Characterization and eradication of the high concentration infection source of oral segment of Candida by infection examination diagnosis system

Purpose: Candida albicans (CA) is a major pathogenic fungus that causes a critical infection and life-threatening disease. Currently, during the treatment of COVID-19 (in ICU), invasive Candidiasis and Candida colonization should be prevented to avoid the increase, in mortality, in SAPSII, and in length of stay. Our aim was to characterize and analyze the infection of Candida albicans (CA) for eradication. Methods: We characterized and analyzed the infection source and infection eld which are carrier eld, diffusion eld, and spread eld, by using a new infection examination (diagnostic) system (IEDS) comprising a new dental formula (measurement analysis) medium (DFM) with time, space, and phase, and the simultaneous relative differential equations (C4RDE). Results: This study showed that CA was not an endogenous microorganism, but a fungus that infected teeth causing dental caries that was successfully eradicated from all teeth of 353 Japanese patients (oral cavity segment). [In the rst examination, the percentages of people with CA (+) was 40.5% (the DMFT: 62.8%) and CA(-) was 56.4% (the DMFT: 47.1%). The difference in DMFT was 15.7% (P<0.01)] (DMFT is decayed, missing, and lled tooth index). Conclusion: Eradication of the infection source (IS) of the oral segment(s) and breaking the infection chain (IC) by IE(D)S will dramatically reduce the threat to the living body by pathogenic microorganisms. And, our methods help to eradicate the infection source (IS), prevent critical infection, and reduce mortality. The IEDS is a powerful tool that can be applied for any microbial infection easily. It is possible for the dentists to characterize CA by IEDS and eradicate CA from the oral cavity. Moreover, CA must consider the phase of the space-time of infection source, infection eld, receptor eld, resistance eld, adhesive colony, xed colony and infection chain. Moreover, CA must consider the phase of microorganisms. If they are neglected, the statistical research


Introduction
Candida albicans (CA) is a fungus that causes a life-threatening infection [1][2][3]. Furthermore, CA colonization on airway is associated with pneumonia, malignant tumors, failure of organ transplantation, ocular opacities, digestive disorders, and respiratory disorders [3][4][5][6][7][8][9][10][11]. Moreover, a recent study [12] suggested an adverse in uence of COVID-19-associated pneumonia with CA, on the oral cavity, pharynx, heart, liver, spleen, central nervous system, urinary organs, eye, skin, vagina, and the endocrine system, any of which can be the cause of the infection [1,2]. This leads to a synergistic bio lm formation which causes virulence [13]. Conventionally, as CA is considered an endogenous microorganism, there are few existing methods of disinfection of CA from a living body or the prevention of transmission to living body.
The medium is observed as the infection source (IS) ( eld), diffusion eld (DF), spread eld (SF), and carrier eld (CF), which comprise the infection eld (IF), and the continuum of the infection eld (IF) is the infection chain (IC). (Figs. 1 and 2, and in Supplementary Notes S1-S10) The elds and IS are expressed by the two equations in C4RDE [14], which describe the active property (active coe cient ε a ) and the decline property (decline coe cient ε d ) for the IF. In addition, the equation

Each eld and IC
In the diffusion eld (DF), microorganisms diffuse from IS by potential energy. The spread eld (SF) is de ned as the spread, without a concentration gradient. The carrier eld (CF) is de ned as the eld in which microorganisms are transferred by carrier (e.g., ventilator, toothbrush, oss, and food). The infection eld (IF) consists of the IS (in the segment) and the three elds (DF, SF, and CF). The continuum of the IF is the IC. In the IF, microorganisms encounter an organ. If the microorganism can adhere (adhesion stage colonization) to the substance that encountered the receptor eld (RcF), it will colonize the substance. In suitable conditions, the proportions of microorganisms increase ( xing stage colonization), the IS will be generated, and the IF will progress. The IF begins from the IS and is infected in three ways (IFF is infection eld function): DF, SF, and CF. In the IS, contaminated substances are generated by the IF.
The IEDS aims to stop the IC by eradication of IS, disinfection of CF, investigation of resistance eld (RsF), and obstruction of RcF-ized for the substance.

Medium (DFM)
A sterilization swab (SS; trace) of the whole tooth without contact with the adjacent tooth ( Fig. 3a) was performed. Then, the swab was applied to the area of dental formula (The trade mark and pat of dental formula, MicroDent Co., Ltd.) on corresponding medium (The CHROMagar TM Cnadida, BD BBL TM , Becton, Dickinson and Company,) (Supplementary Notes S11-S13). 1. The all-teeth examination (ATE) included the swab of all teeth (Fig. 3b).
2. The partial teeth examination (PTE) (tooth unit) included the swab of each tooth. The swab was applied to each area of the DFM (Fig. 3c). If a swab was negative after treatment and disinfection, this con rmed that the IS was tooth caries (Fig 4, 5).  Tables S1-S22). It is thought that the characteristics of IF and IC acquired from space, time, and phase analyses are useful for disinfection, prevention of transmission, and the control of microorganisms; however, they have also suggested the analysis of other microorganisms and viruses.
Treatment based on IEDS revealed that the CA elimination rate in the oral segment is approximately 100% as a result of elimination of dental caries and cleaning (Figs. 4, 5, Table 6, and Supplementary Fig S14).
The combined treatment of dental caries and tooth cleaning by the antifungal agent [15] resulted in the eradication of CA by approximately 100% in the oral segment of the part of the IC.
CA and DMFT index (WHO) is decayed, missing, and lled tooth index.

CA in a dental-caries tooth
The IS is de ned as the space that is colonized (adhesion stage or xing stage) by microorganism (in the shape of microscopic image of caries tooth, multi formation images, such as fungi mycelium and spore, are seen ( Supplementary Fig. 1)). Fields 1. The main RcF is dental caries.
2. DF is about one tooth.
3. SF is not observed in CA; LB is observed.
4. CF is evaluated by the sampling by a swab; therefore, toothbrushes, ventilator, ne droplet nuclei, respiratory drop, ejected contaminated water drops by dental air turbine, and food are considered to be important CFs.
5. The tooth has a powerful RsF. 6. IF is eliminated in the oral segment.
7. IF is mathematically equivalent to any segment. It can be calculated for complicated systems using only two kinds of coe cient (active coe cient ε a and decline coe cient ε d ).
8. The IS is detectable; therefore, it can be completely eradicated. 9. RcF is detectable, and hence, investigation of the IS is easy. Moreover, RcF, which is the risk eld of infection, can be eliminated, and infection can be prevented. 10. As RsF is detectable, the prevention of transmission is possible.
Space analysis by dental formula (Supplementary Notes S18-S24) The local IS indicated that teeth caries included the xing colonization part of the CA (Fig. 4, Tables 3 When CA was present in many teeth, in the oral segment, it appeared to be an endogenous microorganism ( Fig. 3e and 5) [22]. In contrast, when CA infections was present in a few teeth, it was xed to the tooth and caused teeth caries ( Fig. 3d and 4). A CA(+) caries tooth and a CA(-) caries tooth existed in the same mouth. (Supplementary Table S18 3. The phase of microorganisms (Especially, Only phase analysis, It de ne as comparison of multiple microorganisms.) It detects RcF or RsF. An interesting relationship between RsF and Lactobacillus (LB) is indicated ( Supplementary Fig. 5).

Re-infection
Moreover, light adhesion (adhesive colony) was observed in some cases which could lead to re-infection; however, these cases were easily removed.

Discussion
There are many examples that omit the IEDS based on speci c space, time, and phase of the IF, and these are confusing. There are studies describing the involvement of dental caries [15-18]; in contrast, there are studies not related to dental caries [19,20]. It may be the feature of CA that discussion in [3][4][5][6][7][8][9][10][11][12] similar to VAP has generated. In these studies, there was no analysis of the infection caused by CA, the application of the antifungal agent was stopped, and there was no plan against re-infection; moreover, studies have been reported regarding the use of antifungal agents for infection in root canal (Pseudomonas [9] is treated as an important pathogenic bacteria of apical periodontitis), but there is a lack of studies on the propriety of application of the antifungal agent after disinfection, and the technique and extent of maintenance to achieve permanent prevention of transmission and full disinfection. Therefore, these methods have not resulted in the eradication of infection.
In the space analysis of the CA(+) non caries tooth we found that in 32 of 46 adjacent healthy teeth with CA(+), CA was removed by professional cleaning or brushing (after dental caries operation); (Table 5a). In 36 of 57 non-adjacent healthy teeth with CA(+), CA was removed by professional cleaning or brushing; (after dental caries operation) (Table 5b).
These are considered to be light CA infections, which depends on the internal transmission by IF (from the caries teeth), or on the external transmission (food, dust splash, hospital infection, etc.) by IF, and adhesion. Internal transmission was considered for DF that was less than one tooth away (Fig. 2,  Supplementary Figs. 11-13).

Outbreak (of colonization) in organs (tissue, substance) (Supplementary Note S16)
The rst-and second-level CA (from all true healthy teeth and the restored healthy teeth in Table 7) can be mostly disinfected by cleaning; however, at the third level, disinfection cannot be accomplished without treatment. The fourth level is a colony concentration (CC) of 100 or more, and a speci c exponential increase was also observed on the chart (Table 7) The result of CA adhesion in healthy tooth is given in (Table 5). CA was detected on teeth, except for the caries tooth. It was not a xed CA, but an adherent CA. We assumed the possibility of internal transmission from the 243 CA(+) caries tooth to the others. We found that in 32 teeth, adhesive colonization was removed by tooth cleaning. Among the non-adjacent healthy teeth, in which there was a strong possibility of internal transmission and/or external transmission, 36 CA (+) teeth had removed adhesive colonization by tooth cleaning, which accounted to a total of 311 teeth (87.4%; 311/356). Furthermore, the dummy is the result of the external and/or internal transmission; therefore, 10 teeth can be added to the total which resulted in 90.2% (321/356) of the teeth. If the 35 unclear teeth are removed, this becomes 100% (321/321). Therefore, it is thought that caries tooth occurs from a xed CA. The IS is a caries tooth, which was treated with an antifungal agent, resulting in disinfection (Supplementary Note S24). If CC in each dental formula area is 0, it must also be 0 in saliva, in periodontal tissue, and in the air.
The oral segment is the entrance for the living body.
For the oral segment, which is the entrance for the living body, to become CA-free, the infection chain (IC) must be broken in the connected segment that could be infected from the oral segment. There is a strong possibility that the teeth constitute the rst habitat of CA; thus, CA disinfection of the teeth was very effective (Supplementary Notes S26-S27). Moreover, resin lling, arti cial tooth for the nal prosthetics, denture and healthy teeth, which are clear on DFM, also occasionally have adherent CA. The information from IEDS can examine the IR (IR subtracts IS from IC), but in many cases, it is possible to maintain a patient's oral segment CA-free continuously. In the oral segment, which is the entrance to the living body, ref. 2 (P87) suggests food as the source of IF (Japan is an exception [23]), IES shows the speci c dental caries are because of infection from a source other than food. The treatment, based on detailed measurement analysis of the IF, shows that the IC can be eliminated, including internal and external infections from the natural environment.

No endogenous microorganism
In the oral segment, one or more CA-infected teeth may exist. In the case of the latter, two or more IF(s) (and accordingly, DF, SF, and CF) exists in the restricted space of the oral segment; thus, the IF(s) suggest that CA is the causative endogenous microorganism. Importantly, there is a possibility that RsF is powerful in the oral segment. Moreover, it is thought that the reason for the negative value of ATE was the small amount of external transmission.
This may indicate that the prevalence of CA(s) in Japan, such as in food, soil, and water, was very low (Table 1). Furthermore, the Food Sanitation Law of Japan [23] does not recognize the contamination of pathogenic microorganisms. The same is true of the Water Supply Law [24].
The characterizations of CA CA must consider the phase of the space-time of infection source, infection eld, receptor eld, resistance eld, adhesive colony, xed colony and infection chain. Moreover, CA must consider the phase of microorganisms. If they are neglected, the statistical research or the medical treatment etc., the study cannot obtain the correct result.

Conclusion
In the treatment and prevention of infection, speci cation of the characteristics of IS and IF of the microorganisms is the highest priority. The IEDS, including the DFM, indicates the exact DF, SF, CF, RsF, RcF, dental caries time, IC, and other properties of microorganisms such as CA, and can simultaneously show the infection route and infection medium (Fig. 1, 2) (Supplementary Note S28). The surveillance of CA disinfection and the information from IEDS of speci c teeth are very effective in the prevention of infection by CA.
It is necessary to perform dental formula examination of the microorganisms contained in dental caries and group it according to three phases (the infrared analysis method is important for Streptococcus mutans). Accordingly, information from IEDS can prevent infection, while preventing various diseases in other internal organs. Additionally, it is able to perform high-precision dental caries treatment (including operation), and prevent tooth loss. It is necessary to classify and verify the internal transmission from the untreated xing teeth and active internal transmission by dental operation.
The outer CA infection routes (IR) include soil, food, pets, and care, through which progression can occur. Inner infections can transfer from the oral segment to respiratory organs or digestive organs, nasal cavity, and paranasal sinus. Moreover, disinfection of the ventilator used for treatment is important.
As mentioned above, the eld of CA is mainly observed as CF.
The use of the ventilator Although the use of the ventilator through the oral cavity in which dental-caries tooth exists is not recommended, it is inevitable in cases of infection. Therefore, in cases of infection, medical treatment is recommended. IEDS is very effective in prevention of hospital infection. The clinic has always disclosed that treatment data without personal data will be used for research (See Supp. Figure 0), and no participant (patients) has ever objected to this.

Consent for publication
The clinic has always disclosed that treatment data will be used for research (See Supp. Figure 0)