Innate Inspiration: Antifungal Peptides and Other Immunotherapeutics From the Host Immune Response.

The purpose of this review is to describe antifungal therapeutic candidates in preclinical and clinical development derived from, or directly influenced by, the immune system, with a specific focus on antimicrobial peptides (AMP). Although the focus of this review is AMP with direct antimicrobial effects on fungi, we will also discuss compounds with direct antifungal activity, including monoclonal antibodies (mAb), as well as immunomodulatory molecules that can enhance the immune response to fungal infection, including immunomodulatory AMP, vaccines, checkpoint inhibitors, interferon and colony stimulating factors as well as immune cell therapies. The focus of this manuscript will be a non-exhaustive review of antifungal compounds in preclinical and clinical development that are based on the principles of immunology and the authors acknowledge the incredible amount of in vitro and in vivo work that has been conducted to develop such therapeutic candidates.

Ultraviolet A light effectively reduces bacteria and viruses including coronavirus.

Antimicrobial-resistant and novel pathogens continue to emerge, outpacing efforts to contain and treat them. Therefore, there is a crucial need for safe and effective therapies. Ultraviolet-A (UVA) phototherapy is FDA-approved for several dermatological diseases but not for internal applications. We investigated UVA effects on human cells in vitro, mouse colonic tissue in vivo, and UVA efficacy against bacteria, yeast, coxsackievirus group B and coronavirus-229E. Several pathogens and virally transfected human cells were exposed to a series of specific UVA exposure regimens. HeLa, alveolar and primary human tracheal epithelial cell viability was assessed after UVA exposure, and 8-Oxo-2'-deoxyguanosine was measured as an oxidative DNA damage marker. Furthermore, wild-type mice were exposed to intracolonic UVA as an in vivo model to assess safety of internal UVA exposure. Controlled UVA exposure yielded significant reductions in Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Clostridioides difficile, Streptococcus pyogenes, Staphylococcus epidermidis, Proteus mirabilis and Candida albicans. UVA-treated coxsackievirus-transfected HeLa cells exhibited significantly increased cell survival compared to controls. UVA-treated coronavirus-229E-transfected tracheal cells exhibited significant coronavirus spike protein reduction, increased mitochondrial antiviral-signaling protein and decreased coronavirus-229E-induced cell death. Specific controlled UVA exposure had no significant effect on growth or 8-Oxo-2'-deoxyguanosine levels in three types of human cells. Single or repeated in vivo intraluminal UVA exposure produced no discernible endoscopic, histologic or dysplastic changes in mice. These findings suggest that, under specific conditions, UVA reduces various pathogens including coronavirus-229E, and may provide a safe and effective treatment for infectious diseases of internal viscera. Clinical studies are warranted to further elucidate the safety and efficacy of UVA in humans.

Alternative therapy to manage otitis media caused by multidrug-resistant fungi.

During the last few decades, the increase in the incidence of multidrug-resistant (MDR) fungal infections has become an emerging threat to public health. Therefore, it is important to illuminate the usage of alternative therapy to treat MDR fungal infection. This study was carried out to elucidate the usage of plant extract and essential oil, either alone or with other antifungal drugs to treat otitis media caused by MDR fungi. Medicinal plant is a safe and cheap source when compared with chemical antifungal drugs. Twenty-one fungal isolates out of 104 ear swabs from patients suffering from otitis media were characterized using both phenotypic and genotypic methods. The antibiogram typing was used to determine the MDR isolates. The sensitivity of MDR fungal isolates was tested against several plant extracts and essential oils, either alone or with other antifungal drugs. Thyme oil and clove extracts proved to have synergistic effects suggesting their use in the treatment of fungal infections, especially otitis media caused by MDR fungi. The ultrastructure of MDR fungal isolates exhibited a complete destruction post exposure to the used materials when observed under the transmission microscope (TEM). Thyme oil and clove extract were found to be the most effective agents against MDR fungal isolates and they constitute a promising tool for the management of fungal infection causing the otitis media.

[Effect observation of cleaning up the external auditory canal by otoendoscope combined with Clotrimazole Ointment in the treatment of pregnancy with otitis externa mycotica].

Objective:To investigate the clinical effect of cleaning up the external auditory canal under otoendoscope combined with Clotrimazole Ointment in the treatment of pregnancy with otitis externa mycotica.Method:From May 2015 to May 2017,16 cases of pregnant patients(19 ears)with otitis externa mycotica were divided into two groups:pure cleaning up group and cleaning up combined with medication group.In the pure cleaning up group,external auditory canal were only cleaned up under otoendoscope conventionally in 9 patients(11 ears),while in the cleaning up combined with medication group,Clotrimazole Ointment was topically applied after cleaning up the external auditory canal under otoendoscope in 7 patients(8 ears).After treatment of 2 weeks,the clinical curative effect,adverse reaction and average time interval to take effect were compared at the end of treatment.Result:The total effective rate(100%)in cleaning up combined with medication group's was significantly better than that in pure cleaning up group's(81.81%)(P<0.05);The average time interval to take effect in cleaning up combined with medication groupwas significantly shorter than that in pure cleaning up group's[(2.71±0.70)d vs(5.40±1.96)d,P<0.05].After the two-week treatment,there was a four-week follow-up.Only one patient in pure cleaning up group relapsed.After two-week treatment by Clotrimazole Ointment,this patient was cured.Conclusion:Cleaning up the external auditory canal under otoendoscope combined with Clotrimazole Ointment is effective and safe for the treatment of otitis externa mycotica in pregnant women.The addition of topical application of Clotrimazole Ointment further improve the therapeutic efficacy,as compared to the conventional method of cleaning up the external auditory canal under otoendoscope.We suggest clinical application of this method.

Light treatments of nail fungal infections.

Nail fungal infections are notoriously persistent and difficult to treat which can lead to severe health impacts, particularly in the immunocompromized. Current antifungal treatments, including systemic and topical drugs, are prolonged and do not effectively provide a complete cure. Severe side effects are also associated with systemic antifungals, such as hepatotoxicity. Light treatments of onychomycosis are an emerging therapy that has localized photodynamic, photothermal or photoablative action. These treatments have shown to be an effective alternative to traditional antifungal remedies with comparable or better cure rates achieved in shorter times and without systemic side effects. This report reviews significant clinical and experimental studies in the field, highlighting mechanisms of action and major effects related to light therapy; in particular, the impact of light on fungal genetics.

Antimicrobial blue light inactivation of pathogenic microbes: State of the art.

As an innovative non-antibiotic approach, antimicrobial blue light in the spectrum of 400-470nm has demonstrated its intrinsic antimicrobial properties resulting from the presence of endogenous photosensitizing chromophores in pathogenic microbes and, subsequently, its promise as a counteracter of antibiotic resistance. Since we published our last review of antimicrobial blue light in 2012, there have been a substantial number of new studies reported in this area. Here we provide an updated overview of the findings from the new studies over the past 5 years, including the efficacy of antimicrobial blue light inactivation of different microbes, its mechanism of action, synergism of antimicrobial blue light with other angents, its effect on host cells and tissues, the potential development of resistance to antimicrobial blue light by microbes, and a novel interstitial delivery approach of antimicrobial blue light. The potential new applications of antimicrobial blue light are also discussed.

Topical treatment of mycotic rhinitis-rhinosinusitis in dogs with meticulous debridement and 1% clotrimazole cream: 64 cases (2007-2014).

OBJECTIVE To evaluate outcomes for dogs with mycotic rhinitis-rhinosinusitis (MRR) treated by meticulous debridement and topical application of 1% clotrimazole cream and investigate potential prognostic factors that could help predict whether 1 or multiple treatments would be needed for clinical resolution of the condition. DESIGN Retrospective case series. ANIMALS 64 dogs. PROCEDURES Medical records were reviewed to identify dogs treated for MRR by meticulous debridement and topical application of 1% clotrimazole cream. Signalment, clinical signs, previous treatments, CT findings, presence of unilateral or bilateral disease, predisposing factors, number and type of treatments, and complications were recorded. Outcome information was obtained from records or by telephone interview with owners. Association of selected factors with the number of treatments needed for clinical resolution was evaluated. RESULTS Clotrimazole was instilled via the trephination site (n = 42) or under endoscopic guidance (22). Thirteen dogs underwent a 5-minute flush with 1% clotrimazole solution prior to cream application, and 34 received adjunctive oral itraconazole treatment. The MRR was deemed resolved in 58 dogs, and clinical signs persisted in 1 dog. Five dogs died (2 of causes unrelated to MRR) ≤ 1 month after treatment. The first treatment was successful in 42 of 62 (68%) dogs; overall success rate was 58 of 62 (94%). No prognostic factors for the number of treatments needed to provide clinical resolution were identified. Seven dogs with reinfection were successfully retreated. CONCLUSIONS AND CLINICAL RELEVANCE Topical treatment by meticulous debridement and 1% clotrimazole cream application had results similar to or better than those described in other studies of dogs with MRR. Trephination or adjunctive itraconazole treatment did not influence the number of treatments needed for a successful outcome.

Medical diagnostics for indoor mold exposure.

In April 2016, the German Society of Hygiene, Environmental Medicine and Preventative Medicine (Gesellschaft für Hygiene, Umweltmedizin und Präventivmedizin (GHUP)) together with other scientific medical societies, German and Austrian medical societies, physician unions and experts has provided an AWMF (Association of the Scientific Medical Societies) guideline 'Medical diagnostics for indoor mold exposure'. This guideline shall help physicians to advise and treat patients exposed indoors to mold. Indoor mold growth is a potential health risk, even without a quantitative and/or causal association between the occurrence of individual mold species and health effects. Apart from the allergic bronchopulmonary aspergillosis (ABPA) and the mycoses caused by mold, there is only sufficient evidence for the following associations between moisture/mold damages and different health effects: Allergic respiratory diseases, asthma (manifestation, progression, exacerbation), allergic rhinitis, exogenous allergic alveolitis and respiratory tract infections/bronchitis. In comparison to other environmental allergens, the sensitizing potential of molds is estimated to be low. Recent studies show a prevalence of sensitization of 3-10% in the total population of Europe. The evidence for associations to mucous membrane irritation and atopic eczema (manifestation, progression, exacerbation) is classified as limited or suspected. Inadequate or insufficient evidence for an association is given for COPD, acute idiopathic pulmonary hemorrhage in children, rheumatism/arthritis, sarcoidosis, and cancer. The risk of infections from indoor molds is low for healthy individuals. Only molds that are capable to form toxins can cause intoxications. The environmental and growth conditions and especially the substrate determine whether toxin formation occurs, but indoor air concentrations are always very low. In the case of indoor moisture/mold damages, everyone can be affected by odor effects and/or impairment of well-being. Predisposing factors for odor effects can be given by genetic and hormonal influences, imprinting, context and adaptation effects. Predisposing factors for impairment of well-being are environmental concerns, anxieties, conditioning and attributions as well as a variety of diseases. Risk groups that must be protected are patients with immunosuppression and with mucoviscidosis (cystic fibrosis) with regard to infections and individuals with mucoviscidosis and asthma with regard to allergies. If an association between mold exposure and health effects is suspected, the medical diagnosis includes medical history, physical examination, conventional allergy diagnosis, and if indicated, provocation tests. For the treatment of mold infections, it is referred to the AWMF guidelines for diagnosis and treatment of invasive Aspergillus infections. Regarding mycotoxins, there are currently no validated test methods that could be used in clinical diagnostics. From the perspective of preventive medicine, it is important that mold damages cannot be tolerated in indoor environments.

Efficacy of medical grade honey in the management of canine otitis externa - a pilot study.

BACKGROUND: The high prevalence of antimicrobial resistance within otic pathogens has created a need for alternative therapies of otitis externa (OE). Evidence suggests that medical grade honey (MGH) may be effective against drug-resistant pathogens. HYPOTHESIS/OBJECTIVES: The efficacy of a commercial MGH compound was assessed in an open clinical trial. We hypothesized that it would be an effective alternative to conventional treatments. ANIMALS: Client-owned dogs (n = 15) with a confirmed diagnosis of infectious OE were enrolled in this pilot study. METHODS: Dogs were prescribed MGH (1 mL daily per ear) until cure was achieved or for a maximum of 21 d. Evaluation was based on weekly clinical scores, cytological progression and owner assessments of pruritus. Swab samples were submitted for culture and susceptibility testing. MGH was tested for biocidal activity against the bacterial isolates. RESULTS: Medical grade honey promoted rapid clinical progress, with 70% of dogs achieving clinical cure between days 7 and 14 and over 90% having resolved by Day 21. There was a decrease in clinical scores throughout the duration of the trial (P < 0.001) and owner-assessed pruritus also decreased significantly (P < 0.05). In vitro assays of the biocidal activity of MGH showed activity against all bacterial isolates, including meticillin-resistant strains of Staphylococcus pseudintermedius (MRSP) and other species of drug-resistant bacteria. CONCLUSION AND CLINICAL IMPORTANCE: Medical grade honey was successful in both clinical and laboratory settings, thus demonstrating its potential of becoming an alternative treatment for canine OE.

Maggot therapy in treatment of a complex hand injury complicated by mycotic infection.

Complex injuries of the hand remain a therapeutic challenge for surgeons. We present the case of a male who suffered a devastating injury of the hand caused by a conveyor belt. The patient developed a progressive Absidia corymbifera infection of the affected soft tissues. Initial treatments with serial surgical debridement and topical and intravenous itraconazole were unsuccessful in eliminating the infection. We decided to use maggot debridement therapy in a new special design to debride all necrotic, devitalized tissue and preserve only healthy tissue and functioning structures. This maneuverer followed by negative pressure therapy allowed progressive healing. In such complex hand injuries, maggot debridement combined with negative pressure therapy could be considered to achieve effective and considerable results, although future functional morbidity may occur (Fig. 4, Ref. 18).

Haploidentical Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide in a Patient with Chronic Granulomatous Disease and Active Infection: A First Report.

PURPOSE: We describe haploidentical hematopoietic cell transplantation (HCT) with high-dose post-transplant cyclophosphamide (PTCy) in a boy with x-linked chronic granulomatous disease (CGD). METHODS: A persistent and life-threatening fungal infection was the indication for HSCT. Non-myeloablative conditioning with PTCy (50 mg/kg days 3 and 4) was used in the absence of fully matched donors. RESULTS: Engraftment occurred on day 24. The patient experienced Grade 2 graft-versus-host disease of the skin and gastrointestinal tract and CMV infection, both of which were controlled. Chimerism was 100 % at days 30 and 6 months. Cessation of antifungal therapy was consistent with cure of the infection. CONCLUSIONS: Haploidentical HCT with high-dose PTCy for CGD is feasible and succeeded even in the context of active infection.

Fungal Infections of the Spine.

STUDY DESIGN: Review of the literature. OBJECTIVE: To retrospectively examine the frequency of published fungal infections by species and the treatment algorithms used to eradicate the disease. SUMMARY OF BACKGROUND DATA: Fungal infections of the spine present unique challenges to the modern multispecialty treatment team. Although rare in comparison with bacterial infections, fungal infections have been increasing in incidence over the past several decades. Evidences-based practice is limited to referencing smaller case series. METHODS: MEDLINE, Scopus, and EMBASE searches were carried out by one of the authors as well as by the research desk at the University of Miami/Calder Memorial Library. We included peer-reviewed articles published between 1948 and September 2010; case reports, series, and reviews were all examined and compiled into a database. RESULTS: A total of 130 articles, representing 157 cases, were included in the review. Aspergillus (60 cases, 38.2% of the total) and Candida species (36 cases, 22.9% of the total) were the 2 most common organisms. Surgery was associated with a greater survival rate than medical management alone in patients with Aspergillus (26.9% mortality in surgical patients; 60% in medically treated patients) and Candida (0% vs. 28.6%). Overall mortality was 19.3%. The overall recurrence rate was 7.4%. Amphotericin use was associated with a higher mortality rate than azoles. CONCLUSION: Aspergillus is the most common published pathogen in fungal infections of the spine. Recent publications depicting the use of newer antifungal medications such as azoles report higher survival rates. Surgically treated patients in combination with antifungal therapy showed highest frequencies of patient survival in Aspergillus and Candida infections. LEVEL OF EVIDENCE: 3.

Postoperative infections after cytoreductive surgery and HIPEC for peritoneal carcinomatosis: proposal and results from a prospective protocol study of prevention, surveillance and treatment.

The incidence of infectious complications due to several contributory causes is particularly elevated and life-threatening in patients undergoing peritonectomy and HIPEC procedure for peritoneal carcinomatosis. Following a previous experience, we started a prospective protocol study of preoperative screening, perioperative prophylaxis and postoperative surveillance and treatment. A total of 111 patients with peritoneal carcinomatosis of various origin underwent CRS with HIPEC between April 2004 and December 2012. The group was divided into a pilot group of 30 patients (04/04 to 05/08) and a main group of 81 patients (06/08 to 12/12). Overall postoperative morbidity rate was 44%, with 35.8% of symptomatic infections. No post-operative mortality was observed. Microorganisms were isolated in 24 patients (80.0%) in the first group and 54 (66.7%) in the second. They were symptomatic in 18 cases (75.0%) and 25 (46.3%) cases respectively. In addition, 7 invasive candidosis were recorded (25.9%). Colon resection (P = 0.01) and duration of surgery (P = 0.0008) were associated with infection at logistic regression model. Concerning symptomatic infections, only Infection Risk Index (P = 0.009) showed significance at multivariate analysis. Despite a significant incidence of infectious complications, establishment of a prevention, surveillance and treatment protocol lead to a zero mortality rate in the observed patients of our experience. Owing to the obtained results, we suggest the use of a standardized protocol for the prevention, monitoring and treatment in all patients enrolled for cytoreductive surgery and HIPEC.

A review of the mechanism of injury and treatment approaches for illness resulting from exposure to water-damaged buildings, mold, and mycotoxins.

Physicians are increasingly being asked to diagnose and treat people made ill by exposure to water-damaged environments, mold, and mycotoxins. In addition to avoidance of further exposure to these environments and to items contaminated by these environments, a number of approaches have been used to help persons affected by exposure to restore their health. Illness results from a combination of factors present in water-damaged indoor environments including, mold spores and hyphal fragments, mycotoxins, bacteria, bacterial endotoxins, and cell wall components as well as other factors. Mechanisms of illness include inflammation, oxidative stress, toxicity, infection, allergy, and irritant effects of exposure. This paper reviews the scientific literature as it relates to commonly used treatments such as glutathione, antioxidants, antifungals, and sequestering agents such as cholestyramine, charcoal, clay and chlorella, antioxidants, probiotics, and induced sweating.

Corneal ulceration in a LASIK patient due to vitamin a deficiency after bariatric surgery.

PURPOSE: To review the case of a 41-year-old woman who underwent bariatric surgery in 2000. She subsequently underwent laser in situ keratomileusis (LASIK) surgery in 2008 and complained of dry eye since the LASIK surgery. In November 2010, she was diagnosed with a corneal melt and was treated with aggressive lubrication, followed by eventual amniotic membrane placement and a tarsorrhaphy. She then presented for consultation at the Bascom Palmer Eye Institute when she developed a corneal infiltrate. She was diagnosed with fungal keratitis with corneal xerosis. At that time, vitamin A levels were measured and were less than 2 µ/dL. The patient admitted noncompliance with nutritional supplements. METHODS: Case report. RESULTS: The patient was treated with aggressive lubrication and natamycin. Vitamin supplements were restarted, and the patient experienced dramatic improvement in symptoms with resolution of the infection. A central corneal scar with corneal thinning remains. The patient underwent a penetrating keratoplasty for visual rehabilitation. CONCLUSIONS: Patient education with emphasis on compliance with nutritional supplements is essential after bariatric surgery. Consider vitamin A deficiency in the differential diagnosis of dry eye after LASIK surgery.

Treatment of amphibians infected with chytrid fungus: learning from failed trials with itraconazole, antimicrobial peptides, bacteria, and heat therapy.

Amphibian conservation goals depend on effective disease-treatment protocols. Desirable protocols are species, life stage, and context specific, but currently few treatment options exist for amphibians infected with the chytrid fungus Batrachochytrium dendrobatidis (Bd). Treatment options, at present, include antifungal drugs and heat therapy, but risks of toxicity and side-effects make these options untenable in some cases. Here, we report on the comparison of several novel treatments with a more generally accepted antifungal treatment in experimental scientific trials to treat Bd-infected frogs including Alytes obstetricans tadpoles and metamorphs, Bufo bufo and Limnodynastes peronii metamorphs, and Lithobates pipiens and Rana muscosa adults. The experimental treatments included commercial antifungal products (itraconazole, mandipropamid, steriplantN, and PIP Pond Plus), antimicrobial skin peptides from the Bd-resistant Pelophylax esculentus, microbial treatments (Pedobacter cryoconitis), and heat therapy (35°C for 24 h). None of the new experimental treatments were considered successful in terms of improving survival; however, these results may advance future research by indicating the limits and potential of the various protocols. Caution in the use of itraconazole is warranted because of observed toxicity in metamorphic and adult frogs, even at low concentrations. Results suggest that rather than focusing on a single cure-all, diverse lines of research may provide multiple options for treating Bd infection in amphibians. Learning from 'failed treatments' is essential for the timely achievement of conservation goals and one of the primary aims for a publicly accessible treatment database under development.

Fungal malignant external otitis.

OBJECTIVE: To investigate the clinical characteristics and outcome of fungal malignant external otitis (MEO). METHODS: The files of 60 patients treated for MEO in 1990-2008 at a tertiary medical center were reviewed for clinical characteristics and outcome, and findings were compared between patients with fungal and nonfungal infection. RESULTS: Mean duration of follow-up was 4 years. Nine patients (15%) had fungal disease; the main pathogen was Candida spp. Compared with the nonfungal MEO group, patients with a fungal infection were younger at diagnosis (average 68 vs. 74 years, p = 0.01) and had more facial nerve palsies (55% vs. 14%, p = 0.01), fewer positive bacterial cultures at presentation (33% vs. 75%, p = 0.02), and higher rates of surgery (78% vs. 18%, p = 0.0008) and hyperbaric treatment (78% vs. 4%, p = 0.0001). Eighty-nine percent had persistent infection (>2 courses of systemic antibiotics before antifungal treatment) compared with 12% in the nonfungal group (p = 0.0001). Fungal disease was associated with more persistently positive imaging findings (87.5% vs. 25%, p = 0.0001). There was no significant between-group difference in survival. CONCLUSION: Fungal MEO probably occurs secondary to prolonged antibiotic treatment for bacterial MEO. The fungal disease is more invasive than the bacterial disease, although survival is the same. Treatment should be aggressive and hyperbaric oxygen therapy should be considered.

A rare complication of ear piercing: a case of subcutaneous phaeohyphomycosis caused by Veronaea botryosa in China.

We present the third case of phaeohyphomycosis caused by Veronaea botryosa in China and the tenth case worldwide. A 16-year-old Chinese girl developed crusted, verrucous lesions, initially on the left ear and later on the left buttock, within 2-5 months of receiving an ear piercing. Histopathological examination of biopsy specimens confirmed diagnosis of subcutaneous phaeohyphomycosis. Microscopic examination of the colonies recovered in culture from a portion of the biopsy specimen resulted in the identification of Veronaea botryosa based primarily on the presence of two-celled, brownish pigmented, cylindrical conidia produced sympodially from erect conidiogenous cells. The lesions significantly improved with daily oral treatment with itraconazole 400 mg and adjuvant thermotherapy for 6 months. A maintenance therapy with low dose itraconazole was prescribed in order to achieve clinical and mycological cure. A two-year follow-up didn't reveal any recurrence of infection. Our case is the first report of V. botryosa infection associated with a cosmetic procedure, which suggests that skin piercing could precipitate V. botryosa or other dematiaceous, as well as opportunistic fungal infections.

Otitis externa micótica y perforación timpánica: reporte de dos casos / Otomycosis and perforated tympanic membrane: two cases

La otitis externa constituye entre el 5 por ciento y 30 por ciento de las consultas de urgencia ORL y afecta al 10 por ciento de la población al menos 1 vez en la vida. La gran mayoría son de causa bacteriana, sólo el 15 por ciento a 20 por ciento son atribuidas a hongos. Las complicaciones son poco frecuentes alcanzando el 10 por ciento a 20 por ciento de los casos según la serie, siendo la perforación timpánica la más frecuente. Se presenta la evolución y tratamiento de 2 casos.

External otitis constitutes among 5 percent-30 percent of urgency ORL consultations and affects 10 percent of the population at least 1 time in their life. Most of them had a bacterial origin, only 15 percent to 20 percent are attributed to fungi. Otomycosis complications are slightly frequent reaching 10 percent to 20 percent of the cases according to diferent series, being the tympanic perforation the most frequent. We present 2 cases, their evolution and treatment.