A 5-year-old girl suffers from severe encephalitis, who is behind it?

preface

Hereditary CARD9 (caspaserecruitment domain-containing protein 9) deficiency is an autosomal recessive primary immunodeficiency disease in which patients are highly susceptible to deep skin fungal infections
due to immunodeficiency 。 Exophialadermatitidis, E.
d.
dermatitis is a dermatophyte [1], a dark biphasic fungus [2], widely distributed in natural and human living environments, less pathogenic in the normal population, but in immunocompromised people, easy to cause systemic dark filatomycosis infection, the prognosis after involving the central nervous system is poor [2, 4], mortality can be as high as 80%.

This article reported that a 5-year-old female and child with a long-term headache was initially diagnosed with dermatitis external vial mold nervous system infection, and later confirmed as hereditary CARD9 deficiency by whole exome sequencing, aiming to improve clinicians' understanding of the disease, and at the same time improve the microbiology room staff's understanding of rare bacteria, reduce misdiagnosis and missed diagnosis
.

Case history

General information: child, female, 5 years and 11 months, admitted to the hospital for "repeated headache for 4+ months, aggravated by 1+ months", 5 days before admission, monitoring body temperature with transient fever, fluctuating at 37.
5~38.
5 °C, external hospital cranial enhanced MRI indication: multiple abnormal signals in the right basal ganglia area, thalamic area, occipital lobe and bilateral cerebellar hemispheres, with the right occipital lobe as the weight, considering the possibility of inflammatory demyelinating lesions, inclined to vasculitis or acute disseminated encephalomyelitis
.
In order to further clarify the cause, the outpatient clinic was admitted to the Department of Pediatric Neurology of our hospital as "the cause of
intracranial lesions is to be diagnosed".
The patient is generally healthy, with no family history of disease or previous surgery
.

Examination: The child occasionally complained of headache upon admission, mental and appetite were acceptable, no dizziness, vomiting, convulsions, impaired consciousness and impaired limb movement, and no abnormalities
were seen in urine and urine.
Admission examination: body temperature 36.
6°C, heart rate 86 breaths/min, breathing 21 breaths/min, blood pressure 90/58mmHg
.
Clear consciousness, normal gait, no special cardiopulmonary abdominal examination; The muscle strength and muscle tone of the limbs are normal, the ataxia is negative, and the pathological signs and meningeal irritation signs are negative
.
Auxiliary examination on admission: blood routine showed the percentage of eosinophils 0.
1%~15%, the absolute value of eosinophils was 0.
01~1.
27×109/L, and the rest was normal; Autoantibodies showed antinuclear antibody 1:1000, anti-histone antibody 146(+), and the rest of the antibodies were negative
.
Routine EEG shows frequent release
of slow waves in the right occipital and posterior temporal lobes.
Clinically consider the possibility of autoimmune encephalitis, and perform a lumbar puncture to complete cerebrospinal fluid examination
.
Cerebrospinal fluid cytology showed nucleated cells 125×106/L, cerebrospinal fluid biochemistry: protein 674.
5mg/L, sugar 2.
48mmol/L, need to be alert to intracranial infection
.

5 days after admission, the child had a low-grade fever, a body temperature of 37.
5~38.
5 °C, and paroxysmal headache
.
MRI multimodal enhanced scan of brain tumour shows lesions in the right temporoparietal lobe, right basal ganglia, and right thalamus, considering the possibility of
inflammation.
The first cerebrospinal fluid pathogen metagene test results showed the microbial fragments with confidence level A: Aeromonas mild; Microbial fragments with confidence rating B were: Serratia Plymouthi, Acinetosphingosinemonas, Burkholderia cepacia, Acinetobacter baumannii, Staphylococcus epidermidis; Escherichia
coli.
At the same time, autoantibodies were reviewed to show antinuclear antibodies (+,1:1000), anti-histone antibodies 97.
9(+), anti-mitochondrial antibodies M2 type 26.
6(+), anti-Ro~52 antibodies 89.
7(+), anti-SS~A (Ro) antibodies 68.
8(+).

Considering the possibility of intracranial infection with autoimmune encephalitis, no obvious abnormalities
were found in the delivery of autoimmune encephalitis + paraneoplastic syndrome-related antibodies + central nervous system demyelinating-related antibodies + oligoclonal IgG detection.

18 days after admission, the laboratory department reported the critical value of cerebrospinal fluid: yeast-like fungus was found in cerebrospinal fluid culture, and the identification result was external bottle mold dermatitis, fungal G test: 1-3-β-D dextran 333pg/ml
.
Due to the long course of the child's disease, there is currently dermatitis external bottle mold infection, need to be alert to immune function defects, send whole gene exome examination, the results show that there is 1 homozygous mutation
in the CARD9 gene.

The cerebrospinal fluid culture specimens were detected
by the automatic blood culture system BACTECFX of BD Company of the United States and the matching children's blood culture flask.
After cerebrospinal fluid culture is reported positive, oval fungal spores (magnification 10*100) are visible under Gram stain microscope as shown in Figure 1, positive specimens are subcultured with sand weak medium, sand weak medium is purchased from Antu Biotechnology Co.
, Ltd.
, the colony morphology on the medium is shown in Figure 2, the colonies are fluorescently stained and observed under an optical microscope see Figure 3, and the colonies are stained with Medan and observed under an optical microscope see Figure 4
。 Colony identification using VITEKMS automatic microbial mass spectrometry detection system (French bioMérieux), the identification result is Exophialadermatitidis, the confidence of the identification result is 99.
9%, and the VITEK2COMPACT system (French bioMérieux) system and YST identification card are used for strain identification, which shows that the identification accuracy is low and additional tests are required for verification
.
The TDRFUNGUS susceptibility reagent strip of Tiandi Human Biotechnology Co.
, Ltd.
was used for drug susceptibility test, and the results are shown in Table 1
.

Thirty-six days after admission, re-examination of cerebrospinal fluid culture and cerebrospinal fluid metagenes reported the detection of dermatitis external bottle mold
.
The final diagnosis of the child was: 1.
severe encephalitis: dermatitis external bottle mold infection; 2.
hereditary CARD9 deficiency
.

Fig.
1 Dermatitis external bottle mold, cerebrospinal fluid culture after primrose, Gram staining, light microscope 10×100

Figure 2 Dermatitis outer bottle mold, sand weak medium, 35 °C for 48 hours

Fig.
3 Dermatitis external bottle mold, fungal fluorescence staining, fluorescence microscope 10×100 times

Fig.
4 Dermatitis external bottle mold, Medan staining, light microscope 10×40 times

Treatment: The laboratory reported that the identification result of cerebrospinal fluid fungus was dermatitis external bottle mold, because the dermatitis external bottle mold currently has no vertice, according to the in vitro susceptibility test, the MIC value of voriconazole and amphotericin is low
.
Antifungal treatment
with voriconazole (200 mg× 6 tablets, 200 mg, oral Q12 h).

20 days after admission, the child had convulsive seizures, manifested as slightly upward staring of both eyes, no response to exhalation, the duration of about 10 minutes after relief, the child was poor spirit, unable to answer, double pupils to light reflex dull, neck resistance, limb muscle tone is high, oxygen saturation maintenance is not good, so transferred to the children's intensive care unit to continue treatment
。 After entering the PICU, combined with the child complicated with acute kidney injury stage II.
, amphotericin B liposome (10mg/bottle, 10mgivgtt Qd), voriconazole (0.
1g/stick, 0.
1givgtt Q12h), flucytosine (0.
5g×50 tablets, 0.
5g tube feeding Q6h) triple drug antifungal
.

34 days after admission, under the condition of continuous monitoring of liver and kidney function, the dose of liposomal amphotericin B was gradually increased to 60mgivgtt qd, the child was still in a coma, repeated fever, cerebrospinal fluid nucleated cells up to 520×106/L, sugar minimum 0.
06mmol/L reexamination head MRI showed that the scope of brain lesions was progressively expanded, indicating that the current antifungal treatment effect was not good, so adjust the antifungal treatment plan, stop voriconazole, and give posaconazole 18~24mg/ kg.
dtid, amphotericin B is adjusted to intrathecal injection, the first dose is 0.
05~0.
1mg, and then gradually increased to 0.
5~1mg, 2~3 times a week, 5-fluorocytosine continues to be used as before
.

Treatment results, follow-up and prognosis: continue antifungal treatment for 8 months, during which "OMAYA capsule implantation" was performed, regular drainage, the child continued to be in a coma state and had repeated fever, the fungal G test fluctuated at 232~259pg/ml, and respiratory failure occurred 10 months after admission, and finally the child's family gave up treatment and was automatically
discharged.

Case studies

Clinical case studies

How is dermatitis external vial mold central nervous system infection treated?

There was no significant difference
in antifungal activity regardless of source (environmental or clinical and isolate) and isolates (invasive or mucosal).
It has been reported that triazole antifungals such as voriconazole and posaconazole may be more effective against the anti-infective effect of dermatitis external bottle mold; Posaconazole can be used to treat pulmonary cystic fibrosis caused by Extravial Dermatitidis and has been shown to be more effective in combination with amphotericin B [2].

The European Society for Clinical Microbiology and Infectious Diseases (ESCMID) and the European Union of Medical Mycology (ECMM) jointly recommend voriconazole and posaconazole for the treatment of dermatitis external vial central nervous system infiltrative infection, considering that voriconazole brain tissue permeability is good
.
When surgery is not feasible, combination therapy with triazoles and flucytosine may be first-line [14].

Because flucytosine monotherapy is prone to drug resistance, it is often clinically used in combination with amphotericin B, although amphotericin B and its liposomal central nervous system permeability is low, but when there is a central nervous system fungal infection, the combination of amphotericin B and flucytosine is also an effective treatment for central nervous system fungal infections, and in vitro it shows a high synergistic effect on dermatitis external vial mold isolate [15]
.

The child in this case was successively given voriconazole, posaconazole combined with amphotericin B liposome, flucytosine triple antifungal treatment, the effect is still poor, the analysis may be related to
the CARD9 gene mutation of the child.
It is thought that patients with CARD9 deficiency may exhibit impaired
fungal clearance by monocytes, macrophages, and/or microglia at the blood-brain barrier.
On the other hand, CARD9-deficient cells have been shown to require fungal conditioning by serum to effectively kill fungi, and the limited entry of plasma proteins into the central nervous system may be the cause of impaired fungal killing function of the central nervous system in CARD9-deficient patients [16].

Dermatitis exfoliac infection involving the central nervous system has a poor prognosis, chronic progression of infection, only a few cases have been reported, and only deaths have been reported in Asia, the specific mechanism is unknown, and may be related to unequal exposure to
fungi and involvement in different (immune) host factors.

Inspection case studies

For invasive fungal infections, fungal culture remains the gold standard
for the diagnosis of infectious diseases.

The child of the case was admitted to the hospital for "repeated headaches for more than 4 months", and the cause was not found until 18 days after admission, the laboratory department reported the critical value of cerebrospinal fluid: yeast-like fungus was detected in cerebrospinal fluid culture, and the identification result was dermatitis external bottle mold
.
Coupled with the fungal G test: 1-3-β-D dextran 333pg/ml, it was confirmed that the child was encephalitis caused by dermatitis
external bottle mold infection.

Pathogenic examination methods such as fungal culture are currently the main technical means for
clinically confirming dermatitis external bottle mold infection.
Due to the slow growth of in vitro culture of Dermatitis Outer Bottle Medium, it is easy to be covered by fast-growing fungi such as Aspergillus during the culture process, which often leads to clinical missed diagnosis and difficult early diagnosis, it is recommended to extend the laboratory culture time and try to choose special media such as sand weak medium to effectively improve the detection rate of external bottle mold in clinical microbiology laboratories [2,15].

Molecular identification technology, especially DNA sequencing, is an important basis for clinical fungal laboratories to identify dermatitis vial mold infection, and phylogenetic analysis based on ITS sequence can better identify the strains in the genus Vialitis to the species level [4,16-17].

In addition, this case shows that MALDI~TOF is also a recommended identification method
.

The child has a deletion of the CARD9 gene, which predisposes the child to fungi

Due to the long course of the disease and susceptibility to rare bacteria, we sent a whole gene exon test and found that the child had a primary immunodeficiency, that is, hereditary CARD9 deficiency
.
This is an autosomal recessive genetic disorder, and patients with deficiency of this protein are highly susceptible to deep skin fungal infections, Lanternier, Fanny and Pathan, Saad et al.
[1] A correlation study on deep dermatophytosis and hereditary CARD9 deficiency showed that all 17 patients with deep dermatomycosis studied had autosomal recessive CARD9 defects
.
The child in this case had hereditary CARD9 deficiency and was combined with central system infection of Vitis exfoliitis mold, which was consistent with the clinical features
of this immunodeficiency disease.

Why did the first etiological metagenomic test not detect dermatitis external bottle mold?

Metagenomic sequencing has gradually gained clinical attention
for its advantages of not requiring purification culture and being able to quickly and comprehensively display sequence information.
But why hasn't this case been detected? We believe that there may be the following three reasons: 1.
The fungal cell wall is thick, and it is difficult to extract DNA to break the wall, resulting in false negatives; 2.
Improper storage of specimens, resulting in nucleic acid degradation; 3.
The amount of bacteria is too small to detect the fungus
.
Therefore, when we chase new detection methods, we must not forget the traditional classic detection methods, and the culture of pathogenic bacteria is still the gold standard
for the diagnosis of infectious diseases.

Knowledge development

Dermatitis external bottle mold is a dull biphasic fungus [2], formerly named Wangielladermatitidis, and is currently taxonomically classified in Ascomycota, Pezizomycotina, Herpotrichiellaceae, and Exophiala
。 Dermatitis outer bottle mold is widely distributed in the natural environment in moist, rotting soil, plants and other rotting organisms; In recent years, there have also been reports of isolation from human living environments such as dishwashers and saunas [3].

Extravial mold dermatitidis is an opportunistic pathogenic fungus that mainly affects immunocompromised people such as organ transplantation and long-term use of glucocorticoids, causing dark filatomycosis, which can cause systemic infection in some cases and a poor prognosis after involving the central nervous system [2,4].

Dermatitidis was first isolated in 1937 from the facial rash of a Japanese female patient [5].

It was previously thought to occur mainly in East Asia, mainly Japan and China, but in recent years, related case reports have been on the rise worldwide, especially in Europe and North America, suggesting that the bacterium is not a regional pathogenic fungus
.
At present, the distribution characteristics of ecological niches of Vitis exfoliitis in the natural environment are not clear, and some studies have suggested that it may exist in tree decay organisms in tropical rainforests [2]
.
It is thought that infection of superficial tissues such as the skin may be associated with bacterial implantation after trauma, while systemic infection may result from infection after spores are inhaled through the respiratory tract into the bloodstream [2].

In addition, reports of intestinal and surrounding tissue infections have been increasing in recent years, suggesting that dietary intake is also one of the possible routes of transmission [6].

Published case reports are mainly in North America, Europe, and East Asia, and the lungs are the most commonly reported organ affected (27.
90 percent), although the common sites of infection vary by region [6].

The author reviewed the published case reports related to dermatitis external bottle mold, 8 [7-14] cases of central nervous system infection are all from Asia, and the central nervous system manifestation of dermatitis external bottle mold is particularly prominent in China, and its case fatality rate is even as high as 80%.

This difference in clinical manifestations and prognosis may be related to
the virulence of external bottle mold strains of dermatitis in different regions and the local clinical diagnosis and treatment level.

Pathogenic examination methods such as fungal culture are currently the main technical means for
clinically confirming dermatitis external bottle mold infection.
Due to the slow growth of in vitro culture of Dermatitis Outer Bottle Medium, it is easy to be covered by fast-growing fungi such as Aspergillus during the culture process, which often leads to clinical missed diagnosis and difficult early diagnosis, it is recommended to extend the laboratory culture time and try to choose special media such as sand weak medium to effectively improve the detection rate of external bottle mold in clinical microbiology laboratories [2,15].

Molecular identification technology, especially DNA sequencing, is an important basis for clinical fungal laboratories to identify dermatitis vial mold infection, and phylogenetic analysis based on ITS sequence can better identify the strains in the genus Vialitis to the species level [4,16-17].

In addition, this case shows that MALDI-TOF is also a recommended identification method
.

Antifungal treatment options for dermatitis external bottle mold infectious disease are diverse
.
It is generally believed that if the site of dermatitis external bottle mold infection is limited and surgery is indicated, surgical resection of the lesion followed by antifungal therapy can improve prognosis
.
For invasive infections caused by external bottle mold dermatitis, especially bloodstream disseminated infections, systemic antifungal drugs are the mainstay of treatment [18].

However, even after regular antifungal treatment, dermatitis external bottle mold infection still has a high mortality rate (37.
70%)
.

Dermatitis outer bottle mold is more likely to occur in immunodeficiency patients, the mortality rate is high, and the treatment is difficult, so the drug susceptibility monitoring and molecular epidemiological research of related strains need to be strengthened as soon as possible, otherwise it is not conducive to improving the clinical diagnosis and treatment level
of dermatitis external bottle mold infection.

Case summary

The child, a 5-year-old woman, was admitted to the hospital
with "repeated headaches for 4+ months and temperature increase for 5 days".
After admission, cerebrospinal fluid culture detected dermatitis exinophoria, and the child was found to be hereditary CARD9 gene deficiency
by whole gene exon examination.
Extravial mold dermatitis is an opportunistic pathogenic fungus, which mainly invades immunocompromised people such as organ transplantation and long-term use of glucocorticoids, causing dark filatomycosis, and some cases can cause systemic infection, and the prognosis is poor
after involving the central nervous system.
The caspase-recruiting domain egg 9 encoded by the CARD9 gene is an important adaptor protein
.
It mainly mediates inflammatory response and early innate immune response in fungal, bacterial, viral and other infections to play an anti-infection immune role
.
Therefore, when the CARD9 gene is defective, the immune response is suppressed, and the body is vulnerable to fungal infections
.

This case has non-specific clinical manifestations, which makes it difficult for us to diagnose early, and the rapid progression of the disease and the extremely high case fatality rate also increase the difficulty
of treatment.
We have gained some experience in the diagnosis and treatment of this case: 1.
The traditional method is still classic, and ordinary culture is still the standard for the diagnosis of infectious diseases; 2.
For rare bacterial infections, we should trace the source
.
For example, in this case, we found the culprit: hereditary CARD9 gene deficiency
.
3.
Establish effective clinical and laboratory communication, multiple multidisciplinary discussions, and close collaboration between clinical and laboratory can better identify the cause
.
We also reflected on the shortcomings: 1.
The current identification and understanding of rare fungi is not enough; 2.
We should look at new detection methods dialectically, traditional culture technologies cannot be lost, and new molecular diagnostic technologies such as metagenomic sequencing technology should be used well and played just right, so as to better help clinical diagnosis
.

Expert reviews

Gan Jing, Deputy Director of Pediatric Neurology, West China Second Hospital, Sichuan University

The onset of central nervous system fungal infection is often insidious and presents as a chronic or subacute process, and the clinical manifestations are not specific, but the increase in intracranial pressure is more prominent
with the progression of the disease.
In addition, the condition is often fluctuating, and sometimes the illusion of remission occurs after symptomatic treatment alone, but this improvement is often only maintained
for a short time.
The number and morphology of intracranial foci of central nervous system fungal infection vary mainly depending on the degree of immunosuppression of the body and the clear
route of infection.
The use of antifungal drugs in sufficient quantities after diagnosis is an important means of curing fungal infections of the central nervous system, and the main antifungal treatment drugs currently include: polyenes (amphotericin B preparations), triazoles (voriconazole, ixaconazole, itraconazole, posaconazole), echinococcins (caspofungin, micafungin, aniprofen) and flucytosine
。 The distribution of the above drugs in the cerebrospinal fluid is completely different: fluconazole, flucytosine, voriconazole central nervous system permeability is high; Amphotericin B and its liposomal central nervous system permeability are low, but can still be measured in cerebrospinal fluid and are effective in meningitis, and liposomal
amphotericin is recommended when amphotericin doses cannot be tolerated or treatment is ineffective or renal insufficiency.
In addition, surgical treatment is recommended for patients with clear surgical guidelines, and early, sufficient, adequate course of treatment, combined antifungal therapy is continued after surgery, and residual and recurrent central nervous system fungal granulomas require long-term treatment
.

Intractable intracranial hypertension is an important cause of death and various complications in patients with fungal meningitis, long-term drugs are not effective in lowering intracranial pressure, cerebrospinal fluid drainage is more needed to lower blood pressure, and lumbar puncture can be repeated to slowly drain cerebrospinal fluid; However, for patients who cannot control cerebrospinal fluid pressure due to frequent lumbar punctures in the short term, external cerebrospinal fluid drainage is required
.
For patients with refractory intracranial hypertension, Ommaya capsule implantation and drainage is a more convenient and safe procedure with less pain and no herniation, and care should also be taken to avoid secondary infection
.
If adequate antifungal therapy is ineffective with other methods of controlling intracranial pressure, ventricular shunting
is recommended as soon as possible.

Dermatitis external bottle mold central nervous system infection is difficult to diagnose, and easy to misdiagnose, but dermatitis external bottle mold and other fungal infections caused by central nervous system lesions also have something in common, often manifested as fever, headache, vomiting, convulsions, impaired consciousness and local nervous system signs, etc.
, head imaging can be manifested as intracranial proliferative lesions
。 The cranial imaging manifestations of central nervous system infection caused by external bottle mold dermatitis are diverse, and cerebrospinal fluid may decrease sugar and increase protein, but fungal G test may have abnormal findings, these manifestations are correspondingly reflected in this child, but the final diagnosis still depends on fungal culture, so it is recommended that repeated cultures can be performed if conditions and when the condition allows, in order to obtain positive results
.
In addition, there is no clear treatment standard for this disease, and studies have suggested that the combination of amphotericin B and its liposomes, 5-fluorocytosine and itraconazole or voriconazole has a certain effect, but the prognosis of patients with central nervous system infection is poor
.

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