Diagnosis of candidiasis: a comparative review of methods and assessment of resistance

Laboratory diagnostics of candidiasis: analytical accuracy of methods and problems of antifungal resistance

Candidiasis, caused by opportunistic fungi of the genus Candida (in particular C. albicans, C. glabrata, C. tropicalis), represents a significant challenge in clinical medicine, ranging from common mucosal infections to life-threatening invasive candidiasis. Accurate and timely laboratory diagnosis is critical to differentiate colonization from true infection and to ensure effective therapy. This analytical review is devoted to comparing classical and molecular methods, as well as assessing the problem of resistance development.

Comparison of classical and innovative diagnostic methods

The choice of diagnostic test depends on the clinical form of candidiasis (local or systemic). Below is a comparison of the key methods.

1. Microscopy and culture (Culture)

• Microscopy: A rapid, cost-effective method that detects yeast cells and pseudohyphae in a smear. However, its sensitivity depends on the skill of the laboratory technician and the number of fungi in the sample.
• Cultural research (Baksowing): It is the “gold standard” for diagnosing local infections. It allows identification of the species Candida and quantify the presence of the fungus. Culture is critical for further testing.

2. Susceptibility testing (Antimicrobial susceptibility test)

After culture isolation, antifungal susceptibility testing is mandatory. This is especially important given the increasing proportion of non-albicans species Candida, which often exhibit natural or acquired resistance to standard azoles (source: WHO).

Specific laboratory markers C. albicans: The role of serology and PCR

Diagnostic panel for Candida albicans includes both direct methods for detecting the pathogen and indirect (immunological) tests.

1. Serological diagnostics (Candida albicans, IgM, IgG)

Serological testing (antibody testing) is commonly used to assess the body's immune response to the fungus, but its interpretation is complex.

• IgM (Immunoglobulin M): IgM antibodies are produced at the initial stage of infection. Their presence in venous blood may indicate acute or recent infection. However, in immunocompromised patients (risk group for invasive candidiasis), IgM production may be reduced or absent.
• IgG (Immunoglobulin G): IgG antibodies indicate past contact from Candida or chronic infection. Because C. albicans is part of the normal microflora, low titer IgG can be detected in most healthy people. Diagnostically significant is a significant increase in IgG titer in paired sera (after 10-14 days) or exceptionally high titer.

Clinical note: Serological tests (IgM, IgG) are of limited value for the diagnosis of acute invasive candidiasis due to their low speed and specificity. Their use is mostly limited to assessing immune status or monitoring chronic forms (source: Mayo Clinic).

2. Molecular diagnostics (Real-time PCR)

Polymerase chain reaction (PCR) testing is a direct method and has the highest sensitivity and speed.

• PCR (qualitative determination): DNA detection Candida albicans (scrape, ejaculate, test sample). This method is ideal for local diagnostics (urogenital candidiasis, vaginal candidiasis, scraping tests), as it quickly and unambiguously confirms the presence of the fungus in the sample.
• Advantages: High analytical accuracy (Real-time PCR), short lead time (1 day), the possibility of testing from various biological samples (scrape, ejaculate).

Biomarkers and molecular diagnostics of systemic infections

Diagnostics invasive candidiasis is more difficult, as blood cultures often give false-negative results. Here, non-culture methods come to the fore.

1. Detection of β-D-glucan (BDG)

• • β-D-glucan (BDG): Measures the level of a cell wall component of most pathogenic fungi. The BDG test has a high sensitivity (over 70% according to Mayo Clinic), which makes it a valuable tool screening for early detection of invasive candidiasis.
  • Limitation: The test is not specific for Candida and may be positive in other fungal infections or even with the use of certain medical materials.

2. Polymerase chain reaction (PCR)

Molecular methods based on PCR detect Fungal DNA directly in a blood sample, which is a breakthrough in the speed of diagnosis. PCR has a high specificity and allows you to identify the species Candida within hours, not days as with culture. This is critical for initiating empirical therapy when invasive infection is suspected, which significantly reduces mortality (source: FDA).

Standardized criteria for invasive candidiasis

To unify the diagnosis of invasive candidiasis, standardized criteria are used, such as the criteria from ECIL (European Conference on Infections in Leukemia). The diagnosis is classified as possible, probable or confirmed based on a combination of three groups of data:

• Patient risk factors: For example, neutropenia, organ transplantation, prolonged stay in the ICU.
• Clinical signs: Persistent fever that does not respond to antibiotics.
• Mycological evidence: Positive blood culture (confirmed), or positive non-culture test results (BDG, PCR) (probable).

Using these criteria helps clinicians make informed decisions even in the absence of a positive blood culture.

Mechanisms of antifungal resistance and clinical challenges

The main problem of modern mycology is the growing antifungal resistance, especially the most common drug – fluconazole.

• Risk factors: Long-term use of azoles, previous hospital treatment, and the presence of concomitant diseases (e.g., diabetes mellitus).
• Molecular mechanisms of resistance:
  • For Azoles: Mutations in the gene ERG11 (encodes the target enzyme) and activation of efflux pumps (proteins CDR1/CDR2), which actively pump the drug out of the fungal cell. These mechanisms are most characteristic of C. glabrata and C. parapsilosis.
  • For Echinocandins: Mutations in the gene FKS1/FKS2 (encodes an enzyme that synthesizes the cell wall), although resistance to this class of drugs remains rare.

Clinical strategy: If standard therapy does not provide an effect within 72 hours, it is necessary to: 1) confirm the diagnosis using highly sensitive tests (PCR, BDG); 2) obtain the results of the antimycogram from the culture; 3) switch to drugs of other classes, such as echinocandins.

A good laboratory strategy involves using a combination of methods: rapid microscopy for preliminary evaluation, culture for identification and antimycobacterial activity, and the use of BDG or PCR for early screening for systemic forms. This comprehensive approach, guided by the clinical picture, is the basis for effective management of candidal infections.

Sources

1. World Health Organization (WHO). Antimicrobial resistance and its control. Guidelines on surveillance of fungal resistance. 2023.
2. US Food and Drug Administration (FDA). Guidance for Industry on Development of Antifungal Drugs. Recommendations for diagnostic test usage. 2022.
3. Mayo Clinic Laboratories. Fungal Diagnostics Testing Menu. Protocols for $\beta$- D-glucan and culture sensitivity testing.
4. European Conference on Infections in Leukemia (ECIL). Revised guidelines for the diagnosis of invasive fungal disease. 2020.