Trends in Antifungal Drug Susceptibility of Cryptococcus neoformans Obtained 1 Through ...

34 Pages·2011·0.35 MB·English

AAC Accepts, published online ahead of print on 28 March 2011
Antimicrob. Agents Chemother. doi:10.1128/AAC.00048-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

1 Trends in Antifungal Drug Susceptibility of Cryptococcus neoformans Obtained
2 Through Population-based Surveillance, South Africa, 2002-2003 and 2007-2008
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4 Nelesh P. Govendera,b*, Jaymati Patela, Marelize van Wyka, Tom M. Chillerc and
5 Shawn R. Lockhartc for the Group for Enteric, Respiratory and Meningeal disease
6 Surveillance in South Africa (GERMS-SA)
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8 aMycology Reference Unit, National Institute for Communicable Diseases, a Division lo
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9 of the National Health Laboratory Service, Johannesburg, South Africa ed

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10 bFaculty of Health Sciences, University of the Witwatersrand, Johannesburg, South om

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11 Africa tt
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12 cMycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA /a
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14 *Corresponding author: Dr Nelesh P. Govender, Mycology Reference Unit, National .o
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15 Institute for Communicable Diseases, Private Bag X4, Sandringham, 2131, South o
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16 Africa. p
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18 Phone: [phone]. Fax: [phone]. E-mail: [email protected] 1
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20 Running Title: C. neoformans Susceptibility Trends, South Africa e
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22 Word Count (body): 3528
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24 Keywords: Population-Based; Surveillance; South Africa; Cryptococcus;
25 Cryptococcus neoformans; Cryptococcosis; HIV; AIDS; Cryptococcal Meningitis;
C. neoformans Susceptibility Trends, South Africa
1 Susceptibility; Resistance; Serial; Incident; Fluconazole; Voriconazole; Amphotericin
2 B; Itraconazole; Flucytosine; Posaconazole; Clinical and Laboratory Standards
3 Institute; M27-A3; Broth Microdilution, E-test
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Page 2 of 34
C. neoformans Susceptibility Trends, South Africa
1 Abstract
2 Word count: 240
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4 Cryptococcus neoformans is the most common cause of meningitis amongst adult
5 South Africans with HIV/AIDS. Widespread use of fluconazole for treatment of
6 cryptococcal meningitis and other HIV-associated opportunistic fungal infections in
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7 South Africa may lead to the emergence of isolates with reduced fluconazole o
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8 susceptibility. Minimum inhibitory concentration (MIC) testing using a reference lo
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9 broth microdilution method was used to determine if isolates with reduced ed

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10 susceptibility to fluconazole or amphotericin B had emerged amongst cases of om

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11 incident disease. Incident isolates were tested from two surveillance periods (2002-3 tt
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12 and 2007-8) when population-based surveillance was conducted in Gauteng Province, /a
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13 South Africa. These isolates were also tested for susceptibility to flucytosine, a
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14 itraconazole, voriconazole and posaconazole. Serially-collected isolate pairs from .o
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15 cases at several large South African hospitals were also tested for susceptibility to o
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16 fluconazole. Of the 487 incident isolates tested, only 3 (0.6%) demonstrated a p
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17 fluconazole MIC of ≥ 16 µg/ml; all of these isolates were from 2002-3. All incident 3
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18 isolates were inhibited by very low concentrations of amphotericin B, and exhibited 1
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19 very low MICs to voriconazole and posaconazole. Of 67 cases with serially-collected y

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20 isolate pairs, only 1 case was detected where the isolate collected more than 30 days e
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21 later had a fluconazole MIC value significantly higher than the MIC of the
22 corresponding incident isolate. Although routine antifungal susceptibility testing of
23 incident isolates is not currently recommended in clinical settings, it is still clearly
24 important for public health to periodically monitor for the emergence of resistance.
Page 3 of 34
C. neoformans Susceptibility Trends, South Africa
1 Introduction
2
3 Cryptococcus neoformans is the most common cause of meningitis amongst
4 adult South Africans with HIV/AIDS (23). In South Africa, cryptococcal meningitis is
5 often diagnosed amongst HIV-infected patients with advanced immunosuppression
6 and poor prognostic factors such as a high fungal burden (22, 31) and is associated
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7 with high mortality (22). Although the fungicidal combination of amphotericin B and o
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8 flucytosine is recommended for induction treatment (33), flucytosine is not available lo
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9 in countries with a high incidence of cryptococcosis (8), and the use of amphotericin ed

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10 B deoxycholate is limited by toxicity and the need for clinical and laboratory om

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11 monitoring (8). Since 2000, fluconazole has been widely available in South Africa tt
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12 through the Diflucan Partnership Program for treatment of cryptococcal meningitis /a
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13 and oesophageal candidiasis (41). Due to ease of administration and low toxicity, in a
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14 sub-Saharan Africa fluconazole is often first-line treatment for cryptococcal .o
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15 meningitis, despite its fungistatic activity. o
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17 With widespread use of low-dose fluconazole (≤ 200 mg daily) for 3
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18 prophylaxis and treatment of candidiasis and other opportunistic fungal infections 1
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19 which may precede cryptococcal meningitis, it is possible that cryptococcal lineages y

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20 with reduced susceptibility could arise by selective pressure and expand to cause e
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21 incident cryptococcosis amongst persons with HIV/AIDS. However, isolates with
22 reduced fluconazole susceptibility may be more likely to emerge in circumstances
23 where patients have been treated with suboptimal induction-phase regimens
24 (including fluconazole monotherapy ≤ 400 mg daily) and where long-term, low-dose
25 fluconazole (200 mg daily) is prescribed for suppression of disease (10). While we do
Page 4 of 34
C. neoformans Susceptibility Trends, South Africa
1 not yet know enough about the relationship between elevated fluconazole MIC values
2 and patient outcome to warrant routine testing, susceptibility testing of surveillance
3 isolates can give us reliable data for trend analysis.
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5 Long-term prophylaxis, and in some cases induction therapy, of cryptococcal
6 meningitis in South Africa is still largely dependent upon fluconazole. In 2000, South
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7 African Department of Health guidelines recommended a relatively low fluconazole o
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8 dose (400 mg daily), as an alternative to amphotericin B induction-phase treatment lo
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9 (39). Development of resistance to fluconazole would be devastating to the treatment ed

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10 of this disease, and so it is important for public health agencies to monitor for changes om

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11 in susceptibility to this drug. In this study, two methods were used to monitor for tt
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12 changes in fluconazole susceptibility over time. In the first, incident cryptococcal /a
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13 isolates obtained through population-based surveillance from two time intervals a
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14 (2002-3 and 2007-8) in Gauteng Province, South Africa were tested to determine if .o
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15 median MIC values to fluconazole and amphotericin B were elevated or had changed o
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16 over time. In addition, the susceptibility to flucytosine, itraconazole, voriconazole and p
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17 posaconazole was assessed. In the second, serially-collected isolate pairs from cases at 3
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18 several large sentinel hospitals within and outside Gauteng Province were tested for 1
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C. neoformans Susceptibility Trends, South Africa
1 Methods
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3 Population-based surveillance for cryptococcosis
4 Cases of laboratory-confirmed cryptococcosis were reported to the Mycology
5 Reference Unit, National Institute for Communicable Diseases (NICD) in
6 Johannesburg from 1 March 2002 through 28 February 2008. Active population-based
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7 surveillance was restricted to Gauteng Province from March 2002 through February o
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8 2004 (31); from January 2005, surveillance was expanded nationally (20). A case of lo
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9 incident cryptococcosis was defined as the first episode of laboratory-confirmed ed

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10 disease in a patient (encapsulated yeasts observed by microscopic examination of an om

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11 India ink-stained fluid, or a positive cryptococcal antigen test or culture of tt
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12 Cryptococcus species from any body site) diagnosed at a South African clinical /a
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13 laboratory. a
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15 For culture-confirmed cases, cryptococcal isolates were transported to the o
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16 NICD and stored in brain-heart infusion broth with 10% glycerol at -70ºC. At p
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17 enhanced surveillance hospitals, nurse surveillance officers collected detailed case 3
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18 information, including HIV infection status, in-hospital antifungal treatment, and in- 1
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19 hospital outcome (survival or death); surveillance was enhanced at four Gauteng y

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20 hospitals from 2002 through 2008 and at an additional 14 hospitals across South e
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21 Africa in 2005. Isolates were collected with minimal case demographic data at non-
22 enhanced surveillance hospitals. Ethics clearance for surveillance was obtained from
23 the Human Research Ethics Committee (Medical), University of the Witwatersrand,
24 Johannesburg and from other university and provincial ethics committees.
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C. neoformans Susceptibility Trends, South Africa
1 Selection of isolates for antifungal susceptibility testing
2 Incident cases were included if the person had been diagnosed with a first
3 episode of laboratory-confirmed cryptococcosis: (1) at one of four enhanced
4 surveillance hospitals in Gauteng Province, (2) from 1 March 2002 through 28
5 February 2003 (2002-3) or from 1 March 2007 through 28 February 2008 (2007-8),
6 and (3) where the incident isolate was stored by the NICD. We selected cases from
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7 these sites because continuous surveillance had been performed for 6 years. A sub-set o
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8 of incident cases from each surveillance period was selected using a random-number lo
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9 generator. Incident cases were excluded if the isolate was non-viable, contaminated or ed

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10 misplaced after storage, identified as Cryptococcus gattii or another cryptococcal om

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11 species, or if the case patient was known to be HIV-uninfected or had been treated tt
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12 with antifungal drugs which suggested a prior episode of cryptococcosis. /a
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14 Cases with serially-collected isolates more than 30 days apart were selected if .o
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15 (1) the case was diagnosed between 1 January and 31 December 2005 at 18 enhanced o
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16 surveillance hospitals across South Africa, and (2) serially-collected isolate pairs had p
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17 been stored at NICD. We selected cases from 2005 because most patients were treated 3
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18 with low-dose fluconazole induction treatment (≤ 400 mg daily) during this period. 1
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19 Cases were excluded if the isolate was non-viable, contaminated or misplaced after y

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20 storage, or identified as C. gattii or another cryptococcal species. e
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22 Antifungal susceptibility testing
23 Isolates were tested by reference laboratories at the NICD and the Centers for
24 Disease Control and Prevention (CDC). Isolates were sub-cultured at least twice on
25 Sabouraud dextrose agar (Diagnostic Media Products - National Health Laboratory
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C. neoformans Susceptibility Trends, South Africa
1 Service (DMP), Johannesburg, South Africa) after long-term storage to ensure
2 optimal growth and purity. Isolates were confirmed as C. neoformans using standard
3 phenotypic tests, including development of brown-pigmented colonies on Staib’s
4 Niger-seed agar (DMP) and a positive test for urease on urea-containing media
5 (DMP) (32). C. neoformans was distinguished from C. gattii using canavanine glycine
6 bromothymol-blue agar (DMP).
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7 The MIC for six antifungal drugs (amphotericin B, fluconazole, flucytosine, o
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8 voriconazole, posaconazole and itraconazole) was determined for incident isolates. lo
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9 Fluconazole MICs were determined as outlined by the Clinical Laboratory Standards ed

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10 Institute M27-A3 (13) using microbroth dilution panels prepared at the NICD. om

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11 Fluconazole, flucytosine, voriconazole, posaconazole and itraconazole MICs were tt
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12 determined at the CDC for a sub-set of incident isolates using custom microbroth /a
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13 dilution panels prepared as outlined in M27-A3 by TREK Diagnostic Systems, Inc. a
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14 (Cleveland, Ohio, USA) (13). All microbroth dilution panels were inoculated with .o
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15 RPMI 1640 medium (with glutamine and phenol red but without bicarbonate) (13). A o
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16 sub-set of isolates were tested at both laboratories and the results were found to be in p
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17 essential agreement. Serially-collected isolates were tested at the NICD using 3
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18 fluconazole with in-house panels (13). MIC values were determined visually 1
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19 following 72 h of incubation. The quality control isolates Candida parapsilosis ATCC y

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20 22019 and Candida krusei ATCC 6258 were run on all days of testing. The MICs for e
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21 amphotericin B were determined by the Etest (bioMérieux S.A., Marcy ľEtoile,
22 France) on RPMI 1640 plates containing 2% glucose, as recommended by the
23 manufacturer. The Etest has been determined to be more discriminatory than the CLSI
24 method for distinguishing isolates thought to be amphotericin B susceptible vs. non-
25 susceptible based on clinical data (28). Geometric mean MIC values were calculated
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C. neoformans Susceptibility Trends, South Africa
1 for incident isolates for each surveillance period and compared using Student’s t-test.
2 For serially-collected isolates, essential agreement was defined as MIC values within
3 two dilutions of each other. Interpretive breakpoints were not assigned because there
4 are no accepted breakpoints for Cryptococcus with any antifungal drug (12).
5
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C. neoformans Susceptibility Trends, South Africa
1 Results
2
3 Incident cases of cryptococcosis
4 Case selection and demographic characteristics
5 From 1 March 2002 through 28 February 2008, 8439 cases of incident
6 cryptococcosis were detected through population-based surveillance in Gauteng
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7 Province. The inclusion criteria for antifungal susceptibility testing were met by 1033 o
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8 cases of incident disease: 462 in 2002-3 and 571 in 2007-8. Of these cases, 391 and lo
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9 280 from each period, respectively, were randomly selected. A total of 238 cases ed

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10 from 2002-3 and 249 from 2007-8 had viable isolates available for testing. Apart from om

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11 more female patients in the selected group in 2007-8, there were no significant tt
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12 differences in the baseline demographic characteristics of cases with and without /a
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13 viable isolates for each surveillance period (data not shown). Table 1 shows a a
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14 comparison of baseline characteristics of cases with viable isolates from 2002-3 vs. .o
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15 2007-8. Patients were significantly more likely to be treated with amphotericin B than o
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16 fluconazole or no drug in 2007-8 compared to 2002-3 (Table 1). The case-fatality p
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17 ratio was also significantly higher in 2007-8 compared to the earlier period (Table 1). 3
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18 1
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19 Antifungal susceptibility results y

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20 Amphotericin B, flucytosine, itraconazole, voriconazole and posaconazole e
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21 MICs were determined for 237 incident isolates; fluconazole MICs were determined
22 for 487 incident isolates (Table 2 and Table 3). None of these isolates demonstrated
23 fluconazole MICs of ≥ 32 µg/ml. Only 3 of these isolates (0.6%) had elevated
24 fluconazole MIC values (MIC = 16 µg/ml); all of these isolates were from the earlier
25 surveillance period (2002-3) (Table 3). Three additional isolates from the earlier
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