First case of neonatal bloodstream infection caused by Malassezia furfur in mainland China diagnosed via metagenomic next‑generation sequencing: A case report

Introduction

Malassezia furfur (M. furfur) is a lipophilic yeast that is part of the normal human skin microbiota. Under specific conditions, it can transition to a pathogenic form (1,2). This organism possesses unique biological characteristics, notably a lipid-rich cell wall structure that requires exogenous long-chain fatty acids for growth (3). In immunocompetent individuals, M. furfur typically causes superficial infections such as pityriasis versicolor and folliculitis (4). However, in certain populations, particularly neonates, its infection can lead to life-threatening systemic disease (5). The pathogenic mechanisms involve biofilm formation, phospholipase secretion and immune evasion, rendering it a notable opportunistic pathogen in nosocomial infections (1,6,7).

Neonates are susceptible to M. furfur infections because of multiple interacting factors (6), including an underdeveloped skin barrier, characterized by a thin stratum corneum and incomplete intercellular junctions, which provide a physical pathway for pathogen invasion (8). Concurrently, an immature immune system, including reduced neutrophil activity and low complement levels, impairs pathogen clearance (9). Clinical interventions further exacerbate infection risk. For instance, lipid emulsions in parenteral nutrition serve as ideal media for fungal proliferation and central venous catheters (CVCs) offer surfaces for biofilm formation (10). These risk factors are synergistically amplified in preterm infants, positioning them as a high-risk group for invasive Malassezia infections.

Traditional fungal detection methods for M. furfur have notable limitations (11). Routine cultures require lipid supplementation, such as olive oil, and exhibit slow growth, typically taking 5-7 days, thereby resulting in a clinical laboratory detection rate <30%. Morphological identification relies on the characteristic ‘spaghetti and meatballs’ appearance under microscopy, which is often absent in systemic infection samples (12). Serological tests are hindered by immature antibody responses in neonates, leading to low sensitivity (13). These technical constraints contribute to missed or delayed diagnosis in >40% of invasive cases, highlighting key gaps in current diagnostic frameworks (14).

Metagenomic next-generation sequencing (mNGS) offers a promising solution for these diagnostic challenges (15). This technology captures all nucleic acid sequences in a sample without bias, thus eliminating the need for pathogen-specific primers or prior culture (16). Theoretically, mNGS can detect the entire microbial genome of a clinical specimen. Compared with traditional methods, mNGS has distinct advantages for detecting fastidious organisms, as it does not rely on pathogen viability, can identify unculturable microbes and enables simultaneous species identification and resistance gene profiling via bioinformatic analysis (17). Furthermore, the turn-around time is typically shortened to 24-48 h. Multiple studies have demonstrated the efficacy of mNGS in detecting bacterial pathogens in neonatal bloodstream infections (18,19); however, its utility in diagnosing fungal infections, especially lipid-dependent yeasts, remain underexplored (20).

The present study analyzed a representative M. furfur infection case and conducted a systematic review to illustrate the need to incorporate M. furfur detection into the routine fungal pathogen screening spectrum for neonatal bloodstream infections. The technical value of mNGS was evaluated for the detection of this organism. The present case report highlights the diagnostic improvements offered by mNGS over conventional methods, which include timely detection, enhanced capability for identifying polymicrobial infections and the beneficial impact on clinical decision-making. These findings are expected to provide evidence-based guidance for optimizing the diagnostic pathways involved in neonatal invasive fungal infections and to inform revisions of infection control strategies in intensive care units. The present case report provides specific attention to technological innovations in mNGS for detecting lipid-dependent fungi, with the aim of addressing current diagnostic blind-spots in neonatal fungal bloodstream infections.

Case report

This was a retrospective case-based study involving an infant male patient who was admitted to and treated in the neonatal intensive care unit of Jiangmen Central Hospital, Jiangmen, Guangdong, China, and was subsequently diagnosed with bloodstream infection caused by M. furfur.

mNGS

Plasma was isolated from whole blood samples of the infant patient, and nucleic acids were extracted using a magnetic bead-based DNA/RNA Extraction Kit (cat. no. 2005-01; GenK) according to the manufacturer's instructions. Nucleic acid concentration and purity were assessed using an Equalbit 1x dsDNA HS Assay Kit (cat. no. EQ121-01; Vazyme Biotech Co., Ltd.) on a Qubit™ 4 Fluorometer (Thermo Fisher Scientific, Inc.). DNA libraries were constructed using a Pathogen Microbial DNA/RNA Library Preparation Kit (cat. no. 2102 GenK) following the standard procedures recommended by the manufacturer. Library concentration and fragment size were determined using a Qubit fluorometer and an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.), respectively. After quality control, high-throughput sequencing was performed on the Illumina NextSeq™ 550Dx platform (Illumina, Inc.), generating 76-bp single-end reads. Throughout the workflow from nucleic acid extraction to library sequencing, negative template controls (NTCs) were included under a non-host-depletion protocol to monitor potential background microbial signals introduced during nucleic acid extraction, library preparation and DNA sequencing. Raw sequencing data were preprocessed using fastp v0.23.2 (OpenGene; HaploX Ltd.) to remove low-quality reads, adapter-contaminated reads, duplicate sequences and short reads (<50 bp), thereby generating high-quality clean reads. Subsequently, clean reads were aligned to the human reference genome T2T-CHM13 v.2.0 (GCA_009914755.4) using Bowtie v.2.3.5.1. The remaining non-human reads were mapped to a custom-built pathogen database using Burrows-Wheeler Aligner (BWA; v.0.7.17-r1198-dirty) and species annotation was performed using a defined species annotation algorithm. The final pathogen identification and reporting were based on established reporting thresholds in conjunction with clinical data (21). The reference microbial genome database included the complete genome sequences of bacteria, fungi, viruses and parasites.

The criteria for positive detection were as follows: i) At least one species-specific read for the detection of Mycobacterium, Nocardia and Legionella pneumophila; ii) at least three unique reads required for other bacteria, fungi, viruses and parasites; and iii) pathogens were excluded if the ratio of microorganism reads per million of a given sample to the NTC was <10.

Literature search

A literature search was performed using the PubMed database (https://pubmed.ncbi.nlm.nih.gov/advanced/). The search string used the keywords (Malassezia furfur) AND (newborn) AND (neonate) AND (infant) AND (infection) AND [(bloodstream) OR (blood) OR (plasma)] to identify relevant articles. The inclusion criteria were human neonates, confirmed M. furfur infection and reporting of detection methods and outcomes.

Birth history

The neonate was born via spontaneous vaginal delivery at a gestational age of 25 weeks and 2 days with a birth weight of 660 g, length of 32 cm and head circumference of 22 cm. He was admitted to and treated in the neonatal intensive care unit of Jiangmen Central Hospital, Jiangmen, Guangdong, China. There was no premature rupture of membranes (0 h) and no evidence of intrauterine distress (0 h). The fetal heart rate was normal and umbilical cord entanglement was not observed around the neck. The amniotic fluid was clear, and the placenta appeared normal.

Present illness history

At birth, the patient exhibited no spontaneous respiration, generalized cyanosis, poor responsiveness or hypotonia of the four limbs. The heart rate was 80-90 beats per min. Immediate resuscitation measures were initiated, including endotracheal intubation and positive-pressure ventilation using a T-piece resuscitator. Pulmonary surfactant (Curosurf®; 120 mg) was administered for replacement therapy. Following intervention, the neonate's skin color and responsiveness gradually improved, with no signs of oral frothing. The Apgar score at 1 min was 4 (with 2 points deducted for respiration and 1 point each deducted for heart rate, responsiveness, muscle tone and skin color), which improved to 8 at 5 min (2 points deducted for respiration) and 9 at 10 min (1 point deducted for respiration). After obtaining parental consent, the patient was transferred to the neonatal intensive care unit (NICU) for further treatment. Antenatal corticosteroid therapy (dexamethasone sodium phosphate, 10 mg intramuscular injection once daily, administered for 2 consecutive days in January 2025 promoted fetal lung maturation.

Diagnostic and therapeutic course

During hospitalization, the patient experienced a complex clinical course, characterized by immature pulmonary function, recurrent infections, feeding intolerance and inadequate weight gain. As hyaline membrane disease was suspected, the patient received two separate 10-day courses of dexamethasone, one in January and the second 4 weeks later. In early February, a sputum culture revealed methicillin-resistant Staphylococcus aureus, prompting the initiation of linezolid treatment. In mid-February, signs of infection recurred and the blood culture identified Escherichia coli, leading to a change in therapy to a combination of meropenem and linezolid. A follow-up blood culture performed in late February returned negative results.

In early March, after >3 weeks of broad-spectrum antimicrobial therapy, the patient again exhibited clinical deterioration, including decreased responsiveness, abdominal distension, intermittent hypoxia, fever and generalized hyporesponsiveness. In March, CRP levels increased sharply to 84.54 mg/l, prompting the adjustment of antibiotic therapy from meropenem alone to meropenem combined with linezolid. However, no clinical improvement was observed. A few days later, the CRP level had increased further to 156.38 mg/l, accompanied by a marked decrease in the platelet count to 57x109/l, which was not present during the previous clinical course. The patient appeared pale and lethargic, raising concerns regarding a new infectious source or a possible disseminated infection. Given the repeated negative or inconsistent blood and sputum culture results and unclear etiology of the infection, a rare or difficult-to-culture pathogen was suspected. Following consultation with a multidisciplinary team, mNGS analysis of blood samples was performed.

In March 2025, mNGS analysis of a blood sample detected 148 sequencing reads corresponding to M. furfur with a relative abundance of 72.6% (Fig. 1). The total number of bp in the M. furfur reference genome was 8,311,773 and the total length of the detected M. furfur sequences was 10,901 bp, corresponding to a genome coverage of 0.1312% and an average sequencing depth of 1.001x. Because M. furfur is a constituent of the normal skin microbiota in humans and other warm-blooded animals and may colonize from the neonatal period onward, potential skin- or background-derived contamination was carefully evaluated. Importantly, M. furfur was not detected in corresponding NTCs processed using the same sequencing batch. Therefore, the M. furfur signal in the blood samples fulfilled the predefined sample-to-NTC reporting criteria and was not attributable to background NTC contamination.

Figure 1 mNGS detection of Malassezia furfur in the blood sample. (A) mNGS results showing detection of Malassezia at the genus level and M. furfur at the species level. (B) Distribution of reads mapped to the M. furfur reference genome.

The mNGS results were further interpreted in the context of the patient's clinical course. The patient had multiple risk factors for invasive Malassezia infection, including extreme prematurity, extremely low birth weight, prolonged NICU hospitalization, broad-spectrum antimicrobial exposure, lipid exposure and catheter-related risks. At the time of mNGS testing, the patient exhibited clinical deterioration with fever, generalized hyporesponsiveness, abdominal distension, intermittent hypoxia, markedly elevated CRP levels and thrombocytopenia, whereas conventional cultures were negative or inconsistent. These laboratory and clinical findings supported true bloodstream infection rather than incidental skin contamination during blood collection.

After the mNGS results were obtained, empirical antibacterial therapy was discontinued and targeted antifungal treatment with fluconazole along with platelet transfusion support was initiated. Notably, no viable isolate was obtained because additional lipid-supplemented fungal culture using Dixon's or Sabouraud agar with an olive oil overlay was not performed after a positive mNGS result. Cerebrospinal fluid fungal culture was negative, and biochemical analysis revealed no abnormalities. No evidence of disseminated fungal infection was present. By day 6 of fluconazole therapy, CRP levels normalized, body temperature stabilized, mental responsiveness improved, signs of pulmonary infection resolved, feeding tolerance increased and the platelet count gradually returned to normal (Fig. 2). The patient was subsequently managed with low-flow oxygen support and the patient's overall condition stabilized. The key treatment schedule for neonates is shown in Fig. 3.

Figure 2 Dynamic changes in laboratory indicators during hospitalization. Serial changes in WBC, CRP, PLT and HB are shown during the clinical course. WBC, white blood cell; CRP, C-reactive protein; PLT, platelet; HB, hemoglobin; mNGS, metagenomic next-generation sequencing.

Figure 3 Timeline of the diagnostic and therapeutic course of the neonate. The timeline summarizes the major clinical events, microbiological findings and treatment adjustments from birth to discharge. CRP, C-reactive protein; mNGS, metagenomic next-generation sequencing; NIPPV, nasal intermittent positive pressure ventilation.

Literature review results

Initially, the present case report identified 43 relevant articles through a systematic literature search. Based on the inclusion criteria, 28 articles were ultimately included for a detailed analysis of the clinical characteristics (excluding 14 that did not provide specific case details and 1 that did not involve a newborn) and 35 articles for the analysis of risk factors (excluding 8 that did not mention infection-related risk factors). Between 1981 and 2025, 250 cases of M. furfur bloodstream infections in infants were reported globally. The present case report systematically summarized the reported neonatal cases of M. furfur infections (Table I) (22-49), reviewed the principal laboratory diagnostic methods and conducted a comparative analysis of their technical characteristics and clinical applicability (Table II) (12,50-65). In addition, the distribution of different categories of risk factors associated with bloodstream infection is illustrated using brick-shaped bar charts in Fig. 4. Furthermore, publications addressing risk factors related to M. furfur bloodstream infections are systematically summarized and organized in Table III (22-40,42-44,46-51,53,66-71).

Figure 4 Distribution of risk factors associated with neonatal bloodstream infection caused by Malassezia furfur. Brick-shaped bar charts showing the distribution of risk factors reported in the literature. NICU, neonatal intensive care unit; ICU, intensive care unit.

Table I A systematic review of 251 reported cases of M. furfur infection: Demographics, initial presentation, therapy and outcome.

Table I

A systematic review of 251 reported cases of M. furfur infection: Demographics, initial presentation, therapy and outcome.

Author/s, year	No. cases	Neonatal Classification	Sex	Birth weight (g)	GA (weeks)	Symptom	Progression of disease	Supporting therapy	Antifungal therapy	Outcome	(Refs.)
Redline and Dahms, 1981	1	Preterm	F	740	28	Respiratory-distress syndrome	Cardiomegaly, edema and cholestasis	Intralipid	AMB and flucytosine	Deceased	(22)
Powell et al, 1984	5	Preterm (n=4), Term (n=1)	2 M, 3 F	N/A	27-40	Apnea and bradycardia, low-grade fever, interstitial pneumonia, elevated neutrophil band counts and thrombocytopenia	Catheter-related bloodstream infection	Intralipid	N/A	All survived	(23)
Long and Keyserling, 1985	4	N/A	N/A	N/A	N/A	Fever, apnea and bradycardia and thrombocytopenia	N/A	Intralipid	AMB	N/A	(24)
Dankner et al, 1987	5	Preterm (n=4), Term (n=1)	4 M, 1 F	640-3,345	25-38	Thrombocytopenic, fungemia, necrotizing enterocolitis, apnea and bradycardia	Pulmonary vasculitis and splenomegaly	Intralipid	AMB	All survived	(25)
Alpert et al, 1987	7	Preterm (n=5), Term (n=2)	N/A	670-3,400	25-40	Respiratory distress, apnea, bradycardia and thrombocytopenia	Persistent fungemia	N/A	AMB (n=2) and 5-fluorocytocine (n=1)	Survived (n=5), deceased (n=2)	(26)
Aschner et al, 1987	19	N/A	N/A	N/A	N/A	Apnea, bradycardia, pulmonary deterioration and thrombocytopenia	Catheter colonization and fungemia	N/A	N/A	N/A	(27)
Azimi et al, 1988	12	Preterm	N/A	580-1,500 (n=11), 3,000 (n=1)	N/A	Bradycardia, fever, respiratory distress and signs of necrotizing enterocolitis	Catheter occlusion, sepsis and fungemia	Intralipid	N/A	All survived	(28)
Larocco et al, 1988	8	Preterm	3 M, 5 F	700-2,610	25-34	Fever, apnea, bradycardia, temperature instability, hepato-splenomegaly and gastrointestinal bleeding	N/A	Intralipid (n=7)	N/A	Survived (n=6), deceased (n=2)	(29)
Mickelsen et al, 1988	3	Preterm	N/A	700-960	25-27	Thrombocyto penia, leukocytosis and elevated band-cell counts	Fungemia	Intralipid	AMB	All survived	(30)
Surmont et al, 1989	6	Preterm	4 M, 2 F	900-2,200	28-33	Fever, pulmonary infiltrates, leukocytosis, thrombocytopenia, apnea and bradycardia	Fungaemia, cholestasis and BPD	Intralipid	Miconazole (n=5)	Survived (n=5), deceased (n=1)	(31)
Richet et al, 1989	3	Preterm	N/A	<1,000	24-26	Bronchopneumonia associated with respiratory failure, thrombocytopenia and leukocytosis.	N/A	Intralipid	AMB (n=1)	Survived (n=1), deceased (n=2)	(32)
Hruszkewycz et al, 1991	3	Extremely premature	N/A	<1,500	N/A	Bronchopulmonary dysplasia	N/A	Intralipid	AMB	All survived	(33)
Ormälä et al, 1992	12	Preterm	N/A	<1,500	<32	N/A	N/A	N/A	N/A	All survived	(34)
Marcon and Powell, 1992	57	Preterm majority	N/A	N/A	N/A	Fever, apnea, bradycardia and thrombocytopenia	Sepsis and catheter infections	Intralipid	AMB (primary)	Mortality ~10%	(35)
Welbel et al, 1994	5	Preterm	2 M, 3 F	690-1,470	26-31	Fever, apnea	N/A	Intralipid	N/A	All survived	(36)
Taylor et al, 1994	1	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	(37)
Sizun et al, 1994	2	Preterm	N/A	1,400, 2,500	31, 40	Fever, occasionally moderate; a worsening respiratory condition; apnea; and bradycardia	N/A	Intralipid	N/A	Survived (n=1), deceased (n=1)	(38)
Shattuck et al, 1996	26	Preterm	N/A	<1,500	<30	Fungemia	Rare progression to sepsis	N/A	AMB (n=2)	deceased (n=1)	(39)
Chang et al, 1998	15	Preterm	9 M, 6 F	<1,300	24-31	N/A	N/A	Intralipid	AMB (n=10)	Survived (n=14), deceased (n=1)	(40)
Niamba et al, 1998	10	N/A	4 M, 6 F	N/A	N/A	Erythematous papules and pustules on the face and scalp (non-comedonal lesions resembling neonatal acne)	Lesions appeared on 3–21 days of life, with a self-limiting course and resolution within 1–2 weeks	N/A	Topical ketoconazole 2% used in 4 cases (others received no treatment)	All survived	(41)
Chryssanthou et al, 2001	8	Preterm	N/A	660-1,100	23-27	Fever and apnea		Intralipid	AMB, flucytosine and fluconazole	All survived	(42)
Tirodker et al, 2003	4	Preterm	N/A	N/A	N/A	Fever	Sepsis	N/A	N/A	N/A	(43)
Rosales et al, 2004	1	Preterm	Male	567	23	Fever	Meningitis	Intralipid	AMB	Deceased	(44)
McCoy et al, 2011	1	Preterm	N/A	2,250	36	Fever and apnea	Sepsis	Intralipid	AMB	Survived	(45)
Iatta et al, 2014	6	Infant	5 M, 1 F	£1,000g-1,500g	N/A	Severe birth asphyxia, premature birth, esophageal atresia, tetralogy of Fallot	Fungemia	Total lipid parenteral nutrition	L-AMB and FLZ	All survived	(46)
Cornu et al, 2018	2	Preterm	1 M, 1 F	1,460	29.5	Candidemia	N/A	Intralipid	FLZ	All survived	(66)
Chen et al, 2020	20	Preterm	11 M, 9 F	660-1,420	24-31	Apnea, bradycardia and thrombocytopenia	Fungemia	Intralipid	Prophylactic FLZ and AMB	All survived	(47)
Huang et al, 2020	4	Preterm	2 M, 2 F	490-810	23-26	PPROM, RDS, PDA, IVH, pulmonary HTN, intestinal obstruction, congenital pneumonia, BPD, pulmonary hemorrhage	N/A	Parenteral lipid supplements, TPN, intubation and mechanical ventilation	Prophylactic FLZ, L-AmB and AmB-d	All survived	(48)

[i] AMB, amphotericin B; BPD, bronchopulmonary dyplasia; HTN, hypertension; PPROM, preterm premature rupture of membrane; RDS, respiratory distress syndrome; PDA, patent ductus arteriosus; IVH, intraventricular hemorrhage; TPN, total parenteral nutrition; M, male; F, female; N/A, not applicable; FLZ, fluconazole.

Table II Methods for Malassezia detection in clinical samples.

Table II

Methods for Malassezia detection in clinical samples.

Category	Method	Advantages	Limitations	Challenges	(Refs.)
Culture-based methods	Lipid-enriched media (forexample, modified Dixon and Leeming-Notman) • Incubation at 32-37˚C in a humid environment for 7-10 days (slow-growing species such as Malassezia sympodialis may require extended culture) • Morphological identification: creamy to pale-yellow colonies, smooth or wrinkled surface and yeast-like cells under microscopy • Biochemical tests: Tween 80 hydrolysis and urease assay	• Gold standard for species differentiation and obtaining pure isolates for downstream analysis • Enables antifungal susceptibility testing to guide clinical therapy • Morphological observations complement identification through colony and cellular features	• Time-consuming (2-4 weeks), delaying diagnosis and treatment • Stringent lipid requirements may result in missed detections on routine media • Risk of false negatives from improper sampling or transport • Limited resolution for closely related species without molecular confirmation	• High species diversity (>14 known species) challenges discrimination of near-relatives • Risk of environmental contamination and aerosol spread in open culture • Competition from rapid molecular and proteomic methods, despite foundational role of culture	(49-52)
Microscopic examination	• 10% potassium hydroxide preparation to dissolve keratin and reveal yeast forms and pseudohyphae • Gram stain: Gram-positive oval or spherical cells • Giemsa or lactophenol cotton blue staining for enhanced contrast • Examination at 400x to 1000x magnification to identify 3-8 µm yeasts and budding structures	• Rapid and simple with minimal steps, suitable for point-of-care • Low-cost, requiring no specialized equipment • Results available within 30 min, ideal for preliminary screening	• Low sensitivity when fungal burden is low, leading to potential under-detection • Cannot differentiate between Malassezia species • Operator-dependent, with subjective interpretation and potential variability	• Variable sample quality can cause false negatives • Interference from co-existing microbes (bacteria or fungi) • Lack of standardized staining and interpretation protocols	(12,50,53,54)
Molecular methods	• Internal transcribed spacer 1/2 region sequencing for species identification via sequence alignment • 26S rDNA D1/D2 region analysis (for example, RFLP) for interspecies discrimination • Multiplex PCR targeting species-specific genes (for example, β-tubulin, CYP51) for rapid detection	• High resolution distinguishes closely related species that culture cannot separate • Rapid turnaround by extracting DNA directly from clinical samples, bypassing culture delays • Potential for global standardization through sequence databases	• Higher cost for sequencing and primer synthesis compared with that of morphological methods • Requires specialized equipment and bioinformatics expertise, limiting use in resource-poor settings • False negatives owing to low DNA yield or sample degradation • Intraspecies genetic variability necessitates multilocus analysis • Public database inaccuracies and incompleteness can affect identifications • PCR inhibitors (for example, keratin) in clinical samples may impede amplification	• Establishing robust reference databases for all Malassezia spp. remains ongoing • Ensuring quality control and avoidance of contamination in molecular workflows	(55-60)
Mass spectrometry (matrix-assisted laser desorption ionization time-of-flight mass spectrometry)	• Protein extraction from colonies or clinical specimens using formic acid/acetonitrile • Spotting extracts onto a target plate with matrix (for example, α-cyano-4-hydro-xycinnamic acid) • Laser ionization generates PMFs matched against reference spectra	• Rapid results within min, far faster than culture or biochemical assays • High specificity allowing species-level discrimination based on unique PMFs • Automated analysis reduces human error and is amenable to high-throughput screening	• Dependent on comprehensive and up-to-date spectral databases; missing entries can lead to misidentification • Sample preparation protocols must be strictly standardized for reproducible results • High initial investment and maintenance costs may limit adoption in smaller laboratories • Limited discrimination of intraspecies variants without supplementary methods • Potential interference from host proteins or lipids in complex clinical samples	• Continuous database expansion required to include newly described species • Integration with clinical workflows demands validation and quality assurance measures	(35,61-64,71)

[i] RFLP, restriction fragment length polymorphism; PMF, peptide mass fingerprints; PCR, polymerase chain reaction.

Table III Risk factors associated with bloodstream infections caused by M. furfur

Table III

Risk factors associated with bloodstream infections caused by M. furfur

Specific risk factor		(Refs.)
Host-related factors	Extremely low birth weight (<1,000 g)	(22,25,26,28,30-35,42,44,46-48,65,66)
	Prematurity (<32 week gestation)	(22,23,25-27,30-35,38,39,42,44,46,65-67)
	Immature immune system	(22,23,25-28,35,38,42,43,46,48,50,51,65,66)
	Malnutrition	(22,25,30,35,47,48)
	Impaired skin and mucosal barriers	(23,25,28,46,48,65,66)
Iatrogenic/device-related factors	Prolonged use of central venous catheter	(22,23,25-33,35-40,42-44,46-48,50-52,65-70)
	Intravenous lipid emulsion infusion	(22,33,35,38-40,42,44,46-48,51,52,67-70)
	Dependence on total parenteral nutrition	(22,24-33,35,37,39,40,42-44,46-48,51,65,69)
	Prolonged NICU or ICU stay	(22-34,36,40,44,46-48,50,51,65-67)
	Mechanical ventilation	(22,25,27,28,30,32,34,39,42,48,65,66,69)
	Prolonged or broad-spectrum antibiotic exposure	(22,25,27-37,39,42,43,47,48,65,66,71)
Microbial-related factors	Lipid-dependence of Malassezia pachydermatis	(23-27,29-35,46-48,51,67-70)
	Ability to form biofilms on catheter surfaces	(38,46,48,69,71)
	Difficult to detect using conventional culture methods	(22-29,31-36,38,46,50,66,69,71)
Diagnostic and treatment delays	Delayed recognition of fungal infection	(22-29,31,32,35,46,48,50)
	Ineffective empirical antibiotic therapy	(22,25,27-33,35,47,48,66)
	Delay in initiating antifungal therapy	(22,23,25-29,32,35,66)

[i] ICU, intensive care unit; NICU, neonatal ICU.

Discussion

M. furfur is a lipophilic yeast commonly residing on human skin, especially in sebum-rich regions such as the scalp, face and upper torso (72). It exhibits strict requirements for exogenous fatty acids owing to the absence of the acyl-CoA synthase genes necessary for endogenous long-chain fatty acid biosynthesis (73,74). Although M. furfur is typically commensal and contributes to skin microbiota homeostasis, it can undergo pathogenic transformations under certain conditions.

The transition of M. furfur from a benign skin commensal to opportunistic pathogen involves a multifactorial interplay between host immunity, skin and gut barrier integrity, microbial dysbiosis and nutrient-rich external environments (72). In extremely preterm infants, underdeveloped skin, immature immune systems, frequent antibiotic administration and invasive procedures such as CVC insertion are the key drivers of pathogenic conversion. Both the present case report and a previous report underscore these risk factors (8,75). In particular, CVCs serve as entry points for M. furfur into the bloodstream and are recognized as risk factors for catheter-associated Malassezia fungemia (31).

M. furfur should not be regarded solely as a commensal skin infection in the NICU setting. Despite being a part of the normal human skin microbiota, M. furfur is currently recognized worldwide as an opportunistic fungal pathogen in the NICU, particularly among preterm and extremely low birth-weight infants exposed to lipid-containing parenteral nutrition, prolonged CVC placement, broad-spectrum antimicrobial therapy or prolonged hospitalization (75,76). In this high-risk population, M. furfur may cause catheter-related fungemia, persistent bloodstream infections, pulmonary involvement, thrombocytopenia and sepsis-like clinical deterioration. Clinical manifestations are often subtle and non-specific and include temperature instability, respiratory distress, feeding intolerance, apnea, bradycardia, poor peripheral perfusion, lethargy and reduced responsiveness. Laboratory findings may include leukocytosis, elevated CRP levels and thrombocytopenia (8). Because these features overlap with those of Gram-negative bacterial sepsis or other invasive fungal infections, missed or delayed diagnoses remain common. Thus, M. furfur should be actively considered as a differential diagnosis in neonatal bloodstream infections, particularly when routine blood cultures are negative and risk factors such as lipid parenteral nutrition or prolonged catheter use are present.

For the patient of the present case report, M. furfur was confirmed as the causative pathogen using mNGS, highlighting the limitations of currently available conventional microbiological diagnostic methods for neonatal infections.

Although conventional culture remains essential for obtaining viable isolates and performing antifungal susceptibility testing, the capacity to recover M. furfur from clinical specimens is limited (77). Because M. furfur is a lipid-dependent yeast, routine blood culture systems and standard fungal media may fail to support its growth unless appropriate lipid supplementation is provided (50). Lipid-enriched media, such as Dixon's or Sabouraud dextrose agar supplemented with olive oil or long-chain fatty acids, can improve recovery and allow for downstream phenotypic testing. However, culture sensitivity is limited by strict lipid requirements, slow growth and the possibility of overgrowth by faster-growing microorganisms. These limitations contribute to false-negative conventional culture results and the delayed recognition of bloodstream Malassezia infections (53,78).

Microscopic techniques, such as potassium hydroxide preparation combined with India ink staining or Gram staining, offer rapid and convenient diagnostic options. However, their sensitivity is limited, particularly for the detection of organisms in sterile body fluids. These methods cannot provide species-level identification and rely heavily on the skills and experience of laboratory personnel (53).

The (1→3)-β-D-glucan (BDG) assay is commonly employed to evaluate systemic fungal infections (79). However, because the cell wall of M. furfur contains little to no β-D-glucan, BDG testing in such infections frequently yields false-negative results. Therefore, BDG assays should not be solely used to exclude the diagnosis of Malassezia-associated infections (80).

Conventional PCR and reverse-transcription-quantitative PCR exhibit high sensitivity and specificity and can directly detect pathogen DNA or RNA in clinical samples. Nonetheless, these approaches require optimized PCR primer design and specialized technical platforms and currently lack widely available commercial assays or standardized operating procedures (53,56).

Although matrix-assisted laser desorption ionization time-of-flight mass spectrometry theoretically enables rapid and accurate species-level identification, its clinical application for Malassezia species remains limited. Challenges include difficulties in protein extraction from lipid-rich yeasts and inadequate coverage of Malassezia sequences in current databases, preventing their widespread adoption in routine diagnostics (53,61).

As a rapidly advancing untargeted detection technology, mNGS is particularly advantageous for identifying pathogens in difficult, low-abundance infections (81). In the present study, mNGS achieved etiological confirmation when traditional methods failed to yield positive results. However, mNGS application faces several challenges, including its high cost, the need for specialized personnel to interpret the results and incomplete representation of Malassezia sequences in current databases, which increases the risk of misinterpretation due to background contamination.

The present case represents the first neonate with bloodstream infection caused by M. furfur in mainland China identified using mNGS. One culture-confirmed case of neonatal M. furfur fungemia has previously been reported in Taiwan, China (47). The present case differs from the adult pulmonary case reported by Zhang et al (76) in terms of host background, infection site and diagnostic metho. The study by Zhang et al (76) showed that M. furfur infection mainly occurred in preterm infants and immunocompromised adults, that fungemia was the most common clinical form, and that invasive procedures and total parenteral nutrition were the main risk factors. Adult infection is commonly associated with acquired immunosuppression, transplantation, hematological disease or severe gastrointestinal disease, whereas neonatal infection is mainly driven by extreme prematurity, extremely low birth weight, immature skin and mucosal barriers, prolonged NICU hospitalization, central venous catheterization, broad-spectrum antimicrobial exposure and lipid-containing parenteral nutrition. In addition, neonatal manifestations are usually non-specific and may resemble bacterial sepsis, while routine culture may fail to detect M. furfur because of its lipid-dependent growth requirements. The clinical significance of the present case lies in the timely identification of M. furfur in an extremely preterm neonate with sepsis-like deterioration, multiple predisposing factors and negative or inconsistent conventional microbiological findings. This case highlights the value of mNGS in detecting rare lipid-dependent fungal pathogens that may be missed in routine cultures.

Through an in-depth analysis of the present case, combined with a retrospective review of the literature, the present case report summarized all known risk factors associated with M. furfur bloodstream infection in neonates. These include extremely low birth weight (<1,500 g), extreme prematurity (gestational age <28 weeks), immature immune function, CVC placement, prolonged parenteral nutrition containing lipid emulsions, exposure to broad-spectrum antibiotics and compromised skin and intestinal mucosal barrier integrity. These risk factors are prevalent among patients in NICUs, creating a favorable clinical environment for the transition of M. furfur from a commensal organism to a pathogen (76).

Notably, the patient of the present case report exhibited nearly all of the aforementioned high-risk factors as well as typical clinical characteristics, including persistently negative blood cultures, difficulty in localizing the source of infection and lack of response to conventional antibiotic therapy. When facing diagnostic challenges, mNGS enabled rapid and definitive identification of M. furfur, elucidated the causative agent and directly guided antifungal treatment strategies. As a result, the patient's clinical symptoms improved rapidly, culminating in a successful recovery and hospital discharge, thereby affirming the importance of early pathogen identification and timely intervention for improving clinical outcomes.

This case highlights that although invasive M. furfur infections remain under-recognized in routine clinical practice, their pathogenic potential in high-risk NICU patients should not be underestimated. The present case report suggests that clinical infection guidelines should consider including M. furfur in the spectrum of potential pathogens, particularly in cases of neonatal sepsis characterized by negative blood cultures. This inclusion can help achieve a more comprehensive diagnostic framework. Furthermore, the present study reinforces the core principles of the early detection, diagnosis and treatment of neonatal infections. Proactive assessment of infection-related risk factors, coupled with the timely application of advanced diagnostic techniques, such as mNGS, can help overcome the limitations of conventional methods and significantly enhance diagnostic efficiency and precision.

At present, no standardized treatment guidelines specifically targeting M. furfur bloodstream infections are available. Management generally follows the principles established for invasive fungal diseases and is individualized based on the antifungal susceptibility profiles and clinical status of the neonate. Commonly employed antifungal agents include amphotericin B, itraconazole, voriconazole and fluconazole. Amphotericin B demonstrates sensitivity against the majority of Malassezia species but carries a high risk of toxicity, particularly nephrotoxicity, necessitating cautious use (82). Itraconazole and voriconazole exhibit good in vitro activity against M. furfur, although clinical experience with their use in neonates remains limited (51). Fluconazole has been effective in certain cases, although some studies report lower susceptibility of Malassezia species to fluconazole. Given its favorable safety profile, stable plasma concentration and relatively low side effect burden, fluconazole is more commonly used in neonatal populations (47).

In the present case, the patient, an extremely preterm infant with multiple infection-related risk factors, was promptly managed upon diagnosis by discontinuing lipid emulsion support and initiating antifungal therapy with fluconazole. Despite reported variability in M. furfur susceptibility to fluconazole, clinical decision-making in this case was guided by the limited extent of infection, absence of deep organ involvement and preserved hepatic and renal function (47). Fluconazole was selected as the initial treatment owing to its safety and controllability. Following treatment initiation, the patient's body temperature normalized, inflammatory markers decreased, no recurrence of infection was observed and the patient was successfully discharged. This outcome indicates that under specific clinical circumstances, fluconazole may be a viable therapeutic option for neonatal M. furfur bloodstream infections.

As a lipophilic yeast, M. furfur lacks the ability to synthesize long-chain fatty acids and relies entirely on exogenous lipid sources for growth. In the context of parenteral nutrition, especially when lipid emulsions are used, the organism can colonize catheter surfaces and extensively proliferate, thereby leading to fungemia. Therefore, immediate discontinuation of lipid emulsions upon diagnosis or strong suspicion of M. furfur infection is key for halting ongoing pathogen expansion and controlling infection progression. Combining antifungal therapy with cessation of lipid supplementation has been reported to significantly shorten the disease course and improve clinical outcomes (8).

With rapid advancements in mNGS, its application in the diagnosis of infectious diseases has become increasingly widespread. The core advantages of mNGS include its high sensitivity. mNGS can detect extremely low-abundance microbial DNA or RNA, providing supplementary diagnostic yield in scenarios where traditional methods exhibit low detection rates. Unlike targeted PCR or culture-based methods, the broad detection spectrum of mNGS does not rely on predefined pathogenic hypotheses. Instead, it enables unbiased and comprehensive screening of all potential pathogens within a sample. An important utility of this method is the rapid identification of novel or rare pathogens in clinical situations where conventional diagnostic techniques are limited. In such cases, mNGS facilitates the discovery of rare, fastidious or even novel pathogens, thereby providing key information for guiding clinical decision-making (16,83).

Despite these advantages, some limitations of mNGS must not be overlooked. Currently, the cost of mNGS is substantially higher when compared with that of traditional culture or PCR methods, restricting its widespread adoption in frontline clinical practice. Owing to factors such as sample handling, reagent impurities and environmental microbial DNA contamination, mNGS may detect non-pathogenic microorganisms, complicating data interpretation. Additionally, interpretation of mNGS results requires integration of sequencing read counts, relative microbial abundance and clinical context. It demands sophisticated bioinformatic platforms and highly trained personnel for accurate analysis. Furthermore, owing to the lack of antimicrobial susceptibility information as provided by culture-based methods, mNGS cannot directly provide antimicrobial susceptibility profiles, necessitating either additional testing or empiric therapeutic decision-making (17,84). In the present case report, additional lipid-supplemented fungal cultures using Dixon's or Sabouraud agar with an olive oil overlay were not performed after a positive mNGS result and no viable M. furfur isolate was available for phenotypic antifungal susceptibility testing. This represents a limitation of the present case, although the patient displayed favorable clinical and laboratory responses after fluconazole therapy. The mNGS results should not be interpreted in isolation, particularly for M. furfur, a common skin-associated commensal organism that may theoretically be introduced during blood collection or sample processing. Nonetheless, in this case, M. furfur was not detected in the corresponding NTC, whereas the blood sample yielded 148 reads assigned to M. furfur with a relative abundance of 72.6%, arguing against NTC-related background contamination. Moreover, the patient had multiple risk factors for invasive Malassezia infection, including extreme prematurity, extremely low birth weight, prolonged NICU hospitalization, broad-spectrum antimicrobial exposure, lipid exposure and catheter-related risks. The clinical course was also consistent with bloodstream infection, including fever, clinical deterioration, markedly elevated CRP levels, thrombocytopenia, negative or inconsistent conventional cultures and subsequent improvement after antifungal therapy. Collectively, these findings support a true bloodstream infection rather than incidental skin contamination or overinterpretation of the isolated mNGS results. Therefore, mNGS results must not be interpreted in isolation during clinical practice. Comprehensive evaluation combining patient clinical presentations, laboratory findings, imaging studies and results from conventional microbiological tests is essential. For any detected pathogen, a scientific judgment must be made based on infection site, host immune status, sequencing abundance and pathogenic relevance to avoid misdiagnosis, missed diagnosis or overtreatment.

In the present case report, by integrating mNGS findings within the clinical context, M. furfur was successfully identified as the causative pathogen. Targeted antifungal therapy was subsequently implemented, leading to a favorable clinical outcome. This case demonstrates the value of adopting mNGS technology in the management of complex infections in neonates.

The present case report describes the case of a neonatal bloodstream infection caused by M. furfur in mainland China that was identified directly from blood samples utilizing mNGS. Through a systematic review of the literature, the epidemiological characteristics, clinical manifestations, outcomes, predisposing factors and diagnostic approaches associated with neonatal M. furfur infection were summarized. Based on the diagnostic and therapeutic experience from this case, we propose that M. furfur should be considered in the differential diagnosis of neonatal sepsis, particularly among extremely low-birth-weight infants with persistent clinical suspicion of infection despite negative conventional microbiological tests. The application of mNGS may provide important diagnostic support in such cases, but its results should be interpreted in conjunction with overt clinical manifestations, conventional microbiology techniques and patient-specific risk factors.

Acknowledgements

Not applicable.

Funding

Funding: This work was supported by the Science and Technology Project of Jiangmen (grant nos. 2025YL01029 and 2022YL01014) and Guangdong Provincial Clinical Research Center for Laboratory Medicine (grant no. 2023B110008).

Availability of data and materials

The host-depleted mNGS sequencing data generated in the present study have been deposited in the Genome Sequence Archive (GSA) at the National Genomics Data Center (NGDC), China National Center for Bioinformation (CNCB), under GSA accession number CRA044583. The data are publicly available at: https://ngdc.cncb.ac.cn/gsa/browse/CRA044583. The associated BioProject accession number is PRJCA066328, and the corresponding run accession numbers are CRR3191935 and CRR3191936. Human reads and any potentially identifiable information were removed prior to data submission to protect patient privacy. Other data supporting the conclusions of this article are included within the article and its figures and tables. Additional anonymized data are available from the corresponding author.

Authors' contributions

SG designed the present study. SG and LS confirm the authenticity of all the raw data. SG, LS and QF were responsible for data collection and case acquisition. SG, ZL and YL performed and analyzed all the experiments. SG, LS and KL conducted the literature review and analysis. HL made substantial contributions to the acquisition and interpretation of clinical data, including the evaluation of information. KL contributed to the methodological aspects of the study including technical support, interpretation of sequencing results and analysis of data. PK contributed to the conception and design of the study, methodological guidance, interpretation of findings and revision of the manuscript. SG and PK revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was conducted in accordance with the Declaration of Helsinki and was reviewed and approved by the Ethics Committee of Jiangmen Central Hospital [approval no. (2025)242 A]. This was an observational study involving retrospective analysis of anonymized clinical and laboratory data generated during routine medical care. No additional intervention, sampling or follow-up was performed. Given the retrospective study design and the use of anonymized data, the Ethics Committee waived the requirement for informed consent to participate. All patient-related information was anonymized before analysis and publication.

Patient consent for publication

Written informed consent for publication of the clinical details and accompanying images was obtained from the legal guardian of the patient. All patient-related information was anonymized before publication, and no directly identifiable personal data are presented.

Competing interests

The authors declare that they have no competing interests.

References