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A systematic review of fluconazole resistance in clinical isolates of Cryptococcus species

Felix Bongomin1, 2*, Rita O. Oladele1,3, Sara Gago1,4, Caroline B. Moore1,2, and

Malcolm D. Richardson1, 2

1Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Rd, Manchester, M13 9PL, UK2The National Aspergillosis Centre & NHS Mycology Reference Centre-Manchester, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Southmoor Rd, Manchester, M23 9LT, UK3Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, Lagos, Nigeria4Manchester Fungal Infection Group, 1Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, Core Technology Facility Building, 46 Grafton Street, Manchester, M13 9NT, UK

Running head: Fluconazole resistance in Cryptococcus spp.

Keywords: Fluconazole, Resistance, Cryptococcus isolates, relapse, incident, HIV

*Corresponding author: Felix Bongomin, National Aspergillosis Centre, 2nd Floor Education and Research Centre, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Southmoor Road, Manchester, M23 9LT, UK. E-mail: felix.bongomin@manchester.ac.uk

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Abstract

Fluconazole is the most commonly used antifungal agent for both the

treatment of cryptococcal meningitis, and for prophylaxis against the

disease. However, its prolonged use has the potential to exert selection

pressure in favour of fluconazole-resistant strains. We evaluated the

prevalence of fluconazole resistance of Cryptococcus spp. clinical isolates

in 29 studies from 1988 to May 2017 included in EMBASE and MEDLINE

databases. A total of 4,995 Cryptococcus isolates from 3,210 patients

constituted this study; 248 (5.0%) of the isolates were from relapsed

episodes of cryptococcosis were included in this analysis. Eleven (38%) of

the studies used minimum inhibitory concentrations (MICs) breakpoints

of ≥64 µg/mL to define fluconazole resistance, 6 (21%) used ≥32 µg/mL,

11 (38 %) used ≥16 µg/mL, and 1 (3%) used ≤20 µg/mL. Overall, mean

prevalence of fluconazole resistance was 12.1 % (95% confidence interval

(CI): 6.7% - 17.6%) for all isolates (n=4,995). Mean fluconazole

resistance was 10.6% (95% CI: 5.5% - 15.6%) for the incident isolates

(n=4,747), and 24.1% (95% CI: -3.1% - 51.2%) for the relapse isolates

(n=248). Of the 4,995 isolates, 936 (18.7%) had MICs above the

ecological cut-off value. Fluconazole resistance appears to be an issue in

Cryptococcus isolates from patients with relapses. It is unclear whether

relapses occur due to resistance or other factors. There is an urgent need

to establish antifungal breakpoints for Cryptococcus spp.

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Introduction

Cryptococcal meningitis (CM) is the leading cause of meningitis among

persons living with HIV infection 1. The introduction of highly active

antiretroviral therapy (HAART) has led to a substantial reduction in the

global incidence of cryptococcosis. However, CM still accounts for about

15% of AIDS-related mortality 2,3. Rajasingham and colleagues recently

estimated an average global cryptococcal antigenaemia prevalence of 6

% (95% CI 5·8–6·2) among HIV-infected persons with CD4 counts <100

cells/μL. The global burden of the disease reveals an estimated 223,100

annual cases; it being a major problem in developing countries; in fact,

73% of these cases (162,500) are annually diagnosed in sub-Saharan

Africa 2. Furthermore, about 70% (range 56–84%) of people positive for

cryptococcal antigenaemia would progress to develop cryptococcal

disease or die without diagnosis, unless initiated on HAART or pre-

emptive fluconazole 2.

Fluconazole is a potent inhibitor of the fungal cytochrome P450-

dependent lanosterol C14α-demethylase, leading to inhibition of

ergosterol biosynthesis. At doses higher than 800mg/day it shows a dose-

dependent fungicidal activity against Cryptococcus spp. 4,5. Amphotericin

B in combination with flucytosine for induction followed by fluconazole

for consolidation and suppression therapy is the recommended antifungal

regimen for the management of acute CM 6,7. However, in resource-

limited settings where flucytosine is not routinely available, monotherapy

with high dose fluconazole or in combination with amphotericin B for

induction therapy is the treatment of choice 8. Long-term maintenance

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therapy with fluconazole is recommended to prevent relapses following

an episode of CM and in advanced HIV patients with asymptomatic

cryptococcal antigenaemia 9. For patients in whom fluconazole therapy is

contraindicated, itraconazole is an acceptable but less effective

alternative 6.

There is however growing concern regarding high rates of fungal

persistence and recurrent CM 6. Although antifungal drug resistance in

Cryptococcus species is relatively uncommon, the risk of development of

secondary resistance among patients with relapses is a major problem

10,11. Fluconazole-resistant Cryptococcus was first documented in 1993 in

a 30 year old HIV-infected Zambian woman diagnosed with C. gattii

meningitis who had poor clinical and mycological response to

fluconazole12. A few other studies in the 1990s confirmed the occurrence

of fluconazole–resistant Cryptococcus infection especially in patients

infected with HIV and this was linked to long-term suppressive

fluconazole therapy and in patients with recurrent/relapsed cryptococcal

meningitis 13. A more recent study described a 30% [6 of 20 cases]

incidence of fluconazole-resistant Cryptococcus isolates in patients who

had never had prior exposure to fluconazole 11, this is alarming.

This systematic review aims to describe the prevalence and pattern of

fluconazole-resistance in clinical isolates of Cryptococcus spp.

Methods

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The guidance outlined in the Preferred Reporting Items for Systematic

Reviews and Meta-Analyses (PRISMA) statement 14 were observed and

the PRISMA check-list was used to guide this.

Data Sources and search strategies

With the help of a qualified medical librarian, we performed a systematic

primary article search using EMBASE and MEDLINE databases to

identify all articles reporting fluconazole susceptibility testing in clinical

isolates of Cryptococcus spp. between 1988 and May 2017. There was no

language restriction. The last data search was performed on the 31st May

2017. Search terms used included “cryptococcal meningitis”,

“cryptococcosis”, “Cryptococcus”, “HIV”, “fluconazole resistance”,

“fluconazole”, “antifungal drug resistance”, “antifungal susceptibility

testing”.

Inclusion criteria

Randomized clinical trials, experimental studies on clinical isolates, case

series and observational studies were all included.

Exclusion criteria

Single case reports, systematic reviews and animal studies were

excluded.

Data extraction

The first and second authors independently reviewed all available titles

and abstracts, and articles were selected if they fulfilled the inclusion

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criteria above. In cases of doubt, the third author was consulted.

Reference sections of the identified studies were also examined in order

to identify additional relevant studies that were not identified by the

primary literature search (Fig. 1).

Data from selected publications extracted were: 1) study type, 2)

whether the patient case was incident, relapse or both, 3) country the

study was conducted in, 4) the antifungal susceptibility method

performed, 5) the Cryptococcus species isolated 6) the minimum

inhibitory concentration (MIC) cut-off value used to defined resistance,

and 7) the reported prevalence of resistance.

Results

Twenty-nine studies met the inclusion criteria, 21 (72.4%) were

published between 2010 and 2017. Of the 4,995 isolates tested in these

studies, only 248 (5.0%) were from relapse episodes of CM. Ten (34.5 %

%) of the studies were conducted in sub-Saharan Africa, 8 (27.6%) in

Europe, 5 (17.2%) in South America, 4 (13.8%) in Asia, and 2 (6.9 %) in

the United States of America. Half of the studies in sub-Saharan Africa

were conducted in South Africa. With respect to the design of these

published studies, majority (69.0%, n=20) were prospective cohort or

prospective population-based surveillance and the remainder 9 (31.0%)

were retrospective. Table 1 shows a summary of the studies evaluated

for discussion.

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Table 1: Summary of the studies evaluated for discussion

S/N

Author Year Country Type of study Number ofisolates

Population size

Species isolated Method of fluconazole susceptibility testing

MIC Cut-off

FluconazoleResistance (%)

1 Gago et al 15 2017 Spain Retrospective, case series

28 12 C. neoformans grubii (65%) & C. neoformans neoformans (35%)

Broth microdilution (CLSI)

≥64 μg/mL 29

2 Chen et al 16 2017 South Africa

Retrospective,Cohort, relapse isolates

38 18* C. grubii (84%) and C.gattii (16%)

Broth microdilution(CLSI)

≥16 μg/mL 50

3 Kassi et al 17 2016 Ivory coast

Retrospective, multicentric, cohort

363 61 C. neoformans var. neoformans (87.6%)

Broth microdilution(CLSI)

≥16 μg/mL 0.3

4 Herkert et al18 2016 Brazil Prospective 18 18 Cryptococcus gattiisensu lato,

Broth microdilutionCLSI

≥64 μg/mL 0

5 Figueiredo et al19 2016 Brazil Prospective 50 32 78.1% C. neoformans21.9% C. gattii

Broth microdilutionCLSI

≥64 μg/mL 0

6 Cordoba et al20 2016 Argentina

Prospective 707 n/a C. neoformans Broth Microdilution (EUCAST)

≥32 μg/mL 0

7 Hagen et al 21 2016 Denmark Prospective 108 n/a 91.7% C. neoformans6.5% C. gattii

Broth Microdilution(EUCAST)

≥32 μg/mL 2

8 Agudelo et al 22 2015 Colombia

Retrospective, multicentric, cohort

71 33 C. neoformans var grubii Disk diffusion method ≥64 μg/mL 22.7

9 Smith et al 23 2015 Uganda Retrospective, multicentric, cohort

198 198 C. neoformans Broth microdilution(CLSI)

≥64 μg/mL 3

10 Chen et al 24 2015 Taiwan Prospective, cohort 89 89 C. neoformans Broth microdilution(CLSI)

≥16 μg/mL 33.7

11 Van wyk et al 25 2014 South Africa

Prospective, population-based surveillance.

185 89 C. neoformans var. grubii (81%)

Broth microdilution(CLSI)

≥16 μg/mL 11

12 Kammalac et al 26 2014 Cameroon

Prospective, cohort study

146 146 C. neoformans E-tests ≥32 μg/mL 7.3

13 Arsenijevic et al 27* 2014 Serbia Prospective 34 25 C. neoformans Broth microdilutionCLSI

>16 lg ml 1/18#(2.9%)

14 Munivenkataswamy et al. 28 2013 India Prospective, population-based cohort study

12 12 C. neoformans E-test ≥64 μg/mL 10

15 Lee et al 29 2012 Taiwan Retrospective, cohort study

46 46 C. neoformans (89%) C. gattii (11%)

Broth microdilution(CLSI)

≥16 μg/mL 26.1

16 Trpkovic et al 30* 2012 Serbia Prospective 31 31 C. neoformans E-test ≥32 μg/mL 9/31# (29%)17 Govender et al 31 2011 South

AfricaProspective, population-based surveillance

487 487 C. neoformans Broth microdilution,(CLSI)

≥16 μg/mL 0.6

18 Mdodo et al 32 2011 Kenya Prospective, cohort 67 67 C. neoformans (94%)C. gattii (6%)

Broth microdilution(CLSI)

≥32 μg/mL 0

19 Mlinaric-Missoni et al 33 2011 Croatia Retrospective 48 15 C. neoformans Broth microdilution ≥16 μg/mL 0

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(CLSI)20 Jarvis et al 34 2010 South

AfricaProspective, cohort 300 300 C. neoformans E-test ≥64 μg/mL 0

21 Guinea et al 35 2010 Spain Prospective 58 55 C. neoformans Broth microdilution(CLSI)

≥16 μg/mL 3.6%

22 Arechavala et al 36 2009 Argentina

Retrospective, cohort, incident and relapse cases

265 (116/149)

116 C. neoformans Broth microdilution and E-tests

≥64 μg/mL 0/9.1#(0.4)

23 Bii et al 37 2007 Kenya Prospective, cohort 80 80 C. neoformans var. grubii (93.7%);C. neoformans var. neoformans (3.8%);C. gattii (2.5%)

Broth microdilution ≥64 μg/mL 11.2

24 Aller et al 38 2007 Spain Prospective, incident and relapse cases

70 (54/16)

58 C. neoformans Broth microdilution(CLSI)

≥16 μg/mL 17.4%

25 Bicanic et al 39 2006 South Africa

Prospective, cohort, relapse cases

32 27 C. neoformans E-tests ≥64 μg/mL 43.75

26 Perkins et al 40 2005 Spain Prospective 317 n/a C. neoformans Broth Microdilution (EUCAST)

≥16 μg/mL 46.6%

27 Sar et al 10 2004 Cambodia

Prospective, cohort 402 402 C. neoformans (98.5%) Broth microdilution (NCCLS) and E-tests

≥32 μg/mL 2.5/14# (14.9)

28 Brand et al 41 2001 USA Prospective, population-based surveillance

732 (368/364)#

732 C. neoformans Broth microdilution(CLSI)

≥64 μg/mL 1.1

29 Casadevall et al 42 1993 USA Retrospective, case series, relapse cases

13 5 C. neoformans var. neoformans Broth macrodilution(CLSI)

≥20 μg/mL 0

# Two cohorts of patients, incident and relapse prevalence were treated differently.*Possible overlap of isolates. n/a – population size not mentioned Abbreviations: MIC. Minimum inhibitory concentration. CLSI. Clinical and Laboratory Standards Institute. NCCL. National Centre for Clinical Laboratory. EUCAST. The European Committee on Antimicrobial Susceptibility Testing

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The clinical isolates in all these studies were obtained from HIV-infected

patients diagnosed with cryptococcal disease. C. neoformans was the

most common species isolated in all the studies. While 26 (89.7%) of the

studies were conducted on isolates from incident episodes of

cryptococcosis, 3 (10.3%) were from relapse/recurrent isolates, and 2

(7.0%) was from a mixture of both. Most of the studies, (n=22, 75.9%),

used broth microdilution MIC determination technique with either the

Clinical Laboratory Standards Institute (CLSI) or The European

Committee on Antimicrobial Susceptibility Testing (EUCAST) alone or in

combination with E-test (n=3, 13.6%) for fluconazole susceptibility

testing. Eleven (38%) of the studies used MIC breakpoints of ≥64 µg/mL

to define fluconazole resistance, 6 (21%) used ≥32 µg/mL, 11 (38 %) used

≥16 µg/mL, and 1 (3%) used ≤20 µg/mL.

Fluconazole resistance ranged from 0% to 50%. In 7 (24.1%) studies, all

isolates were fully susceptible to fluconazole. The highest resistance

rates were reported in South Africa (43.6% and 50%) 16,39 followed by

Spain (29% and 46.6%) 15,40, Taiwan (33.7% and 26.1%) 24,29 while, two

studies from the US revealed rates of 0% and 1.1% 41,42 (Table 1).

Overall, mean prevalence of fluconazole resistance was 12.1 % (95%

confidence interval (CI): 6.7% - 17.6%) for all isolates (n=4,995). Mean

fluconazole resistance was 10.6% (95% CI: 5.5% - 15.6%) for the incident

isolates (n=4,747), and 24.1% (95% CI: -3.1% - 51.2%) for the relapse

isolates (n=248) Fig.2. Of the 4,995 isolates, 936 (18.7%) had MICs

above the epidemiological cut-off value (ECV).

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Figure 2: Prevalence of fluconazole resistance in 4,995 clinical isolates. The mean prevalence was 12.1 % (95% CI: 6.7% - 17.6%) for all isolates, 10.6% (95% CI: 5.5% - 15.6%) for the incident isolate, and 24.1% (95% CI: -3.1% - 51.2%) for the relapse isolates.

Discussion

In this systematic review, we evaluated 29 studies reporting fluconazole

resistance in 4,995 clinical isolates of Cryptococcus spp. from HIV-

infected individuals. There are no accepted clinical breakpoints to define

fluconazole resistance in Cryptococcus isolates, thus there was use of

different breakpoints for fluconazole susceptibility in the reviewed

studies. To facilitate detection of emerging resistance to antifungals,

Pfaller et al 43 established a MIC of 8mg/L as an ECV for fluconazole for

C. neoformans using CLSI. Extrapolating this ECV, 936 (18.7%) isolates

from 25 studies with available MIC50 and MIC90 or MIC range had MICs

above 8mg/L and thus were resistant to fluconazole. This percentage is

higher than the one reported by Pfaller (4.8%) and might be an indication

of Cryptococcus resistance emergence.

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Over the years there has been a gradual increase of fluconazole

resistance in clinical isolates of C. neoformans. The first susceptibility

data reported from the US in 1993 demonstrated zero (0%) resistance in

13 isolates 42; however 8 years later another American multicentre study

with 732 isolates demonstrated 1.1% resistance 41. It will be interesting

to know if this upward trend is still persisting. The picture is essentially

the same in South Africa where in 2010 using the fluconazole E-test

gradient strip, the resistance was 0% amongst 300 isolates 34; the next

year it increased to 0.6% in 487 isolates 31, then 11% (2014) in 185

isolates 25. A recent publication by Chen and colleagues (2017)

demonstrated 50% in 38 isolates in South Africa 16. It is imperative to

state that an earlier report by Bicanic and colleagues (2006) revealed

that 76% of isolates had reduced susceptibility to fluconazole with 43.8%

of them completely resistant, however the numbers of isolates were just

32 and there were in CM relapses cases 39. Interestingly, it was the only

South African study that used fluconazole MICs ≥64 µg/mL. The picture

is also appears same for Taiwan 29 (Table 1). However, this might be due

to the low MIC (≤16 µg/mL) cut-off used compared to other studies in

this review where resistances were 10% or less when a break point of

≤32 µg/mL is used 29. Arechavala and colleagues from Argentina showed

a 9% emergence of fluconazole resistance in relapse isolates that were

fully susceptible to fluconazole at the time of diagnosis of the index

episode of CM 36. Recurrent cryptococcal diseases is currently thought to

be associated with antifungal resistance, poor compliance, changes in

immune function or a combination of these circumstances 42,44.

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An earlier study has shown that fluconazole suppression therapy reduced

the In vitro resistance of C. neoformans to fluconazole appeared to be

linked to extended maintenance treatments, isolates of incident episodes

of cryptococcal meningitis cases from HIV-infected patients from

Cambodia showed a steep rise in fluconazole resistance from 2.5% in the

first year to 14% in the second year of active laboratory based

surveillance 10.

Antifungal susceptibility tests have become essential tools to guide the

treatment of fungal diseases, to know the local and global disease

epidemiology, and to identify resistance to antifungals45. The EUCAST

and CLSI are responsible for the establishment and validation of MIC

interpretive breakpoints for antimicrobial susceptibility testing.

Antifungal susceptibility testing and molecular strain typing if possible

should always be performed on relapse isolates to investigate whether

the increase in resistance is due to selection pressure or whether it is a

new strain 42,46. However, the unavailability of MIC interpretive

breakpoints for any antifungal against Cryptococcus spp. together with

discrepancies between the available methods, makes it difficult to

correlate in vitro MICs and clinical outcome when a single episode is

tested 11. Most currently published studies however use previously

established fluconazole breakpoints, that is, MICs of ≤8 µg/ml as

susceptible, 16 to 32 µg/ml as dose-dependent susceptible, and ≥64

µg/ml as resistant 23,28,34. MIC values obtained by both broth micro- and

macro-dilution techniques have been shown to have good agreement 47.

Studies have shown that MICs can be potential predictors of the clinical

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response to fluconazole therapy and may aid in the identification of

patients who will not respond to fluconazole therapy, with therapeutic

failure observed in patients who were infected with isolates for which

fluconazole MICs were ≥16 μg/ml 48.

A major limitation in this review was the fact that due to the differences

in the methods of fluconazole susceptibility testing methods, MIC

breakpoint values and study design, a meta-analysis could not be

conducted, which would have helped to identify the predictors or drivers

of resistance.

Unlike polyenes and echinocandins, the mechanism of azole (e.g.

fluconazole) resistance is fairly well understood. One or more of the four

mechanisms contributes to azole resistance: 1) development of active

efflux pump resulting into decreased drug concentration at the site of

action, 2) alteration of drug target site (the enzyme lanosterol C14α-

demethylase), 3) up-regulation of the target enzyme, and 4) utilisation of

latter sterols of the ergosterol pathways negating the action of azoles 49.

Genetic mutations in ERG11, CDR and MDR genes in Candida spp. and

Cyp51A and Cyp51B genes in Aspergillus spp. have been well described

as the molecular mechanisms to azole resistance 50. In Cryptococcus spp.,

fluconazole resistance phenotype has been associated with point

mutation in the ERG11 gene responsible for the amino acid substitution

G470R, G484S, and expression of genes that encodes for efflux

mechanisms15,51. Finally, intrinsic fluconazole hetero-resistance in

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Cryptococcus spp. can lead to fluconazole resistance by selection from

hetero-resistant clones after induction by exposure to fluconazole 52.

Our analysis suggests that fluconazole resistance is a relatively common

event in relapse episodes of CM. However, as shown by the negative

lower 95% CI of the mean fluconazole resistance in the relapse isolates,

it is most likely that relapse of cryptococcal meningitis is multi-factorial

and not solely dependent on fluconazole resistance. Due to the limitations

of the currently available methods for in vitro fluconazole susceptibility

testing in Cryptococcus, the impact of this resistance on clinical

outcomes of patients is not fully understood. There is therefore a clear

need to establish break points for fluconazole testing in Cryptococcus

spp.

Transparency Declaration

All authors report no conflicts of interest relevant to this article.

Acknowledgement

We would like to thank Dr. Stephen Woods, a senior medical librarian at

the Academy Library of Wythenshawe Hospital, Manchester University

NHS Foundation Trust for helping us with literature search. The NC3Rs

training fellowship support Dr. Sara Gago’s work NC/P002390/1. The

Global Action Fund for Fungal Infections (GAFFI) supports Felix

Bongomin.

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References

1. Britz E, Perovic O, Von Mollendorf C, et al. The epidemiology of meningitis among adults in a south African province with a high HIV prevalence, 2009-2012. PLoS One. 2016;11(9):2009-2012. doi:10.1371/journal.pone.0163036.

2. Rajasingham R, Rachel MS, Benjamin JP, et al. Global Burden of Disease of HIV-Associated Cryptococcal Meningitis: an Updated Analysis. Lancet Infect Dis. 2017;3099(17):1-9. doi:10.1016/S1473-3099(17)30243-8.

3. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TAM. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. Aids. 2009;23(4):525-530. doi:doi: 10.1097/QAD.0b013e328322ffac.

4. Richardson K. The discovery and profile of fluconazole. J Chemother. 1990;2(1):51-54. http://www.ncbi.nlm.nih.gov/pubmed/2332784.

5. Kartalija M, Kaye K, Tureen JH, et al. Treatment of experimental cryptococcal meningitis with fluconazole: impact of dose and addition of flucytosine on mycologic and pathophysiologic outcome. J Infect Dis. 1996;173(5):1216-1221. http://www.ncbi.nlm.nih.gov/pubmed/8627075.

6. Perfect JR, Dismukes WE, Dromer F, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2010;50(3):291-322. doi:10.1086/649858.

7. Day JN, Chau TTH, Wolbers M, et al. Combination Antifungal Therapy for Cryptococcal Meningitis. N Engl J Med. 2013;368(14):1291-1302. doi:10.1056/NEJMoa1110404.

8. Mayanja-Kizza H, Oishi K, Mitarai S, et al. Combination therapy with fluconazole and flucytosine for cryptococcal meningitis in Ugandan patients with AIDS. Clin Infect Dis. 1998;26(6):1362-1366. doi:10.1086/516372.

9. Rapid advice: diagnosis, prevention and management of cryptococcal disease in HIV-infected adults, adolescents and children. World Health Organization HIV/AIDS program. http://apps.who.int/iris/handle/10665/44786. Published 2011. Accessed August 20, 2017.

10. Sar B, Monchy D, Vann M, Keo C, Sarthou JL, Buisson Y. Increasing in vitro resistance to fluconazole in Cryptococcus neoformans Cambodian isolates: April 2000 to March 2002. J Antimicrob Chemother. 2004;54(2):563-565. doi:10.1093/jac/dkh361.

11. Cheong JWS, McCormack J. Fluconazole resistance in cryptococcal disease: emerging or intrinsic? Med Mycol. 2013;51(3):261-269. doi:10.3109/13693786.2012.715763.

12. Peetermans W, Bobbaers H, Verhaegen J, Vandepitte J. Fluconazole-resistant Cryptococcus neoformans var gattii in an AIDS patient. Acta Clin Belg. 1993;48(6):405-409.

283

284285286287

288289290

291292293294

295296

297298299300

301302303304

305306307

308309310311

312313314315316

317318319320

321322323

324325326

Page 16 of 20

13. Venkateswarlu K, Taylor M, Manning NJ, Rinaldi MG, Kelly SL. Fluconazole tolerance in clinical isolates of Cryptococcus neoformans. Antimicrob Agents Chemother. 1997;41(4):748-751.

14. Moher D, Shamseer L, Clarke M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015;4(1):1. doi:10.1186/2046-4053-4-1.

15. Gago S, Serrano C, Alastruey-Izquierdo A, et al. Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain. Mycoses. 2017;60(1):40-50. doi:10.1111/myc.12543.

16. Chen Y, Farrer RA, Giamberardino C, et al. Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii. MBio. 2017;8(2):1-18. doi:10.1128/mBio.00166-17.

17. Kassi FK, Drakulovski P, Bellet V, et al. Molecular epidemiology reveals genetic diversity among 363 isolates of the Cryptococcus neoformans and Cryptococcus gattii species complex in 61 Ivorian HIV-positive patients. Mycoses. 2016;59(12):811-817. doi:10.1111/myc.12539.

18. Herkert PF, Hagen F, de Oliveira Salvador GL, et al. Molecular characterisation and antifungal susceptibility of clinical Cryptococcus deuterogattii (AFLP6/VGII) isolates from Southern Brazil. Eur J Clin Microbiol Infect Dis. 2016;35(11):1803-1810. doi:10.1007/s10096-016-2731-8.

19. Figueiredo TP, De Lucas RC, Cazzaniga RA, et al. Antifungal susceptibility testing and genotyping characterization of cryptococcus neoformans and gattii isolates from hiv-infected patients of Ribeirão Preto, São Paulo, Brazil. Rev Inst Med Trop Sao Paulo. 2016;58. doi:10.1590/S1678-9946201658069.

20. Córdoba S, Isla MG, Szusz W, Vivot W, Altamirano R, Davel G. Susceptibility profile and epidemiological cut-off values of Cryptococcus neoformans species complex from Argentina. Mycoses. 2016;59(6):351-356. doi:10.1111/myc.12479.

21. Hagen F, Hare Jensen R, Meis JF, Arendrup MC. Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark. Mycoses. 2016;59(9):576-584. doi:10.1111/myc.12507.

22. Agudelo CA, Muñoz C, Ramírez A, et al. Response to therapy in patients with cryptococcosis and AIDS: Association with in vitro susceptibility to fluconazole. Rev Iberoam Micol. 2015;32(4):214-220. doi:10.1016/j.riam.2014.07.006.

23. Smith KD, Achan B, Hullsiek KH, et al. Cryptococcus neoformans in Uganda. Antimicrob Agents Chemother. 2015;59(12):7197-7204. doi:10.1128/AAC.01299-15.Address.

24. Chen Y-C, Chang T-Y, Liu J-W, et al. Increasing trend of fluconazole-non-susceptible Cryptococcus neoformans in patients with invasive cryptococcosis: a 12-year longitudinal study. BMC Infect Dis.

327328329

330331332

333334335336

337338339

340341342343

344345346347348

349350351352353

354355356357

358359360361

362363364365

366367368

369370371

Page 17 of 20

2015;15(1):277. doi:10.1186/s12879-015-1023-8.

25. Van Wyk M, Govender NP, Mitchell TG, Litvintseva AP. Multilocus sequence typing of serially collected isolates of cryptococcus from HIV-infected patients in South Africa. J Clin Microbiol. 2014;52(6):1921-1931. doi:10.1128/JCM.03177-13.

26. Kammalac Ngouana T, Dongtsa J, Kouanfack C, et al. Cryptoccocal meningitis in Yaoundé (Cameroon) HIV infected patients: Diagnosis, frequency and Cryptococcus neoformans isolates susceptibility study to fluconazole. J Mycol Med. 2015;25(1):11-16. doi:10.1016/j.mycmed.2014.10.016.

27. Arsic Arsenijevic V, Pekmezovic MG, Meis JF, Hagen F. Molecular epidemiology and antifungal susceptibility of Serbian Cryptococcus neoformans isolates. Mycoses. January 2014:n/a-n/a. doi:10.1111/myc.12171.

28. Munivenkataswamy R, Gopi A, Mohammed Usman S, Jagadeesh. Human immunodefeciency virus associated cryptococcal meningitis at a tertiary care centre: Diagnostic tools and Antifungal susceptibility testing. J Clin Diagnostic Res. 2013;7(8):1623-1625. doi:10.7860/JCDR/2013/6147.3271.

29. Lee C-H, Chang T-Y, Liu J-W, et al. Correlation of anti-fungal susceptibility with clinical outcomes in patients with cryptococcal meningitis. BMC Infect Dis. 2012;12(1):361. doi:10.1186/1471-2334-12-361.

30. Trpković A, Pekmezović M, Barać A, Crnčević Radović L, Arsić Arsenijević V. In vitro antifungal activities of amphotericin B, 5-fluorocytosine, fluconazole and itraconazole against Cryptococcus neoformans isolated from cerebrospinal fluid and blood from patients in Serbia. J Mycol Med. 2012;22(3):243-248. doi:10.1016/j.mycmed.2012.06.002.

31. Govender NP, Patel J, Van Wyk M, Chiller TM, Lockhart SR. Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates obtained through population-based surveillance in South Africa in 2002-2003 and 2007-2008. Antimicrob Agents Chemother. 2011;55(6):2606-2611. doi:10.1128/AAC.00048-11.

32. Mdodo R, Moser SA, Jaoko W, et al. Antifungal susceptibilities of Cryptococcus neoformans cerebrospinal fluid isolates from AIDS patients in Kenya. Mycoses. 2011;54(5):438-442. doi:10.1111/j.1439-0507.2010.01946.x.

33. Mlinaric-Missoni E, Hagen F, Chew WHM, Vazic-Babic V, Boekhout T, Begovac J. In vitro antifungal susceptibilities and molecular typing of sequentially isolated clinical Cryptococcus neoformans strains from Croatia. J Med Microbiol. 2011;60(10):1487-1495. doi:10.1099/jmm.0.031344-0.

34. Jarvis JN, Meintjes G, Williams Z, Rebe K, Harrison TS. Symptomatic relapse of HIV-associated cryptococcal meningitis in South Africa: the role of inadequate secondary prophylaxis. S Afr Med J. 2010;100(6):378-382.

35. Guinea J, Hagen F, Peláez T, et al. Antifungal susceptibility, serotyping, and genotyping of clinical Cryptococcus neoformans isolates collected

372

373374375376

377378379380381

382383384385

386387388389

390391392

393394395396397

398399400401402

403404405406

407408409410411

412413414

415416

Page 18 of 20

during 18 years in a single institution in Madrid, Spain. Med Mycol. 2010;48(7):942-948. doi:10.3109/13693781003690067.

36. Arechavala AI, Ochiuzzi ME, Borgnia MD, Santiso GM. Fluconazole and amphotericin B susceptibility testing of Cryptococcus neoformans: Results of minimal inhibitory concentrations against 265 isolates from HIV-positive patients before and after two or more months of antifungal therapy. Rev Iberoam Micol. 2009;26(3):194-197. doi:10.1016/j.riam.2009.02.001.

37. Bii CC, Makimura K, Abe S, et al. Antifungal drug susceptibility of Cryptococcus neoformans from clinical sources in Nairobi, Kenya. Mycoses. 2007;50(1):25-30. doi:10.1111/j.1439-0507.2006.01293.x.

38. Aller AI, Claro R, Castro C, Serrano C, Colom MF, Martín-Mazuelos E. Antifungal Susceptibility of Cryptococcus neoformans Isolates in HIV-Infected Patients to Fluconazole, Itraconazole and Voriconazole in Spain: 1994–1996 and 1997–2005. Chemotherapy. 2007;53(4):300-305. doi:10.1159/000102585.

39. Bicanic T, Harrison T, Niepieklo A, Dyakopu N, Meintjes G. Symptomatic relapse of HIV-associated cryptococcal meningitis after initial fluconazole monotherapy: the role of fluconazole resistance and immune reconstitution. Clin Infect Dis. 2006;43(8):1069-1073. doi:10.1086/507895.

40. Perkins A, Gomez-Lopez A, Mellado E, Rodriguez-Tudela JL, Cuenca-Estrella M. Rates of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var. neoformans. J Antimicrob Chemother. 2005;56(6):1144-1147. doi:10.1093/jac/dki393.

41. Brandt ME, Pfaller MA, Hajjeh RA, et al. Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates in the United States: 1992 to 1994 and 1996 to 1998. Antimicrob Agents Chemother. 2001;45(11):3065-3069. doi:10.1128/AAC.45.11.3065-3069.2001.

42. Casadevall A, Spitzer ED, Webb D, Rinaldi MG. Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole. Antimicrob Agents Chemother. 1993;37(6):1383-1386. doi:10.1128/AAC.37.6.1383.Updated.

43. Pfaller MA, Castanheira M, Diekema DJ, Messer SA, Jones RN. Wild-type MIC distributions and epidemiologic cutoff values for fluconazole, posaconazole, and voriconazole when testing Cryptococcus neoformans as determined by the CLSI broth microdilution method. Diagn Microbiol Infect Dis. 2011;71(3):252-259. doi:10.1016/j.diagmicrobio.2011.07.007.

44. Govender N, Meintjes G, Bicanic T, et al. Guideline for the prevention, diagnosis and management of cryptococcal meningitis among HIV-infected persons: 2013 update. South Afr J HIV Med. 2013;14(2):76. doi:10.7196/sajhivmed.930.

45. Alastruey-Izquierdo A, Melhem MSC, Bonfietti LX, Rodriguez-Tudela JL. Susceptibility Test for Fungi: Clinical and Laboratorial Correlations in Medical Mycology. Rev Inst Med Trop Sao Paulo. 2015;57(Suppl 19):57-

417418

419420421422423424

425426427

428429430431432

433434435436437

438439440441

442443444445

446447448449450

451452453454455

456457458459

460461462

Page 19 of 20

64. doi:10.1590/S0036-46652015000700011.

46. Pfaller MA, Messer SA, Boyken L, et al. Global Trends in the Antifungal Susceptibility of Cryptococcus neoformans (1990 to 2004). J Clin Microbiol. 2005;43(5):2163-2167. doi:10.1128/JCM.43.5.2163.

47. Datta K, Jain N, Sethi S, Rattan A, Casadevall A, Banerjee U. Fluconazole and itraconazole susceptibility of clinical isolates of Cryptococcus neoformans at a tertiary care centre in India: A need for care. J Antimicrob Chemother. 2003;52(4):683-686. doi:10.1093/jac/dkg399.

48. Aller AI, Martin-Mazuelos E, Lozano F, et al. Correlation of fluconazole MICs with clinical outcome in cryptococcal infection. Antimicrob Agents Chemother. 2000;44(6):1544-1548. doi:10.1128/AAC.44.6.1544-1548.2000.

49. Kanafani ZA, Perfect JR. Resistance to Antifungal Agents: Mechanisms and Clinical Impact. Clin Infect Dis. 2008;46(1):120-128. doi:10.1086/524071.

50. Pam VK, Akpan JU, Oduyebo OO, et al. Fluconazole susceptibility and ERG11 gene expression in vaginal candida species isolated from lagos Nigeria. Int J Mol Epidemiol Genet. 2012;3(1):84-90.

51. Rodero L, Mellado E, Rodriguez AC, et al. G484S Amino Acid Substitution in Lanosterol 14-α Demethylase (ERG11) Is Related to Fluconazole Resistance in a Recurrent Cryptococcus neoformans Clinical Isolate. Antimicrob Agents Chemother. 2003;47(11):3653-3656. doi:10.1128/AAC.47.11.3653-3656.2003.

52. Sionov E, Chang YC, Garraffo HM, Kwon-Chung KJ. Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence. Antimicrob Agents Chemother. 2009;53(7):2804-2815. doi:10.1128/AAC.00295-09.

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Figure legends

Fig.1: Study selection criteria according to PRISMA flow diagram

Fig.2: Figure 2: Prevalence of fluconazole resistance in 4,995 clinical isolates. The mean prevalence was 12.1 % (95% CI: 6.7% - 17.6%) for all isolates, 10.6% (95% CI: 5.5% - 15.6%) for the incident isolate, and 24.1% (95% CI: -3.1% - 51.2%) for the relapse isolates.

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