The First Case Of Chromoblastomycosis Caused By Fonsecaea Nubica In The North Of China And Literature Review

BackgroundChromoblastomycosis(CBM)is a chronic cutaneous and subcutaneous infection caused by black fungi,and predominantly found in tropical and subtropical regions.The mean causative agents of CBM are Fonsecaea spp.and Cladophialophora spp.As for Fonsecaea species,Fonsecaea pedrosoi,Fonsecaea monophora,Fonsecaea nubica and Fonsecaea pugnacius are all potential etiologic pathogens of CBM.F.nubica was a new species firstly described in 2010 by Najafzadeh et al.This species is morphologically similar to F.pedrosoi and F.monophora and is only distinguishable using multilocus molecular data including AFLP profles,sequences of the ribosomal internal transcribed spacers(ITS),and partial sequences of the cell division cycle(CDC42),?-tubulin(TB2)and actin(ACT1)genes.By now there are more than 40 strains of F.nubica reported in the literatures.These strains were most commonly isolated from Brazil and China,while there were also strains isolated from Africa,France,Australia,Thailand,Japan,Korea and Bangladesh.CBM is often caused by trauma inoculation through the broken skin.It has also been reported to be related to insect bites.It occurs in the exposed parts of the body,mainly at the extremities of the limbs,and is more common in the lower legs and feet.CBM are frequently found in farmers,laborers and persons walking barefoot.CBM can occur at all ages,mainly in 41~70 years old.It predominated in males.Sclerotic bodies are the hallmark for the diagnosis of CBM.The diagnosis of CBM caused by F.nubica was confirmed according to the results of clinical manifestation,histopathology,mycological examinationand,and DNA sequencing.Due to diversity in the etiological agents,bioavailablity of the antifungals,and variation in clinical manifestation,there isn't such a ”gold” treatment for CBM,which is a big challenge for clinicians.Objective1.To summarize the clinical and experiment characteres of one case of Chromoblastomycosis caused by Fonsecaea nubica.2.To review all cases of Chromoblastomycosis caused by Fonsecaea nubica reported in domestic or abroad.Methods1.Clinical Material: We diagnosed a case of chromoblastomycosis due to Fonsecaea nubica in a 75-year-old Chinese male,withing red plaques on his right back for 15 months,who had a history of trauma.2.Histopathological examination:Specimens were obtained from the lesion of the patient's right back,and were fixed with formalin solution.The specimens were sent to the pathology department for HE staining,PAS staining and Grocott's methenamine staining.The specimens were observed under the microscope.3.Fungal culture: The isolate from skin lesion was grown on Sabouraud's glucose agar at 28? for 14 day,and colonies can be observed.4.Direct fungal microscopic examination(10%KOH): Specimens were obtained from the colonies,and then make smear.The specimens were observed under the microscope.5.Fungal fluorescence staining:Slide culture was conducted on potato dextrose agar at 28 C for 7 days.The smear specimens of slide culture stained with Evans blue plus calcofluor were observed under fluorescence microscope in white,ultraviolet and merged light.6.Scanning electron microscopy: The specimen was fixed at 2.5% glutaraldehyde at 4°C for 4 hours and dehydrated with ethanol in gradient concentration(50%,70%,90% and 100%)in the meantime,which is observed under scanning electron microscope.7.Molecular biology methods: DNA of the colony was extracted,amplified using polymerase chain reaction(PCR)and sequenced of internal transcribed spacer(ITS)and actin(ACTI).PCR products were sent to Tsingke Biotech for Sanger sequencing service.8.Vitro antifungal susceptibility testing: Antifungal susceptibility testing was carried out with broth microdilution method according to the Clinical and Laboratory Standards Institute(CLSI)guidelines(M38-A2 document).Candida parapsilosis(ATCC 22019)was used as quality control.MIC values of the following antifungal agents were tested: itraconazole terbinafene amphotericin B and fluconazole.9.Phylogenetic trees: Phylogenetic trees of Fonsecaea nubica based on confidently aligned internal transcribed spacer(ITS)sequences constructed with neighbor joining implemented in MEGA 6.0.Cladophialophora bantiana and Cladophialophora emmnonsii were taken as the outgroup.10.Review the reported cases of chromoblastomycosis caused by Fonsecaea nubica in domestic or abroad.Literatures were searched from China National Knowledge Infrastructure,Wanfang Database,PubMed,MEDLINE and Elsevier.We We analyzed the history,clinical manifestations,histopathological examination,direct fungal microscopic examination,fungal culture,scanning electron microscopy,molecular Identification,vitro antifungal susceptibility testing and treatment of cases.Results1.Clinical Material: A 75-year-old male,native and resident of Henan Province,presented to the Dermatology Clinic of First Affiliated Hospital of Zhengzhou University for erythematous plaque on his right back for15 months.The patient was stabbed by branches.Physical examination revealed a well-defined,irregularly shaped,red plaque with clear margin,40 mm×10mm in size on his right back.There were no scales,swelling,suppuration or fluctuation with no regional lymphadenopathy.A case of Chromoblastomycosis caused by Fonsecaea nubica was diagnosed with the combination of Clinical features,histopathological examination,mycology Examination,and Molecular biology methods.The patient was cured with oral itraconazole capsule for 16 weeks,and combined with excisional surgery.No recrudescence was found after 8-month follow-up.2.Histopathological examination showed pseudoepitheliomatous hyperplasia in the epiderm,a granulomatous response in the derm,and round,thick-walled,brownish,septate muriform cells in multinucleated giant cells.Grocott's methenamine(GMS)and periodic acid–Schiff(PAS)stain were positive.3.Fungal culture: slowly growing,black and velvety colonies.4.Direct fungal microscopic examination(10%KOH): brown-separated hyphae.5.Fungal fluorescence staining:numerous pale brown,septate conidiophores with Rhinocladiella type conidia.6.Scanning electron microscopy: septate conidiophores with Rhinocladiella type conidia.Conidiogenous cells were oval,located at the apex of the conidiophore,with prominent denticles.7.Molecular biology methods: The nucleotide sequence of our strain was compared with the GenBank database using the BLAST algorithm.The PCR products revealed 100% identity with F.nubica strain CBS 269.64(GenBank accession number:MH988741).8.Vitro antifungal susceptibility testing: MICs of itraconazole,terbinafine,amphotericin B and fluconazole were 0.5?g/mL,0.5?g/mL,8?g/mL,and 32?g/mL respectively.The MICs of itraconazole and terbinafine were lower than that of amphotericin B and fluconazole.9.Phylogenetic trees: Phylogenetic trees of Fonsecaea nubica based on confidently aligned internal transcribed spacer(ITS)sequences constructed with neighbor joining implemented in MEGA 6.0.Origin and location of strains were indicated next to the strain number.10.Through literature searching,a total 5 cases of CBM caused by F.nubica were included in this study.1Caes from Japan,1 case from France,1 case from Bangladesh and 2 cases from guangdong,China.The patients' average age is 52.6 years old,with a minimum age of 32 years and a maximum age of 66 years.Mean duration of the infection was 5.1 years,with the shortest duration of 1 year and the longest duration of 10 years.The skin lesions of 2 patients located in the lower limb,2 patients located in the upper limb,and 1 patient located in the ear.5 cases all had a history of trauma.Lesions of 5 cases were plaques.One case presented abnormalities in the left wrist and left elbow joints.Histopathological examination was performed in all 5 cases.Hematoxylineosin(HE)staining of 4 cases showed granulomatous inflammatory changes and sclerotic bodies.Direct fungal microscopy was performed in 2 cases,1 cases showed sclerotic bodies.5 cases of fungal culture showed black,gray,olivaceous,and velvety colonies.5 cases of fungal microscopy showed dark brown,brown,light brown,seperate hyphae,and light brown,conidia with septum.conidiophores were olivaceous brown,terminal or intercalary.Conidiogenous cells were pale brown,unicellular,broadly clavate,singly or in short chains,with prominent denticles and dark scars.All the 5 cases sequenced the ITS sequence of rDNA,and the pathogens were identified to be F.nubica.2 Cases carried out In vitro antifungal susceptibility testing.The MICs of 1 case is in descending order:echinocandins(caspofungin,micafungin and anidulafungin),amphotericin B,voriconazole,azoles(itraconazole and posaconazole).The MICs of 1 case is in descending order is fluconazole,amphotericin B,terbinafine and itraconazole.1 case didn't mentioned treatment.4 Cases were treated with itraconazole,terbinafine,itraconazole combined with itraconazole,and the lesions of 4 cases improved.Conclusions1.A case of Chromoblastomycosis caused by Fonsecaea nubica was diagnosed with the combination of Clinical features,histopathological examination,mycology Examination,and Molecular biology methods.2.Up to now,5 cases of Chromoblastomycosis caused by Fonsecaea nubica were reported in domestic and foreign literatures.we present the sixth case of Chromoblastomycosis caused by Fonsecaea nubica in domestic and foreign and the first case of Chromoblastomycosis caused by Fonsecaea nubica in Northern China.