Characterization of ocular and nasopharyngeal microbiome in allergic rhinoconjunctivitis.

BACKGROUND: Allergic rhinoconjunctivitis (ARC) is a prevalent allergic condition in the pediatric population. Microbial dysbiosis has increasingly been recognized to influence on host immunity and allergic diseases. However, the microbial profile of ARC has not been characterized. This cross-sectional study aims to evaluate the changes in nasal and ocular surface microbiome of children with ARC. METHODS: Ocular and nasopharyngeal swabs were collected from controls and pediatric ARC cases for 16S rRNA amplicon sequencing. The bacterial community profile was analyzed. The correlation of the microbial diversity with the ARC-related clinical scores was studied. RESULTS: A total of 23 patients with ARC and 17 healthy controls were recruited;30 were ocular samples (15 controls vs 15 ARC), while 40 were nasal samples (17controls vs 23 ARC) The alpha diversity of nasopharyngeal microbiome was significantly higher in ARC patients than healthy controls (P < 0.01), but not for ocular microbiome. The clinical scores in all subjects were negatively correlated with the Shannon diversity for ocular (P = 0.014) and positively correlated with nasopharyngeal (P = 0.010) microbiome. While the ocular microbiome remained significantly distinct from nasopharyngeal microbiome in terms of both alpha and beta diversity in both healthy subjects and ARC patients, significant differences of relative abundance of certain phyla (Bacteroidetes, Cyanobacteria, and Deinococcus-Thermus) and genera (Dolosigranulum and Moraxella) between nasal and ocular surfaces were only detected in healthy controls, but not in the ARC subjects, suggesting the microbial composition at both body sites becoming more similar at disease state. CONCLUSION: This study reported (a) a higher alpha diversity in ocular than nasopharyngeal microbiome in both ARC patients and controls, and (b) nasopharyngeal microbiome became more diverse in ARC patients than in controls. Our results suggested an interaction of the microbiome between ocular and nasal compartments in patients with ARC.

[Clinical profile of sensitization to fungi in Medellin, Colombia]. / Perfil clínico de sensibilización a hongos en Medellín, Colombia.

BACKGROUND: Fungi sensitization correlates with patologies like asthma, rhinitis, conjunctivitis and dermatitis. In general it is associated with other sensitizations. In Medellin the multisystemic pattern is associated with fungi sensitization. OBJECTIVE: To determine the clinical pattern of the fungi sensitization in Medellin. MATERIAL AND METHOD: We reviewed the medical records of 897 patients younger than 70 years old with fungi sensitization in the prick test during the period 1 January 2011 to 31 March 2014 in the allergy facility from 2 clinics. We evaluate 16 fungi and the presence of allergic diseases as well as the environmental conditions. RESULTS: Form 897 prick test, 12.8% were positive to fungi, and the most frequent was Candida albicans with 30.4%. Rhinitis were present in 100% of patients, asthma in 46.1%, conjunctivitis in 92.2% and the multisystemic pattern in 9.6%. The multisystemic pattern was associated with younger age with a risk of 9,259, 95% CI: 1.14-74.95 and with Aspergillus fumigatus sensitivity with a risk of 4.381, 95% CI: 1,116-17,204. CONCLUSIONS: The pattern of sensitivity was higher with Candida albicans. Most patients were polysensitized. The multisystemic pattern was associated with been younger and with Aspergillus fumigatus sensitivity. From the findings of this study, allergens like Candida and Aspergillus fumigatus should be tested in Medellin.

Antecedentes: la sensibilización a hongos se relaciona con enfermedades alérgicas como: asma, rinitis, conjuntivitis y dermatitis. En general, se asocia con sensibilidad a otros alérgenos. El patrón multisistémico se asoció en Medellín con la sensibilización a hongos. Objetivo: determinar el perfil clínico de la sensibilización a hongos en la ciudad de Medellín, Colombia. Material y método: estudio observacional, descriptivo y retrospectivo consistente en la revisión de historias clínicas de pacientes menores de 70 años de edad con sensibilidad a hongos en las pruebas de neumoalergenos realizadas entre el 1 enero de 2011 y el 31 de marzo de 2014 en la consulta de alergología de dos clínicas. Se evaluaron 16 hongos y la coexistencia de enfermedades alérgicas y las condiciones ambientales. Resultados: de 897 pruebas de prick realizadas, 12.8% resultaron positivas a alguno de los hongos, el más frecuente fue Candida albicans con 30.4%. La rinitis se encontró en 100% de los pacientes, asma en 46.1%, conjuntivitis en 92.2% y el patrón mutisistémico en 9.6%. El patrón mutisistémico se asoció con ser joven con riesgo de 9.259, IC 95%: 1.14-74.95 y a estar sensibilizado a Aspergillus fumigatus con un riesgo de 4.381, IC 95%: 1.116-17.204. Conclusiones: el patrón de sensibilidad mostró mayor sensibilidad a Candida. La mayoría de los pacientes está polisensibilizada. El patrón multisistémico es más frecuente en niños, se relaciona con sensibilidad a Aspergillus. Por los hallazgos de este estudio, alérgenos como Candida y Cladosporium deberían evaluarse en Medellín.

Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis.

OBJECTIVES: To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibody assay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detecting Chlamydia trachomatis in these conditions. METHODS: Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: a vernal keratoconjunctivitis group (group A) and a control group (group B). The polymerase chain reaction and a direct fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves, and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curves were plotted using a categorical variable with only two possible outcomes (positive and negative). RESULTS: Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. All patients in group A with positive polymerase chain reactions also presented with positive direct fluorescent antibody assays. CONCLUSION: The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection was confirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and by positive polymerase chain reactions in 20% of these patients. The direct fluorescent antibody assay detected Chlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although the diagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients. Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggest considering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergic disease.

Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis

OBJECTIVES: To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibodyassay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detectingChlamydia trachomatis in these conditions. METHODS: Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: avernal keratoconjunctivitis group (group A) and a control group (group B). The polymerase chain reaction and adirect fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves,and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curveswere plotted using a categorical variable with only two possible outcomes (positive and negative). RESULTS: Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. Allpatients in group A with positive polymerase chain reactions also presented with positive direct fluorescentantibody assays. CONCLUSION: The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection wasconfirmed by positive direct fluorescent antibody assays in 49.4 percent of vernal keratoconjunctivitis patients and bypositive polymerase chain reactions in 20 percent of these patients. The direct fluorescent antibody assay detectedChlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although thediagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients.Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggestconsidering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergicdisease.

Aspergillus keratitis in vernal shield ulcer--a case report and review.

An unusual case of vernal shield ulcer with superadded fungal keratitis caused by Aspergillus fumigates is reported. A 26-year-old man, a known case of vernal keratoconjunctivitis (VKC) presented with the complaint of diminution of vision in the right eye. Patient was on topical steroids and anti-allergic treatment for the past two months. In the right eye, a shield ulcer with an elevated plaque was seen. Scrapings from the right cornea revealed fungal filaments on a wet KOH mount and culture revealed growth of Aspergillus fumigatus. The patient was diagnosed as VKC with shield ulcer with secondary fungal keratitis. The patient was treated with topical cyclosporine, topical moxifloxacin, topical natamycin, and topical amphotericin eye drops. The patient responded well and finally recovered to a best spectacle-corrected visual acuity of 20/20 at the end of nine months. The chronic ocular surface changes and induced inflammation in VKC, and the instillation of topical steroids for therapy, may create an environmental milieu favorable for fungal keratitis. Microbiological evaluation should be considered, even in cases of suspected sterile keratitis, to prevent possible worsening of an associated infective corneal condition. This warrants patient education, periodic reviews and a very cautious approach to indiscriminate use of topical corticosteroids in cases of VKC with shield ulcer. In the event of any secondary fungal infection, use of steroid sparing topical agent, for example cyclosporine may be considered.

Staphylococcus aureus accelerates an experimental allergic conjunctivitis by Toll-like receptor 2-dependent manner.

Allergic conjunctivitis is an inflammatory eye disease mediated by Th2-type cytokines and Staphylococcus aureus (S. aureus) colonization has been suggested as playing a role. This study used an experimental allergic conjunctivitis model to determine whether colonization by S. aureus affects the development of allergic conjunctivitis and modifies the immune response to OVA allergen. Mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. Of interest, inoculation of S. aureus markedly accelerated the signs of allergic conjunctivitis and was associated with higher levels of IgE Ab in serum. In addition, mice inoculated with S. aureus had more IL-4, IL-5, IL-13 and eotaxin secretion than non-inoculated group. In contrast, inoculation of TLR2(-/-) mice with S. aureus had no effect on severity of allergic conjunctivitis. The findings suggest that activation of TLR2 signal by S. aureus induces Th2-type immune responses and accelerates experimental allergic conjunctivitis.

Preliminary evidence of the efficacy of probiotic eye-drop treatment in patients with vernal keratoconjunctivitis.

BACKGROUND: Probiotics have been shown to improve allergic inflammation. The aim of this study was to evaluate the efficacy of Lactobacillus Acidophilus eye-drops in controlling signs and symptoms of vernal keratoconjunctivitis (VKC). METHODS: Seven patients (mean age 11.8 +/- 4.3; five M, two F) with mild to moderate VKC were included in the study. Lactobacillus Acidophilus diluted in saline solution (2 x 10(8) CFU/ml) was administrated as eye-drops four times daily for 4 weeks in both eyes. Clinical signs (conjunctival hyperemia, chemosis, secretion, Trantas dots, superficial punctuate keratitis) and symptoms (itching, photophobia, burning, tearing) were evaluated and scored from 0 to 3 at baseline, after 2 and 4 weeks of treatment. Total sign (TSS) and symptom (TSyS) scores were calculated. Conjunctival impression cytology was performed in three patients at baseline and after 4 weeks of treatment, in order to evaluate the expression of ICAM-1 and TLR-4. RESULTS: In the six out of seven patients who completed the study, symptoms were significantly improved after both 2 weeks (TSyS: baseline 6.7 +/- 0.9 vs 4.1 +/- 1.2; p = 0.017) and 4 weeks (TSyS: baseline 6.7 +/- 0.9 vs 3.6 +/- 1.2, p = 0.011) of treatment. A significant improvement of clinical signs was observed after 4 weeks of treatment (TSS: baseline 7.5 +/- 1.6 vs 3.9 +/- 1.7, p = 0.034) but not after 2 weeks of treatment (TSS: baseline 7.5 +/- 1.6 vs 5.3 +/- 1.5; NS). In particular, photophobia was significantly reduced (2 +/- 0.6 vs 1 +/- 0.3; p = 0.023) at 2 weeks, while at 4 weeks the scores for itching (1.8 +/- 0.3 vs 1 +/- 0.3), tearing (1.6 +/- 0.4 vs 0.8 +/- 0.2), conjunctival hyperemia (2.3 +/- 0.2 vs 1.4 +/- 0.5) and chemosis (1.2 +/- 0.4 vs 0.4 +/- 0.4) were significantly lower compared to baseline. A down-regulation of ICAM-1 and TLR-4 was observed in two patients showing clinical improvement after 4 weeks of treatment. CONCLUSION: Our open pilot study showed that 1-month treatment with probiotic eye-drops improves signs and symptoms in patients with VKC. Additional double-blind controlled clinical trials with a larger sample of patients are needed to confirm the effects of topical Lactobacilli on VKC patients.

Microbiota conjuntival em pacientes com alergia ocular / Conjunctival microbiota in patients with ocular allergy

OBJETIVO: Avaliar a presença de microbiota aeróbia da conjuntiva de portadores de alergia ocular e comparar a um grupo controle. MÉTODOS: Foram examinados 133 pacientes no período de abril a junho de 2001 divididos em 2 grupos. O grupo A foi composto de 63 portadores de conjuntivite alérgica (sem uso de medicação) e o grupo B de 70 pacientes do ambulatório geral (controle). Foram coletadas amostras do fundo de saco conjuntival do olho direito de todos os pacientes e o material foi semeado em meios sólidos de cultura (ágar sangue, chocolate e Sabouraud). RESULTADOS: No grupo A, 30 culturas (47,7 por cento) foram positivas e no grupo B, 6 (8,6 por cento). Sete bactérias foram isoladas no grupo A e 4 no B. A análise estatística revelou associação significante entre a positividade dos cultivos e conjuntivite alérgica. CONCLUSÃO: Microbiota bacteriana foi mais freqüentemente encontrada nos pacientes com alergia ocular.

[Conjunctival microbiota in patients with ocular allergy]. / Microbiota conjuntival em pacientes com alergia ocular.

PURPOSE: To evaluate de presence of conjunctival aerobic microbiota in patients with ocular allergy as compared to a control group. METHODS: One hundred and thirty-three patients were evaluated from April to June 2001 and divided into 2 groups. Sixty-three patients with allergic conjunctivitis (without medication) were in group A and 70 patients from the general outpatient clinic were in group B (control group). Samples from the conjunctival sac of the right eye were collected and cultured in solid media (blood, chocolate and Sabouraud agar). RESULTS: In group A, 30 cultures (47.7%) were positive and 6 (8.6%) in group B. Seven bacteria were isolated from group A and 4 from group B. Statistical analysis revealed significant association between positive cultures and allergic conjunctivitis. CONCLUSION: Bacterial microbiota was more frequently found in patients with ocular allergy.

[The relationship between Staphylococcus aureus and atopic keratoconjunctivitis].

PURPOSE: To investigate the role of Staphylococcus aureus in patients with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) complicated by atopic dermatitis (AD). SUBJECTS AND METHODS: Microbiological culture from the conjunctival sac of acute exacerbated patients with VKC and AKC complicated by AD was performed. The subjects were 29 patients (31 eyes) with VKC and AKC who showed acute exacerbated clinical symptoms of conjunctivitis. Antigen specific IgE antibodies to staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) in serum were also examined. In this study, the patients were divided into two groups: An AD group consisting of 22 patients with VKC and AKC complicated by AD, and a control group consisting of 8 patients with allergic conjunctivitis without AD. We also examined SEA/SEB specific IgE antibody in tears from the 5 patients who underwent bacteriological examinations of the conjunctival sac at the same time. RESULTS: Twenty-seven out of 31 eyes were gram-positive in the bacteriological culture from the conjunctival sac (87.1%). Staphylococcus aureus was detected in 21 out of the 27 eyes (77.8%). In the AD group, 2 of the 22 cases were gram-positive for serum SEA specific IgE antibodies, 2 cases of the 22 cases were gram-positive for SEB specific IgE antibodies, and 9 cases were gram-positive for both SEA and SEB specific IgE antibodies. Serum SEA and SEB specific IgE antibodies were all gram-negative in the control group. Either SEA or SEB specific IgE antibody in tears was detected in all of the above 5 patients who underwent bacteriological examinations of the conjunctival sac, and 4 of the 5 were gram-positive. Staphylococcus aureus was isolated in 3 out of the 5 patients, and 1 case was gram-negative. CONCLUSION: Staphylococcus is one of the exacerbating factors in VKC and AKC. It is important to evaluate both bacteriological examinations of the conjunctival sac and SEA/SEB specific IgE in tears.

No apparent association between periocular and ocular microcolonization and the degree of inflammation in patients with atopic keratoconjunctivitis.

BACKGROUND: The cause of the chronic inflammation in atopic keratoconjunctivitis (AKC), the ocular manifestation of atopic eczema/dermatitis syndrome, is largely unknown. OBJECTIVE: To investigate the possibility that microorganisms may be important in the inflammatory activity in AKC. METHODS: Fifteen patients with AKC participated in the study. The presence of aerobic bacteria and fungi was related to the severity of clinical signs, the numbers of inflammatory cells in tears and conjunctival biopsies, and the concentration of various cytokines in tears. In addition, serological evidence for IgE sensitization to Staphylococcus aureus B antigen and Malassezia sympodialis antigen was investigated. Twelve healthy subjects were included for control purposes. RESULTS: The patients exhibited moderate clinical signs of AKC. No relation was found between the severity of AKC and the presence of microorganisms, despite the fact that S. aureus was frequently isolated. AKC patients showed significantly higher levels of IFN-gamma, TNF-alpha (tumour necrosis factor-alpha), IL-2, IL-4, IL-5 and IL-10 than controls. An association was found between conjunctival signs and the levels of all cytokines except IL-5. CONCLUSION: We found no evidence to suggest that periocular and ocular microcolonization are related to inflammatory parameters in AKC. However, confirmation of the present results in a longitudinal study with repeated clinical examinations and samplings in the same individual is required before the contribution of S. aureus to on-going inflammation in AKC can be dismissed.

Penicillium keratitis in vernal Keratoconjunctivitis.

We report a case of penicillium keratitis in vernal shield ulcer in the absence of corticosteroid use. This report illustrates super-added infection in vernal shield ulcer by an organism which is otherwise innocuous and forms a part of the normal ocular flora.

Influence in the quality of life of the respiratory patients by environmental control and the acaricide Frontac®.

The present prospective, open, observational study assessed the effect of the use of standard environmental control measures together with the application of an acaricide-containing aerosol of esbiol/benzyl benzoate/piperonyl butoxide/2-phenylphenol (Frontac) on several clinical and environmental parameters in patients suffering from perennial allergic rhinoconjunctivitis related to house dust mites (HDM). Fifty-five patients were enrolled and studied for one year. Both symptoms and the patients' quality of life (evaluated by the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ)) were assessed at several clinic visits. The amount of HDM antigen was semi-quantitatively estimated using the guanine test. We found a significant decrease (p < 0.001) in most nasal symptoms scores: sneezing, runny nose, stuffy nose and itching. The overall quality of life (QoL) score also showed a significant improvement during the study period; the mean score decrease found was 0.86 (CI 95 %, 0.54-1.17) (p < 0.001). Five of the seven domains included in the questionnaire decreased significantly: non-hayfever symptoms; practical problems; nasal symptoms; eye symptoms, and emotions. These QoL score decreases were found during the first 3 months and later remained stable at lower levels than at baseline. The content of guanine in dust samples decreased significantly from 2.17 0.75 to 1.43 0.68 (p < 0.001). No toxic adverse events were recorded. In conclusion, these results show an improvement in the quality of life and a clinical benefit of the long-term use of the new chemical acaricide tested, together with environmental measures, in the home of patients suffering from allergic respiratory pathology.

Mycotic keratitis in non-steroid exposed vernal keratoconjunctivitis.

PURPOSE: To report a patient with vernal keratoconjunctivitis who developed mycotic keratitis in absence of known risk factors. METHODS: A 17-year-old male suffering from vernal keratoconjunctivitis presented with infective keratitis. The patient had been treated in the past with topical antihistaminics and vasoconstrictors. The patient had not been exposed to topical steroids in 2 years of follow-up. He did not have dry eye or corneal micro or macroerosions prior to the development of infective keratitis. Corneal scrapings were obtained and subjected to KOH wet mount smear, calcofluor and Grams stain as well as bacterial culture sensitivity and fungal culture. RESULTS: Clinical diagnosis of mycotic keratitis in association with vernal conjunctivitis was supported by microbiological investigations. KOH wet mount and calcofluor staining showed presence of filamentous septate hyphae while fungal culture showed growth of aspergillus fumigatus. Antifungal therapy was initiated in the form of topical natamycin 5% suspension to which the patient responded and recovered 6/6 final visual acuity. CONCLUSION: The authors wish to conclude that patients suffering from vernal keratoconjunctivitis, even in the absence of corneal involvement, steroid exposure and trauma, may be at increased risk of developing keratomycosis.

Method for detection of conjunctival chlamydial infection and allergic conjunctivitis.

In this study 104 adult patients with chronic conjunctival irritation without obvious diagnosis were investigated by conjunctival scrapings. The smears were stained with May-Grünwald-Giemsa or fluorescein-conjugated monoclonal antibodies prepared against Chlamydiae. In 45 cases it was possible to make a correct diagnosis of Chlamydia infection (29%) or allergy (20%).

Bacterial keratitis associated with vernal keratoconjunctivitis.

We cared for two patients with longstanding vernal keratoconjunctivitis who had bacterial corneal ulcers in each eye. Both patients were young, black, and had histories of atopy. The patients came for treatment with acute symptoms of pain, redness, and reduced vision in the affected eye. On examination in each case we found an epithelial defect associated with dense stromal infiltration, a calcific plaque in the bed of the ulcer, and a severe anterior chamber reaction, including a hypopyon in two cases. Cultures of corneal scrapings from all four eyes were positive for Staphylococcus aureus, and three of the four infections were polymicrobial. All four eyes responded rapidly to intensive topical antibiotic therapy, debridement of the calcific plaque, and subsequent treatment with topical corticosteroids and/or cromolyn sodium. Bacterial keratitis can occur in patients with vernal keratoconjunctivitis, especially those with vernal corneal ulcers. The abnormalities of ocular immune mechanisms found in patients with vernal keratoconjunctivitis may predispose them to bacterial keratitis.

Role of Staphylococcus aureus in chronic allergic conjunctivitis.

A study was undertaken to test the hypothesis that Staphylococcus aureus colonization of the lid margins could determine the expression of allergic eye disease in atopic patients. The authors compared lid isolates of S. aureus from 23 adults who had both atopic dermatitis and chronic conjunctivitis and isolates from 14 patients who had atopic dermatitis but who lacked ocular disease. No significant difference was found in either the staphylococcal protein A or hemolytic toxin production by isolates from the two disease groups, and there was no difference between groups in the quantity of serum IgG nor IgE antibodies to staphylococcal ribitol-teichoic acid. In seven patients with chronic allergic conjunctivitis who were challenged with staphylococcal protein A or heat-killed S. aureus, delayed-type hypersensitivity was not enhanced. These results suggest that although S. aureus colonization of the lids is common in atopic patients, neither the pattern of toxin production nor humoral or cell-mediated immunity to S. aureus play a role in the expression of chronic allergic conjunctivitis.

[Allergic conjunctivitis. Treatment and course]. / Conjuntivitis alérgica. Tratamiento y evolución.

A group of 74 patients with allergic conjunctivitis from the out patient service of allergy of the Hospital Pediátrico Provincial Docente José Luis Miranda, in Santa Clara, were studied. In this study are stressed the importance of the search for etiologic agents influencing the disease and the used diagnostic methods. Finally, the used treatments are pointed out. The most frequent were the strepto-staphylo-gamma vaccines, preventive measures, antibiotic drops, environmental measures and sodium cromoglycate drops. Treatment was dynamic, individual and associated with the ophthalmology and pediatric allergy specialties.