Ketorolac-fluconazole: A New Combination Reverting Resistance in Candida albicans from Acute Myeloid Leukemia Patients on Induction Chemotherapy: In vitro Study

Figures & data

Table 1 Clinical Data of 170 Patients with Newly Diagnosed Acute Myeloid Leukemia

Character	N (%)
Gender	72 F/98 M
Median Age in years (range)	49 (18–69)
AML Diagnosis
AML (M0) Undifferentiated acute myeloblastic leukemia	11 (6.47)
AML (M1) Acute myeloblastic leukemia with minimal maturation	33 (19.41)
AML (M2) Acute myeloblastic leukemia with maturation	49 (28.82)
AML (M4) Myelomonocytic	51 (30)
AML (M5) Monocytic	19 (11.18)
AML (M6) Acute erythroid leukemia	4 (2.36)
AML (M7) Acute megakaryocytic leukemia	3 (1.76)
Cytogenetic/Molecular Risk
Favorable risk	29 (17.05)
Intermediate risk	64 (37.67)
Unfavorable risk	31 (18.23)
Not available	46 (27.05)
Neutropenic Fever	116 (68.23)
Response of Induction Chemotherapy
Complete remission (CR)	81 (47.64)
Partial remission (PR)	20 (11.76)
Resistant disease (RD)	46 (27.05)
Undefined response	23 (13.52)

Table 2 Interpretative Breakpoints of Disk Diffusion Method for Fluconazole, Itraconazole, Miconazole, and Voriconazole²⁶

Antifungal	Abbr.	Conc./Disc	Zone Diameter in mm
			S	I	R
Fluconazole	FU	25 µg	≥19	15–18	≤14
Itraconazole	IT	10 µg	≥23	14–22	≤13
Miconazole	MIC	10 µg	≥20	12–19	≤11
Voriconazole	VRC	1 µg	≥17	14–16	≤13

Abbreviations: S, susceptible; I, intermediate; R, resistant.

Table 3 Gene-specific Primers Used for RT- qPCR³¹

Gene	Primer Sequence
CDR1	F: 5′-- TGCCAAACAATCCAACAA--3′ R: 5′--CGACGGATCACCTTTCATACGA--3′
CDR2	F: 5′-- AAGGTTT TGATGCTACTGC--3′ R: 5′--GTCGGACATGTGGCTCAAA--3′
MDR1	F: 5′--GTGTTGGCCCATTGGTTTTCAGTC--3′ R: 5′--CCAAAGCA GTGGGGATTTGTAG--3′
ACT1	F: 5′--AAGAATTGATTTGGCTGGTAGAGA--3′ R: 5′-TGGCAGAAGATTGAGAAGAAGTTT--3′

Table 4 Resistance Patterns of 50 C. albicans Using a Kirby–Bauer Disk Diffusion Method for Azoles

Total (n= 50)	Fluconazole (25 μg/Disc)N (%)	Miconazole (10 μg/Disc)N (%)	Voriconazole (1 μg/Disc)N (%)	Itraconazole (10 μg/Disc)N (%)
Sensitive (S)	3 (6)	3 (6)	3 (6)	3 (6)
Intermediate (I)	0 (0)	10 (20)	0 (0)	10 (20)
Resistant (R)	47 (94)	37 (74)	47 (94)	37 (74)

Note: Data are presented as number (%) of isolates resistant to antimicrobial indicated.

Table 5 In vitro Interaction of Ketorolac with Fluconazole Against Resistant C. albicans

Mean MIC (μg/mL)		Mean MIC (μg/mL)		Mean FICI	IN
Alone		Combination
Ketorolac	Fluconazole	Ketorolac	Fluconazole
>10	>160	2.5	0.62	0.25	SYN

Abbreviations: IN, interpretation; SYN, synergism.

Table 6 The Level of the Efflux Pumps Gene Expression in Resistant and Sensitive Strains in the Human Study Group

Gene	Mean ±SE	Median	P-value
CDR1
Resistant strains	11.16±0.9	11.02	0.006**
Sensitive strains	0.13±0.12	0.13
CDR2
Resistant strains	0.33±0.07	0.1	0.078*
Sensitive strains	0.02±0.003	0.02
MDR1
Resistant strains	7.81±0.96	5.88	0.004***
Sensitive strains	0.37±0.003	0.04

Notes: *Nonsignificant (P>0.05), **significant (P<0.05), ***highly significant (P<0.005).

Figure 1 Efflux pump gene expression of C. albicans isolates in relation to the MIC of fluconazole.

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