Smartphone-assisted mobile fluorescence sensor for self-calibrated detection of anthrax biomarker, Cu2+, and cysteine in food analysis.

Dipicolinic acid (DPA), as a biomarker for Bacillus anthracis, is highly toxic at trace levels. Rapid and on-site quantitative detection of DPA is essential for maintaining food safety and public health. This work develops a dual-channel self-calibrated fluorescence sensor constructed by the YVO4:Eu and Tb-ß-diketone complex for rapid visual detection of DPA. This sensor exhibits high selectivity, fast response time, excellent detection sensitivity, and the detection limit is as low as 4.5 nM in the linear range of 0-16 µM. A smartphone APP and portable ultraviolet lamp can assemble a mobile fluorescence sensor for on-site analysis. Interestingly, adding Cu2+ ions can quench the fluorescence intensity of Tb3+. In contrast, the addition of cysteine can restore the fluorescence, allowing the accurate detection of Cu2+ ions and cysteine in environmental water and food samples. This work provides a portable sensor that facilitates real-time analysis of multiple targets in food and the environment.

Characterization of Ba813 harbouring Bacillus cereus in patients with haematological malignancy and hospital environments at a medical centre in Japan.

Introduction. Bacillus cereus harbouring Ba813, a specific chromosomal marker of Bacillus anthtacis, is found in patients with severe manifestations and causes nosocomial outbreaks.Aim. We assessed the genetic characteristics and virulence of Ba813(+) B. cereus in a hospital setting.Methodology. Three neutropenic patients with haematological malignancy developed B. cereus bacteraemia within a short period. Fifteen B. cereus were isolated from different sites in a haematology ward. A total of 18 isolates were evaluated for Ba813- and B. anthracis-related virulence, food poisoning-related virulence, genetic diversity, bacteria motility and biofilm formation.Results. Ba813(+) B. cereus was detected in 33 % (1/3) of patients and 66 % (9/15) of the hospital environment. The 18 strains were divided into 2 major clusters (clade 1 and clade 2), and 14 strains were classified into clade 1. All Ba813(+) strains, including four sequence types, were classified into clade 1/the cereus III lineage, which is most closely related to the anthracis lineage. Two strains belonging to clade 1/non-cereus III carried the B. anthracis-associated cap gene, but not Ba813. B. cereus, including Ba813(+) strains, had significantly lower prevalence of enterotoxin genes than clade 2 strains. In clade 1, B. cereus, Ba813(+) strains showed significantly higher swimming motility and biofilm formation ability than Ba813(-) strains.Conclusion. Ba813(+) B. cereus, which are genetically closely related to B. anthracis, were abundant in a haematological ward. Ba813(+) B. cereus with high motility and biofilm formation abilities may spread easily in hospital environments, and could become a hospital-acquired infection.

Sporadic human cutaneous anthrax outbreak in Shaanxi Province, China: report of two cases from 2018

ABSTRACT China's compulsory annual livestock anthrax vaccination policy has remarkably reduced but not completely eradicated human anthrax infections. Herein we describe a sporadic human cutaneous anthrax outbreak involving two cases in 2018 in Shaanxi Province, both involving herdsman who dealt with unvaccinated and potentially sick cattle. Both patients showed Bacillus anthracis-positive blister smear and blood culture. Treatment with penicillin was followed by uneventful recovery for both. The prompt performance of the prophylactic measures successfully interrupted the further transmission of this sporadic human cutaneous anthrax outbreak.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil

BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Detection of Bacillus anthracis in animal tissues using InBios active anthrax detect rapid test lateral flow immunoassay.

The Active Anthrax Detect (AAD) Rapid Test lateral flow immunoassay is a point-of-care assay that was under investigational use for detecting Bacillus anthracis capsular polypeptide (polyglutamic acid) in human blood, serum and plasma. Small sample volumes, rapid results and no refrigeration required allow for easy use in either the field or laboratory. Although the test was developed for use in suspect cases of human inhalation anthrax, its features also make it a potentially powerful tool for testing suspect animal cases. We tested animal tissue samples that were confirmed or ruled out for B. anthracis. The AAD Rapid Tests were also deployed in the field, testing animal carcasses during an anthrax outbreak in hippopotami (Hippopotamus amphibius) and Cape buffalo (Syncerus caffer) in Namibia. Evaluation of all samples showed a specificity of 82% and sensitivity of 98%. However, when the assay was used on specimens from only fresh carcasses (dead for <24 h), the specificity increased to 96%. The AAD Rapid Test is a rapid and simple screening assay, but confirmatory testing needs to be done, especially when the age of the sample (days animal has been deceased) is unknown. SIGNIFICANCE AND IMPACT OF THE STUDY: In countries where anthrax is endemic, many human outbreaks are often caused by epizootics. Earlier detection of infected animals may allow for identification of exposed people, early implementation of prevention and control methods, and ultimately lessen the number of people and animals affected. Detection of Bacillus anthracis in animal tissues using a simple, rapid and field-deployable method would allow for faster outbreak response. We evaluated a simple sample collection and processing method for use with the Active Anthrax Detect Rapid Test lateral flow immunoassay to screen dead animals for anthrax.

Ántrax cutáneo, último brote diagnosticado en Chile / Cutaneous anthrax, the last outbreak diagnosed in Chile

Resumen El ántrax, es una zoonosis causada por una bacteria generadora de esporas, llamada Bacillus anthracis. En forma natural tiene una distribución global, con una predilección en zonas agrícolas con pocas normativas de sanidad pública veterinaria. El contagio humano ocurre por el consumo de carnes de animales enfermos, por contacto a través de una puerta de entrada en la piel o por la inhalación de esporas de productos derivados del animal afectado (lana, cuero, huesos). La infección en los seres humanos compromete con mayor frecuencia la piel, seguido por el tracto gastrointestinal y los pulmones. El control de la enfermedad se basa en la prevención, de allí la importancia de la vigilancia en la detección de casos y brotes. Presentamos el último brote de ántrax cutáneo diagnosticado en Chile con descripción de dos primeros casos clínicos del brote.

Anthrax is a zoonosis caused by a spore-forming bacterium, called Bacillus anthracis. Naturally it is of global distribution, with a predilection in agricultural zones with few norms of public veterinary health. Human contagion occurs through the consumption of diseased animal's meat or through a doorway into the skin or through the spores inhalation of products derived from the affected animal (wool, leather, bones). The most frequent infection in humans occurs in the skin, followed by the gastrointestinal tract and lungs. We present the last outbreak of cutaneous anthrax diagnosed in Chile with a description of the first two clinical cases of the outbreak. Control disease is based on prevention, hence the importance of surveillance in detecting cases and outbreaks.

Injectional anthrax in human: A new face of the old disease.

Unusual human behavior leads to the emergence of new forms of infectious diseases and new routes of infection. In recent years, a new form of anthrax, called injectional anthrax, emerged and was related to 2 human anthrax outbreaks in Europe. The infection was caused by heroin contaminated with anthrax spores. The new form of anthrax differs from the earlier known "natural" forms of the disease in symptoms, length of the incubation period and recommended treatment. Despite medical treatment, the mortality rate in injectional anthrax is about 35%. This article presents an overview of the forms of anthrax infection in humans, with focus on injectional anthrax syndrome, as well as actual recommendations for treatment, including antibiotic therapy, surgery and possibilities of administering anthrax antitoxin. As a source of contamination of heroin have not been identified and new cases of injectional anthrax might occur again in any country in the future.

Genome sequencing of two Bacillus anthracis strains: a virulent strain and a vaccinal strain

ABSTRACT Bacillus anthracis strain SPV842_15 was isolated from bovine fetus, while B. anthracis strain Brazilian vaccinal was recovered from a commercial vaccine. We report here the genome sequences of both strains. The SPV842_15 genome is composed of a single circular chromosome with a length of 5,228,664 base pairs, and comprises 5911 coding sequences. In turn, the Brazilian vaccinal genome remains in 201 contigs with 5733 coding sequences. Both genomes have an overall C + G content of 35.4%, and 11 genes encoding the ribosomal RNAs (rRNAs) 5S, 16S and 23S. Only the plasmid pX01 sequence, which carries genes for toxins synthesis, was detected and completely assembled for both strains. These plasmids have a length of 181,684 base pairs and a C + G content of 32.5%. These genomic data generate insights about vaccinal B. anthracis virulence.

Diagnosis of cutaneous anthrax in resource-poor settings in West Arsi Province, Ethiopia.

INTRODUCTION: Cutaneous anthrax is a zoonotic disease caused by the spore-forming bacterium Bacillus anthracis, which typically presents with ulcers after contact with animals or animal products, and is rarely seen in high-income countries but is common in those with low- and middle-incomes. Objective. The aim of this study is to show the main clinical characteristics of cutaneous anthrax in endemic areas. MATERIAL AND METHODS: The study describes the main clinical characteristics of cutaneous anthrax in eight patients (six female and two male, age range 1 - 56 years) admitted to the rural General Hospital of Gambo, West Arsi Province of Ethiopia from 2010-2013. RESULTS: In all cases, lesions began as an erythematous papule located on exposed sites (n=7 head; n=1 thigh) and subsequently became a necrotic black eschar surrounded by an edematous halo. Two patients presented with painful ipsilateral adenopathy near the black eschar. Four patients developed a malignant pustule on the suborbital region of the face. Patients responded positively to treatment, and the lesions resolved, leaving eschars. However, one patient suffered the loss of an eyeball, and another died 12 hours after starting treatment. CONCLUSIONS: Physicians working in rural areas of resource-poor settings should be trained in the clinical identification of cutaneous anthrax. Early antibiotic treatment is essential for decreasing morbidity and mortality.

Milk-originated Bacillus cereus sensu lato strains harbouring Bacillus anthracis-like plasmids are genetically and phenotypically diverse.

Bacillus cereus sensu lato is widely distributed in food products, including raw and processed milk. Plasmids often determine bacterial virulence and toxicity, but their role in the evolution of B. cereus sensu lato is only partly known. Here, we observed that nearly 8% of B. cereus sensu lato isolates were positive for pXO1-like plasmids and 12% for pXO2-like plasmids in raw and ultra-heat-treated (UHT) milk from one dairy plant. However, pXO1-like plasmids were significantly more frequent in raw milk, while pXO2-like plasmids were more frequent in processed milk. Strains from raw and UHT milk were enterotoxigenic, with up to one-fifth of the isolates being psychrotolerant. Phylogenetic assessment using multi-locus sequence typing revealed a polyphyletic structure for these bacilli, with distinct groups of cold-adapted isolates and pathogenic strains (including emetic B. cereus). Populations corresponding to both sampling sites exhibited significant linkage disequilibrium and the presence of purifying selection. The far-from-clonal population structure indicated the presence of sequence types or ecotypes adapted to specific conditions in the dairy industry. A high recombination-to-mutation ratio suggested an important role for horizontal gene transfer among B. cereus sensu lato isolates in milk.

Virulence plasmid stability in environmentally occurring Bacillus anthracis from North East Turkey.

The Bacillus anthracis virulence plasmid pXO2, which encodes for a polypeptide capsule, can be lost during long term laboratory storage. To determine if pXO2 is lost in nature we screened B. anthracis isolates obtained from B. anthracis spores from contaminated animal burial sites in Turkey for their ability to express a capsule upon primary culture. A total of 672 B. anthracis colonies were examined of which ten produced a mixed mucoid (capsule +ve)/non-mucoid (capsule -ve) phenotype and a further one colony yielded non-mucoid colonies upon repeated culture. Screening by PCR using pXO2 specific primers revealed that seven of these isolates had eliminated the plasmid. Of the four colonies which were positive by PCR, one regained the ability to express a capsule upon repeated culture suggesting that the defect was reversible. This is an important observation as capsule expression is a principal marker of virulence and in the absence of PCR serves as a key diagnostic marker. The results of this preliminary study suggest that pXO2 is lost in nature and that further studies are need to determine the mechanisms by which this occurs.

Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho.

INTRODUCTION: Bacillus anthracis is the causative agent of anthrax, a disease endemic in regions of Northern Cape Province and Kruger National Park of South Africa. Accurate identification of virulent B. anthracis is essential but challenging due to its close relationship with other members of B. cereus group. This study characterized B. anthracis and Bacillus species that were recovered from animals and the environment where animals died of anthrax symptoms in southern Africa using a polyphasic approach. METHODOLOGY: For this purpose, 3 B. anthracis and 10 Bacillus isolates were subjected to microbiology tests, BiologOmniLog identification system (Biolog), 16S ribosomal RNA (rRNA) sequence analysis, polymerase chain reaction (PCR) detection of protective antigen (pag) and capsule (cap) regions, and real-time PCR using hybridization probes targeting chromosomal, pag, and capC genes. RESULTS: The Bacillus isolates were non-hemolytic, non-motile, and susceptible to penicillin, which is typical of B. anthracis, but resistant to gamma phage, unlike typical B. anthracis. The Biolog system and 16S rRNA gene sequence analysis identified most of the Bacillus isolates as B. endophyticus (7 of 10). Conventional PCR revealed that most of the Bacillus isolates contained capBCA gene regions. This highlights the limitation of the specificity of conventional PCR and the fact that the real-time PCR is more specific and reliable for anthrax diagnosis. CONCLUSIONS: Real-time PCR, 16S rRNA sequencing, and confirmatory microbiology tests including phage resistance distinguished Bacillus isolates from B. anthracis in this study. Identification of B. anthracis should be done using a polyphasic approach.

Cutaneous anthrax: evaluation of 28 cases in the Eastern Anatolian region of Turkey.

CONTEXT: Anthrax is an endemic disease in developing countries. Human cases are usually associated with animal products. About 95% of naturally acquired cases are cutaneous anthrax. OBJECTIVE: In this study, cutaneous anthrax cases from the Elazig province (the Eastern Anatolian region) of Turkey seen in our hospital within a 6-year period were evaluated with respect to epidemiological and clinical features, diagnosis, treatment and outcome. METHODS: Twenty-eight patients with cutaneous anthrax observed between January 2009 and December 2014 were investigated retrospectively. The diagnosis of cutaneous anthrax was based on detailed history, dermatologic findings, including painless, ulcers covered by a characteristic black eschar and/or microbiological procedures, including Gram stain and culture of materials obtained from the lesions. RESULTS: Of the 28 patients followed up with cutaneous anthrax diagnosis, 14 (50%) were female and 14 (50%) were male. The mean age of the cases was 39.6 years (age range 17-65 years). The patients have an incubation period in the range of 1-9 days (mean 4.6 ± 0.5 days). The cases were seen between April and November of each year during the study period. Twenty-three cases (82%) had a history of contact with animals or animal products. Twenty patients (71.4%) showed malignant pustules and eight (28.6%) malignant edema. Bacillus anthracis was isolated in three cases (10.7%) and Gram stain smear were positive in five cases (17.8%). All patients were treated successfully with penicillin or ciprofloxacin. Systemic corticosteroids were added to the antibiotic treatment in six patients with malignant edema. Sepsis no developed in patients, all the cases recovered. CONCLUSION: Anthrax is still a serious public health problem in Turkey. Cutaneous anthrax must always be kept in mind when characteristic lesions such as a painless ulcer with vesicles, edema, and a history of contact with animals or animal products are observed in an individual. Early and correct diagnosis significantly affects course of the disease. Protective precautions such as vaccination of animals against anthrax and education of the population would reduce the incidence of the disease.

Historical evolution of human anthrax from occupational disease to potentially global threat as bioweapon.

PURPOSE: Anthrax is caused by Bacillus anthracis, which can naturally infect livestock, wildlife and occupationally exposed humans. However, for its resistance due to spore formation, ease of dissemination, persistence in the environment and high virulence, B. anthracis has been considered the most serious bioterrorism agent for a long time. During the last century anthrax evolved from limited natural disease to potentially global threat if used as bioweapon. Several factors may mitigate the consequences of an anthrax attack, including 1. the capability to promptly recognize and manage the illness and its public health consequences; 2. the limitation of secondary contamination risk through an appropriate decontamination; and 3. the evolution of genotyping methods (for microbes characterization at high resolution level) that can influence the course and/or focus of investigations, impacting the response of the government to an attack. METHODS: A PubMed search has been done using the key words "bioterrorism anthrax". RESULTS: Over one thousand papers have been screened and the most significant examined to present a comprehensive literature review in order to discuss the current knowledge and strategies in preparedness for a possible deliberate release of B. anthracis spores and to indicate the most current and complete documents in which to deepen. CONCLUSIONS: The comprehensive analysis of the two most relevant unnatural anthrax release events, Sverdlovsk in the former Soviet Union (1979) and the contaminated letters in the USA (2001), shows that inhalational anthrax may easily and cheaply be spread resulting in serious consequences. The damage caused by an anthrax attack can be limited if public health organization, first responders, researchers and investigators will be able to promptly manage anthrax cases and use new technologies for decontamination methods and in forensic microbiology.

Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

Evaluation of the relationship between the Adenosine Triphosphate (ATP) bioluminescence assay and the presence of Bacillus anthracis spores and vegetative cells.

BACKGROUND: The Adenosine triphosphate (ATP) bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. METHODS: Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface) of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. RESULTS: When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU) for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. CONCLUSIONS: This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event.

Retrospective review of the case of cutaneous anthrax-malignant pustule from 1995 in 15-year old girl.

A 15-year-old girl was admitted to our Department with cutaneous lesion resembling black eschar. Anamnesis revealed that before getting ill she was wearing pullover made of rough sheep's wool and ornaments made of leather like straps. Cutaneous anthrax was confirmed by identification of B. anthracis in specimens from weeping ulceration, culture from black eschar, thermoprecipitation test, and bioassay on guinea pig. The girl was treated with crystalline Penicillin. She responded well to the therapy and recovered after 28 days. What attracts attention in presented case is the fact that the girl didn't belong to high risk group of human anthrax, which might lead to misdiagnosis. In 1990-1999, Poland there were reported 22 cases of anthrax - it was almost exclusively cutaneous form. In the years following 1999 antrax was reported even less often - in the period 1991-2013 it was recorded a total of 26 cutaneous anthrax cases.

The development of surface-enhanced Raman scattering as a detection modality for portable in vitro diagnostics: progress and challenges.

This perspective provides an overview of the diverse surface-enhanced Raman scattering (SERS)-based sensor platforms that have been developed for in vitro diagnostic applications. To provide focus, protein and nucleic acid detection assays based on the principle of extrinsic SERS sensing are emphasized, as well as their potential for translation to fully integrated point-of-care (POC) test platforms. The development of intrinsic SERS sensors, which are predicated on the direct detection of analytes by laser excitation, entails unique opportunities and challenges deserving of their own attention. As the robust sensing of disease pathogens and cancers in both clinical facilities and limited resource settings is the targeted objective of many next-generation biosensors, the majority of the research progress summarized here centers on SERS sensors developed for the rapid, sensitive and selective detection of disease-causing pathogens and biomarkers. In our effort to communicate a realistic assessment of the progress that has been made and the challenges that lie ahead, we avoid an overtly optimistic appraisal of the current status of SERS diagnostics that does not tacitly acknowledge the difficulties inherent in aligning SERS-based technologies alongside ELISA and PCR technologies as a complementary method for bioanalyte detection possessing unique advantages.

Evaluation of cutaneous palpebral anthrax.

CONTEXT: Anthrax is a rare disease caused by Bacillus anthracis. Antrax is zoonotic disease and is often encountered in persons engaged in animal husbandry. Cutaneous anthrax is approximately 95% of anthrax in humans. Palbebral involvement is rare. OBJECTIVE: In this study, we aimed to evaluate the clinical presentation, diagnosis and treatment of cases with cutaneous palpebral anthrax. METHODS: In this study, the patients diagnosed of cutaneous palpebral anthrax between January 2000 and December 2012, were investigated and evaluated, retrospectively. Cutaneous palpebral anthrax was diagnosed by the presence of typical anthrax lesion and/or observation of gram-positive encapsulated bacilli in gram prepations and/or culture positive of samples taken from lesions. In the cases who were culture-negative and without bacilli in gram-staining, the diagnosis was based on the presence of characteristic clinical presentation with a history of severe scarring formation, swelling, black eschar and positive response to the treatment. RESULTS: A total of 21 patients with cutaneous palpebral anthrax admitted to the two hospitals between January 2000 and December 2012. Eight patients were male (38.1%) and 13 patients were female (61.9%), and the mean age was 31 ± 21.2 (range 1-82 years). The most common symptoms on admission to the hospital were swelling and redness on the skin. Periorbital lesions were in the right eye in 14 cases and the most common eyelid involvement was seen in upper eyelid with 15 cases. The diagnosis was based on isolation of bacteria in five (23.8%) cases, detection of gram-positive bacilli in direct examination of characteristic lesion material in six (28.5%) cases. Ten (47.7%) cases were diagnosed by the characteristic appearance of the lesion. Malignant pustule was seen in all of our patients and seven cases (33.4%) had malignant edema. In the treatment, penicilin was used for 10 (47.7%) cases, ampicillin-sulbactam for five (23.8%) cases and, ciprofloxacin for three (14.3%) cases. Cicatricial ectropion was observed in 10 (47.7%) patients, lagophthalmos developed in four (19%) patients, and corneal scar in two (9.5%) patients. The distribution of the cases did not differ by the year but showed a density in the months from July to September (62.7%). CONCLUSION: Early diagnosis and high dose antibiotic treatment can facilitate the treatment and prevent development of eyelid complications including cicatricial ectropion, corneal scars and palpebral symphysis. Prolonged follow-up is necessary in patients who develop complications and surgical intervention.