Exploring the immunogenicity of Rv2201-519: A T-cell epitope-based antigen derived from Mycobacterium tuberculosis AsnB with implications for tuberculosis infection detection and vaccine development.

Research dedicated to diagnostic reagents and vaccine development for tuberculosis (TB) is challenging due to the paucity of immunodominant antigens that can predict disease risk and exhibit protective potential. Therefore, it is crucial to identify T-cell epitope-based Mycobacterium tuberculosis (MTB) antigens characterized by specific and prominent recognition by the immune system. In this study, we constructed a T-cell epitope-rich tripeptide-splicing fragment (nucleotide positions 131-194, 334-377, and 579-643) of Rv2201 (also known as the 72 kDa AsnB)from the MTB genome, ultimately yielding the recombinant protein Rv2201-519 in Escherichia coli BL21 (DE3). Subsequently, we gauged the recombinant protein's ability to detect tuberculosis infection through ELISpot and assessed its immunostimulatory effect on mouse models using flow cytometry and ELISA. Our results indicated that Rv2201-519 possessed promising sensitivity; however, the sensitivity was lower than that of a commercial diagnostic kit containing ESAT-6, CFP-10, and Rv3615c (80.56 % vs. 94.44 %). The Rv2201-519 group exhibited a propensity for a CD4+ Th1 cell immune response in inoculated BALB/c mice that manifested as higher levels of antigen-specific IgG production (IgG2a/IgG1 > 1). In comparison to Ag85B, Rv2201-519 induced a more robust Th1-type cellular immune response as evidenced by a notable rise in the ratio of IFN-γ/IL-4 and IL-12 cytokine production and increased CD4+ T cell activation with a higher percentage of CD4+IFN-γ+ T cells. Rv2201-519 also induced a higher level of IL-6 compared with Ag85B, a higher percentage of CD8+ T cells specific for Rv2201-519, and a lower percentage of CD8+IL-4+ T cells. Collectively, the current evidence suggests that Rv2201-519 could potentially serve as an immunodominant protein for tuberculosis infection screening, laying the groundwork for further evaluation in recombinant Bacillus Calmette-Guérin (BCG) and subunit vaccines against MTB challenges in future studies.

A novel multistage antigens ERA005f confer protection against Mycobacterium tuberculosis by driving Th-1 and Th-17 type T cell immune responses.

Introduction: Tuberculosis (TB) is a major threat to human health. In 2021, TB was the second leading cause of death after COVID-19 among infectious diseases. The Bacillus Calmette-Guérin vaccine (BCG), the only licensed TB vaccine, is ineffective against adult TB. Therefore, there is an urgent need to develop new effective vaccines. Methods: In this study, we developed a novel multistage subunit vaccine (ERA005f) comprising various proteins expressed in metabolic states, based on three immunodominant antigens (ESAT-6, Rv2628, and Ag85B). We utilized the E. coli prokaryotic expression system to express ERA005f and subsequently purified the protein using nickel affinity chromatography and anion exchange. Immunogenicity and protective efficacy of ERA005f and ERA005m were evaluated in BALB/c mice. Results: ERA005f was consistently expressed as an inclusion body in a prokaryotic expression system, and a highly pure form of the protein was successfully obtained. Both ERA005f and ERA005m significantly improved IgG titers in the serum. In addition, mice immunized with ERA005f and ERA005m generated higher titers of antigen-specific IgG2a than the other groups. Elispot results showed that, compared with other groups, ERA005f increased the numbers of IFN-γ-secreting and IL-4-secreting T cells, especially the number of IFN-γ-secreting T cells. Meanwhile, ERA005f induced a higher number of IFN-γ+ T lymphocytes than ERA005m did. In addition, ERA005f improved the expression of cytokines, including IFN-γ, IL-12p70, TNF-α, IL-17, and GM-CSF and so on. Importantly, both ERA005f and ERA005m significantly inhibited the growth of Mtb. Conclusion: The novel multistage antigen ERA005f elicited a strong antigen-specific humoral response and Th-1 and Th-17 cell-mediated immunity in mice. Meanwhile, it can effectively inhibit H37Rv growth in vitro, and represents a correlate of protection in vivo, indicating that ERA005f may exhibit excellent protective efficacy against Mycobacterium tuberculosis H37Rv infection. Our study suggests that ERA005f has the potential to be a promising multistage tuberculosis vaccine candidate.

A novel multi-component protein vaccine ECP001 containing a protein polypeptide antigen nPstS1 riching in T-cell epitopes showed good immunogenicity and protection in mice.

Tuberculosis (TB) is an infectious disease that seriously affects human health. Until now, the only anti-TB vaccine approved for use is the live attenuated Mycobacterium bovis (M. bovis) vaccine - BCG vaccine, but its protective efficacy is relatively low and does not provide satisfactory protection against TB in adults. Therefore, there is an urgent need for more effective vaccines to reduce the global TB epidemic. In this study, ESAT-6, CFP-10, two antigens full-length and the T-cell epitope polypeptide antigen of PstS1, named nPstS1, were selected to form one multi-component protein antigens, named ECP001, which include two types, one is a mixed protein antigen named ECP001m, the other is a fusion expression protein antigen named ECP001f, as candidates for protein subunit vaccines. were prepared by constructing one novel subunit vaccine by mixing or fusing the three proteins and combining them with aluminum hydroxide adjuvant, and the immunogenicity and protective properties of the vaccine was evaluated in mice. The results showed that ECP001 stimulated mice to produce high titre levels of IgG, IgG1 and IgG2a antibodies; meanwhile, high levels of IFN-γ and a broad range of specific cytokines were secreted by mouse splenocytes; in addition, ECP001 inhibited the proliferation of Mycobacterium tuberculosis in vitro with a capacity comparable to that of BCG. It can be concluded that ECP001 is a novel effective multicomponent subunit vaccine candidate with potential as BCG Initial Immunisation-ECP001 Booster Immunisation or therapeutic vaccine for M. tuberculosis infection.

Immunogenicity and efficacy analyses of EPC002, ECA006, and EPCP009 protein subunit combinations as tuberculosis vaccine candidates.

Tuberculosis (TB) is the leading cause of death from infectious diseases worldwide, and developing a new TB vaccine is a priority for TB control. Combining multiple immunodominant antigens to form a novel multicomponent vaccine with broad-spectrum antigens to induce protective immune responses is a trend in TB vaccine development. In this study, we used T-cell epitope-rich protein subunits to construct three antigenic combinations: EPC002, ECA006, and EPCP009. Fusion expression of purified protein EPC002f (CFP-10-linker-ESAT-6-linker-nPPE18), ECA006f (CFP-10-linker-ESAT-6-linker-Ag85B), and EPCP009f (CFP-10-linker-ESAT-6-linker-nPPE18-linker-nPstS1) and recombinant purified protein mixtures EPC002m (mix of CFP-10, ESAT-6, and nPPE18), ECA006m (mix of CFP-10, ESAT-6, and Ag85B), and EPCP009m (mix of CFP-10, ESAT-6, nPPE18, and nPstS1) were used as antigens, formulated with alum adjuvant, and the immunogenicity and efficacy were analyzed using immunity experiments with BALB/c mice. All protein-immunized groups elicited higher levels of humoral immunity, including IgG and IgG1. The IgG2a/IgG1 ratio of the EPCP009m-immunized group was the highest, followed by that of the EPCP009f-immunized group, which was significantly higher than the ratios of the other four groups. The multiplex microsphere-based cytokine immunoassay revealed that EPCP009f and EPCP009m induced the production of a wider range of cytokines than EPC002f, EPC002m, ECA006f, and ECA006m, which included Th1-type (IL-2, IFN-γ, TNF-α), Th2-type (IL-4, IL-6, IL-10), Th17-type (IL-17), and other proinflammatory cytokines (GM-CSF, IL-12). The enzyme-linked immunospot assays demonstrated that the EPCP009f- and EPCP009m-immunized groups had significantly higher amounts of IFN-γ than the other four groups. The in vitro mycobacterial growth inhibition assay demonstrated that EPCP009m inhibited Mycobacterium tuberculosis (Mtb) growth most strongly, followed by EPCP009f, which was significantly better than that of the other four vaccine candidates. These results indicated that EPCP009m containing four immunodominant antigens exhibited better immunogenicity and Mtb growth inhibition in vitro and may be a promising candidate vaccine for the control of TB.

A Multistage Antigen Complex Epera013 Promotes Efficient and Comprehensive Immune Responses in BALB/c Mice.

Tuberculosis (TB) remains a serious global health problem. Despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, the primary factor for the TB pandemic and deaths is adult TB, which mainly result from endogenous reactivation of latent Mycobacterium tuberculosis (MTB) infection. Improved new TB vaccines with eligible safety and long-lasting protective efficacy remains a crucial step toward the prevention and control of TB. In this study, five immunodominant antigens, including three early secreted antigens and two latency associated antigens, were used to construct a single recombinant fusion protein (Epera013f) and a protein mixture (Epera013m). When formulated with aluminum adjuvant, the two subunit vaccines Epera013m and Epera013f were administered to BALB/c mice. The humoral immune responses, cellular responses and MTB growth inhibiting capacity elicited after Epera013m and Epera013f immunization were analyzed. In the present study, we demonstrated that both the Epera013f and Epera013m were capable of inducing a considerable immune response and protective efficacy against H37Rv infection compared with BCG groups. In addition, Epera013f generated a more comprehensive and balanced immune status, including Th1, Th2 and innate immune response, over Epera013f and BCG. The multistage antigen complex Epera013f possesses considerable immunogenicity and protective efficacy against MTB infection ex vivo indicating its potential and promising applications in further TB vaccine development.

Development of an animal model for rosacea­like skin lesions caused by Demodex.

To develop an animal model of rosacea-like skin lesions caused by Demodex mites, a suspension of Demodex mites was injected into the skin of Japanese rabbits. The pathology of the skin lesion was assessed using H&E staining after 4 weeks of modeling. The skin lesions observed after 4 weeks were further treated with the recombinant bovine basic fibroblast growth factor (rbFGF) gel. Untreated lesions in the same rabbit were considered as the blank control. Erythema papules were observed in the model rabbit skin and could be observed most clearly in the 2nd week. Lumpy foreign bodies, telangiectasia and granuloma-like structure were observed in the model rabbit in the 1st, 2nd, and 3rd weeks, respectively. An organized granuloma-like structure was observed in the 4th week. The color of the skin lesions became lighter than that of the self-control after 4 weeks of rbFGF treatment. In conclusion, the model of Demodex-induced rosacea-like skin lesions can be developed through intradermal injection of suspension of Demodex mites into Japanese rabbits. The model can mimic the phenotype of skin lesions and histopathological manifestations in the Demodex mite-positive patient with rosacea.

High Immunogenicity of a T-Cell Epitope-Rich Recombinant Protein Rv1566c-444 From Mycobacterium tuberculosis in Immunized BALB/c Mice, Despite Its Low Diagnostic Sensitivity.

The discovery of immunodominant antigens is of great significance for the development of new especially sensitive diagnostic reagents and effective vaccines in controlling tuberculosis (TB). In the present study, we targeted the T-Cell epitope-rich fragment (nucleotide position 109-552) of Rv1566c from Mycobacterium tuberculosis (MTB) and got a recombinant protein Rv1566c-444 and the full-length protein Rv1566c with Escherichia coli expression system, then compared their performances for TB diagnosis and immunogenicity in a mouse model. The results showed that Rv1566c-444 had similar sensitivity with Rv1566c (44.44% Vs 30.56%) but lower sensitivity than ESAT-6&CFP-10&Rv3615c (44.4% Vs. 94.4%) contained in a commercial kit for distinguishing TB patients from healthy donors. In immunized BALB/c mice, Rv1566c-444 elicited stronger T-helper 1 (Th1) cellular immune response over Rv1566c with higher levels of Th1 cytokine IFN-γ and IFN-γ/IL-4 expression ratio by ELISA; more importantly, with a higher proliferation of CD4+ T cells and a higher proportion of CD4+ TNF-α+ T cells with flow cytometry. Rv1566c-444 also induced a higher level of IL-6 by ELISA and a higher proportion of Rv1566c-444-specific CD8+ T cells and a lower proportion of CD8 + IL-4 + T cells by flow cytometry compared with the Rv1566c group. Moreover, the Rv1566c-444 group showed a high IgG secretion level and the same type of CD4+ Th cell immune response (both IgG1/IgG2a >1) as its parental protein group. Our results showed the potential of the recombinant protein Rv1566c-444 enriched with T-Cell epitopes from Rv1566c as a host T cell response measuring biomarker for TB diagnosis and support further evaluation of Rv1566c-444 as vaccine antigen against MTB challenge in animal models in the form of protein mixture or fusion protein.

Construction and immunogenicity of a T cell epitope-based subunit vaccine candidate against Mycobacterium tuberculosis.

Despite antibiotic treatment and Bacille Calmette-Guérin (BCG) vaccination, Mycobacterium tuberculosis remains a major public health burden in most developing countries. Therefore, developing an improved vaccine is high priority. In this study, we cloned the genes of the immunodominant antigen of M. tuberculosis viz. its 38-kDa antigen (Pst homolog) (Rv0934, PstS1), and its T cell epitopes (amino acid [aa]169-405 and [aa]802-1119), which we termed PstS1p. Prokaryotic expression showed that the two recombinant proteins were mainly in the form of inclusion bodies. We also evaluated the immunity and immunogenicity of PstS1 and PstS1p. Both PstS1 and its T cell epitopes elicited significantly higher antigen-specific immunoglobulin G (IgG) antibodies in mouse serum, indicating that they enhanced antibody response. They also elicited the T helper 1 (Th1)-type response and promoted CD4+ T cell proliferation. Compared to PstS1, PstS1p promoted stronger cell-mediated immune response. These data indicate that PstS1p is highly immunogenic in mice, and may be a promising candidate vaccine for controlling tuberculosis.

Chlamydia trachomatis glycogen synthase promotes MAPK-mediated proinflammatory cytokine production via TLR2/TLR4 in THP-1 cells.

AIMS: To investigate the roles and mechanisms of C. trachomatis glycogen synthase (GlgA) in regulating the inflammatory response in THP-1 cells. MAIN METHODS: In this work, after THP-1 cells were stimulated with GlgA, transcript and protein expression levels were measured by qRT-PCR and ELISA, respectively. Western blotting and immunofluorescence were used to determine the signaling pathway involved in the inflammatory mechanism. KEY FINDINGS: GlgA elicited the expression of interleukin-8 (IL-8), interleukin-1beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) in THP-1 cells, and the blockade of TLR2 and TLR4 signaling abrogated the induction of IL-8, TNF-α and IL-1ß expression. Similarly, IL-8, IL-1ß and TNF-α secretion was reduced by transfection with a dominant negative plasmid (pDeNyhMyD88). Moreover, Western blotting and immunofluorescence experiments further validated that MAPKs and NF-кB signaling are involved in the transcription and translation of these cytokines. Treatment of the cells with ERK and JNK inhibitors dramatically attenuated the induction of IL-8, IL-1ß and TNF-α. SIGNIFICANCE: These results suggest that GlgA contributes to inflammation during C. trachomatis infection via the TLR2, TLR4 and MAPK/NF-кB pathways, which may enhance our understanding of the pathogenesis of C. trachomatis.

Demodex-induced Lupus miliaris disseminatus faciei: A case report.

RATIONALE: Lupus miliaris disseminatus faciei (LMDF) is an inflammatory granulomatous skin disease without a clear etiology that frequently involves the middle area of the face and the upper eyelids. Pathological features of the disease include caseation necrosis and epithelioid granuloma. Consensus treatment for LMDF is currently unavailable. PATIENT CONCERNS: A 47-year-old Chinese female patient who presented with facial pruritic, erythematous papules 8 months before this study. She was diagnosed with skin tuberculosis at another hospital and given antituberculosis medication. However, the treatment was not efficacious. DIAGNOSES: In this study, the diagnosis of Demodex-induced LMDF was made by a dermatologist according to physical examination, skin biopsy pathology, and microscopic examination. INTERVENTIONS: The patient was given ornidazole tablets (500 mg twice a day) and recombinant bovine basic fibroblast growth factor gel (0.2 g/cm twice a day) for an 8-week period. OUTCOMES: Eight weeks after the treatment, the facial erythematous papules were improved, and no new skin lesions were observed. The patient showed no signs of recurrence during the 6-month follow-up. LESSONS SUBSECTIONS: This case showed that ornidazole combined with recombinant bovine basic fibroblast growth factor gel might be useful in treatment of Demodex-induced LMDF. In addition, the results suggested that pathological caseation necrosis was caused by a series of inflammatory and immune responses to Demodex infection.

Improved telangiectasia and reduced recurrence rate of rosacea after treatment with 540 nm-wavelength intense pulsed light: A prospective randomized controlled trial with a 2-year follow-up.

The aim of the present study was to evaluate the clinical efficacy and safety of 540 nm-wavelength intense pulsed light (IPL) for the treatment of telangiectasia in late-stage rosacea. Between July 2013 and January 2016, patients with rosacea who tested positive for Demodex folliculorum were recruited. Patients received anti-mite therapy and were then randomly apportioned to receive either three 540 nm-IPL treatments at 4-week intervals (IPL group), or no treatment (control group). Telangiectasia was assessed by the same clinician at baseline and at follow-up intervals over 2 years, where ≥90% clearance of telangiectasia was considered to indicate effective treatment. The rates of effective treatment, improvement (≥30% clearance) and recurrence (original or neo-location) were compared in both groups. After 33 patients were lost during follow-up, the IPL and control groups were comprised of 107 and 120 patients for the final analysis, respectively. The rates of effective treatment and total efficacy in the IPL group (66.36 and 95.33%, respectively) were found to be significantly higher compared with those of the control group (0 and 30.83%, respectively). By contrast, the rates of recurrence were found to be lower in the IPL group (8.41%) compared with the control group (48.33%). Redness-to-blisters associated with IPL treatment (9.7% of analyzed patients) subsided within one week and hyperpigmentation (1.9%) within 3 months. To conclude, treatment with 540 nm-IPL improved facial telangiectasia in late-stage rosacea that remained after sequential anti-mite therapy and effectively reduced the recurrence of rosacea. The present study was registered into the Chinese Clinical Trial Registry under the title 'Sequential therapy for mites folliculitis' (Trial registration number: ChiCTR-IPR-15006451; approved May 27, 2015).

Identification of HeLa cell proteins that interact with Chlamydia trachomatis glycogen synthase using yeast two­hybrid assays.

Chlamydia trachomatis (C. trachomatis) is the leading cause of bacterial sexually transmitted diseases and infectious diseases that cause blindness. The pathophysiology of chlamydial infections is poorly understood, but secreted proteins have emerged as key virulence factors. C. trachomatis glycogen synthase (GlgA) is a chlamydial secretory protein, which localizes in the lumen of chlamydial inclusion bodies and the cytosol of host cells. In order to improve understanding of the roles of GlgA in chlamydial pathogenesis, four proteins that interact with GlgA, Homo sapiens CXXC finger protein 1, prohibitin (PHB), gelsolin­like actin­capping protein and apolipoprotein A­I binding protein were identified using yeast two­hybrid assays. The functions of these proteins are complex, and preliminary results suggested that PHB interacts with GlgA. However, further studies are required to determine the specific interactions of these proteins with GlgA. The findings of the present study may provide a direction and foundation for future studies focusing on the mechanism of GlgA in C. trachomatis infection.

Effect of recombinant bovine basic fibroblast growth factor gel on repair of rosacea skin lesions: A randomized, single-blind and vehicle-controlled study.

The aim of the present study was to assess the effect of topical use of recombinant bovine basic fibroblast growth factor (rbFGF) gel on the repair of facial skin lesions in patients with rosacea. In the present single-blind study, a total of 1,287 patients with Demodex mite-induced rosacea who received treatment with ornidazole tablets were randomized to rbFGF gel treatment group (n=651) or control group (n=636) without revealing the group identity. Patients in the treatment group were treated with topical application of rbFGF gel over the skin lesions (0.2 g/cm2) for up to 8 weeks, whereas patients in the control group received gel vehicle treatment unless ulceration occurred. Skin lesions of all patients were scored prior to and following treatment with rbFGF gel and subjected to histological analysis. All patients were followed up for 6 months. Significant improvement in the total effective rates for erythema, papules, desquamation and dryness were observed in the rbFGF treatment group. At the end of the 2, 4 and 6 months of follow-up, the total effective rates for patients in the treatment group were significantly higher than those in the control group (81.67 vs. 28.84%; 85.11 vs. 40.81%, and 96.56 vs. 55.82%, respectively). Following treatment for 6 months, none of the patients in the rbFGF group exhibited ulceration or scar formation. In the control group, 61% of patients experienced exacerbation of skin lesions, of which, 12% exhibited ulceration and were treated with rbFGF gel to prevent scar formation. Histological analysis revealed gradual reduction in epidermal hyperplasia and resolution of dermal edema in skin lesions treated with rbFGF gel. In conclusion, rbFGF gel may improve the repair of facial rosacea skin lesions in patients treated with anti-Demodex.

Vaccination with MIP or Pgp3 induces cross-serovar protection against chlamydial genital tract infection in mice.

Previously we reported that recombinant Chlamydia muridarum macrophage infectivity potentiator (MIP) provided partial protection against C. muridarum genital tract infection in mice. On the other hand, Chlamydia trachomatis plasmid encoded Pgp3could induce the protection against C. muridarum air way infection. This study aimed to evaluate the immunogenicity of MIP and Pgp3 from C. trachomatis serovar D and further investigate whether MIP and Pgp3 provide cross-serovar protection against C. muridarum genital tract infection in mice. Our results showed that vaccination by any regimen, including MIP alone, Pgp3 alone or MIP plus Pgp3, induced specific serum antibody production and Th1-dominant cellular responses in mice. Live chlamydial shedding from the vaginal and inflammatory pathologies in the oviduct markedly reduced. However, MIP + Pgp3 vaccination did not provide better protection than the single immunization. In conclusion, this study demonstrated that both MIP and Pgp3 can induce cross-serovar protective against chlamydial genital tract infection, and provided the guide for the development of optimal multisubunit vaccines against C. trachomatis infection.

Macropinocytosis activated by oncogenic Dbl enables specific targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells.

BACKGROUND: Successful implementation of gene therapy heavily relies on efficiently delivering genetic materials and specific targeting into cells. Oncogene-driven endocytosis stimulates nutrient uptake and also develops an endocytosis-mediated defense against therapeutic agents. Cell-penetrating peptides, typically HIV-Tat, are well known for efficient delivery of nucleic acid drugs but lack targeting specificity. Various passive targeting strategies were pursued to enhance the tumor targeting efficiency; however, they are still limited by complicated cellular endocytosis routes and the heterogeneity of cancer types. METHODS: Tat/pDNA complexes were noncovalently compacted and their physiochemical properties were determined. The siRNA pool and pLV-RNAi-GFP lentivirus were used to knock down dbl oncogene (originally isolated from diffuse B-cell lymphoma) expression, and its overexpression was performed by plasmid transient transfection. The cellular uptake of fluorescent ligands was quantified by confocal imaging and flow cytometry analysis. The transgene efficiency was determined by the Luciferase expression assay. Rho GTPase activation was checked by the GST-Rho GTPase-binding domain pull-down assay. RESULTS: pGL3 plasmid DNA was noncovalently compacted with the Tat peptide into nano-size complexes at high N/P ratios. Macropinocytosis, a clathrin- and caveolin-independent endocytosis process, was shown to contribute to the uptake of middle-sized (∼600 nm) Tat/pGL3 complexes. Cell-type-specific variation in macropinocytosis was essentially controlled by the action of the Dbl oncogene. Onco-Dbl presentation constantly induced a high level of macropinocytosis activity in ovarian cancer cells. Onco-Dbl overexpression hyperstimulated macropinocytosis enhancement in cells mainly through actin cytoskeleton reorganization mediated by the PH domain and Rac1 activation. The Dbl-driven Rho GTPase signaling collectively determined the cell-type-specific macropinocytosis phenotype. CONCLUSION: Such an aspect can be exploited to selectively confer targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells. Our work provides a novel alternative for targeted delivery of cell-penetrating peptide-based nucleic acid drugs into certain tumor types if specific endocytosis pathways are used.

Treatment of mites folliculitis with an ornidazole-based sequential therapy: A randomized trial.

OBJECTIVE: Treatment of Demodex infestations is often inadequate and associated with low effective rate. We sought to evaluate the efficacy of an ornidazole-based sequential therapy for mites folliculitis treatment. METHODS: Two-hundred patients with mites folliculitis were sequentially treated with either an ornidazole- or metronidazole-based regimen. Sebum cutaneum was extruded from the sebaceous glands of each patient's nose and the presence of Demodex mites were examined by light microscopy. The clinical manifestations of relapse of mites folliculitis were recorded and the subjects were followed up at 2, 4, 8, and 12 weeks post-treatment. RESULTS: Patients treated with the ornidazole-based regimen showed an overall effective rate of 94.0%. Additionally, at the 2, 4, 8, and 12-week follow-up, these patients had significantly lower rates of Demodex mite relapse and new lesion occurrence compared with patients treated with the metronidazole-based regimen (P < 0.05). CONCLUSION: Sequential therapy using ornidazole, betamethasone, and recombinant bovine basic fibroblast growth factor (rbFGF) gel is highly effective for treating mites folliculitis.