Season, Weather, Arthritis, Dermatitis, and Cardiovascular Abnormalities – Systemic Diseases Caused by Foot Fungal Infection and Its Secreted Proteins, Case Report

The cause of many rheumatic diseases is still unknown. Some Infections might play a role, but the causative evidence is far from definitive. In this arthritic case, an association between a chronic foot fungal infection with Aspergillus sydowii (Aspsy) and arthritis was initially suggested when the treatment of foot hyperkeratosis-like lesions (Xiangya lesions) provoked multiple joints arthritis symptoms. In order to find the association of fungal infection and arthritis, data from scrupulous observations of plantar lesions, arthritic symptoms, and weather features in events of the fungal infection relapses, foot lesion manipulation, or subcutaneous injection of fungal secreted proteins were gathered and analyzed in three years. secreted proteins may mediate the fungal pathogenicity. Effective treatments of the fungal infection improved arthritis and dermatitis. These pathological characteristics have not been described before and could be a new disease, or one of the unknown pathogenic mechanisms for some known rheumatic diseases, such as rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. The results of this research may provide an insight into a novel pathogenic mechanism for some chronic arthritis and may shed light on further clinical studies on the pathogenesis and environmental factors of some rheumatic diseases. Abstract Background, The cause of many rheumatic diseases is still unknown. Some Infections might play a role, but the causative evidence is far from definitive. In this arthritic case,

an association between a chronic foot fungal infection with Aspergillus sydowii (Aspsy) and arthritis was initially suggested when the treatment of foot hyperkeratosis-like lesions (Xiangya lesions) provoked multiple joints arthritis symptoms. In order to find the association of fungal infection and arthritis, data from scrupulous observations of plantar lesions, arthritic symptoms, and weather features in events of the fungal infection relapses, foot lesion manipulation, or subcutaneous injection of fungal secreted proteins were gathered and analyzed in three years.
Case presentation Of the patient, relapses of the fungal infections on Xiangya lesions often occurred after rainy and humid days in winter and spring. Significant relapses of the infection aggravated the symptoms of arthritis within a few days, and the symptoms gradually improved in 2-3 weeks after the remission of fungal infection by topical antifungal treatment. Also, repeated trimming/debriding Xiangya lesion or subcutaneous injection of fungal secreted proteins also induced the arthritis symptoms similar to those of foot fungal infections. Arthritis Dermatitis, bradycardia, hypertension, and elevated blood monocytes were concurrent abnormalities. Topical methotrexate on the fresh trimmed plantar lesions was able to prevent and relieve arthritis.
Conclusions Active fungal infections on plantar Xiangya lesions were associated with cold and humid weather in winterspring or partial lesion debridement. The active fungal infections induced and exacerbated arthritis, dermatitis, and cardiovascular abnormalities. Fungal secreted proteins may mediate the fungal pathogenicity. Effective treatments of the fungal infection improved arthritis and dermatitis. These pathological characteristics have not been described before and could be a new disease, or one of the unknown pathogenic mechanisms for some known rheumatic diseases, such as rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. The results of this research may provide an insight into a novel pathogenic mechanism for some chronic arthritis and may shed light on further clinical studies on the pathogenesis and environmental factors of some rheumatic diseases. Keywords: Arthritis of fungal protein, Aspergillus skin infection, rheumatic diseases pathogenesis, methotrexate, case report  infections on the right plantar heel. Sudden onset of pain on the right plantar heel during routine recreational walking on Day 0 had led to fatigue for 2-3 days, otherwise normal, and the pain relieved at rest. Local examination revealed normal skin, only reddish and slightly elevated area approximately 2.5-centimeter diameter, warm and tender by palpation. The partial surface layer of the skin over the lesion, 0.8x1.0 centimeter, was removed on Day 5, topical ciclopirox cream was started to apply on the lesion twice daily, and the fungal lesion became dark red pigmented a day after (not blooding) (Fig. 1D).
Multiple joints stiffness, pain, and tenderness were recorded according to a visual analog scale 0 as no pain to 10 as the most severe pain possible [7] and displayed in B and C, in    Treating fungal infection on the right heel with topical ciclopirox ointment * Fungal secreted proteins in the culture medium were used in Trial 1 or after purified and concentrated in PBS in Trial 2. ** Intracellular fungi produced fungal secreted proteins in hyperkeratotic lesions.      Background Introduction Rheumatic arthritis, such as rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis, are lifelong maladies with genetic and environmental factors playing a significant role in their pathogenesis. The environmental factors may consist of environmental hazards, dysbiotic conditions, and infections of bacteria, fungi, or viruses [1,2]. Nonetheless, causative evidence of the environmental factors is far from definitive.
One of the difficulties for proving environmental pathogenic factors and mechanisms is the lack of evidence of infecting agents that are persistently present and mediate the pathogenic effects throughout the fluctuating course of the chronic diseases. Also, season and weather are known affecting chronic arthritic symptoms of many patients, but the underlying mechanism is still a mystery [3,4,5].
Chronic foot fungal infections are prevalent in the general population [6]; Aspergillus species have emerged as a common pathogen of foot fungal infections [7]. Despite the high prevalence of cutaneous fungal infections, their pathogenic effects on human health and systemic diseases have not been adequately studied and are mostly unknown.
Increasing research evidence might have implied that Aspergillus infection may associate with rheumatic diseases [8,9,10], but the underlying causal relationship has yet to explore.
For the same case, we have recently found that fungus Aspergillus sydowii causes both extracellular and intracellular infections of foot skin [11]. The Aspergillus infected anucleated human skin keratinocytes, formed the fungal-human composite cells (zombie cells), and reanimated them to grow and proliferate to form hyperkeratosis-like lesions, Xiangya lesion, which resists antifungal drugs and produces abundant fungal proteins. An association between the fungal infection and arthritis was initially suspected when arthritis symptoms occurred during the treatment of long-time foot fungal infections and hyperkeratosis-like lesions. In this report, the weather, the recurrence of fungal infections, and the symptoms of chronic arthritis during the three years were dynamically observed and analyzed. Seemingly harmless plantar fungal infections and Xiangya lesions were found responsible for causing chronic arthritis, dermatitis, and cardiovascular abnormalities.  For single measurement, the intra-class correlation coefficient (ICC) was 0.924, and 95% confidence interval was 0.890 to 0.953, for the average of 25 measurements had higher intra-rater reliability with ICC 0.997, and 95% confidence interval 0.995-0.998, which means highly reproducible.

Patient Information and History
Fungal secreted proteins: Primary culture of skin lesions and subsequent fungal cultures were carried out using human cell culture medium (a chemically defined medium, ATG293, Shenzhen Yisheng Branch Co., Ltd., China). After the fungal culture, the medium was collected by centrifugation at 4000 rpm (3000 g) for 15 minutes and filtered twice through a 0.22μM cut-off syringe filter. Subcutaneous injection used the filtered medium. The medium above was concentrated 33 times on the Amicon Ultra-15 centrifugal filter device (Minipore, UFC 901096) by centrifugation and used for protein quantification on SDS-PAGE.
The concentrated proteins were applied to SDS-PAGE and visualized by coomassie blue stain, about thirty protein bends ranging from 5kD to 200kD of molecular weight were detected and quantified with gel image analysis software; as a result, equivalent concentrations of the proteins in the original medium were 0.01 to 1.5μg/mL. Concentrated protein above was diluted with phosphate-buffered saline (PBS) to about five times the concentration of the original medium, and then further sterilized filtering through a syringe filter with a cut-off size of 0.2μM (17597-K, Sartorius, Germany) and stored in a refrigerator at 4 °C before used for subcutaneous injections.
The subcutaneous tests and observations: In this study, the subcutaneous injection of fungal secreted protein to human subjects (researchers) followed current guidelines for skin prick tests that are allergic to air allergens [13,14]. In the three years, two focused observational trials had been conducted to examine the relationship of foot fungal infections and arthritis, starting with subcutaneous injection of fungal secreted proteins for 46 days in the first trial and nine days in the second trial. For the first trial, 800-1000μL of culture medium from 3 fungal strains and the second trial 200μL of the concentrated fungal secreted proteins were given daily by subcutaneous injections on the front of the thighs, which was the most convenient for self-administering the injections and for observing local reactions. On the trials, the assessments of finger stiffness were measured with calipers five times a day at wakeup, 12, 16, 20, and 0 o'clock, meanwhile taking the blood pressure and heart rate. Joint pain and press pain of joints, including joints of hand digitals, wrists, elbows, knees, ankles, and lumber, was self-examined and recorded in visual analog scale (VAS) once daily [15]. Liquid cyanoacrylate glue (superglue) was directly applied to the surface of fresh trimmed lesions and polymerized in a minute to form a thin film isolating atmosphere from lesion cells [11].

Case Presentation
Weather and relapses of fungal infection: Ten relapses of plantar fungal infection were photo-recorded from May 1 of 2015 to April 30 of 2018, and all occurred at the hyperkeratosis-like Xiangya lesion. Eight of the ten relapses occurred between October to March, correlating with winter and early spring, and the rest two occurred in August and September of 2017 in a period the subject worked and lived in air-conditioning facilities for more than 20 hours a day. Compared to the 3-year average weather, the 7-day weather during the relapses was higher pressure (p < 0.0005), lower temperature (p < 0.01), rainy day (p < 0.01) and higher humidity (p < 0.025) ( Table 1).

Fungal infection and arthritis:
Some of the recurrences of the fungal infections were barely noticed and were observed only in the photo records review. In the significant relapses, the infection caused topical inflammatory symptoms, such as red, warm, pain, and swollen, and 3-5 day fatigue, and followed by the onset of arthritis symptoms in days, including stiffness, pain, tenderness and swollen around the joints of fingers, toes, limbs and lumbar ( Fig. 1 and 2), and cutaneous nodules on side of finger. Arthritis lasted for the entire course of the fungal infections. Dermatitis and cardiovascular abnormalities concurred with arthritis. Although no anti-arthritis remedy was given, arthritis symptoms faded in a week to two weeks after plantar inflammation subsided.
Serum galactomannan (GM) was 0.04. The rest laboratory tests, including the rest of the blood cell count, liver and kidney function tests, rheumatoid factor, anti-CCP antibody, erythrocyte sedimentation rates, and C-reactive protein were all in the normal range during relapses or remissions. The X-ray showed the asymmetric increased thickness of soft tissues on the affected fingers and interphalangeal joints and did not reveal any bone and cartilage deformities on the joints of the hands and fingers (data not shown).

Fungal secreted proteins and arthritis:
The amount of fungal secreted proteins for subcutaneous injection was equivalent to 1mL fungal culture medium (either straight medium or 200µL five times concentrated protein solution in PBS, which consisted of approximately 30 secreted protein peptides at a concentration from 0.01 to 1.5µg/mL. The causative protein(s) seemed rather stable as that the protein solution of the injections was stored at 4°C for 14 months and still induced arthritis.
The injections on the front of thighs caused local skin reactions, red, swollen, pain, and tender in areas about 4-8 centimeters in diameters starting in 3-4 hours and lasting for 2-4 days (Fig. 1E). This reaction was similar to the appearance of the skin reaction of the fungal infection or trimming Xiangya lesion. Arthritis symptoms measuring by finger stiffness started in 2-3 days after the initial injection, and in 7-9 days, they quickly improved after the injections ceased ( Fig. 3B and C). Once-daily of the injection for nine consecutive days induced arthritis symptoms for 15 days (Fig. 3). Prolonged period injections extended the duration of arthritis accordingly (Fig. 4A). Elevated blood monocytes count, 0.65x10 9 /L, also occurred. The secreted protein(s) that causes arthritis were very potent and should need no more than 2µg/day in each subcutaneous injection, which is 100,000 times less than the amount 200mg of anti-TNFα antibody used one dose per injection in arthritis treatment.
Subcutaneous injections to lab mouse: Before self-experimentation with subcutaneous injections of fungal proteins, 100μL of culture medium from 6 fungal strains' cultures and fresh medium as control were given subcutaneously to seven groups of three mice in each group for eight consecutive days, and observations continued for ten additional days after the injections. No apparent adverse effect or skin and joint abnormalities resulted from the injections were observed (data not shown).

Xiangya lesion and arthritis:
Overgrowth of plantar hyperkeratosis-like Xiangya lesion rendered walking pain beneath the lesion and warranted to trim the lesion for pain relief every two months or so. Repeatedly trimming/debriding the lesions led to lesion cell rapid growth and local stimulation sensations for 2-3 days by fungal proteins [11] and followed by the arthritis symptoms in 2 days (Fig. 4D). Topical methotrexate applying twice daily on the lesion immediately after trimming/debriding provided local symptom relief as well as prevented the aggravations of arthritis symptoms (Fig. 4D). Similarly, covering the trimmed lesions with cyanoacrylate glue also had the effect of local symptom relief.
Dermatitis: Dermatitis was a concurrent skin manifestation associated with the relapses of arthritis caused by the fungal infection of the foot and activation of the Xiangya lesion, but it was less apparent when the subcutaneous injection of the fungal proteins was from the cultures. Dermatitis of the skin at the exterior side of the right ankle, above the left knee, the back of finger joints, auricles, and behind auricles gradually emerged following the relapses of the foot fungal infections and arthritis and lasted longer (Fig. 1). The skin started with red and itch, gradually became hyperkeratotic, rough, and pigmented. There were bumpy nodules on or near the arthritic joints, some pain in the active phase of arthritis. Eventually, in months after the arthritis symptoms subsided, peeling skin on dermatitis sites occurred, and the skin returned to a regular appearance in the absence of any topical treatment to dermatitis. Nevertheless, some residual of finger stiffness, dermatitis on the back of fingers, finger nodules, and high blood pressures stayed into the summer and throughout the year.
Cardiovascular abnormalities: Edema, high blood pressure, and bradycardia were found to be associated with skin fungal infections (Fig. 4 A and B). The edema manifested as swollen digital joints, finger stiffness, and pitting edema on the plantar soles, especially in the morning or after a one-hour walk (Fig. 4). Its appearance coincided with arthritis, but lasted longer and became less severe and eventually disappeared in late summer and autumn.
An increase of blood pressure (BP), especially with systolic pressure, was found to associate with foot fungal infection and fungal secreted proteins in the two trials, where the 30-day average BP (systolic/diastolic pressure) increased from 129/83mmHg in the early phase of the first trial (N = 150) to 150/89 mmHg at the end of the second trial (N = 150) over 1.5 years (Fig. 4A and B). The 30 day average of BP was at 147/83mmHg in four months without using anti-hypertension medications after the second trial ended. Systolic pressure increased more than diastolic pressures, which indicated a decrease of vascular compliance for the underlying cause, possibly due to atherosclerosis, maybe, a mechanism similar to that of the nodule formation on the fingers. Nevertheless, more research will need for the cause of hypertension.
The heart rate of the subject gradually decreased from 70-80 beats per minute to below 60 beats per minute in two trials (Fig. 4). It was sinus bradycardia on an electrocardiogram. After the first trial, the daily average of heart rate slowly returned to 65-75 beats/minute (5 measurements/day, seven days) in 4 months, and after the second trial, 65-70 beats per minute (5 measurements/day, seven days) in 4 months and 70-82 beats per minute (5 measurements/day, 7 days) in 8 months with a remission of the fungal infection.
Methotrexate: Topical 0.2% Methotrexate ointment on freshly trimmed hyperkeratotic lesions twice daily for seven days showed slow growth of the lesion, relief of topical irritation and preventing arthritis symptoms induced by trimming/debriding the Xiangya lesions (see Fig. 4 D). Methotrexate might also reduce the arthritis symptoms after applied to the lesions of the fungal infection. However, methotrexate did not seem to cure the Xiangya lesion, to reduce the lesion size in the long term, or to prevent relapses of fungal infection after ceased.
Treatment: Active fungal infections subsided after treatments with lesion debridement and topic antifungal drugs, namely terbinafine, ketoconazole or ciclopirox, with or without methotrexate and achieved remissions in 3-5 weeks. Arthritis and dermatitis had not been treated for their symptoms and were improved after active fungal infection retreated (Fig.   2). All topical drugs, including terbinafine, fluconazole, clotrimazole, ketoconazole, ciclopirox, urea, and salicylic acid ointments, or oral terbinafine and fluconazole did not The Xiangya lesion is an intracellular fungal infection of the skin epithelium consisting of composite human-fungal cells, zombie cells that express fungal proteins [11]. After trimming/debriding the lesion, the remaining lesion cells multiply more rapidly, produce abundant secreted proteins of Aspsy, which disperse into the systemic circulation and lead to arthritis and dermatitis. Fungi are ecological species that modify their phenotypes and genotypes in adapting environmental and nutritional conditions [16,17], which might lead to changes in its secreted proteins according to the locations or the depth into deeper tissues of the human body. It could be conceivable if such infection of Aspergillus progressing from skin epithelium to under dermis tissue, blood vessels, muscles, and bones would cause hosts' remote tissue damages accordingly as the diseases progress.
The mechanism of methotrexate, a disease-modifying anti-rheumatic drug (DMARD) in arthritis treatment, is considered acting on the human immune system but is still not clear how it works exactly [18,19]. Topical use of methotrexate can prevent arthritis symptom attack or relieve arthritis symptoms in 14 days. The mechanism of its effect is more likely due to its inhibitory effect on fungal protein productions rather than to modulate the host immune system.
Aspergillus sydowii, widely present in soil and a known pathogen of sea fan corals [20], has been reported in foot infections cross continents [21,22,23], together with other pathogenic Aspergillus species [24], are emerging as common causative agents in foot infections [7], and recent researches have implicated potential associations of Aspergillus infections with rheumatic diseases [8,25]. On another aspect, the clinic characteristics of Availability of data and materials: The author guaranty materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes upon request.
Competing interests: The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.     infections was retrospectively observed on the right small toe (Fig. 1-A2).  CARE-checklist_Zhu_20200113.pdf