Summary
Introduction:
The prevalence of azole resistance in C parapsilosis is very low in most parts of the world. However, South Africa has reported an exceptionally high prevalence of azole resistance in C parapsilosis strains isolated from candidaemia cases. We aimed to determine the possible molecular mechanisms of fluconazole resistance in C parapsilosis isolates obtained through surveillance at a large neonatal unit at a South African academic hospital.
Methods:
We sequenced the ERG11 and MRR1 genes of C parapsilosis isolates recovered from cases of neonatal candidemia, followed by microsatellite genotyping. A total of 73 isolates with antifungal susceptibility results were analysed.
Results:
Of these, 57 (78%) were resistant, 11 (15%) susceptible dose-dependent and 5 (7%) susceptible. The most commonly identified amino acid substitution within the ERG11 gene was Y132F in 68% (39/57) of fluconazole-resistant isolates and none in susceptible isolates. Three amino acid substitutions (R405K, G583R and A619V) and 1 nucleotide deletion at position 1331 were identified within MRR1 gene in 19 (26%) isolates. Microsatellite genotyping grouped isolates into four clusters (50 isolates). Cluster 1 accounted for 23% (17/73) of all cases, cluster 2 for 22% (16/73), cluster 3 for 14% (10/73) and cluster 4 for 10% (7/73). We found an association between cluster type and fluconazole resistance (P-value = .004). Isolates harbouring the Y132F substitution were more likely to belong to a cluster than non-Y132F isolates.
Conclusion:
Fluconazole resistance in C parapsilosis strains from a single South African neonatal unit was associated with cluster type and predominantly driven by Y123F amino acid substitutions in the ERG11 gene.
Keywords: Candida parapsilosis, candidemia, ERG11, fluconazole resistance, MRR1, South Africa
1 |. INTRODUCTION
Candidemia is associated with high morbidity and mortality in neonatal units.1,2 Candida parapsilosis is the second most common non-albicans Candida species in South African public sector hospitals and a common cause of candidemia in neonates, accounting for 53% of cases.3 The prevalence of azole resistance in South African C parapsilosis strains is exceptionally high (58%–63%) versus other parts of the world (1%–7.7%).3–7 Mechanisms leading to azole resistance are better characterised in Candida albicans than in other Candida species. Resistance may occur in one of the following ways: (a) point mutations in ERG11 gene, resulting in reduced azole binding affinity; (b) overexpression of the drug target (ERG11) gene which encodes for 14α demethylase; (c) overexpression of the genes encoding multidrug transporters [ATP binding cassettes (ABC) and major facilitator superfamily (MFS)]; (d) overexpression of CDR1 and CDR caused by activating mutations in the transcription factor genes TAC1 leading to efflux of all azole antifungal agents8; (e) overexpression of an MFS transporter (MDR1) caused by activating mutations in the transcription factor genes MRR1 leading to efflux of fluconazole and voriconazole9–11; and (f) mutations in ERG3 gene resulting in the inactivation of sterol desaturase. A number of studies from the United States of America, United Kingdom, Brazil, Portugal, South Korea and India have recently explored the possible mechanisms of azole resistance in C parapsilosis strains.12–17 Souza et al (2015) demonstrated that not only does upregulation of MDR1 and amino acid substitutions in ERG11 lead to resistance to fluconazole but upregulation of ERG11 and CDR1 are involved as well.14 The high prevalence of fluconazole resistance in South African C parapsilosis bloodstream isolates provides a unique opportunity to investigate the mechanisms of fluconazole resistance in our setting. We describe the possible molecular mechanisms of fluconazole resistance in C parapsilosis isolates causing invasive neonatal infections at Chris Hani Baragwanath Academic Hospital, Soweto, South Africa.
2 |. MATERIALS AND METHODS
We conducted laboratory-based surveillance at Chris Hani Baragwanath Academic Hospital through the TRAC-SA programme from 2009 to 2010. Reporting laboratories submitted bloodstream Candida isolates to the National Institute for Communicable Diseases (NICD) in Johannesburg with corresponding laboratory reports containing patient demographic data. A case of candidemia was defined as a person of any age admitted to any hospital with first isolation of any Candida species from blood culture. For the purposes of this molecular sub-study, a neonate was defined as a patient aged ≤ 28 days admitted to a neonatal unit at Chris Hani Baragwanath Academic Hospital. Any patient beyond this age was excluded. Chris Hani Baragwanath Academic Hospital is the third largest hospital in the world with approximately 3200 beds. The neonatal unit at this hospital forms part of the department of paediatrics, which has 408 beds. In 2000, 5645 children were admitted and the death rate was 9%. Half the admitted children were neonates. Incidence of neonatal candidemia was calculated using the annualised number of neonatal cases as a numerator and the annual number of admissions to the neonatal unit as a denominator. The annualised number of cases was also used to calculate the proportion of positive neonatal blood cultures that were attributed to Candida species at the same hospital.
Candida isolates were submitted to National Institute for Communicable Diseases (NICD) from the laboratory at Chris Hani Baragwanath Academic Hospital. Phenotypic identification was initially performed using ChromAgar Candida medium (Mast Diagnostics, Merseyside, UK), Vitek 2 YST, API 20C Aux and API ID 32C (bioMérieux, Marcy l’Etoile, France). Minimum inhibitory concentrations (MIC) against nine antifungal agents were determined and categorised using Clinical and Laboratory Standards Institute (CLSI) M60 interpretive breakpoints. C parapsilosis ATCC 22019 and C krusei ATCC 6258 strains were included in quality control (QC) runs on all days of testing and MICs were consistently found to be within QC range. Sequencing of the internal transcribed spacer (ITS) domain of the multi-copy ribosomal RNA gene was performed to confirm species identification using universal primers.18 Isolates that were confirmed as C parapsilosis sensu stricto were then genotyped by polymorphic microsatellite marker analysis.19 To screen for mutations that confer resistance to azole antifungals, we sequenced the ERG11 gene and transcription factor gene MRR1 of C parapsilosis isolates using a previously described method.13 The deduced nucleotide sequences were then compared to C parapsilosis 22019 ATCC wild-type ERG11 and CDC317 wild-type MRR1 strain. Pearson’s chisquare test or Student’s t test was used to compare categorical or continuous variables. Analysis was performed with Stata version 14.0 (StataCorp). Approval for laboratory-based surveillance was obtained from the Human Research Ethics Committee (Medical), University of the Witwatersrand, Johannesburg.
3 |. RESULTS
During the surveillance period, 204 cases of candidemia occurred among infants aged < 1 year admitted to Chris Hani Baragwanath Academic Hospital. Of these, 69% (140/204) of cases occurred among neonates. C parapsilosis accounted for 59% (82/140) of cases of neonatal candidemia at this hospital, followed by C albicans and Candida glabrata accounting for 31% (44/140) and 6% (8/140), respectively. Of the 82 neonatal C parapsilosis cases, 73 cases had viable isolates; the remaining nine were no longer viable for further molecular characterisation. The incidence of neonatal candidemia at Chris Hani Baragwanath Hospital (23 440 neonatal unit admissions) was 49 cases per 10 000 admissions in 2009. During the same year, Candida species accounted for 8% (115/1376) of positive blood cultures in this hospital’s neonatal unit.
Using ITS gene sequencing, all 73 isolates were confirmed as C parapsilosis sensu stricto. Of these, 78% (57/73) were resistant to fluconazole, 15% (11/73) were susceptible dose-dependent (SDD) and 7% (5/73) were susceptible (Table 1). Two ERG11 amino acid substitutions, Y132F and R398I, were identified in 82% (47/57) of fluconazole-resistant isolates. The most commonly identified substitution was Y132F in 68% (39/57) of fluconazole-resistant isolates and none in susceptible isolates. Ninety-two per cent (36/39) of Y132F mutations were in combination with the R398I substitution. Three amino acid substitutions, R405K, G583R and A619V, and one nucleotide deletion at position 1331 which caused a frameshift mutation were identified within MRR1 gene in 19 of 73 (26%) isolates. The missense mutation, G583R, was exclusively identified in three (4%) resistant isolates. Another substitution, A619V was identified in four (5%) resistant and two (3%) SDD isolates. One nucleotide deletion at position 1331 was identified in one SDD and one resistant isolate.
TABLE 1.
Antifungal susceptibility profile of Candida parapsilosis isolates from cases of neonatal candidaemia, TRAC-SA [2009–2010 (n = 73)]
| Number of isolates with MIC (mg/L) | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Antifungal agents | <0.008 | 0.015 | 0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | >64 | Susceptible | Susceptible dose-dependent | Resistant | Wild type | Non-wild type |
| Amphotericin B | 1 | 35 | 37 | - | - | - | 36 (49) | 37 (51) | |||||||||||
| Fluconazole | 1 | 1 | 3 | 11 | 10 | 26 | 19 | 2 | 5 (7) | 11 (15) | 57 (78) | 5 (7) | 68 (93) | ||||||
| Voriconazole | 2 | 2 | 1 | 13 | 5 | 16 | 10 | 21 | 3 | 26 (36) | 31 (42) | 16 (22) | 18 (25) | 55 (75) | |||||
| Itraconazole | 4 | 23 | 50 | 4 | 1 | 26 (36) | 47 (64) | 0 (0) | 71 (97) | 2 (3) | |||||||||
| Posaconazole | 3 | 8 | 14 | 21 | 27 | - | - | - | 11 (15) | 62 (85) | |||||||||
| Caspofungin | 1 | 1 | 1 | 34 | 39 | 73 (100) | 0 (0) | 0 (0) | 34 (47) | 39 (53) | |||||||||
| Micafungin | 1 | 2 | 2 | 16 | 52 | 73 (100) | 0 (0) | 0 (0) | 73 (100) | 0 (0) | |||||||||
| Anidulafungin | 1 | 1 | 1 | 2 | 56 | 11 | 1 | 72 (99) | 1 (1) | 0 (0) | 72 (99) | 1 (1) | |||||||
| Flucytosine | 40 | 30 | 3 | 73 (100) | 0 (0) | 0 (0) | 73 (100) | 0 (0) | |||||||||||
Note: A wild-type isolate was defined as an isolate with a minimum inhibitory concentration ≤ published epidemiologic cut-off value for the specified agent using the Sensititre YeastOne method.
Four clusters comprising of 50 isolates were identified in this hospital by microsatellite genotyping. Cluster 1 accounted for 23% (17/73) of all cases, cluster 2 for 22% (16/73), cluster 3 for 14% (10/73) and cluster 4 for 10% (7/73). Twenty-three isolates (32%) did not belong to a cluster (Figure 1). Compared to non-cluster isolates, a larger proportion of isolates belonging to clusters were associated with fluconazole resistance (15/23 [65%] versus 41/50 [82%], P-value = .004). All isolates belonging to cluster 4, a majority of cluster 1 (13/17; 76%), cluster 2 (14/16; 87%) and cluster 3 (7/10; 70%) isolates were resistant. Isolates with Y132F substitution had a 39% higher odds of belonging to a cluster than non-Y132F isolates (odds ratio 1.39; 95% CI, 0.48–4.00; P-value = .6) (Table 2).
FIGURE 1.
Dendrogram generated by UPGAM based on microsatellite analysis results showing clustering of fluconazole-resistant Candida parapsilosis strains from a single neonatal unit
TABLE 2.
Fluconazole-resistant Candida parapsilosis isolates with ERG11 Y132F substitution by cluster type and year (n = 39)
| MIC (g/mL) | ||||
| Isolate ID | Fluconazole | Voriconazole | Year isolated | Microsatellite cluster |
| MRU05 | 16 | 0.25 | 2009 | 1 |
| MRU09 | 16 | 1 | 2010 | 3 |
| MRU10 | 32 | 0.25 | 2010 | NC |
| MRU14 | 16 | 0.5 | 2010 | 3 |
| MRU15 | 16 | 0.25 | 2010 | NC |
| MRU16 | 32 | 1 | 2010 | NC |
| MRU17 | 32 | 0.5 | 2010 | NC |
| MRU18 | 16 | 0.25 | 2010 | NC |
| MRU20 | 16 | 0.25 | 2010 | 3 |
| MRU22 | 32 | 1 | 2010 | 2 |
| MRU25 | 16 | 0.12 | 2010 | 3 |
| MRU28 | 16 | 1 | 2010 | 3 |
| MRU30 | 16 | 0.12 | 2009 | 2 |
| MRU34 | 16 | 0.25 | 2009 | 2 |
| MRU36 | 32 | 1 | 2010 | 1 |
| MRU37 | 16 | 0.25 | 2009 | 1 |
| MRU39 | 16 | 0.5 | 2009 | 2 |
| MRU41 | 32 | 0.5 | 2009 | 1 |
| MRU42 | 32 | 0.5 | 2009 | 2 |
| MRU43 | 64 | 0.5 | 2009 | 2 |
| MRU47 | 32 | 1 | 2009 | 2 |
| MRU48 | 32 | 1 | 2010 | 2 |
| MRU50 | 16 | 0.5 | 2009 | 1 |
| MRU51 | 16 | 0.5 | 2009 | 1 |
| MRU52 | 32 | 1 | 2009 | 2 |
| MRU55 | 32 | 0.5 | 2009 | 1 |
| MRU56 | 16 | 2 | 2009 | NC |
| MRU58 | 32 | 1 | 2009 | 4 |
| MRU59 | 16 | 1 | 2009 | 4 |
| MRU60 | 32 | 1 | 2009 | 4 |
| MRU61 | 16 | 1 | 2009 | NC |
| MRU63 | 32 | 1 | 2009 | 4 |
| MRU64 | 32 | 1 | 2009 | NC |
| MRU66 | 16 | 1 | 2009 | NC |
| MRU67 | 16 | 1 | 2009 | NC |
| MRU68 | 16 | 0.5 | 2009 | 1 |
| MRU70 | 32 | 0.015 | 2009 | 1 |
| MRU72 | 32 | 1 | 2009 | 4 |
| MRU73 | 64 | 2 | 2009 | NC |
Abbreviation: NC, Non-cluster isolates.
4 |. DISCUSSION
Candida parapsilosis has emerged as the most common cause of nosocomial fungal bloodstream infections in neonates in South Africa.3,7 During the 2009–2010 surveillance period, we demonstrated that C parapsilosis was the major cause of candidemia at Chris Hani Baragwanath Academic Hospital neonatal unit. Almost 80% of isolates were resistant to fluconazole. The predominant amino acid substitution was Y132F in the ERG11 gene, accounting for 68% of fluconazole-resistant isolates. We found an association between fluconazole resistance mutations and cluster types. Y132F isolates were more likely to belong to a cluster than non-Y132F isolates. Gain-of-function mutations in MRR1 gene (G583R and A619R) accounted for a small proportion of fluconazole resistance.
The prevalence of fluconazole-resistant C parapsilosis in other parts of the world including the Latin America, USA (7.7%) and Asia Western Pacific (6%) is much lower (1%–6%).4–6,20 To date, South Africa has reported the highest fluconazole resistance in C parapsilosis (58%–63%) globally.3,7 The current study reports fluconazole resistance of 78%, which is the highest to date to be reported in South Africa compared to previous studies. In recent years, studies have shown that point mutations within the ERG11 gene are major drivers of fluconazole resistance in Candida species. Similarly, our data showed point mutations within ERG11 gene constitutes a majority (47/57; 82%) of mutations in fluconazole-resistant isolates. Of particular interest, Y132F substitution was found to be a major contributor to fluconazole resistance in South African C parapsilosis isolates, accounting for 68% (39/57) of the fluconazole-resistant isolates, which is considerably higher than reported in other countries. Recent studies showed that Y132F substitution accounts for 31%–57% of fluconazole-resistant C parapsilosis isolates world-wide. This amino acid substitution Y132F was found exclusively in fluconazole-resistant isolates and none in susceptible and SDD isolates.12–14,21 Our study findings confirm that Y132F is the predominant mechanism driving fluconazole resistance in C parapsilosis. Y132F has also been reported in fluconazole-resistant C albicans, C tropicalis and C auris isolates, though South African C auris strains have the F126T substitution.22–24 The presence of Y132F and other substitutions at this position in C albicans and C tropicalis has been associated with reduced susceptibility to azole antifungals.20,21 Another substitution, R398I was identified in 92% (36/39) of Y132F isolates. This was a similar occurrence in other studies, suggesting that R398I alone cannot drive or facilitate fluconazole resistance in C parapsilosis isolates but may function to enhance resistance or to stabilise the conformational change derived from the other substitution.12,13,15,25 Further analysis needs to be undertaken to ascertain the role played by R398I point mutation in fluconazole resistance.
Sequence analysis of the transcription factor gene MRR1 revealed three amino acid substitutions G478R, G583R and A619V which were exclusively identified in both SDD and resistant isolates. Although our study did not explore the effects of these substitutions on expression of MDR1, previous studies have shown that these substitutions have some form of gain-of-function activity in fluconazole-resistant C parapsilosis isolates. Initial studies indicate these substitutions change fluconazole susceptibility in C parapsilosis isolates as they were identified exclusively in SDD and resistant isolates.13,26 Branco et al (2015) successfully demonstrated that G583R confers hyperactivity to MRR1 transcription factor leading to upregulation of MDR1 drug transporter.16 In the present study, A619V was found in 2 SDD and 4 resistant isolates. This was similar to an observation in a multicentre US study, where this substitution was identified in a resistant isolate with 16-fold increased levels of MDR1 and in an SDD isolate with 10-fold increased levels of MDR1. The authors suggested that A619V has a moderate gain-of-function activity.13 Although we did not assess the expression levels of MDR1 in the current study, based on the expression results of the same SNPs from other studies, our findings confirm that MDR1 is an important role player in fluconazole resistance in C parapsilosis.
Microsatellite analysis showed that Y132F isolates were more likely to belong to a cluster than non-Y132F isolates, suggesting that C parapsilosis strains with or without a Y132F substitution isolated in this particular hospital’s neonatal unit were equally responsible for candidemia outbreaks over an 18-month period. The fact that it appeared in multiple clusters also suggests that the mutation was acquired independently in multiple isolates rather than exclusively through clonal transmission. The presence of Y132F substitution in small clonal clusters in hospitals or communities from the US and South Korea studies may suggest that C parapsilosis isolates harbouring this substitution may have a competitive advantage over non-Y132F fluconazole-resistant isolates in a hospital setting.13,15 The same Y132F substitution has been detected in clonal C auris strains from Pakistan, India and Venezuela.24 This is concerning because both C parapsilosis and C auris have been implicated in nosocomial outbreaks of candidaemia in hospital settings, and they are resistant to fluconazole which is a treatment of choice for invasive candidiasis particularly in resource-limited countries where echinocandins are not available for public sector hospitals. As such, it is important to conduct ongoing surveillance for fluconazole-resistant non-albicans Candida species because of their ability to persist in the hospital environment, medical devices, hands of healthcare workers and eventually causing outbreaks.
A number of limitations were identified in this study: first, we only analysed isolates and data from a single centre to establish the association between clusters and mutations. Therefore, the data may not be representative of other medical centres in the country or region. Second, we had very limited demographic, clinical and treatment data for these cases. Lastly, we did not undertake quantitative real-time PCR to determine the expression levels of ERG11 and MDR1 in isolates with SNPs in the ERG11 gene and MRR1 transcription factor gene.
5 |. CONCLUSIONS
We demonstrated that fluconazole resistance in C parapsilosis strains from a single South African neonatal unit was significantly associated with clusters and predominantly driven by Y123F amino acid substitutions in the ERG11 gene. Although a large number of isolates had the Y132F substitution, it was not found exclusively in a single cluster.
ACKNOWLEDGEMENTS
We acknowledge the technical assistance provided by staff members of the National Institute for Communicable Diseases (Centre for Healthcare-Associated Infections, Antimicrobial Resistance and Mycoses). We specifically acknowledge personnel at the neonatal unit and NHLS microbiology laboratory at Chris Hani Baragwanath Academic Hospital for participating in the TRAC-SA surveillance project.
Members of the TRAC-SA surveillance group: NHLS Chris Hani Baragwanath: Jeannette Wadula; van Rensburg and partners: Chris Janse van Rensburg; NHLS Groote Schuur: Andrew Whitelaw; Ampath National Laboratory Service: Inge Zietsman (co-principal investigator), Norman Miller, Peter Smith, Johan van Greune, Adrian Brink; NHLS Steve Biko Pretoria Academic: Anwar Hoosen, NHLS Charlotte Maxeke Johannesburg Academic: Olga Perovic; NHLS Dr George Mukhari: Maphoshane Nchabaleng; NHLS Tygerberg: Heidi Orth; NHLS Inkosi Albert Luthuli: Yacoob Coovadia; NHLS Universitas: Loekie Badenhorst; Lancet laboratories: Johan Moolman, AK Peer, Chetna Govind; NHLS Helen Joseph/Rahima Moosa: Ranmini Kularatne, Barry Bhagoobhai; Vermaak and partners: Ben Prinsloo; NHLS Grey’s: Sumayya Haffejee; NHLS Greenpoint: John Simpson; NHLS Rob Ferreira: Greta Hoyland; PathCare laboratories: Marthinus van Schalkwyk; NHLS East London: Glenda Bowie; NHLS Mthatha: Patricia Hanise, Sandeep Vasaikar; NHLS Pelonomi: Linda Wende; US Centers for Disease Control and Prevention: Tom Chiller, Angela Ahlquist-Cleveland, Shawn Lockhart; National Institute for Communicable Diseases: Jaymati Patel, Nelesh P. Govender (principal investigator).
Funding information
National Institute for Communicable Diseases; National Research Fund Thuthuka grant number: 99302 awarded to REM; TRAC-SA project was funded by an investigator-initiated research grant from Pfizer (to NPG and ILZ). The external funders had no role in study design, implementation or data collection, analysis and reporting.
Footnotes
CONFLICT OF INTEREST
None.
DISCLAIMER
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the US Centers for Disease Control and Prevention (CDC).
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