A nationwide multicenter study of the Cochlin tomo-protein detection test: clinical characteristics of perilymphatic fistula cases

Abstract

Objective: To investigate the positive rate for the Cochlin tomo-protein (CTP: an inner ear-specific protein) detection test among patients with inner ear-related clinical manifestations and evaluate the clinical characteristics of definite perilymphatic fistula (PLF).

Methods: We have performed an ELISA-based CTP detection test using middle ear lavage (MEL) samples from 497 cases of suspected PLF enrolled from 70 clinical centers nationwide between 2014 and 2015. In addition to the CTP-positive rate, audio-vestibular symptoms were compared between CTP-positive and -negative cases.

Results: 8–50% of patients in category 1 (trauma, middle and inner ear disease cases), and about 20% of those in categories 2, 3 and 4 (external origin antecedent events, internal origin antecedent events, and without antecedent event, respectively) were positive for CTP. In category 1 cases, the earlier tested samples showed a higher CTP-positive rate, whereas no differences were observed in categories 2, 3 or 4. The characteristic clinical features in the earlier tested cases were nystagmus and fistula sign in CTP test-positive cases in category 1, and streaming water-like tinnitus in those in categories 2, 3 and 4.

Conclusion: The present study clarified that CTP detection test-positive patients exist at considerable rates among patients with inner ear-related manifestations.

Chinese abstract

目的: 探讨对具有内耳相关临床表现的病人所进行的Cochlin断层蛋白 (CTP: 内耳特有性蛋白) 检测的阳性率, 并评估确定的外淋巴瘘 (PLF) 的临床特征。

方法: 我们对从2014年至2015年间在全国范围70个临床中心招募的疑有PLF的497个病例所得到的中耳灌洗 (MEL) 样品进行了基于ELISA的CTP检测试验。除CTP阳性率外, 对音频前庭症状在CTP阳性和阴性病例之间进行了比较。

结果: 类别1 (创伤、中耳和内耳病例) 中的8-50％的患者, 以及类别2、3和4 (分别指: 外部起源前因事件、内部起源前因事件、以及没有先前事件) 中的约20％, 都呈CTP阳性。在1类病例中, 较早测试的样品显示较高的CTP阳性率, 而在类别2、3或4中没有观察到差异。早期测试病例中的特点性临床特征是, 在类别1中, CTP测试阳性病例中的眼球震颤和瘘迹象, 以及在类别2、3和4中的流水样耳鸣。

结论: 本研究说明, 在内耳相关表现的患者中, CTP检测阳性患者占相当大的比例。

Keywords:

• Perilymphatic fistula
• perilymph
• Cochlin isoform
• hearing loss
• vestibular symptom
• vertigo
• tinnitus
• ELISA
• biomarker

Introduction

Perilymphatic fistula (PLF) is defined as an abnormal communication between the fluid (perilymph)-filled space of the inner ear and the air-filled space of the middle ear and mastoid, or cranial spaces. The primary manifestations of perilymph fistulization are sudden, progressive or fluctuating sensorineural hearing loss, vertigo and/or dizziness. However, the clinical manifestations of PLF vary widely and it is, therefore, very difficult to make a definite diagnosis of PLF.

The conventional gold standard for PLF detection is the intraoperative visualization of perilymph leakage, which ostensibly confirms the existence of PLF. However, this confirmation is invasive and difficult to perform in cases with mild–moderate hearing loss or idiopathic cases without any antecedent event. Further, the surgical procedure itself invites seepage and bleeding, which accumulates in the concave-shaped round and oval window niches, and this can be misinterpreted as perilymph leakage.

Based on proteomic analysis, we have identified an isoform of Cochlin, Cochlin tomo-protein (CTP), [1] as a perilymph-specific protein that is not expressed in the blood, CSF or saliva [2]. The detection of CTP in the middle ear is indicative of the presence of a PLF and perilymph leakage. We have recently established a highly reliable ELISA-based CTP detection test. We performed a multicenter investigator-initiated clinical trial of this CTP detection test involving 70 hospitals nationwide. In this study, we aimed to elucidate the proportion and clinical characteristics of the CTP test-positive cases among PLF-suspected cases and those with PLF-related disorders.

Subjects and methods

Subjects

This study included of 497 cases registered from 70 hospitals (as listed below in the Acknowledgements) between 2014 and 2015. The subjects included (1) PLF-suspected cases caused by trauma, or middle and inner ear diseases and (2) patients with some inner ear-related manifestations.

The study protocol for middle ear lavage (MEL) sampling of the patients was reviewed and approved by the Ethics Committee of each collaborative institution, and written informed consent was obtained from all subjects.

Diagnostic criteria and categorization

The diagnostic criteria for PLF used in this study (based on the criteria of the Intractable Hearing Loss Research Committee of the Ministry of Health and Welfare, Japan revised in 2016) are shown in Table 1, where a definite diagnosis can be made with the visual identification of fistula between the middle and inner ear by microscope or endoscope, or the detection of a perilymph-specific protein, such as CTP, from MEL.

Table 1. Diagnostic criteria for PLF (based on the criteria of the Intractable Hearing Loss Research Committee of the Ministry of Health and Welfare, Japan revised in 2016).

A. Symptoms

Hearing impairment, tinnitus, aural fullness, vestibular symptoms are observed in cases who had preceding events as listed below:

(1) Coexisting or pre-existing middle and/or inner ear diseases, (trauma, cholesteatoma, tumor, anomaly, SCCD, etc.) middle and/or inner ear surgeries

(2) Barotrauma caused by antecedent events or external origin (e.g., blasting, diving or flying, etc.)

(3) Barotrauma caused by antecedent events of internal origin (e.g., nose-blowing, sneezing, straining or carrying heavy objects, etc.)

B. Laboratory findings

(1) Microscopic/endoscopic inspection

Visual identification of fistula(s) between middle and inner ear by microscope or endoscope. Fistulas can develop at the cochlear window, vestibular window, fracture site, microfissure, malformation, destruction in bony labyrinth caused by inflammation, etc.

(2)  Biochemical test

Perilymph-specific protein is detected from the middle ear

C. Reference

(1) A perilymph specific protein; e.g., Cochlin-tomoprotein (CTP) detection test

After myringotomy, the middle ear is rinsed with 0.3 ml saline three times, the fluid recovered (middle ear lavage (MEL)) and tested by polyclonal antibody ELISA

The cutoff criteria: ∼0.4 < CTP negative; 0.4 ≦ CTP <0.8 intermediate; 0.8 ≦ CTP positive

(2) Idiopathic cases may exist

(3) Following symptoms and/or test results may be observed:

1. Streaming water-like tinnitus or feeling of running water in the middle ear

2. Popping sound can be heard at the onset

3. Nystagmus and/or vertigo induced by pressure application to the middle ear (Hennebert's phenomenon, fistula sign)

4. Imaging studies may show a fistula in the bony labyrinth or pneumolabyrinth

5. Progression of hearing impairment, tinnitus, aural fullness may be acute, progressive, fluctuating or recurrent

6. The main complaints can be vestibular symptoms without hearing impairment

D. Differential diagnosis

Inner ear diseases with known causes, such as viral infection, genetic, vestibular schwannoma, etc.

E. Diagnosis

Probable PLF: Only symptoms listed in A

Definite PLF: Symptoms and laboratory findings listed in B

The categorization for PLF-related disorders used in this study is given in Table 2. According to these criteria, we classified the enrolled patients into four categories: category 1 includes trauma, and middle and inner ear diseases; category 2 includes barotraumas caused by antecedent events of external origin; category 3 includes barotraumas caused by antecedent events of internal origin and category 4 consists of cases with no apparent antecedent event (idiopathic). Among the 497 participants, 125 were classified into category 1 (among which the cause was cholesteatoma in 50 cases, stapes injury in 23 cases, head trauma in 18 cases, traffic accident in 14 cases, post-middle ear surgery in 8 cases, inner ear malformation in 8 cases and Eustachian tube insufflation in 4 cases). Twenty-eight cases had antecedent events of external origin, such as diving, flying, elevators or loud noise (category 2), while 77 cases had antecedent events of internal origin such as nose-blowing, sneezing, straining or carrying heavy objects, vomiting, coughing, delivery or running (category 3). Further, 192 cases did not experience any antecedent events prior to the onset of cochleo-vestibular symptoms (category 4: idiopathic cases). We excluded 75 participants from further analysis as the dates of onset of their symptoms were unclear. We counted the numbers of samples in relation to the onset to MEL sampling interval, and found that there were peaks at 0–10 days and 41–50 days. As the onset to MEL sampling interval might affect the CTP test results through natural healing of the fistula, we compared the CTP test-positive rates and patient profiles of the cases with an interval of less than 30 days (Group A: category 1; Group C: categories 2, 3 and 4) and more than 30 days (Group B: category 1; Group D: categories 2, 3 and 4) between onset and MEL sampling (explained below).

Table 2. Categorization of PLF.

Category 1: linked to trauma, middle and inner ear diseases, middle and/or inner ear surgeries

Category 2: linked to barotrauma caused by antecedent events of externalorigin (such as flying or diving)

Category 3: linked to barotrauma caused by antecedent events of internalorigin (such as straining, sneezing or coughing)

Category 4: has no apparent antecedent event

The ELISA-based CTP protein detection test

MEL samples for the ELISA-based CTP protein detection test were taken from the participants as follows: (1) following myringotomy, (2) the middle ear was washed three times with 0.3 ml saline, (3) the fluid was recovered and red blood cells and white blood cells were removed from MEL by centrifugation (2000g, 3 min.) and (4) the supernatant was then collected and frozen [3].

Prior to this multicenter study, we developed an ELISA-based CTP detection kit using anti-CTP polyclonal antibodies in collaboration with IBL (Immuno-Biological Laboratories, Inc., Gunma, Japan). As there is no gold standard for the detection of perilymph leakage at present without the detection of an obvious fistula by microscopic or endoscopic observation, we collected MEL to compare two middle ear conditions as positive and negative controls. One is surgically created PLF in which the perilymph leaks out during cochlear implantation surgery. The other is presumably a ‘normal middle ear’ condition without perilymph leakage, such as conductive hearing loss due to anomalous ossicles, or MEL obtained prior to cochlear implant insertion. Based on ROC analysis with the Youden index for CTP, we defined the cutoff criteria as: ∼0.4 ng/ml < CTP negative, 0.4 ≦ CTP <0.8 ng/ml intermediate and 0.8 ng/ml ≦ CTP positive [4].

We performed this clinical trial using this novel ELISA-based CTP detection kit with the help of SRL, Inc. (SRL Inc., Tokyo, Japan). The ELISA test is automated, and the testing conditions are normalized according to the standard ELISA protocol, with test results directly delivered to physicians to avoid any bias related to the testing procedure.

Statistical analysis

The proportion of CTP-positive cases and onset-MEL sampling interval were analyzed. For statistical analysis, Fisher’s exact test was used, and a p value of <.05 was considered to be statistically significant. Statistical analyses were performed using JMP pro version12.1.0. (SAS Institute, Cary, NC).

Results

Causes and CTP test-positive rates for suspected PLF patients caused by the trauma, or middle and inner ear diseases (category 1)

The CTP-positive rate for the PLF-suspected cases caused by trauma, or middle and inner ear diseases (category 1) was 43% for those with stapes injury, 17% for head trauma, 29% for traffic accident, 50% for Eustachian tube insufflation, 8% for cholesteatoma, 50% for inner ear malformation and 25% for post-middle ear surgery (Supplementary Tables 1 and 2). We compared the CTP test-positive rates of the cases with an interval of within 30 days (Group A) and more than 30 days (Group B) between onset and MEL sampling for four causes (stapes injury, head trauma, traffic accident and Eustachian tube insufflation). Group A had a higher positive rate (Fisher’s exact test, p = .025), indicating that the onset to MEL sampling interval affects the CTP-positive rate in category 1 cases (Figure 1). We then analyzed differences in patient symptoms and signs between the CTP-positive and intermediate/negative-cases in each group. In Group A, nystagmus (Fisher’s exact test, p = .0017) and fistula sign (Fisher’s exact test, p = .018) were more frequently observed in CTP-positive cases (Figure 2 and Supplementary Table 2-(1)), whereas there were no significant differences in patient symptoms and signs in Group B (Supplementary Table 2-(2)).

Figure 1. The proportion of CTP-positive, -intermediate and -negative cases according to the onset-MEL sampling interval for each cause in category 1 cases, showing that samples taken earlier within 30 days had a higher positive rate (see Supplementary Table 1 for details).

Figure 2. Clinical characteristics and symptoms in CTP test-positive cases classified as category 1 and tested within 30 days of onset (Group A). Nystagmus and fistula sign were more frequently observed in CTP-positive cases (see Supplementary Table 2-(1) for details).

CTP-positive rates in patients with antecedent events and idiopathic cases (categories 2, 3 and 4)

Among the 28 cases with antecedent events of external origin, such as diving, flying, elevators or loud noise (classified into category 2), 14% were positive, 14% were intermediate and 71% were negative according to the results of the CTP detection test (Supplementary Table 3). Among the 77 cases with antecedent events of internal origin, such as nose-blowing, sneezing, straining or carrying heavy objects, vomiting, coughing, delivery or running (classified into category 3), 23% were positive, 18% were intermediate and 58% were negative. Among the 192 cases without any antecedent events (classified into category 4), 19% were positive, 21% were intermediate and 60% was negative. A comparison of the CTP-positive rates in each category revealed no significant differences (Fisher’s exact test, p > .05).

Figure 3. The proportion of CTP-positive, -intermediate and -negative cases in categories 2, 3 and 4, indicating that approximately 20% of patients with inner ear-related manifestations were CTP positive. No significant differences in CTP-positive rate were found among the categories. Further, no significant differences were found in positive rate between the samples tested within or after 30 days (see Supplementary Table 2 for details).

In order to examine the CTP-positive rates in respect to the onset-MEL sampling interval, we compared the positive rates between the samples taken within 30 days (Group C) and more than 30 days (Group D) of onset in each category. However, there were no statistically significant differences in positive rates between Groups C and D (Fisher’s exact test, p > .05) (Figure 3).

Clinical characteristics and symptoms in the CTPtest-positive cases classified as categories 2, 3 and 4

We next sought to elucidate the patient profiles of the CTP test-positive cases in Groups C and D (Supplementary Tables 4 and 5). We compared the proportions of CTP-positive, -intermediate and -negative cases for each characteristic symptoms and sign, and the results are summarized in Supplementary Tables 4 and 5.

Figure 4. Clinical characteristics and symptoms in CTP test-positive cases classified as categories 2, 3 and 4, and tested within 30 days of onset (Group C). No significant differences were found in the positive rates among males and females, onset age, severity of hearing loss, presence/absence of vestibular symptoms, nystagmus, fistula sign or popping sound. A significant difference was found in streaming water-like tinnitus in category 4 cases and in total cases (see Supplementary Table 4 for details).

In Group C, likewise in Group D, there were no significant differences in the CTP test-positive rates among males and females, onset age or severity of hearing loss. We next analyzed the presence/absence of symptoms and signs, such as vestibular symptoms, nystagmus, fistula sign, popping sound or streaming water-like tinnitus. In the CTP test-positive cases in Group C, the positive rate of those with vestibular symptoms was 50%, that for nystagmus was 56%, fistula sign was 0%, popping sound was 3% and streaming water-like tinnitus was 25% (Figure 4). In the CTP test-positive cases in Group D, the positive rate of those with vestibular symptom was 78%, nystagmus was 71%, fistula sign was 6%, popping sound was 14% and streaming water-like tinnitus was 18%. We next analyzed the differences in patient symptoms and signs between the CTP-positive and intermediate/negative-cases in each group. The only symptom which showed a significant difference was streaming water-like tinnitus in total cases (Group C) (Supplementary Table 4-(4)) (Fisher’s exact test, p = .0354). In category 4 cases who are tested within 30 days (Supplementary Table 4-(3)), streaming water-like tinnitus was observed at a significant rate (Fisher’s exact test, p = .0464), and more CTP-positive cases tended to be observed among profound hearing loss patients and elderly patients, but these trends were not statistically significant.

Discussion

The real clinical picture of PLF is still unclear as the conventional gold standard for PLF detection is the intraoperative visualization of perilymph leakage. However, this confirmation process is invasive and difficult to perform in cases with mild–moderate hearing loss or idiopathic cases without any antecedent event. In addition, there are no physiological or biochemical diagnostic tests for PLF that possess the proper specificity or sensitivity. This has resulted in the very existence of idiopathic PLF being regarded as controversial or questioned [4–7].

CTP, identified in 2009, is an inner ear-specific protein and regarded as a useful diagnostic marker for PLF. Its characteristics have already been reported in detail [1–3,8]. We initially developed a Western blotting-based CTP detection test for PLF diagnosis, and improved the throughput and sensitivity of this test by using an ELISA-based CTP detection kit.

The most prominent result of the present study was the clarification that CTP detection test-positive patients exist at considerable rates among patients with or without antecedent events. Since CTP-positive cases were found in all categories of patients (regardless of antecedent events), it is possible that an antecedent event is not a single cause of PLF and a considerable portion of idiopathic cases with cochleo-vestibular symptoms may be the result of PLF or other undetected pathophysiological conditions.

Middle ear disease (cholesteatoma, tumor, etc.), ear surgery (stapes, cochlear implantation, etc.) and trauma (head injury [9] or stapes injury [3,10], etc.) are well accepted as causes of PLF. Among these cases, each of these causes should be discussed separately; e.g., 50 of 128 cases were related to cholesteatoma, where the CTP detection test can be utilized for the staging of fistula depth induced by chronic inflammation (in preparation). In the case of post-stapes surgery hearing loss due to dislocation of the piston, the CTP detection test could be used to identify perilymph leakage prior to exploratory tympanotomy [11], while in the case of direct middle ear trauma, the CTP test was useful in deciding the surgical treatment [3].

It is noteworthy that the onset-MEL sampling interval affected the CTP test-positive rate in cases with middle ear disease or trauma. When comparing the positive rates between the samples taken within or after 30 days of onset, samples taken earlier showed a higher positive rate (Figure 1, Supplementary Table 1). Causes such as stapes injury, head trauma, traffic accident and Eustachian tube insufflation are the precise types of event that might cause perilymph leakage; therefore, it is reasonable that earlier testing should show higher CTP-positive rates as the PLF can heal naturally over time and the leaked perilymph washed out through the Eustachian tube. On the other hand, in categories 2, 3 and 4, there were no such differences in the positive rates between the early and late onset-MEL sampling intervals (Figure 3, Supplementary Table 3). The exact reason for these differences is unknown; however, this might reflect the fact that leakage in categories 2, 3 and 4 can be intermittent or recurrent.

One of the main purposes of the present study was to elucidate the clinical characteristics of the CTP test-positive cases. In category 1, nystagmus and fistula sign were more frequently observed in those cases who were tested within 30 days of onset (Figure 2). When those signs are observed after traumatic events, it is likely that the patient has PLF. There were no significant differences in positive rates among males and females, onset age, severity of hearing loss, presence/absence of vestibular symptom, nystagmus, fistula sign or popping sound in all categories or groups. The only symptom which showed a significant statistical difference was streaming water-like tinnitus in Group C and category 4 patients who were tested within 30 days of onset (Figure 4, Supplementary Table 4-(3), (4)). As streaming water-like tinnitus is a rare symptom, this could be a clue to the diagnosis of PLF. This type of tinnitus has been a well-known symptom of PLF in national studies in Japan, but is not well reported in the literature from other countries.

Some limitations of the present study warrant discussion. The inclusion criteria of this study were wide enough to enroll many types of patients in one study design, but may have contained some selection bias with regard to the participant enrollment process. As doctors select the participants for the CTP detection test based on pre- and/or co-existing ear conditions, preceding barotraumatic events and characteristic symptoms, some patient selection bias is inevitable. We are now conducting a prospective study to delineate the real clinical picture of CTP-positive ear diseases. Another limitation is associated with the CTP ELISA test. Since CTP is a novel biomarker and MEL is also a newly identified substance for sampling from the human body, we have not fully elucidated all the factors that might induce false-positive or -negative results.

Conclusion

We have reported a large scale, multicenter investigator-initiated trial of the CTP detection test. This is the largest study conducted so far for PLF diagnosis. We could identify CTP-positive cases among a considerable portion of patients with inner ear-related manifestations with or without antecedent events. Our results indicate that PLF should be considered as a cause of cochleo-vestibular diseases which have been previously neglected or misdiagnosed.

Acknowledgements

This work was supported by the Ministry of Health and Welfare, Japan (http://www.mhlw.go.jp/english/) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (http://www.mext.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank Mr. Koichi Toyoda, Industry-University Cooperation Advisor at Saitama Medical University for helping us to establish this clinical trial.

We thank the participants of the consortium for the Multicenter investigator-initiated trial of the CTP detection test: Dr. Yuichiro Horibe (Aichi Medical University), Dr. Teruyuki Satoh (Akita University), Dr. Kan Kishibe (Asahikawa Medical University), Dr. Nobuyoshi Tsuzuki (Japanese Red Cross Ashikaga Hospital), Dr. Shin-ichi Haginomori (Osaka Medical College), Dr. Yuko Kataoka (Okayama University), Dr. Yuko Suzuka (Kanazawa Medical University), Dr. Hitoshi Sugimoto (Kanazawa University), Dr. Kenji Ishii (Kamio Memorial Hospital), Dr. Seiya Goto (Kariya Toyota General Hospital), Dr. Hiroshi Shinohara, Dr. Hironari Shimizu and Dr. Saeko Yoshida (Kawakita General Hospital), Dr. Susumu Baba (Kansai Medical University), Dr. Hiroshi Hosono and Dr. Hajime Sano (Kitasato University), Dr. Rie Kanai (Medical Research Institute Kitano Hospital), Dr. Toru Seo (Kinki University), Dr. Norio Yamamoto (Kyoto University), Dr. Masako Masuda (Kumamoto University), Dr. Ryouta Mihashi (Kurume University School of Medicine), Dr. Katsumasa Takahashi (Gunma University), Dr. Takashi Nabekura (Miyazaki Prefectural Miyazaki hospital), Dr. Taisuke Kobayashi (Kochi Medical School), Dr. Hidekane Yoshimura, Dr. Satoshi Iwasaki and Dr. Yoh-ichiro Iwasa (International University of Health and Welfare MITA Hospital), Dr. Yutaka Isogai (Speech, Language and Hearing Center, International University of Health and Welfare Clinic), Dr. Takayuki Morikawa (Komatsuzaki Hospital), Dr. Koichiro Wasano (Japanese Red Cross Shizuoka Hospital), Dr. Masayuki Shirakura (Jichi Medical University Saitama Medical Center), Dr. Sei Kobayashi (Showa University), Dr. Hiroaki Suzuki (Shinshu University) Dr. Kunihiko Makino and Dr. Kana Lee (Shin-Suma General Hospital), Dr. Itomi Nishijima (Sannoh Hospital), Dr. Koichi Kure (Seirei Yokohama Hospital), Dr. Kenichi Ando (Takayama Red Cross Hospital), Dr. Shintaro Fujimura, Dr. Tsuyoshi Kojima, Dr. Tetsuro Kobayashi and Dr. Ryusuke Hori (Tenri Hospital), Dr. Akinobu Kakigi (Tokyo University), Dr. Kyoko Shirai and Dr. Noriko Nagai (Tokyo Medical University), Dr. Yoshihiro Noguchi, Dr. Ken Kitamura, Dr. Yoshiyuki Kawashima and Dr. Masatoki Takahashi (Tokyo Medical and Dental University), Dr. Manabu Komori (The Jikei University School of Medicine), Dr. Fumie Kaneko, Dr. Hana Mori and Dr. Hiroshi Sunose (Tokyo Women's Medical University, Medical Center East), Dr. Masanori Ishii (Japan Community Healthcare Organization Tokyo Shinjuku Medical Center), Dr. Yurika Kimura (Tokyo Metropolitan Geriatric Hospital), Dr. Sota Yamaguchi (Toho University Ohashi Medical Center), Dr. Noriaki Takeda (Tokushima University School of Medicine), Dr. Takashi Kashiwagi and Dr. Satoru Fukami (Dokkyo Medical University), Dr. Yasuomi Kunimoto, Dr. Junko Kuya and Dr. Hiroaki Yazama (Tottori University), Dr. Fujisaka Michirou (Toyama University), Dr. Tadao Yoshida (Nagoya University Graduate School of Medicine), Dr. Toshimitsu Nemoto (Japanese Red Cross Narita Hospital), Dr. Makoto Miura and Dr. Saki Yabuuchi (Japanese Red Cross Society Wakayama Medical Center), Dr. Nozomu Wakayama (Nippon Medical School Musashikosugi Hospital), Dr. Kunihiro Mizuta (Hamamatsu University School of Medicine), Dr. Takeshi Morita (Hyogo Prefectural Amagasaki Hospital), Dr. Akira Sasaki (Hirosaki University Graduate School of Medicine), Dr. Tetsuko Ueno (Fukuoka University School of Medicine), Dr. Yukio Nomoto (Fukushima Medical University), Dr. Shigehiro Ueyama (Funai Ear Nose Throat Clinic), Dr. Yasuo Hosoda, Dr. Makiko Ohtani and Dr. Takeo Nonoda (Hosoda Ear Clinic), Dr. Shinya Morita (Hokkaido University Graduate School of Medicine), Dr. Hiroshi Sakaida (Mie University Graduate School of Medicine), Dr. Yuko Hata and Dr. Katsuhiro Tsutsumiuchi (Mitsui Memorial Hospital), Dr. Keiji Matsuda and Dr. Takahiro Nakajima (Miyazaki University), Dr. Toshinori Kubota (Yamagata University Faculty of Medicine), Dr. Kazuma Sugahara (Yamaguchi University Hospital), Dr. Shuichiro Endo and Dr. Kyousuke Hatsushika (University of Yamanashi), Dr. Makito Tanabe (Yamamoto Ear Surgicenter), Dr. Shigeki Tsuchihashi and Dr. Masanobu Hiraoka (Wakayama Medical University)

Disclosure statement

Saitama Medical University has the patent for this CTP ELISA test. The development of this CTP ELISA test was performed in collaboration with Biomedical Laboratories under license from and with the technical assistance of Saitama Medical University with royalties.

The authors did not receive funding from SRL (Special Reference Laboratories) Inc.

This relationship had no influence on results.

Additional information

Funding

This work was supported by the Ministry of Health Labour and Welfare, Japan (http://www.mhlw.go.jp/english/) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (http://www.mext.go.jp/english/).