Fluconazole and itraconazole susceptibility of clinical isolates of Cryptococcus neoformans at a tertiary care centre in India: a need for care

K. Datta1,2,*, N. Jain1, S. Sethi1, A. Rattan2, A. Casadevall3 and U. Banerjee1

1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110029; 2 Microbiology NDDR, Ranbaxy Research Laboratories, Gurgaon 122001, Haryana, India; 3 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Received 3 October 2002; returned 9 February 2003; revised 30 June 2003; accepted 2 July 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: In cryptococcosis, fluconazole is a standard prophylactic, therapeutic and maintenance option, particularly in the expanding HIV/AIDS group. However, its excessive use may lead to resistance in Cryptococcus neoformans. Variations in clinical response to fluconazole have already been noted elsewhere, and cases of post-therapy relapse are not uncommon. To assess azole antifungal susceptibility profiles of clinical cryptococcal isolates in India, the All India Institute of Medical Sciences (AIIMS) has recently initiated preliminary studies using NCCLS M27-A.

Materials and methods: Twenty-eight randomly chosen AIIMS clinical isolates (spanning 1997–2000), 16 isolates from other institutions in North India, and six reference strains of C. neoformans were subjected to susceptibility testing to fluconazole and itraconazole.

Results: Among clinical isolates, susceptibilities to fluconazole and itraconazole were 84.1% and 93.2%, respectively. MICs for all clinical isolates were 0.25–32 mg/L for fluconazole and <0.03–0.25 mg/L for itraconazole. MIC50 and MIC90 values for fluconazole were 4 and 16 mg/L, respectively, and those for itraconazole were 0.032 and 0.125 mg/L, respectively. Out of 28 AIIMS clinical isolates, 22 had minimum fungicidal concentrations (MFCs) of fluconazole at ³128 mg/L. Moderately high fluconazole MICs (16–32 mg/L) were observed in 16% of clinical isolates—probably the first such report from India. MIC/MFC ratios for fluconazole and itraconazole were 1:32 or more in 16 AIIMS clinical isolates, indicating possible azole tolerance. There was good agreement between MIC values obtained by the micro- and macro-broth dilution techniques of M27-A compared in this study.

Conclusions: The observed MIC data warrant continued surveillance of susceptibility values of clinical cryptococcal isolates in India.

Keywords: cryptococcosis, MICs, azole antifungals, clinical isolates, India


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cryptococcus neoformans is an encapsulated yeast and opportunistic pathogen.1 In different studies worldwide, cryptococcosis has been seen to occur in approximately 3–30% of patients immunocompromised as a result of HIV/AIDS, haematological or metabolic diseases leading to immune-impairment, and therapeutic immune-suppression.1 In some regions, including India, a considerable proportion of people with no apparent immune impairment have also been afflicted with this disease.2 It is almost uniformly fatal if not diagnosed and treated early, and even with early institution of therapy, certain patient populations may not respond.1

Until now, the major antifungals available for therapy of cryptococcosis are limited to amphotericin B, 5-fluorocytosine and fluconazole. However, the side effects associated with administration of amphotericin B (infusion-related events and hepatotoxicity) and fluorocytosine (myelodepression) may restrict their use. Azoles, on the other hand, offer a safer and efficacious option. There is a great deal of global experience with fluconazole in the treatment of cryptococcosis;3 clinical data on the use of the newer azoles (itraconazole and voriconazole) in cryptococcosis are somewhat more scarce.1,4 One major concern with azole use has been the possible emergence of clinical resistance,5 as has been seen in other yeasts, notably Candida.6 Clinical resistance may be the result of infection with a resistant strain, or a relapse of infection after antifungal-mediated in vivo selection of a more resistant phenotype or strain, particularly in the immunocompromised patients.6,7 In the pre- and early AIDS era, despite widespread use of various azoles, the apparent incidence of direct azole resistance was low. However, as the AIDS pandemic has continued, with prolonged and profound immunosuppression combined with the frequent use of azoles for prophylaxis and/or for therapy of oropharyngeal thrush in these high-risk patients, there have been increasing case reports of clinical relapse isolates of C. neoformans with lower susceptibilities to fluconazole than those in the original isolates.1,5

Current practices of anti-cryptococcal therapy in India generally include amphotericin B, alone or with flucytosine (5-fluorocytosine), and sometimes followed by fluconazole. Administration of this therapeutic regimen has usually, but not always, resulted in survival of the patient.2 The All India Institute of Medical Sciences (AIIMS), being a referral tertiary care centre, encounters a large number of patients with varying degrees of immunosuppression, including HIV/AIDS.2,8 Worldwide, standard therapeutic practice in the HIV/AIDS group includes prophylactic or suppressive therapy with fluconazole.4,9 In India, use of fluconazole prophylaxis is not very common, and there has been no report yet on cryptococcosis being clinically unresponsive to fluconazole therapy. However, this agent is easily available in the market, and there is no regulation moderating its usage in various institutions. Therefore, as already seen in candidiasis,10,11 the concerns about emergence of clinical resistance to antifungals also hold true for cryptococcosis. In this study, an attempt was made to look retrospectively at the trend of in vitro azole susceptibility with randomly chosen clinical isolates from AIIMS and other centres of North India, using the standardized technique M27-A of the National Committee for Clinical Laboratory Standards (NCCLS, Wayne, PA, USA).12 This study was a part of a larger study being carried out at AIIMS and the Albert Einstein College of Medicine to characterize cryptococcal isolates from Indian patients.

In this study, we have also compared the results of fluconazole susceptibility of some isolates by both the macro-broth dilution procedure and its micro-broth dilution modification as mentioned in the M27-A document. In order to establish intra- and inter-laboratory reproducibility of results, the tests have been independently carried out in two different laboratories (AIIMS and Ranbaxy) by three investigators (K.D., N.J. and S.S.).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cryptococcal strains and isolates

This study utilized cryptococcal strains (reference ATCC strains and clinical isolates) maintained in the Laboratories for Clinical Mycology and Investigation of Infections in Immunocompromised Patients, Department of Microbiology, AIIMS. Clinical isolates were obtained from patient samples routinely sent to the laboratory for isolation and identification; 28 (of a total of 44) clinical isolates for this study were randomly selected from ones dated between 1997 and 2000 (a subset—approximately 45%—of all cryptococcal isolates obtained at AIIMS during this period). The rest of the cryptococcal isolates, as well as the Quality Control (QC) isolates for susceptibility testing (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258), were obtained from the mycopathogen culture collection of the Ranbaxy Research Laboratories, Gurgaon, Haryana.

Azole antifungal agents

Fluconazole was obtained from Ranbaxy Research Laboratories, and itraconazole, from Lee Pharma, Hyderabad, Andhra Pradesh, India. Powdered forms of the antifungals were dissolved in appropriate solvents—distilled water for fluconazole; di-methyl sulphoxide (Sigma) for itraconazole—to make stock solutions following the recommendations of the M27-A protocol. For use, these agents were diluted so as to have final concentrations of 64 and 16 mg/L, respectively in RPMI 1640 (Life Technologies); a range of dilutions was obtained by two-fold serial dilutions.12

In vitro antifungal susceptibility testing

In vitro susceptibility of yeast isolates to fluconazole and itraconazole was tested using the micro-broth dilution and macro-broth dilution technique as described in the M27-A protocol with RPMI 1640 buffered to pH 7 using MOPS (Sigma).12 The MICs were estimated as defined in M27-A, as the lowest concentration that substantially inhibits the growth of the organism.12 As a quality control measure, the results were considered valid only when the MIC values of the QC isolates fell within the pre-specified ranges: C. parapsilosis ATCC 22019 (fluconazole, 2–8 mg/L; itraconazole, 0.06–0.25 mg/L) and C. krusei ATCC 6258 (fluconazole, 16–64 mg/L; itraconazole, 0.12–0.5 mg/L).

Determination of minimum fungicidal concentration (MFC)

The MFC is defined as the minimum concentration at which the growth of the yeast is completely inhibited. This was determined by spotting on Sabouraud’s Dextrose Agar (HiMedia, Mumbai, India) plates 0.02 mL of the yeast suspension from each well or tube having a concentration equal to and upwards of the MIC. The plates were incubated for 48–72 h and the appearance of growth was noted.

Analysis of susceptibility results

The MIC values obtained in the micro-broth dilution procedure were analysed by the WHONET 5.0 software (WHO, Geneva, Switzerland); this software is designed by and freely available from the World Health Organization as a part of its antimicrobial resistance monitoring programme. It analyses laboratory susceptibility testing data and generates values of clinically significant parameters reflecting the susceptibility pattern, namely: percent susceptible; percent resistant; MIC50 (the median MIC values); and MIC90 (the concentration maximum within which 90% of tested isolates have their MICs). The susceptibility and resistance breakpoints for a given antifungal in WHONET 5.0 have been defined, as per M27-A, as MIC values of isolates from patients who have responded and not responded, respectively, to a therapeutic regimen of that antifungal. For fluconazole, the respective breakpoints are 8 and 64 mg/L, and for itraconazole, 0.125 and 1 mg/L. The isolates with values in between these breakpoints are considered to be of intermediate susceptibility.12 The variations in MIC values obtained using the micro- and macro-broth dilution techniques were analysed using a General Linear Model Repeated Measures test with Repeated Contrasts in SPSS 7.5 for Windows (SPSS Inc., Chicago, IL, USA).

Ethical considerations

The study presented herein involves routine laboratory clinical isolates and is not directly related to investigations in humans or animals, and contains no easily identifiable patient information. The ethical standards of the respective institutions as directed by the institutional review committees have been adhered to.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Susceptibility to fluconazole and itraconazole

The results of the susceptibility testing of clinical isolates by the micro-broth dilution method have been summarized in Table 1. In this study, none of the isolates tested showed true resistance to the azole antifungals tested. However, the clinical isolates were not seen to be universally susceptible (according to NCCLS breakpoints) to fluconazole and itraconazole (respectively, 84.1% and 93.2% susceptible); intermediate susceptibility to fluconazole was observed in 16% of clinical isolates. To our knowledge, this is the first report from India on higher fluconazole MIC values in clinical cryptococcal isolates. Breakpoint MIC correlations with clinical outcome have been established for candida infections,12 and a high MIC usually gives an indication for increasing strength and/or dosing of the drug for optimum effect. MIC50s of fluconazole and itraconazole (4 and 0.032 mg/L, respectively) were within limits of susceptibility, but the MIC90 value of 16 mg/L of fluconazole indicates the trend that at least 10% of the isolates may tend towards higher MIC values (>=16 mg/L) of this agent.


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Table 1. MIC and MFC value ranges (mg/L) for 44 clinical isolates for fluconazole and itraconazole
 
Minimum fungicidal concentrations

The observations of MFCs and MIC/MFC ratios have been summarized in Table 1. The in vitro MFC is not a therapeutically significant parameter, but it may be an important indicator of altered drug action on the organism. Out of 44 isolates in this study, 28 (63.6%, comprising 22 AIIMS clinical isolates, and six from other institutions) isolates had fluconazole MFCs at >=128 mg/L; itraconazole MFCs spanned a wide range (0.03–8 mg/L). Though fluconazole has mostly fungistatic action in vivo, even at therapeutic doses, the fact that the MFCs of fluconazole in 16 AIIMS isolates (36.4%) were seen to have values ³32 times the MIC (Table 1), even in susceptible isolates, seems to indicate the possible occurrence of an azole tolerance phenomenon, a harbinger of future resistance. In itraconazole also, the MIC/MFC ratio was seen to be ³1:32 in 19 out of 44 isolates, (43.2%, comprising 16 AIIMS isolates and three from other institutions) (Table 1), and 12 of these were isolates which also had a high fluconazole MIC/MFC ratio (data not shown). The MIC/MFC ratio may not be significant in situations where both MIC and MFC values are low (as with itraconazole); yet, even in the absence of in vitro resistance to either azole agent, this observation needs to be considered, since azole cross-resistance is known to occur in many Candida clinical isolates,11 and also in C. neoformans. It would be worthwhile to investigate the clinical outcome in patients whose isolates have a high MIC/MFC ratio, in order to assess the clinical correlation of this parameter.

Significance of observations in azole susceptibility

The present retrospective study was a part of an effort at AIIMS to attempt to monitor the trend of antifungal susceptibilities in clinical cryptococcal isolates and to provide essential basal-level susceptibility values of primary isolates. To our knowledge, it was the first systematic attempt of this kind in Indian institutions. Testing of antifungal susceptibility is not yet a routine procedure in Indian institutions because of various considerations. However, the benefits of susceptibility testing cannot be overlooked in the light of three facts: (a) escalation of yeast, including cryptococcal, infections in the ever-widening group of immunosuppressed patients;9 (b) inability of current antifungal treatments to clear cryptococcal infection in certain patient populations, particularly those with HIV/AIDS, and incidences of post-therapy relapse;1 (c) observed correlation between enhanced in vitro MIC and clinical failure of an antifungal agent in yeast infections such as candidiasis and cryptococcosis.13 Antifungal susceptibility testing may, therefore, serve two important purposes—monitoring of the pattern of susceptibility of clinical isolates to existing antifungals and generation of a database, which may be of critical importance in prevention of any sudden emergence of drug-resistant pathogens, and also in studies of the cause–effect relationship of such patterns.

Worldwide, fluconazole is used as a standard prophylaxis and therapy of cryptococcosis, and also to prevent relapse in immunosuppressed patients.1,4,9 In a developing country like India, the prophylactic use of fluconazole has not been very frequent even in HIV-infected or neutropenic patients mainly because of issues related to cost and patient compliance. Itraconazole, though effective in vitro (as seen from its low MICs and MFCs), has restricted clinical efficacy owing to high protein binding (99.8%), low oral bio-availability and erratic pharmacokinetics.14 This, as well as its high cost, precludes its routine use in cryptococcosis in India. Because of these reasons, azole resistance has not yet been envisaged to be a problem. But, even in such a situation, higher azole MIC values and high MIC/MFC ratios were observed. We do not yet know the clinical significance of these observations. However, these observations considered together underscore the need for initiating a programme of routine susceptibility testing in cryptococcosis for surveillance purposes. Monitoring susceptibility patterns at referral centres having specific expertise would be extremely useful and may even guide treatment decisions, as happens in bacterial infections. For instance, strains with fluconazole MICs of 16–32 mg/L or more may not respond consistently to this drug treatment alone with standard dosing schedules.

Comparison of macro- and micro-broth procedures (susceptibility data not shown)

Another objective for this study was to assess the relative merits of the micro and macro methods in the setting of Indian institutions (though in the global literature the micro/macro comparisons are well established), in order to determine the feasibility of adoption of a particular method for our laboratories. In our hands there was good agreement between the two methods, with the difference in results for each isolate not being significant at 95% confidence interval (P = 0.899). The micro method is more attractive because of its obvious ease of use, but it has been our experience that the use of 96-well microtitre plates sometimes proves too costly for peripheral health care centres, and the visual scoring system of the NCCLS method too confusing for the relatively less trained personnel working there. The macro method is less costly, but its performance may prove too cumbersome. These are a few of the reasons why most Indian institutions have not taken up the responsibility of carrying out antifungal susceptibility testing, though often it has been felt very necessary.

Suitability of the testing methodology in the Indian setting

The M27-A reference standard method was developed through a consensus process to generate a greater degree of intra- and inter-laboratory agreement of results, ensured by adherence to strict quality control measures, such as use of QC isolates. It also offers the flexibility of choice of a method, or of development of a new method, so long as the results for the QC isolates are concordant with those by the reference method. Therefore, factors such as ease of performance, economy, or rapid results influence the adoption of a particular method at a given laboratory. However, compliance to the QC guidelines, required to maximize reproducibility of results, demands experience, training and resources. Therefore, at present in India, antifungal susceptibility testing should be carried out in reference laboratories only.

The currently established guidelines have noted reservations about testing of cryptococcal isolates, since the medium (RPMI 1640) and the testing conditions (temperature, time) have been optimized for Candida species and may not be optimal for C. neoformans;12 for example, the growth of C. neoformans is slower in RPMI 1640. It would be beneficial to identify cryptococcal strains that can serve as the QC isolates, thereby obviating the need for the present Candida QCs.

Conclusion

This study was a pilot study that yielded some interesting results on azole susceptibility patterns, and it merits a more concerted prospective follow-up with inclusion of a larger number of samples from different institutions of the country, and in-depth analysis of trends in different patient populations.


    Acknowledgements
 
We acknowledge the help received from Dr Sandip Sinharay of Educational Testing Services, Princeton, NJ, for the statistical analysis and are grateful to Drs Tania Sorell and Tania Pfeiffer of the Women’s and Children’s Hospital, Adelaide, Australia, for the gift of the reference strains of C. neoformans, ATCC 34871, 34878, 34880, 28957 and 90112, and to Dr Arunaloke Chakraborty of the Post Graduate Institute of Medical Education and Research, Chandigarh, for the gift of ATCC 90126, and a clinical isolate (MCCL 250012). Part of the work presented herein is part of the PhD thesis work of K.D. at AIIMS, and the research fellowship from CSIR for K.D. towards the initial part of this study is gratefully acknowledged. Part of this work was presented as a poster at the 5th International Conference on Cryptococcus and Cryptococcosis in Adelaide, March 2002. Part of this study was supported by DBT grant no. BT/PR1690/MED/09/260/99; U.B. and A.C. acknowledge support from the AIDS International Training and Research Program (NIH D43-TW01403) of the Albert Einstein College of Medicine (Program Director: Vinayaka Prasad).


    Footnotes
 
* Corresponding author. Tel: +1-718-430-2372; Fax: +1-718-430-8968; E-mail: kdatta@aecom.yu.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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14 . United States Food and Drug Administration. (2002). Hepatotoxicity labelling for Sporanox (itraconazole) Oral Solution. [Online.] http://www.fda.gov/cder/foi/label/2002/20657s8lbl.pdf (29 June 2003, date last accessed).