itraconazole, the MIC50 and MIC90 values of 58 non-C. albicans to other azoles were two to threefold higher than C. albicans. Miconazole, amphotericin B, nystatin, and 5-flucytosine had good in vitro antifungal activity for all isolates.Conclusion: The study provides valuable data on the species distribution and antifungal susceptibility of oropharyngeal Candida isolates from geographically diverse areas of China. C. albicans remains the most common species but with increasing rates of azoles resistance.

Species distribution and in vitro antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis

BackgroundIn Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis.MethodsA total of 296 clinical oral yeasts were isolated from 292 HIV-infected patients with oropharyngeal candidiasis at the Muhimbili National Hospital, Dar es Salaam, Tanzania. Identification of the yeasts was performed using standard phenotypic methods. Antifungal susceptibility to fluconazole, itraconazole, miconazole, clotrimazole, amphotericin B and nystatin was assessed using a broth microdilution format according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI; M27-A2).ResultsCandida albicans was the most frequently isolated species from 250 (84.5%) patients followed by C. glabrata from 20 (6.8%) patients, and C. krusei from 10 (3.4%) patients. There was no observed significant difference in species distribution between patients with primary and recurrent oropharyngeal candidiasis, but isolates cultured from patients previously treated were significantly less susceptible to the azole compounds compared to those cultured from antifungal naïve patients.ConclusionC. albicans was the most frequently isolated species from patients with oropharyngeal candidiasis. Oral yeast isolates from Tanzania had high level susceptibility to the antifungal agents tested. Recurrent oropharyngeal candidiasis and previous antifungal therapy significantly correlated with reduced susceptibility to azoles antifungal agents.

Good evidence to support efficacy of antifungals for Candida treatment in HIV infection

Data sources Medline and EMbase and the Cochrane Library to April 2000.Study selection Articles were only included if they were written in English and the study was performed in humans. A scoring system for quality was used and evidence was graded as good, fair, poor or insufficient. Studies that had no control or comparison group were excluded, as were studies of non-HIV/AIDS populations. Studies reporting mixed-site candidiasis were also excluded if oropharyngeal candidiasis was not reported separately.Data extraction and synthesis Study design, sampling, study group characteristics, interventions and reported outcomes were extracted.Results For prevention, six trials met inclusion criteria but there was insufficient evidence to draw conclusions about nystatin, clotrimazole, amphotericin B suspension, ketaconazole or itraconazole. One study demonstrated that nystatin pastilles (at 200 000 and 400 000 IU/day) was effective in the prevention of new or recurrent oropharyngeal candidiasis. There is good evidence, however, that fluconazole is also effective (doses were 50–1000 and 150–200 mg/day). Nausea is a common side effect of fluconazole, but this was reported to be tolerable.For treatment, 12 trials were included. None addressed the efficacy of amphotericin B suspension. Evidence for the effectiveness of the other antifungal agents was good: fluconazole, ketaconazole and itraconazole showed similar efficacy and were more effective than clotrimoxazole or nystatin, particularly when mycological and relapse rates were considered. Mild adverse events, mainly gastrointestinal problems, rarely resulted in discontinuation of treatment.Conclusions There is evidence that antifungal drugs are effective in the prevention and treatment of oropharyngeal candidiasis in HIV-infected people. The need remains, however, to improve the evidence base. Larger, better-defined studies are required to address issues such as immunological status, viral load, history of previous candidiasis, past exposure to antifungals, drug interactions and antiretroviral therapy. Studies should also include compliance-monitoring, fungal speciation and susceptibility testing.

Azole resistance in Candida spp

The emergence of azole-resistant Candida spp. is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis in HIV-infected patients. Several mechanisms can cause this resistance. An important mechanism of azole resistance is reduced intracellular accumulation of the drug. Among the multidrug efflux transporters, ABC transporters and the major facilitator superfamily are reported to cause the resistance. Erg11p, sterol C14 alfa-demethylase, is a target of azole derivatives. It was reported that ERG11 over-expression had only a modest effect on the development of azole resistance. However, mutations in the ERG11 gene can cause the resistance, probably by reducing binding of azole to the target enzyme. We sequenced the ERG11 gene in a high-level azole resistant C. albicans strain, Darlington, and found that two amino acid substitutions, Y132H and I471T, had been encoded in the Darlington ERG11 gene. To assess the significance of these substitutions, we replaced one of the two copies of ERG11 gene in anazole-susceptible strain of C. albicans with a copy of the Darlington ERG11 and this resulted in a modest increase in azole resistance. Furthermore, to estimate the effect of Y132H and I471T individually, ERG11 genes with either or both mutations were expressed in S. cerevisiae. The I471T substitution, not previously described, conferred azole resistance when overexpressed alone and increased this resistance when added to the Y132H substitution. Alterations in the sterol biosynthetic pathway are another resistance mechanism. Inhibition of 14 alfa-demethylase by azole results not only in ergosterol depletion but also in accumulation of methylated sterol 14 alfa-methylergosta-8, 22(28)-dien-3 beta, 6 alfa-diol. We deleted the ERG3 gene, which encodes a sterol 5, 6-desaturase, in C. albicans, and the deletion resulted in reduced susceptibility of the mutant to azoles.Sterol analysis revealed that erg3 mutant lost both ergosterol and diol when cultured with fluconazole.

Low levels of antigenic variability in fluconazole-susceptible and -resistant Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.

Three serial isolates of Candida albicans were obtained by direct swab or by oral saline rinses from each of five human immunodeficiency virus-infected patients with recurrent oropharyngeal candidiasis. Genotyping techniques confirmed the presence of a persistent strain in multiple episodes from the same patient, which was different from the strains isolated from other patients. Fluconazole susceptibility was determined by both an agar dilution method and the National Committee for Clinical Laboratory Standards macrobroth procedure. In four of these patients the strains developed fluconazole resistance, and in one patient the strain remained susceptible. The different isolates were propagated as yeast cells on a synthetic medium, and their cell wall proteinaceous components were extracted by treatment with beta-mercaptoethanol. Protein and mannoprotein components present in the extracts were analyzed by electrophoresis, immunoblotting, and lectin-blotting techniques. The analysis showed a similar composition, with only minor qualitative and quantitative differences in the polypeptidic and antigenic patterns associated with the cell wall extracts from serial isolates from the same patient, as well as those from different strains isolated from different patients. Use of monospecific antibodies generated against two immunodominant antigens during candidiasis (enolase and the 58-kDa fibrinogen-binding mannoprotein) demonstrated their expression in all isolates tested. Overall, the antigenic makeup of C. albicans strains remained constant during the course of infection and was not affected by development of fluconazole resistance. In contrast to previous reports, the low degree of antigenic variability observed in this study may be due to the fact that the isolates were obtained from a highly homogeneous population of patients and to the uniformity in techniques used for the isolation, storage, and culture of the different strains, as well as extraction methodologies.

Nosocomial fungal infections: candidemia

Candida species are frequently encountered as part of the human commensal flora. Colonization mostly precedes candidemia and is an independent risk factor for the development of candidemia. Genotyping methods showed the similarity between colonizing and infecting strains, thus making endogenous origin likely, though exogenous sources like total parenteral nutrition also have been described. Health care workers (HCWs) play an important role in the transmission of yeasts. Candida species are frequently isolated from the hands of HCWs and can be transmitted from hands to patients. Granulocytopenia and damage of the mucosal lining resulting from intensive chemotherapy due to cancer, the increasing use of broad spectrum antibiotics, and the use of intravenous catheters are other important risk factors for the development of candidemia. Candidemia is associated with a high mortality and prolonged hospitalization. Therefore, and because of the high frequency of dissemination, all candidemias should be treated. Amphotericin B was considered the standard drug for the systemic treatment of candidemia. Fluconazole has been shown to be an effective and safe alternative in non-neutropenic patients. 5-Fluorocytosine has been used in combination with amphotericin B in the treatment of deep-seated infections. Liposomal formulations of amphotericin B and other new antifungal drugs currently are under investigation. C. albicans is the most frequently isolated Candida species, although the proportion of infections caused by non- C. albicans species is increasing. Also, there are reports of development of resistance to amphotericin B. C. lusitaniae is known for primary resistance and the development of resistance to amphotericin B. Development of resistance to fluconazole is mainly seen in AIDS patients with recurrent oropharyngeal candidiasis who receive longer courses of therapy.

Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR.

Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908-910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1, YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDR genes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps.

Clinical evaluation and microbiology of oropharyngeal infection due to fluconazole-resistant Candida in human immunodeficiency virus-infected patients.

Signs and symptoms of oropharyngeal candidiasis (OPC) were correlated with microbiology and clinical response to fluconazole in a cohort of patients with advanced human immunodeficiency virus (HIV) infection and recurrent OPC. Sixty-four HIV-infected patients with a median CD4 cell count of < 50/mm3 (range, 3-318/mm3) who presented with OPC were enrolled in a longitudinal study. Specimens for cultures were taken weekly until clinical resolution. Therapy with fluconazole was increased weekly as required to a maximum daily dose of 800 mg until resolution of symptoms and oral lesions. Resistant or dose-dependent susceptible yeasts, defined as a minimum inhibitory concentration of > or = 16 micrograms/mL, were detected in 48 (31%) of 155 episodes. Clinical resolution with fluconazole therapy occurred in 107 (100%) of 107 episodes with susceptible yeasts vs. 44 (92%) of 48 episodes with resistant or dose-dependent susceptible strains (P = .008). Patients from whom fluconazole-resistant yeasts were isolated required longer courses of therapy and higher doses of fluconazole for response, but overall, excellent responses to fluconazole were seen in patients with advanced HIV infection.

Detection and Significance of Fluconazole Resistance in Oropharyngeal Candidiasis in Human Immunodeficiency Virus-Infected Patients

The epidemiology and clinical significance of fluconazole resistance were assessed in a cohort of advanced human immunodeficiency virus (HIV)-infected patients with recurrent oropharyngeal candidiasis. Fifty patients were prospectively evaluated using a novel method of detecting fluconazole resistance with chromogenic media containing fluconazole; results were confirmed with macrobroth testing. Resistant yeasts, defined as MICs > or = 8 micrograms/mL, were detected in 16 (32%) of 50 patients: 7 (14%) had resistant Candida albicans, 7 (14%) had resistant non-C. albicans yeast, and 2 (4%) had mixed resistant yeasts. MICs were > or = 32 in 11 of 16 isolates. Previous fluconazole use and severe immunosuppression were risk factors for resistance. However, 5 of 26 patients had resistant isolates with no prior fluconazole use, and all were severely immunosuppressed. Despite the high prevalence of resistance, 48 patients clinically responded to fluconazole. Fluconazole-resistant C. albicans and non-C. albicans yeast infections are common in patients with advanced immunodeficiency, but clinical efficacy of fluconazole remains high.

Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting.

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.

Fluconazole- and itraconazole-resistant Candida albicans strains from AIDS patients: multilocus enzyme electrophoresis analysis and antifungal susceptibilities

Multilocus enzyme electrophoresis and in vitro susceptibility testing with a broth microdilution method were used to analyze Candida albicans strain diversity in four AIDS patients with recurrent oropharyngeal candidiasis who successively developed clinical resistance to fluconazole (FCZ) and itraconazole (ITZ). One to ten colonies per sample were randomly chosen from oral washings collected before the initial FCZ treatment and just before every other antifungal treatment; a total of 98 isolates were analyzed. Multilocus enzyme electrophoresis analysis revealed 14 different electrophoretic types (ETs). Statistical analysis of genetic distances showed that C. albicans isolates clustered into five subpopulations (I to V). In each subpopulation, isolates are closely related, and genetic distances between subpopulations I to IV are short. In contrast, subpopulation V, which contained isolates typed as ET8 and ET14, is strongly divergent from the others; these isolates may represent atypical C. albicans isolates. Only one patient was infected with a single strain during the course of azole therapy; for the three remaining patients, variants of the same strain and different strains were concurrently isolated. Clinical FCZ resistance was clearly correlated with in vitro data for three patients. Moreover, MICs of ITZ increased during FCZ therapy, and MICs of ITZ which were > or = 1.56 micrograms/ml were found when clinical ITZ resistance occurred; isolates from subpopulation V showed the highest MICs of ITZ. Because of the emergence of clinical ITZ resistance after clinical FCZ resistance, the feasibility of long-term azole therapy for mucosal candidiasis in AIDS patients is questioned.

Fluconazole once a week as secondary prophylaxis against oropharyngeal candidiasis in HIV‐infected patients: A double‐blind placebo‐controlled study

To assess the efficacy and tolerance of fluconazole (150 mg oral dose, once a week) in the prevention of recurrent oropharyngeal candidiasis in patients with moderate to severe human immunodeficiency virus (HIV) infection. A randomised, double-blind, placebo-controlled trial. Eighty-four patients with moderate to severe HIV infection who had successfully completed two to four weeks treatment with fluconazole for oropharyngeal candidiasis were randomly allocated to receive either placebo or fluconazole. Pre-treatment clinical and laboratory characteristics were similar in the two groups. Success was classified as absence throughout the course of treatment of clinical evidence of oropharyngeal candidiasis, and failure as recurrence or relapse of symptomatic oropharyngeal candidiasis. Of 73 evaluable patients the median time to relapse was > or = 168 days in the fluconazole group and 37 days in the placebo group (P < 0.0001). One patient in the placebo group and 18 patients in the fluconazole group completed six months' treatment without clinical relapse (P < 0.001). Fluconazole was well tolerated and prevented clinical relapse of oropharyngeal candidiasis in 71% of patients who completed three months of treatment (95% confidence interval [CI], 55-86) and 58% (95% CI, 41-75) who completed six months of treatment.

Thrush Can Be Prevented in Patients With Acquired Immunodeficiency Syndrome and the Acquired Immunodeficiency Syndrome—Related Complex

Recurrent oropharyngeal candidiasis is common in patients with acquired immunodeficiency syndrome and the acquired immunodeficiency syndrome-related complex. It causes local pain and discomfort, loss of taste, and aversion to food and may lead to secondary complications. We examined, in a double-blind study, whether recurrent thrush could be prevented by prophylaxis. Twenty-five patients with one to four previous thrush episodes who had no thrush at the outset of the study were randomized to receive 100 mg of fluconazole or placebo daily for 12 weeks. If thrush occurred, prophylaxis was stopped and patients were treated conventionally, after which prophylaxis was resumed. After the randomized study, some patients were given continuous fluconazole (open phase). In the randomized study, thrush occurred in eight of 13 placebo-treated patients and none of 12 fluconazole-treated patients. Possible side effects were not different between the groups. Dermatophytosis and onychomycosis and cryptococcuria also improved in the fluconazole-treated patients, and fungal colonization was significantly decreased. One episode of thrush occurred in the open phase in an intermittently compliant patient (group total, 71.5 patient-months of fluconazole treatment); in contrast, the 25 patients also had had two episodes of Candida esophagitis, three of cryptococcosis, and 13 of dermatophytosis before entry. Subsequent to entry in the randomized trial, in 92.3 patient-months without fluconazole, there were 35 episodes of thrush, one of esophagitis, one of cryptococcemia, and one of dermatophytosis, and preexisting dermatophytosis and onychomycosis were unchanged or worsened. Individual patients observed with and without fluconazole treatment also showed its efficacy. In conclusion, thrush can be prevented in patients with acquired immunodeficiency syndrome and the acquired immunodeficiency syndrome-related complex with negligible toxic effects. Larger trials to confirm prevention of all mycoses with prophylaxis should be considered.