Sir,Injury and trauma, often used interchangeably, represent a major health problem worldwide. Everyday around the world almost 16,000 people die from various injuries. Injuries represent 12% of the global burden of disease. and therThe improved survival and functional outcome among injured patients in developed countries can be partly attributed to high-cost equipment and technology. Much of this high-end technology is unaffordable and unavailable to victims from developing nations. However, much improvement in the outcome of trauma patients has come from improvements in the organization of trauma care services in the form of developing trauma systems in given geographical areas. The improvement and organization of trauma services or trauma systems is a cost effective way of improving patient outcome and is achievable in almost all settings.5 Proper The organization of a trauma system has four impact pillars: organization of pre-hospital care facilities, hospital networking, communication systems, and organization of in-hospital care (acute care and definitive care). An integrated approach is required at all levels: human resources (staffing and training), physical resources  and the process (organization and administration). Compared to the western world, the trauma care services in India lack each of the elements listed above. Most of the physical resources for in-hospital care in terms of infrastructure and equipment are already available at secondary and tertiary care hospitals and need moderate upgrades. Therefore, the thrust areas in the field of trauma services are as follows:Provide physical resources for pre-hospital care and communication systems.Provide well-trained staff at all levels of care from pre-hospital to definitive trauma care. Providers should be well trained and should understand the critical needs of a trauma victim. Skill-based training programs for doctors as well as paramedical staff in Acute Life Support (ALS) procedures are needed.Organize and integrate pre-hospital services with definitive care facilities  so that a patient is shifted to an appropriate facility in the shortest possible time.The Government of India has planned this organization in an apex to the base format. The establishment of the Jai Prakash Narain Apex Trauma Center (JPNATC) at the All India Institute of Medical Sciences in New Delhi is a step forward in providing an apex institution for quality trauma patient care facilities, which will act as a role model for other institutions and centers providing trauma care in the country. More than providing the best patient care facilities, the role of this apex trauma center has been envisaged as an apex research and training institution that will help the nation's administrators formulate policies regarding the organization of trauma care facilities throughout the country.It should be once again emphasized that the establishment of innumerable trauma centers with heavy financial burden should not be the goal of policy makers. Instead, upgrading existing hospital infrastructure to treat severely injured patients should be undertaken. Training of manpower in acute care and pre-hospital services should be a priority. Proper organization and administration of trauma services along with legislative backup will go a long way in strengthening India's essential trauma care services.


Bacterial plaque has been implicated as the primary etiological factor in the initiation and progression of periodontal disease. Anatomic factors such as cervical enamel projections, enamel pearls, and developmental grooves are often associated with advanced, localized periodontal destruction. Enamel projections and pearls are mostly associated with molars. Enamel projections in the furcation areas of molars have no true attachment and are therefore highly susceptible to the creation of a deep periodontal pocket. A close association has been reported in the past between enamel projection and furcation involvement. Here, we present a case report with bilateral cervical-enamel projection and its management by flap operation with odontoplasty, and regenerative procedure by placement of graft in the osseous defect. Decreased pocket depth and a gain in clinical attachment level were observed on follow-up. Periodontal disease is not a single entity nor is there a single causative factor that elicits a consistent periodontal response. The primary cause of gingival inflammation is bacterial plaque along with other predisposing factors. Dental plaque has been implicated as the primary etiological factor in periodontal disease. Host response to this etiological factor presents a wide range of responses. The irreversible damage follows the form of inflammatory periodontal disease when etiological factors remain undetected or ignored and their effects, over a period of time, surpass the resistance capacity of the host. It is of paramount importance to disclose and reverse the factors producing the changes early in the genesis of the disease process.Factors propagating plaque retention and accumulation include calculus, topography of the root, inadequate restoration, overhangs, food impaction, orthodontics appliances, tooth position, iatrogenic factors, extraction of third molars, periodontal pockets, and dental caries.Anatomic aberrances like palatoradicular grooves, cervical enamel projection, enamel pearls, cysts, foreign bodies, paltal rugae, etc.Habits and self-inflicted injuries.Mechanical factors include improper tooth brushing, use of abrasive dentrifices and other oral hygiene modalities like dental floss and oral lavage. Thermal and radiation factors include tissue burn due to hot food, electrosurgery, and biochemical factors include injury due to use of dental materials, tobacco, and chemical dessicants.Additional local factors contributing to inflammatory and degenerative results include:Developmental abnormalities such as cervical enamel projection, enamel pearl, or palatogingival grooves, may predispose the affected area to plaque accumulation, making oral hygiene procedure, scaling, and root planing difficult, and consequently, cause periodontal breakdown. “Cervical enamel projections (CEPs) are the focal apical extension of the coronal enamel beyond the normally smooth cervical margin on to the root of the tooth”. They are flat ectopic deposits of enamel that are triangular in shape and tapering in form, extending apically into the furcation area.Cervical enamel projections on molars have been defined as a dipping of the enamel from the cemento-enamel junction toward the furcal area of molars.2 Strong The current article reviews the literature, stresses the importance of contributing factors in the initation and progression of periodontal disease, and presents a case report with bilateral cervical enamel projection and its successful management.st half of the 19th century when detailed descriptions of enamel pearls and cervical enamel projections were presented. A treatise on the Comparative anatomy of the teeth (1840) with exquisite illustrations clearly indicates the presence of enamel extensions on both maxillary and mandibular molar in both humans and primates.[Interest in root enamel goes back to the 1primates.Linderer's article et al.[6In 1967, a study by Leib et al.–67Masters and Hoskins suggesteGrade I - The enamel projection extends from the cementoenamel junction of the tooth toward the furcation entrance.Grade II - The enamel projection approaches the entrance to the furcation. It does not enter the furcation, and therefore, no horizontal component is present.Grade III - The enamel projection extends horizontally into the furcation.The reported prevalence of CEPs has varied significantly among studies; overall mean values have ranged from 8.6 to 32.6% in molars.–3The prevalence of CEPs varied between the first, second, and third molars.A higher prevalence of CEPs was found in the mandibular molars than in the maxillary molars.The prevalence is highest for mandibular and maxillary second molars.Cervical enamel projection has been reported on maxillary central incisors and premolars.Found frequently on buccal surface.Grade II enamel projection has higher prevalence.Zee and Bratthal have stuAtkinson first meMasters and Hoskins later stThe enamel projection may be eliminated down to the crestal bone level by ‘saucerization. OsteoplaA 24 year-old boy reported to the Department of Periodontics at the Government Dental College and Hospital, Aurangabad, with the chief complaint of bleeding gums. A diagnosis of localized chronic periodontitis was made on the basis of history and clinical and radiological examination results. Clinical examination showed deep periodontal pockets on the lingual surface with Grade II furcation involvement in the right and left mandibular first molars. Radiographic examination showed cervical enamel projection; phase I therapy was completed. Open flap debridement revealed Grade II cervical enamel projection on the lingual surfaces of #36 and #46. The cervical enamel projection was eliminated down to the crestal bone level by odontoplasty followed by a regenerative procedure. The sixth month's follow-up of the patient showed clinical attachment gain and decreased periodontal probing depth Figures –8.Cervical enamel projection is the most common anatomical variation in molar teeth. Cervical enamel extensions occur at the mid-cervical line and consist of a V-shaped, smooth or rough extension of the enamel over the root. Cervical enamel extensions are associated with chronic periodontal infection in furcal areas and with inflammatory cysts known as buccal bifurcation cysts. The buccal bifurcation cyst is most commonly seen in younger children. CEPs can be detected on routine examination with an explorer or probe, radiograph or during surgical intervention.et al.[A higher prevalence of CEPs was found in mandibular molars than in maxillary molars. The prevet al. reportedet al. reportedet al. have repet al. ThereforCEPs are probably related to more rapid progression of pocket formation because of their anatomy and location. The enamel covering of the CEP precludes an organic connective tissue attachment due to which a hemidesmosomal attachment probably exists in the region of the CEP, which may be less resistant to breakdown by bacterial plaque. Once breakdown occurs, rapid progression of the disease becomes more likely because the projection morphology of the cervical enamel allows the retention of microbial dental plaque. In addition, the region's inaccessibility to cleansing and its proximity to the furcation could predispose the pocket to further furcation invasion.A few reports have provided some guidelines for the treatment of multirooted teeth with furcation involvements associated with prominent enamel projections. One of the techniques is the incorporation of projection flattening or removal, into surgical procedures such as the modified Widman flap or the exicisional new attachment procedure combined with furcationplasty. This would contribute to the promotion of collagen fiber attachment in grade I and shallow grade II furcal lesions. In addition, opening the furca for access to plaque control with special brushes or cleaners is another adjunctive approach for the management of grade III and grade IV furcal lesions.In the present case, the most viable therapeutic approach was thought to be an exploratory surgical procedure to determine the nature of the entity and to render appropriate treatment. Intraoral periapical radiographs showed cervical enamel projections extending below the crest of the bone. Open flap debridement revealed grade II cervical enamel projection with the lingual surface of the right and left mandibular first molars. The actual treatment performed was uncomplicated and consisted primarily of the removal of the anomaly along with a regenerative procedure. This resulted in a smooth surface of the hard tooth structure, which allowed proper adaptation of the soft tissue flap. A reduction in inflammation and probe penetration occurred after periodontal therapy.Cervical enamel projections might be considered a secondary etiological factor in periodontal breakdown and attachment loss. Although at greater risk for breakdown, mandibular teeth with CEPs should be considered good candidates for regenerative procedures that target bifurcation defects.


Mathematical models of infection that consider targeted interventions are exquisitely dependent on the assumed mixing patterns of the population. We report on a pilot study designed to assess three different methods  for obtaining contact data relevant to the determination of these mixing patterns.65 adults were asked to record their social encounters in each location visited during 6 study days using a novel method whereby a change in physical location of the study participant triggered data entry. Using a cross-over design, all participants recorded encounters on 3 days in a paper diary and 3 days using an electronic recording device (PDA). Participants were randomised to first prospective recording method.P < 0.001) and composition , P < 0.001) and the total number of reported contacts was observed, highlighting the importance of sampling study populations based on household characteristics as well as age. New contacts were still being recorded on the third study day, but compliance had declined, indicating that the optimal number of sample days represents a trade-off between completeness and quality of data for an individual.Both methods captured more contacts than a pre-study questionnaire, but ascertainment using the paper diary was superior to the PDA . Paper diaries were found more acceptable to the participants compared with the PDA. Statistical analysis confirms that our results are broadly consistent with those reported from large-scale European based surveys. An association between household size , The study's location-based reporting design allows greater scope compared to other methods for examining differences in the characteristics of encounters over a range of environments. Improved parameterisation of dynamic transmission models gained from work of this type will aid in the development of more robust decision support tools to assist health policy makers and planners. Mathematical models of infection are useful decision support tools for health policy makers choosing between alternative interventions to limit the spread of infectious diseases . IncreasEuropean researchers have developed paper diary tools to estimate the number and intimacy of conversational and physical encounters between individuals . By thesThe timeliness and hence completeness of recording using paper diaries has been questioned in other applications. Stone and colleagues, using a paper diary electronically instrumented to capture opening and closure, were able to demonstrate a marked discrepancy between reported and actual compliance with completion of a pain diary . DiariesThe primary aim of this study was to compare three different methods for the recording of social contacts likely to be sufficient for transmission of respiratory pathogens. Timeliness and completeness of entries in paper diary tools was compared to those entered in a hand-held electronic diary (PDA). We also assessed the ability of a questionnaire administered before study commencement to predict social encounters. Furthermore, we employed an explicitly location-based design across all three methods  to aid in construction of bi-partite network models, in which interactions are characterised by the places in which they occur. Statistical characteristics of the recorded interactions were used for methodological assessment and comparison with European and other studies.65 adult participants were asked to complete a pre-entry questionnaire detailing their anticipated activities and social contacts over three defined days . They were then randomised to first use either a paper diary or personal digital assistant (PDA). Participants recorded their conversational or touch encounters over the three days before switching to the alternative method for the same days in the subsequent week. The study day commenced on waking and ended with sleep. The day of study commencement was not randomised, but varied with the day of enrolment between participants.Diary Tools in the Additional file The diaries were designed to explicitly allow recording of social encounters within the context of locations visited by participants on a given study day . Written informed consent was obtained from all subjects prior to participation.Statistical Analyses in the Additional file The number and type  of contacts made was described across the whole dataset, for each category of location and for each participant. The data on both total recorded encounters and uniquely named contacts on a given day was modelled using a negative-binomial regression model . The model was used to identify key participant  and methodological  predictors of observed social encounters. Observations were grouped by participant using the 'cluster' command to ensure accurate reporting of variance, given repeated measures on the same individuals over time. Predictors, including interaction terms, found to be significant (P < 0.05) in univariate analyses (see section Gross ascertainment differences between prospective diaries (paper and PDA) were assessed using the Bland-Altman test.Assuming that the paper diary would capture a similar number of contacts to previous studies  [65 adult participants took part in the study. 54 were female. A large proportion were university employees engaged in health-related research. Others outside the academic sector became aware of the study by word of mouth. 13 of the subjects were aged 20-29 years, 14 were 30-39 years, 22 were 40-49 years, 11 were 50-59 years and 5 were older than 60 years. The participants lived in households containing between 1 and 6 individuals (median of 3), of which half contained at least one member less than 18 years of age. As can be seen from Figure Almost 13,000 contact episodes were recorded across all three methods: 5,544 in the paper diary, 4,392 in the PDA and 3,035 in the pre-entry questionnaire. Approximately half of all encounters recorded by any means involved some form of physical contact. 55% of all reported contacts were made with members of the same sex. As seen in previous studies [As repeated encounters with the same nominated individual were common, similar comparisons were made using the number of uniquely identified contacts. Table Statistical Analyses in the Additional file P = 0.034) or pre-entry questionnaire  compared to the paper diary was predictive of fewer recordings. Wednesday was used as the reference for the effect of day, and study day 1 (for a given method) as the reference for the effect of study day.Results from the negative-binomial model are shown in Tables P = 0.004, Wilcoxon matched-pairs signed-rank test for difference). These differences between reporting sources were significant in a multivariate negative-binomial regression model . There was, however, a significant reduction in recording by the third study day in a model that also took day of the week into account .Participant age and sex were not found to be significant in the univariate analysis. However, household size and presence or absence of a child (<18 years) were found to be significant explanatory variables. No evidence for an interaction between household size and presence of a child was found, despite household size being significantly associated with presence of a child  all resided in households whose size was at or above the 75Non-saturation of contacts over recording days in the Additional file The number of new individuals encountered over successive study days was examined using the paper diary record, as this was the most complete. Participants recorded a median of 33 different nominated contacts range 6-115) over the 3 days. As can be observed in Figure 15 over tFurther to the difference in number of named contacts between Wednesday and Sunday, a change in the location of contacts made on these days was also observed Figure . Again, Participants reported no difficulty in remembering to take either the paper or electronic diary with them each day. Almost all reported their estimates of encounters as 'about right' by either means. There was, however, a distinction in self-reported ease of use with the majority of respondents 63%) describing the paper diary as 'easy' to use, with contrasting divided opinions on the PDA (30% 'difficult' and 35% 'easy'). Despite this perceived difficulty, good within-subject concordance was found between the two prospective reporting sources (correlation coefficient 0.69) indicating that both tools are adequate Figure . Consist% describMore participants reported filling out the diary 'within 1 hour' when using the paper form (32%) rather than the PDA (16%). Most entries were made 'several hours later' regardless of the type of diary , with more participants delaying data entry to the end of the day with the PDA (23%) compared with the paper record (13%). This self-report was concordant with the actual time from encounter to data entry as automatically captured by the electronic diary, with a mean delay of 3.6 hours (median <1 hour). Again, the timeliness of data entry did not significantly affect the number of named individuals recorded by either reporting source .This study used an explicitly location-dependent ascertainment method to capture social encounters. We were convinced of the validity of participants' self-reported encounters relevant to respiratory pathogen transmission by the rich narrative thread describing the day's activities captured within the diaries. More encounters were captured using the paper diary than other methods, with participants consistently reporting ease of use and timely data entry by this means. Moreover, respondents uniformly underestimated social encounters when asked to predict their contacts over the survey period (pre-entry questionnaire), justifying the need for contemporaneous diary recording.The study's crossover design reduced between-individual variation when comparing the number of recorded contacts across prospective  methods. Potential biases due to training, learning or 'burn-out' effects were minimised by ensuring that all participants were recruited and enrolled by a single research assistant (PMN) and by randomising subjects to commence recording using either the paper or electronic diary. Future protocols will additionally include randomisation of the first recording day , to miniSeveral factors may have contributed to the relatively poor acceptance of the electronic diary in this study compared with others, despite the relatively high representation of health-based researchers in this convenience sample who might be anticipated to be familiar with similar recording tools. Firstly, our location-based methodology may place a greater burden on participants than other study protocols. Secondly, the custom-built software designed for this study could potentially have been made more accessible with participant input over a longer development phase, although the close correlation observed between paper and PDA recordings for each subject suggested that most participants persisted with the electronic diary in spite of perceived difficulty. With the increasing use of 'smartphone' technologies, development of high-quality PDA-style software presents as an emergent challenge to field-based researchers. In relation to the study's objectives, the fact that respondents reported longer delays in entering contacts into the PDA went against the rationale for its use in other settings , as imprDefinitive validation of recorded contacts by some form of external non-participant based observation would be desirable, but aside from privacy concerns, would conceivably change behaviour. As with other studies to date designed to measure the social interactions relevant to respiratory disease transmission, this study was not able to capture illness or exposure events. Mikolajczyk et al  investigThe diary method developed for this protocol differed from tools used in other studies as location was the focal point for defining and describing each new set of recorded encounters. In consequence, and in contrast to earlier work , repeateIn an analysis of the POLYMOD data, Kretzschmar et al  have claThe usefulness of recording the intensity of contact is confirmed by consistent observation of closer mixing in home and social settings than work environments . HoweverWith others, we observed clear differences between weekend and weekdays, both in the number and location of encounters. Due to budgetary and resource limitations, we restricted collection to three study days, choosing to have only one weekend day (Sunday). The absolute number of contacts observed was similar to findings from the European POLYMOD surveys . ClearlyOur results confirm the importance of household-level mixing to providing opportunities for the spread of infection both within and between age groups. Further data collection is required to supplement this pilot study in order to aid parameterisation of models describing heterogeneity of population mixing in the Australian urban context. How these results compare to similar European studies will be of interest.Models of infectious disease spread are of use to policy makers aiming to predict the likely impact of interventions to reduce disease transmission targeted at specific age groups or social settings. Discussion of the possible benefit of school closure to mitigate the spread of pandemic influenza is a timely example and reflects current uncertainty regarding the contribution of spread in different settings to outbreak dynamics ,24.We have reported both mean and median encounters in Figure Variation in behaviour is likely to explain, at least in part, differences in the number of secondary transmission events between individuals. Published stochastic models  have exaWe have reported on the use of three different diary tools for the recording of social encounters relevant to respiratory pathogen transmission. Our results indicate that prospective diary tools provide more complete data than a pre-entry questionnaire. The study's location-based reporting design allows greater scope compared to other methods for examining differences in the characteristics of encounters over a range of environments.Now that we are confident of the quality of our data collection tool, further analysis of the detailed time-use information gathered will be undertaken, with a focus on how different measures of 'contact-intensity' may influence the characterisation of key locations for contact mixing. Improved parameterisation of dynamic transmission models gained from work of this type will aid in the development of more robust decision support tools to assist health policy makers and planners.PDA: personal digital assistant;The authors declare that they have no competing interests.JMcC, JMcV and KF performed the statistical analyses and interpreted the results. PN coordinated the study, recruiting all subjects and collating the data. JMcC, JMcV, PP, GR and TN conceived the study and designed the diary collection tools. JMcV and JMcC wrote the initial drafts of the manuscript. All authors contributed to and approved the final manuscript.Vaccine & Immunisation Research Group, Murdoch Childrens Research Institute & Melbourne School of Population Health, The University of Melbourne, Victoria, Australia School of Behavioural Science, The University of Melbourne, Victoria, Australia The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/166/prepubSupplementary Material. Supplementary Material for Comparison of three methods for ascertainment of contact information relevant to respiratory pathogen transmission in encounter networksClick here for file


A 26-year-old man presented with an irreducible right inguino-scrotal swelling and fecal discharge from the scrotum. Exploratory laparotomy and inguinal exploration revealed that the caecum, appendix, and terminal ileum had herniated into the scrotum and had perforated through the skin forming a fecal fistula. The herniated gangrenous bowel was resected and a stoma fashioned. Spontaneous entero-scrotal fistulae are very rare and eight pediatric cases have been mentioned in literature till date. We report the first case of true sliding hernia presenting as spontaneous entero-scrotal fistula in an adult. Complications like incarceration, strangulation, and obstruction of the herniae are hence not uncommon in surgical practice in such countries.Inguinal herniae are quite common in surgical practice. However, sliding herniae are rare with an incidence of 2 to 5%. The exac3, serum urea (48mg/dl), serum creatinine (0.8mg/dl), serum sodium, potassium 132 and 3.5 mEq/L, respectively. A plain abdominal radiograph of the abdomen revealed pneumoperitoneum. Further radiological investigations were limited to an ultrasound in the Emergency department showing free intraperitoneal air and fluid along with the confirmation of bowel loops in the inguinal hernia. Dye studies to identify the source of the fistula were withheld as the obstructed bowel contents in the strangulated inguinal hernia was the obvious source of the fecal fistula and the patient was planned for exploration. One unit of fresh whole blood was transfused prior to surgery. The patient underwent exploratory laparotomy through a vertical midline incision that revealed moderate amount of pus and dirty flakes contaminating the peritoneal cavity. The caecum with the appendix had traversed through right inguinal canal to lie in the scrotum. The caecum had perforated through the right scrotal skin with fecal discharge. The herniated bowel was gently maneuvered out of the scrotum and brought into the abdomen [A 26-year-old farmer presented to the Emergency department with acute abdomen, fecal discharge from the right inguinoscrotal region and shock. The patient elaborated the presence of a reducible swelling involving the right ingunoscrotal region since 5 years that became painful and irreducible the week prior to admission. Gradually abdominal distension, pain, vomiting, and obstipation ensued. Foul smelling discharge emanated from the right scrotal region 3 days back. The patient was dehydrated, had a feeble pulse (112/min), and was tachypneic (26/min). His blood pressure was 70/52 mm Hg. Clinical examination of the abdomen revealed it to be grossly distended and tender. Diffuse guarding, rigidity, and a tympanic abdomen pointed towards peritonitis. The scrotal area was found to be stained with greenish material with fecal odor. A closer examination revealed fecal material flowing out of an opening at the root of the right scrotum, the surrounding skin being inflamed and macerated. The ingu abdomen . A resecrum urea 8mg/dl, sSliding hernia (synonym: hernia en glissade) is a result of slipping of the parietal peritoneum due to elongated mesentery. In the rAn English literature search was done in Pubmed/MEDLINE and Ovid databases using the keywords “enterocutaneous fistula,” “complications of inguinal hernia”, “scroto-fecal fistula,” and “strangulated hernia”. Enterocutaneous fistulae due to previous surgery and Crohn's disease were excluded from the articles generated.et al.[et al.[et al.[Till date most cases of inguinal herniae leading to scrotal fecal fistula have been described in the pediatric age group.–4 There et al. reportedl.[et al. in theirl.[et al. There hal.[et al. In the pl.[et al.Strangulated hernial contents perforate when the twisted and thinned out bowel gives way. The ensuing leaking bowel contents macerate surrounding tissue, suppurate through the fascial planes, and finally end up forming a fistulous communication with the skin. The skin of the inguino-scrotal skin being thin probably favors fistula formation.th postoperative day. Greater skin loss would require skin grafting as a delayed procedure.In such patients, immediate surgical management is warranted as the fecal contents could destroy large areas of both skin and surrounding tissue. The paediatric series of inguino-scrotal fistulae show that the testis on the side of the hernia was either non-viable or required removal in two cases. Large areas of devitalized skin and subcutaneous tissue need to be debrided and the adjoining testis checked for viability. The scrotal wound, in our patient, was left to heal by secondary intention as primary suturing was not appropriate. Daily wound care resulted in healthy granulation and secondary suturing was performed by mobilizing scrotal skin on the 6Differential diagnosis of the acute scrotum includes acute torsion of the testes, torsion of the testicular appendage, acute epididymo-orchitis and Fournier's gangrene. In most of the cases a thorough clinical examination easily clinches the diagnosis. Torsion of the testes presents as an extremely tender testicle. Pain not relieved by elevation of the affected testis, a normal urinalysis and blood count differentiate it from acute epididymo-orchitis. Doppler evaluation of the testicular vasculature might be required in equivocal cases. Torsion of the testicular appendage presents similar to torsion of the testis and is frequently discovered at surgical exploration. Fournier's gangrene is a severe polymicrobial infection of the scrotal skin with sparing of the testes. In the initial stages, the scrotal skin is extremely painful, edematous, and inflamed. In advanced cases, necrosis of the scrotal skin ensues shamefully exposing the testes. Patients with spontaneous enterocutaneous fistulae should be evaluated for Crohn's disease. Spontaneous enterocutaneous fistula in patients with tuberculosis of the bowel has been reported in literature in areas where the disease is endemic.Vigorous resuscitation to correct fluid and acid-base deficits is of prime importance. Exploration is inevitable in most of the cases ensuring correction of shock and electrolyte imbalances present. Difficulties during inguinal dissection must be anticipated. Care must be taken to identify the contents of the hernia, most importantly the bladder, to avoid inadvertent injury. General condition of the patient guides primary bowel anastamosis or fashioning a stoma. Increasing experience with laparoscopic surgery has led to reports of the laparoscopic management of enterocutaneous fistulae in the setting of Crohn's disease. The feasPlain radiography of the abdomen is useful in confirming the presence of obstruction or perforation. Dye studies to confirm the origin and communication of the fistulae are required especially in the setting of Crohn's disease. Intra-luminal dye studies are generally avoided in the acute setting as they are time consuming and serve little purpose. In our case, the obvious fecal material exuding from the fistulous tract and the presence of pneumoperitoneum warranted exploration. The increasing use of computed tomography scans in the surgical emergency can with some accuracy identify the loop of bowel that has herniated into the inguinal canal and give information on the condition of the strangulated contents. The presence of bowel wall edema and non-enhancement of the bowel wall following intravenous contrast portend a gangrenous bowel.Most of the spontaneous entero-scrotal fecal fistulae have been reported from resource poor countries like Nigeria,–8 PakistThis report highlights the need for an early diagnosis followed by immediate surgical management of inguinal hernia. Standards of care have improved in countries like India yet such complications are not uncommon. Native indigenous treatment of inguinal swellings in the rural areas, poverty, and ignorance on the part of the patient from seeking proper medical care are the main causes for such presentation. Spontaneous entero-scrotal fistula is a rare presentation in the adult and reflects the state of health care in the developing countries. Timely elective surgery saves the patient not only from such complications but also saves money spent on treatment and less loss of working days.


An integrated computer system consisting of an expandableionanalyzer and a PC has been used to automate concentrationmeasurements with ion-selective electrodes (ISEs). The ionanalyzerdetermines ionic concentrations using a reference electrodecoupled with an ISE. The measurements and calibrations arecarried out in a thermostated sample changer equipped with a flowcell. The data obtained by the ionanalyzer are sent via a standardRS 232-C interface to a microcomputer. In this paper, we describethe automatic data acquisition system and the subsequent processingof the measurements. One program (Transorion) automaticallycollects the measurements carried out by the ionanalyzer, giving areal-time graphic representation of the measurement on thecomputer screen. A second program (Graforion) facilitates themanagement of the data stored by the first program, and listing andgraphics of these can be obtained on the printer/plotter. The methodhas been used to study continuous concentration changes in anaqueous solution of potassium iodide.


The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes.The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test.+ cells was preserved. Furthermore, the observed increase of CD4+ lymphocytes could be ascribed to the accumulation of CD4+ cells with effector memory phenotype.KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECsThe decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease. Li. Liet aler et al . This diiu et al , we morets Table , which i+ effector memory cells in the peripheral blood. On the contrary, the number of newly produced B cells is precociously reduced and this may represent a warning signal anticipating the profound defects of humoral immunity, which normally characterize the later stages of the disease. Therefore, we are currently following patients at later stages of the disease in order to investigate modifications of newly produced B and T lymphocytes in the course of the therapy.Based on these preliminary observations we suggest that the production of new T lymphocytes is normal in CLL at the very early disease stage; the presence of CD4 lymphocytosis can be partially ascribed to the accumulation of CD4The authors declare that they have no competing interests.LI was the principal investigator and takes primary responsibility for the paper. MM and GR recruited the patients. MC, CG, CZ and CL performed the laboratory work for this study. LI, MM, LC and GR wrote the manuscript and participated to the discussion. All authors read and approved the final manuscript.


Despite being reported rarely, renal cell carcinoma is the third most frequent neoplasm to metastasize to the head and neck region preceded only by breast and lung cancer. Little information exists regarding the presentation and work-up of metastatic renal cell carcinoma in the oral cavity.We report the case of a 63-year-old Caucasian man presenting with an oral cavity lesion that was painful and that had grown substantially over several months. Biopsy resulted in persistent bleeding requiring cautery and manual pressure. Immunoperoxidase testing was necessary to make the diagnosis of metastatic renal cell carcinoma and rule out other clear cell carcinomas of salivary gland origin.Metastatic renal cell carcinoma is part of the differential diagnosis for patients presenting with a new head or neck lesion in the setting of a history of kidney cancer. The physician needs to be prepared for the increased risk of bleeding and understand the importance of immunohistochemical staining to differentiate between metastatic renal cell carcinoma and malignancies of salivary origin. Unfortunately, the prognosis is invariably poor in these patients. Metastatic lesions of the oral cavity are extremely rare, accounting for approximately 1% of all malignant oral tumors. Renal cell carcinoma (RCC) is the third most frequent neoplasm to metastasize to the head and neck region preceded only by breast and lung cancer. It accounts for nearly 3% of all adult malignancies and is the most lethal urologic cancer. Approximately one-third of patients present with metastatic disease and 40% to 50% will develop distant metastases (asynchronous metastatic disease) after the initial diagnosis. The expected 5- and 10-year survival rates for these patients are 5–30% and 0–5%, respectively. The most common sites of metastasis include the lungs, regional lymph nodes, bone, liver, adrenal glands, contralateral kidney and brain [Despite being reported infrequently, head and neck region metastases may be linked to RCC in up to 8–15% of cases . The nosA 63-year-old Caucasian man presented to his primary care physician with a 6-month history of intermittent right anterior neck and intraoral pain. The patient noted a tongue mass, which had grown substantially over the last several months. The mass made eating difficult at times and resulted in one episode of mild oral bleeding that resolved spontaneously. He was referred to our institution's department of otolaryngology/head and neck surgery for further evaluation.The patient's past medical history is significant for RCC of the right kidney diagnosed 4 years prior and treated with right radical nephrectomy. An appropriate work-up at that time included a CT scan of the chest, abdomen, and pelvis and liver functions tests, all of which were negative for metastatic disease. He did not follow-up with his urologist as recommended after the surgery.The physical exam revealed an erythematous, indurated 3 cm mass in the right anterior floor of mouth region that was tender to palpation. It was not fixed to the mandible and appeared vascular. The neck exam was positive for a 3 cm firm mass in the right thyroid lobe with no pathologic lymphadenopathy otherwise.Biopsy of his anterior floor of mouth lesion was notable for persistent bleeding and revealed clear cell carcinoma, consistent with the patient's previous history of renal cell cancer Figure . HistoloOriginal surgical, pathology and postoperative records were eventually obtained revealing the discovery of suspicious lymph nodes near the renal hilum during his original nephrectomy. The resected lymph nodes were found to harbor metastatic carcinoma and the patient was referred to a medical oncologist at that time to discuss additional therapeutic options. Unfortunately, he did not seek further care for his metastatic RCC. After receiving the news of his biopsy results in our clinic, the patient decided to return to his original institution for further care and died of metastatic disease within several months.Metastatic tumors to the oral cavity are extremely rare. Therefore, existing literature is based largely on sporadic case reports. Possible routes of metastasis to the oral cavity include arterial, venous and lymphatic circulations. In the case of head and neck metastasis without lung involvement, several theories exist to address a route of dissemination that avoids pulmonary vascular filtration. These include spread via Batson's venous plexus or through the thoracic lymphatic duct. Batson's paraspinal plexus is a valveless, venous system extending from the skull to the sacrum allowing tumor emboli to bypass the pulmonary venous system with minimal resistance resulting in metastasis to the head and neck region in the absence of obvious lung lesions .Reports of RCC to the head and neck region involve the nose, tongue, paranasal sinuses, larynx, mandible, temporal bone, thyroid gland, and parotid glands . The locMetastatic RCC will often behave similarly to that of the primary renal lesion in terms of morphology and histology. The risk of bleeding after fine needle aspiration biopsy of RCC in the kidney is well documented, with up to 90% of patients bearing evidence of perinephric bleeding on CT imaging and 5–7% developing clinically significant hemorrhage [Histologically, differentiating among clear cell tumors with conventional light microscopy can be challenging. It can be especially difficult to distinguish between RCC metastasis and clear cell malignancies of salivary glands. Clear cell carcinomas of salivary gland origin are usually nests of clear cells divided by thin, fibrous connective septa and irregular vascular tissue. Immunohistochemical staining helps in this distinction, with RCC metastasis exhibiting focal cytokeratin positivity  and a strong reaction for vimentin .Treatment of renal adenocarcinoma metastasis to the head and neck is directed mainly toward palliation . ExcisioOf interest in this patient is whether other measures taken at the time of the initial diagnosis and treatment period may have slowed disease progression or prolonged survival. Certainly, the finding of lymph node involvement portends a poor prognosis with 5- and 10-year survival rates similar to those with systemic metastasis . An extended lymph node dissection has been suggested to benefit patients with early, isolated or microscopic involvement of the nodes. However, this remains controversial with a lack of randomized data to support any survival advantage [Immunologic therapy following radical nephrectomy in the setting of metastatic disease may improve time to progression in properly selected patients. Prospective trials have demonstrated that, in patients with synchronous metastatic disease, cytoreductive nephrectomy and a systemic cytokine give a distinct survival advantage over those treated with immunotherapy alone (17.4 versus 11.7 months in patients with an Eastern Cooperative Oncology Group performance status of 0–1) [Finally, newer agents targeting the VEGF pathway such as bevacizumab and sorafenib may provide hope for patients with metastatic RCC. Early trials have shown a prolongation of progression-free survival with the use of these targeted molecular therapies in cytokine-refractory patients .RCC has been shown to metastasize to the head and neck region in rare instances. Therefore, the work-up of a new oral or neck lesion in light of a history of RCC should include metastatic RCC as part of the differential diagnosis. The physician needs to be prepared for the increased risk of bleeding involved in the biopsy of RCC metastasis. Should the biopsy specimen reveal clear cell carcinoma of the mouth, it is vital to perform immunohistochemical staining to differentiate between metastatic RCC and malignancies of salivary origin. If a diagnosis of metastatic RCC is established, additional therapeutic options, including immunotherapy, tyrosine kinase inhibitors, and participation in a clinical trial, should be discussed with the patient despite the poor overall prognosis.CT: computed tomography; RCC: renal cell carcinoma; VEGF: vascular endothelial growth factor.The authors declare that they have no competing interests.TW collected and interpreted all of the urologic data pertinent to the case report and was the major contributing author of the final written report. NA collected and interpreted all of the otolaryngologic data pertinent to the case including the pathology specimens deemed Figures Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Delleman Oorthuys syndrome (oculocerebrocutaneous syndrome) is a rare, congenital sporadic disorder affecting the skin and central nervous system. We present the case of a one-month-old male who presented with an orbital cyst in the left eye since birth along with other manifestations of this syndrome. The manifestations of this syndrome resemble other developmental disorders like Goldenhar and Goltz syndrome. Conservative management of the orbital cyst in these cases have been described. The need to diagnose this rare congenital anomaly with cerebral malformations as a separate entity is crucial in the management of these children. This entA one-month-old infant of healthy parents with a 39-year-old father and 26-year-old mother presented to us with left-sided ocular swelling which was first noticed at birth and had increased to the presenting size in one month . The chiA large cystic swelling in the child's left orbit was positive for transillumination test. Skin over the swelling was hypoplastic with prominent veins. There was no other area of skin hypoplasia elsewhere in the body. Multiple cystic appendages were present in the periorbital region. No globe was appreciated on the left side. Examination of the right eye was normal. B-scan ultrasonography of the left orbit showed fluid-filled cystic areas with no evidence of globe.Systemic evaluation including neurological examination revealed no abnormality. Developmental milestones were normal for age.On axial computed tomographic scan there was anophthalmia with a hypodense cystic area in the left orbit. This area had no intracranial communication . There wThe patient underwent a simple cyst aspiration (with 20 mL of fluid drained). There was a recurrence of cystic swelling at one month follow-up, and repeat cyst aspiration was done with addition of ethanolamine oleate sclerotherapy (0.8 mL). Excision of periocular skin appendages was done simultaneously. This was followed by placement of a conformer in the empty socket. No recurrence of cyst was noticed in one year of follow-up .Oculocerebrocutaneous syndrome (OCC), a rare genetic disorder is characterized primarily by an orbital cyst with periorbital skin appendages which may be associated with anopthalmia or microphthalmia, major cerebral malformation, and focal dermal hypoplasia or aplasia.–4 The orNeurological abnormalities associated with OCC syndrome may include malformations of the ventricular system in the brain like colpocephaly, multiple fluid-filled cystic cavities within the cerebral and cerebellar cortex, agenesis of corpus callosum, and hydrocephalus.–4 AffectThe cutaneous features include underdevelopment or absence of skin in certain localized regions . Characteristic of this syndrome are pink-colored or flesh-colored outgrowths of skin (cutaneous tag) within certain facial areas, most commonly the periocular area.Lesions in the syndrome correspond to a disruption of embryonic development at a very early stage. Microphthalmos with cyst is related to incomplete closure of the embryonic fissure, normally occurring at five to six weeks, with cysMost of the cases reported were sporadic with no reports of affected siblings. The inheritance pattern of OCC is variable, but the most common hypothesis put forward for this syndrome is mosaicism. However,Delleman Oorthuys syndrome shows overlapping clinical features with Goldenhar syndrome and Goltz syndrome. However, in the Goldenhar syndrome, the characteristic feature is of epibulbar dermoid and skin tags in the preauricular region with vertebral anomalies and absence of cerebral cyst. Goltz syndrome is an X-linked dominant syndrome, occurring only in females , may have microphthalmos and coloboma and focal dermal hypoplasia but the other characteristic feature is of polysyndactyly and poor dendition.Management of orbital cyst in previously reported cases was by aspiration of cyst followed by dissection and removal of cyst from surrounding periocular structures. We used We also recommend a detailed neurological workup in these children; and if a cerebral or cerebellar manifestation such as hydrocephalus is present, it warrants urgent neurological opinion for surgery to prevent further neurological damage. Patients with congential cystic eye with periocular skin appendages with cerebral malformation should be assessed by the ophthalmologist, neurophysician and pediatrician in close coordination for proper management of these patients.


The end-Permian biotic crisis (∼252.5 Ma) represents the most severe extinction event in Earth's history. This paper investigates diversity patterns in Anomodontia, an extinct group of therapsid synapsids , through time and in particular across this event. As herbivores and the dominant terrestrial tetrapods of their time, anomodonts play a central role in assessing the impact of the end-Permian extinction on terrestrial ecosystems. Taxonomic diversity analysis reveals that anomodonts experienced three distinct phases of diversification interrupted by the same number of extinctions, i.e. an end-Guadalupian, an end-Permian, and a mid-Triassic extinction. A positive correlation between the number of taxa and the number of formations per time interval shows that anomodont diversity is biased by the Permian-Triassic terrestrial rock record. Normalized diversity curves indicate that anomodont richness continuously declines from the Middle Permian to the Late Triassic, but also reveals all three extinction events. Taxonomic rates (origination and extinction) indicate that the end-Guadalupian and end-Permian extinctions were driven by increased rates of extinction as well as low origination rates. However, this pattern is not evident at the final decline of anomodont diversity during the Middle Triassic. Therefore, it remains unclear whether the Middle Triassic extinction represents a gradual or abrupt event that is unique to anomodonts or more common among terrestrial tetrapods. The end-Permian extinction represents the most distinct event in terms of decline in anomodont richness and turnover rates. The most severe extinction event in Earth's history at the Permian-Triassic boundary  had a major influence on the diversity of life This study focuses on diversity patterns of Anomodontia, an extinct group of terrestrial tetrapods, throughout the Permian and Triassic periods and in particular across the end-Permian extinction event. Anomodonts belong to the diverse clade of Therapsida and constituted the major primary consumers among vertebrates of their time. Therefore, they play a central role for assessing the impact of the end-Permian extinction on terrestrial ecosystems. A well-documented, cosmopolitan fossil record reflects their great taxonomic and morphological diversity, ranging from small burrowing forms to large grazers, which is unparalleled by any other clade of Permian-Triassic terrestrial tetrapods Diversity studies can be performed on a global or local scale, with both approaches having advantages and disadvantages Lystrosaurus there are three distinct species, L. curvatus, L. maccaigi, and L. hedini, that are known from below as well as above the Permian-Triassic boundary Methods to estimate global biodiversity patterns can be divided into taxonomic, phylogenetic, and morphological approaches. Phylogenetic diversity measures use a phylogenetic tree topology to infer extended time ranges via ghost lineages, assuming the same age of sister taxa The present investigation utilizes quantitative methods to assess the taxonomic diversity of anomodonts at the genus and species level. Taxonomic diversity has been extensively studied for many clades. Early studies on the global taxonomic diversity of tetrapods in general and across the PTB were undertaken by Pitrat The goal of the present study is a global taxonomic diversity assessment  to be performed at genus and species levels. Subsequently, the results are tested for a possible correlation with the Permian-Triassic terrestrial rock record to account for potential sampling biases. Ultimately, this study aims to provide new insights into the taxonomic patterns of extinction, recovery, and diversification of the most diverse and dominant clade of Permian-Triassic terrestrial herbivores and thus the impact of the end-Permian extinction on the continental vertebrate ecosystem.The diversity assessment of the present study is based on a dataset that has recently been assembled as part of a review of the stratigraphic correlation, composition, and similarity of global anomodont-bearing tetrapod faunas from the Permian and Triassic The current study makes a number of assumptions that the reader needs to be aware of, including the uncertainty regarding the correlation of the LVFs to the SGCS. This concerns the analysis at the stage-scale as well as at the numerical time scales using one and five million-year intervals, respectively (see below). Furthermore, the stratigraphic ranges of the anomodont taxa are not necessarily precisely known, because not all occurrences are tied to measured stratigraphic sections. The listed stratigraphic ranges  are takeTapinocephalus AZ) and Gamkan II (Pristerognathus AZ) to reflect the finer stratigraphic resolution of the South African Karoo Basin and correlated strata. The duration of the LVFs varies between 1.5 and 6.5 Ma with a mean duration of 3.19 Ma (3.71 Ma in the Triassic vs. 2.58 Ma in the Permian). To avoid a bias of the diversity patterns by the unsteady duration of the sampling intervals, the third and fourth sets of time bins uses one million year and five million year intervals, respectively. The stratigraphic framework is based on the most recent Geologic Time Scale The present dataset incorporates a total of 128 species in 68 genera from 77 faunal assemblages, spanning a time period of 42 Ma . The focbL), taxa that have their first occurrence within an interval and cross its top boundary (NFt), and those that cross the bottom and top boundaries of an interval (Nbt). The fifth variable is represented by taxa that are known before and after an interval but not within it, also known as Lazarus taxa. For anomodonts this variable is negligible and not further considered. Various combinations of these variables result in different richness counts that respond differently to biases (see below). For example, the total diversity is given as NTOT = NbL+NFt+Nbt+NFL.Among diversity assessments, richness is usually calculated on the basis of up to five variables A large number of potential sampling biases have been proposed to effect diversity assessments, of which some or all have previously been investigated for selected datasets. These include a possible taxonomic bias, time scale bias, collection bias, rock record bias, population bias, research effort bias, and taphonomic bias. With respect to a taxonomic bias, Wagner et al. TOT) is investigated in the framework of four divergent sets of time bins, i.e. stages, LVFs, as well as one and five million year intervals (see above). However, with respect to the Middle to Late Triassic the 1-Ma-scale utilizes a higher stratigraphic resolution than is provided by the current correlation scheme. Therefore, it needs to be clarified that the richness curves during the Triassic are artificially high (see Foote TOT−NFL) and one by calculating the estimated mean standing diversity In addition, at the 1-Ma-scale two further diversity assessments were performed, one excluding singletons  within an interval.Regarding taphonomic biases, it is well established that different environments and preservational conditions can result in different relationships between live richness and death richness In contrast, a research effort bias and most significantly an available rock record bias have previously been reported to have an effect on diversity assessments. For example a positive correlation of rock outcrop area, rock volume, or number of formations with taxonomic richness has been observed for a number of samples O and rE). Thereby, the origination rate (rO) is calculated as the number of originating taxa (NFL+NFt) divided by the total number of taxa (NTOT) within one interval. For extinctions, the per-taxon rate (rE) represents the number of taxa becoming extinct (NFL+NbL) divided by the total number of taxa (NTOT) within one interval. At the stage level, origination and extinction rates per interval were further divided by the duration of each stage (Δt). Van Valen metrics for origination and extinction were also calculated but are not illustrated as they show a matching pattern to the per-taxon rates bt, NbL, and/or NFt values of zero. In addition, the rates of diversification (rD = rO−rE) and turnover (rT = rO+rE) were calculated to establish the overall change in the composition of anomodont faunas throughout the Permian and Triassic.Origination and extinction rates were calculated for the international marine stages and the one million year intervals using Sepkoski's TOT−NFL) and the estimated mean standing diversity (EMSD) at the 1-Ma scale only.Anomodonts experienced three distinct extinctions during the Permian and Triassic, at the end of the Guadalupian, the end of the Permian, and close to the Anisian-Ladinian boundary. This is indicated by analysis of the raw richness data at the stage-, LVF-, 5-Ma- and 1-Ma-scales, including investigations of the total richness at all scales and furthermore total richness without singletons (NTOT) increases from initially four genera and five species to twelve genera and thirteen species in the late Guadalupian (Gamkan I LVF). In the early Lopingian (Gamkan II LVF), genus and species diversity is cut in half, but thereafter increases continuously until it reaches its maximum of 20 genera and 41 species in the latest Permian Platbergian LVF (Changhsingian). At the end of the Permian, global anomodont diversity collapses down to only two genera and nine species in the earliest Triassic Lootsbergian LVF (Induan and early Olenekian), suggesting a major impact of the end-Permian extinction on terrestrial tetrapod ecosystems. Recovery of the global anomodont diversity after the PTB lasted several million years, but diversity increased continuously to reach a second peak of twelve genera and 23 species in the Perovkan LVF (late Anisian and early Ladinian). Thereafter, anomodont diversity rapidly collapsed for a third time at the Anisian-Ladinian boundary and remained low, not exceeding three genera and species until the extinction of the clade in the late Triassic (Norian).Anomodont diversity at the LVF-scale  shows thThe diversity pattern at the LVF-scale changes slightly when corrections for the unequal durations of the LVFs are made . First, Anomodont diversity at the stage-scale  was alsoIn contrast, the diversity pattern at the 5-Ma-scale  depicts TOT) of anomodonts . Moreover, a statistically significant positive correlation between the number of taxa and the number of formations per interval is apparent from scatter plots as well as Spearman's ρ values , 5. The To investigate to what extent the terrestrial rock record throughout the Permian and Triassic influences the observed diversity pattern of anomodonts, the total diversity and the total diversity without singletons was divided by the number of species-informative anomodont faunas at the stage-scale and at the1-Ma-scale, respectively . These nIn conclusion, the general pattern of the normalized curves implies a rapid initial diversification to the overall maximal genus and species richness at the end of the Middle Permian, followed by a continuous decline in diversity interrupted by the previously observed peaks in the latest Permian and during the Middle Triassic.Taxonomic rates (origination and extinction) are important aspects in investigating the diversification and extinction of a clade, and respond quite differently to biases in the fossil record. Most importantly, Foote The per-species origination and extinction rates as well as the resulting diversification and turnover rates are illustrated at the stage-scale and1-Ma-scale . The samDiversity curves of the raw data indicate that anomodonts experienced three distinct diversifications with subsequent extinction events during their evolutionary history in the Permian and Triassic. The observed extinctions are at the end of the Guadalupian, at the end of the Permian, and close to the Anisian-Ladinian boundary. The former two of these extinction events have previously been documented on the basis of the tetrapod fossil record There is some variation in the observed diversity patterns based on the different diversity measures applied. Given the stratigraphic framework used as basis for this study, the diversity curve at the 1-Ma-scale shows an increased stratigraphic resolution and consequently a more accurate reflection of taxonomic richness, at least for the Permian. Moreover, except for the time-corrected richness curves at the LVF-scale and the stage-scale, the results of the diversity assessments on the different time scales are consistent in the recovery of the three events, respectively, suggesting a real signal at the end of the Guadalupian. Alternatively, the failure to recover the end-Guadalupian event at the time-corrected LVF-scale and the stage-scale might suggest that the detection of this event at the remaining scales potentially represents a relic of an apparent rather than a genuine increased stratigraphic resolution. Analyses on the genus and species level likewise reveal coherent patterns. The exclusion of singletons at the 1-Ma-scale results in lower richness peaks in the Middle and Late Permian but has no impact on the Triassic pattern. Diversity curves based on the estimated mean standing diversity (EMSD) recover the previously observed overall pattern of anomodont diversity but partially distort the relation between Permian and Triassic richness. This is the result of fundamental differences between the terrestrial rock records of these two periods. The overall maximum of anomodont diversity in the Triassic can be interpreted as a relic of the coarse correlation of global anomodont faunas, which are at the scale of the LVFs, whereas the Permian and earliest Triassic record is continuous and much better resolved as a result of the excellent sequence in South Africa. This includes the taxonomic uncertainty for Triassic anomodonts as well as the limited knowledge about the actual stratigraphic ranges of the Middle and Late Triassic anomodont taxa, causing an artificially high overall diversity during this time, which is indicated by the plateau in the Middle Triassic at the 1-Ma-scale . Thus, tThe impact of the end-Permian extinction on terrestrial tetrapod ecosystems has been hotly debated in the last decades. Early workers rejected the idea of an end-Permian extinction event on land simultaneous and similar in force to the marine realm, but rather argued for a gradual extinction of terrestrial tetrapods The analysis of raw diversity data has often been challenged In an attempt to correct for this bias imposed by the variable rock record, the diversity curves of anomodonts were normalized by dividing the richness counts by the number of formations per time interval. These normalized diversity curves display previously observed elements but generally show a pattern that is quite different from that of the raw data. It reflects a rapid initial diversification to overall maxima of the genus and species richness at the end of the Middle Permian, being followed by a continuous decline in diversity only broken up by the peaks in the latest Permian and during the Middle Triassic . AlthougIt is important to note that diversity patterns and taxonomic rates (origination and extinction) respond differently to heterogeneity in the fossil record The pattern of origination and extinction of anomodonts, as described above, reveals that the raw species originations and extinctions  closely In conclusion, the pattern portrayed by the taxonomic rates of anomodonts indicates distinct changes in diversification and turnover dynamics throughout the Permian and Triassic. Anomodonts confirm the previously noted pattern that the end-Guadalupian and end-Permian extinctions were not simply driven by increased rates of extinction but also by low origination rates . However, this pattern is not evident at the final decline of anomodont diversity during the Middle Triassic. In contrast, taxonomic rates are equally unstable, throughout the entire period and the observed extinction in the Middle Triassic is not evident from the taxonomic rates alone. For this reason together with the apparent influence of a bias in the terrestrial rock record on the raw diversity curves, it remains unclear whether the Middle Triassic extinction represents an actual event or if it is a relic of our limited knowledge of the fossil record during this time. In any case, on the basis of the normalized diversity curves in combination with the pattern of taxonomic rates, it can be concluded that anomodonts experienced three distinct periods of diversification and at least two extinction events, i.e. the end-Guadalupian and end-Permian events, before their final decline towards the end of the Triassic. The end-Permian extinction represents the most distinct event in terms of decline in anomodont richness and turnover rates. Only further improvements to the stratigraphic framework and additional investigations on the diversity patterns of other Permian-Triassic terrestrial tetrapods will be able to reveal if the mid-Permian event corresponds to the end-Guadalupian event of the marine realm and whether the mid-Triassic decline of anomodont diversity represents a gradual or abrupt event that is unique to anomodonts or more common among terrestrial tetrapods. Ultimately, the incorporation of a phylogenetic perspective with the consideration of ghost-lineages at the species level will be an essential step to improve our understanding of the actual survivorship of anomodonts across the Permian-Triassic extinction event.Text S1Explanation of stratigraphic ranges of anomodont species from left to right as displayed in (0.03 MB DOC)Click here for additional data file.


Hepatitis A virus (HAV) strains detected in environmental and clinical samples were analysed to characterize the genotypes of HAV circulating in Greece. Fifty (50) sewage samples were collected from Patras (South-Western Greece) and Alexandroupolis (North-Eastern Greece) from 2007 until 2009, accordingly. The clinical samples derived from an HAV outbreak involved populations from three neighbouring prefectures of North-Eastern Greece . HAV particles were detected by nested RT-PCR, using a previously validated set of primers to amplify a 290-bp fragment encompassing the 5'-NTR. Positive HAV samples were confirmed by sequencing of the PCR product. To determine the relatedness between the different isolated sequences, a phylogenetic tree was constructed.Results showed a 100% prevalence of genotype I, and particularly subgenotype IA. The analyzed HAV strains were closely related between them with the percentage of nucleotide identity ranging between 96% and 100%.The study revealed the major prevalence of circulating strains of IA genotype in Greece and underlined the usefulness of molecular methods for the detection and typing of viruses in both environmental and clinical samples. The present study is, to our knowledge, the first in Greece to depict the simultaneous molecular characterization of HAV strains isolated from both clinical and environmental samples. The Hepatitis A virus (HAV) is responsible for around half the cases of hepatitis diagnosed worldwide and is recognized currently as one of the most important human food-borne pathogens, as it is the cause of most outbreaks reported in the Western world. It is not possible to distinguish HAV strains by serotyping, but seven genotypes can be differentiated with molecular methods . HAV infAn epidemiological shift, from high to low prevalence, has been observed in recent decades in the countries of Southern Europe, including Greece. Consequently, the Mediterranean basin as a whole should no longer be considered as an endemic area ,6. StudiThe aim of the present study was to correlate HAV isolates from clinical and environmental samples by applying molecular methods in order to reveal the prevalence of genotypes of HAV in Greece. HAV strains from environmental sewage samples, analysed over a 2-year period (2007-2009), were collected from the cities of Patras and Alexandroupolis. Clinical HAV strains were collected during a major HAV outbreak among patients with acute hepatitis, from the hospitals of the cities of Alexandroupolis, Komotini and Xanthi.A total of one hundred 100) sewage samples were collected from the biological treatment plants of two large Greek towns, Patras (SW Greece) and Alexandroupolis (NE Greece), from 2007 until 2009. Samples were collected at the entry-point of the Patras' biological treatment plant, which receives sewage from a population of about 250,000 inhabitants. Sewage samples were also collected from both the entry and exit-points of the treatment plant of Alexandroupolis, a city of approximately 50,000 inhabitants. Sewage sampling, concentration and HAV detection were performed according to previously described protocols 0 sewage .Serum samples from hospitalized HAV outbreak cases were collected and analysed for HAV as previously described . A totalViral nucleic acids were extracted using the QIAamp Viral RNA mini-kit (Qiagen), in line with the manufacturer's instructions. Reverse transcription polymerase chain reaction (RT-PCR) and nested PCR techniques were used for the detection of HAV, according to previously published protocols . A 290-bhttp://www.ebi.ac.uk. The neighbour-joining method was applied for the phylogenetic tree analysis, the reliability of which was assessed by bootstrap resampling , using MEGA 4.0.2 software. The HAV genotype was determined by comparing the different sequences of the Greek strains included in the phylogenetic analysis with the reference sequences of different HAV genotypes.All positive samples  were confirmed by sequencing of the PCR product. The purified PCR products of the clinical HAV strains were sequenced by Lark Technologies , and the environmental strains by VBC-Biotech (Austria). The nucleotide sequences obtained were analyzed by BLAST N program at the NIH website , and were compared with each other and with other published sequences deposited in the GenBank database. Multiple alignments were performed using Clustal W2 software HAV was detected in four sewage samples  collected from the inlet of the biological treatment plant of Patras and in one sample  collected from the entry-point of the treatment plant of Alexandroupolis. Sera from eight hospitalized patients with acute hepatitis were collected and analyzed for the detection of HAV genome .Sequence analysis of the nested PCR products of 5'NTR region, showed high degree of identity among environmental and clinical samples. Comparison of the nucleotide sequence of PAT73  with the sequences of the other sewage samples from Patras , the sewage sample from Alexandroupoli (ALEef) and the clinical strains from the cities of Komotini , Xanthi  and Alexandroupoli , showed 96%-100% similarity Figure . ComparaThe hepatitis A virus still represents a major public health problem. HAV is a significant cause of morbidity and socioeconomic losses in many parts of the world, while it can result in fulminant hepatitis and death, albeit rarely . HAV hasThe hepatitis A vaccine is included in the Greek National Immunization Program (GNIP) and universal vaccination is recommended for all children over 12 months of age . With reIn conclusion, this study - the first in Greece to depict the simultaneous molecular characterization of HAV strains isolated from both clinical and environmental samples - revealed the major prevalence of circulating strains of IA genotype in Greece. Further environmental surveillance could be used in order to enrich the poor existing clinical data from Greece and evaluate the prevalence of HAV in the environment as well as in the community.Environmental surveillance could prove to be a valuable strategy in the study of prevalence and of the incidence of various pathogens, especially when there is a lack of sufficient clinical data. This lack is mainly due to the fact that most infections develop asymptomatically in children and to the problematic reporting of hepatitis A cases through the surveillance system in Greece.The authors declare that they have no competing interests.PK carried out the sequence alignments, constructed the phylogenetic trees and participated in the writing of the manuscript. PZ participated in the molecular analyses and helped to draft the manuscript. SF and IM collected the samples and participated in the viral concentration, nucleic acids extraction and nested PCRs. AV was responsible for setting up and coordinating the study, and drafted the manuscript. All authors read and approved the final manuscript.


The mechanisms of immune response are structured within a highly complex regulatory system. Genetic associations with variation in the immune response to rubella vaccine have typically been assessed one locus at a time. We simultaneously assessed the associations between 726 SNPs tagging 84 candidate immune response genes and rubella-specific antibody levels. Blood samples were obtained from 714 school-aged children who had received two doses of MMR vaccine. Associations between rubella-specific antibody levels and 726 candidate tagSNPs were assessed both one SNP at a time and in a variety of multigenic analyses.Single-SNP assessments identified 4 SNPs that appeared to be univariately associated with rubella antibody levels: rs2844482 (p = 0.0002) and rs2857708 (p = 0.001) in the 5'UTR of the LTA gene, rs7801617 in the 5'UTR of the IL6 gene (p = 0.0005), and rs4787947 in the 5'UTR of the IL4R gene (p = 0.002). While there was not significant evidence in favor of epistatic genetic associations among the candidate SNPs, multigenic analyses identified 29 SNPs significantly associated with rubella antibody levels when selected as a group (p = 0.017). This collection of SNPs included not only those that were significant univariately, but others that would not have been identified if only considered in isolation from the other SNPs.For the first time, multigenic assessment of associations between candidate SNPs and rubella antibody levels identified a broad number of genetic associations that would not have been deemed important univariately. It is important to consider approaches like those applied here in order to better understand the full genetic complexity of response to vaccination. The importance of developing protective humoral immunity following vaccination is widely recognized, as those who fail to respond are at increased risk of contracting the disease if exposed. Rubella is well controlled via vaccination programs in industrialized countries, but epidemics of the disease occasionally occur in developing countries and both rubella virus infection and congenital rubella syndrome remain a major health concern around the world ,2. UnderOur group and others have shown that polymorphisms in the human leukocyte antigen (HLA) region, as well as SNPs in cytokine and cytokine receptor genes, are associated with differences in a variety of immune responses to rubella vaccine, but do not explain all of the variance in immune responses seen within the population -16. StudWhile the role of cytokines in antiviral immune responses has been established, little is known about how other gene families control immune responses to rubella virus. Studies in a variety of other models  have also recently demonstrated the importance of innate and vitamin receptor genes in regulating immune responses -23. In tThe discovery of genetic variations caused by single nucleotide polymorphisms (SNPs) has led to population-based immunogenetic studies intended to elucidate the potential relationship between host genomic variation and immune response . A high In this study, we searched for evidence of multigenic associations among a broad collection of SNPs identified from non-HLA genes that are candidates for being immunologically relevant to the development of a humoral response to rubella vaccination after two doses of measles-mumps-rubella vaccine. Due to the large number of genes in immune response gene families, we selected a group of key genes for each family, which, based on biologic plausibility and the published literature, are highly likely to be involved in regulating rubella immunity. We were particularly interested in assessing the degree to which the inclusion of multilocus associations would identify important genetic variation that contributes to rubella-specific antibody levels. To date, no such data have been published to address this issue.We attempted to genotype a total of 768 SNPs in a cohort of 738 children who had received two doses of rubella vaccine. After SNPs with call rates below 90%, HWE p-values less than 0.001 or minor allele frequencies less than 5% were excluded from analysis, a total of 726 remained, for an overall genotyping success rate of 94.5%. Subject exclusions were made on the basis of poor DNA quality (n = 6), complete genotyping failure (n = 4) and call rates below 95% (n = 14). These exclusions removed 24 subjects, leaving 714 subjects for analysis. Data from these individuals are summarized in Table Figure Our omnibus test of association between the number of positively associated alleles carried across all of these candidate SNPs and the observed antibody levels suggested that, even with the high number of null SNPs apparent from the univariate analyses, there was a significant association between this collection of SNPs and rubella antibody after performing a randomization test to account for multiple comparisons p = 0.029). In order to narrow the list of SNPs that contributed to this omnibus association, we performed a stepwise selection procedure. This procedure identified a list of 29 SNPs that all contributed to the genetic association with rubella antibody levels when considered simultaneously in the same regression model, with a global p-value obtained via a randomization test of 0.017. These SNPs are illustrated in Table 9. In ord-5 for the combination of rs10489626, an intronic SNP in IL12RB2, and rs1420094, a 3' flanking SNP in IL18R1, was actually less extreme than what would be expected if there were no pair-wise epistatic associations present.The plot in Figure As with the assessment of all possible pair-wise interactions, the use of recursive partitioning algorithms did not yield strong evidence in favor of major epistatic interactions. After forming the recursive partitioning tree, an assessment of the degree of pruning required to avoid over-fitting indicated that the best-fitting tree was one with a single node, with the split being on rs7801617. Because of this finding, further analyses seeking additional multi-allelic epistatic effects were not pursued.Because of the regulatory complexity of immune responses to complex antigens, there is almost certainly a diversity of genes that together influence the immune response to vaccination. While there are many genes that are strong candidates, the bulk of those that have an effect on the levels of rubella-specific antibody raised in response to immunization have yet to be identified. In this study, we examined a broad collection of SNPs that tag 84 genes that have been identified as potentially playing a role in the immune process. We have performed an array of analyses in an attempt to identify potential multigenic associations between these candidate SNPs and the observed levels of rubella-specific antibody levels following MMR-II vaccination.As we have reported previously, a number of SNPs and HLA alleles have already been identified as being univariately associated with variation in responses to rubella immunization -16. In tWe performed two separate analyses in our search for epistatic effects among the 726 SNPs. We first assessed the significance of all possible two-way combinations of SNPs . While there were more SNP pairs with p-values between 0.01 and 0.0001 than would have been expected under the null hypothesis, we found that the smallest p-value was actually larger than what would be expected under the hypothesis that no SNP pairs were significantly associated with rubella antibody levels. Likewise, as we performed a recursive partitioning analysis, we found that the best-fitting classification tree had only one node. This provided further evidence against the presence of major epistatic associations being in effect among the SNPs from the candidate genes under study.While the evidence for epistasis among the candidate SNPs is not strong, our analysis supports the concept that considering the broader collection of a large number of SNPs does provide additional insight into the genetic control of rubella antibody levels. By assessing all SNPs in a single model we were able to identify a broader collection of candidates than was possible when each SNP was considered individually. While the univariate tests implicated SNPs in the LTA, IL6 and IL4R genes, the analysis of all SNPs identified not only these genes but also a broader collection of genes that appear to contribute to variation in rubella antibody levels. The 29 SNPs that were jointly associated with humoral immunity are shown in Table While there is evidence in favor of there being a major genetic component to the observed levels of rubella-specific antibodies following vaccination , the curThe genes that have been implicated as being associated with rubella antibody levels were selected for study because of their general relevance to viral immunity. The humoral immune response to rubella vaccine reflected in assayed antibody levels is the cumulative result of the actions and interactions of multiple genes and pathways. Those SNPs that were identified as being important for humoral immunity to rubella are included in Table This manuscript outlines the first analysis of its kind, an assessment of the associations between a large collection of SNPs from immunologically relevant genes and rubella antibody levels. This analysis made it possible to examine all SNPs we genotyped on a well-characterized cohort of study participants and assess the likelihood that the univariate SNP results were significant on their own. This examination also made it possible to study a variety of types of associations between candidate SNPs and rubella antibody levels, both epistatic and otherwise. When complicated analytical approaches were employed, we used resampling methodologies to obtain statistical significance levels that were not over-trained to the data . This stThe implications of this work are two-fold. First, it presents a novel methodological approach that can provide additional insights into candidate genetic associations when analyzing similar data in other studies. When searching for associations between genes that are part of a complex system and outcomes that are potentially influenced by genetic variation in this system, it is important to consider more than just simple single-SNP assessments. Second, it is clear that the "dominant allele" model of genetic regulation of complex system processes such as immune response is both uncommon and too limiting. At least for those viral vaccines we have studied , variation in immune responses are clearly the result of small contributions from many genes and sets of genes acting in concert. These data provide motivation to move beyond simple univariate associations to the types of analyses illustrated in this report in order to identify significant multigenic associations otherwise unobservable by simple univariate models. The value of approaches such as this include the ability to more clearly identify and model the genetic determinants of immune response to viral vaccines. The findings from these models can then be used to inform the development of next-generation vaccines.The characteristics of the 738 healthy children and young adults (age 11 to 19 years) who participated in this study have previously been described . Study sRubella-specific IgG antibodies after two doses of the rubella vaccine were detected in serum by a whole rubella virus-specific chemiluminescent immunoassay  according to the manufacturer's instructions. The limit of detection for this assay was 0.5 IU/ml and the coefficient of variation in our laboratory was 6%.http://www.hapmap.org)[http://pga.mbt.washington.edu/)[http://egp.gs.washington.edu/)[2 <0.90 for Caucasians. Using these criteria, we selected 768 potential SNPs in our candidate genes of interest , Seattleton.edu/), and NIEton.edu/) as sourcton.edu/) to identOur genotyping methods were previously described in detail . BrieflyWe used PCR-based TaqMan assays  as the secondary platform to genotype SNPs that failed genotyping on the Illumina platform. All assays were performed according to the manufacturer's instructions, and the results were analyzed on the ABI Prism 7900 using Sequence Detection Software (Applied Biosystems).Illumina 10% GenCall scores >0.4 and call rates >90% were used as thresholds for the initial laboratory quality control. The data from genotype calls made by BeadStudio 2 software were transferred to SAS for further analysis, where call rates for each subject, and for each SNP, were estimated. Participants' genotypes were used to estimate allele frequencies for each SNP, and departures from Hardy-Weinberg equilibrium (HWE) were assessed using a Pearson goodness-of-fit test or, for SNPs with a minor allele frequency of less than 5%, a Fisher exact test .th and 75th percentiles, of the antibody levels were summarized overall, and for the categories of the descriptive variables. Comparisons of the antibody levels across the different categorizations of the descriptive variables were achieved via analysis of variance (ANOVA), with rubella antibody levels being analyzed on the logarithmic scale to correct for data skewness. All analyses were adjusted for the following set of covariates potentially associated with the measured rubella-specific antibody titers: age at enrollment, race, gender, age at first rubella vaccination, age at second rubella vaccination, and cohort status.The purpose of the efforts reported here was to determine the multigenic contributions of SNPs from the candidate genes involved with immunity that were selected for this study. Prior to performing statistical comparisons, the characteristics of study participants were categorized and examined in relationship to the measured rubella-specific IgG antibody levels (represented as IU/ml). The median, and the 25While the focus of this effort was on the multigenic contributions of these SNPs to variation in rubella-specific antibody levels, a test of association was obtained for each genotyped SNP that met quality control criteria in our study subjects to examine the ordinal genetic association between each of the SNPs of interest while adjusting for the descriptive variables using linear regression. A quantile plot of the overall distribution of single-SNP p-values was extracted and summarized for all SNPs.The primary interest was to identify the degree to which combinations of SNPs might provide novel insight into the potential control of rubella-specific antibody levels. Therefore, a series of multigenic analyses were carried out. We first assessed the degree to which individual SNPs provided additive information into the levels of rubella-specific antibody titers. We applied two approaches to identify these multi-allelic associations. The first was a single omnibus test of association where we identified the positively associated allele of each SNP and tallied the number of positively associated alleles of the candidate SNPs within each individual and assessed the association between this count variable and rubella antibody levels while controlling for the descriptive variables. The second approach again forced the inclusion of descriptive covariates, but relied on a stepwise model selection paradigm for identification of SNPs to be included. In this stepwise process, we added or removed a single ordinal SNP variable at a time until only SNPs that contributed to an association with rubella antibody levels remained in the regression model, using p-values for entrance or removal of 0.05. For each of these methods, we applied randomization tests to account for the fact that we identified the "positively associated allele" for each SNP within the same dataset in which we were performing the test of significance, and to account for the model selection approach we used to identify the model of candidate SNPs . These rIn order to search for potential combinations of SNPs that were jointly associated with antibody levels, we applied the approach advocated by Marchini et al , in whicAdditionally, we used recursive partitioning techniques to search for potential higher-order interactions among the candidate SNPs that might influence the observed levels of rubella-specific antibody levels. For a quantitative phenotype, these methods recursively search for optimal combinations of splits of explanatory variables such that the differences in the average levels of the phenotype among the identified groups are maximized ,48. We uDr. Poland is the chair of a safety evaluation committee for novel non-rubella vaccines undergoing clinical studies by Merck Research Laboratories.VSP participated in conception and design of the study. He oversaw statistical analyses, prepared the initial draft of the manuscript and finalized the manuscript after the writing and editing that was done by the co-authors. RAV participated in the conception and design of the study. He participated in the statistical analyses and made critical revisions to the manuscript for important intellectual content focused on statistical analysis and interpretation of the results. MMO performed statistical analyses and critically revised the manuscript for important intellectual content focused on statistical analysis. IGO participated in the selection of candidate genes and SNPs. She critically revised the manuscript for important intellectual content, focusing primarily on the genetic and immunologic aspects. GAP participated in conception and design of the study. He oversaw study conduct as the holder of the grants that supported the research effort. He provided critical revisions to the manuscript for important intellectual content, focusing primarily on the broad implications of the research efforts. All authors have read and approved the final manuscript.VSP Dr. Pankratz is an Associate Professor of Biostatistics with training in statistical genetics. His interests lie in the study of genetic associations with a variety of phenotypes. He has worked with the Mayo Vaccine Research Group since 1998, studying the genetics of immune responses following vaccination.RAV Mr. Vierkant is an Assistant Professor of Biostatistics with extensive experience in the design and analysis of genetic association studies. He has worked with the Mayo Vaccine Research Group since 1998, studying the genetics of immune responses following vaccination.MMO Ms. O'Byrne is a Statistical Programmer Analyst in the Division of Biomedical Statistics and Informatics with extensive experience in analyzing data, and in preparing code to run a broad variety of statistical analyses, both novel and standard.IGO Dr. Ovsyannikova is an Associate Professor of Medicine with a broad background in immunology and vaccinology. She has extensive experience in the design and conduct of genetics studies that focus on the contribution of candidate genes to differences in a variety of measures of post-vaccination immune response.GAP Dr. Poland is a Professor of Medicine, Molecular Pharmacology & Experimental Therapeutics, Infectious Diseases. He has led the Mayo Clinic Vaccine Research Group since 1988 and helped to quantify the degree of genetic contribution to variation in a number of post-vaccination immune response measures. The efforts of the group he leads have focused in recent years on the study of genetic associations with a broad range of immune response measures raised by a variety of vaccines.Supplementary Table: Genes included in the analysis, along with the numbers of genotyped SNPs of various types.Click here for file