Incident cryptococcal isolates obtained through population-based surveillance in South Africa maintained low MIC values to the first-line antifungal drugs fluconazole and amphotericin B over a 6-year period, and very low MICs were determined for newer drugs, such as voriconazole and posaconazole. Despite infrequent use of these agents in South Africa, a small number of isolates were determined to have elevated MIC values to itraconazole and/or flucytosine. Nonsusceptibility to fluconazole, as defined by a 4-fold increase in the MIC (10
), was detected in only 1 case with serially collected isolates.
Historically, the determination of the MIC in the laboratory has been the method of choice for monitoring antifungal resistance. Although a standardized antifungal drug susceptibility testing method has been developed (13
), antifungal resistance in Cryptococcus
is difficult to define in the laboratory due to the absence of interpretive breakpoints. Attempts to correlate MIC with clinical outcome have produced mixed results. For example, in a small case series, an incident isolate with a fluconazole MIC of ≥16 μg/ml was associated with subsequent clinical failure among 5 of 25 (20%) patients (2
); however, this finding was not replicated in other studies (15
). Nevertheless, if it is performed consistently over time, MIC testing can indicate shifts in susceptibility among isolates at a population level.
The distribution of 200-mg fluconazole tablets through the Diflucan Partnership Program in South Africa has doubled from approximately 1.8 million doses in 2002 to 3.8 million doses in 2008 (Pfizer, South Africa, personal communication). Despite this, the finding that almost all incident cryptococcal isolates had a fluconazole MIC of ≤16 μg/ml was not surprising. A large global study found that the fluconazole MIC of incident isolates did not change substantially over a 15-year period (1990-2004), despite increased use of fluconazole (35
). In addition, Brandt et al. tested 143 incident C. neoformans
(serotype A) isolates from the same population-based surveillance system in Gauteng Province (2002-2003) with the reference broth microdilution method; the MIC range, MIC50
, and MIC90
for fluconazole were 0.5 μg/ml to 8 μg/ml, 2 μg/ml, and 4 μg/ml, respectively, which are almost identical to the results that we obtained (9
). In contrast, a report from Cambodia described incident isolates with increased fluconazole MICs over a 2-year period when they were tested with the Etest method (38
). The findings from the latter study are difficult to explain. Fluconazole-resistant Candida
species have been shown to emerge in areas where primary fluconazole prophylaxis is used as a preventative strategy (3
), but Candida
is a colonizer that can replicate as a commensal in the host and can inhabit many different body sites to avoid maximum exposure to antifungal drugs. Dormant cryptococcal strains, which are hypothesized to have established a latent infection many years previously, reactivate primarily in the milieu of advanced HIV-associated T-cell immunodeficiency and cause disseminated disease (18
). Even in the setting of primary fluconazole prophylaxis, incident C. neoformans
isolates with reduced susceptibility to fluconazole have been infrequently documented (4
); this may be directly related to the fact that they do not actively replicate in the host as commensal organisms prior to onset of disease.
Voriconazole and posaconazole have consistently been shown to have good activity against C. neoformans
). However, the use of voriconazole and posaconazole is still restricted to salvage settings (33
), as no clinical trials have been undertaken to compare these agents to first-line drugs and these agents remain prohibitively expensive for use in resource-limited settings. In our study, the three incident isolates with reduced fluconazole susceptibility (MIC = 16 μg/ml) had relatively low MICs to voriconazole. However, fungistatic azole drugs would still not be the first choice for treatment of incident cryptococcosis.
Even in the absence of interpretive breakpoints, MIC testing may be more helpful to document the emergence of resistance over time among patients with serially collected isolates if the same test method is used and isolate pairs are tested in parallel. In our surveillance, we found that, in most cases, serially collected isolates displayed fluconazole MIC results within 2 dilutions, indicating essential agreement. Similarly, a Ugandan study which compared fluconazole broth microdilution MICs of serially collected isolates from 17 patients found no evidence of a stepwise increase in MIC over a 2- to 10-week period (37
). In contrast, a prospective, observational study from Cape Town, South Africa (2003 to 2005), found that 16 of 20 (80%) patients with culture-confirmed relapse disease had isolates with reduced fluconazole susceptibility, as determined by the Etest method (6
). Bicanic et al. suggested that the high prevalence of fluconazole nonsusceptibility among Cape Town isolates was associated with low-dose fluconazole induction treatment (400 mg daily) and concurrent rifampin use (6
). A Cambodian study also reported that the MIC to fluconazole, as determined by Etest, increased significantly from the year 2000 to 2002 (38
). In contrast, we found only one case where the serially collected isolate had an MIC value significantly elevated above the MIC of the incident isolate, despite most patients receiving low-dose fluconazole induction treatment (400 mg daily). Similarly, Brandt et al., who also determined the fluconazole MIC for isolates from Gauteng surveillance (2002-2003) serially collected more than 30 days apart, detected an increase in fluconazole MIC values of at least 3 log2
dilutions over time for only 2 of 30 cases (9
Differences in MIC testing methods may have contributed to some of these reported differences. Several reports indicate that Cryptococcus
MIC values to the azoles that have been generated by Etest are higher than those generated by broth microdilution testing when they are performed in parallel (15
). While we used a broth dilution method for MIC determination, Bicanic et al. used the Etest method, where endpoint determination for azoles is technically more difficult to establish and may be more subjective (6
). There is still some question as to whether the results of various testing methodologies can be directly compared. In addition, Bicanic et al. defined resistance to fluconazole as a single (relapse episode) isolate with an MIC of ≥16 μg/ml; isolate pairs were not tested in parallel (6
We believe that there are other, more common reasons for recurrent disease, such as nonadherence to suppressive fluconazole treatment (14
), development of the immune reconstitution inflammatory syndrome (IRIS) following initiation of cART (7
), or suboptimal induction-phase treatment. Recently, Jarvis et al. described patients with symptomatic relapse disease at the same Cape Town hospital in 2007-2008 when amphotericin B induction-phase treatment and antiretroviral treatment were the standard of care (24
). Of the 69 relapse episodes that were detected over this 2-year period, most were due to IRIS (45%) or nonadherence to or nonprescription of fluconazole maintenance treatment (43%) (24
). In contrast to the earlier Cape Town study, very few isolates with elevated fluconazole MICs (MIC ≥ 16 μg/ml, determined by the Etest) were detected from this group of patients.
In our study, there were some cases where the MIC value of the second isolate compared to that of the incident isolate dropped when both were tested in parallel using the same broth microdilution method, a phenomenon that has been documented previously (10
). Although we do not currently understand this mechanism, recent work by Desnos-Ollivier and colleagues indicates that at least 20% of C. neoformans
infections may be comprised of multiple strains and genotypes (16
). By testing only selected subpopulations cultured from the original clinical specimen, other strains contributing to disease in a given patient may be missed (16
Major challenges to improving management of patients with cryptococcosis include (i) preventing cryptococcosis by early diagnosis of HIV infection and timely initiation of cART well before the CD4+
T-cell count falls below 200 cells/ml, (ii) diagnosing cryptococcal meningitis early using strategies such as screening high-risk patients (with CD4+
T-cell counts below 100 cells/ml) with the cryptococcal antigen test (21
), (iii) improving access to first-line antifungal drugs such as amphotericin B and flucytosine, (iv) improving management of raised intracranial pressure (5
), (v) facilitating access to cART soon after diagnosis of cryptococcosis, and (vi) reducing the high mortality rate by optimal management of patients during and after hospital admission. cART improves long-term survival if patients survive the first episode of cryptococcal meningitis (27
). Recognizing the need to optimize management of the first episode and improve survival rates, South African clinicians have treated an increasing proportion of patients with amphotericin B deoxycholate since 2005 (19
Although the cryptococcal isolates in this study were obtained through active population-based surveillance, this study has several limitations. First, cases of incident cryptococcosis were drawn from a relatively small geographic area (four hospitals in Gauteng Province). However, we selected these sites because long-term trends could be examined and because we expected that fluconazole nonsusceptibility was more likely to emerge in settings where patients were likely to have previously received fluconazole for other indications. Second, the sample may have been underpowered to detect a small change in MIC50s and MIC90s between the two surveillance periods. Third, patient follow-up was limited to the duration of hospital admission; hence, any association between MIC and outcome was unlikely to be meaningful. Fourth, we lacked sufficient clinical and laboratory data to make the distinction between persistence and relapse among cases with serially collected isolates.
In conclusion, we have found no evidence for the emergence of resistance to fluconazole among incident cryptococcal isolates in South Africa. However, fungicidal agents should still be preferentially selected for induction treatment when they are available. Similarly, only one case with serially collected isolates was associated with significantly changed fluconazole MIC values, suggesting that clinical attention needs to be focused on other more common causes of recurrence.