Candidemia in Brazilian neonatal intensive care units: risk factors, epidemiology, and antifungal resistance

Abstract

Candidemia is responsible for substantial morbidity and mortality in neonatal intensive care units and represents a challenge due to the complexity of hospitalized neonates, the deficiency in approved and precise diagnostic techniques, and the increasing number of species resistant to antifungal agents. Thus, the objective of this study was to detect candidemia among neonates evaluating the risk factors, epidemiology, and antifungal susceptibility. Blood samples were obtained from neonates with suspected septicemia, and the mycological diagnosis was based on yeast growth in culture. The fungal taxonomy was based on classic identification, automated system, and proteomic, when necessary molecular tools were used. The in vitro susceptibility tests were performed according to the broth microdilution method from Clinical and Laboratory Standards Institute. Statistical analysis was performed using the R software version R-4.2.2. The prevalence of neonatal candidemia was 10.97%. The major risk factors involved were previous use of parenteral nutrition, exposure to broad-spectrum antibiotics, prematurity, and prior use central venous catheter, but only this last was statistically associated with mortality risk. Species from Candida parapsilosis complex and C. albicans were the most frequent. All isolates were susceptible to amphotericin B, except C. haemulonii that also exhibited elevated MICs to fluconazole. C. parapsilosis complex and C. glabrata exhibit the highest MICs to echinocandins. Considering these data, we emphasize that an effective management strategy to reduce the impact of neonatal candidemia should involve the knowledge of risk factors, rapid and precise mycological diagnostic, and tests of antifungal susceptibility to help in the selection of an appropriate treatment.

Introduction

The ubiquitous yeasts belonging to the Candida genus are opportunistic pathogens which, despite treatment with antifungal drugs, can cause fatal invasive infection in immunocompromised people, including critically ill neonates. In this context, candidemia, characterized as the presence of Candida species in bloodstream, is the leading cause of invasive fungal infections in neonatal intensive care units (NICUs) and is responsible for substantial morbidity and mortality in newborns complicating the clinical course [1, 2].

Considering the NICU environment, the major risk factors associated with candidemia development are prematurity, low birth weight, immunosuppressive conditions (including immaturity of skin and gastrointestinal tract), the prolonged hospital stay, total parenteral nutrition (TPN), the use of intensive care devices (mechanical ventilation, central venous access), and exposure to medications that promote fungal growth (postnatal corticosteroids and broad-spectrum antibiotics) [3–5].

In this context, the economic impact of these infections is also important, considering that hematogenous Candida infections have been associated with increased costs of care and prolonged hospitalization time [6].

The epidemiology of this type of invasive infection is changing; although C. albicans remains the most common fungal agent from neonatal candidemia, many studies detected an increasing number of non-C. albicans species (NCAs) as etiological agent, including C. parapsilosis, C. guilliermondii (current name Meyerozyma guilliermondii), C. tropicalis, and C. glabrata [1, 7]. Understanding the epidemiology of Candida species is critical, as health professionals often begin empiric therapy before antifungal susceptibilities are known [8].

Furthermore, notable differences in Candida species distribution can be observed between different hospital units and also dependent on the predisposing conditions of the patients infected and the antifungal agents they receive as prophylaxis or therapy [9].

Due to the complexity of neonatal patients at risk for candidemia, the deficiency in approved sensitive and precise diagnostic techniques, and the increasing number of species that are resistant and refractory to commonly used therapies, this opportunistic mycosis represents a challenge for diagnosis and treatment [1, 10].

Thus, the objective of this study was to detect candidemia among neonates hospitalized in NICUs evaluating the risk factors and antifungal susceptibility of the etiological agents, contributing with important epidemiological and clinical data for effective medical strategies.

Material and methods

Ethics approval

Before conducting this study, the project was evaluated and approved by the Research Ethics Committee from the Instituto de Medicina Integral Professor Fernando Figueira (IMIP) registered under CAAE: 0246.0.099.000–10.

Definitions

The newborns included in the study were less than 28 days of birth and hospitalized in NICUs, and each case of yeast fungemia was confirmed by the isolation of yeast species from blood cultures.

Infants with a birth weight less than 2.500 g were classified as low birth weight and less than 1.500 g of very low birth weight (VLBW), and less than 1.000 g were considered as extremely low birth weight infants (ELBW) [11]. Prematurity was defined as a gestational age of less than 37 weeks [12].

Collection and manipulation of clinical samples

Blood samples were obtained between March 2010 and December 2014 from neonates with suspected septicemia hospitalized in NICUs from three hospitals in the state of Pernambuco, Brazil.

The blood to be analyzed was collected after medical request. Then, the samples were sent to Medical Mycology Laboratory at the Federal University of Pernambuco (UFPE) and processed for culture on Sabouraud dextrose agar (SDA) medium and agar brain heart infusion (BHI) both supplemented with chloramphenicol (50 mg/L) contained in Petri dishes and maintained at 30 °C and 37 °C for a period of 15 days. After the growth of the yeast colonies, they were purified and subsequently identified.

Culture purification

The yeasts were purified from colony fragments suspended in sterile distilled water plus 50 mg/L of chloramphenicol. From this suspension, 0.2 mL was seeded by depletion on the surface of SDA with antibiotic contained in Petri dishes. After this procedure, the colonies were transferred to tubes containing SDA with added yeast extract [13, 14].

Identification of the etiologic agents

The identification was based on the analysis of parameters as colony macroscopy (edges, texture, color, and growth time), microscopy (somatic and reproductive structures), and physiological and biochemical characteristics (assimilation of carbon and nitrogen compounds, fermentation of carbon sources, acetic acid production, and production of urease) [13, 14]. The etiologic agents were also identified using the automated system VITEK 2 ID-YST® (bioMérieux), and posteriorly, the taxonomy was confirmed by mass spectrometry analysis.

Proteomic taxonomy was conducted with direct analysis by mass spectrometry (MALDI-TOF Autoflex III Bruker Laser nd:yag smartbeam, Bruker Daltonics Inc., USA/Germany). The spectra for determining the protein profile of the isolates were obtained using a nitrogen laser (337 nm). The mass variation between 2000 and 20,000 Da was recorded using linear mode with a pulse of 104 ns at a voltage of + 20 kV. Final spectra were generated by adding 20 laser shots accumulated per profile, and 50 profiles were produced per sample. The list of peaks obtained was exported to the database of the MALDI Biotyper system, version 3.0, and analyzed by the SARAMIS™ software (Spectral Archiving and Microbial Identification System, AnagnosTec, Postdam-Golm, Germany, www.anagnostec.eu) where the final identifications were achieved [15, 16].

The species identified as belonging to C. parapsilosis complex, C. haemulonii, and C. pelicullosa (current name Wickerhamomyces anomalus) by the classic, automated taxonomy, and proteomic were also submitted to molecular identification.

For molecular taxonomy, the total genomic DNA was extracted with the commercial kit PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, USA). C. parapsilosis complex and C. haemulonii were submitted to genetic sequencing. The ITS region of the rDNA was amplified and sequenced using ITS1 and ITS4 primers [17] on the automated ABI 3130 genetic analyzer (Applied Biosystems, USA). The sequences were assembled and edited using Sequencher DNA Sequence Assembly Software 4.1.4 (Gene Codes Corporation, EUA) and compared with sequences deposited in public genomic databases (GenBank, NCBI, USA, and CBS database, The Netherlands). W. anomalus were evaluated and compared using species-specific primers (P. anom-F 5′-GAG GGT GGT GGC TTA CCT CT-30 and P. anom-R 50-AAA ATA CCT CTT CTA AAC CTG AG-30) [18].

Patient profile evaluation

Patient data were collected after confirmation of candidemia from medical records. Patient characteristics obtained included gestational age and sex, birth weight, reasons for NICU admission, previous diseases, predisposing risk factors such as surgery, administration of intravenous nutrition, use of antibiotics and immunosuppressive medications, and use of medical invasive devices.

Data of clinical and microbiological evolution including details of the antifungal drugs used (dosing regimen and duration) and outcome were also collected.

In vitro antifungal susceptibility

The in vitro susceptibility tests were performed according to the method of broth microdilution using 96-well microtiter plates in accordance with the standards described in the documents M27-A3 and M60-Ed2 from the Clinical and Laboratory Standards Institute [19, 20].

American Type Culture Collection (ATCC) strains (C. krusei ATCC6528, C. parapsilosis ATCC 22,019, and C. tropicalis ATCC750) were included in the essays for quality control. Stock solutions using pure powders of amphotericin B (United Medical), fluconazole (Pfizer), micafungin (Astellas Pharma), caspofungin (Sigma-Aldrich), and anidulafungin (Pfizer) were prepared using dimethyl sulfoxide (DMSO) as diluent. To evaluate the minimum inhibitory concentration (MIC), each drug was used at the following concentrations: amphotericin B (0.03 to 16 μg/mL), micafungin (0.01 to 8 μg/mL), caspofungin (0.01 to 8 μg/mL), anidulafungin (0.01 to 8 μg/mL), and fluconazole (0.12 to 64 μg/mL). The antifungal agents MIC were determined by visual observation after 24 h. The MIC for amphotericin B was represented by 100% fungal growth inhibition; for echinocandins and fluconazole, the MIC corresponded to 50% of growth inhibition in relation to the positive control. MIC breakpoints were interpreted according to CLSI document M60-Ed2 [20]. For amphotericin B and species that breakpoints were not available, the epidemiological cutoff value was employed [21, 22].

Statistical analysis

Data were analyzed using R software version R-4.2.2. Pearson’s chi-square test or the Fisher exact test (when the first was not possible) was used to verify the association of categorical variables. Odds ratios (ORs) were calculated using a contingency table comparing the candidemia risk factors with the neonate outcome (death). ORs were calculated in multivariate models and were considered significant at a p value of < 0.5.

Results

During the study period, from 401 newborns with septicemia suspicion evaluated, a total of 44 infants with candidemia were identified, which corresponds to an estimated prevalence of 10.97%. The demographic and clinical characteristics from the affected neonates, the microbiological identification of the isolated yeasts, antifungal therapy used, and outcome are shown in Table 1. The neonates were more likely to be male (61.7%). The mean gestational age of patients overall was 30.84 weeks, and the birth weight average was 1380.72 g. C. parapsilosis complex was the predominant species (38.6%), followed by C. albicans (31.8%), W. anomalus (11.3%), M. guilliermondii (4.5%), C. glabrata (4.5%), C. haemulonii (4.5%), C. tropicalis (2.3%), and C. famata (current name Debaryomyces hansenii) (2.3%). From the 44 affected babies, 9 (20.4%) evolved to death due to clinical worsened associated with the fungemia. All neonates that did not received antifungal therapy (6.8%) died.

Table 1.

Characteristics of neonates diagnosed with fungemia, etiological agent identification, antifungal treatment, and outcome

Registered number	Gender	Gestational age (weeks)	Birth weight	Etiological agent identified	Antifungal therapy	Clinical evolution
6394	F	34	2150 g	Candida albicans	FLZ	Clinical improvement
6395	F	27	1020 g	C. albicans	None	Death
6396	M	30	1130 g	C. albicans	FLZ/AMB	Death
6397	F	37	2360 g	C. albicans	FLZ/AMB	Clinical improvement
6399	M	32	2450 g	C. albicans	FLZ	Clinical improvement
6400	F	30	955 g	C. albicans	FLZ/AMB	Clinical improvement
6401	M	33	1196 g	C. albicans	FLZ	Clinical improvement
6402	M	30	940 g	C. albicans	FLZ/AMB	Clinical improvement
6410	F	32	1360 g	C. albicans	FLZ/AMB	Clinical improvement
6414	M	29	1470 g	C. albicans	FLZ/AMB	Clinical improvement
6431	M	31	1420 g	C. albicans	FLZ/AMB	Clinical improvement
85	M	30	966 g	C. albicans	FLZ/AMB	Clinical improvement
105	F	32	1125 g	C. albicans	FLZ/AMB	Clinical improvement
140	M	27	700 g	C. albicans	FLZ/AMB	Death
6398	F	34	1848 g	C. parapsilosis	FLZ	Clinical improvement
6404	M	26	700 g	C. orthopsilosis	FLZ	Death
6405	M	33	1090 g	C. orthopsilosis	FLZ	Clinical improvement
6406	F	38	3200 g	C. parapsilosis	FLZ	Death
6407	M	32	1740 g	C. metapsilosis	FLZ/AMB	Clinical improvement
6408	M	30	1064 g	C. metapsilosis	FLZ	Clinical improvement
6409	M	29	735 g	C. parapsilosis	FLZ/AMB	Death
6411	M	31	1460 g	C. parapsilosis	FLZ/AMB	Clinical improvement
6412	F	33	1024 g	C. parapsilosis	FLZ/AMB	Death
6429	F	25	810 g	Lodderomyces elongisporus	None	Death
6430	M	32	1532 g	C. parapsilosis	FLZ	Clinical improvement
57	F	29	1525 g	C. orthopsilosis	MICA/FLZ/AMB	Clinical improvement
76	F	27	1260 g	C. metapsilosis	FLZ/AMB	Clinical improvement
83	M	28	775 g	C. parapsilosis	FLZ/AMB	Clinical improvement
87	M	30	1915 g	C. parapsilosis	FLZ	Clinical improvement
89	M	35	1635 g	C. parapsilosis	None	Death
145	F	36	2530 g	C. parapsilosis	MICA/FLZ/AMB	Clinical improvement
6279	F	29	980 g	Wickerhamomyces anomalus	FLZ	Clinical improvement
6280	M	31	1690 g	W. anomalus	FLZ	Clinical improvement
6281	F	34	1390 g	W. anomalus	FLZ	Clinical improvement
6283	M	26	830 g	W. anomalus	FLZ/ AMB	Clinical improvement
6345	M	37	3460 g	W. anomalus	FLZ/AMB	Clinical improvement
6403	M	36	2080 g	Meyerozyma guilliermondii	FLZ	Clinical improvement
63	M	29	1345 g	M. guilliermondii	FLZ	Clinical improvement
6392	F	31	1140 g	C. glabrata	FLZ	Clinical improvement
86	F	30	2295 g	C. glabrata	AMB	Clinical improvement
123	M	26	660 g	C. haemulonii	AMB/FLZ	Clinical improvement
129	M	28	1020 g	C. haemulonii	AMB/FLZ	Clinical improvement
6413	M	31	1400 g	C. tropicalis	AMB	Clinical improvement
91	M	27	925 g	Debaryomyces hansenii	AMB	Clinical improvement

M, male; F, female; FLZ, fluconazole; AMB, amphotericin B; MICA, micafungin

Most affected neonates made previous use of TPN, and all of them were prior exposure to broad-spectrum antibiotics for prophylaxis or therapy. The majority were considered VLBW (weight between 1000 and 1500 g) and made prior use of central venous catheter (CVC). Major risk factors for candidemia and the association with death outcome are shown in Table 2; only the use of CVC was considered significant for mortality.

Table 2.

Risk factors for candidemia in neonates from Brazilian’s intensive care units and association with mortality risk

Risk factor	Number of neonates	Percentage	Odds ratio (95% CI)	p value
Prematurity	41	93.18%	0.48 (0.04, 6.04)	0.505**
Low birth weight	11	25%	0.27 (0.03, 2.46)	0.405**
Very low birth weight	18	40.91%	0.75 (0.16, 3.51)	1**
Extremely low birth weight	12	27.27%	2.70 (0.58, 12.5)	0.22**
Respiratory distress syndrome	34	77.27%	0.28 (0.06, 1.35)	0.18**
Parenteral nutrition	43	97.72%	-	-
Central venous catheter	23	52.27%	10.67 (1.20, 94.74)	0.02**
Prior use of broad-spectrum antibiotics	44	100%	-	-

(-): not possible to calculate; **: p-value obtained by Fisher exact test

The laboratorial diagnosis was based on detection of fungal growth in culture. Macroscopically, colonies were white to cream colored, with a creamy aspect, smooth texture, and regular edges. According to their physiological and biochemical characteristics, identified by the automatic system VITEK 2 and MALDI-TOF MS, the isolates were identified as C. parapsilosis complex (17) and C. albicans (14), followed by W. anomalus (5), M. guilliermondii (2), C. glabrata (2), C. haemulonii (2), C. tropicalis (1), and D. hansenii (1). After, W. anomalus and C. haemulonii species had their taxonomy confirmed by molecular tools. Species from C. parapsilosis complex were also differentiated by molecular biology being identified as C. parapsilosis sensu stricto (10), C. orthopsilosis (3), and C. metapsilosis (3), and one isolate was identified by genomic sequencing as the ascomycetous Lodderomyces elongisporus (deposited in GenBank database with the accession number OP501930), result that differed from proteomic taxonomy that is previously identified as C. parapsilosis sensu stricto. The ITS sequence of C. haemulonii (register 123) was also deposited at the GenBank database having the accession number KJ934715.

The MICs of the 44 isolates against the antifungal agents tested are presented in Table 3. The antifungal susceptibility test to micafungin and caspofungin was not performed to all yeasts because of laboratorial problems associated with lack of raw material. All isolates were susceptible to amphotericin B, considering the cutoff value of MIC ≥ 2 μg/mL, except C. haemulonii strains that also demonstrated elevated MICs to fluconazole. Approximately 42.8% of C. albicans were resistant to fluconazole and 21.4% were considered dose-dependent. Species from C. parapsilosis complex showed the highest MICs in front of echinocandins, especially anidulafungin. One strain of C. glabrata showed in vitro resistance in front of the three echinocandins tested.

Table 3.

Minimum inhibitory concentration (MIC) of yeast species isolated from neonates hospitalized in intensive care units front of amphotericin B, fluconazole, micafungin, anidulafungin, and caspofungin

Register	Species	Minimum inhibitory concentration (MIC, µg/mL)
		Amphotericin B	Fluconazole	Micafungin	Anidulafungin	Caspofungin
6394	Candida albicans	0.06 (S)	0.5 (S)	NP	0.12 (S)	NP
6395	C. albicans	0.12 (S)	64 (R)	NP	0.01 (S)	NP
6396	C. albicans	0.12 (S)	64 (R)	NP	0.12 (S)	NP
6397	C. albicans	0.03 (S)	8 (R)	NP	0.06 (S)	NP
6399	C. albicans	0.03 (S)	0.5 (S)	NP	0.01 (S)	NP
6400	C. albicans	0.03 (S)	4 (SDD)	NP	0.12 (S)	NP
6401	C. albicans	0.03 (S)	4 (SDD)	NP	0.12 (S)	NP
6402	C. albicans	0.25 (S)	64 (R)	NP	0.06 (S)	NP
6410	C. albicans	0.06 (S)	64 (R)	NP	0.06 (S)	NP
6414	C. albicans	0.12 (S)	0.25 (S)	NP	0.06 (S)	NP
6431	C. albicans	0.06 (S)	4 (SDD)	NP	0.01 (S)	NP
85	C. albicans	0.5 (S)	1 (S)	0.01 (S)	0.01 (S)	0.01 (S)
105	C. albicans	0.25 (S)	64 (R)	0.01 (S)	0.03 (S)	0.03 (S)
140	C. albicans	0.25 (S)	0.5 (S)	0.01 (S)	0.01 (S)	0.03 (S)
6398	C. parapsilosis	0.03 (S)	0.12 (S)	NP	4 (I)	NP
6404	C. orthopsilosis	0.03 (S)	0.25 (S)	1 (S)	4 (I)	0.25 (S)
6405	C. orthopsilosis	0.06 (S)	0.25 (S)	1 (S)	2 (S)	0.25 (S)
6406	C. parapsilosis	0.03 (S)	2 (S)	2 (S)	8 (R)	0.5 (S)
6407	C. metapsilosis	0.03 (S)	2 (S)	1 (S)	2 (S)	0.125 (S)
6408	C. metapsilosis	0.03 (S)	1 (S)	2 (S)	0.25 (S)	0.25 (S)
6409	C. parapsilosis	0.25 (S)	2 (S)	2 (S)	8 (R)	1 (S)
6411	C. parapsilosis	0.06 (S)	0.12 (S)	1(S)	8 (R)	0.25 (S)
6412	C. parapsilosis	0.5 (S)	0.5 (S)	2 (S)	8 (R)	1 (S)
6429	Lodderomyces elongisporus	0.25 (S)	0.25 (S)	1 (S)	0.25 (S)	0.25 (S)
6430	C. parapsilosis	0.03 (S)	0.5 (S)	1 (S)	0.12 (S)	0.25 (S)
57	C. orthopsilosis	0.25 (S)	0.25 (S)	0.03 (S)	0.03 (S)	0.25 (S)
76	C. metapsilosis	0.03 (S)	0.5 (S)	0.25 (S)	1 (S)	0.5 (S)
83	C. parapsilosis	0.5 (S)	1 (S)	0.06 (S)	1 (S)	0.25 (S)
87	C. parapsilosis	1 (S)	2 (S)	0.5 (S)	4 (S)	0.5 (S)
89	C. parapsilosis	1 (S)	1 (S)	1 (S)	2 (S)	0.5 (S)
145	C. parapsilosis	0.5 (S)	1 (S)	1 (S)	1 (S)	1 (S)
6279	Wickerhamomyces anomalus	0.03 (S)	4 (S)	NP	0.03 (S)	NP
6280	W. anomalus	0.03 (S)	2 (S)	NP	0.01 (S)	NP
6281	W. anomalus	0.03 (S)	2 (S)	NP	0.01 (S)	NP
6283	W. anomalus	0.03 (S)	4 (S)	NP	0.12 (S)	NP
6345	W. anomalus	0.12 (S)	4 (S)	NP	0.06 (S)	NP
6403	Meyerozyma guilliermondii	0.03 (S)	0.5 (S)	NP	1 (S)	NP
63	M. guilliermondii	0.25 (S)	32 (DD)	0.12 (S)	0.12 (S)	0.12 (S)
6392	C. glabrata	0.03 (S)	0.5 (SDD)	NP	0.03 (S)	NP
86	C. glabrata	0.5 (S)	16 (SDD)	0.5 (R)	0.5 (R)	0.5 (R)
123	C. haemulonii	8 (R)	32 (NE)	0.03(NE)	0.01 (NE)	0.25 (NE)
129	C. haemulonii	8 (R)	32 (NE)	0.03 (NE)	0.01 (NE)	0.25 (NE)
6413	C. tropicalis	0.12 (S)	4 (SDD)	NP	0.06 (S)	NP
91	Debaryomyces hansenii	0.25 (S)	2 (NE)	1 (NE)	1 (NE)	1 (NE)

NP, not performed; S, susceptible; R, resistant; SDD, susceptible-dose-dependent; I, intermediate; NE, breakpoint or epidemiological cutoff value not established

Discussion

Candidemia is an important cause of elevated morbidity and mortality in NICUs. Therefore, to reduce the occurrence and improve the prognostic, it is extremely necessary for physicians and microbiologists to promote the prevention, early detection, and prudent management of candidemia in high-risk newborns [7].

In our study, the prevalence of bloodstream infection by Candida sp. in Brazilian’s NICU was of approximately 11% with a female to male ratio of 1:1.58. Various studies in intensive care units among pediatrics and neonatal population show a candidemia incidence range of 7.7–39% [23–26]. The isolation rates depend on the percentage of preterm babies, use of broad-spectrum antibiotics, and level of care in the NICU.

We emphasize that the majority of the babies diagnosed were premature (93.17%), and some other neonatal problems, especially related to prolonged stay in NICU, may have contributed to the occurrence of the candidemia. Most affected neonates made previous use of TPN (97.7%), and all of them were prior exposure to broad-spectrum antibiotics for prophylaxis or therapy. The majority were considered VLBW and approximately 52.3% made prior use of CVC. Some previous studies showed that those risk factors are the ones that are most commonly associated with candidemia development in newborns [3, 7, 27]. Lamba et al. [27] identified similar risk factors for neonatal candidemia; the most common were low birth weight, prolonged use of broad-spectrum antibiotics, and presence of CVC. Chen et al. [4] in a study conducted in a Chinese NICU identified that CVC (58%) and TPN (84.1%) were significant predisposing factors for the development of neonatal candidemia that was similar to our results. Candida species have a great capacity to adhere and form biofilms on the surface of foreign materials, which is associated with high virulence and protects the yeast cell from antifungal agents’ penetration and immune responses [4]. These virulence factors may explain why invasive and indwelling medical devices, as CVC and TPN, have been described as risk factors associated to candidemia.

Additionally, in our study, when the neonatal candidemia risk factors were associated with mortality risk, there was no statistically significant value, except the use of CVC, probably because of the sample size.

Recent changes in candidemia epidemiology have been documented, including an increasing proportion of neonatal candidiasis by NCAs, especially by C. parapsilosis complex [28, 29]. In our research, species from this complex were the most isolated etiological agent of candidemia representing 38.6% of cases, followed by C. albicans (31.8%). Similar data was reported in an American study of neonatal and pediatric candidemia conducted by Harrington et al. where the most prevalent species were C. parapsilosis (34.7%) and C. albicans (32.7%) [2]. Asadzadeh et al. [28] reported that in Kuwait’s NICUs, C. albicans was the most common species from candidemia during 2010 and 2011; however, C. parapsilosis surpassed C. albicans during 2012 to 2014. The main reservoir of C. parapsilosis in NICU environment remains unknown, but transmission has been associated with hands of health care workers or with contaminated catheter or parenteral nutrition solution. Furthermore, this yeast has the ability to form biofilm on the surface of intravascular devices [29, 30].

Since 2005, C. parapsilosis was recognized as a complex represented by three cryptic species, C. parapsilosis sensu stricto, C. metapsilosis, and C. orthopsilosis [31], and is only possible to differentiate them with proteomic and/or molecular methods [32]. In this study, we observed a prevalence of C. parapsilosis sensu stricto that is similar with other researches [32, 33]. However, among the isolates, a yeast primarily misidentified as C. parapsilosis by VITEK 2® and MALDI-TOF MS had the taxonomy confirmed as L. elongisporus after genomic sequencing. According to Ahmad and colleagues [34], L. elongisporus is genetically closely related to C. parapsilosis complex species. This yeast has been described as emerging fungal pathogen, and human fungemia caused by this fungus has rarely been reported [35–37].

The third most isolated species in this study was W. anomalus which was associated with an outbreak in NICU with five affected babies that was previously reported [18]. Infections caused by this yeast are rare, especially in Brazilian’s NICU [18]. However, reports have indicated cases of outbreaks in other countries attributable to this species in premature neonates that need intensive care [38, 39].

Another interesting data in this study was the detection of two cases of disseminated hematogenous infection by C. haemulonii in premature infants. Candidemia caused by this yeast has also rarely been described in humans, being limited to a few sporadic cases in adults and endemic and restricted occurrences in neonates [40, 41]. This species belongs to C. haemulonii complex that is represented by C. haemulonii sensu stricto, C. duobushaemulonii, and C. haemulonii var. vulnera and related species as C. auris and C. pseudohaemulonii. Recently, this complex has attracted attention due to reduced susceptibility to azoles and amphotericin B [42].

C. glabrata, M. guilliermondii, D. hansenii, and C. tropicalis were responsible for approximately 13.6% of the cases. In general, non-C. albicans species represented 68.1% of the fungemia isolates; other researchers conducted in NICUs also found a prevalence of NCAs [7, 43].

In addition to the determination of risk factors for candidemia among neonates and the correct pathogen taxonomy, it is crucial to identify antifungal resistance in yeasts species, in order to help in the choice of the most adequate treatment. The in vitro antifungal susceptibility test showed that all yeasts were sensitive to amphotericin B, except the two C. haemulonii isolates that also exhibited high MICs to fluconazole. Resistance to amphotericin B is unusual among the major candidemia species [25, 27, 44]. However, corroborating with our study, Ben-Ami and colleagues [45] published that clinical isolates of C. haemulonii had high MICs for amphotericin B and azoles, whereas echinocandin MICs were within the susceptible range. The importance of these resistance profiles for treatment strategies among neonates remains to be determined [45].

Among C. albicans isolates, resistance to fluconazole was detected in 42.8% of the strains and dose-dependent MIC in 21.4%. In line with our study, Yenisehirli et al. [46] published a resistance rate of 34% to fluconazole among C. albicans clinical isolates. The possibility of reduced susceptibility of C. albicans to fluconazole may be due to widespread and prophylactic use of this antifungal agent [44].

Echinocandins emerged as alternative to yeasts that were resistant to azoles, but recently, there has been an emergence of Candida species that are resistant to these antifungal agents [47]. Among the clinical species isolated, we verified that the higher MICs against echinocandins were from C. parapsilosis complex and C. glabrata, including four strains of C. parapsilosis sensu stricto and one of C. glabrata that were considered resistant to anidulafungin. However, Papp et al. [48] affirm that patients with candidemia by C. parapsilosis usually respond well to echinocandin treatments, even with the high MIC values. In concern to C. glabrata resistance, many authors have already described the decreased susceptibility to echinocandins in clinical isolates [47, 49].

Determining the neonatal candidemia risk factors and the use of proteomic and molecular tests to species identification and analyzing the in vitro antifungal susceptibility are important tools not only as a subsidy for therapeutic strategies but also for observing the epidemiological trend in the occurrence of species and antifungal resistance in a given region. Rare species as L. elongisporus and C. haemulonii were isolated in Brazilian’s Northeast NICUs and antifungal resistance is a constant challenge. Thus, appropriate measures must be taken to reduce the incidence and consequences of this fungal infection, and an effective and safe management strategy should involve the rapid and precise mycological diagnostic with the correct taxonomy and laboratory tests of drug susceptibility to help in the selection of an appropriate treatment.

Data Availability

The datasets generated and/or analyzed during the current study are not publicly available to protect our participants’ sensitive data but are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.