Unlike other pathogenic yeasts, Candida auris has evolved its ability to spread directly in the population. It can survive on the surface of cold, hard and dry objects, and even some chemical detergents cannot inactivate it. Contact with pathogen carriers and contaminated objects may cause infection, and the risk of in-hospital infections in medical institutions is particularly high. Once a patient is infected, Candida auris can be transferred to major organs through the blood and proliferate in them in large quantities. The characteristics of multidrug resistance make doctors have very few choices.
The deadly fungus is now in China, the source is unclear
In 2018, nine years after the first report in Japan, Chinese clinical workers were isolated from the alveolar lavage fluid of a 76-year-old female patient with hypertension and nephrotic syndrome in Beijing, and named this strain. In the following years, Candida auris was also reported in various parts of China. On April 20 this year, Professor Huang Guanghua's team from Huashan Hospital Affiliated to Fudan University, a top infectious academia in China, published an invited editorial at (IF: 5.882), which for the first time recorded the disease of Candida auricida in China.
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2018
In 2018, Professor Wang Hui's team at the Third Hospital of Peking University reported for the first time that China's first Candida auris strain () was isolated from a 76-year-old female patient with hypertension and nephrotic syndrome, and was identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and verified using internal transcriptional spacer (ITS) sequencing analysis, indicating that this strain belongs to the South Asian evolutionary branch;
2018
In the same year, the Beijing Military Region General Hospital reported two blood infections caused by Candida fluconazole-resistant auricles, and the patient was a neonatal in the Neonatal Intensive Care Unit (NICU);
2018
Later that year, in a retrospective study, the First Affiliated Hospital of China Medical University reported 15 cases of Candida auricular colonization and infection. The isolates of these 15 cases were initially mistaken for Candida himulus ( ). All Candida auris strains isolated from the hospital were resistant to fluconazole, but had lower MICs to other tested antifungal drugs;
2019
In 2019, reports of Candida auris appeared in Taiwan, which was isolated from a 55-year-old man with diabetes and pemphigus vulgaris. This strain had a lower MIC (8 mg/L) for fluconazole and a relatively higher MIC (4-8 mg/L) for amphotericin B;
2020
In 2020, the First Affiliated Hospital of Xiamen University reported a blood infection case of Candida auricida, which was from a 67-year-old man with a 10-year history of gastric ulcer and diabetes. This is also the first time that Candida auric infection has been reported in southern China;
2021
In January 2021, Beijing Hospital reported a case of Candida auricida infection in NICU neonates and named the strain;
2021
Later in January of the same year, the Hong Kong Department of Health reported the isolation and genome sequencing results of 19 Candida auris strains obtained from 15 patients in a public hospital in Hong Kong. Based on ITS and genomic sequence analysis, all 19 strains were identified as South Asian branchlets;
2021
In February 2021, the First Affiliated Hospital of China Medical University once again reported new cases of candida auris.
The bacterial strains and the strains isolated from Hong Kong belong to the South Asian branch, all other C.auris isolates in mainland China belong to the South African branch. Therefore, there are at least two different genetic evolutionary branches of Candida auris in mainland China (South Asia and South Africa).
Figure 3 Reported cases and branches of Candida auris in China
The article points out that so far, Candida auric infection in China has the following characteristics:
Most cases are associated with superficial tissues (such as the urinary tract), while blood or deep tissue and organ infections are observed with lower frequency;
Most hospitals in China reported sporadic Candida auricida cases, rather than cluster infections, with two group infections of Candida auricida in only two hospitals (Shenyang and Hong Kong);
Most of the C. auris clinical isolates reported in China are only resistant to fluconazole, although some areolates show higher MICs for amphotericin B and echinocin, but this may be due to recent evolutionary adaptation to antifungal drug use. F126L or mutation of the antifungal protein Erg11 can be observed in all fluconazole-resistant isolates in China;
Most cases of candida auris in China occur in ICU patients aged 55 and older, who usually have a history of underlying diseases such as diabetes and/or hypertension;
Prone to Candida auric colonization or infection appears to be a general correlation with prolonged hospitalization and/or antifungal drug treatment.
At present, China's clinical and basic research on Candida auris is very limited, and many outstanding issues still need to be resolved. For example, Candida auricida cannot be isolated and identified through traditional microbiological identification techniques, which makes it impossible to diagnose infections caused by Candida auricida in a timely manner.
To this end, the Clinical Microbiology Group of the Testing Branch of the Chinese Medical Association published the "Expert Consensus on Diagnosis, Treatment and Control of Candida auricida infection" in the Journal of Clinical Testing in August 2020, giving clear guidance on the isolation and identification of Candida auricida. We excerpted the core part of the "Consensus" on isolation and identification.
Identification of threats—Microbiological identification of Candida auris
Tips for suspected Candida auris
Genetic analysis shows that the bacteria is closely related to Candida Portugal and Candida Citrus.
Recommended 1
The possibility of Candida auris should be considered in the following situations:
(1) Candida does not form pseudohyphae at 25 ℃, and can grow at 42 ~ 45 ℃;
(2) Vitek YST: identified as Candida similon, Candida Portuguese, C., Candida anonymous;
(3) API 20 CAUX: identified as yeast japonica ( ) (but no characteristic red pigment), candida sake (C. sake), or the identification results are uncertain ();
(4) API: identified as Candida abortion;
(5) BD Candida Phoenix identification card: identified as Candida ximulus and Candida streptomorph;
(6): Identified as Candida albicans, Candida tropicalis, Candida periarthritis, Candida anonymous, Candida Portuguese, and Candida Jiyemon;
(7) RapID Yeast Plus: identified as Candida virginia;
(8) Biomery mass spectrometry: identification as Candida albicans and Candida himulus or the identification results are uncertain;
(9) Candida is resistant to azoles or to amphotericin B. Literature reports that 93% of C. auris clinical isolates are resistant to fluconazole, 33% are resistant to voriconazole, and 35% are resistant to amphotericin B.
Separation and identification technology
The morphology of this bacteria under the microscope has no characteristic suggestion, and bacterial species identification is required after the colony is pure. Correct identification requires personnel to be aware of the situation, sufficient experience, and quality control, and the results are credible, repetitive and comparable.
2.1 Traditional Methods
Recommended 2
Vitek 2 YST 8.01 version can identify Candida auricida; the Candida himulus and C. identified in this version need to further determine whether Candida auricida is. However, the BD Candida Phoenix Identification Card, Biomerie API 20C, RapID Yeast Plus cannot identify the bacteria.
Recommended 3
The chromogenic medium can be preliminarily identified, but cannot be final. Candida auris colonies are white or creamy on the sapphire agar medium, pink or beige on the plate, and can change to light pink or purple on the 4th day of culture. Candida chromogenic agar may initially distinguish Candida citrus from Candida auricida. Candida auris can grow larger colonies within 24 hours, and the colony color changes from colorless to pink or beige within 24 to 48 hours. The bacteria grow well at 42 °C (while Candida similone cannot grow at 42 °C), and 0.01% cycloheximide does not grow. Because of the diverse colors and no characteristics, the color-developing medium cannot be finalized.
2.2 Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (-time of mass, MALDI-TOF MS)
Recommended 4
The bacteria can be identified by a Brook mass spectrometer, MALDI CA (version 4) or RUO database (2014 and above). The accuracy of RUO database to identify Candida auris is 75.4% to 100%. Ethanol/formic acid extraction is recommended for sample treatment.
Recommended 5
BioMerrier mass spectrometer, MS IVD version 3.2 (IVD) database of this version is not currently approved by the U.S. Food and Drug Administration) or MS 4.14 and above (RUO) to identify the bacteria. The accuracy of IVD 3.2 of the biomerier mass spectrometer Vitek MS was 96.7% and the accuracy of the identification of Candida auricida in the RUO database was 93.4%. Direct coating is recommended for sample treatment.
2.3 Molecular Biology Methods
Recommended 6
The 26S rDNA D1/D2 region or internal transcriptional spacer (,ITS) sequence was used as the target for species identification, and the primer sequence was as follows. ITS Upstream Primers: 5′
-CGG-3′, downstream primers: 5′--3′; 26S rDNA upstream primers: 5′
-AAAG-3′, downstream primers: 5′--3′.
PCR amplification and sequencing are performed based on the above primers, and the results must be reproducible, verifiable and comparable. Compared with the gene sequences of any branch of Candida auris in the database, the sequence is more than 98%, which is considered to be the same strain.
Recommended 7
For Candida himulus and Candida Portuguese, which are similar to Candida auricida, real-time PCR (real-time PCR) method can be considered when identifying Candida auricida. Candida auricida specific primers: F: 5'-A-3'; R: 5'
-GCGAC-3'. Candida auricida and related species (Candida auricida, Candida himulus, Candida Portuguese, ) specific upstream primers: 5′
--3'; downstream primers: 5'--3'.
Reason: et al. used this system to identify 140 clinically isolated fungal strains (including 44 Candida auricus, 7 Candida similon, 6 Candida Portuguese, 6, 73 other yeast-like fungi and 4 filamentous fungi) and the results were 100% consistent with the rDNA-based sequencing results.
Molecular biological detection of original specimens
Recommended 8
The Candida auricida ITS2 gene target was used to detect Candida auricida in clinical samples by real-time fluorescence PCR (probe method). The primer and probe sequences were as follows. ITS2 gene-specific primers F: 5′-T-3′, R: 5′--3′, probe P: 5′
-FAM--ZEN-IFQ-3'. Gene-specific primers F: 5'--3', R: 5'--3', probe P: 5'
--CA-IRQ-3'.
Reason: Leach et al. used real-time fluorescence PCR to detect the sensitivity of clinical samples between 89% and 100%. Ahmad et al. further evaluated the skin samples of 247 confirmed patients, confirming that the method has good diagnostic performance (sensitivity 93.6%, specificity 97.2%). Centers for Disease Control and Prevention (
) This system is recommended for the PCR method for identification of Candida auricida in clinical specimens.
Antifungal drug sensitivity test
When it is clear that infections caused by Candida auris, the results of the drug sensitivity test can guide clinical treatment.
4.1 Indications for the drug sensitivity test of Candida auricida
Recommended 9
Drug sensitivity tests were performed on all Candida auris isolates. In non-infection, the isolation of multidrug-resistant Candida auricida has sensory control significance.
Recommended 10
When Candida auricida causes an aggressive infection and previous antifungal treatments are not effective, drug sensitivity tests must be performed.
Recommended 11:
Candida auris causes ear infections, and drug sensitivity tests are performed when antifungal empirical treatment is not performed, targeted treatment is prepared, or if the empirical treatment is not effective.
4.2 Test drugs, methods and parameters
Commonly used antifungal sensitivity test methods include micro broth dilution method (CLSI-BMD), instrument method ( , Vitek 2) and gradient dilution method (E-test) methods. These methods have differences in the minimum inhibitory concentration ( , MIC) results when detecting Candida auris. The MICs of amphotericin B were detected with Vitek 2, while the MIC values were lower. Methods Data for detecting antifungal drug sensitivity showed that fluconazole had high MIC values, but other azoles had very low MIC values. MICs for CLSI-BMD detection of caspofungin may be increased pseudo-in, and some studies have also shown inter-laboratory differences in CLSI-BMD and methods for detection of caspofungin sensitivity. Some scholars recommend the CLSI-BMD and E-test methods for antifungal sensitivity tests of Candida auris.
Recommended 12
The MIC of 5-fluorocytosine, azoles, echinocin, and amphotericin B was tested using the CLSI macro broth dilution method as the reference method. The operating and interpretation standard is CLSI M27 Ed4.
Recommended 13
When using the E-test method, the MIC of voriconazole and caspofungin can be detected. The MIC results of the E-test method for detecting amphotericin B are lower than those of the reference method.
Recommended 14
When using biomerier Vitek AST YS07 card, MICs of 5-fluorocytosine, fluconazole, voriconazole, caspofungin, and micafungin can be detected. The MIC results of the detection of amphotericin B are higher than those of the reference method, and the results are unreliable.
Recommended 15
When using Thermo Fisher® Y010, MICs of 5-fluorocytosine, fluconazole, voriconazole, itraconazole, posaconazole, caspofungin, micafungin, anirifungin, and amphotericin B can be detected.
Recommended 16
There are currently no reports of ATB 3 for the detection of Candida auricida resistance. If used, verification and comparison are required.
Recommended 17
Candida auric trial epidemiological value reference website
, see Table 1.
4.3 Drug resistance mechanism detection
In fluconazole-resistant Candida auricular strains, the incidence of amino acid substitutions F126L, Y132F and K143R encoded by the ERG11 gene is much higher than that of the sensitive strains. In addition, since the lanosterol 14α-demethylase () encoded by ERG11 is the main target for the action of azoles, upregulation of ERG11 expression can also lead to the need for higher concentrations of drugs to bind to ERG11, thereby causing drug resistance. The FKS1 gene is involved in encoding 1,3-β-D-glucan synthase, which is also a target for echinocin to play a role. The hot spot encoding S639F mutation in FKS1 may lead to a reduced affinity of Candida auricin drugs and thus lead to drug resistance.
Homology research technology
Recommended 18
Homology analysis is required when aggregation or fulminant appearance of Candida auris occurs.
Recommended 19
According to the literature, the following homology analysis techniques are recommended.
(1) Pulse field gel electrophoresis (-field gel, PFGE): Korean scholars use PFGE technology to analyze the homology of Candida auris. The judgment criteria are: if the band size and number between the strains are the same, it is considered to be the same type; if the band changes of 1 to 3 bands are considered to be highly similar types; if the band changes of 4 or more bands are considered to be different types.
(2) Multi-locus sequence typing ( , MLST): 4 internal fragments of the housekeeper gene (RPB1, RPB2, ITS and D1/D2) were amplified by PCR and their sequences were determined to analyze strain mutations. Using this method, Korean scholars identified the ST2-type popular strain, and Indian scholars successfully distinguished 104 Candida auricida strains from India, South Africa, Brazil, Japan and South Korea and constructed phylogenetic trees.
(3) M13 DNA PCR: et al. used M13 DNA PCR technology to genotype 12 strains of Candida auricida, and successfully distinguished 10 different genotypes of Candida auricida from India and 1 strain from Japan and Korea.
(4) Whole genome sequencing (whole, WGS): Among all molecular typing technologies for pathogen traceability, WGS has the highest resolution due to its wide analysis range. SNP is the most extensive variant in the genome, representing genetic differences between different individuals. Genome-wide SNP analysis (wgSNP) provides more SNP sites, and the bacterial types can be divided more carefully by comparing SNP information.
Write it later
The threat of fungi has often not been paid attention to, and we are far from knowing the emerging fungi threats.
What is the original source of Candida auris in China? Did it originate from other countries, and if so, how did it come to China? What measures should be taken to prevent clustered infections and in-hospital or in-hospital transmission? Is the current application of antifungal drugs in clinical environments related to the emergence of Candida auricida? Does C.auris' virulence and antifungal resistance develop rapidly? We still have no answers to these questions.
At a time when the novel coronavirus is raging, it is particularly necessary to pay attention to fungal infections, especially fungal infections in hospitals. When severely ill patients are dependent on medical machinery such as ventilators and their immune system is suppressed, they will appear extremely fragile. Current research shows that Candida auris can survive on various surfaces such as metals, plastics, fabrics, paper, etc., and resist a variety of common cleaners and disinfectants. Under the threat of the new crown epidemic, disposable protective equipment such as masks, protective clothing, and face screens are facing a shortage. Medical staff often have to reuse these equipment, which also creates more favorable conditions for the spread of Candida auricida.
In addition, Professor Huang Guanghua's team also pointed out in the study that Candida similone is a species closely related to Candida auris and is usually resistant to a variety of antifungal drugs. Compared with Candida auric infection, Candida ximus is much more frequently observed in the clinical environment of China. Candida ximus may be more likely to become a "super fungus" in China?
To address these pressing problems, more efforts are needed to study these emerging pathogens, which will rely heavily on clinical, experimental, and collaborative collaboration among basic researchers.
