Trends in Antifungal Drug Susceptibility of Cryptococcus ...aac.asm.org/content/early/2011/03/28/AAC.00048-11.full.pdf · Trends in Antifungal Drug Susceptibility of ... C. neoformans

Trends in Antifungal Drug Susceptibility of Cryptococcus neoformans Obtained 1

Through Population-based Surveillance, South Africa, 2002-2003 and 2007-2008 2

3

Nelesh P. Govendera,b*

, Jaymati Patela, Marelize van Wyk

a, Tom M. Chiller

c and 4

Shawn R. Lockhartc for the Group for Enteric, Respiratory and Meningeal disease 5

Surveillance in South Africa (GERMS-SA) 6

7

aMycology Reference Unit, National Institute for Communicable Diseases, a Division 8

of the National Health Laboratory Service, Johannesburg, South Africa 9

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

Africa 11

cMycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA 12

13

*Corresponding author: Dr Nelesh P. Govender, Mycology Reference Unit, National 14

Institute for Communicable Diseases, Private Bag X4, Sandringham, 2131, South 15

Africa. 16

17

Phone: +27 11 555 0353. Fax: +27 11 555 0435. E-mail: [email protected] 18

19

Running Title: C. neoformans Susceptibility Trends, South Africa 20

21

Word Count (body): 3528 22

23

Keywords: Population-Based; Surveillance; South Africa; Cryptococcus; 24

Cryptococcus neoformans; Cryptococcosis; HIV; AIDS; Cryptococcal Meningitis; 25

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

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C. neoformans Susceptibility Trends, South Africa

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Susceptibility; Resistance; Serial; Incident; Fluconazole; Voriconazole; Amphotericin 1

B; Itraconazole; Flucytosine; Posaconazole; Clinical and Laboratory Standards 2

Institute; M27-A3; Broth Microdilution, E-test 3


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Abstract 1

Word count: 240 2

3

Cryptococcus neoformans is the most common cause of meningitis amongst adult 4

South Africans with HIV/AIDS. Widespread use of fluconazole for treatment of 5

cryptococcal meningitis and other HIV-associated opportunistic fungal infections in 6

South Africa may lead to the emergence of isolates with reduced fluconazole 7

susceptibility. Minimum inhibitory concentration (MIC) testing using a reference 8

broth microdilution method was used to determine if isolates with reduced 9

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

incident disease. Incident isolates were tested from two surveillance periods (2002-3 11

and 2007-8) when population-based surveillance was conducted in Gauteng Province, 12

South Africa. These isolates were also tested for susceptibility to flucytosine, 13

itraconazole, voriconazole and posaconazole. Serially-collected isolate pairs from 14

cases at several large South African hospitals were also tested for susceptibility to 15

fluconazole. Of the 487 incident isolates tested, only 3 (0.6%) demonstrated a 16

fluconazole MIC of ≥ 16 µg/ml; all of these isolates were from 2002-3. All incident 17

isolates were inhibited by very low concentrations of amphotericin B, and exhibited 18

very low MICs to voriconazole and posaconazole. Of 67 cases with serially-collected 19

isolate pairs, only 1 case was detected where the isolate collected more than 30 days 20

later had a fluconazole MIC value significantly higher than the MIC of the 21

corresponding incident isolate. Although routine antifungal susceptibility testing of 22

incident isolates is not currently recommended in clinical settings, it is still clearly 23

important for public health to periodically monitor for the emergence of resistance. 24


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Introduction 1

2

Cryptococcus neoformans is the most common cause of meningitis amongst 3

adult South Africans with HIV/AIDS (23). In South Africa, cryptococcal meningitis is 4

often diagnosed amongst HIV-infected patients with advanced immunosuppression 5

and poor prognostic factors such as a high fungal burden (22, 31) and is associated 6

with high mortality (22). Although the fungicidal combination of amphotericin B and 7

flucytosine is recommended for induction treatment (33), flucytosine is not available 8

in countries with a high incidence of cryptococcosis (8), and the use of amphotericin 9

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

monitoring (8). Since 2000, fluconazole has been widely available in South Africa 11

through the Diflucan Partnership Program for treatment of cryptococcal meningitis 12

and oesophageal candidiasis (41). Due to ease of administration and low toxicity, in 13

sub-Saharan Africa fluconazole is often first-line treatment for cryptococcal 14

meningitis, despite its fungistatic activity. 15

16

With widespread use of low-dose fluconazole (≤ 200 mg daily) for 17

prophylaxis and treatment of candidiasis and other opportunistic fungal infections 18

which may precede cryptococcal meningitis, it is possible that cryptococcal lineages 19

with reduced susceptibility could arise by selective pressure and expand to cause 20

incident cryptococcosis amongst persons with HIV/AIDS. However, isolates with 21

reduced fluconazole susceptibility may be more likely to emerge in circumstances 22

where patients have been treated with suboptimal induction-phase regimens 23

(including fluconazole monotherapy ≤ 400 mg daily) and where long-term, low-dose 24

fluconazole (200 mg daily) is prescribed for suppression of disease (10). While we do 25


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not yet know enough about the relationship between elevated fluconazole MIC values 1

and patient outcome to warrant routine testing, susceptibility testing of surveillance 2

isolates can give us reliable data for trend analysis. 3

4

Long-term prophylaxis, and in some cases induction therapy, of cryptococcal 5

meningitis in South Africa is still largely dependent upon fluconazole. In 2000, South 6

African Department of Health guidelines recommended a relatively low fluconazole 7

dose (400 mg daily), as an alternative to amphotericin B induction-phase treatment 8

(39). Development of resistance to fluconazole would be devastating to the treatment 9

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

in susceptibility to this drug. In this study, two methods were used to monitor for 11

changes in fluconazole susceptibility over time. In the first, incident cryptococcal 12

isolates obtained through population-based surveillance from two time intervals 13

(2002-3 and 2007-8) in Gauteng Province, South Africa were tested to determine if 14

median MIC values to fluconazole and amphotericin B were elevated or had changed 15

over time. In addition, the susceptibility to flucytosine, itraconazole, voriconazole and 16

posaconazole was assessed. In the second, serially-collected isolate pairs from cases at 17

several large sentinel hospitals within and outside Gauteng Province were tested for 18

susceptibility to fluconazole. 19


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Methods 1

2

Population-based surveillance for cryptococcosis 3

Cases of laboratory-confirmed cryptococcosis were reported to the Mycology 4

Reference Unit, National Institute for Communicable Diseases (NICD) in 5

Johannesburg from 1 March 2002 through 28 February 2008. Active population-based 6

surveillance was restricted to Gauteng Province from March 2002 through February 7

2004 (31); from January 2005, surveillance was expanded nationally (20). A case of 8

incident cryptococcosis was defined as the first episode of laboratory-confirmed 9

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

India ink-stained fluid, or a positive cryptococcal antigen test or culture of 11

Cryptococcus species from any body site) diagnosed at a South African clinical 12

laboratory. 13

14

For culture-confirmed cases, cryptococcal isolates were transported to the 15

NICD and stored in brain-heart infusion broth with 10% glycerol at -70ºC. At 16

enhanced surveillance hospitals, nurse surveillance officers collected detailed case 17

information, including HIV infection status, in-hospital antifungal treatment, and in-18

hospital outcome (survival or death); surveillance was enhanced at four Gauteng 19

hospitals from 2002 through 2008 and at an additional 14 hospitals across South 20

Africa in 2005. Isolates were collected with minimal case demographic data at non-21

enhanced surveillance hospitals. Ethics clearance for surveillance was obtained from 22

the Human Research Ethics Committee (Medical), University of the Witwatersrand, 23

Johannesburg and from other university and provincial ethics committees. 24

25


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Selection of isolates for antifungal susceptibility testing 1

Incident cases were included if the person had been diagnosed with a first 2

episode of laboratory-confirmed cryptococcosis: (1) at one of four enhanced 3

surveillance hospitals in Gauteng Province, (2) from 1 March 2002 through 28 4

February 2003 (2002-3) or from 1 March 2007 through 28 February 2008 (2007-8), 5

and (3) where the incident isolate was stored by the NICD. We selected cases from 6

these sites because continuous surveillance had been performed for 6 years. A sub-set 7

of incident cases from each surveillance period was selected using a random-number 8

generator. Incident cases were excluded if the isolate was non-viable, contaminated or 9

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

species, or if the case patient was known to be HIV-uninfected or had been treated 11

with antifungal drugs which suggested a prior episode of cryptococcosis. 12

13

Cases with serially-collected isolates more than 30 days apart were selected if 14

(1) the case was diagnosed between 1 January and 31 December 2005 at 18 enhanced 15

surveillance hospitals across South Africa, and (2) serially-collected isolate pairs had 16

been stored at NICD. We selected cases from 2005 because most patients were treated 17

with low-dose fluconazole induction treatment (≤ 400 mg daily) during this period. 18

Cases were excluded if the isolate was non-viable, contaminated or misplaced after 19

storage, or identified as C. gattii or another cryptococcal species. 20

21

Antifungal susceptibility testing 22

Isolates were tested by reference laboratories at the NICD and the Centers for 23

Disease Control and Prevention (CDC). Isolates were sub-cultured at least twice on 24

Sabouraud dextrose agar (Diagnostic Media Products - National Health Laboratory 25


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Service (DMP), Johannesburg, South Africa) after long-term storage to ensure 1

optimal growth and purity. Isolates were confirmed as C. neoformans using standard 2

phenotypic tests, including development of brown-pigmented colonies on Staib’s 3

Niger-seed agar (DMP) and a positive test for urease on urea-containing media 4

(DMP) (32). C. neoformans was distinguished from C. gattii using canavanine glycine 5

bromothymol-blue agar (DMP). 6

The MIC for six antifungal drugs (amphotericin B, fluconazole, flucytosine, 7

voriconazole, posaconazole and itraconazole) was determined for incident isolates. 8

Fluconazole MICs were determined as outlined by the Clinical Laboratory Standards 9

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

Fluconazole, flucytosine, voriconazole, posaconazole and itraconazole MICs were 11

determined at the CDC for a sub-set of incident isolates using custom microbroth 12

dilution panels prepared as outlined in M27-A3 by TREK Diagnostic Systems, Inc. 13

(Cleveland, Ohio, USA) (13). All microbroth dilution panels were inoculated with 14

RPMI 1640 medium (with glutamine and phenol red but without bicarbonate) (13). A 15

sub-set of isolates were tested at both laboratories and the results were found to be in 16

essential agreement. Serially-collected isolates were tested at the NICD using 17

fluconazole with in-house panels (13). MIC values were determined visually 18

following 72 h of incubation. The quality control isolates Candida parapsilosis ATCC 19

22019 and Candida krusei ATCC 6258 were run on all days of testing. The MICs for 20

amphotericin B were determined by the Etest (bioMérieux S.A., Marcy ľEtoile, 21

France) on RPMI 1640 plates containing 2% glucose, as recommended by the 22

manufacturer. The Etest has been determined to be more discriminatory than the CLSI 23

method for distinguishing isolates thought to be amphotericin B susceptible vs. non-24

susceptible based on clinical data (28). Geometric mean MIC values were calculated 25


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for incident isolates for each surveillance period and compared using Student’s t-test. 1

For serially-collected isolates, essential agreement was defined as MIC values within 2

two dilutions of each other. Interpretive breakpoints were not assigned because there 3

are no accepted breakpoints for Cryptococcus with any antifungal drug (12). 4

5


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Results 1

2

Incident cases of cryptococcosis 3

Case selection and demographic characteristics 4

From 1 March 2002 through 28 February 2008, 8439 cases of incident 5

cryptococcosis were detected through population-based surveillance in Gauteng 6

Province. The inclusion criteria for antifungal susceptibility testing were met by 1033 7

cases of incident disease: 462 in 2002-3 and 571 in 2007-8. Of these cases, 391 and 8

280 from each period, respectively, were randomly selected. A total of 238 cases 9

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

more female patients in the selected group in 2007-8, there were no significant 11

differences in the baseline demographic characteristics of cases with and without 12

viable isolates for each surveillance period (data not shown). Table 1 shows a 13

comparison of baseline characteristics of cases with viable isolates from 2002-3 vs. 14

2007-8. Patients were significantly more likely to be treated with amphotericin B than 15

fluconazole or no drug in 2007-8 compared to 2002-3 (Table 1). The case-fatality 16

ratio was also significantly higher in 2007-8 compared to the earlier period (Table 1). 17

18

Antifungal susceptibility results 19

Amphotericin B, flucytosine, itraconazole, voriconazole and posaconazole 20

MICs were determined for 237 incident isolates; fluconazole MICs were determined 21

for 487 incident isolates (Table 2 and Table 3). None of these isolates demonstrated 22

fluconazole MICs of ≥ 32 µg/ml. Only 3 of these isolates (0.6%) had elevated 23

fluconazole MIC values (MIC = 16 µg/ml); all of these isolates were from the earlier 24

surveillance period (2002-3) (Table 3). Three additional isolates from the earlier 25


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surveillance period had elevated itraconazole MIC values (MIC ≥ 1 µg/ml). As 1

expected, all incident isolates were inhibited by low concentrations of amphotericin B 2

(MIC90 = 0.19 µg/ml). Similarly, the MICs for voriconazole and posaconazole were 3

low; for all tested isolates, the MIC was ≤ 0.25 µg/ml and ≤ 0.5 µg/ml for 4

voriconazole and posaconazole respectively. Despite no flucytosine use in South 5

Africa during the surveillance period, 17 of 237 (7%) isolates had MIC values of 8 6

µg/ml or 16 µg/ml. There was no difference in MIC50 and MIC90 between the two 7

surveillance periods for any of the antifungal drugs tested (Table 2). There was no 8

association between incident fluconazole MIC and in-hospital outcome (data not 9

shown). The geometric mean fluconazole MIC value for isolates collected from 10

2002-3 was 2.3 µg/ml while the geometric mean MIC value for the isolates collected 11

from 2007-8 was 2.1 µg/ml (not statistically significant; p= 0.1). 12

13

Cases with serially-collected isolates 14

Case selection and demographic characteristics 15

From 1 January through 31 December 2005, 1538 cases of incident 16

cryptococcosis were detected at 18 enhanced surveillance hospitals in seven South 17

African provinces. The criteria for susceptibility testing were met by 67 cases 18

diagnosed at 11 enhanced surveillance hospitals in six provinces. The mean age of 19

case patients was 33 years (SD ±9.2 years) and 42 (63%) were female. Seventy four 20

percent (40/54) of patients received fluconazole monotherapy; 38 patients (95%) were 21

treated with fluconazole ≤ 400 mg per day in hospital for a median of 8 d (range, 1 to 22

32 d). Only 4 (6%) patients were receiving combination antiretroviral treatment 23

(cART) at the time of incident diagnosis; a further 6 (8%) patients were known to 24


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have initiated cART post-diagnosis. The median time between the serially-collected 1

isolates was 70 d (range, 32 to 238 d). 2

3

Antifungal susceptibility results 4

Amongst the cases with serially-collected isolates, the fluconazole MIC50 and 5

MIC90 for the incident isolates was 2 µg/ml and 16 µg/ml respectively, and for the 6

isolates collected >30 d later was 2 µg/ml and 8 µg/ml respectively. The fluconazole 7

MIC remained the same for 34 (51%) isolate pairs, increased by 1 log2 dilution for 11 8

(16%) isolate pairs, and increased by 2 log2 dilutions for 7 (10%) isolate pairs (Table 9

4). For 1 case, the MIC increased significantly from 2 µg/ml to 32 µg/ml. This patient 10

had been treated with low-dose fluconazole monotherapy at diagnosis of incident 11

cryptococcosis; the second serial isolate was collected 5 months later. No clinical data 12

were available for the second episode. The fluconazole MIC decreased for 14 (21%) 13

pairs (Table 4). The incident isolates from ten cases displayed elevated MIC values 14

(16 µg/ml to 64 µg/ml), with six of these producing MIC values of 64 µg/ml. Isolates 15

collected more than 30 d after the incident isolate displayed elevated MIC values 16

amongst 12 cases, with only two isolates having MIC values of 64 µg/ml. Of note, 17

amongst four cases where there was not essential agreement between the serially-18

collected isolates, the MIC dropped significantly; from 64 µg/ml to 1 µg/ml (1 case), 19

from 64 µg/ml to 2 µg/ml (2 cases) and from 32 µg/ml to 0.5 µg/ml (1 case). 20


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Discussion 1

Incident cryptococcal isolates obtained through population-based surveillance 2

in South Africa maintained low MIC values to the first-line antifungal drugs 3

fluconazole and amphotericin B over a six-year period, and very low MICs were 4

determined for newer drugs such as voriconazole and posaconazole. Despite 5

infrequent use of these agents in South Africa, a small number of isolates were 6

determined to have elevated MIC values to itraconazole and/or flucytosine. Non-7

susceptibility to fluconazole, as defined by a four-fold increase in MIC (10, 33), was 8

detected in only 1 case with serially-collected isolates. 9

10

Historically, the determination of the MIC in the laboratory has been the 11

method of choice for monitoring antifungal resistance. Although a standardised 12

antifungal drug susceptibility testing method has been developed (13), antifungal 13

resistance in Cryptococcus is difficult to define in the laboratory due to the absence of 14

interpretive breakpoints. Attempts to correlate MIC with clinical outcome have 15

produced mixed results. For example, in a small case series, an incident isolate with a 16

fluconazole MIC of ≥ 16 µg/ml was associated with subsequent clinical failure among 17

5 of 25 (20%) patients (2); however, this finding was not replicated in other studies 18

(15, 25). Nevertheless, if performed consistently over time, MIC testing can indicate 19

shifts in susceptibility amongst isolates at a population-level. 20

21

The distribution of 200 mg fluconazole tablets through the Diflucan 22

Partnership Program in South Africa has doubled from approximately 1.8M doses in 23

2002 to 3.8M doses in 2008 (personal communication, Pfizer, South Africa). Despite 24

this, the finding that almost all incident cryptococcal isolates had a fluconazole MIC 25


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of ≤ 16 µg/ml was not surprising. A large global study found that the fluconazole 1

MIC of incident isolates did not change substantially over a 15-year period (1990-2

2004) despite increased use of fluconazole (35). In addition, Brandt et al. tested 143 3

incident C. neoformans (serotype A) isolates from the same population-based 4

surveillance system in Gauteng Province (2002-3) with the reference broth 5

microdilution method; the MIC range, MIC50 and MIC90 for fluconazole was 0.5 6

µg/ml to 8 µg/ml, 2 µg/ml and 4 µg/ml, which is almost identical to the results that we 7

obtained (9). In contrast, a report from Cambodia described incident isolates with 8

increased fluconazole MICs over a 2-year period when tested with the E-test method 9

(38). The findings from the latter study are difficult to explain. Fluconazole-resistant 10

Candida species have been shown to emerge in areas where primary fluconazole 11

prophylaxis is used as a preventative strategy (3), but Candida is a coloniser that can 12

replicate as a commensal in the host and can inhabit many different body sites to 13

avoid maximum exposure to antifungal drugs. Dormant cryptococcal strains, which 14

are hypothesised to have established a latent infection many years previously, 15

reactivate primarily in the milieu of advanced HIV-associated T-cell 16

immunodeficiency and cause disseminated disease (18). Even in the setting of 17

primary fluconazole prophylaxis, incident C. neoformans isolates with reduced 18

susceptibility to fluconazole have been infrequently documented (4, 29); this may be 19

directly related to the fact that they do not actively replicate in the host as commensal 20

organisms prior to onset of disease. 21

22

Voriconazole and posaconazole have consistently been shown to have good 23

activity against C. neoformans (1, 9, 11, 36). However, the use of voriconazole and 24

posaconazole is still restricted to salvage settings (33), as no clinical trials have been 25


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undertaken to compare these agents to first-line drugs and these agents remain 1

prohibitively expensive for use in resource-limited settings. In our study, the three 2

incident isolates with reduced fluconazole susceptibility (MIC of 16 µg/ml) had 3

relatively low MICs to voriconazole. However, fungistatic azole drugs would still not 4

be the first choice for treatment of incident cryptococcosis. 5

6

Even in the absence of interpretive breakpoints, MIC testing may be more 7

helpful to document the emergence of resistance over time amongst patients with 8

serially-collected isolates if the same test method is used and isolate pairs are tested in 9

parallel. In our surveillance, we found that, in most cases, serially-collected isolates 10

displayed fluconazole MIC results within two dilutions, indicating essential 11

agreement. Similarly, a Ugandan study which compared fluconazole broth 12

microdilution MICs of serially-collected isolates from 17 patients found no evidence 13

of a stepwise increase in MIC over a 2 to 10-week period (37). In contrast, a 14

prospective, observational study from Cape Town, South Africa (2003-5) found that 15

16 of 20 (80%) patients with culture-confirmed relapse disease had isolates with 16

reduced fluconazole susceptibility as determined by the E-test method (6). Bicanic et 17

al. suggested that the high prevalence of fluconazole non-susceptibility amongst Cape 18

Town isolates was associated with low-dose fluconazole induction treatment (400 mg 19

daily) and concurrent rifampicin use (6). A Cambodian study also reported that the 20

MIC to fluconazole as determined by E-test increased significantly from year 2000 to 21

2002 (38). In contrast, we found only one case where the serially-collected isolate had 22

an MIC value significantly elevated above the MIC of the incident isolate, despite 23

most patients receiving low-dose fluconazole induction treatment (400 mg daily). 24

Similarly, Brandt et al., who also determined the fluconazole MIC for serially-25


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collected isolates more than 30 d apart from Gauteng surveillance (2002-3), detected 1

an increase in fluconazole MIC values of at least 3 log2 dilutions over time for only 2 2

of 30 cases (9). 3

4

Differences in MIC testing methods may have contributed to some of these 5

reported differences. Several reports indicate that Cryptococcus MIC values to the 6

azoles that have been generated by E-test are higher than those generated by broth 7

microdilution testing when performed in parallel (15, 17, 30, 34, 40). While we used 8

a broth dilution method for MIC determination, Bicanic et al. used the E-test method, 9

where endpoint determination for azoles is technically more difficult to establish and 10

may be more subjective (6). There is still some question as to whether the results of 11

various testing methodologies can be directly compared. In addition, Bicanic et al. 12

defined resistance to fluconazole as a single (relapse-episode) isolate with an MIC of 13

≥ 16 µg/ml; isolate pairs were not tested in parallel (6). 14

15

We believe that there are other, more common reasons for recurrent disease 16

such as non-adherence to suppressive fluconazole treatment (14), development of the 17

immune reconstitution inflammatory syndrome (IRIS) following initiation of cART 18

(7, 26) or suboptimal induction-phase treatment. Recently, Jarvis et al. described 19

patients with symptomatic relapse disease at the same Cape Town hospital in 2007-8 20

when amphotericin B induction-phase treatment and antiretroviral treatment were 21

standard of care (24). Of the 69 relapse episodes that were detected over this 2-year 22

period, most were due to IRIS (45%) or non-adherence or non-prescription of 23

fluconazole maintenance treatment (43%) (24). In contrast to the earlier Cape Town 24


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study, very few isolates with elevated fluconazole MICs (MIC ≥ 16 µg/ml determined 1

by the E-test) were detected from this group of patients. 2

3

In our study, there were some cases where the MIC value of the second isolate 4

dropped compared to the incident isolate when both were tested in parallel using the 5

same broth microdilution method, a phenomenon that has been documented 6

previously (10). Although we do not currently understand this mechanism, recent 7

work by Desnos-Ollivier and colleagues indicates that at least 20% of C. neoformans 8

infections may be comprised of multiple strains and genotypes (16). By testing only 9

selected subpopulations cultured from the original clinical specimen, other strains 10

contributing to disease in a given patient may be missed (16). 11

12

Major challenges to improve management of patients with cryptococcosis 13

include (i) prevention of cryptococcosis by early diagnosis of HIV infection and 14

timely initiation of cART well before the CD4+ T-cell count falls below 200 cells/ml, 15

(ii) early diagnosis of cryptococcal meningitis using strategies such as screening high-16

risk patients (with CD4+ T-cell count below 100 cells/ml) with the cryptococcal 17

antigen test (21), (iii) improving access to first-line antifungal drugs such as 18

amphotericin B and flucytosine, (iv) improving management of raised intracranial 19

pressure (5), (v) facilitating access to cART soon after diagnosis of cryptococcosis, 20

and (vi) reducing the high mortality rate by optimal management of patients during 21

and after hospital admission. cART improves long-term survival if patients survive 22

the first episode of cryptococcal meningitis (27). Recognising the need to optimise 23

management of the first episode and improve survival rates, South African clinicians 24


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have treated an increasing proportion of patients with amphotericin B deoxycholate 1

since 2005 (19). 2

3

Although the cryptococcal isolates in this study were obtained through active 4

population-based surveillance, this study has several limitations. First, cases of 5

incident cryptococcosis were drawn from a relatively small geographic area (four 6

hospitals in Gauteng Province). However, we selected these sites because long-term 7

trends could be examined and because we expected that fluconazole non-8

susceptibility was more likely to emerge in settings where patients were likely to 9

receive prior fluconazole for other indications. Second, the sample may have been 10

under-powered to detect a small change in MIC50 and MIC90 between the two 11

surveillance periods. Third, patient follow-up was limited to duration of hospital 12

admission; hence, any association between MIC and outcome was unlikely to be 13

meaningful. Fourth, we lacked sufficient clinical and laboratory data to make the 14

distinction between persistence and relapse amongst cases with serially-collected 15

isolates. 16

17

In conclusion, we have found no evidence for the emergence of resistance to 18

fluconazole amongst incident cryptococcal isolates in South Africa. However, 19

fungicidal agents should still be preferentially selected for induction treatment when 20

available. Similarly, only one case with serially-collected isolates was associated with 21

significantly changed fluconazole MIC values, suggesting that clinical attention needs 22

to be focused on other more common causes of recurrence. 23


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Acknowledgements 1

The findings and conclusions in this presentation/report are those of the author(s) and 2

do not necessarily represent the official position of the CDC. 3

4

This work was presented, in part, at the International Society for Human and Medical 5

Mycology (ISHAM) 2009 conference, Tokyo, Japan, 25-29 May 2009, Abstract 6

number: EP02-5, and the Federation of Infectious Disease Societies of Southern 7

Africa (FIDSSA) conference, Sun City, South Africa, 23-25 August 2009. 8

9

We acknowledge the technical assistance provided by Thokozile Gloria Zulu, 10

Nevashan Govender, Penny Crowther and Cheryl Cohen (NICD) and Naureen Iqbal, 11

Benjamin Park and Joyce Peterson (CDC). We appreciate the critical review of this 12

manuscript by Mary E. Brandt (CDC). We thank all laboratory and clinical personnel 13

throughout South Africa for contributing to surveillance. 14

15

Members of the Group for Enteric, Respiratory and Meningeal disease Surveillance in 16

South Africa (GERMS-SA), 2005-8: 17

S. Vasaikar (Eastern Cape); N. Janse van Rensberg, A. Möller, P. Smith, A.M. 18

Pretorius (Free State); K. Ahmed, A. Hoosen, R. Lekalakala, P. P. Sein, C. Feldman, 19

A.S. Karstaedt, O. Perovic, J. Wadula, M. Dove, K. Lindeque, L. Meyer, G. 20

Weldhagen (Gauteng); S. Harvey, P. Jooste (Northern Cape); D. Cilliers, A. Rampe 21

(North West Province); W. Sturm, T. Vanmali, P. Bhola, P. Moodley, S. Sithole, H. 22

Dawood (KwaZulu Natal); K. Hamese (Limpopo); K. Bauer, G. Hoyland, J. Lebudi, 23

C. Mutanda (Mpumalanga); R. Hoffmann, S. Martin, L. Liebowitz, E. Wasserman 24

(Western Cape), A. Whitelaw (Western Cape); A. Brink, I. Zietsman, M. Botha, X. 25


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Poswa, M. da Silva, S. Budavari (Ampath laboratories); C. Heney, J. Smit (Lancet 1

laboratories); M. Senekal (Pathcare laboratories); A. Schuchat, S. Schrag (CDC); K.P. 2

Klugman (Emory); C. Cohen, L. Dini, L. de Gouveia, J. Frean, S. Gould, K. Keddy, 3

K.M. McCarthy, J. Patel, S.T. Meiring, E.G. Prentice, V.C. Quan, J. Ramalivhana, A. 4

Sooka, A. von Gottberg and N.P. Govender (National Institute for Communicable 5

Diseases) 6

7

Members of the Gauteng Cryptococcal Surveillance Initiative Group, 2002-4: 8

K. Ahmed, L. Bhagoobhai, M.E. Brandt, A. Brink, H.H. Crewe-Brown, M. Da Silva, 9

M. Dove, G. Elliot, I. Ganchi, S. M. Gould, R.A. Hajjeh, C. Heney, A.S. Karstaedt, 10

H.J. Koornhof, A. Hoosen, T. Makhanya, M.R.B. Maloba, L. Marcus, M. Mason, 11

K.M. McCarthy, N. Mhlongo, S.A. Mirza, P. Moeng, J. Morgan, M. Nchabeleng, S. 12

Nkomo, B. Olivier, W. Owen, S. Peter, E. van Schalkwyk, J. van den Ende, N. 13

Xundu, R. Zulch, B. Arthington-Skaggs, N. Iqbal, K. Stamey, D.W. Warnock, K.A. 14

Wannemuehler 15

16

Conflicts of interests: Nelesh P. Govender is the recipient of a research grant from 17

Pfizer South Africa. 18

19

Funding source: From 2002 through 2004, this study was funded through a 20

cooperative agreement between the National Health Laboratory Service and the CDC, 21

Atlanta, Georgia. From 2005 through 2006, the study was partially funded by the 22

United States Agency for International Development’s Antimicrobial Resistance 23

Initiative, transferred via a Cooperative Agreement U60/CCU022088 from the CDC, 24

Atlanta, Georgia. From 2005 through 2008, the study was also partially supported by 25


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the CDC, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention 1

(NCHHSTP), Global AIDS Program (GAP) Cooperative Agreement 2

U62/PSO022901. The contents are solely the responsibility of the authors and do not 3

necessarily represent the official views of the CDC. 4

5


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3:74-7523


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Table 1: Comparison of selected case patients (with isolates available for MIC determination) diagnosed in each surveillance period: 1 March 1

2002 through 28 February 2003 and 1 March 2007 through 28 February 2008, n=487 2

3

Surveillance period Characteristic

2002-2003 (n=238) 2007-2008 (n=249)

p*

Gender (n=487) n=238 n=249

Male 115 (48) 98 (39)

Female 123 (52) 151 (61)

0.051

Age category (n=482) n=235 n=247

<15 years 4 (2) 1 (0.4)

15-24 years 21 (9) 15 (6)

25-44 years 178 (76) 194 (79)

45-64 years 32 (13) 35 (14)

>64 years 0 (0) 2 (0.6)

0.279


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Baseline CD4+ T-cell count (n=243) n=71 n=172

0-50 cells/mL 53 (73) 108 (63)

51-100 cells/mL 13 (18) 35 (20)

101-200 cells/mL 2 (4.5) 24 (14)

>200 cell/mL 3 (4.5) 5 (3)

0.040

Specimen from which diagnosis was made

(n=487)

N=238 n=249

Cerebrospinal fluid 231 (97) 216 (87)

Blood 3 (1) 33 (13)

Other 4 (2) 0 (0)

<0.001

Induction-phase antifungal treatment

(n=474)

n=238 n=236

Amphotericin B 38 (16) 95 (40)

Fluconazole 193 (81) 122 (52)

<0.001


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No treatment recorded 7 (3) 19 (8)

Outcome at the end of hospital admission

(n=474)

n=238 n=236

Died 62 (26) 84 (36)

Survived 176 (74) 152 (64)

0.024

*Chi-squared test – Mantel-Haenszel or Fisher’s exact test 1


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Table 2: Minimum inhibitory concentrations of 6 antifungal drugs for incident cryptococcal isolates from 2 surveillance periods: 1 March 2002 1

through 28 February 2003 and 1 March 2007 through 28 February 2008, n=237* 2

3

Minimum Inhibitory Concentration (mg/L) for:

Isolates from 2002-2003

n=119*

Isolates from 2007-2008

n=118*

Antifungal drug

Range MIC50 MIC90 Range MIC50 MIC90

Amphotericin B 0.012-0.38 0.094 0.19 0.008-0.94 0.094 0.19

Flucytosine 0.25-16 1 4 0.05-8 1 2

Fluconazole* 0.5-16 1 2 0.25-8 1 2

Voriconazole 0.008-0.25 0.015 0.06 0.008-0.25 0.015 0.03

Posaconazole 0.03-0.5 0.12 0.25 0.03-1 0.06 0.12

Itraconazole 0.03-1 0.12 0.25 0.015-0.5 0.06 0.12

*Fluconazole MIC results: n=238 (2002-2003) and n=249 (2007-2008), total n=487 4


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Table 3: Fluconazole minimum inhibitory concentrations for incident cryptococcal isolates from 2 surveillance periods: 1 March 2002 through 1

28 February 2003 and 1 March 2007 through 28 February 2008, n=487 2

3

Fluconazole MIC (mg/L) 2002-2003, n (%) 2007-2008, n (%) Total

0.25 0 (0) 1 (1) 1 (0.4)

0.5 13 (5) 14 (5) 27 (6)

1 39 (16) 53 (21) 92 (19)

2 99 (42) 97 (39) 196 (40)

4 68 (29) 70 (28) 138 (28)

8 16 (7) 14 (6) 30 (6)

16 3 (1) 0 (0) 3 (0.6)

Total 238 249 487


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Table 4: Fluconazole minimum inhibitory concentrations (MIC) for serial isolates collected more than 30 days apart from 67 cases of 1

cryptococcosis, 2005. 2

Fluconazole MIC for isolate collected ≥ 30 days after incident isolate (µg/ml) Fluconazole MIC for

incident isolate (µg/ml) 0.5 1 2 4 8 16 32 64

Number of

isolates

0.5 0 1 0 0 0 0 0 0 1

1 0 3 2 1 0 0 0 0 6

2 0 3 13 5 1 0 1 0 23

4 0 1 2 11 1 4 0 0 19

8 0 0 0 1 5 2 0 0 8

16 0 0 0 0 1 1 0 1 3

32 1 0 0 0 0 0 0 0 1

64 0 1 2 0 0 0 2 1 6

Number of isolates 1 9 19 18 8 7 3 2 67

3


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