Diagnosis and treatment of oral fungal infections caused by genetic defects (with a flow chart for the diagnosis and treatment of chronic mucocutaneous candidiasis)

preface

Chronicmucocutaneous candidiasis (CMC) is a rare and special type of candidal infection characterized by increased susceptibility of the skin, mucous membranes, and nails to candidaspp.
and dermatophytes ^[1].

CMC occurs in patients
with various acquired or inherited immunodeficiency disorders.
Gainof function GOF of signaltransducer and activator of transcription 1 (STAT 1) is autosomal dominant and is the most common genetic cause of
CMC.
The family of signal transduction and transcription protein activators (STAT) is a key signaling molecule
downstream of interferon (IFN), cytokines, growth factors, and hormones.
Most STAT1GOF mutations are located in STAT1's coiled-coil-domains and DNA-binding domains, and mutations that occur in the DNA linkerdomains are rarely reported in the literature ^[2].

。 The clinical presentation of patients with STAT1GOF mutations is heterogeneous, but nearly all patients present with symptoms of CMC and are often associated with other infectious (bacterial, fungal, or viral infections) and noninfectious diseases (autoimmune/inflammatory, aneurysm, and tumors) ^[3].

The most common manifestation of CMC is oral candidiasis, which often develops early and
persists due to long-term chronic infection of candida and immunodeficiency.
Chronic proliferative candidiasis is a proliferative lesion of the mucous membrane caused by candidal infection, with a potential malignant risk, which is a rare and severe type of oral candidiasis, but is a common local manifestation
in patients with CMC.
Due to the atypical clinical manifestations, and the insufficient understanding of CMC by clinicians, and its pathogenesis is relatively complex, diagnosis and treatment are more difficult, so the condition is often delayed, resulting in the risk of oral mucosal cancer, increased risk of other serious infections throughout the body, and even life-threatening in severe cases
.

Case history

The patient, a 19-year-old male, came to the Department of Oral Mucosa in December 2021
with tongue cracks accompanied by hardness for 5 years and pain for 1 year.
In the past 5 years, the patient has repeatedly complained of tongue cracks, but they were misdiagnosed as grooved tongues and were not treated
.

Clinical oral examination found that the patient's tongue had different shades of grooves, and the tongue was obviously hyperplasia and enlargement, nodular, and hard texture (Figure A).

Saliva samples from patients were sent to the laboratory for fungal culture, and the results showed that Candida albicans + and the number of colonies > 200/mL (Figure B), suggesting a diagnosis of oral candidiasis
.
Later, the pathology of the back of the tongue showed chronic inflammation of the epithelial mucosa, epithelial hyperplasia, microabscess formation on the surface, and PAS staining showed that Candida hyphae invaded the epithelium, and the pathological diagnosis was chronic proliferative candidiasis (Figure C).

The patient had no abnormalities in the four items of complete blood analysis, thyroid function, biochemistry, immunoglobulin, infectious diseases, etc.
, and the patient had no systemic diseases or external triggers, so it was considered that the patient had the possibility
of genetic defects related to fungal infection.
Therefore, second-generation gene exome sequencing was performed on the peripheral blood of the patient to screen for potential pathogenic gene locus, and the results showed that the patient had mutations in the linker region of STAT1 gene (c.
1666C>T, p.
Leu556Phe), which was found
for the first time at home and abroad.
Further first-generation sequencing verification was performed on the saliva samples of parents without fungal infection as a control, and it was confirmed that heterozygous mutations in this gene were the cause of chronic candidal infection in patients (Figure D), so the patient was finally diagnosed with chronic mucocutaneous candidiasis
.

We first treated the patient with antifungal (oral fluconazole capsules + mycin tablets) for 2 months, the tongue lesions basically subsided, but there were still nodules in the middle of the dorsal tongue, at this time, the patient's saliva sample was sent to the laboratory for fungal culture, and the results showed that no candida growth was
seen 。 We subsequently treated the residual lesions of the dorsal tongue with ALA-PDT (photodynamic), that is, 20% 5-aminolevuleric acid (ALA) was applied to the residual lesions for 2 hours, and then irradiated with a photodynamic laser therapy instrument (LH-600 type, 635mm, 600mW, 1 spot every 1.
5 minutes, the diameter of the spot is 1cm, the light source is about 1.
5cm away from the lesion), and the tongue nodule has basically subsided (Figures E and F).

After the patient was given 2~4% sodium bicarbonate mouthwash to prevent the recurrence of the disease, it is still under
further follow-up.

Fig.
A Oral examination of tongue lesions in patients (initial diagnosis)

Microbiological examination: saliva specimen received by the laboratory (fungal culture medium identification)

S.
sand medium results: Candida +, > 200 pcs/mL; Komaga medium identification result: Candida albicans
.

Figure B Saliva fungal culture examination

Pathological examination: pathology taken from the back of the tongue shows chronic inflammation of the epithelial mucosa, epithelial hyperplasia, microabscess formation on the surface, PAS staining shows that Candida hyphae invade the epithelium, and pathological diagnosis is chronic proliferative candidiasis
.

Fig.
C Pathological examination of the tongue (PAS stain)

First-generation sequencing test verified that the patient had a heterozygous mutation
in the linker region of STAT1 gene (c.
1666C>T, p.
Leu556Phe) in the control of his parents without fungal infection.

Figure D Generation 1 sequencing verification results

Figure E Clinical manifestations of the tongue (after antifungal therapy)

Figure F Comparison chart before and after treatment

Case studies

Clinical case studies

Treatment challenges and strategies for chronic proliferative candidiasis that often occurs in patients with chronic mucocutaneous candidiasis?

Chronic proliferative candidiasis, although rare, is one of the most severe types of oral candidiasis and carries a risk of cancer
.
This type may be accompanied by abnormal epitheliosis to varying degrees, and some cases can progress to oral squamous cell carcinoma
.
In the 2017 World Health Organization (WHO) classification of head and neck tumors, chronic candidiasis was listed as a potential malignant disease of
the oral cavity.
The long antifungal treatment period of chronic proliferative candidiasis and the tendency to relapse pose great challenges
to clinical diagnosis and treatment.

There is currently no ideal treatment
for chronic proliferative candidiasis.
These include removal of possible predisposing factors (e.
g.
, smoking), antifungal therapy, topical retinoid, and surgical treatment
.
Antifungal therapy is a common treatment for chronic proliferative candidiasis, but the response is often suboptimal
.
Because the disease has a risk of cancer, and clinically common cases due to poor antifungal treatment effect, photodynamic (PDT), laser, cryogenic and other therapies are also used to effectively treat
residual lesions resistant to antifungal therapy.

In this patient, because the genetic defect cannot be removed, after aggressive treatment, on the basis of the resolution of the lesion, some precautions should be taken to prevent the recurrence
of proliferative candidiasis.
Such as the use of 2% ~ 4% sodium bicarbonate mouthwash gargle, administration of thymus peptide and other drugs to improve autoimmunity
.

Inspection case studies

Further investigation and diagnosis of chronic mucocutaneous candidiasis?

Chronic mucocutaneous candidiasis is a group of rare and specific syndromes associated with candidal infection, typically characterized by long-term or recurrent candidiasis of the skin, nails, and mucous membranes, mostly caused by Candida albicans, often with endocrine system disease or immunodeficiency
.
Diagnosis of chronic mucocutaneous candidiasis requires a combination of history, clinical presentation, laboratory tests, and biopsy
.

Basic laboratory tests include: mycological examination (10% KOH smear microscopy of oral exfoliated cells, saliva or gargle-containing Candida culture identification), complete blood cell analysis, biochemistry, anti-HIV antibody detection, immunoglobulin quantification, etc.
; According to the possible clinical classification, serum electrolytes, endocrine indicators (thyroid function, parathyroid hormone, cortisol, etc.
) can be further examined, and genetic testing can be performed
.
In addition, chronic mucocutaneous candidiasis is often manifested in the oral cavity as chronic proliferative candidiasis, and its typical histopathological manifestations can be seen with obvious epithelial hyperplasia, neutrophil infiltration to form microabscesses, and PAS staining can detect invading hyphae in the keratinized layer
.

Knowledge expansion

Diagnosis and treatment of chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis is a group of uncommon and idiosyncratic syndromes associated with candidal infection, typically presenting with prolonged or recurrent candidiasis
of the skin, nails, and mucous membranes.
The pathogenesis is thought to be mainly caused by immunodeficiency, and some patients are related
to genetic factors.
Because its diagnosis requires a combination of history, clinical manifestations, laboratory tests, and biopsy, and chronic proliferative candidiasis that often occurs in patients with chronic mucocutaneous candidiasis is a refractory candidiasis infection, the diagnosis and management of this disease is a major challenge
for clinicians.
Our department is an advanced position in the diagnosis and treatment of oral candidiasis at home and abroad, and has published many papers to describe its diagnosis and treatment methods [^4,5], and summarized the diagnosis and treatment methods of the disease into a diagnosis and treatment flow chart, in order to further standardize the diagnosis and treatment of the disease ^[6].

Flow chart for diagnosis and treatment of chronic mucoscutaneous candidiasis ^[6].

Antimicrobial effect of photodynamic therapy

Photodynamic therapy is a non-invasive new technique whose action is based on topical or systemic application of photosensitizers
.
The photosensitizer can accumulate in large quantities in the diseased tissue, and then a specific wavelength of light is applied to irradiate the target site and activate the photosensitizer, and the excited photosensitizer transmits energy to the surrounding oxygen, and then generates highly active single state oxygen, which oxidizes with adjacent biological macromolecules, resulting in cytotoxic effects, which in turn leads to cell damage and even death
.

Photodynamic therapy is currently recognized as a precise targeted therapy that effectively kills pathogenic microorganisms, and is also known as antibacterial photodynamic therapy (aPDT)
because of its inactivation effect on pathogenic microorganisms 。 At present, there are few international reports on the clinical application of photodynamic therapy in the treatment of proliferative candidiasis, and the Department of Oral Mucosa of our hospital applied for a new technology and new therapy project in 2020, exploring the efficacy and safety of photodynamic therapy for the treatment of chronic proliferative candidiasis single-arm clinical trial, and the results showed that photodynamic treatment of chronic proliferative candidiasis can effectively regress the lesions resistant to antifungal therapy, improve the surface hyperplasia of lesions, and reduce the lesion area ^[7].

。 The treatment of ALA-PDT in this case has also achieved good therapeutic effect, and the patient's nodular hyperplasia and lesions have basically subsided, and further follow-up is still under
follow-up.

Case summary

This case is a young male, the clinical manifestations are mainly tongue cracks and nodule thickening, due to the patient's young age of onset, good general condition, and clinical manifestations similar to the performance of the oral mucosal benign disease grooved tongue, easy to be misdiagnosed as benign grooved tongue, delayed diagnosis and treatment
.
Diagnosis of chronic mucocutaneous candidiasis requires a combination of clinical history, clinical presentation, laboratory tests, and biopsy
.
In this case, the clinician first discovered suspicious nodule thickening and suspected oral candidiasis, and then performed saliva fungal culture to find Candida albicans to diagnose oral candidiasis; Combined with the clinical manifestations of the pathology of the tongue, it was diagnosed as the most rare type of oral candidiasis, that is, chronic proliferative candidiasis; Then, combined with the basic test items found no abnormalities, and the patient had no systemic diseases and no external triggers, the patient was subjected to genetic testing, and through sequencing and verification, it was found that the patient had a heterozygous mutation in the linker region of the STAT1 gene (c.
1666C>T, p.
Leu556Phe), which was found
for the first time at home and abroad.
Patients are eventually diagnosed with chronic mucocutaneous candidiasis
.
After basic antifungal therapy with antifungals, ALA-PDT (photodynamic) therapy was applied to residual lesions and necessary measures were taken to prevent recurrence
.

The case reminds us that if young patients have tongue cracks from childhood and adolescence, there is a gradual thickening and hardening of the performance, even if there are no symptoms, it should also be paid attention to, can not directly diagnose benign oral mucosal disease grooved tongue, should be pathological examination to exclude the possibility of chronic proliferative candidiasis, and then screen for possible gene mutations through gene sequencing to determine whether there is a possibility of chronic mucosal cutaneous candidiasis, and patients should be given corresponding treatment measures, For chronic proliferative candidiasis, antifungal agents combined with PDT can be used, and patients can be provided with genetic and disease prevention counseling and guidance
.

Expert reviews

Sun Zhiqiang, Deputy Chief Physician, Department of Clinical Laboratory, Peking University Stomatological Hospital

Chronic mucocutaneous candidiasis is a rare group of special syndromes associated with candidal infection and is often first seen in dentistry
because almost 100% of patients have prolonged or recurrent oral candidiasis.
However, at present, stomatologists have insufficient understanding of the disease, and the diagnosis of the disease often depends on the combination of clinicians, laboratory departments and pathologists, so it often causes misdiagnosis or missed diagnosis of the disease and delays the condition
.
Patients with chronic mucocutaneous candidiasis often develop chronic proliferative candidiasis in the oral cavity, which is a proliferative lesion of the mucous membrane caused by candidal infection, which has a potential malignant risk, so the delay in diagnosis and treatment has serious
consequences.

In this case, it is rare that clinicians can accurately identify the rare type of oral candidiasis, that is, the atypical clinical manifestations of proliferative candidiasis, and make an accurate diagnosis through further microbiological screening and pathological diagnosis
.
In addition, the heterozygous mutations of the STAT1 gene linker region (c.
1666C>T, p.
Leu556Phe) found in the genetic screening of this patient have not been reported at home and abroad, which is an innovative new discovery
.
Clinicians have adopted novel treatment methods, namely antifungal agents combined with ALA-PDT to treat the clinical manifestations of proliferative candidiasis, control the disease in time, and give patients measures to prevent recurrence, which provides an important reference
for clinicians in the treatment and prevention of such patients.

This case suggests that clinicians should pay attention to the integration and communication with laboratory departments and pathologists in clinical work, jointly make decisions on the diagnosis and treatment of diseases, and reduce the missed diagnosis and misdiagnosis
of the disease.

References:

1.
Martinot M， Korganow AS， Wald M， et al.
Case Report： A New Gain-of-Function Mutation of STAT1 Identified in a Patient With Chronic Mucocutaneous Candidiasis and Rosacea-Like Demodicosis： An Emerging Association[J].
Frontiers in Immunology， 2021， 12：760019.

2.
Zhang W， Chen X， Gao G， et al.
Clinical Relevance of Gain- and Loss-of-Function Germline Mutations in STAT1： A Systematic Review[J].
Frontiers in Immunology， 2021， 12：654406.

3.
Depner M， Fuchs S， Raabe J， et al.
The Extended Clinical Phenotype of 26 Patients with Chronic Mucocutaneous Candidiasis due to Gain-of-Function Mutations in STAT1[J].
Journal of Clinical Immunology， 2016， 36(1)：73-84.

4.
LIU Xuanling, HUA Hong.
Chronic mucocutaneous candidiasis[J].
Beijing Journal of Stomatology, 2006, 14(1):3.

5.
Liu X， Hua H.
Oral manifestation of chronic mucocutaneous candidiasis： seven case reports[J].
Journal of Oral Pathology & Medicine， 2007， 36(9)：528-532.

6.
Hua Hong, Gao Yan.
80 cases of oral mucosal disease[M].
First edition.
Beijing:Peking University Medical Press,2019.
]

7.
Zhang Wenqing.
A retrospective cohort study of oral chronic proliferative candidiasis and an exploratory single-arm clinical trial on the efficacy and safety of photodynamic therapy adjuvant therapy[D].
Peking University.
2022.