Xiqing Li

Successful treatment for chromoblastomycosis caused by Fonsecaea monophora: a report of three cases in Guangdong, China

Fonsecaea pedrosoi is the most prevalent aetiological agent of chromoblastomycosis. Fonsecaea monophora is a new species segregated from Fonsecaea pedrosoi. Herein, we report on three cases of chromoblastomycosis caused by F. monophora that were successfully treated with terbinafine and/or itraconazole. Clinical characteristics and mycological parameters are described. Two of the three patients underwent combination therapy with itraconazole and terbinafine during early stages of treatment and were completely healed in a relatively short course of treatment.

Synergistic effects of terbinafine and itraconazole on clinical isolates of Fonsecaea monophora

Our objective was to develop new approaches to the chemotherapy of invasive infections caused by Fonsecaea monophora. The in vitro effects of a combination of terbinafine with itraconazole on 18 clinical isolates were evaluated using a checkerboard microdilution method. The mode of interaction between the two drugs on the 18 isolates was analyzed using fractional inhibitory concentration index (FICI) analysis. FICI analysis demonstrated that 12 (67%) were synergistic, 4 (22%) were additive, and 2 (11%) were indifferent, with no antagonism being observed. The minimal inhibitory concentrations (MICs) obtained with the terbinafine-itraconazole combination were within levels that can be achieved in plasma at clinically relevant doses. Our results indicate the terbinafine-itraconazole combination may be an effective therapy for Fonsecaea monophora infection, which should be tested in clinical setting with patients with this disease.

Chromoblastomycosis in Mainland China: A Systematic Review on Clinical Characteristics

Chromoblastomycosis is one of the most frequent chronic infections caused by melanized fungi. In order to evaluate the clinical characteristics of chromoblastomycosis in Mainland China, we performed an evidence-based review of published literature. PubMed and Chinese-language database of CNKI, VIP and Wanfang data during January 1990–August 2011 were searched. Epidemiology, clinical features, laboratory findings, therapy and prognosis were analyzed. Cladophialophora carrionii was the most common causative agent in the north of the Mainland China, and Fonsecaea monophora and F. pedrosoi were the most common agents in the southern part of the Mainland China. Infection commonly initiated after the etiologic agents gain entrance through puncture wounds and more common involved extremities of the males. Skin lesions were found in different sites, like the extremities, buttocks, trunk and face, and presented diversity morphology. There were about seven different clinical types found in Mainland China: plaque type, tumoral type, cicatricial type, verrucous type, pseudo-vacuole type, eczymatous type and mixed type of lesions. The success of treatment for chromoblastomycosis was related to the causative agent, the clinical form and severity of the lesions. Most of the patients could be treated successfully with the physical treatment, chemotherapy and/or combination therapy. The itraconazole, terbinafine or a combination of both were commonly medication for these mycosis patients. Physical methods were usually indicated to support chemotherapy with some severe forms and long-lasting cases. Photodynamic therapy has been extended from the oncological field to that of antimicrobial chemotherapy in these years. We applied it on some recalcitrant cases of chromoblastomycosis and found its good clinical response, and hopeful it could be a promising therapy in near future.

A refractory case of chromoblastomycosis due to Fonsecaea monophora with improvement by photodynamic therapy

Chromoblastomycosis is one of the most frequently encountered mycoses in tropical and temperate regions caused by the implantation of the infectious structures and one which is associated with low cure and high relapse rates. The etiologic agents play a critical role affecting clinical outcome and in southern China, Fonsecaea pedrosoi and F. monophora are the main causative agents of chromoblastomycosis. We treated, for two years, a 55-year-old male patient with chromoblastomycosis caused by F. monophora with itraconazole and terbinafine, two antifungals recommend in earlier papers in the literature but without any positive response. As a result we introduced the photodynamic therapy (PDT) employing 5-aminolevulinic acid (ALA) irradiation. The lesions were improved after two periods of ALA-PDT treatment, each consisting of exposures at weekly intervals for 5 weeks but new lesions developed with the cessation of ALA-PDT treatment. Thereafter, positive clinical improvement was obtained when voriconazole at 200 mg was combined with terbinafine at 250 mg in treating the patient. The in vitro susceptibility of the F. monophora isolate to terbinafine, itraconazole, and voriconazole was assessed and the fungus was found to be sensitive to all three, with the minimal inhibitory concentrations of 0.125, 1, 0.0625 μg/ml, respectively. However, the determination of in vitro susceptibility profiles may not predict clinical response.

Chromoblastomycosis caused by a meristematic mutant of Fonsecaea monophora

We report the first case of chromoblastomycosis caused by a meristematic mutant of Fonsecaea monophora in an 81-year-old Cantonese male. The patient had a seven-month history of a red plaque with thick yellow crust and purulent exudates on the back of his right hand. Microscopic examination of direct smears revealed brown sclerotic cells and black colonies were recovered in culture from samples of the purulent exudantes. The microscopic appearance of this isolate was quite different from that of other Fonsecaea species, exhibiting mere meristematic growth. The rDNA ITS sequence data confirmed that this isolate was a mutant of Fonsecaea monophora. The patient showed good response to treatment with itraconazole and complete healing was achieved without relapse after long-term follow-up.

Cutaneous protothecosis: two new cases in China and literature review

Protothecosis is a rare infection caused by Prototheca spp., which are achlorophyllous algae found in trees, various aquatic sources such as lakes and rivers, sewage treatment plants, soil, and household garbage. They can infect both humans and animals. Human protothecosis is rare, and most cases are found in immunocompromised patients. In mainland China, only four cases caused by Prototheca spp. have been reported. In general, clinical manifestations of reported cases can be classified into three clinical forms: cutaneous (66%); olecranon bursitis (15%); and disseminated or systemic infections (19%). Currently, Prototheca zopfii, P. wickerhamii, P. stagnora, P. ulmea, and P. blaschkeae are classified to the genus. Among these, only the former two have been reported to cause infections in humans, with P. wickerhamii being most common. In this article, two cases of cutaneous protothecosis caused by P. wickerhamii will be presented. Both patients responded well to treatment with intravenous voriconazole (VRC), although one died later from other complications.

RNAi-mediated silencing of fungal acuD gene attenuates the virulence of Penicillium marneffei

A number of pathogens, most of them intracellular, employ the glyoxylate cycle in order to ingest fatty acids as carbon sources as a way of coping with nutrient deprivation during the infection process. Isocitrate lyase, which is encoded by the pathogens acuD gene, plays a pivotal role in the glyoxylate cycle, which has been implicated in fungal pathogenesis. In this study, the acuD gene of Penicillium marneffei was knocked down using siRNA expressed by a filamentous fungi expression system. The acuD siRNA reduced the acuD genes mRNA and protein expression by 21.5 fold and 3.5 fold, respectively. When macrophages were infected with different transformants of P. marneffei, the knockdown of acuD expression with RNA interference was lethal to the pathogens. In addition, the secretion of tumor necrosis factor-alpha and interferon-gamma from the infected macrophages was reduced. Moreover, the RNAi-mediated silencing of acuD expression reduced the fungal burden in the nude mice infected with P. marneffei; inhibited the inflammatory response in the lungs, livers, and spleens during the chronic phase instead of the acute phase of infection; and thus prolonged survival of the infected animals. Collectively, our data indicate that the RNAi-mediated silencing of acuD expression could attenuate virulence of P. marneffei. The endogenous expression of the delivered siRNA vector could be used to evaluate the role of functional genes by continuous and stable expression of siRNA.

The role of melanin pathways in extremotolerance and virulence of Fonsecaea revealed by de novo assembly transcriptomics using illumina paired-end sequencing

Melanisation has been considered to be an important virulence factor of Fonsecaea monophora. However, the biosynthetic mechanisms of melanisation remain unknown. We therefore used next generation sequencing technology to investigate the transcriptome and digital gene expression data, which are valuable resources to better understand the molecular and biological mechanisms regulating melanisation in F. monophora. We performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of parent (CBS 122845) and albino (CBS 125194) strains using the Illumina RNA-seq system. A total of 17 352 annotated unigenes were found by BLAST search of NR, Swiss-Prot, Gene Ontology, Clusters of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) (E-value <1e‒5). A total of 2 283 unigenes were judged to be the differentially expressed between the two genotypes. We identified most of the genes coding for key enzymes involved in melanin biosynthesis pathways, including polyketide synthase (pks), multicopper oxidase (mco), laccase, tyrosinase and homogentisate 1,2-dioxygenase (hmgA). DEG analysis showed extensive down-regulation of key genes in the DHN pathway, while up-regulation was noted in the DOPA pathway of the albino mutant. The transcript levels of partial genes were confirmed by real time RT-PCR, while the crucial role of key enzymes was confirmed by either inhibitor or substrate tests in vitro. Meanwhile, numbers of genes involved in light sensing, cell wall synthesis, morphology and environmental stress were identified in the transcriptome of F. monophora. In addition, 3 353 SSRs (Simple Sequence Repeats) markers were identified from 21 600 consensus sequences. Blocking of the DNH pathway is the most likely reason of melanin deficiency in the albino strain, while the production of pheomelanin and pyomelanin were probably regulated by unknown transcription factors on upstream of both pathways. Most of genes involved in environmental tolerance to oxidants, irradiation and extreme temperatures were also assembled and annotated in transcriptomes of F. monophora. In addition, thousands of identified cSSR (combined SSR) markers will favour further genetic linkage studies. In conclusion, these data will contribute to understanding the regulation of melanin biosynthesis and help to improve the studies of pathogenicity of F. monophora.

Role of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways in regulating replication of Penicillium marneffei in human macrophages.

Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To elucidate the mechanisms involved, we investigated the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) pathways in cytokine expression, phagosome-lysosome fusion and replication of P. marneffei in P. marneffei-infected human macrophages. Analysis of both ERK1/2 and p38 showed rapid phosphorylation in response to P. marneffei. Using specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that ERK1/2 and p38 were essential for P. marneffei-induced tumor necrosis factor-α production, whereas p38, but not that of ERK, was essential for IL-10 production. Furthermore, the presence of PD98059 always decreased phagosomal acidification and maturation and increased intracellular multiplication of P. marneffei, whereas the use of SB203580 always increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that a proper balance of between ERK1/2 and p38 may play an important role in controlling the replication of P. marneffei. Our findings further indicate a novel therapeutic avenue for treating P. marneffei by stimulating ERK1/2 or activating ERK1/2-dependent mechanisms.

A chronic chromoblastomycosis model by Fonsecaea monophora in Wistar rat

Chromoblastomycosis is a chronic, cutaneous and subcutaneous infection characterized by verrucose lesions. It is primarily caused by Fonsecaea monophora in southern China and responds poorly to available therapies. In order to investigate the pathogenicity of Fonsecaea monophora, we established a chronic chromoblastomycosis animal model in Wistar rats. Suspensions of 2x10(6) cfu conidia and hyphal fragments were intracutaneous (ic) and subcutaneous (sc) on either side of the backs of the rats. Small nodules were formed at the inoculation sites within the first week after inoculation. By the second week, the nodules had enlarged and become soft, and pus could be aspirated from them. In the fourth week, the nodules in the ic inoculated group had ulcerated and sclerotic bodies were observed in smears prepared from all animals. In the 3rd month after inoculation, the nodules at the sites of the ic inoculation had become flat, with a thin black crust on the surface. For the ic inoculated group, sequential biopsy studies revealed extensive necrosis with neutrophil infiltration and sclerotic bodies and some debris of fungi in the 1st month, sclerotic bodies inside multinucleated giant cells by the 2nd month and widespread granulomatous inflammations in the 3rd month.