Chromoblastomycosis: A case series from Eastern China.

Chromoblastomycosis (CBM) is a chronic fungal infection of the cutaneous and subcutaneous tissues caused by brown pigmented fungi. Fonsecaea monophora is one of the most common pathogens of CBM in China. Most formal cases have been reported from Southern China, however, the infection is not uncommon in Eastern China where very few case series are available. To describe the clinical aspects of CBM, we report a series of 11 cases between 2018 and 2021 at a single medical center in Eastern China. The patients were predominately male (n = 9) and the disease duration ranged from 3 months to 20 years. Plaque type lesions were the most common clinical manifestations. There were 7 cases of mild forms and 3 cases of severe forms. Among the 3 severe cases, one case gave up treatment due to economic poverty; one case did not respond to a 1-year systemic treatmen; one case was cured by combination therapy of 10 months. Other cases were cured by treatment with antifungal agents. All cases of direct mycological examination were positive. All isolates were identified by morphology and sequencing of the the ITS regions of ribosomal DNA, Ten were F. monophora and 1 was Cladophialophora carrionii. All cases had been evaluated at other clinics, where 8 cases were misdiagnosed as other diseases. As a neglected tropical disease (NTD), CBM is still a major challenge in the field of dermatology, especially in its severe clinical forms. As an effective and simple diagnostic method of CBM, direct microscopic examination should be further promoted in rural hospitals.

Introduction

Chromoblastomycosis (CBM) is an implantation mycosis, consisting of chronic cutaneous and subcutaneous lesions that develop at the site of previous transcutaneous trauma [1]. CBM is strongly associated with agricultural activities and the lack of protection, which further underscores the occupational nature of this disease [2]. It can lead to chronic persistent infections and may cause an incapacity for labor in some severe clinical forms. Although known for 100 years, CBM still poses a therapeutic challenge to clinicians due to its recalcitrant nature and common relapse after treatment [3].The species of the genus Fonsecaea are the most common etiological agent of CBM, and comprises four associated species: F. pedrosoi, F. monophora, F. nubica and F. pugnacious [4-7].Cladophialophora carrionii was the most common causative agent in the north of the Mainland China, and F. monophora and F. pedrosoi were the two most common agents in the southern part of Mainland China [8]. A comprehensive evaluation using molecular sequencing data showed that F. monphora is the most prevalent pathogen of CBM in Guangdong, Southern China [9]. We have carried out a clinical research project on cutaneous and subcutaneous infectious diseases at Department of Dermatology of Hangzhou Third People’s Hospital between 2018 and 2021 and found 11 cases of CBM. To evaluate the clinical characteristics of CBM in Zhejiang, Eastern China, we analyzed their clinical features, mycological findings, pathogens, comorbidities, treatments, and outcomes.

Methods

Ethics statement

This study was approved by the ethics committee of Hangzhou Third People’s Hospital and the study participants were informed about the study procedures and written informed consent was obtained.

A prospective descriptive study of patients with clinically suspected CBM or other important cutaneous and subcutaneous infectious diseases was performed at the medical mycology laboratory in the Hangzhou Third People’s Hospital between January 2018 and December 2021. Age, sex, and occupation, and clinical data, such as comorbidities, traumatic history, duration of the lesions, and clinical type of skin lesions were recorded. The diagnosis of these cases was made by clinical observation, histopathology, mycological examination and nontuberculous mycobacteria (NTM) culture. The diagnosis of CBM was based on the definitions described and proposed by Carrión in 1950; in which the lesions are divided into five different types (verrucous, nodular, plaque, cicatricial and tumoral) [10]. CBM lesions were graded according to the criteria proposed by Queiroz-Telles et al [11]. This study was approved by the ethics committee of our hospital and the study participants were informed about the study procedures and written informed consent was obtained.

Sample collectiont

We referred to the flowchart for laboratory diagnosis of sporotorichosis that was proposed by Orofino Costa et al [12], and developed a standardized operation process. For open lesions, including ulcer or abscess with fluctuation, tissue fluid was taken for microscopic examination and culture. For closed lesions without fluctuation, only tissue culture was performed. A sterilized lancet was used for scraping the surface of the lesions, principally those that were highly pigmented and known as black dots. Pus samples were collected by squeezing the skin lesions. In most cases, skin biopsies were performed and portions of the specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin & eosin (H&E) and periodic acid-Schiff (PAS) or acid-fast staining. Other fresh portions were directly inoculated onto Sabouraud dextrose agar (SDA) and Lowenstein-Jensen solid medium.

Etiological detection

KOH or fluorescent staining preparations of scales and pus that were obtained from the lesions were examined under a microscope for muriform bodies or septate brown pigmented hyphae, hyaline spores or hyphae. Separate scales or pus samples were used for fungal or NTM culture. Portions of skin biopsies and debris were inoculated onto culture media to recover the etiologic agent. The primary isolation of the fungus was performed on agar slants of SDA containing chloramphenicol (CMP, 0.125 g/l) and that were incubated at 25°C for two weeks. Another primary isolation of the NTM was performed on Lowenstein-Jensen solid medium and incubated at 25°C for four weeks. The fungal isolates were initially identified by slide-culture microscopy and NTM isolates were primarily identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik MALDI Biotyper). The final identification of the isolated agent was performed by sequencing of the ITS 1 and ITS 4 regions of rDNA or 16S rRNA and compared with sequences deposited in GenBank by Blast program.

Results

In this study, a total of 100 cases of important cutaneous and subcutaneous infectious diseases were found, including 45 cases of sporotrichosis, 29 cases of NTM infection, 11 cases of CBM, 7 cases of Majocchi’ s granuloma, 4 cases of hyalohyphomycosis, 2 cases of candida infection, 1 case of phaeohyphomycosis and 1 case of Talaromyces (Penicillium) marneffei infection (Fig 1). A total of 11 patients with CBM came from Zhejiang province and their clinical data are summarized in Table 1. The age ranged from 39 to 83 years (mean of 62.9 years). There were six farmers, three carpenters, one gardener, and one retired teacher who planted a variety of flowers at home. The duration of CBM ranged from 3 months to 20 years (mean of 6.5 years), but most of the patients suffered from the infection for more than 1 year (75%). Six patients (54.5%) either could not recall or gave a negative history of trauma prior to their manifestations. Among five cases with a history of trauma, three cases were caused by a wooden spike prick and two cases caused by insect bite. Diabetes was the most common comorbidity. Three patients (27.2%) had diabetes, two patients had chronic kidney disease, one patient had a diffuse pulmonary interstitial fibrosis with hypertension, and one patient had heart disease. In 63.6% of cases (7/11), the lesions were localized to 1 site and the most common afflicted site was the upper limb (7 cases), followed by the lower limb (2 cases), the buttocks (1 case), and the trunk (1 case). The most common clinical variety of described lesions was plaque (63.6%, 7/11) followed by verrucous (18.2%, 2/11), nodular and cicatricial (9.1% each). In addition, according to their severity, lesions’ grade showed three cases of severe form (Fig 2A–2C), especially case 1 who had lost the capacity for labor, seven cases of mild form (Fig 3A, 3C and 3E), and one case of moderate form. All patients reported that pain worsened as the course of disease increased. Before their referral to our clinic, all cases had been evaluated at other clinics, eight cases were misdiagnosed as other types of diseases, of which sporotrichosis and eczema were the most common diagnoses, followed by lupus vulgaris and neurodermatitis.

Fig 1

Number of cases with important cutaneous and subcutaneous infectious diseases between 2018 to 2021.

Note: We carried out NTM culture at the end of 2020.

Table 1

Clinical and fungal results of eleven cases of chromoblastomycosis.

Case	Date	Age(years)/sex	Occupation	trauma	Clinical features/site/grade	Duration(m)	Treatment regimen	outcome	Pathogen
1	2018.2	61/M	Farmer	No	Verrucous/right ankle/severe	180	Lost to follow-up	No	F. monophora
2	2018.3	68/M	Carpenter	Yes	Cicatricial/left forearm/moderate	84	TBF (0.25 g/day) for 5 months	Cured	C.carrionii
3	2018.8	39/M	Farmer	No	Verrucous/buttocks/severe	240	ITZ (0.2 g/day) for 12 months	Failure	F. monophora
4	2019.1	56/M	gardener	Yes	Plaque/left forearm/mild	12	ITZ (0.2 g/day) fo r4 months	Cured	F. monophora
5	2019.7	62/M	Carpenter	Yes	Plaque/trunk/severe	168	ITZ (0.2–0.4 g/day) for 10 months + PDT	Cured	F. monophora
6	2019.10	71/F	Farmer	No	Plaque/right leg/mild	18	ITZ (0.2 g/day) for 3 months	Cured	F. monophora
7	2020.3	83/M	gardener	No	Plaque/left wrist/mild	5	ITZ (0.2 g/day) for 3 months	Cured	F. monophora
8	2020.5	70/F	Farmer	No	Plaque/right forearm/mild	120	ITZ (0.4 g/day) for 3 months	Cured	F. monophora
9	2021.3	57/M	Farmer	No	Plaque/left wrist/mild	18	ITZ (0.4 g/day) for 5 months	Cured	F. monophora
10	2021.5	68/M	Farmer	Yes	Nodular/left forearm/mild	3	ITZ (0.2 g/day) for 3 months	Cured	F. monophora
11	2021.8	57/M	Carpenter	Yes	Plaque/right wrist/mild	8	ITZ (0.4 g/day) for 4 months	Cured	F. monophora

F, female; M, male; ITZ, itraconazole; TBF, terbinafine; m, month; PDT, photodynamic therapy

Fig 2

Severe form: verrucous skin lesions on the right ankle in case 1 with a loss of labor activities (A). Verrucous lesions on the buttocks in case 3. The lesions were unresponsive to itraconazole (200 mg per day) for one year (B). Clinical picture of case 5 with extensive erythematous plaque lesions (C), clinical cure after 10 months of combination therapy (D).

Fig 3

Mild form: Clinical picture of case 4, before treatment (A), and during 30 months of post-treatment follow-up (B). Clinical picture of case 8, before treatment (C), and during 14 months of post-treatment follow-up (D). Clinical picture of case 9, before treatment (E), and during 8 months of post-treatment follow-up (F).

All cases of direct mycological examination were positive, and skin scrapings or pus that were treated with 10% KOH revealed muriform cells with or without germinated hyphae (Fig 4A and 4B). Histopathology proved the characteristic brownish thick-walled sclerotic bodies being demonstrable in 5 cases either within or outside the giant cells (Fig 4C). Pathogens were isolated from 11 cases. One pathogen were identified as C. carrionii (Fig 4D) and ten as F. pedrosoi (Fig 4E and 4F) based on their morphological characters. Based on the sequences of the ITS1-5.8S-ITS2 region of rDNA, 10 F. pedrosoi isolates reidentified as F. monophora.

Fig 4

Mycological features of CBM.

Pigmented muriform cell with obvious septation (A) and brown muriform cell with germinated hyphae (B) (10% KOH, ×400). Mixed granuloma with sclerotic body (PAS, ×400) (C). Slide culture on potato dextrose agar at 25°C on day 8 of C.carrionii (D) and F. monophora(E) (×400). F. monophora colonies on SDA at 25°C on day 7 (F).

Finally, ten cases received systemic treatment, and case 1 gave up treatment due to economic poverty. The seven mild cases were cured by treatment with itraconazole 200–400 mg per day, with a treatment course that ranged from 3 months to 5 months (Fig 3B, 3D and 3F). Case 2 was cured by treatment with terbinafine 250 mg per day for 5 months. However, it should be noted that treatments of case 7, case 8 and case 9 have beed briefly interrupted due to the interference of comorbidity. Case 3 had been treated with itraconazole (200 mg per day) for one year, but no improvement was observed. Currently, case 3 is receiving a new treatment plan. Case 5 was cured by combined itraconazole and photodynamic therapy for 10 months (Fig 2D). In our study, the total rate of complete remission was 90% (9/10) in our study.

Discussion

In China, chromoblastomycosis is primarily caused by C. carrionii in the north and by F. monophora and F. pedrosoi in the southern and eastern parts of the country [13], especially in Guangdong Province and Shandong Province of Mainland China [8]. F. pedrosoi and C. carrionii infections are normally observed in tropical and subtropical areas of endemicity around the world [14]. These are brown pigmented fungi living as saprophytes on plants or vegetable debris in the soil [15]. Zhejiang is located in the southeast coast of China and in the middle of a subtropical zone, with monsoon humid climate. C. carrionii occurs in semiarid areas, whereas F. pedrosoi is associated with humid climates [14]. In terms of geography and climate, Zhejiang is suitable for the survival of these brown pigmented fungi, especially the genus Fonsecaea. Molecular phylogeny has facilitated the identification of cryptic species within the F. pedrosoi species complex. The isolates identified as F. pedrosoi morphologically now belong to F. pedrosoi sensu strico, F. monophora [16], and F. nubica [7]. F. pedrosoi and F. nubica are strictly associated with chromoblastomycosis, whereas F. monophora is also involved in phaeohyphomycosis of the brain and other organs [7]. In clinical practice, both F. pedrosoi and F. monophora are easily misidentified because both have similar morphological features. Although it may have limitations, the ITS region can confirm the species in most of the circumstances. These limitations may be overcome by the sequencing of other loci and/or by using other molecular methods. The combination of CDC42, ACT1, BT2, lactase (Lac), homogentisate (HmgA), and polyketide synthase (PKS1) (the first three genes are the most common) with ITS sequencing can increase the accuracy [17]. Based on our study, F. monophora is the dominant pathogen in CBM cases found in Zhejiang. F. monophora isolates exhibited high genetic diversity [18]. and we hypothesized that many Fonsecaea isolates might have been misidentified due to the lack of molecular tools in the past. Thus, a long-term surveillance and molecular identification of all species in Fonsecaea isolates from CBM cases are necessary for epidemiology.

In agreement with previous literature, we found that the identified CBM cases are prevalent among old male farmers. Chromoblastomycosis is considered an occupational disease worldwide that affects farm laborers, gardeners, lumberjacks, vendors of farm products, and other workers exposed to contaminated soil and plant materials [14]. On rare occasions, wood fragments containing muriform cell-like structures were histopathologically observed in patients with CBM [19,20]. In our series, carpenter was the second most common occupation. CBM is usually gradual and indolent in nature. As with sporotrichosis, CBM is difficult to eradicate, GRAPH 1 suggests that CBM has been in a stable sporadic state. At the time of presentation, the duration of the disease varied from 20 days to 35 years [3]. Most of the studied cases were chronic, with a mean duration of 6.5 years. The lower limbs are the areas of the body that would be most likely to be in contact with fungi contaminated material [21]. In most series, there has been a predilection for the lower legs [2,3,21,22]. However, in Australia, the upper limbs were the most affected parts by CBM [23], which is also observed in our cohort, where the hand, wrist, and forearm were predominantly affected. The difference might be attributable to differences in economic structures among these countries. Rural workers in Eastern China tend to wear protective clothing and shoes that cover the legs but not the hands and forearms. Consistent with the results from previous reports [21,22], any history of trauma was recalled only in 45.5% of the cases. This may be related to a trauma insensitivity when engaged in agricultural activities. Except for three cases of infection via penetration of wood splinters, two cases had history of insect bite which may be a route of inoculation of soil or vegetative matter contaminated by brown pigmented fungi. Recently, CBM has been increasingly reported in immunocompromised individuals, while diabetes is the most common comorbidity in Taiwan [18]. In our series, diabetes, and kidney health problems were the predisposing conditions.

The clinical features of CBM are the manifestation of disease evolution. Initially, the lesion is a small, nonpruriginous with erythematous papules, then the lesions progress into an erythematous plaque, with or without scales or ulcerations, and with a well-defined border. Later, the plaque expands centrifugally and develops an irregular verrucous or papillomatous surface [24]. The most frequent clinical aspect of CBM is different according to various reports. However, verrucous lesions [21,25,26], nodular lesions [25,27], cicatricial lesions [2], and plaque-like lesions [3] are respectively predominant. In our study, plaque lesions were the most common, followed by verrucous lesions. According to Queiroz-Telles et al [11], lesions can be graded as mild forms (a solitary plaque or nodule measuring less than 5 cm in diameter); moderate forms (solitary or multiple lesions which may be nodular, verrucous or plaque types, existing alone or in combination, covering one or two adjacent cutaneous regions, measuring less than 15 cm in diameter); and severe forms (any type of lesion alone or in combination, covering extensive cutaneous regions whether adjacent or non-adjacent). Severe lesions tend to respond slowly or even become non-responding to antifungal drugs. Table 1 shows that mild form is the most common in our study. Obviously, cases with severe forms had a longer disease course.

Chromoblastomycosis (CBM) is a neglected tropical disease (NTD) [12]. Its diagnostic characteristic is the presence of brown muriform cells in the infected tissue, which can be demonstrated using potassium hydroxide (KOH) mount and hematoxylin and eosin staining [28]. The sensitivity of direct examination ranges from 90 to 100% and this method is fast, easy, and inexpensive [14]. In the current study, microscopy showed muriform cells with or without germinating hyphae in the lesion scales of all patients. The tissular response is not specific in CBM specimens, and it may be similar to that of the tissue reactions observed for most implantation mycoses [14]. Pires et al. found two main types of granulomatous tissue reactions: suppurative granuloma with abundant fungal cells, mostly from verrucous lesions, and tuberculoid granuloma, with few parasites, from plaque and atrophic lesions [29]. Histopathology showed that muriform cells are present in 45.4% of the cases, while it was noted be 100% in the cases reported by Mead and Ridley in 1957 [30], and in 12 of the 13 cases that were reported by Leslie and Beardmore in 1979 [31]. CBM lesions are chronic, indolent, and clinically polymorphic. CBM can mimic a wide spectrum of diseases with infectious and noninfectious causes [14], such as cutaneous tuberculosis, leprosy, leishmaniasis, sporotrichosis, mycetoma, psoriasis, and malignancies such as verrucous carcinoma and cutaneous lymphoma [32]. Our study confirmed that 72.7% (8/11) had been misdiagnosed. This suggests the importance of improving the diagnostic ability of clinicians in rural areas, especially when using a direct microscopic examination. The fluorescent reagent was demonstrated to increase the sensitivity of the detection of many fungi, but the utility in the case of pigmented fungi is not helpful when compared with KOH.

CBM is difficult to treat and is associated with low cure rates and high relapse rates, especially in chronic and extensive cases. The treatment choice and the results depend on the etiological agent, size, extent of the lesions, topography, and the presence of complications [33]. Treatment may be divided into three groups; physical treatment, chemotherapy and combination therapy [11]. Patients showing severe and advanced clinical forms of disease require a long duration of continuous systemic antifungal treatment [34,35]. Itraconazole is the most common choice of first-line agents [18] and was the most common choice of first-line agent in our practice (200–400 mg/day). Our study showed that mild cases respond well to systemic agents, with a treatment duration varying from 3-5months. Case 3 treatment suggested that severe lesions tend to respond slowly or even become non-responding to antifungal drugs. The 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) directly inactivate F. monophora through a ROS dependent oxidative damage and indirect activation of macrophages [36]. Case 5 treatment suggested that the combination of antifungal drugs and PDT is effective for severe forms. CBM mainly affects agricultural workers and rural population who are often at the bottom of society and unable to bear the burden of long-term treatment. In our series, it is a pity that case 1 lost the capacity to work due to the long-term lack of effective treatment. The burden and medical impact of this implantation mycosis are certainly underestimated. The use of protective equipment such as gloves, shoes, and adequate clothes may be a key in reducing the risk of infection [14].

In conclusion, CBM is a neglected fungal disease that mainly affects low-income agricultural workers and rural population. The high misdiagnosis rate of chromoblastomycosis is a major challenge in dermatology, especially for rural hospitals. The diagnosis of CBM requires laboratory confirmation by direct mycological examination and/or histopathology. The visualization of muriform cells in clinical samples is a cornerstone of this disease diagnosis. Direct microscopic examinations should be further promoted in rural hospitals. The treatment choice and results also depend on the patients’ lesion grade. For severe forms, high doses and a long treatment and combination therapy are the first treatment choices. Apart from morphological identification, molecular analysis is important, especially for Fonsecaea spp. F. monophora is identified as the major pathogen affecting the hands and forearms of patients in Zhejiang. However, due to the study small case series, the results may have some limitations.

1 Jul 2022

Dear Mr xia,

Thank you very much for submitting your manuscript "Chromoblastomycosis: A case series from eastern China" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

This relatively small case series has merit as this disease has rarely been diagnosed from this part of China before, cases came forward as part of a campaign, the initial diagnosis was made with KOH, not biopsy, and most of the isolates were identified as F monophora.

The article would be stronger if these points were emphasised (along with the long period of missed diagnosis in many of them). It would also be stronger if a proper description of the campaign to find cases was described, and the results of this campaign in terms other NTDs described. how many cases of leprosy, sporotrichosis, mycetoma, cutaneous mycobacterial infections etc were found alongside chromoblastomycosis.

The article requires extensive revision and better quality pictures (and preferably before and after treatment pictures) to be acceptable for publication. Please attend to the referees comments.

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This relatively small case series has merit as this disease has rarely been diagnosed from this part of China before, cases came forward as part of a campaign, the initial diagnosis was made with KOH, not biopsy, and most of the isolates were identified as F monophora.

The article would be stronger if these points were emphasised (along with the long period of missed diagnosis in many of them). It would also be stronger if a proper description of the campaign to find cases was described, and the results of this campaign in terms other NTDs described. how many cases of leprosy, sporotrichosis, mycetoma, cutaneous mycobacterial infections etc were found alongside chromoblastomycosis.

The article requires extensive revision and better quality pictures (and preferably before and after treatment pictures) to be acceptable for publication. Please attend to the referees comments.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Please explain in more detail what is meant by a consultation campaign.were there regional

organised clinics specifically seeking cases of chromoblastomycosis or skin disease in general. We

need to know a bit more about this recruitment method.

Was the mycological work, including direct microscopy carried out in a single laboratory ?

Reviewer #2: Suggestions and or comments were attached

Reviewer #3: The authors retrospectively reviewed 11 cases of CBM in one center of Eastern China in three years.

Reviewer #4: The objective can be more clearly stated. It is a prospective study but in the introduction, it stated that "In order to evaluate the clinical characteristics of CBM in Zhejiang,eastern China,we reviewed 11 cases..," which may imply that it was a retrospective study instead.

In page 2, paragraph 1, the author stated: "Patients were recruited between January 2018 and December 2021 and during consultation campaigns conducted in Zhejiang Province.." What was meant by consultation campaigns? Were these "information campaigns" conducted in other clinics so that these kinds of cases were referred to the Hangzhou Third people's Hospital? It may good to describe further the "consultation campaign" and referral system followed since this would give readers an idea on the actual catchment area of the facility. Is Hangzhou Third people's Hospital a specialty hospital?

Since it is a case series, conducted prospectively, it would have been better if pictures of individual cases were taken, and sizes of lesions were recorded. It would be interesting to note if cases with smaller lesion sizes responded to therapy faster and more completely, compared to those with larger and more disseminated lesions.

Was surgery an option in any of the cases? If not, why not?

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Please explain the following phrase - consolidate treatment due to the interference of comorbidity.

Kidney trouble – better chronic renal disease.

Reviewer #2: The use of lesions severity graduation would be this article more interesting for clinicians

Reviewer #3: The study indicated that the most common pathogenic fungus was F.monophora.

Reviewer #4: Figure 1 clinical photos are not clear. It would be good to indicate which particular cases these were.

Since it is a case series, it would be good to include before and after treatment pictures as well.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The authors argue for improving diagnostic ability in rural areas to reduce the risk of misdiagnosis. It

would, be useful here to discuss possible methods of doing this.

Please mention other forms of treatment such as oral terbinafine or heat therapy.

It would be useful in the discussion to on the distribution and case load of chromoblastomycosis in

China. How common is it ? Are there any explanations for the differ ent distribution of organisms ?

Why is F monophora a dominant cause in China ? Is this a true geographic variation or is this because

without molecular tools most such strains have been misidentified as F pedrosoi in the past ?

Reviewer #2: Suggestions and or comments were attached

Reviewer #3: The authors should indicate the limitations of this case series, such as the limited number of the patients, single center study, etc.

Reviewer #4: The conclusions can be improved to refer more to the cases seen in the facility.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: There are some changes needed to the English. Please mention that chromoblastomycosis is

formally designated by WHO as an NTD

Reviewer #2: (No Response)

Reviewer #3: None

Reviewer #4: Page 4, paragraph 1: associated condition, not predisposing

Page 3, paragraph 1: 8 cases were misdiagnosed; Page 4, paragraph 2: 7 cases were diagnosed. Please clarify.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: Suggestions and or comments were attached

Reviewer #3: The novelty of this study is relatively low.

Reviewer #4: 1. There may be a need to edit the article for it to be publishable.

2. These are the points that I think are good take-aways from this article

a. All cases were initially evaluated in other clinics before the information campaign and 8 were misdiagnosed. This highlights the importance of educating primary physicians or those who usually initially manage these cases, on recognizing chromoblastomycosis and other skin neglected tropical diseases.

b. All 11 cases were diagnosed with chromoblastomycosis after doing a simple, easy, non-invasive, inexpensive, out-patient procedure, the KOH smear. This illustrates that KOH smear may even be more sensitive in catching these cases compared to punch biopsy.

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Reviewer #1: No

Reviewer #2: Yes: Flávio Queiroz-Telles

Reviewer #3: No

Reviewer #4: Yes: Maria Christina Filomena Batac

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23 Jul 2022

Submitted filename: response.docx

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16 Aug 2022

Dear Mr xia,

Thank you very much for submitting your manuscript "Chromoblastomycosis: A case series from eastern China" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please remove the term dematiaceous and replace with brown pigmented - throughout the manuscript, including the abstract.

There are still some errors of english, which should be improved before final publication. For example 'histopathology, not histopathology of biopsies'

In the abstract just state how many males, not a ratio.

The authors have added helpful information on the other similar presentations. Do they have any sense of trends over time? Is CBM diminishing in frequency, or static? And how does it compare to sporotrichosis, for example.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ahmed Fahal, FRCS, FRCSI, FRCSG, MS, MD, FRCP(London)

Section Editor

PLOS Neglected Tropical Diseases

Ahmed Fahal

Section Editor

PLOS Neglected Tropical Diseases

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Please remove the term dematiaceous and replace with brown pigmented - throughout the manuscript, including the abstract.

There are still some errors of english, which should be improved before final publication. For example 'histopathology, not histopathology of biopsies'

In the abstract just state how many males, not a ratio.

The authors have added helpful information on the other similar presentations. Do they have any sense of trends over time? Is CBM diminishing in frequency, or static? And how does it compare to sporotrichosis, for example.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The authors have answered my queries

Reviewer #2: The author added the reviwers suggestions or comments to the current manuscript version

Reviewer #4: The paper described that aside from chromoblastomycoses, other subcutaneous mycoses were diagnosed during the same 3-year period of the study. It is good to disclose if these have been described or will be described in another paper, (and include proper citations).

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The authors have answered my queries

Reviewer #2: The author added the reviwers suggestions or comments to the current manuscript version

Reviewer #4: Pictures had no accompanying descriptive texts.

It will be good to compare the frequency of cases of subcutaneous mycoses diagnosed over the years in their facility. Has there been an increase in diagnosis due to greater awareness of the disease entities? Or after they have adapted this "routine flowchart" in diagnosing subcutaneous diseases?

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The authors have answered my queries

Reviewer #2: This paper is relevant for the Public Health because chromoblastomycosis is one of the few fungal infections, officially recognized as NTD by the WHO

Reviewer #4: (No Response)

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Nil needed

Reviewer #2: Please note that "dematiaceous fungi "is an obsolete term. "Melanized fungi" is week stablished denomanation for all chromoblastomicosis and phaeohyphomiycosis etiological agents

Reviewer #4: Abstract: Since the sample being described is small, only 11, then it is better to just say outright how many are males and how many are females, instead of giving the ratio of 4.5:1

Terms: histopathology, not histopathology of biopsies

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have answered my queries

Reviewer #2: I suggested a mino revision

Reviewer #4: The flow of the narrative can be improved. Although much improved from the first draft, it needs more editing to be publishable.

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Reviewer #1: No

Reviewer #2: Yes: Flavio Queiroz-Telles

Reviewer #4: Yes: Maria Christina Filomena Batac

Figure Files:

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

25 Aug 2022

Submitted filename: response(R2).docx

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7 Sep 2022

Dear Mr xia,

We are pleased to inform you that your manuscript 'Chromoblastomycosis: A case series from eastern China' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

David W. Denning

Guest Editor

PLOS Neglected Tropical Diseases

Ahmed Fahal

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

None

14 Sep 2022

Dear Mr Xia,

We are delighted to inform you that your manuscript, "Chromoblastomycosis: A case series from eastern China," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases