A Series of 35 Cutaneous Infections Caused by Mycobacterium marinum in Han Chinese Population

Cutaneous Mycobacterium marinum infection is an increasingly infectious disease presenting unique diagnostic and therapeutic challenges. The aim of this study was to evaluate the differences in time to treatment among patients with different types of skin lesions and who were treated with single or multidrug therapies. In addition, the clinical characteristics of M. marinum infection were explored and the mechanism of the host immune responses was investigated. The electronic medical records of 35 patients with M. marinum infection were reviewed. The clinical characteristics, histopathological and laboratory data, and treatment outcomes were analyzed. Immunohistochemical analysis was performed to clarify the immune mechanisms induced by M. marinum infection in 9 patients and 5 healthy controls. Of the 35 patients, 25 (71.4%) had lesions with sporotrichoid patterns. The duration of patients with sporotrichoid lesions or treatment with multiple drugs was longer, although differences were not significant, possibly due to the small cohort. However, this trend was also observed in previous studies, making it worthy of further attention. Expression levels of cytokines (IFN-γ, IL-4, IL-9, and FOXP3) were significantly upregulated in the patient specimens, whereas there were no significant differences in IL-17 and IL-22 expression levels between the patient and control groups.


Introduction
Mycobacterium marinum is a slow-growing nontuberculous Mycobacterium (NTM) that was frst isolated and identifed from fsh by Aronson in 1926 [1][2][3].Later, in 1954, M. marinum was described as a human opportunistic pathogen by Collins et al. [3,4].Exposure to fsh tanks and handling of fsh are known risk factors for M. marinum infection, especially for individuals with minor skin trauma prior to contact with the infection source [5].M. marinum infection usually presents as warty nodules or plaques, occasionally with a sporotrichoid pattern, which may progress to shallow crusting, ulceration, and eventual scar formation [6,7].Bacteremia resulting from M. marinum infection most commonly occurs in immunocompromised patients but is very rare [8].Overall, cases of M. marinum infection are sporadic with an annual incidence of 0.27 cases per 100,000 population in the United States [9].
Te diagnosis and treatment of M. marinum infection remain challenging, as the clinical presentation is often insidious and nonspecifc.A diagnosis of M. marinum infection can be suspected by a history of contact with contaminated water or fsh and confrmed by histologic evaluation and mycobacterial cultures.However, there is no consensus on an optimal antimicrobial regimen or treatment duration for M. marinum infection.

Study Approval and Patient Consent.
Te study protocol was approved by the Ethics Committee of Shandong Provincial Hospital of Dermatology (approval no.20181218KYKT001) and conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki.Prior to inclusion in this study, written informed consent was obtained from all subjects.

Case and Data
Selections.Tis study was conducted at the Department of Dermatology, Shandong Provincial Hospital of Dermatology (Jinan, Shandong, China).Te study cohort consisted of 40 subjects, which included 35 patients with newly diagnosed cutaneous M. marinum infection and 5 uninfected healthy individuals as a control group.Normal control tissue specimens were obtained from the limbs of healthy individuals using a surgical scalpel under local anesthesia.Te specimens were spindle-shaped and measured 1 × 0.5 × 0.5 cm in size.All M. marinum infections were confrmed by culture or quantitative real-time PCR (qPCR).Te following data were collected from electronic medical records: patient sex and age, history of aquatic exposure, lesion site and clinical type, culture method, qPCR results, Ziehl-Neelsen staining results, type of antimicrobial therapy, and treatment outcome.Te time to diagnosis was defned as the time from symptom onset to confrmation of M. marinum infection.Te outcome was defned as cure with no sign of infection at the end of the follow-up period.

Histopathology and Laboratory Tests.
All skin tissue specimens were fxed in a 10% bufered neutral formalin solution and cut into sections, which were separately stained with hematoxylin-eosin and fuorochrome staining for acidfast bacilli (AFB).DNA was extracted from fresh tissue samples, and detection was performed concurrently using both qPCR and the Mycobacterium marinum PCR Kit (CAT#: 13-67830y, YaJi Biological, China).

Data Analysis. Data analysis was performed using
Microsoft Excel (Microsoft Corporation, Seattle, WA, USA), and statistical analyses were performed using R software v3.6.1 (https://cran.r-project.org/bin/windows/base/old/3.6.1/).Continuous variables are presented as the mean, median, and interquartile range (IQR), while categorical variables are presented as the number and percentage.Continuous variables were analyzed with the Student's t-test.A two-sided probability (p) value < 0.05 was considered statistically signifcant.

Clinical Characteristics.
Tissue specimens collected from 35 patients from November 1, 2018, to January 31, 2020, were positive for M. marinum infection.Te patient characteristics are summarized in Table 1.All 35 patients were ethnic Chinese and included 6 (17.1%) males and 29 (82.9%)females with a mean age of 54.2 (median, 55; IQR, 49-59) years.Of these 35 patients, 19 (54.3%) had a history of a puncture wound from a fsh bone, and 13 (37.1%)were employed in the fshing industry or other aquatic-related jobs.Te median time from disease onset to confrmation was 3 (IQR, 2.0-4.0)months.None of the patients developed lung abnormalities, as confrmed by chest X-rays, or received immunosuppressant therapy.

Results of Cytokines by IHC.
IHC analysis was performed using specimens collected from 9 patients and 5 healthy controls to clarify the involvement of cytokines during cutaneous M. marinum infection.Te expression levels of FOXP3 (p < 0.05), IL-9 (p < 0.05), IFN-c (p < 0.01), and IL-4 (p < 0.05) were signifcantly increased in all lesions of M. marinum infection as compared to the normal skin specimens, while there was no signifcant diference in the expression levels of IL-22 and IL-17 between the patient and control specimens (Figure 4)

Discussion
In the present study, the clinical features, histopathologic and laboratory data, treatment regimens, and clinical   outcomes of 35 cases of M. marinum infection were systematically reviewed.In addition, the molecular mechanism underlying the regulation of cytokines involved in M. marinum infection was explored.
Diagnosis of M. marinum infection remains challenging due to the failure to record histories of aquatic exposure, unusual clinical manifestations, and empiric antibiotic treatment prior to culture.In this study, only 19 (54.3%) cases were associated with puncture wounds from fsh bones, while the exposure histories of the remaining 16 cases were unknown.Moreover, in clinical practice, skin infection of M. marinum is easily confused with sporotrichosis.Tese factors largely account for the difculty with diagnosis.
Species-specifc PCR has been recently applied in clinical practice for the detection of M. marinum infection.In previous studies, PCR was performed to confrm a diagnosis of M. marinum infection in 34.8%-100% of cases [10,[18][19][20].In the present case series, diagnoses of all 35 patients were confrmed either by culture (26 positive, 74.3%) or qPCR analysis (17 positive, 48.6%), indicating that culture combined with qPCR analysis is more accurate for the identifcation of M. marinum infection.
To the best of our knowledge, no systematic study has investigated the involvement of cytokines in the acquired immune response to M. marinum infection.In this study, the expression levels of IFN-c, L-4, IL-9, and FOXP3 were signifcantly increased in patients with M. marinum infection.IFN-c reportedly plays a protective role in the early stages of infection by contributing to the host immune response against pathogens [23].Moreover, the frequency of NTM infection is reportedly increased by IFN-c signaling and transduction defciencies [24].Meanwhile, FOXP3 impairs the immune response against bacilli, thereby facilitating replication [25].Activation of IL-4 enhances antimycobacterial responses, leading to increased production of infammatory cytokines upon subsequent exposure.Interestingly, even though IL-4 polarizes macrophages towards the M2 phenotype, which typically promotes mycobacterial growth, an enhanced immune response still persists [26].However, IL-9 has the ability to mitigate the detrimental impact of IL-4 on the development of cytotoxic T lymphocytes induced by M. leprae [27].IL-4 and IL-9 produced by immune cells are both positively and negatively regulated in response to M. marinum infection [25].Notably, there were no signifcant diferences in the expression levels of IL-17 and IL-22 between the patient and control groups in this study, possibly due to the limited number of specimens, whereas previous studies reported that IL-17 and IL-22 acted to inhibit the progression of cutaneous Mycobacterium infections [23,28].Hence, additional studies are needed to clarify these discrepancies.Tese results suggest that M. marinum infection may cause complex immune responses involving multiple T cell subsets such as T1, T2, T9, Treg, T17, and T22.

Conclusion
Here, the clinical characteristics, diagnoses, and treatment regimens of 35 cases of M. marinum infection were systematically reviewed, and the involvement of various cytokines was investigated.Timely and accurate diagnosis and treatment are crucial for successful treatment of cutaneous infections caused by M. marinum, but they can be particularly challenging in primary hospitals.Te duration of patients with sporotrichoid lesions or multidrug treatment was longer, consistent with previous studies, and deserves further attention.An inherent limitation to the present study was the small sample size.Terefore, additional studies with larger sample sizes are needed to further investigate the roles and regulatory mechanisms of cytokines in M. marinum infection.

Figure 1 :
Figure 1: Nodules on fngers and back of hand in patient with M. marinum skin infection.

Figure 3 :Figure 4 :
Figure 3: Factors afecting treatment outcomes in M. marinum infections.(a) Correlation between number of drugs and duration of treatment, p > 0.05.(b) Correlation between sporotrichoid lesions and duration of treatment, p > 0.05.
°C in a non-CO 2 incubator for 7 days.Based on the results of drug sensitivity testing, 26 patients with positive cultures received treatment, while 9 patients had negative culture results.Appropriate drugs were administered after confrmation of the pathogen by qPCR analysis.

Table 1 :
Te characteristics of patients with M. marinum infection.