Asthma is recognized as intimately related to immunologic factors and inflammation, although there are likely multiple phenotypes and pathophysiologic pathways. Biomarkers of inflammation may shed light on causal factors and have potential clinical utility. Individual and population genetic factors are correlated with risk for asthma and improved understanding of these contributions could improve treatment and prevention of this serious condition.A population-based sample of 108 children with clinically defined asthma and 216 control children were recruited from a small community in the northern plains of the United States. A complete blood count, high sensitivity C-reactive protein, total IgE and specific antibodies to 5 common airborne antigens (CAA), in addition to basic demographic and anthropomorphic data were obtained. Logistic regression was primarily used to determine the association between these humoral factors and risk of asthma.The body mass index (BMI) of those with asthma and their total leukocyte counts, percentage of eosinophils, and levels of total IgE were all greater than corresponding control values in univariate analysis. The presence of detectable, specific IgE antibodies to five common airborne antigens was more likely among cases compared with controls. In multivariate analysis, total IgE was independently associated with asthma; but not after inclusion of a cumulative measure of specific IgE sensitization.Many previously reported associations between anthropomorphic and immune factors and increased risk of asthma appear to be also present in this American Indian population. In this community, asthma is strongly associated with sensitization to CAA. Asthma has long been recognized as intimately related to immunologic factors and inflammation . ClinicaThe complex interplay between the immune system and environmental exposures precludes any simplistic models of asthma pathophysiology; and makes inferences from epidemiologic data difficult. Thus the differing ecological and socio-economic environment of populations can produce characteristic profiles of various immune and inflammatory measures . The “hyLastly, the population genetics of a community sets the stage for this complex interaction between physiology and environment. Differences in immune response and inflammatory status have been seen in comparisons between ethnic groups , 12 and For all of the above reasons, we sought to obtain additional information from an American Indian population, comparing humoral immune factors among those with and without asthma.The measures reported here derive from a case–control study of the environmental and genetic influences on risk of asthma among an American Indian population in north-central United States. Most primary medical care for this community of predominantly tribal members is provided mainly by federal funding to the Indian Health Service (IHS) and a tribal health department. Specialty pulmonology or allergy medical care is a minimum of 160 miles from the demographic center of the community.th and 4th lowest per capita income counties in America respectively. In addition, over 20 % have less than a high school education [This community is located in the north central portion of South Dakota in an area covering 4266 square miles, which is larger than the state of Connecticut. The area’s population is about 8500, resulting in a population density of between 2 and 3 people per square mile. Most live in cluster housing near small towns or in cluster sites far removed from basic services. Beyond federally supported work in health care and education, ranching and farming provide the bulk of employment. None the less, 33 and 42 % of two counties within this community have incomes below the poverty line, making them the 11ducation .Cases were ascertained through automated query of the IHS electronic medical records system, searching for an inclusive array of ICD-9 codes between 493.00 and 493.92, in addition to codes 786.07 (wheezing) and V17.5 (family history of asthma). The search was limited to individuals between and including the ages of 6 and 17 years. Additional cases were sought by contact with local non-IHS providers. This identified over 700 individuals, who were then consented for further review of medical records to determine potential eligibility.a diagnosis of asthma on at least 2 occasions by more than one provider during the past 2 years.refills of asthma treatment medications on at least 2 occasions during the past 2 years.If spirometry had been conducted, there was an improvement in forced expired volume at one second (FEV1) of 20 % with use of albuterol metered-dose inhaler (MDI) or nebulizerCase definition criteria required at least 2 of the following 3 criteria:Birthweight less than 2500 gNeonatal ventilator treatmentHospitalization at birth greater than 15 days.Congenital heart anomaly requiring surgeryDiagnosis of cystic fibrosisCongenital lung, diaphragm, chest wall, or airway anomalyDiagnosis of pneumonia, pertussis, or tuberculosis within the past yearCongenital muscular disorderExclusionary criteria were:It was found that many of the potential cases initially identified had been assigned an ICD-9 code indicating “asthma” by the pharmacist filling a prescription for a bronchodilator, although the prescribing physician had not indicated a diagnosis of typical “asthma” and was intending to ameliorate the bronchospastic component of a pulmonary infection. These latter children did not meet diagnostic criteria; but did require considerable recruiter effort to contact parents and determine their status via medical record review.No diagnosis of asthma by any provider during the past 2 years.No prescriptions of any asthma meds during the past 2 years.If spirometry had been conducted, an FEV1 greater than 80 % of the predicted value For each case, two controls were initially recruited by identifying the two children born the day after and before the index case and contacting the parents for consent to review medical records for possible inclusion. As the study progressed, this methodology proved too time consuming and many controls were later recruited from previously identified families with children born almost exclusively (>99 %) within 6 months of the index case. Initial recruitment was concentrated on cases and later focused on controls, thus the inherent bias of controls being older at the time of exam  was increased, such that all but 5 (2.3 %) of the pairs were examined within one year of each other. Controls met the same exclusionary criteria as cases in addition to:Consenting cases and controls were then examined according to study protocol, which included anthropomorphic measures, spirometry, salivary DNA collection and a non-fasting blood draw. Environmental measures of home air quality and dust exposure were made.complete blood count (CBC) done at the local IHS laboratory using Abbott Laboratories dedicated reagents on an Abbott CELL-DYN Ruby, Abbott Laboratories, Abbott Park, IL. These were accomplished within 8 h of drawing.high sensitivity C-reactive protein (CRP) done at Sanford Laboratories, Rapid City, SD, using a particle enhanced immunoturbidimetric assay, , on a Roche Cobas 6000 instrument.total IgE was done at Sanford Laboratories, Sioux Falls, SD by Fluorescent Enzyme Immunoassay,  on the Phadia ImmunoCAP 250 instrument.allergen specific IgE sensitive to dog and cat dander, A alternata mold, D farinae mite, and B germanica cockroach antigens. These measures were also conducted at Sanford Laboratories, Sioux Falls, SD by the same technique as the total IgE, using ImmunoCAP dedicated reagents, catalog numbers 14-4110-01, 14-4109-01, 14-4106-01, 14-4108-01, 14-4224-01 respectively, Phadia AB, Uppsala, Sweden).The inflammatory and immune biomarkers reported here are as follows:A questionnaire collected social, demographic, and medical history from both cases and controls, most of which will be reported in the future. One question: “Has a medical person ever said that your child had hay fever or seasonal allergies?” sought to gauge atopic symptomatology.t-test for continuous variables. Univariate and multivariate logistic regression was used to explore effects of multiple biomarkers on risk of asthma. Specific IgE levels were analyzed both as a dichotomized variable (above or below the detection threshold) and as a continuous variable, with those below the detection limit (0.35 kUA/L) assigned a value of 0.35 divided by the √2, as is commonly done [p < 0.05.With the exception of age, the majority of variables measured had non-normal distributions (e.g. skewedness >1) and were natural log transformed prior to hypothesis testing. All of the resulting distributions, except those representing a quantitative value for specific IgE sensitization, exhibited a skewedness of less than 0.685 after transformation. The distribution of specific IgE values remain significantly skewed, even after log-transformation (maximum skewedness of 3.6 for Alternaria), probably due to the large proportion of measures below the detection limit. Descriptive statistics report median, minima and maxima, and 95 % confidence intervals for non-normal variables. Cases and controls were compared using McNemar chi-square statistics for discrete variables and paired nly done . All modThe characteristics of cases and controls are shown and compared in Table The significant difference in age between cases and controls is probably due to a number of factors, as noted in the methods section. The mean difference in age is approximately 4 months; but for this reason all logistic regression models were adjusted for age.Notable differences between case and control groups include body mass index (BMI), white blood cell count (WBC), percent eosinophils, total IgE, and specific IgE levels over the detectable limit for all tested antigens. Among controls the geometric mean total IgE was 88.23 kIU/L and the median was 89 kIU/L, with an interquartile range of 35.5 to 229. There was no significant statistical association between a history of hayfever or seasonal allergy and either atopy (as defined by at least one of the five specific IgE assays) or total IgE. Among cases, 61 % had at least one specific IgE above the detectable limit, compared with only 28 % of controls.p = 0.001).Table Results of multivariate logistic regression models are found in Table p = 0.001); and negatively correlated with % eosinophils ; but notably not correlated with cumulative sensitization . Positive correlations are also seen between total IgE and cumulative BMI, total IgE and CSV , BMI and WBC ; and total IgE and % eosinophils .Correlation between certain variables is present. Not surprisingly age is positively correlated with BMI , as also reported in studies of European , 23–25, The level of high sensitivity CRP did not show any association with case–control status. This finding is unexpected in light of various reports in the literature , 35, altThe present study also documents increased levels of all leukocytes and specifically the proportion of eosinophils among those with asthma. However, these measures are not found to be independent factors in models adjusting for total IgE or a cumulative measure of IgE antibodies targeting putative asthma triggers. Whether this represents different aspects of a common inflammatory factor, or a more direct influence of IgE on eosinophils (or vice versa) is not clear. The literature has focused primarily on the presence of total leukocytes and eosinophils in bronchial washings; but there are reports of increased numbers of both elements in peripheral circulation as well , 37–39.In the present study, IgE antibodies specific for the five common environmental antigens tested are strongly associated with asthma. This is apparent for each of the five antigens when analyzed as a binary variable (above or below the limit of detection); and not surprisingly, when a cumulative variable incorporating all of the antigens was used. The increased risk of asthma in the presence of specific IgE antibodies is commonly reported in the literature  and is fp = 0.07) when a cumulative measure of sensitization was excluded from the model. The increased risk of asthma in the presence of obesity is well established in the literature [Although obviously not a humoral factor, univariate analyses showed obesity was also positively associated with asthma risk in this cohort, with each BMI unit contributing approximately 5 % additional risk. The contribution of BMI was attenuated in multivariate analyses; but was still suggestive (terature , 43; andterature . This apterature .While there are clearly numerous social and environmental factors that influence the risk of asthma and its complications, there also appear to be ethnic and genetic influences at play , 47. DueEvidence from this case–control study among an American Indian population shows strong, independent association between increasing levels of specific IgE sensitization to 5 common environmental antigens and asthma, even after adjustment for other widely accepted risk factors, such as obesity and total IgE levels. The presence of elevated total IgE levels is associated with asthma risk as expected, when there is no adjustment for specific IgE sensitization. In light of evidence that asthma risk is influenced by genetic variants, the current results complement our understanding of asthma, especially as it relates to ethnicity.BMI, body mass index; CAA, common airborne antigens; CRP, C-reactive protein; CSV, cumulative sensitization variable; FEV1, forced expired volume at one second; GM, geometric mean; ICD-9, International Classification of Diseases, ninth edition; IgE, total immunoglobulin E; MDI, metered-dose inhaler; WBC, white blood cell


The prevalence of mitochondrial disease has proven difficult to establish, predominantly as a result of clinical and genetic heterogeneity. The phenotypic spectrum of mitochondrial disease has expanded significantly since the original reports that associated classic clinical syndromes with mitochondrial DNA (mtDNA) rearrangements and point mutations. The revolution in genetic technologies has allowed interrogation of the nuclear genome in a manner that has dramatically improved the diagnosis of mitochondrial disorders. We comprehensively assessed the prevalence of all forms of adult mitochondrial disease to include pathogenic mutations in both nuclear and mtDNA.Adults with suspected mitochondrial disease in the North East of England were referred to a single neurology center from 1990 to 2014. For the midyear period of 2011, we evaluated the minimum prevalence of symptomatic nuclear DNA mutations and symptomatic and asymptomatic mtDNA mutations causing mitochondrial diseases.The minimum prevalence rate for mtDNA mutations was 1 in 5,000 , comparable with our previously published prevalence rates. In this population, nuclear mutations were responsible for clinically overt adult mitochondrial disease in 2.9 per 100,000 adults.Combined, our data confirm that the total prevalence of adult mitochondrial disease, including pathogenic mutations of both the mitochondrial and nuclear genomes , is among the commonest adult forms of inherited neurological disorders. These figures hold important implications for the evaluation of interventions, provision of evidence‐based health policies, and planning of future services. Ann Neurol 2015 Ann Neurol 2015;77:753–759 Mitochondrial disorders are frequently multisystemic in nature and cause significant morbidity and mortality.Accurate assessment of mitochondrial disease prevalence has proven difficult in the past. Meticulous clinical and biochemical characterization of patients remains fundamental to diagnostic yield. Yet diverse and expanding clinical features, variable genotype–phenotype correlates, and the complex structure of referral pathways have all impeded attempts to gauge the true epidemiological impact of mitochondrial disease. Recent advances in diagnostic techniques in tandem with streamlining of referral pathways in the United Kingdom and deployment of extensive family tracing have improved case ascertainment and permitted, for the first time, recording of the minimum prevalence of adult mitochondrial disease, to include pathogenic mutations in both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA).Adult cases (>16 years old) with suspected mitochondrial disease were ascertained following referral to a single specialist mitochondrial center in the North East of England between 1990 and 2011. Referral patterns to this center are drawn mainly from tertiary neurology and neuro‐ophthalmology sources, but historical links with other specialties likely to encounter mitochondrial disease  account for other common routes of referral. The Newcastle Mitochondrial Centre is among only 3 highly specialized, national referral centers for mitochondrial disease in the United Kingdom.Those individuals with pathogenic mtDNA or nDNA mutations, or those with unidentified nuclear mutations, but pathological multiple mtDNA deletions evident in muscle, who were alive at the midyear period of 2011, were identified. Those with multiple mtDNA deletions in muscle were only included when there was no evidence of other muscle pathology, the mtDNA deletions had been independently verified using 2 different techniques , and the clinical and biochemical (>3% cyclooxygenase‐deficient fibers) features were consistent with a diagnosis of mitochondrial disease. Affected individuals were included if they lived within the geographical boundary of the North East of England government office region that is defined by 6 postcodes  and includes County Durham, Northumberland, Newcastle upon Tyne, Sunderland, North Tyneside, Gateshead, Stockton‐on‐Tees, South Tyneside, Middlesbrough, Redcar and Cleveland, Darlington, and Hartlepool. Comprehensive pedigree analysis and family tracing was performed, identifying other clinically affected individuals and also unaffected family members at risk of disease. In contrast to our previous study,Adopting our previous ascertainment criteria,Comprehensive pedigree data were collected from all affected individuals, to define individuals at risk for development of mitochondrial disease. We included both adults and children, as previously reported,Population census data for the midyear period of 2011, as specified by the Office for National Statistics (ONS) of the United Kingdom,−5), whereas the prevalence of all pathogenic mutations in both nDNA and mtDNA is 23 in 100,000 .The minimum point prevalence of clinically affected adults with mitochondrial disease attributable to either the mitochondrial or nuclear genome is 12.5 in 100,000  = 11.1–14.1 × 10−5), in this population  were affected by 1 of the 3 common Leber hereditary optic neuropathy (LHON)−5, 95% CI = 1.5–2.7 × 10−5), m.3460G>A , and m.14484T>C . The most common, pathogenic mtDNA point mutation identified was the m.3243A>G mutation . Thirty‐one symptomatic adults harboring single large‐scale mtDNA deletions were also identified . Five individuals  were clinically affected by the m.8344A>G mutation, manifesting as myoclonic epilepsy with ragged‐red fibers (MERRF).The regional adult population (>16 years old) figure for midyear 2011 was 2,134,449,−5; see Table The midyear 2011 population of adults and children in North East England was 2.6 million.−5; Table SPG7 (spastic paraplegia 7) and dominant mutations in PEO1  were the most common pathogenic nDNA mutations identified in clinically affected individuals . Mutations in OPA1 (optic atrophy 1) caused multisystemic neuromuscular disease in 8 adults in addition to visual failure . Only autosomal recessive POLG (polymerase gamma) mutations were identified in the region, and these caused clinically overt disease in 6 individuals. A further 3 individuals were found to harbor a single pathogenic mutation. Dominant mutations in RRM2B (ribonucleotide reductase M2 B [TP53 inducible]) caused clinically overt disease in 5 adults . Only 5 clinically affected adult patients with progressive external ophthalmoplegia (PEO) and multiple mtDNA deletions in muscle remained genetically undetermined . The remaining 6 clinically affected individuals harbored mutations in the following nuclear genes: DNM2 , SDHA , ETFDH , and TRIT1 , SLC25A4 , POLG, PEO1, OPA1, POLG2, RRM2B, TK2 (thymidine kinase 2), DGUOK (deoxyguanosine kinase), MPV17 , MGME1 , DNA2 (DNA replication helicase/nuclease 2), SPG7, and AFG3L2 (AFG3‐like AAA ATPase 2),SPG7  and PEO1 , followed by OPA1  and RRM2B  have emerged as major causes of adult mitochondrial disease. Recessive mutations in POLG  are also emerging as an important cause of mitochondrial disease in the North East of England. Delineation of an at‐risk group of individuals harboring single pathogenic mutations in POLG is important, even in nonconsanguineous families. Given the relatively common prevalence of pathogenic POLG mutation heterozygosity in European populations and the potential severity of the phenotype due to recessive mutations, the provision of genetic counseling to individuals and their partners should be made, even where the risks to future offspring are small. Less than 10% of adult patients with PEO and muscle restricted mtDNA deletions remain genetically undetermined, reflecting recent advances in diagnostic yield from implementation of next generation sequencing and deep clinical phenotyping of all patients attending our center.Mutations in nDNA genes affect function of the mitochondrial respiratory chain in a variety of ways, including: synthesis, assembly, or chaperoning of individual components of the mitochondrial respiratory chain; synthesis, maintenance, repair, and translation of mitochondrial DNA; and the processes of mitochondrial fission and fusion, which maintain mitochondrial networks. Nuclear mutations account for overt disease in 2.9 per 100,000 of the adult population in the North East of England, approximately one‐third of the prevalence for mtDNA mutations. To date, 14 nuclear encoded genes, −5) are potentially at risk of nDNA‐related mitochondrial disease (Supplementary Table).Inherent difficulties exist in the calculation of those at risk of nDNA‐related mitochondrial disease because of the heterogeneous nature of inheritance patterns of nDNA mutations, variability in disease penetrance and clinical expression, and with the recent advent of next generation sequencing, a recognition of the rapidly expanding number of nDNA mutations causing mitochondrial disease. Accepting such limitations, and implementing the same criteria to determine individuals at risk of mtDNA‐related mitochondrial disease, we would conservatively estimate that a minimum of 5.9 in 100,000 (154 individuals; 95% CI = 5.0–6.9 × 10Comparison with point prevalence figures for mitochondrial disorders in other regions remains difficult. These current results for mtDNA mutations are similar to that reported previously in adult patients with mitochondrial disease in the North East of England a decade earlier and as such act as comparative data.It is clear from this detailed population study of adult patients with mitochondrial disease that physicians recognize the significant risk of disease posed to individuals who have family members diagnosed with transmissible forms of mitochondrial disease. Moreover, we would suggest that family history alone serves as a surrogate marker for the development of mtDNA‐related mitochondrial disease.This rigorous cross‐sectional study benefiting from improved case ascertainment and diagnosis over the past decade indicates that adult mitochondrial diseases are among the most prevalent groups of inherited neurological disorders, affecting up to 1 in 4,300 adults in the United Kingdom. Although we have documented a relatively stable prevalence rate for mtDNA mutations over time, it seems likely that an improved understanding of nuclear phenotypes and advances in next generation sequencing technologies including whole exome and whole genome sequencing are likely to lead to an increased recognition of mitochondrial disease attributable to mutations of the nuclear genome. However, given that <10% of nuclear phenotypes in this cohort remain genetically undetermined, this figure is unlikely to change significantly, unless the perceived phenotypic spectrum is also expanded. Combined, these findings will facilitate more robust assessment of the level of services and financial investment required to provide early detection, prevention, and treatment of mitochondrial diseases.G.S.G. and A.M.S. are joint first authors.Nothing to report.Supporting Information Table 1.Click here for additional data file.


Background: Superior mesenteric artery (SMA) syndrome has been reported as an uncommon condition of external vascular compression of the SMA particularly after rapid weight loss, body casts, or after corrective surgery for spinal deformities, usually within the first few weeks after surgery.Methods: This is a retrospective report of a case of a non-verbal autistic female patient who started to develop SMA syndrome at the age of 16, 4 years after posterior spinal fusion surgery for scoliosis. She was treated conservatively by increasing oral caloric intake, which resulted in increased body weight and relief of symptoms.Results: Seen at 10 years’ follow up, the patient is doing well, and is functional within the limits of her suboptimal cognitive and verbal conditions. She maintains good trunk balance with solid spinal fusion and intact instrumentation at latest follow up.Conclusion: Spinal surgeons should maintain a high index of suspicion for diagnosis of SMA syndrome even years after scoliosis surgery, especially for patients with communication problems, like the case we present here. Appropriate conservative measures can succeed in relieving the symptoms, increasing body weight, and preventing complications including the risk of death. Superior mesenteric artery syndrome (SMAS) is an uncommon condition of the gastrointestinal (GI) tract with external vascular compression of the third part of the duodenum causing partial or complete obstruction secondary to decreased aortomesenteric angle and distance to 6–16° and 2–8 mm, respectively . It has We present here a case of SMAS, where the symptoms ensued 4 years after surgery.2), underwent instrumented posterior spinal fusion for progressive scoliosis  , 10, 12,.7% , 10,Symptoms of SMAS typically develop within a few days following scoliosis surgery , 22, 23.In this report, we present a case of SMA syndrome with a subtle and unclear clinical presentation. Departing from the typical symptoms of SMA syndrome, known to be vomiting 92.9%), abdominal pain (57.1%), distension (42.9%), bilious vomiting (35.7%) , and hyp2.9%, abdA staged procedure, the lumbar modifier of B or C as opposed to A, body mass index (BMI) < 25th percentile, and increased stiffness of the thoracic curve are the most predictive of the development of SMAS after spinal deformity correction [Barium Upper Gastrointestinal (GI) series was the key to diagnosis in our patient after exclusion of other differential diagnoses. The traditional diagnostic method for SMAS is the barium upper GI series, with four diagnostic criteria –28. MagnThe lack of high indices of clinical suspicion can cause diagnostic delays , 25, 30 The importance of maintaining a high index of clinical suspicion for SMAS cannot be overemphasized, specifically in patients who are at higher risk and who are unable to communicate. Although the onset of SMAS reported after scoliosis surgery did not exceed a few weeks, our patient had onset of GI-related symptoms, in the form of weight loss, and possible abdominal pain as late as 4 years after surgery. SMAS should take its place on the list of differential diagnosis even in the presence of non-GI-related symptoms, especially in nonverbal patients, to avoid diagnostic delay and prevent complications.NA, MK and JPD declare no conflict of interest in relation with this paper.


Galeocerdo cuvier), seven lemon (Negaprion brevirostris), and six Caribbean reef (Carcharhinus perezi) sharks tagged with acoustic transmitters were monitored within the MCE using an array of acoustic receivers spanning an area of 1,060 km2 over a five year period. Receivers were concentrated around prominent grouper FSAs to monitor movements of sharks in relation to these temporally transient aggregations. Over 130,000 detections of telemetered sharks were recorded, with four sharks tracked in excess of 3 years. All three shark species were present within the MCE over long periods of time and detected frequently at FSAs, but patterns of MCE use and orientation towards FSAs varied both spatially and temporally among species. Lemon sharks moved over a large expanse of the MCE, but concentrated their activities around FSAs during grouper spawning and were present within the MCE significantly more during grouper spawning season. Caribbean reef sharks were present within a restricted portion of the MCE for prolonged periods of time, but were also absent for long periods. Tiger sharks were detected throughout the extent of the acoustic array, with the MCE representing only portion of their habitat use, although a high degree of individual variation was observed. Our findings indicate that although patterns of use varied, all three species of sharks repeatedly utilized the MCE and as upper trophic level predators they are likely involved in a range of interactions with other members of MCEs.Understanding of species interactions within mesophotic coral ecosystems  lags well behind that for shallow coral reefs. MCEs are often sites of fish spawning aggregations (FSAs) for a variety of species, including many groupers. Such reproductive fish aggregations represent temporal concentrations of potential prey that may be drivers of habitat use by predatory species, including sharks. We investigated movements of three species of sharks within a MCE and in relation to FSAs located on the shelf edge south of St. Thomas, United States Virgin Islands. Movements of 17 tiger ( Abundance and distribution of prey may vary both spatially and temporally and have been shown to dramatically influence movements of predators ,14. FishNegaprion brevirostris; tiger, Galeocerdo cuvier; and Caribbean reef, Carcharhinus perezi) on the Puerto Rico-Virgin Islands platform. Our goals were to: 1) quantify spatio-temporal patterns of MCE reef use for each species of shark, and 2) examine temporal orientation of sharks towards grouper FSAs off St. Thomas, USVI.The insular slope reefs of Puerto Rico and the United States Virgin Islands (USVI) are predominantly MCEs  and are Montastraea spp. [This study was conducted within the MCE located along the southern insular shelf edge of the Puerto Rico-Virgin Islands platform, spanning ~100 km from St. John, USVI, to Vieques, Puerto Rico and adjacent to the 4000 m deep Virgin Islands Trough . This weaea spp. ,25.2 area permanently closed to fishing [2 of dense coral [Epinephelus guttatus) for spawning from December to February [2 of MCE and is closed to fishing between 1 February and 30 April, which coincides with yellowfin (Mycteroperca venenosa) and Nassau (Epinephelus striatus) grouper spawning season but also hosts other aggregating species including yellowmouth , and tiger grouper (M. tigris), cubera (Lutjanus cyanopterus) and dog (L. jocu) snapper and Bermuda chub (Kyphosis sectatrix) [Haemulon album) .The MCE includes two fishing closure areas 12 km south of St. Thomas: the Hind Bank Marine Conservation District (MCD) and the Grammanik Bank . Hind Ba fishing  and encose coral . The HinFebruary . Grammanctatrix) ,28,29. EA total of 30 sharks of three species: 17 tiger, seven lemon, and six Caribbean reef, were caught on bottom longlines (soak times: 3–4 hours) set adjacent to the Hind Bank MCD and Grammanik Bank FSAs. Longlines were 366 m long with 25 360-cm long gangions (180 cm of 6-mm polypropylene rope and 180 cm of 3-mm stainless steel cable) terminating in a 16/0 recurved hook. Sharks were brought on board the fishing vessel using a customized slide that allowed easy retrieval and release of sharks with minimal handling. A seawater hose was placed in the mouth of sharks to irrigate the gills while sharks were measured , sexed, and tagged with a conventional ID dart tag.Acoustic transmitters, V16-3H,  were used for sharks >150 cm and V13-1H (13 x 36 mm) for sharks <150 cm. Transmitters emitted unique coded acoustic signals at 69 kHz with delays of 40–120 seconds and had an estimated battery life of 550–2350 days. Transmitters coated with a 50:50 mixture of paraffin:beeswax to smooth edges and to make the transmitters inert, were surgically implanted into the body cavity . The sur2  that included 127 different locations was used to monitor shark and grouper movements around FSAs (78 locations) and at distant locations within the MCE between 2007 and 2011, encompassing an area of approximately 1,060 km2 . ReceiveTo quantify size of the MCE habitat used by individual sharks, minimum convex polygons (MCP), modified to exclude land  were constructed for each shark as determined by location of all acoustic receivers on which an individual was detected during the study . The relationship between shark size and size of MCP and also the relationship between track duration (i.e. number of days tracked) and size of MCP were examined for tiger and lemon sharks using linear regression. Limited sample sizes for Caribbean reef sharks did not allow examination of these relationships. The extent of detections from all individuals of a given species was used to construct MCPs for each of the three species of shark.To further characterize use of the MCE we quantified three features of the detection history for each individual: 1) “track duration” (span of days between first and last detections on receivers within the entire array); 2) temporal pattern of “presence” (number of consecutive days detected on at least one receiver within the array) and 3) temporal pattern of “absence” (number of consecutive days not detected within the array). Patterns of presence and absence together constituted discrete alternating intervals of presence (detected x number of days in a row) and absence (no detections over a span of x number of days in a row) exhibited by each individual. A sequence of consecutive days present within the array was considered a unique “presence value” and each sequence of consecutive days not detected was treated as a unique “absence value”, both measured in days. To examine patterns of presence/absence histograms depicting frequency of presence values and of absence values were constructed for each individual. Additionally, average presence values and absence values were calculated for individuals and these values combined to obtain an average presence value and an average absence value for each of the three species as a whole. Average presence values and absence values were compared among species using a one-way ANOVA and Tukey’s post-hoc test. To determine if absence values conformed to a random distribution, the frequencies of absence values were compared to a Poisson distribution using a chi square test.Because of the proximity of receivers at FSAs within the Hind Bank MCD and Grammanik Bank and the mobility of telemetered sharks, individuals were frequently detected on multiple receivers at FSAs on the same day, often within a short time. Therefore, for purposes of monitoring presence of sharks at FSAs, the Hind Bank MCD and Grammanik Bank FSAs were considered a single FSA and data from receivers in proximity to both FSAs was combined for analyses directed at association of sharks with FSAs. The combined Hind Bank/Grammanik Bank spawning sites are hereafter referred to as the FSA.The relationship between occurrence of sharks at spawning sites and the grouper spawning activity was examined by comparing occurrence of sharks at the FSA on a monthly basis during grouper spawning months (December—May) and non-spawning months (June—November). To quantify occurrence of sharks at the FSA on a monthly basis, a “residence index” was calculated for each individual shark for each month tracked . ResidenActivity near FSAs was also examined by calculating presence and absence values at the FSA site using the same criteria for calculation of presence and absence used for the entire MCE . Mean presence and absence values at the FSA during grouper spawning were compared with mean values for non-spawning season for each shark using a paired t-test.To determine if sharks disproportionately visited any of the three distinct spawning sites , the percentage of days a given species was detected during the spawning season was calculated for each receiver in each of the three spawning areas by dividing the number of days sharks were detected on a receiver by the total number of days that the receiver was listening during the spawning season. The percentages were arcsine transformed and compared among the three spawning areas using a one-way ANOVA. All statistical analyses were evaluated at a significance level of 0.05.This study was conducted with approval from the US Virgin Islands Department of Planning and Natural Resources Division of Fish and Wildlife scientific collection permit (STT-011-07) and National Marine Fisheries Service (SHK-LOA-07-05). At the time this field work  was performed, the University of the Virgin Islands did not have an Animal Welfare Committee and an institutional permit was not required. Rather, all animal handling procedures were conducted using guidelines established by the American Fisheries Society and American Society of Ichthyology and Herpetology, and all efforts were made to minimize animal stress and suffering.The 30 sharks  implanted with transmitters generated over 130,000 unique detections during the five years of the study. Eighteen sharks  were detected a sufficient number of times to provide data suitable to characterize and compare their movements, with 10 individuals monitored more than two years and six individuals with tracks approaching or exceeding three years . The sha2) and tiger sharks (20.3–387.5 km2). There was a large amount of overlap of activity space among the three species and among individual sharks with no evidence of partitioning of space within the MCE.Use of the MCE varied considerably among both individuals and species on spatial and temporal scales and in terms of association with FSAs. For both lemon and tiger sharks, some individuals were detected on receivers throughout nearly the entire acoustic array, with MCPs nearly as large as the complete array, but other individuals were detected primarily on receivers near the FSA, with much smaller MCPs . One lem2) and location, both near their tagging sites and the FSA. Although average MCP size for tiger sharks (186.4 ± 129.8 km2) was larger than that of lemon sharks (152.69 ± 122.52 km2), the difference was not significant . Linear regression revealed a significant positive relationship between track duration and size of MCPs for tiger sharks (p = 0.03), but not for lemon sharks (p = 0.96). The relationship between shark size and MCP size was not significant for either tiger (p = 0.69) or lemon sharks (p = 0.06). At the species level, both tiger and lemon sharks ranged over nearly the entire area of MCE monitored by the acoustic array , whereas Caribbean reef sharks were detected over a substantially smaller area (roughly 8 km2) around the Grammanik Bank FSA , indicating that arrival and departure of sharks from the MCE was not random. Presence values (number of consecutive days detected on receivers) for sharks within the MCE was highly variable among individuals, and also varied among species. Average presence value for Caribbean reef sharks (25.2 ± 0.6 d) was significantly greater than those for lemon (2.2 ± 0.6 d) and tiger sharks (1.8 ± 0.2 d) (p < 0.0001), however average presence values of lemon and tiger sharks did not differ significantly (p = 0.67). Although presence values of lemon and tiger sharks were low, the pattern of MCE use for all individuals of both species was characterized by brief periods of presence on the reef followed by slightly longer periods without detections, when they may have been absent from the reef.For 17 of the 18 sharks analyzed, the distribution of the number of days between successive days detected within the MCE array differed from a random distribution   (p < 0.0p = 0.001). There was no significant difference between average absence times for lemon and tiger sharks (p = 0.507). Presence and absence values combined indicated that Caribbean reef sharks were both present and absent for substantially longer periods of consecutive days than either lemon or tiger sharks.Absence values (number of consecutive days not detected) also varied among species, with the average absence time of Caribbean reef sharks (35.3 ± 5.7 d) significantly greater than those of lemon (10.6 ± 2.2 d) and tiger sharks (8.4 ± 2.4 d)   occurred on consecutive days (i.e. no gaps between days detected), whereas only 52.8 ± 16.2% of detections occurred on consecutive days for lemon sharks and 44.8 ± 17.7% for tiger sharks. There were also major differences in the number of days that sharks were absent between detections, as indicated by the much higher proportion of absence values that fell between 1–7 d for tiger (42.1 ± 26.7%) and lemon (30.9 ± 24.3%) sharks in comparison to Caribbean reef sharks (2.6 ± 2.5%).p = 0.247) indicating that treatment of the MCD and Grammanik Bank as a single FSA for analyses was appropriate. Combining all monthly residence indices for all years for each species yielded a total of 12 monthly average residence indices for each species over the five year span of the study (p = 0.008) for lemon sharks, but not for tiger or Caribbean reef sharks (p > 0.05). Additionally, the average number of days per month that lemon sharks were detected at the FSA over the five year span of tracking (4.9 ± 2.3 d) was significantly greater than during the non-spawning season (1.4 ± 0.43 d) (p = 0.011). There was no significant difference between FSA residence indices during spawning and non-spawning seasons for tiger sharks (p = 0.411). Residence indices of Caribbean reef sharks were not analyzed because only two sharks were tracked.The degree to which sharks were associated with the FSA site during the grouper spawning season (Dec–May) varied distinctly among species. Lemon sharks demonstrated a marked increase in activity centered in the vicinity of the FSA during grouper spawning months, whereas tiger and Caribbean reef sharks did not exhibit orientation towards the FSA in relation to grouper spawning. There was no significant difference in presence values for lemon sharks at the three discrete spawning areas (he study . Residenp = 0.384). Similarly, the average absence value for lemon sharks at the FSA site during non-spawning (29.5 ± 11.5 d) was more than three times the value recorded during spawning season (8.0 ± 2.1 d), but the difference was not significant (p = 0.148). Average residence index at the Hind Bank MCD for lemon sharks (9.2 ± 1.8) was significantly higher than at the Grammanik Bank (2.9 ± 0.87) (p = 0.016), suggesting that lemon sharks had a greater affinity for the red hind spawning site. Peak detections of lemon sharks at the FSA occurred in January and February, whereas at the El Seco spawning site 95.2% of detections occurred in March. Partitioning receiver detections as a proportion of total detections into spawning and non-spawning seasons clearly illustrated the difference in behavior of lemon sharks during the two seasons, suggesting that grouper spawning has a very strong influence on pattern of MCE use for this species  reported in a number of studies [Although small sample size precludes robust conclusions about use of the MCE by Caribbean reef sharks, both individuals tracked behaved similarly and occupied small activity spaces, each roughly 5.5 km studies ,33–36. C studies , and juv studies ,38. Ther studies  and occa studies ,40. Move studies ,38.There were similarities in spatial and temporal patterns of use of the MCE for lemon and tiger sharks. Both species moved extensively throughout the MCE and were detected on a large majority of receivers within the array. However, lemon sharks exhibited conspicuous seasonal movement patterns with a prolonged presence on the reef and in proximity to the FSA during grouper spawning season followed by long periods of absence during non-spawning. Tiger sharks were the least site attached of the sharks tracked, with little seasonal variation in their movement patterns, and were characterized by relatively short visits to the MCE interspersed with slightly longer absences.Presence and absence values were similar for tiger and lemon sharks, however; the temporal patterns of their use of the MCE revealed marked differences. For example, all lemon sharks tagged were detected on acoustic receivers nearly 1,000 times and tracked for greater than 100 d, generating an average of approximately nine detections per day, whereas tiger sharks were detected an average of only 2.5 times per day. Additionally, the maximum number of consecutive days that a lemon shark was absent (133 d) from the acoustic array was substantially shorter than the maximum absence recorded for a tiger shark (272 d), and lemon sharks were typically absent for less than one week at a time, compared with higher and much more variable absence values for tiger sharks. Temporal presence/absence patterns of lemon sharks were closely associated with their orientation towards the FSA, typified by prolonged presence during spawning season, and long absences during non-spawning; a trend consistent among individuals. Temporal patterns of MCE use for tiger sharks were highly variable among individuals and punctuated by both short presence and absence values.A number of the lemon sharks tracked were present within the MCE and at the FSA for extended periods of time during grouper spawning season over consecutive years. Although lemon sharks are capable of relatively long distance movements ,42, lemoTiger sharks are known to cover great distances, including crossing ocean basins, and even on short time-scales may range over thousands of km ,32,43–46The three species of sharks oriented to the FSA differently, with only lemon sharks showing clear patterns of movement related to the FSA. Although Caribbean reef sharks are piscivorous ,54, theyTiger sharks also demonstrated a lack of orientation to the FSA during the grouper spawning season. Although tiger sharks have been reported to remain in specific locations for short periods of time (days to weeks) in association with seasonally abundant prey ,32,44–46Negaprion acutidens), which are thought to have learned to return to a particular area where food is spatially and temporally predictable [Lemon sharks exhibited a marked orientation to the FSA during grouper spawning season, a pattern that occurred consistently during each of the five years of the study. The chronic nature of this association is evidenced by increased presence of individual lemon sharks at the FSA during each grouper spawning season for as long as four consecutive spawning seasons. Additionally, although only two lemon sharks were detected at the El Seco site, over 95% of their detections were recorded during peak spawning of tiger grouper, the predominant grouper species spawning at that location . Tiger gdictable . The obsdictable ,51,63,64The MCE on the Puerto Rico-Virgin Islands platform was used extensively, year round by all three species of sharks studied. However, each species exhibited different patterns of use of the MCE and orientation towards spawning sites. Caribbean reef sharks were detected throughout the year within a small area exclusively within the FSA site. They exhibited long residence times but occasionally were absent from the array for over a year before returning. Tiger sharks used the majority of the MCE habitat monitored and were typically detected within the acoustic array over short number of consecutive days throughout the year, interspersed by short absences. Lemon sharks also used nearly the entire MCE habitat monitored by the acoustic array, but exhibited a strong association with the FSA and El Seco site during the grouper spawning season. Both the frequency and duration of visits to the MCE by all three species suggest that these species are regularly present on the reef and are likely involved in a wide array of ecological interactions within the MCE, but variable in nature.The high degree of orientation of lemon sharks to spawning sites demonstrates the influence of prey on movements of predators and illustrates the potential of sharks to interact with fishes at spawning sites. The strong association of lemon sharks with multiple spawning sites emphasizes the potential importance of spawning events in shaping ecosystem dynamics of MCEs and also illustrates the potential for predation on commercially important fishes, such as groupers, to influence their populations and to warrant consideration in efforts to manage these stocks.


A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance. Ebolavirus, Marburgvirus, and Cuevavirus. Five different ebolaviruses have been identified. They have at least 30% sequence divergence from virus to virus and include Ebola virus (EBOV), Sudan virus (SUDV), Taï Forest virus (TAFV), Reston virus (RESTV), and Bundibugyo virus (BDBV). Two individual viruses, Marburg virus (MARV) and Ravn virus (RAVV), are members of a single species of Marburgvirus.Filoviruses are highly pathogenic viruses that can cause outbreaks with significant disease and high lethality. Based on revised filovirus naming standards Cuevavirus, in Spain The filovirus genome is nonsegmented, negative stranded RNA that contains seven genes encoding the nucleoprotein (NP), the viral proteins VP24, VP30, VP35, and VP40, the glycoprotein (GP), and the RNA-dependent RNA polymerase (L) protein. Recent outbreaks and newly discovered filoviruses have highlighted the geographic range and diversity of the filoviruses, including the identification of RESTV as a highly pathogenic virus of cynomolgus macaques In regions of the world endemic for the malaria parasite, infection with highly pathogenic viruses can be misdiagnosed, leading to delays in appropriate treatment and improper patient isolation. This misdiagnosis can result in an incomplete understanding of the pathogens circulating in a particular region, impacting preparation for an outbreak response. A recently published study found that most of the acute patients admitted to the Lassa Fever Ward in Sierra Leone, a region hyperendemic for Lassa fever and malaria, were Lassa fever virus (LASV) antigen negative Real-time PCR and pathogen-specific ELISAs are commonly used for pathogen detection and biosurveillance studies. While these assays, especially real-time PCR, are highly sensitive and specific, screening multiple samples for an unknown pathogen can be expensive, time consuming, and limited for broad pathogen screening studies due to small sample volume. One solution to this is the use of multiplexed real-time PCR assays One approach to minimize unwanted sequencing and add specificity to the assay is the use of specific sequence capture probes (SCPs) followed by identification by NGS  plasma samples and human sera samples.The samples from rhesus macaques used in this study were archived and were not collected for the purpose of this study. The NHP experiment and procedures were approved by the USAMRIID Institutional Animal Care and Use Committee (IACUC) and was carried out in compliance with the regulations outlined in the USDA Animal Welfare Act (PHS Policy) and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and all animal work done adhere to the conditions specified in the Guide for the Care and Use of Laboratory Animals . Animals were given enrichment (including toys and mirrors) regularly as recommended by the Guide for the Care and Use of Laboratory Animals. Food was provided , and animals were checked at least daily according to the protocol. All efforts were made to minimize painful procedures; the attending veterinarian was consulted regarding painful procedures, and animals were anesthetized prior to phlebotomy. Following the development of clinical signs, animals were checked multiple times daily. When clinical observations and scores of animals reached defined levels based on the approved IACUC protocol , animals were euthanized by exsanguination following deep anesthesia and administration of a pentobarbital-based euthanasia solution to minimize pain and distress. All animals were housed at USAMRIID.De-identified human sera samples from individuals infected with BDBV in the Democratic Republic of the Congo (DRC) were used in this study. These samples were determined by the institutional Office of Human Use and Ethics to be Not Human Subject Research (HP-12-15). All samples were collected and de-identified in the DRC. The samples had indirect identifiers when we received them that prevented us from matching samples to the data that was collected. None of the samples were collected for this study; this study was an additional use of the samples collected for another purpose. Informed and written consent was obtained, and author JJM was involved in the collection of the samples.Viruses used in this study include the ebolaviruses SUDV, EBOV, RESTV, TAFV, and BDBV. MARV variants included Musoke, Ci67, and Angola. All viruses are maintained at USAMRIID, and IRB approval was not required for use. For each of the filoviruses, total RNA was purified from the supernatant of virus-infected cells using TRIzol . Total nucleic acid from 100 µl cell culture supernatant was isolated directly from the TRIzol mixture using the EZ1 Virus 2.0 kit  and the EZ1 robot (Qiagen) according to the manufacturer's directions. Nucleic acid was eluted in a final volume of 60 µl elution buffer.Archived NHP plasma samples were used in this study. Animals were challenged by the intramuscular route with approximately 300 plaque forming units (PFU) of EBOV H. sapiens-tc/COD/1995/Kikwit, and longitudinal samples were taken over the course of the infection. Whole blood collected into K2EDTA tubes or serum clot activator tubes with gel separator  was centrifuged at room temperature to separate plasma/sera in accordance with manufacturer's guidance. RNA samples were prepared using a 1 part sample: 3 parts reagent ratio into TRIreagent LS . Aliquots were prepared and stored at −80°C until analysis. Serum viremia for NHP samples were titrated by plaque assay using Vero E6, a 1% SeaKem agarose  primary overlay, and a 5% neutral red secondary overlay. Log dilutions of sera were prepared and plated in replicates of three to six, and a tissue-culture EBOV seed was included in half-log dilutions as a positive control. Human serum samples from individuals infected with BDBV were also used in this study. Reference materials, including aliquots of diagnostic samples, were obtained through a collaboration with National Institute of Biomedical Research (French acronym INRB) in Kinshasa. Virus from potentially positive samples was amplified by infecting a monolayer of Vero E6 cells and looking for cytopathic effects (CPE).Q value of <40 cycles.Total nucleic acid from 17.5 µl human serum samples and 17.5 µl of cell culture supernatant from cell cultures with CPE was isolated by TRIzol extraction (elution volume of 70 µl AE buffer) using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's directions. For the NHP samples, 25 µl plasma was extracted with TRIzol and eluted in 100 µl AE buffer. Presence of virus for the human samples was determined using established real-time PCR assays for the SUDV, EBOV, RESTV, TAFV, BDBV, RAVV,and the MARV variants Musoke, Ci67, and Angola, and the presence of EBOV was determined in the NHP samples as previously described in silico analyses, panels of 15 probes for each filovirus were designed to capture approximately 100 bases of viral sequence. Probes were synthesized by Integrated DNA Technologies , and full sequences are available from Pathogenica, Inc, upon request. Please see Reference sequences for the filoviruses SUDV (GenBank # NC_006432), EBOV (GenBank # NC_002549), RESTV (GenBank # NC_004161), TAFV (GenBank # NC_014372), BDBV (GenBank # NC_014373), and MARV variants Musoke (GenBank # NC_001608), Ci67 (GenBank # EF446132), and Angola (GenBank # NC_DQ447660.1) were analyzed to identify conserved regions for probe hybridization that would also contain viral sequence that would differentiate the filovirus isolates during sequence analysis. Pathogenica's probe design algorithms can analyze hundreds or thousands of genomes simultaneously to determine which regions to target to maximize the information contained in the sequences. In addition, the probe design algorithm allows for the production of a multiplex set of non-interacting probes. These are specifically selected to provide discrimination between closely related viruses that differ by 1 or more bases at multiple loci within the viral genome. Based on the For the initial probe evaluation, total RNA from the cell culture supernatant of cells infected with stock virus was amplified from 50 ng total nucleic acid using the Quantitect Whole Transcriptome Amplification Kit (Qiagen) according the manufacturer's protocol. Probe was hybridized to 50 ng amplified cDNA by adding 3 nM probe in 1.5x ampligase buffer  and heating the reaction to 94°C for 2 minutes and then to 60°C (0.1°C/sec temperature decrease) with a 60 min hold at 60°C. Target sequence was captured within the probe by adding 2 units AmpliTaq Stoffel Fragment (Life Technologies) and 4 units Ampligase (Epicentre) to the reaction and incubating at 60°C for 60 min with a final concentration of 0.4 µM dNTPs .taq (Life Technologies) using probe-specific primers designed to amplify the captured region [0.4 µM forward (5′-CAGATGTTATCGAGGTCCGAC) and reverse (5′-GGAACGATGAGCCTCCAAC) primers]. Reactions were amplified at 95°C for 3 min, 40 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, and a final hold at 72°C for 10 min on the MJ Mini  or T100 thermocycler (Bio-Rad). Following the PCR amplification of the captured sequences, amplicons were resolved on a 2% agarose gel and purified using the QIAQuick Gel Extraction Kit (Qiagen). Expected amplicon sizes were from 180 to 200 base pairs. Samples were quantified using the Bioanalyzer  with the DNA 1000 kit.Noncircular probes and background DNA were removed by heating the reaction to 94°C for 2 min, ramping to 37°C, and adding 10 units Exonuclease I  and 50 units exonuclease III (New England Biolabs) to the reaction. Samples were incubated at 37°C for 30 min followed by enzyme inactivation at 94°C for 15 min. Captured sequence was amplified by Platinum All of the filovirus probes were initially evaluated using amplified, strain specific filovirus cDNA. Individual probes that yielded a PCR product of the appropriate size were pooled into the final filovirus probe panel. This panel was further evaluated with each of the amplified filovirus cDNAs using the protocol described above.Gel-purified amplicons were sequenced on the MiSeq or the GAIIx platform  using the 2×150 cycle sequencing kit. Libraries were prepared using the TruSeq DNA Sample Preparation Kit (Illumina) with single (NHP samples) or dual (human samples) indices. The resulting sequencing reads were analyzed using CLC Genomics Workbench . Reads were trimmed for quality, and the primer sequences used for the capture sequence amplification were trimmed. Reads were further trimmed based on length, removing reads less than 100 bases. The trimmed and filtered reads were then mapped to a reference library containing the targeted capture sequences and each filovirus genome . Mapping settings included 70% of the read had to match the reference sequence by at least 80% identity, and nonspecific read mappings were ignored. Reads that mapped to the respective filovirus strain was normalized by determining the percentage of the total sequencing reads following trimming and filtering that successfully mapped. For the human samples, 5 no template controls (NTCs) were included, and a sample was called positive if the percentage of mapped reads or the total number of reads was greater than the NTC average plus three times the standard deviation of the NTCs.Multiplex SCP assays can target divergent genomic signatures for strain identification and differentiation by capturing a conserved signature within a probe followed by sequencing. The 3′ and 5′ probe arms contain sequences that are complementary to the viral sequence flanking the filovirus-specific target sequence, allowing the conserved sequence to be captured for identification by sequencing. To identify these filovirus-specific target signatures, the filovirus genomes for SUDV, EBOV, RESTV, TAFV, BDBV, and the MARV variants Musoke, Ci67, and Angola were aligned. Multiple sequences within each viral genome were identified for probe design, each having a conserved, filovirus-specific sequence. This conserved sequence had to be flanked by additional sequence such that the probe's complementary arms would hybridize at >60°C. This analysis resulted in a panel of 15 sequence capture probes per viral strain.An upfront amplification protocol prior to sequence capture was developed and optimized in order to maximize sensitivity from limited sample volumes. Initially, using five of the SUDV DxSeq probes, a comparison of cDNA generation using Superscript II (Life Technologies) and Qiagen's Whole Transcriptome Amplification (WTA) kit resulted in increased sensitivity, measured by amplicon signal, using the WTA kit (data not shown). Using these same probes, optimization of cDNA concentration showed 50 ng cDNA had increased amplicon yield (data not shown). Testing of all of the filovirus probes individually for generation of appropriately sized amplicon under these conditions allowed for a downselection to the final filovirus DxSeq panel development. Specific information regarding location of the probes within this final panel, including the capture sequence locations, can be found 5 to 1.1×107 pfu/ml. Overall, the pooled probe panel showed very high specificity for the respective filovirus with little cross-reactivity among the different variants  of the samples from the Lassa Fever Ward in Sierra Leone, a region endemic for Lassa fever and malaria, were negative for both of these pathogens. Some of these negative samples showed evidence of an acute infection with, among other viruses, the filoviruses EBOV and MARV Burkholderia pseudomallei from B. malleiHaving the capability to screen a clinical or environmental sample for multiple viruses or bacteria of interest within a single reaction would be a significant improvement for pathogen identification and biosurveillance. Screening a sample for multiple targets can become expensive when testing for a single target at a time, and large scale screening for multiple pathogens could be limited by a small sample volume. Multiplexing assays can address these concerns, and many multiplexed real-time PCR assays have been developed. For example, one assay can identify four different dengue virus serotypes NGS is another technology that can theoretically identify any pathogen in a sample, but this lack of specificity can be a hurdle for getting FDA approval for a diagnostic. Targeted sequencing gives specificity to NGS, allowing many targets to be assayed simultaneously in a single tube reaction. Commercially available panels with >400 probes are available, and the underlying technology has been demonstrated capable of assaying >10,000 loci simultaneously Testing of human and NHP clinical samples using this panel resulted in positive detection of BDBV and EBOV, respectively. While not as sensitive as real-time PCR, the filovirus SCP panel correctly identified EBOV in the three experimentally infected NHPs having the highest viral loads, and BDBV was correctly identified in multiple human clinical sera and cell culture amplified samples. In terms of assay sensitivity, this panel correctly identified the targeted virus at high concentrations, but there was a loss of detection at the samples containing the lower amounts of virus as determined by real-time PCR. This lower sensitivity near the limit of detection likely resulted from the inherent nature of NGS analysis and barcode demultiplexing, reflected in higher background signals among the negative and NTC samples. A previous study found an ∼0.3% read misidentification rate for single indexed samples Overall, we developed an expandable, broad filovirus detection panel that could aid in biosurveillance and diagnostics. While not as sensitive or rapid as real-time PCR, there is added benefit of being able to screen multiple samples for a large number of pathogens using a small sample volume. Regional-specific, syndromic, or biothreat detection panels could be developed by incorporating additional SCPs, ultimately leading to a broad pathogen detection system for use in biosurveillance.Figure S1Read mapping to the SUDV genome shows specific reads mapping to the targeted capture sequence regions. The combined filovirus probe panel was evaluated using RNA from multiple filoviruses. Shown is the mapping of the reads against the SUDV genome using SUDV RNA is the starting material. There were 569,003 reads (90.05%) of 631,885 post-trimming and filtering reads that mapped to the SUDV reference genome.(TIF)Click here for additional data file.Table S1Filovirus probe hybridization and capture sequence.(DOCX)Click here for additional data file.Table S2Detailed read mapping for the nonhuman primate clinical sera samples using the filovirus probe panel.(DOCX)Click here for additional data file.Table S3Detailed read mapping for the human clinical sera samples using the filovirus probe panel (run 1).(DOCX)Click here for additional data file.Table S4Detailed read mapping for the human clinical sera samples using the filovirus probe panel (run 2).(DOCX)Click here for additional data file.


Birth synchrony is well documented among ungulates and is hypothesised to maximize neonate survival, either by minimizing the risk of predation through predator swamping or by synchronising birthing with increased seasonal food availability. We used encapsulated vaginal implant transmitters to locate and capture neonatal moose calves and document the seasonal and diel timing of parturition in two adjacent study areas with different predation pressure in central Ontario, Canada. We tested the hypothesis that predation promotes earlier and more synchronous birth of moose calves. Across both areas, proportionately more births occurred during the afternoon and fewer than expected occurred overnight. Mean date of calving averaged 1.5 days earlier and calving was also more synchronous in the study area with heavier predation pressure, despite average green-up date and peak Normalized Difference Vegetation Index date occurring 2 days later in this study area than in the area receiving lighter predation pressure. We encourage analysis of data on timing of parturition from additional study areas experiencing varying degrees of predation pressure to better clarify the influence of predation in driving seasonal and diel timing of parturition in temperate ungulates. Birth synchrony is well documented among ungulates , 3, 4, 5Alces alces) populations exhibits relatively low variation across >15° of latitude [Despite the potential influence of timing of parturition on calf survival, most studies have failed to detect an influence of inter-annual variation in environmental conditions on timing of moose parturition , 17; butlatitude , 17.Canis spp.) and American black bears (Ursus americanus) tend to be less active [There is ongoing debate regarding the relative importance of various selective pressures in shaping timing and synchrony of ungulate births within and among species. As part of a broader assessment of moose viability near the southern extent of their range in Ontario , 19, 20,s active , 28, 29.2) and the other in the southeastern portion of Wildlife Management Unit 49  (Acer saccharum), poplar (Populus spp.), American beech (Fagus grandifolia), yellow birch , eastern hemlock (Tsuga canadensis), spruce (Picea spp.) and fir (Abies spp.); the forest composition in WMU49 was similar, although with lower abundance of hemlock and more developed and agricultural land and habitat fragmentation [Canis lycaon), eastern coyotes (Canis latrans var.) and American black bears. Wolves were relatively abundant in APP (2.3–3.0/ 100 km2 [2 (95% CI 21–66) and 32 bears/100 km2 (95% CI 15–57) for WMU49 and APP respectively [We studied moose in 2 nearby study sites in central Ontario, Canada –one in the western portion of Algonquin Provincial Park (APP)  . The oveentation . The stuentation ). Potent 100 km2 ) but unc 100 km2 . Black bectively .®; Bayer Inc., Etobicoke, Ontario, Canada) at approximately 0.2 mg/kg [During 2007–2009 we used encapsulated vaginal implant transmitters  to locate and capture calves from a random sampling of cows in both study areas. From January through March, 2007–2009 we captured 119 different adult female moose via darting or aerial net gunning and fitted them with either VHF or GPS radiocollars . Prior to deployment, VITs were cold sterilized for 24h in Nolvasan solution, rinsed with sterile saline, and stored individually in sterile Whirl-Pak® bags. Each VIT was placed in a sterile disposable, lubricated speculum  which was then inserted in the vagina to the cervix. VITs were held in place at the cervix by a 19 mm diameter PVC tube as the speculum was slowly pulled back. Disposable speculums were discarded, and the smaller diameter PVC tube washed with soap and water and cold sterilized in Nolvasan solution between deployments. VITs were typically shed from moose at parturition and the pulse rate changed from 40 PPM to 80 PPM when the temperature dropped below 32°C. We recovered most expelled VITS from within or immediately proximate to obvious calving beds and were only aware of two cases were VITs were expelled prior to the onset of parturition. In addition to the pulse rate change, the VITs emitted a coded pulse that enabled us to determine how long each had been shed, in increments of 30 minutes, for periods of up to 5.5 days. During the course of the study we documented a few cases where VITs had their temperature sensitive mortality switches reset by exposure to direct sunlight. VITs that had been reset by the sun were detected through repeat monitoring. For example, in rare cases we would discover that a VIT known to have been out for multiple days was suddenly sending a signal indicating more recent expulsion. Checking the timing code for VITs that had been “reset” in this manner indicated they had all been reset during the warmest part of the day and this was only ever documented for VITs shed in open meadows and exposed to direct sunlight–whereas most calving sites were in forested cover  where resetting of the timer was not a risk.To avoid pseudoreplication, only a single calving date was recorded in cases where twinning was documented. All capture and handling methods followed the guidelines of the American Society of Mammalogists  and wereWe aerially radio-tracked moose that had been fitted with VITs biweekly following capture until early-May, when daily monitoring began. At the onset of calving these moose were checked daily from the air or by a ground crew to determine calving status. When a VIT was shed, a crew of 2–4 located the calving site using a hand-held antenna and portable receiver. Calf handling and subsequent monitoring are described elsewhere .t-test assuming unequal variances to test whether mean calving dates differed between APP and WMU49. We used this test because it is robust to slight departures from normality [Ks = 0.102, P = 0.50) and only slightly skewed in the right tail in WMU49 .n = 7 additional data points) encountered without use of VITs that we were confident were < 24 hours old at time of encounter. This determination was based published criteria [We successfully recorded the date of parturition of 77 calves born to cows fitted with VITs . Calving was also more synchronous in APP, with calving dates being significantly more variable in WMU49 .Across both study areas the first date of successful calving was May 8, with an overall mean date of calving of May 13 . Eighty P = 0.0294; Based on the coded pulse emitted by the VITs, and excluding cases where field personal did not record the coded signal indicating time of expulsion, or where warm temperatures apparently reset the coded timer in the VITs, we recorded precise time of birth for 39 moose calves . The timing of birth was not uniform throughout the 24–hr period; mean calving time was 14:49 with an angular dispersion of r = 0.299 , May 13 in WMU49 (mean = 13.9)) were earlier than documented in northwestern Ontario , QuébecWe also observed later and less synchronous parturition of moose calves in the study area with lighter predation pressure (WMU49) than observed in nearby APP which, at only 50 km to the east, had similar habitat and climatic features. Wolves and black bears were the only significant predators of moose during this study. Our assertion that predation pressure was lighter in WMU49, despite potentially higher bear densities there, was based primarily on the results of a companion study of calf mortality ; howeverAmong black bears, large males may be more likely to prey on ungulates , 49, incThat predation pressure differed between the two areas was also consistent with the marked differences in defensive behavior exhibited by cows in the 2 study areas. Maternal defense of calves was almost absent in WMU49, with cows generally retreating when they first observed our approach to their calf . In APP,Ovis canadensis) captured from established populations in Utah, Alberta, Montana, and Colorado and translocated into two separate populations in northern Utah adjusted timing and synchrony of parturition to the environmental conditions of their release sites within 5 years of reintroduction [Algonquin Provincial Park was established as a protected area in 1893. However, given that wolves were lethally controlled in APP until 1958  it is nooduction .The mean calving date in APP was only 1.5 days earlier than in WMU49, and similar to the discussion above, some might find it difficult to envision such a subtle difference in parturition date conferring much of a selective advantage in terms of predator avoidance. However, calves during our study gained mobility quickly after birth and by ca. 3 days of age were often difficult to capture by hand on the ground . FurtherUrsus arctos) from much of their original distribution in North America has produced a patchy distribution in the primary predators of moose across the continent. They suggested that the continental synchrony in onset and duration of parturition among North American moose, despite spatially variable predation pressure, indicates moose are tracking long-term patterns of climate to time reproduction [n = 18; WMU49 = 14.9 kg, n = 30; t 18, 30 = 0.880, P = 0.383) were all similar between areas indicating no apparent differences in condition of females between areas.Bowyer et al.  noted thoduction . Howeveroduction , 56. Furoduction ; yet moooduction , moose ioduction . Pregnanoduction , twinninoduction , and wein = 84) the mean age of cows from APP was 4.8 ± 0.42 versus 4.0 ± 0.40 years in WMU49 ..n = 84) n WMU49 n = 84) tAlthough birth dates are well documented for many ungulate species, given the recent advent of VITs, there are few data on precise diel timing of births. Small sample sizes precluded our testing for differences in diel timing of births between study areas, but in pooling study areas we documented a disproportionate number of births in the afternoon. Among mammals the onset of labour is immediately preceded by a sharp increase in free circulating oestrogens . The proThroughout much of the year wolves tend to be less active during mid-day and most active during crepuscular and dark periods , 27, 29.Alternatively, the disproportionate number of afternoon births may simply reflect selection for birthing during the warmest time of day, as temperatures occasionally reached freezing over night during calving in this study and neonatal calves can be sensitive to exposure during harsh weather , 68. HowS1 Table(DOCX)Click here for additional data file.


P = 0.3, 0.7, 0.8 after 6, 72, and 288 hours, respectively). A statistically significant difference was found after 72 hours for TNF-α (P = 0.01), for GSH after 6 hours (P < 0.01), and after 6 and 72 hours for LOOH . Oxidative stress occurred at earlier time points and was pore accentuated HWM versus LWM and prodromal to TNF-α increase.The relationship between mesh weight and host tissue reaction has, so far, not been fully investigated. Lightweight meshes (LWM) are thought to give less inflammatory response compared with heavyweight meshes (HWM). The present study is a randomized, controlled, double-blind clinical trial performed in 61 patients who underwent an elective inguinal hernioplasty. The primary outcome of the study was to investigate the relationship between total amount of prosthetic material (polypropylene), immunological reaction, and oxidative stress. The study was double-blinded. Sixty-one patients were recruited for the study and randomly assigned to 2 groups (groups A and B). Levels of inflammation markers (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) and oxidative stress markers (reduced glutathione [GSH] and lipid hydroperoxides [LOOH]) were determined preoperatively and after undergoing inguinal hernioplasty , respectively, with LWM and HWM. There was no significant difference in IL-6 levels between HWM and LWM . The impressive development of prosthetic hernia repair over the last 20 years has led industry and surgeons to research on new kinds of meshes. In the last 6 to 7 years, LWM have been introduced in clinical practice on the assumption that a lightweight prosthesis could reduce local complications, such as discomfort and chronic pain, or inguinal impairment, that had been referred to heavyweight meshes (HWM).,11,12 However, published studies have succeeded in demonstrating a lower incidence of discomfort and pain using LWM, but some studies have indicated a higher incidence of hernia recurrence,13; despite these results, some controversies still remain and the general interest for LWM has remained.,15The notable development and diffusion of prosthetic surgery of the abdominal wall over the last few years has led to the introduction of lightweight meshes (LWM). followed by an incorporation of them into the area of implant, with a limited fibroblastic response  and a strong scar tissue. The inflammatory response seems to be characterized by increased levels of interleukin (IL)-6 and C-reactive protein,16 associated with other modifications of inflammatory serum markers . The immunologic reaction to polypropylene was already studied in previous published reports. However, in the present study we studied different cytokines such as IL-6 and tumor necrosis factor-α (TNF-α) compared with the previously studied types ,10,19,20 and how these variables are related to oxidative stress. IL-6 and TNF-α were separately studied in different groups and in a smaller cohort of patients, comparing traditional surgery with prosthetic repair for inguinal hernia. The main conclusion of these studies was that polypropylene mesh induces an inflammatory response that was quantitatively relevant with respect to traditional techniques. In some other reports, Di Vita et al stated that such an immunologic response was proportional to the quantity of polypropylene inserted and additionally postulated that such an increase of cytokines  could be correlated to the incidence of local complications due to meshes. IL-6 is often induced together with TNF-α in many inflammatory conditions. However, whether IL-6 plays a pro- or anti-inflammatory role in local inflammation is not clear. Although it is commonly believed that IL-6 acts as an inducer of inflammatory genes, a recent report about IL-6 (−/−) mice indicates a crucial anti-inflammatory role by controlling the level of proinflammatory cytokines.Immunological response seems to play a major role in the complex mechanisms of repair following mesh implantation. To this regard, the polypropylene prostheses, which are more frequently used, induce a rapid and useful acute inflammatory response,,14,15 few on the comparison between lightweight and heavyweight,,21 and to the best of our knowledge, none on the oxidative stress induced by the mesh. Moreover, only one study has been published that clearly correlates the immunological reaction to the amount of prosthetic material. Whether the immunologic reaction depends on pore size and texture instead of materials remains questionable and controversial data have been published in the literature.,22There are few studies on the immunological reaction to polypropylene meshes,The aim of this research was to evaluate a possible relationship between the amount of implanted polypropylene and immunological reactions as well as postoperative oxidative stress, and thus to evaluate, if present, the differences in the biological reaction and biotolerability between LWM and HWM on a statistically significant number of patients with a randomized prospective clinical trial.22.1 Regarding inclusion and exclusion criteria for the study, all patients affected by primary inguinal hernia between 18 and 90 years, not previously operated with implantation of a prosthetic mesh, were enrolled. Patients affected by diabetes, cirrhosis, any chronic inflammatory disease or under corticosteroids, and/or immunosuppressive therapies (neoplastic patients) were excluded from the study. A total of 61 patients were recruited. Two patients were, after being enrolled, excluded due to the discovery of previous operations with mesh implants and an anamnestic positivity for immunological disorders (multiple sclerosis), one patient did not return after the operation to complete the requested protocol of blood analyses. These 3 patients had previously been randomly assigned to LWM. Therefore, in the end 29 patients were enrolled in LWM, whereas there were 32 in HWM. This study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. The patients were not informed as to which prosthesis was implanted. The ethics committee of Policlinico-Vittorio Emanuele University Hospital of Catania approved the study, informed consent form, and the workflow of the research on March 2010, study code CH001GENI. Then the study was registered at http://clinicaltrials.gov/show/NCT01090284 (registration number assigned NCT01090284). The study ended in December 2011.Between March 2010 and December 2011, 64 patients were prospectively recruited and randomized in a double blind manner for the present study. Three patients (dropout rate 4.68%) were excluded from the study . The recruitment of patients took place following the random order in which they were referred to the hernia service of the General Surgery and Week Surgery Unit of the University Hospital of Catania which is a large, high complexity university hospital. After physical examination confirming the diagnosis of inguinal hernia and once surgery had been indicated, informed consent was obtained from the patient by means of a standardized form. Hernia type was registered for each patient according to Rutkow classification.2.22 of polypropylene); for HWM, on the contrary, the mesh was of the “heavyweight” type (220 g/m2). Simple randomization was used by flipping a coin.Patients were randomly assigned to 2 groups , patient 2 (HWM): in LWM inguinal hernioplasty surgery was carried out with the use of the so-called “lightweight” type , as well as kind of implanted mesh. Although anamnestic data of patients were collected by surgeons, the statistical evaluation was carried out by another investigator.2.32) (content of polypropylene). They did not differ for stiffness or size. For each patient, a preoperative blood test was carried out to determine the basal levels of IL-6, TNF-α, reduced glutathione (GSH), and lipid hydroperoxides (LOOH). All these components were determined on blood samples, respectively preoperatively, 6 hours after the operation, on 3rd and 12th postoperative days. All samples were frozen for reference. No perioperative preparation (including medications) was performed in all patients as for our guidelines. All the patients underwent an open local anesthesia  prosthetic inguinal hernia repair as gold standard technique, a published variant of Trabucco's repair with the apposition of one or more plugs. Mesh fixation was performed by suturing mesh flaps around spermatic cord through 2/0 polypropylene (Prolene) stitches . Wound size was about 10 cm and was closed by intradermic reabsorbible suture with 3/0 polyglactin 910 (Vicryl) (Ethicon). All surgical procedures were performed by the same experienced surgeon . All patients received postoperatively, ceftriaxone (1 g/die for 4 days), tramadol , when requested by the patient to control postoperative pain. Patients were monitored in an ambulatory manner for 12 days and complications were recorded.The 2 types of meshes had the same pore size, the same texture (monofilament polypropylene meshes), and came from the same manufacturer . They differed only in weight .According to previous studies,2.6,30 Briefly, sample was incubated with anti-IL-6 or anti-TNF-α, anti-rabbit immunoglobulin G, and horseradish peroxidase conjugates, in successive order. Absorbance at 450 nmol/L was measured, and cytokine concentrations were calculated from a standard curve generated with purified IL-6 or TNF-α. Each measurement was performed in triplicate and averages were reported.Cytokines were measured by commercially available enzyme-linked immunosorbent assay kits . The assay was performed in accordance with the protocol provided by the manufacturer and as previously described.2.7,31 The intra-assay coefficient of variation is 4%, whereas the interassay coefficient of variation is 5.6%.Blood levels of total thiol groups (GSH) were measured in 200 μL of blood samples using a spectrophotometric assay based on the reaction of thiol groups with 2,2-dithio-bis-nitrobenzoic acid at λ=412 nm. The limit of detection for this assay is approximately 15 nmol/L.2.82+ to Fe3+ in the presence of xylenol orange at λ=560 nm. The assay mixture contained 100 mmol/L xylenol orange, 250 mmol/L ammonium ferrous sulfate, 90% methanol, 4 mmol/L butylated hydroxytoluene, and 25 mmol/L H2SO4. After a 30-minute incubation at room temperature, the absorbance was measured using a U2000 Hitachi spectrophotometer . Calibration was obtained using hydrogen peroxide (0.2–20 mmol/L).,33 The limit of detection for this assay is approximately 0.25 nmol/L.LOOH levels were evaluated following the oxidation of Fe2.9t test and ANOVA (with Bonferroni correction for multiple comparisons), as appropriate. Univariate linear regression analyses were performed to identify independent predictors of serum inflammatory marker levels as continuous dependent variables. In these models, we selected as independent variables such as age, body mass index (BMI), operative time, mesh type, and number of plugs. When more than one variable was found significant at univariate analysis , multivariate analysis was performed to assess which was independently associated with serum inflammatory marker levels. All statistical tests were 2-tailed and a P ≤ 0.05 was considered significant. Data were entered into Microsoft Excel for Windows . Statistical analysis was performed using SPSS for Windows release 17.0 .Baseline characteristics are presented as mean and standard deviation. Assumption of normal distribution of cytokines and oxidative stress markers was tested by Shapiro-Wilk test and parametric tests were used. Statistical differences between groups (light vs heavy) at different time after operation  were tested by the 33.1P = 0.013 and P < 0.034)  . This trend was also confirmed by TNF-α data even if gaining significance only after 72 hours ; there was also a statistical significance related to operative time (P = 0.013), type of mesh , and number of plugs (P = 0.001) (see Table P = 0.048) (see Table P = 0.01). On univariate linear regression, this tendency  was confirmed depending mainly on age (P = 0.08) and mesh type (P = 0.06∗). Also, for LOOH we observed a higher increase after 6 and 72 hours for HWM . On univariate analysis, the factor mainly responsible for this difference, also this time, was mesh type (P = 0.02)  prospective randomized clinical trial. Our work confirmed some previously published data, showing that reaction to polypropylene correlates positively not only with contact area (number of plugs) but also with overall amount of implanted material (mesh type). Regarding the statistically significant difference in our cohort it was found only on the third postoperative day for TNF, but this was mostly due to the size of our cohort of patients mainly conditioned by costs of reactants for immunological determinations. Univariate analysis confirmed this tendency giving significance for number of plugs while losing it for BMI. Taking together these data can affirm that also in a prospective clinical trial the intensity of immunologic reaction such as oxidative stress seems to be positively related to increase of overall polypropylene amount and not only with the extension of the contact area. As far as we know this is the only study investigating immunologic reaction on 60 patients, testifying to the difficulties of conducting such a study on large cohorts of patients. In addition, our recruitment was prospective and unlike other studies, all selection biases were preventively eliminated. On the contrary, as regards oxidative stress, the study succeeded in finding such a correlation between increase of mesh weight and oxidative stress, indirectly measured by means of antioxidant system consumption (GSH decreases and LOOH increases). The clinical significance of these data is still unclear; it remains to be elucidated whether our results are related to a higher risk of development of local complications and modification of meshes. As regards clinical practice, LWM could be considered less invasive (possibly inducing less immunologic reactions and oxidative stress) compared with HWM, even if this advantage can mean a high incidence of recurrences. Both oxidative stress and immunologic reaction seem to be linked in experimental models. Moreover, we could postulate that to reduce oxidative stress induced by polypropylene, L-carnitine could be added to the mesh to reduce oxidative effects, even if clinical experiences are needed. On the contrary, the stronger immunologic reaction  due to polypropylene implantation, following some clinical studies attributing an antiseptic role to the increase of such cytokines, could explain the rare occurrence of polypropylene infections. Results of this study should be considered in light of the limitation that we were not able to collect data of 3 patients, and this may have led to selection bias. Moreover, the limited number of patients included in the trial may have weakened some results .Our data show a correlation between number of plugs (polypropylene content) and immunologic reaction related to TNF-α on the third postoperative day. Our data also demonstrate an increase of immunologic reaction proportional to the prolongation of operative times and patient body weight and significantly related to mesh weight  and even number of plugs . A previous work by Di Vita,39 and antioxidative systems,41 in current research. Taking into account the supposed role of oxidative stress on the evolution of local complications, we hope that our study will stimulate a new research field on the evolution of prosthetic materials in abdominal wall surgery, maybe stimulating industries to produce not only “less inflammatory” but also “less oxidative” meshes. Our study does not take into due account the genetic variability of enrolled patients. In fact, it is known that polymorphisms in IL-6 gene may results in a different inflammatory response under certain stressful condition. However, the blinded randomization may in part mitigate such bias. Finally, our sample size was sufficient to find the differences between groups of interest, but an increased number of subjects enrolled in the study may have strongly emphasized also a linear association .To the best of our knowledge this is the first trial investigating the correlation between polypropylene meshes for inguinal hernia repair and oxidative stress, considering the interest for oxidative stress2. The oxidative stress is strongly related to the quantity of polypropylene for inguinal hernia repair, and is prodromal to TNF-α increase. HWM implantation causes a stronger immunologic reaction and oxidative stress in the host compared with LWM. In particular, our data suggest that LOOH and GSH are influenced by the mesh type, whereas TNF-α level is depending on the plug numbers.In conclusion, the immunologic reaction to polypropylene after inguinal hernioplasty is directly related not only to the contact area with the host but mainly to the overall amount of material per cmAs regards the clinical significance of such data we can, at the moment, only speculate; this aspect should be further investigated maybe with studies on larger cohorts of patients.