Breakthrough Candidemia Due to Multidrug-Resistant Candida glabrata during Prophylaxis with a Low Dose of Micafungin

Abstract

We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-β-d-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

TEXT

In recent years, the epidemiology of candidemia in Latin American hospitals has been changing, with a trend toward an increase in the incidence of candidemia due to Candida glabrata, as documented in recently published multicenter studies (1, 2, 3). C. glabrata is naturally less susceptible to azoles than other Candida species (4), and this has led to the expanded use of echinocandins for fungemia treatment caused by this Candida species (5, 6, 7). This class of drug is now reported as the first-line therapy for candidemia in several guidelines of medical societies (5, 6, 7).

Acquired echinocandin resistance among Candida spp. is largely infrequent. Yet, episodes of invasive infection due to echinocandin-resistant Candida isolates are increasingly being reported in U.S. and European medical centers (8, 9, 10, 11). We describe one case of C. glabrata breakthrough candidemia documented in a patient with Burkitt lymphoma and prolonged neutropenia who received antifungal prophylaxis with low doses of micafungin (50 mg/day) in a tertiary care hospital in Curitiba, Paraná, Brazil. This is the first documented case of multidrug resistance among clinical strains of C. glabrata from a Latin American medical center.

Case.

A 25-year-old man was diagnosed with stage IV adult sporadic Burkitt lymphoma in November 2011, and due to profound neutropenia, he received fluconazole prophylactically at doses of 200 mg/day for 20 days (27 November to 16 December), followed by micafungin at doses of 50 mg/day for 12 days (17 December to 28 December). In January 2012 (3 January), the patient was admitted for a third round of chemotherapy treatment with a cyclophosphamide, vincristine, doxorubicin, dexamethasone (hyper-CVAD) regimen, having presented with profound neutropenia since December 2011. Levofloxacin and sulfamethoxazole-trimethoprim were initiated as prophylaxis therapy. Due to prolonged neutropenia, antifungal prophylaxis was restarted with micafungin at doses of 50 mg/day (posaconazole is not available in Brazil). At day 12 of hospitalization, the patient developed fever during a neutropenic period, without a defined focus of infection, and cefepime treatment was initiated empirically. A blood culture was collected and revealed growth of Candida (isolate 1), despite 24 days of exposure to micafungin. Initially, the decision was made to increase the micafungin dose to 100 mg/day. The patient continued to have a fever for 6 days after initiation of the micafungin treatment (100 mg/day), with sequential blood cultures positive for Candida (isolates 2 to 5). Possible infectious foci for persistent fungemia (endocarditis, hepatosplenic candidiasis, and fungal meningitis) were ruled out by clinical, imaging, and laboratory tests. Echinocandin resistance was suspected, and a conventional formulation of amphotericin B was initiated with good clinical response. Blood cultures were negative after 5 days of treatment.

A total of five clinical Candida isolates were obtained from sequential blood cultures collected during breakthrough candidemia that occurred during antifungal prophylaxis with micafungin (50 mg/day). All isolates tested were identified as Candida glabrata by internal transcribed spacer (ITS) sequencing, as previously described by our group (12, 13).

Antifungal susceptibility testing was performed using the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) document M27-A3 (14), using current CLSI MIC interpretative criteria (CLSI, M27-S4) (15). The antifungal compounds were kindly provided as pure powders by their manufacturers. All five C. glabrata bloodstream isolates tested exhibited resistance to fluconazole (FLC), voriconazole (VRC), and all echinocandins. The MIC values for echinocandins were 16- to 33-fold higher than those for the control strain (ATCC 90030). Table 1 summarizes the MIC values obtained for the five C. glabrata isolates for the six antifungal agents tested.

TABLE 1.

In vitro activities of six antifungal agents and GS inhibition profiles for echinocandin drugs against five C. glabrata isolates harboring FKS mutations

Strain/mutation	FKS2 hot spot genotype	MIC (mg/liter) for:						IC50 (ng/ml) for:
		AMB	FLC	VRC	ANF	CSF	MCF	ANF	CSF	MCF
ATCC 90030	FKS1/FKS1 (wild type)	0.25	4	0.03	0.06	0.03	0.03	46.1	29.7	15.29
8622 A	fks1/fks1 (S663F)	0.25	>64.0	4.0	1.0	1.0	0.5	2,655	1,263	61,760
8622 B	fks1/fks1 (S663F)	0.25	>64.0	4.0	1.0	1.0	0.5	NDa	ND	ND
8622 C	fks1/fks1 (S663F)	0.25	>64.0	4.0	1.0	1.0	0.5	ND	ND	ND
8622 D	fks1/fks1 (S663F)	0.25	>64.0	4.0	1.0	1.0	0.5	ND	ND	ND
8622 E	fks1/fks1 (S663F)	0.25	>64.0	4.0	1.0	1.0	0.5	ND	ND	ND

^aND, not determined.

DNA sequencing analyses of the two hot spot (HS) regions of the drug target genes FKS1 and FKS2 were performed on the five C. glabrata clinical isolates, as previously described (16). The two HS regions of the FKS1 gene corresponded to nucleotides (nt) 1873 to 1902 (amino acids [aa] 625 to 633) and nt 4018 to 4041 (aa 1340 to 1347) of the published strain C. glabrata ATCC 90030 (DNA Data Bank of Japan [DDBJ] accession no. HM366440.1), and the two HS regions of the FKS2 gene corresponded to nt 1975 to 2001 (aa 659 to 667) and nt 4122 to 4143 (aa 1374 to 1381) of the published strain C. glabrata ATCC 90030 (DDBJ accession no. HM366442.1). The sequencing of the HS regions of the FKS genes revealed only a nonsynonymous mutation in HS1 in the FKS2 gene, which led to an S663F amino acid substitution (Table 1).

We performed an in vitro glucan synthase inhibition assay, as previously described (17, 18, 19). The echinocandin inhibition parameter 50% inhibitory concentration (IC₅₀) was determined for the wild-type strain (C. glabrata ATCC 90030) and the 8622A clinical isolate (Table 1). The fks mutant enzyme extracted from the clinical isolate showed significantly higher IC₅₀ values than did the corresponding enzyme isolated from the wild-type strain (58-, 43-, and >4,000-fold on average for anidulafungin [ANF], caspofungin [CSF], and micafungin [MCF], respectively) (Table 1 and Fig. 1). These results confirm that the point mutation S663F encoded in the FKS2 gene of C. glabrata yielded a 1,3-β-d-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs, resulting in elevated MICs with a strong potential for clinical failure. In the present investigation, a patient with Burkitt lymphoma and severe neutropenia that was expected to persist for a longer period of time was subjected to antifungal prophylaxis with micafungin at a dosage of 50 mg daily. The patient developed C. glabrata breakthrough candidemia after 19 and 24 days of exposure to fluconazole and micafungin, respectively. In vitro antifungal susceptibility testing confirmed the resistance to fluconazole, voriconazole, and all echinocandins.

FIG 1.

Echinocandin inhibition profiles for product-entrapped 1,3-β-d-glucan synthase enzyme complexes assessed by the incorporation of [³H]glucose into radiolabeled product. Titration curves are shown for anidulafungin (ANF), caspofungin (CSF), and micafungin (MCF) for the wild type and clinical isolate 8622A of C. glabrata.

The epidemiologic impact of using an echinocandin for antifungal prophylaxis in neutropenic patients in terms of promoting the development of multidrug-resistant Candida isolates remains unclear, but it is concerning. Prominent C. glabrata echinocandin resistance has recently been reported by Alexander et al. (8) and Pfaller et al. (9), whose studies suggested significant emergence of multidrug resistance over time to both azoles and echinocandins in C. glabrata isolates.

As echinocandins are more frequently used for the treatment of invasive candidiasis, sporadic cases of echinocandin breakthrough candidemia associated with nonsusceptible Candida isolates and treatment failure have been reported (10, 11, 19). In the present study, all five C. glabrata isolates recovered from sequential blood cultures collected during prophylaxis therapy with micafungin showed increases in the echinocandin MIC values, and all of them showed a point mutation in HS1 in the FKS2 gene that led to a S663F amino acid substitution, which has been previously described as being involved in treatment failure and echinocandin resistance in C. glabrata isolates (20). Notably, other studies have shown that a point mutation in the same position of the FKS2 gene, though with a different amino acid substitution (S663P), is the most frequently observed mutation related to echinocandin resistance in C. glabrata isolates (21, 22). It is important to note that in our case, in accordance with previous publications, the long-term echinocandin exposure (24 days) resulted in the development of FKS gene mutations and echinocandin resistance (10). On the other hand, one case of rapid echinocandin resistance in C. glabrata was reported by Lewis et al. (23) in a patient without previous or prolonged echinocandin exposure (8 days of treatment).

Our findings emphasize that the widespread use of echinocandins may increase the occurrence of echinocandin resistance, especially when using low doses, as in the present case in which the neutropenic patient was exposed to 50 mg daily. In this scenario, the use of echinocandin as prophylactic therapy should be recommended cautiously in patients with a high risk of candidemia development, now that previous exposure to echinocandins has been clearly recognized as a risk factor for the development of resistance.

Nucleotide sequence accession numbers.

The sequences generated in this study have been deposited in the GenBank (NCBI) database under accession numbers KF211452, KF211447, KF211442, KF211437, KF211456, KF211448, KF211438, KF211443, KF211455, KF211449, KF211444, KF211439, KF211454, KF211450, KF211445, KF211440, KF211453, KF211451, KF211446, KF211441, KF305827, KF305828, KF305829, KF305830, and KF305831.