Genotypic diversity and clinical outcome of cryptococcosis in renal transplant recipients in Brazil

Figures & data

Table 1. Demographic and clinical characteristics of 60 renal transplant recipients infected by C. neoformans/C. gattii species complexes.

Characteristics	Value, % (no. of patients) n = 60
Age, average years (range)	49 (21–71)
Male	63.3 (38)
Ethnicity
White	60 (36)
Non-white	40 (24)
Retransplant^a	6.7 (4)
Donor type
Deceased	60 (36)
Living	40 (24)
Immunosuppressive induction therapy	40 (24)
Immunosuppressive agents received^b
Prednisone	100 (60)
Tacrolimus	56.7 (34)
Mycophenolic acid^c	55 (33)
Azathioprine	26.7 (16)
Cyclosporine A	18.3 (11)
Rapamycin	8.3 (5)
Prior rejection	40 (24)
Diabetes mellitus	26.7 (16)
Active cytomegalovirus infection	25 (15)
Hepatitis C infection	18.3 (11)
Time to onset of infection after transplant, average months (range)	30.5 (13 days to 17 years)
Sites of involvement
CNS	83.3 (50)
Pulmonary	50 (30)
Skin, soft-tissue, or osteoarticular	20 (12)
Fungemia	38.3 (23)
Disseminated infection^d	63.3 (38)
Renal failure at baseline^e	56.7 (34)
Serum cryptococcal antigen titre, median (range)^f	1:1024 (0–1:1024)
Change in immunosuppression at diagnosis^g	78.6 (44)
Antifungal therapy
Amphotericin B alone	60 (36)
Amphotericin B + 5FC	18.1 (11)
Amphotericin B + fluconazole	8.3 (5)
Fluconazole	3.3 (2)
None	10 (6)
Mortality at 90 days	45 (27)

Note: CNS, central nervous system; 5FC, 5-flucytosine.

^aIndicates prior receipt of a renal transplant.

^bImmunosuppressive agent that remained unchanged within 3 months of the onset of cryptococcosis.

^cIncludes mycophenolate mofetil or mycophenolate sodium.

^dDefined as the involvement of at least two noncontiguous organ systems or the presence of fungemia.

^eIndicates creatinine ≥2 mg dL⁻¹ at the time of diagnosis of infection.

^fData available for 35 patients.

^gData available for 56 patients.

Figure 1. Molecular type distribution of 82 clinical isolates of C. neoformans/C. gattii species complexes (A) cultured from 60 renal transplants recipients (B).

Figure 2. Phylogenetic relationships as inferred from a maximum likelihood analysis of CAP59, LAC1, PLB1, SOD1, URA5, TEF1 and IGS1 sequences from 82 strains of C. neoformans and C. gattii from transplant patients and 63 reference strains, covering the main molecular types described. The numbers close to the branches represent indices of support (maximum likelihood/neighbor-joining) based on 1000 bootstrap replications. The branches with bootstrap support higher than 70% are indicated in bold.

Figure 3. Median-joining haplotype network of 144 isolates of C. neoformans/C. gattii species complexes (81 isolates originated in this study in addition to 63 reference strains recovered from literature), covering all the concatenated loci CAP59, LAC1, PLB1, SOD1, URA5, TEF1 and IGS1 sequences. The isolates are coded, and their frequencies are represented by (A) fluconazole MIC ≥ than 16 mg l⁻¹ from transplant recipients isolates or (B) 90-days mortality of transplant recipients. The size of the circumference is proportional to the haplotype frequency. The black dots (median vectors) represent unsampled or extinct haplotypes in the population. Further information about isolate source and GenBank accession number can be found in the Supplementary Table 1.

Table 2. Comparisons of infections due to C. neoformans and C. gattii complex in 60 renal transplant recipients.

	Univariate analysis			Multivariate analysis
Characteristics (N°. patients) n = 60	C. neoformans infection % (N^o. of patients) n = 54	C. gattii infection % (N^o. of patients) n = 6	P-value	OR	95% CI	P-value
Age, average years (range)	49 (21–69)	50.5 (32–71)	0.796
Male	66.7 (36)	33.3 (2)	0.179
White	61.1 (33)	33.3 (2)	0.223
Living in the capital before infection	27.8 (15)	6.3 (1)	1.0
Northeast of Brazil as place of birth	31.5 (17)	50 (3)	0.390
Retransplant^a	3.7 (2)	33.3 (2)	0.046
Deceased donor type	57.4 (31)	83.3 (5)	0.387
Induction immunosuppressive therapy	38.9 (21)	50 (3)	0.675
Prior rejection	38.9 (21)	50 (3)	0.675
Cytomegalovirus infection	25.9 (14)	16.7 (1)	1.0
Time to onset of infection after transplant, average months (range)	30 (13 days–17 years)	37.5 (188 days–7 years)	0.667
Sites of involvement
CNS (56)	90.6 (48)	66.7 (2)	0.293
Pulmonary (59)	48.1 (26)	80 (4)	0.353
Skin, soft-tissue, or osteoarticular	16.7 (9)	50 (3)	0.088
Fungemia	35.2 (19)	66.7 (4)	0.191
Disseminated infection^b	61.1 (33)	83.3 (5)	0.4
Renal failure at baseline^c	53.7 (29)	83.3 (5)	0.221
CNS image abnormality (37)	20 (7)	0 (0)	1.0
Diffuse infiltrate in lung image (27)	37.5 (9)	0 (0)	0.529
Serum cryptococcal antigen titre ≥ 1:512 (31)	57.1 (16)	100 (3)	0.265
Mean duration of hospitalization, ±SD (range), days	36.8 ± 38.19 (1–245)	9.33 ± 11.89 (8–33)	0.087
Duration of antifungal induction therapy, average days (range) (54)	26.3 (1–219)	16 (4–28)	0.504
Total duration of antifungal therapy, average days (range) (51)	115 (1–635)	288.5 (193–384)	0.453
Fluconazole MIC ≥ 16 mg l^−1	7.4 (4)	66.7 (4)	0.002	12.62	1.97–80.99	0.008
Mortality at 90 days	42.6 (23)	66.7 (4)	0.394

Note: CNS, central nervous system; SD, standard deviation; MIC, minimum inhibitory concentration.

^aIndicates prior receipt of a renal transplant.

^bDefined as the involvement of at least two noncontiguous organ systems or the presence of fungemia.

^cIndicates creatinine ≥2 mg dL⁻¹ at the time of diagnosis of infection.

Table 3. Fluconazole MIC distribution for 82 isolates of C. neoformans/C. gattii species complexes tested.

						No. of isolates for which the MIC (mg l^−1) was^a:
Molecular type or specie	Number of isolates	Mean mg l^−1	Interval mg l^−1	MIC50 mg l^−1	MIC90 mg l^−1	≤0.12	0.25	0.5	1	2	4	8	16	32	≥64
All isolates	82	8.70	0.25–64	8	16		1		3	11	20	34	9	2	2
C. neformans	72	6.18	0.25–16	8	8		1		3	10	19	33	5
VNI	51	7.22	0.25–16	8	8		1			2	15	28	5
VNII	14	2.50	1–8	2	4				3	8	2	1
VNB	6	6.86	4–8	8	8						2	4
VNI/II	1										1
C. gattii – VGII	10	25.82	4–64	16	64						1	1	4	2	2

Note: MIC, minimum inhibitory concentration.

^aThe modal MIC for each distribution is underlined.

Figure 4. Kaplan–Meier analysis of 12 weeks survival of 60 renal transplant recipient infected by C. neoformans/C. gattii species complexes according to fluconazole MIC ≥ 16 mg l⁻¹ (n = 8) or MIC < 16 mg l⁻¹ (n = 52).

Table 4. Variables associated with 30-day mortality after cryptococcosis in 60 renal transplant recipients.

	Univariate analysis			Multivariate analysis
	Survival, %	Death, %
Variables (N°. patients) n = 60	n = 37	n = 23	P-value	OR	95% CI	P-value
Age, mean, ±SD, years (60)	48.1 ± 12.51	49.6 ± 12.94	0.657
Male (38)	56.8	73.9	0.180
Induction immunosuppressive therapy (24)	27	60.9	0.009
Deceased donor (36)	48.6	78.2	0.023
Prior rejection (24)	43.2	34.8	0.515
Cytomegalovirus infection^a (7)	13.5	8.7	0.697
Receipt of a calcineurin-inhibitor agent^b (45)	64.9	91.3	0.021
Receipt of a tacrolimus^b (34)	45.9	73.9	0.034
Duration of symptoms before diagnosis, mean, ±SD (56)	35.4 ± 42.69	29.2 ± 78.74	0.705
Time to diagnose after admission, mean, ±SD (57)	4.7 ± 9.09	3.6 ± 4.65	0.585
Sites of involvement
CNS^c (50)	89.2	89.5	1
Pulmonary^d (30)	40.5	68.2	0.04
Skin or soft-tissue (12)	24.3	13	0.34
Disseminated infection (38)	54.1	78.3	0.059
Fungemia (23)	18.9	69.6	<0.001	3.79	1.03–13.91	0.044
C. gattii infection (6)	5.4	17.4	0.191
Non-VNI genotype (20)	32.4	34.5	0.851
Creatinine at admission ≥2 mg dL^−1 (34)	51.4	65.2	0.292
Somnolence at admission^e (19)	27.3	58.8	0.029
Confusion at admission^e (19)	30.3	52.9	0.118
Headache at admission^e (30)	72.7	35.3	0.01	0.13	0.04–0.42	0.001
Intracranial hypertension^f (16)	48.1	33.3	0.7
Respiratory failure^b,g (7)	6.7	40	0.08
CSF cell count^b, mean, ±SD, mm^3 (42)	138.5 ± 250.04	101.1 ± 92.8	0.664
CSF glucose ratio^b, mean, ±SD, mg dL^−1 (41)	47.3 ± 24.63	39.2 ± 31.89	0.420
Positive CSF India ink^h (23)	42.4	64.3	0.170
CSF antigen titre >1:512^i (24)	43.3	84.6	0.012
Positive pulmonary culture^j (9)	33.3	100	0.021
AMBd as primary therapy^k (53)	97.3	100	1
Combination therapy^k (16)	24.3	41.2	0.208
Change in immunosuppressive regime after infection^c (44)	80.6	75	0.737
Discontinuation of tacrolimus after infection^l (24)	41.4	80	0.025
Graft loss within 30 days^m (7)	13.5	100	0.028
Fluconazole MIC ≥ 16 mg l^−1 (8)	25	75	0.045

Note: OR, odds ratio; CI, confidence interval; SD, standard deviation; CNS, central nervous system; CSF, cerebrospinal fluid; AMBd, amphotericin B deoxycholate; MIC, minimum inhibitory concentration.

^aInfection occurring within 6 months of the onset of cryptococcosis.

^bAt cryptococcosis diagnosis.

^cData was available for 56 patients.

^dData was available for 59 patients.

^eData was available for 50 patients.

^fData was available for 36 patients.

^gData was available for 30 patients.

^hData was available for 47 patients.

ⁱData was available for 43 patients.

^jData was available for 13 patients.

^kData was available for 54 patients, at least 2 consecutive days of the same antifungal therapy.

^lData was available for 44 patients.

^mData was available for 39 patients.

Data availability

The sequences of all newly identified allele types have been submitted to the MLST database (http://mlst.mycologylab.org) and GenBank (https://www.ncbi.nlm.nih.gov/genbank/).