The aim of this article is to provide an updated review of their different clinical presentations, to suggest a correct approach to their diagnosis and to discuss the usefulness of both old and new diagnostic tools, including fecal biomarkers, atopy patch tests, endoscopy, specific IgG and IgG4 testing, allergen-specific lymphocyte stimulation test (ALST) and clinical score (CoMiss). Food allergy (FA) is defined as an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food . Based oIgE-mediated FA are the most common. They are easily characterized by the presence of specific serum IgE (sIgE) or a positive skin prick test (SPT). They occur most frequently in the first years of life, giving rise to urticaria/angioedema, oral allergic syndrome, rhinitis, or acute asthma and anaphylaxis .Non-IgE FA are characterized by cutaneous reactions , respiratory reactions (such as Heiner’s syndrome) or gastrointestinal reactions, which we will discuss in more detail below .Non-IgE and mixed FA are less understood, despite their frequency: a Swedish population study showed that 36% of 118 children diagnosed with cow’s milk allergy (CMA) by an oral challenge test were negative to specific IgE and SPT for cow’s milk [The diagnosis of non-IgE and mixed FA is mainly clinical and is not always easy. In contrast with IgE FA, the onset of symptoms is delayed and they may have a chronic presentation, making their association with the allergen less evident . FurtherOral food challenge (OFC) is considered the gold standard for the diagnosis of IgE and non-IgE FA . This coThe diagnosis of some forms of non-IgE FA, and especially mixed FA, also requires an endoscopic examination to reveal any eosinophilic infiltration of gastrointestinal tissue.Recent years have seen rising interest in non-IgE FA. A search for “non IgE mediated food allergy” on Pubmed revealed 9 articles published in 2000 and 11 in 2001, compared with 66 and 67 articles respectively for 2018 and 2019. This has resulted in a large increase in knowledge of many of its clinical and non-clinical aspects. The aim of this article is thus to provide an updated review on the different clinical pictures of non-IgE and mixed gastrointestinal FA in the first years of life. It also focuses on the role of both old and new diagnostic tools, including fecal biomarkers, atopy patch tests, endoscopy, Immunoglobulin G, Immunoglobulin G4 , allergen specific lymphocyte stimulation test (ALST), clinical score, and other novel and future tests for the diagnosis of non-IgE and mixed FA. Celiac disease, although classified as a non-IgE-mediated food allergy, is not included in this review.www.pubmed.gov) and Embase databases (www.embase.com). Searches were not restricted by language of publication, publication type or study design, but had to have been published in the last 10 years. However, we also considered earlier relevant studies and guidelines by looking through the references of the reviews and clinical studies published on this topic. This search found 2176 articles in PubMed and 2800 in Embase. Total non-overlapping record identified in PubMed and/or Embase for each category was 4145. Of these articles 189 were considered useful and are cited in the references. The search strategies and results in PubMed (MedLine) and EMBASE are detailed in Search strategy. A comprehensive search was conducted in September 2020 using MEDLINE via PubMed  system was used to generate questions in regard to three of the topics: endoscopy and food challenge, atopy patch test and food challenge, and clinical score and food challenge. Further details on the methods and results are given in the respective paragraphs.Non-IgE gastrointestinal FA present with specific, well-characterized clinical pictures, such as food protein-induced allergic proctocolitis (FPIAP), food protein-induced enterocolitis syndrome (FPIES) and food protein-induced enteropathy syndrome (FPE), eosinophilic gastrointestinal disorders (EGIDs) including eosinophilic esophagitis (EoE), allergic eosinophilic gastroenteritis (AEG) and eosinophilic colitis (EC) , or withNotwithstanding a lack of prevalence studies, FPIAP is believed to be the most common non-IgE FA. It manifests in the first months of life with bloody stools in an otherwise seemingly well infant, in whom other causes of bleeding  have been excluded . In a reA recent study of 257 infants with FPIAP showed even less optimistic data: only 60% of children developed tolerance in the first year of life, although 99% did so within 3 years .SPT and sIgE tests for milk are usually negative. However, about 20% of children with FPIAP may show sensitization or develop IgE-mediated allergy to offending foods over time . EndoscoFor these reasons, it has been suggested, for otherwise seemingly well infants with suspected FPIAP, to wait 2–4 weeks for spontaneous resolution without initiating an elimination diet ,18. If sAn elimination diet  usually leads to regression of symptoms within 3–5 days, although some children may require a few weeks before any improvement is seen . An OFC FPIES usually presents acutely, although a chronic form has also been described. Acute FPIES is characterized by bouts of vomiting 1–4 h after ingestion of the food responsible . RepetitAny food can induce FPIES, but the most common causes vary by age and location. Rice and oats have emerged as the most common triggers in the USA, followed by cow’s milk, soya, egg, fish, fruits, and vegetables . In ItalA variety of protocols have been proposed in relation to OFCs. A recent International Consensus suggests administering a dose of 0.06 to 0.6 g  of the food protein per kilogram of body weight, in three equal doses over 30 min . It is gThe severity of chronic FPIES symptoms depends on the amount of food trigger continuously present in the diet. With low doses , they manifest as intermittent vomiting and/or diarrhea and failure to thrive, without dehydration or metabolic acidosis. More regular intake  is associated with intermittent but progressive vomiting and diarrhea , sometimes with dehydration and metabolic acidosis, and in about 50% of cases failure to thrive. Vomiting should regress within 3 days of excluding the responsible food, and its reintroduction may be followed by the sudden onset of acute clinical signs of FPIES. Without a confirmation challenge, the diagnosis of chronic FPIES remains presumptive .The incidence of FPE is unknown, although it seems to be less common than 20 years ago, and today is rather rare. It manifests with chronic diarrhea and consequent failure to thrive in the first 9 months of life . It mostEGIDs are chronic diseases characterized by a range of gastrointestinal symptoms, eosinophilic infiltration of the gastrointestinal tract and, sometimes, peripheral eosinophilia. Diagnosis requires the exclusion of other causes of eosinophilic infiltration and the involvement of other organs.EoE may Start in the First Years of Life. In a multicentric study of 705 patients with EoE, about half were under 11 years of age. In this subgroup the median age of diagnosis was 3 years and median age of the onset of symptoms 1.1 years (interquartile range 0.4–3 years) . In the The most common symptoms of EoE, such as dysphagia and food impaction, increase with age and are more common during adolescence. Concomitant atopic conditions should increase the suspicion of EoE . It is dFood can be a trigger for children with EoE, especially in the early years of life. Studies showed that about 70% of children were allergic to one or more food, above all cow’s milk, egg, wheat and soya. The younger the age, the more foods may be responsible ,39.AEG is much less common than EoE. It affect both adults and children, and is rarely seen in the first year of life ,41. In yEC is the least common form of EGIDs , althougEven if some studies found that more than half of cases of EC coexist with an allergy to cow’s milk protein, soya, or peanuts, the elimination diet is not usually sufficient to treat it . The IgENon-IgE FA occur with less specific symptoms. These can include repeated regurgitation, vomiting, crises of crying, gas, poor growth, constipation or diarrhea, and it is not always possible to frame them in one of the clinical pictures listed above. There also seem to be some differences in the clinical features and laboratory findings in different ethnic groups and geographical regions ,54. In aNon-IgE FA are characterized by intestinal inflammation and increased permeability, which leads to migration of granulocytes and eosinophils to the intestinal lumen. Due to the lack of reliable diagnostic tests, there is growing interest in finding fecal biomarkers. Several studies have investigated the use of various fecal biomarkers for diagnosis, such as fecal calprotectin (FC), α-1 antitrypsin (AT), β-defensin, tumor necrosis factor-α (TNF-α), fecal IgA, eosinophil-derived neurotoxin (EDN) and eosinophilic cationic protein (ECP).FC is an S-100 group cytosolic protein. This group comprises calcium- and zinc-binding proteins, thereby depriving microorganisms of these trace elements and inhibiting some zinc-dependent enzymes . FC is iOther intestinal proteins , as well as FC, have been studied in non-IgE FA, offering surrogate markers of the cellular response.To investigate the use of FC in FA (particularly CMA) in infants, we selected 6 studies conducted in children with non-IgE FA and 3 studies conducted in a population with both IgE-mediated and non-IgE-mediated forms . As was Baldassarre et al. reported significantly higher FC values in patients than controls, with values dropping by 50% after the elimination diet .Beser et al. enrolled 32 infants under two years of age diagnosed with IgE and non-IgE mediated CMA by OFC. They found higher FC levels in the non-IgE mediated group, suggesting a possible use for this biomarker in the diagnosis and monitoring of non-IgE mediated gastrointestinal forms of CMA .A prospective study conducted by Trillo Belizon et al. reported statistically higher FC values in infants aged 1 to 12 months diagnosed with non-IgE mediated CMA than in both infants for whom such a diagnosis had been excluded and healthy controls. Furthermore, there was a progressive decline in FC values after one to three months of a cow’s milk elimination diet, with significant differences in patients with gastrointestinal symptoms such as diarrhea or rectal bleeding. The authors concluded that an FC value <138 μg/g permits the exclusion of a diagnosis of non-IgE mediated CMA, with a sensitivity of 95% and a specificity of 78.57% .In a study of 46 children with allergic colitis suggestive of CMA, Lendvai-Emmert et al. found considerably lower FC values in children who had followed a strict cow’s milk elimination diet for 3 months compared to their value at diagnosis, thereby indicating FC as a useful parameter for the diagnosis of CMA .Prikhodchenko et al. monitored FC values in 18 children with FPE and 20 healthy age-matched children over the course of the disease. The mean FC concentration was higher in children with FPE than in the control group, but dropped significantly during the course of the disease. The authors concluded that FC shows promise for monitoring the course of FPE and evaluating treatment efficacy in children with FPE .However, other studies reported results conflicting with those cited above. Some studies did not find any statistically significant difference between FC values on diagnosis of CMA and after a normal diet without cow’s milk, or in comparison with healthy controls ,76.The use of FC as a biomarker of intestinal response to OFC has been investigated in very few studies. Berni-Canani et al. investigated the presence of subclinical intestinal inflammation in response to challenge testing of an amino acid-based formula under study in 60 infants aged ≤4 years with both IgE and non-IgE mediated CMA. FC and ECP were measured both before and 7 and 14 days after the challenge. Their values were unchanged in all patients, thereby demonstrating their optimal clinical tolerance of the formula .p values were significantly different between geometric means of FC values in non-IgE-mediated forms in comparison with IgE-mediated forms (18% versus 15%).Merras-Salmio et al. found higher FC values in patients following an elimination diet with a positive challenge than in patients with a negative challenge (39 children) towards cow’s milk protein or in the controls (22 children), demonstrating the presence of mild inflammation of the intestinal mucosa during the challenge. The Mann-Whitney Fecal biomarkers can indicate the degree of intestinal inflammation. Quantification of fecal eosinophils, above all EDN and ECP, reveals the extent of eosinophilic gastrointestinal inflammation, thus making them a non-invasive clinical biomarker. The feces of patients with FPIES, FPE and FPIAP show high levels of EDN, which remain stable at room temperature for at least 7 days, with matching histologic evidence of eosinophilic allergic colitis . Kalach In a child with FPIES, Wada et al. found an increase in TNF-α following sequential measurement prior to hydrolysate challenge and a paradoxical reduction during the challenge. After the challenge, it rose again for one month. Similar results were observed with fecal IgA, which dropped during the challenge, whilst fecal EDN rose during the challenge. The authors concluded that the sequential measurement of fecal TNF- α, together with other markers of intestinal inflammation, could offer a sensitive and non-invasive method to evaluate non-IgE mediated forms of CMA .In a more recent study of eight patients with FPIES and 12 age-matched healthy infants, Wada et al. determined FC, EDN and fecal IgA levels before and after the OFC, finding a significant increase in all three fecal biomarkers in all patients after ingestion of the causative food. However, FC and fecal IgA levels were much lower than EDN, and the authors suggest that fecal EDN testing after ingestion of the causative food may serve as a useful diagnostic marker of FPIES .p < 0.05). The authors found the best diagnostic performance in a combination of FC and EDN (88.9% and 84%) respectively and concluded that FC and EDN are reliable tools in differentiating between FPIAP and gastrointestinal functional disorders in infants [Very recently, Rycyk et al. measured simultaneous FC, EDN and TNFα in 34 infants with gastrointestinal bleeding and 25 control group infants with functional gastrointestinal disorders. FPIAP was diagnosed by open OFC in 27 infants, and the offending food was identified as cow’s milk in 23 and hen’s eggs in 4 patients. Children with FA demonstrated significantly higher FC and EDN levels than the controls  of 68% and a negative predictive value (NPV) of 83% for occult blood in the diagnosis of FPIAP [In conclusion, the results from the available literature do not permit us to make any recommendations concerning the use of FC in the diagnosis of non-IgE FA. Further studies are necessary, involving an adequate number of participants with uniform characteristics such as age, nutrition, and duration of elimination diet, and, above all, the use of clearly defined reference values and FC cut-off times. The latter is a problem of great importance, given that whilst for adults and children over 4 years of age there is a well-defined cut-off value for FC (50 mg/kg), values in children under 4 years are considerably higher, and no cut-off values have been established for infants under one year of age ,87,88,89The measurement of food-specific IgG and IgG4 antibody levels is often proposed for the diagnosis of non-IgE mediated FA, but the results are currently still uncertain. We performed a search on PUBMED and Embase to establish the diagnostic usefulness of IgG and IgG4 testing in this type of allergy, particularly in pediatric age see . None ofStapel et al.  pointed The situation for EoE could be different. Recent data have highlighted the presence of high titers of specific food IgG4 antibodies in sera and esophageal tissue biopsy specimens from adults with EoE ,96. The Schuyler AJ et al.  demonstrThe allergen-specific lymphocyte stimulation test (ALST), also called lymphocyte proliferation or transformation test, has also recently been used to improve the diagnostic work up in non-IgE FA. We selected nine articles from the PubMed and Embase search see .The ALST analyzes lymphocyte proliferation and cytokine production in a culture of peripheral blood mononuclear cells after stimulation with food antigen for 3–5 days. Response is typically reported as the percentage of stimulated cells (stimulation index). Although this test has long been used in the diagnosis and research of disorders associated with immune diseases , its role in allergic diseases is still uncertain ,100.Two studies showed the diagnostic utility of ALST in neonates and infants with non-IgE GI symptoms after ingestion of cow’s milk formula ,102, butCMA can cause functional bowel disorders, which can create difficulty in managing pediatric surgical patients who also have CMA.Ikeda K et al.  examinedYagi H et al.  evaluateKajita N et al. , in a reOverall, all these studies showed that ALST could be a useful tool in the diagnosis of non-IgE mediated gastrointestinal FA, given that it can be performed regardless of the patient’s clinical condition and hence enables early diagnosis. Nevertheless, there are a number of limitations to its use in children: the use of antigens that are not yet standardized, the significant amounts of peripheral blood necessary for the test and the relatively long culture times (5–7 days) .To try to improve these limits, Yagi H et al.  evaluateIn conclusion, the possibility of using specific biomarkers in the diagnosis of non-IgE mediated FA is still uncertain. While ALST and IPAST appear very promising in this regard, further studies are needed for both tests to improve standardization, to enable their use for as many antigens as possible and to better understand the mechanisms underlying the expression of cytokines and/or their receptors.The atopy patch test (APT) is an in vivo test that aims to reproduce the allergic reaction by application of the suspected allergen to the skin. It mimics the cell-mediated immune responses in which T cells play a prominent role, such as in non-IgE FA. APT has been included as a potential test to assess suspected FA in subjects with clinical signs of FPIES, FPIAP, FPE and EGID, as well as those with less specific symptoms .APT is performed by applying the suspected food allergen to healthy untreated skin .The diagReactions are traditionally classified as + in the presence of erythema, slight infiltration and, possibly, papules; ++ in the event of erythema, infiltration, vesicles and papules; and +++ for intense erythema with infiltrate and coalescing vesicles. They are negative if the skin is unaffected, and doubtful in the event of faint erythema only .APT is not recommended for the routine diagnosis of FA . CertainWe evaluated the accuracy of APT compared to OFC using a PICO system. All eligible studies had to meet the inclusion criteria: pediatric patients, FA adequately confirmed by OFC, specific results in relation to the accuracy of APT. Data extraction was developed on the inclusion criteria, taking in consideration the best available evidence. Studies from which it was impossible to extract data on the specificity and sensitivity of APT were excluded. Two reviewers screened all abstracts and full-text articles independently. Any disagreement was resolved by a third party.A total of 56 articles were identified by the literature search. Two of these were systematic reviews ,113, andArticles were screened, but no relevant studies were found in addition to those already included in the systematic reviews, and only two studies published after 2017 were eligible according to the inclusion criteria.The methodological quality of one of the systematic reviews  is low. The second systematic review  showed aMost of the included studies investigated both IgE-mediated and non-IgE FA. Subgroup analyses were conducted in relation to the patients’ age and clinical signs. The gastrointestinal symptoms analyzed were: vomiting, regurgitation, diarrhea, abdominal pain, constipation, hematochezia and failure to thrive. In other cases, the enrolled patients had a specific diagnosis of enterocolitis, enteropathy, gastroesophageal reflux (GER) or FPIES. Subgroup analyses for gastrointestinal allergic symptoms indicated high specificity (91.5%) and low sensitivity (57.4%) for APT. The results showed that FA cannot be ruled out completely in the event of a negative APT, while its high specificity means that a positive APT indicates a high risk of FA.It should perhaps be emphasized that four studies in the above systematic review recruited patients with a non-IgE mediated reaction, a negative SPT and negative specific IgE to the suspected foods. One of these was a retrospective study on FPIES,  while twOur search identified two relevant studies published after the 2017 systematic review. In July 2017, Gonzaga TA et al.  evaluateThe second recent study, by Sirin Kose S et al.,  aimed toThe discussed systematic review and our own search excluded studies without an appropriate diagnosis of FA, namely those that did not compare APT with OFC. For this reason, EoE was not included in the list of food allergies, because the diagnosis must be confirmed by the number of eosinophils in the esophageal biopsy specimen .There is little literature evidence in relation to APT and EoE, with only two studies from the same group evaluated. The first, published in 2007, is a prospective study , while tIn conclusion, APT can be included in the diagnostic workup because it is a safe, specific diagnostic test that could point to a possible FA, especially in children with non IgE-mediated gastrointestinal symptoms . The predictive capacity of APT can therefore be improved by combining it with negative sIgE or SPT measurement. However, several aspects require further investigation, especially to enable the better definition and standardization of the technique.Diagnosis of non-IgE FA in clinical practice is challenging, due to the lack of pathognomonic non-invasive laboratory tests. Many non-IgE and mixed FA such as EoE and FPIAP have typical histological findings which confirm the diagnosis and point to the best treatment ,123. HowMoreover, with the exception of EoE, these investigations supply data that are not easy to interpret, and hence do not change the patient’s elimination diet, the timing of trigger food reintroduction or any strong suspicions of a different diagnosis, such as autoimmune enteropathy, tufting enteropathy, microvillus inclusion disease, or congenital disaccharide deficiencies, in the case of persistent symptoms.We aimed to compare the diagnosis of non-IgE and mixed FA based on histology and elimination diet vs. OFC. Only a handful of studies satisfied the above-mentioned diagnostic work-up . After eRectal bleeding is common in not-sick newborns and infants . A recenWith persistent bleeding, a diagnosis of FPIAIP should be confirmed by avoidance diet and oral food reintroduction at home, or OFC under supervision if SPT and sIgE tests for food are positive. However, in clinical practice if symptoms disappear and the infant is well, the confirmatory oral provocation test may be overcome in the first months of life. It should be periodically performed over the first year of life to test the acquisition of tolerance.Given the age of presentation and the favorable course in the majority of cases, biopsies are generally not recommended, except in cases of unusual or abnormal symptoms such as constipation, diarrhea with mucus-streaked stools but without grossly visible bleeding, or severe rectal bleeding complicated with anemia despite a cow’s milk elimination diet.In EoE the problem is far more complex. EoE is a chronic esophageal inflammatory disease characterized clinically by symptoms of esophageal dysfunction and histologically by eosinophil-predominant inflammation . When EoThe search strategy  identified only one study. Yukelsen et al . investiThese results lead to various observations. First, this study showed the existence of a relationship between GERD and allergic disease. Second, it underlined that while OFC and allergy testing can identify many patients with allergic disease, endoscopy enables the diagnosis of EoE.The detection of eosinophilic infiltration (>15/hpf) in at least one esophageal biopsy is the diagnostic hallmark of EoE (35). The recent Joint Recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition report a diagnostic algorithm addressing gastroesophageal reflux disease (GERD) management in clinical practice .Upper GI endoscopy with biopsies should be performed in cases of persistent symptoms, such as crying, vomiting, anemia, feeding problems and/or failure to thrive, to identify and characterize esophagitis or enteropathy.Poddar  et al. pThe diagnosis of CMA in the first year of life is often challenging because its presentation is non-specific, especially in non-IgE mediated and mixed forms. A clinical score, the CoMiSS (Cow’s Milk related Symptom Score), has recently been proposed. According to the authors, the CoMiSS should be used as an awareness tool to help recognize the symptoms of CMA in infants and young children ,130.In order to review the diagnostic performance of CoMiSS and any other clinical scores, we carried out a PUBMED and Embase search using the terms listed in p = 0.001). A more than 50% reduction in a baseline score ≥12.0 was the best predictor of a positive challenge [In the first study, Vandenplas and colleagues evaluated the diagnostic performance of CoMiSS in relation to an open challenge performed after 1 month of elimination diet (extensive hydrolysate). A total of 116 infants with symptoms compatible with mild to moderate non-IgE CMA were included. The challenge was performed in 73% and was positive in 69% of the infants. The study showed a reduction in the CoMiSS during the elimination diet, and found that it was correlated to the challenge result. The average score reduction was 8.07 points; the challenge was positive in 80% of patients in whom the score was reduced to 6.0 points or less, but only in 48% if it remained ≥7  in this larger cohort did .In another study of 226 healthy, mostly  breastfed Polish infants of the same age, the median and average scores were 4.0 and 4.7 respectively, while the 95th percentile was 11.0. Only 11 infants (4.9%) scored ≥12.0 .So, the next question was: what is the clinical usefulness of CoMiSS? In particular, what is its predictive value when compared to the OFC in non IgE mediated CMA diagnosis?Chakrabarty and Rigley evaluated the diagnostic performance of CoMiSS in two small studies, comparing it with the outcome of the elimination diet . Interestingly, significantly reduced values were found at the end of the observation period ,135.Bajerova et al. used CoMiSS to identify patients at risk of CMA. The authors suggested that lower cut-offs  would increase the sensitivity of the method in children with non-specific symptoms of milk protein allergy .None of the above studies reported the number of cases with sIgE and/or SPT.Prasad carried out a study on 83 patients aged between 0 and 24 months with symptoms suggestive of CMA. A score >12.0 was obtained in 60 patients (72%). CMA was confirmed in 70 patients by OFC (performed in only 56% of cases) or ImmunoCAP. In detail, in 78.6% of patients with CoMiSS >12.0 and in 15% of patients with a value ≤12.0, the CoMiSS showed a PPV of 93% and an NPV of 33%. According to the authors the low NPV was probably because many children were already on the elimination diet and this would have led to a reduction in the score .In two Italian studies, CoMiSS was compared with response to the elimination diet . In the A meta-analysis of 3 studies to investigate the usefulness of CoMiSS as a predictor of CMA as confirmed by open challenge  found thKose and Seda evaluated the response to the elimination diet according to the CoMiSS score in 112 children diagnosed with CMA, egg allergy or both. OFC confirmed the diagnosis in 46 patients (41%), in whom the modification of the score during the 1-month elimination diet was assessed. A ≥50% reduction corresponded to a sensitivity of 83.7%, 84.6% and 87.5% for milk allergy, egg allergy and both, respectively. According to the authors, this value could be employed as a cut-off for the diagnosis of the corresponding allergies ,146.A very recent study evaluated 168 children with a baseline score ≥12 who were started on elimination diet for 4 weeks: children who responded to the diet also underwent the open challenge. This study has two important strengths: the large number of children enrolled and the diagnostic confirmation, in all the “responders” to the diet, by open challenge. The allergy was confirmed in 54.2% patients; the ROC curve showed that the best cut-off for CoMiSS was 12.5, which corresponded to a sensitivity of 64.8% and a specificity of 54.4%. The study also showed that some symptoms, such as skin involvement, were more frequently observed in children with confirmed allergy whose score was significantly higher. The authors therefore concluded that the systematic evaluation of symptoms associated with CoMiSS can aid the selection of infants who might benefit from an elimination diet ,148.A collaborative study by Belgian and Spanish authors on children aged <6 months assessed the variability of the score when calculated by a pediatrician and by parents, as well as day to day variability when evaluated by parents over 3 consecutive days. The data suggest that CoMiSS can be calculated by parents, before medical consultation, without the need for special training. In the Spanish arm of the study, the diagnostic performance of the score was also compared in relation to OFC: 10 out of 13 children (76%) with a score ≥10 and 7 out of 8 with a score >12 were diagnosed as allergic to CM by OFC ,150.Finally, CoMiSS was recently included in a computer-based algorithm in which a score ≥12 increases the likelihood of the diagnosis and supports a dietary prescription for babies, whether exclusively breast-fed or not . The scoIn conclusion, 14 studies have evaluated the diagnostic efficacy of the CoMiSS. Of these, 9 were prospective studies, and 5 enrolled less than 50 children. Only about half the studies reported the percentage of positive specific IgE and/or SPT in the study population; in these studies, the vast majority seemed to be non-IgE FA. The diagnostic efficacy of CoMiSS compared to OFC was evaluated in 13 studies. In the 3 studies  which used a CoMiSS cut-off value of >12, the PPV was between 80 and 100%. The vast majority of studies found a reduction in CoMiSS after elimination diet and that a >50% reduction in CoMiSS was predictive of a subsequent positive OFC.Although further studies are needed to validate CoMiSS in the diagnostic workup of CMA and, possibly, other types of FA, and to define the optimal cut-off values, it can already be considered a useful tool, especially for suspected non-IgE mediated FA. As also affirmed by other authors, it should also be used to monitor response to therapeutic interventions such as the elimination diet, but at present it is not sufficient in itself to diagnose FA and cannot replace the OFC .Recent years have seen great interest in the search for biomarkers that, supported by clinical evidence, could facilitate the diagnostic path for non IgE-mediated and mixed FA. However, results have been poor . LaboratThere is growing evidence that the microbiome contributes to the development and presentation of allergic diseases. It seems that gut dysbiosis likely precedes the development of food allergy, and the timing of dysbiosis appears to be critical . SpecifiOther studies have shown that activation of the innate immune response underlies the pathogenetic signs of these diseases. Mehr et al. used RNA sequencing and bioinformatic approaches to analyze whole blood from children with FPIES before OFC and during any acute reactions . PatientSimilarly, Schouten et al.  found a A recently proposed ALST measures interleukin 2 α-receptor mRNA expression within 24 h, using a small amount of peripheral blood. However, tests like these need further study to adapt their use to as many allergens as possible and to better understand the mechanisms underlying the expression of both cytokines and their receptors .The basophil activation test (BAT) is a laboratory test for the in vitro simulation of an in vivo allergenic challenge, using cytofluorometric evaluation of basophil activation markers (CD63 and CD203c). The BAT is mainly used for IgE-mediated FA, but can also be used for non IgE-mediated allergies, albeit with a lower diagnostic accuracy; it is in fact one of the few in vitro techniques currently available for this kind of allergic reaction . Indeed,Some promising results are also arriving from the instrumental diagnostics field. To support the diagnosis of non IgE-FA in symptomatic individuals, a recent study proposed the use of abdominal ultrasound and Doppler imaging to evaluate intestinal vessel density (VD) . The autIn contrast, infectious diseases do not produce the same ultrasound evidence. Moreover, patients with delayed allergy showed a larger VD in the ileum and jejunum than did the other two groups. These parameters could therefore be used to distinguish a non IgE-mediated FA from a severe infection. This non-invasive examination is suitable for use in children, but as with all ultrasound procedures, it is operator-dependent. In any case, given the low number of cases included in the clinical study and the variation in the participants’ ages, further investigation is needed .Finally, genetics could be used to identify individuals affected by or at risk of numerous disorders, including allergic diseases. A large number of genes have been identified by genome-wide association studies for food allergy . AllergiClassical epigenetic mechanisms, including DNA methylation and histone modifications, have been shown to be involved in the development of IgE-mediated food allergies such as CMA ,172,173.In recent years, a common inflammatory pathway has been hypothesized for allergic diseases, characterized by a “type-2 inflammation” involving different cells besides the classic Th2 cells. These cells, from both the innate and adaptive systems, produce a unique Th set of so-called ‘type-2′ cytokines, the effectors of the allergic response ,175,176.Interestingly, a different DNA methylation profile of Th1 and Th2 cytokine genes and achievement of tolerance has been demonstrated in children with IgE mediated food allergy . This suIn addition to the classic mechanisms discussed above, other epigenetic mechanisms have also been proposed in non-IgE-mediated food allergy. It has been reported that post-transcriptional control elements such as miRNAs may be involved in the pathogenesis of non-IgE delayed cow’s milk hypersensitivity , suggestAlthough epigenetic research is still in its infancy, especially in the field of non-IgE mediated food allergies, it may have several promising clinical applications, ranging from prevention to early prediction of the success of a given therapeutic strategy. Finally, ongoing progress in molecular biology and omics sciences  may offer new insights into non-IgE food allergies .Non-IgE mediated and mixed FA constitute a heterogeneous group of diseases arising through immunological mechanisms that are not yet well understood. In clinical practice, diagnosis generally relies on a compatible clinical history and the resolution of symptoms upon the elimination of the presumed triggering antigens. Diagnostic confirmation, however, requires a different approach in the different clinical pictures. An OFC or home reintroduction of food may be attempted in many cases, while some cases, endoscopy and biopsy of the affected intestinal tract is also essential for diagnosis. Promising new diagnostic tools to facilitate diagnosis are being studied, with encouraging results in some cases, such as CoMiSS, LSTs and IPAST. Further studies are still necessary to fully understand the physiopathology of these diseases and, consequently, improve their diagnosis and prognosis.


Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation. Septins are GTP-binding proteins that polymerize into heterooligomeric core complexes, and further assemble into higher-order structures, including filaments, rings, and cages . The sepSEPT7 plays important roles during cell division, cytokinesis, and neuronal differentiation. For example, depletion of SEPT7 leads to microtubule destabilization as well as cytokinesis defects in fibroblasts . SEPT7 iSEPT7 is a key component of most types of septin filaments . SeptinsPhosphorylation is an important mechanism in the regulation of septin function. During the epididymal transition of spermatozoa, defective SEPT4 phosphorylation results in a defective membrane diffusion barrier . In the Xenopus embryos, knockdown of SEPT7 or SEPT2 causes defects in ciliogenesis [The primary cilium is a microtubule-based organelle that protrudes from the surface of most mammalian cells. It plays important roles in development and differentiation. Disruption of cilium structure or function causes many diseases, including obesity, blindness, polycystic kidney disease, mental retardation, and polydactyly . The priogenesis . In mammogenesis . Thus, sogenesis ,25,26.In this study, we aimed to explore whether SEPT7 phosphorylation influences septin filament assembly and ciliogenesis. We found that PKA increased SEPT7 phosphorylation at the Thr. Overexpression of phospho-memetic SEPT7 reduced filament assembly, suggesting that SEPT7 phosphorylation at Thr197 is important for the assembly of SEPT7 filaments. We also found that PKA interacted with SEPT7 through the GTP-binding domain. Furthermore, we found that PKA-mediated phosphorylation of SEPT7 reduced the frequency of ciliated cells and reduced the ciliary length. These findings suggest that SEPT7 phosphorylation disrupts septin filament assembly and ciliogenesis.3VO3  as indicated for analysis of protein expression.Human SEPT7 and PKA were amplified from a human RNA panel  and cloned into pFLAG-CMV-2, pEGFP-C3, and pCDNA3-HA vectors, as described previously . All conFor immunoprecipitation analysis, 2 μg of indicated antibodies was incubated with Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 15 min on a rotator, and the cell lysates were immunoprecipitated with the bead‒antibody complex at 4 °C overnight on a rotator. The samples were washed three times with a wash buffer . The precipitates were mixed with SDS sample buffer, then boiled for 10 min. For Western blotting, the proteins were adjusted to an equal amount of protein, electrophoresed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto PVDF membranes . The blots were then incubated with an anti-FLAG , anti-Myc , anti-GFP , anti-HA , anti-PKA  antibody, anti-SEPT4 , anti-SEPT6 , anti-SEPT7 , or antiphosphothreonine  antibody. Immunofluorescence analysis of filament formation followed.For the immunofluorescence analysis, the plasmids were transfected into NT2/D1 cells or 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific). After 24 h, NT2/D1 cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) permeabilized with 0.1% triton X-100 in PBS, and blocked with the antibody diluent  for 1 h. NT2/D1 cells or 293T cells were washed three times with PBS. The filament could then be visualized using fluorescence microscopy .http://www.ebi.ac.uk/, accessed on 25 January 2020). The accession numbers for SEPT7 proteins of different species were as follows: Homo sapiens , Sus scrofa (pig) (XP_003134838.1 isoform X1), Canis lupus familiaris (dog) (XP_022283282.1), Felis catus (domestic cat) (XP_003982994.1), Mus musculus (house mouse) (NP_033989.2), Rattus norvegicus (brown rat) (NP_072138.2), Bos taurus (cattle) (NP_001001168.1), Pongo abelii (Sumatran orangutan) (NP_001126872.1), Xenopus laevis (African clawed frog) (NP_001086183.1), Xenopus tropicalis  (XP_002939459.1), and Danio rerio (zebrafish) (NP_001242958.1). The accession numbers for SEPTIN groups of Homo sapiens were as follows: SEPT1 (NP_443070.1), SEPT2 (NP_001008491.1), SEPT3 , SEPT4 , SEPT5 (NP_002679.2), SEPT6 , SEPT7 , SEPT8 , SEPT9 , SEPT10 , SEPT11 (NP_060713.1), and SEPT14 (NP_997249.2). The above information was provided by the National Center for Biotechnology Information (NCBI) database .The human SEPT7 orthologous proteins were used for multiple sequence alignment by the ClustalW2 program  for 1 h in the dark. The nuclei were stained simultaneously with 4′,6-diamidino-2-phenylindole . After extensive washing, the cover slips were mounted on glass slides in 50% glycerol. Primary cilia were imaged with an Axio Imager M2 fluorescence microscope  and captured using ZEN Pro software (Zeiss). Primary cilia images were generated, and the length of cilia was measured from z-stacks using add-on features of the ZEN Pro software.p-values were considered significant at * p < 0.05; ** p < 0.01; and *** p < 0.001.Data were expressed as the mean ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA), combined with Tukey’s multiple comparison test for posterior comparisons. Homo sapiens, the predicted phosphorylation site is highly conserved , thus disrupting septin filaments in the sperm annulus . GBD is onserved B. Thus, PRKACA), was co-transfected with wild-type SEPT7 or T197A mutant followed by an examination of the phosphorylation status of SEPT7. Overexpression of PKACA2 increased the Thr phosphorylation of SEPT7, and this signal was reduced in the T197A mutant of the 293T and NT2/D1 cell lines  to understand whether phosphatase regulated SEPT7‒SEPT7 interaction. We found that Na3VO4 did not interfere with the interaction between GFP-SEPT7 and FLAG-SEPT7  in the early stages of embryogenesis disrupts the assembly of septin filament formation, thus leading to the dissociation of the septin complex. Our previous work shows that phosphorylation of a germ-cell-specific SEPT12 leads to a complete loss of the septin ring at the sperm annulus, illustrating the important roles of septin phosphorylation in the regulation of septin assembly and the formation of higher-order structures. The phosphorylation site of SEPT12 is located in the GBD, and this domain is conserved among several septins, including SEPT7. We thus checked whether phosphorylation of SEPT7 affected the assembly of the septin filament. Indeed, SEPT7 phosphorylation in the GBD led to the defective polymerization of septin filaments. Thus, phosphorylation of septin in the GBD might affect septin filament formation. However, it is still unclear whether other septins, in addition to SEPT7 and SEPT12, also show a similar phenotype, and this hypothesis still needs to be tested in the future. It has been shown that the septin complexes regulate cilium length and ciliogenesis. Ablation of any of these septins’ expression by RNA interference inhibits ciliogenesis. Post-translational modifications play an important role in the regulation of septin‒septin interactions and control the formation of a high-order septin complex. These modifications include SUMOylation, acetylation, ubiquitination, and phosphorylation. Phosphorylation of the terminal subunit septin, Cdc11, disrupted neck filaments and influenced higher-order septin architecture . PhosphoXenopus epidermis, SEPT7 forms a ring at the base of motile cilia. Knockdown of SEPT7 leads to fewer and shortened cilia [Xenopus embryos [Depletion of SEPT7 results in the concomitant loss of SEPT2 and SEPT9 expression of the complex ,31. In ted cilia . Althouged cilia . Hedgeho embryos . Interes embryos . In the  embryos ,34, and  embryos ,36,37. I embryos ,39. In tIn summary, we found that SEPT7 was phosphorylated by PKA. PKA-mediated SEPT7 phosphorylation impeded SEPT7‒SEPT7 interaction, but did not affect the SEPT2-6-7 or SEPT4-6-7 complexes. In addition, phosphorylation of SEPT7 was reduced upon serum starvation, during which primary cilium started to grow. The T197E mutant inhibited ciliogenesis upon serum starvation. Thus, the phosphorylation status of SEPT7 is essential for septin filament formation and ciliogenesis.


The conventional pedicle-screw-based dynamic stabilization process involves dissection of the Wiltse plane to cannulate the pedicles, which cannot be undertaken with minimal surgical invasion. Despite some reports having demonstrated satisfactory outcomes of dynamic stabilization in the management of low-grade spondylolisthesis, the extensive soft tissue dissection involved during pedicle screw insertion substantially compromises the designed rationale of motion (muscular) preservation. The authors report on a novel method for minimally invasive insertion of dynamic screws and a mini case series.The authors describe innovations for inserting dynamic screws via the cortical bone trajectory (CBT) under spinal navigation. All the detailed surgical procedures and clinical data are demonstrated.A total of four  patients (mean age 64.75 years) with spinal stenosis at L4–5 were included. By a combination of microscopic decompression and image-guided CBT screw insertion, laminectomy and dynamic screw stabilization were achieved via one small skin incision (less than 3 cm). These patients’ back and leg pain improved significantly after the surgery.This innovative dynamic screw stabilization via the CBT involved no discectomy , no interbody fusion, and little muscle dissection (not even of the Wiltse plane). As a minimally invasive surgery, CBT appeared to be a viable alternative to the conventional pedicle-screw-based dynamic stabilization approach. Various fusion techniques have been accepted as the surgical management for disc degenerative disease or spondylolisthesis, including anterior, posterior, transforaminal, and extreme lateral lumbar interbody fusion  –4. In thPedicle-screw based dynamic stabilization systems use elastic materials, for example, a synthetic cord-and-spacer or metallic spring, instead of a rigid metallic rod, to connect between individual pedicle screws . PlacemeThe authors report in this technical note an innovative method to insert four dynamic screws via a 3-cm skin incision to neutralize a degenerative spine or to stabilize low-grade spondylolisthesis. Under image guidance, the screws were placed along the cortical bone trajectory (CBT) and assembled with dynamic cords. The surgery involved microscopic decompression of the bilateral neuroforamen and dynamic screw insertion, but no discectomy  or interbody fusion procedures. Compared to the standard minimally invasive TLIF, the novel technique preserved the intervertebral disc and required less muscle dissection. Although the theoretical benefit of reducing ASD requires longer-term follow up to confirm, this technique spared arthrodesis and was done with minimal soft tissue or facet destruction. This is the first report to describe dynamic screw placement via the CBT.Four consecutive patients , with an average age of 64.75 years, who underwent minimally invasive dynamic screw stabilization with the Dynesys Dynamic Stabilization  system were retrospectively analyzed.After general anesthesia, the patient who had a L4–5 herniated disc with bilateral foraminostenosis Fig.  was put After decompression, the O-arm  was brought into the room to acquire images for navigated screw placement. The reference pin was inserted into the left posterior superior iliac crest. The acquired images were transferred to the Stealth Station  for spinal navigation. Prior to the beginning of instrumentation, we used an image-guided ball tip probe to confirm the accuracy of navigation and locate the entry points. The entry points of CBT screws are generally over the cephalad lateral part of the pars interarticularis, slightly caudal to the sulcus of the facet complex. An image-guided high-speed drill was used to break through the cortex. The trajectory was chosen, using the navigation system, to allow the screw to course through the dense cortical bone with the screw tip barely penetrating the cortex of the vertebral body laterally. A 5.2 mm image-guided cannulated tap was used to create the screw tract. The Dynesys screws  were subsequently placed under navigation. A total of four CBT screws were inserted into the L4 and L5 bodies with bi-cortical purchase  and she was discharged from the hospital on post-operation day 5.A 58-year-old male presented with low back pain and left sciatica for 6 months. The symptoms were on and off several times and it got worse prior to a visit to our clinic.His radiographic evaluations demonstrated a huge L4–5 ruptured disc with caudal migration and left L4–5 foramen compression. Since his symptoms were compatible with nerve root compression by the L4–5 ruptured disc, surgical decompression, removal of sequestrated disc fragments and dynamic stabilization were recommended. The post-operative fluoroscopy demonstrated that 3.1 degree motility was preserved at post-op 12 months  for lumbar degenerative disease , 9, 10. The CBT used for screw insertion was first published by Santoni et al. in 2009 for posterior midline lumbar fusion surgery. Compared with the traditional pedicle-screw trajectory used, several biomechanical studies have demonstrated the superiority of CBT screws, which could yield a 30% increase in pullout strength and a 71% increase in insertional torque , 18. MorDynamic screw stabilization could be achieved with minimally invasive surgery via the CBT by a combination of microscopic decompression and spinal navigation. This innovative dynamic screw stabilization involves no discectomy, no interbody fusion, no facet violation, and little muscle dissection (not even of the Wiltse plane). As a minimally invasive surgery, CBT appeared to be a viable alternative to the conventional pedicle-screw-based dynamic stabilization. However, long term studies are needed to investigate such a minimally invasive surgical strategy.


Scaphoid fractures are most often treated with a single headless compression screw. However, intercarpal Kirschner wire (K-wire) might be added to improve stability and fracture outcomes. This study will determine if there is a difference in treatment outcome (union rate and time to union) between scaphoid fracture fixations using a single headless compression screw with and without augmentation using a intracarpal intramedullary K-wire.We conducted a retrospective review of patients who underwent surgery for isolated scaphoid fractures over a 15 years period from December 2000 to December 2015. Only patients who underwent open surgery with bone grafting were included. They were divided into a group treated with a single screw fixation, and another group treated with screw and K-wire fixations.Forty-four (58.7%) patients had single screw fixation and 31 (41.3%) had screw augmented with K-wire fixation. The overall union rate was 88.0%, with an overall mean time to union of 5.3 months. There was no difference in union rate (p=0.84) and time to union (p=0.66) between the single screw group and combined screw and K-wire group. Univariate analysis found that older age  had a significant effect on union rate. Regression model showed that age had a significant effect on months to union.In open fixation of scaphoid fractures with compression screw and bone grafting, union rate and time to union is comparable whether or not screw fixation was augmented with an intracarpal K-wire. There was no increased risk of complications associated with augmented screw. Age of patient affected time to union and union rate. The incidence of scaphoid fracture varies between 29 to 43 per 100,000 each year in the general population3, and can be as high as 121 per 100,000 in the military population4. Scaphoid fractures commonly occur due to a fall on outstretched hand with a hyperextended wrist. It is commonest in young men aged 15 to 29, who also have the highest incidence of non-union, and longest time to union5. A majority (64%) of scaphoid fractures occur at the waist, and a small percentage (4.6%) occur at the proximal pole6. Although most scaphoid fractures heal uneventfully, 5%–10% of scaphoid fractures develop non-union9. Common risks for non-union include displaced scaphoid fractures, proximal pole fractures, and avascular necrosis (AVN)11.The scaphoid is the most frequently fractured carpal bone, and accounts for 50% to 80% of all carpal fractures in young people12. Surgical fixation can be performed with headless compression screws, Kirschner wires (K-wires), and plates17. However, they are now most often treated with a single headless compression screw19.Scaphoid fractures can be treated conservatively or surgicallyet al20. performed a biomechanical study and found that an intercarpal K-wire (scaphocapitate) added more stability to a long screw fixation of scaphoid but not significantly. Allon et al21. believed that stabilising the construct after screw fixation of scaphoid with intercarpal scapho-capitate, and sometimes scapho-lunate K-wires, may improve union rate. However, they found no significant difference in union rate, time to union, complication rate, and post-operative range of motion. They concluded that intercarpal K-wires can be safely used to augment scaphoid screw fixation21.With cannulated screw fixation, K-wires can be used to augment screw fixation of the scaphoid. There have been two papers published on augmenting scaphoid screw fixations with K-wires, but they are intercarpal, not intracarpal. Dodds It is not known if augmenting scaphoid screw fixation with intracarpal intramedullary K-wires would improve stability and improve union rate, or the additional drilling and presence of additional hardware would affect fracture union. As we could not find any papers looking at the effect of intracarpal K-wires being used to augment scaphoid screw fixation, we decided to look at our cases to evaluate if this would have any effect on fracture outcomes. As such, we performed an audit on all open scaphoid headless compression screw fixations with bone graft, to determine if there is a difference in fracture outcome (union rate and time to union).The study was approved by the hospital’s ethics committee . A retrospective review of patients who underwent surgery for isolated scaphoid fractures over a 15-year period from December 2000 to December 2015 were identified using hospital records. There were a total of 136 patients of which 93 had open surgery, and 43 minimally invasive surgery . In the open surgery group, 87 had bone grafting. We excluded 6 patients, with double screw fixation and 6 patients with avascular necrosis from this group of 87 patients, giving a final study sample size of 75 patients.23. Post-operative data was based on patients’ last documented visit.All data extracted from patient charts and electronic records were recorded on a standardised form in Microsoft Excel. Patients’ demographic data, radiographs, operative records, and clinical data were obtained. Post-operative radiographs were typically performed weekly or bi-weekly for the first six weeks, and monthly thereafter until the fracture had united radiographically. Fracture union was identified based on the presence of trabecular bridging across fracture site in at least two scaphoid views with no gaps seen in any of the radiographic viewsDescriptive statistics were used to describe the participants’ socio-demographic characteristics and clinical data at baseline. For continuous data, mean and standard deviation were calculated. For categorical data, counts and percentages were used. Independent t-test was used to compare continuous variables between the two groups. Chi square and Fisher’s exact test (if count less than 4) were for categorical outcome variables. This was to check for any differences in socio-demographic data between both groups at baseline. Chi square test and odds ratio were used to compare union rate, which was a categorical data. T-test was used to compare time to union, which was a continuous data.Univariate analysis was performed on the dataset to identify potential factors that could affect union rate and time to union, and the significant factors included in the regression model analysis for months to union. For the univariate analysis, Pearson correlation was used to compare two continuous variable, ANOVA test was used to compare three or more groups with continuous variables, t-test was used to compare two groups with continuous variables, and chi-square test was used to compare between two categorical data. A p-value of <0.05 was considered statistically significant.There were 44 (58.7%) patients with single screw fixation , and 31 (41.3%) patients with augmented screw with intracarpal K-wire fixation . There were no differences in the demographic, radiographic, and operative data between both groups .The overall union rate was 88.0%, with an overall mean time to union of 5.3 months. The odds ratio for recovery using combined screw and K-wire was 0.87. The union rate using single screw was 88.6%, and 87.1.% for combined screw and K-wire. Mean time to union for single screw was 5.2 months, and 5.7 months for combined screw and K-wire. There was no statistical difference in union rate and time to union between the single screw group, and augmented screw group.Age had a significant effect on union rate using univariate analysis . Patients who had scaphoid union were older , compared to those who had non-union . Only age was significant in the regression model with respect to time to union  .There was no significant difference in post-operative outcomes between both groups. Patients returned to work after 1.3 months and 1.7 months in the single screw group, and the augmented screw group, respectively (p=0.35). The follow-up duration in the single screw group and the augmented screw group was 12.7 months and 17.2 months, respectively, but the difference was not significant (p=0.17). Grip strength (p=0.86), wrist flexion (p=0.99), and wrist extension (p=0.34) were similar between both groups .Three patients in the single screw group had complications (7.3%). One patient had screw protrusion which required a secondary procedure to remove the screw, one patient had the screw displacement and required revision surgery, and one patient had hypertrophic scarring of the wound which was treated conservatively. In the augmented screw group, two had revision surgery due to the persistent non-union (6.5%). Ten out of 31 (32.3%) patients in the combined screw and K-wire group underwent removal of K-wire after the scaphoid had healed.24, whereas screw fixations often involve the placement of one screw25. Double screw fixation has been reported but the numbers are low27. In our own centre, the rate of double screw fixation was 4.4% (6/136 patients).Fixations with K-wires or headless screws are well established methods of fixation of scaphoid fractures. Proponents of K-wire fixation often utilise multiple K-wireset al28 would insert a second mini-screw into the scaphoid if he deemed the fixation to be insufficiently stable. If that is not possible due to insufficient space in the scaphoid or poor bone quality, he would insert an intercarpal K-wire or mini-screw from the distal scaphoid into the capitate to reduce micro-motion at the fracture site. He would keep this intercarpal fixation until the fracture has healed before removing at about 3-6 months. The use of an intercarpal K-wire as an addition to the rigid construct afforded by headless compression screws in scaphoid fractures has been investigated both in the laboratory and in the clinical setting21. However, both studies were not able to conclusively prove that the additional K-wire constructs improve biomechanical stability or improve union rate.Fracture site stability is important for scaphoid healing. In his surgical protocol, Slade Despite the prevalent use of K-wires during scaphoid screw fixation, the results of augmenting screw fixations of scaphoid with intracarpal K-wires, has never been specifically investigated. It is not known if these intracarpal intramedullary K-wires which cross the fracture site with the screw would improve stability and improve union rate, or would the additional drilling and presence of additional hardware affect fracture union.21. However, it is not clear what functional outcome, and possible complications would ensue if they are kept for as long as six months28. Additional, intercarpal K-wires may potentially cause permanent damage to the midcarpal joint cartilage. However, intracarpal intramedullary K-wires may reduce the amount of bone interface available for healing at the fracture site29. Intercarpal K-wires, on the other hand, generally do not cross the fracture site and thus, the additional K-wire should have no direct interference with fracture healing.In our centre, 41% (31/75) of open scaphoid screw fixation with bone graft had intracarpal intramedullary K-wires. This is a sizeable proportion which had this augmented fixation. A 1mm K-wire is inserted before screw insertion. It is then bent and cut just under the skin as they are part of the fixation construct and need to be kept until fracture healing. As these K-wires do not cross any carpal joints, wrist mobilisation is not restricted. Intercarpal K-wires might need protection lest the wire break in the joint from motion. As such, final functional outcome may also be affected if intercarpal K-wires are kept for too long. Evidence shows that if the wires are removed early at six weeks, there would be no significant effect on final wrist motion32. Our union rate is comparable with what is published in contemporary literature.Our union rate for the single screw, and augmented screw groups were 89% and 87%, respectively. Published systematic reviews have reported union rates of 80% to 88%, and 84% to 92% for internal fixation with non-vascularised bone grafts and vascularised bone grafts, respectivelyOur results show that the addition of intracarpal K-wire does not improve the union rate or reduce the time to union. However, we acknowledge that this is a retrospective study and we believe a prospective study could be done to validate this outcome.Our study did not show any increased in the risk of complications with the additional intracarpal K-wire, other than the fact that patients may require a secondary procedure to remove it. This may require a slightly longer period of follow-up although the difference was not found to be statistically significant in our study. We think the presence of hardware outside the bone may cause these patients to need a slightly longer time off work, but again, in our study the difference in time off work between the groups was not statistically significant. It should also be noted that 61% (19/31 of the patients) of them did not have their intramedullary K-wire removed. Those who had the K-wire removed could probably feel the bent tip of the wire under the skin.14. A possible reason for this could be compliance issues in the more active and younger patients during their rehabilitation. Unfortunately, we do not have any data on the rehabilitation aspect to study this. In terms of time to union, our regression model found that older patients did have longer time to union as expected. Our study used plain radiographs to determine the union of scaphoid fracture. Although Computed Tomography (CT) scans and Magnetic Resonance Imaging (MRI) may provide better evidence of union34, it is not routinely used in our clinical setting. Plain wrist radiographs have been found to be adequate and reliable in diagnosing union postoperatively36. In addition, our results apply only to open scaphoid fixations with bone grafts and may not necessarily be valid for cases that were performed without bone grafts or performed through minimally invasive techniques.Our study found that age was a significant factor affecting the rate of union in scaphoid screw fixations with bone grafting, where younger patients would have slightly lower union rates. This is quite surprising as most would expect union rates to be lower in older patientsIn open fixation of scaphoid with compression screw and bone grafting, our retrospective review has shown that union rate and time to union is comparable whether the screw fixation was augmented with an intracarpal K-wire or not. There are no increased complications associated with the additional K-wire although K-wire removal may be required in some patients after fracture healing. Our audit also found that older age was a significant factor with improved union rate, but increased time to union.


When analyzing the variable “Fracture Torque,” although it was verified that the replica screws (Iconekt® and Exaktus®) had a lower maximum torque, 65.11 Nm and 62.89 Nm, respectively, compared to the original Mis® brand (70 Nm and 69 Nm), there were no statistically significant differences p > 0.05. Nonoriginal screws did not present different fracture resistances compared to the original Mis® brand screws. The fracture site of Iconekt® screws showed a different pattern compared to the other brands.Micromovements of the implant-abutment connection influence peri-implant bone preservation. The maximal torque after a cycle of implant prosthetic screw tightening using original components of different manufacturers and replicas produced by other companies is evaluated and quantified in this study. A total of 30 Mis Seven® standard platform implants and 30 interfaces were used, and 30 standard platform screws were tested, 10 Mis®, 10 Iconekt®, and 10 Exaktus®. The screws were tightened with an MIS® torquemeter until their respective fracture, and the fracture point was measured through the equipment's load cell, CS-Dental Testing Machine®. The screws were analyzed under an Olympus® SZ61 microscope. The fracture points were recorded and compared among all samples. To compare the mean values of the fracture torques, t-tests were performed using the reference values associated with each brand and the sample results. The variable “Place of Fracture” between the original Mis® brand and the Exaktus® replica compared to the Iconekt® replica presented a statistically significant difference ( The use of dental implants dates back approximately 1300 years to the Mayan civilization, when people replaced missing teeth with tooth-shaped shells [Nowadays, with the phenomenon of osseointegration well implemented in implantology, the use of implants has been increasing, and it is possible to rehabilitate almost all types of dental absences , 3.Several problems have arisen with the use of dental implants, with screw losses proving very common complications, particularly in the first designs of components for dental implants. Thus, other prosthetic systems, special screws, and torque wrenches have been investigated and developed, subsequently reducing the rates of such failures –6.Regardless of the tolerance between the interface/implant parts, the screw and the level of torque used play an important role in the stability of the union when subjected to cyclical loads. The greater the torque applied to the retaining screw and prosthetic interface, the smaller the micromovement between the parts , 8.The bonding force starts when the bolt is tightened using a tool. The screw head contacts the inside of the prosthetic interface as well as the screw threads with the internal threads of the implant. In the tightening process, the screw is subjected to tension and stretches, pressing the two parts against each other (prosthetic interface and implant), thus establishing what is known as the bonding force –11.Loosening or fractures of the prosthetic interface screw can present a severe complication, as it is necessary to remove the restoration to gain access to the screw. As a result, cemented restorations can be damaged or destroyed in this process .There is no consensus in the literature on screw loosening, with reports of loosening rates ranging from 2% to 40%. The most likely cause of most screw loosening is improper screw tightening , 14.Torque control is the primary method used in dentistry for tightening prosthetic interface screws, and manufacturers specify the torque at which screws need to be tightened to achieve an optimal preload , 16.The longevity and success of implant rehabilitations are related not only to the stability of the implant but also to a correct and efficient union between the prosthetic interface and the implant, with the prosthetic screw being a crucial element in maintaining this union , 18.The aim of this study is to evaluate the fracture resistance of original and nonoriginal screws, as well as to verify their fracture pattern, clarifying if the use of nonoriginal brands has the same clinical performance when compared with original brands, in respect to resistance to maximum torsion.A total of 30 Mis Seven® standard platform implants and 30 interfaces were used, and 30 standard platform screws were tested, 10 Mis®, 10 Iconekt®, and 10 Exaktus®.The implants with the prosthetic interface fixed with the screw to be tested were mounted on a base and correctly stabilized .The screws to be tested were subjected to a torsional force and manual tightening with an MIS torquemeter  until thThe equipment used for the study was a CS-Dental Testing Machine® , model ID1-BAD . The CS-The screws to be tested were subjected to a torsional force with a torque wrench until fracture occurred. A brand-new torque wrench was also used every time a prosthetic screw was tested. The fracture points were automatically recorded in the machine and compared.The screws were analyzed under an Olympus® SZ61  microscope to view the fracture pattern at the macroscopic level.A standard laboratory protocol was established and applied at the Institute for Research and Advanced Training in Health Sciences and Technologies (IINFACTS-CESPU) to test all the selected samples.All prosthetic screws from the manufacturers were labeled with a serial number and control date: 10 Mis® prosthetic screws, 10 Iconekt® prosthetic screws, and 10 Exaktus® prosthetic screws.The presence of anomalies and defects was assessed with a stereoscope , and a 90× magnifier was used to evaluate any changes in the surfaces.The prosthetic abutment was coupled to the implant analog (of the corresponding brands) with a prosthetic screw with a respective manual key. The prosthetic screw was tightened by hand until there were no gaps between the two parts. A brand-new manual screwdriver was used every time a prosthetic screw was tested.The two parts were placed in a load cell and connected to the CS-Dental Testing Machine®.The screws to be tested were subjected to a torsional force with a torque wrench until fracture occurred. A brand-new torque wrench was also used every time a prosthetic screw was tested.The fracture points were automatically recorded in the machine and compared between all samples.The fractured parts were analyzed under an optical microscope to observe microscopic fracture characteristics.For the organization and description of the data used, descriptive statistics was used. Absolute and relative frequencies were used for the analysis of variables.The Kolmogorov–Smirnov test was used to verify the existence of normal distribution of continuous variables. For continuous variables without normal distribution destruction, the Kruskal–Wallis test was used to verify statistical differences. In the case of categorical variables, the chi-square test was used.Statistical analysis was performed using IBM SPSS®  version 22.0 software.After performing the mechanical stress tests on the screws, it was found that, in the MIS® brand, 9 of the 10 screws tested fractured, and all fractures occurred in the arm area Figures . The aveIn the macroscopic analysis using a 90× magnification, it was found that the screw had a clean finish  with no In the Iconekt® brand, 8 of the 10 screws tested fractured, and all the fractures occurred in the first turn of the thread Figures .The mean fracture torque was 65.11 Ncm with a standard deviation 11.02 Ncm.Upon observation with the microscope, there was a clean finish on the outside of the screw head ; swarf cIn the Exaktus® brand, all the 10 screws tested fractured: nine in the thread area of the first turn and one in the arm Figures .The mean fracture torque was 62.89 Ncm with a standard deviation of 5.78 Ncm.Upon observation with the microscope, it was verified that the screw had a clean finish  with no In the statistical analysis, the variables “fracture torque” and “fracture site” were studied. The variable “fracture torque” in the various brands did not have a normal distribution, so a non-parametric Kruskal–Wallis test was used, and there were no statistically significant differences in the means of fracture torque between the various brands.In the analysis by fracture site, the chi-square test was used, and there were statistically significant differences (< 0.001) between the MIS® and Iconekt® brands.With the use of dental implants with screw-retained prosthetic rehabilitations, a correct torque on the screw is essential so that an ideal preload occurs in the implant union-prosthetic interface.Loosening or fracture of the prosthetic screw is related to an implant-prosthetic interface mismatch, and the presence of a gap between the implant and the prosthetic interface can cause unfavorable stress distributions within the connection components, the implant, and the bone. Screw loosening or fractures are common complications in implant restorations. It is speculated that the gap between the implant and the prosthetic interface has a significant influence on these findings . Many auAccording to the work of Piermatti et al. , the strThe aim of this study was to test the same screws, but from different manufacturers, with the originals and replicas made by other companies. The material used in the manufacture of the screws was the same in all brands studied: titanium 6Al4 V.There are no previous studies comparing original and replica screws in the field of dentistry. The results obtained show statistically significant differences when analyzing the variable “Place of Fracture” between the original Mis® brand and the Exaktus® replica compared to the Iconekt® replica.In the 10 Mis® screws and in 9 of the 10 Exaktus® screws tested, the fracture site was in the arm, whereas for Iconekt®, all 10 screws fractured in the thread area first. These results are surprising since the material they are made of and the morphologies are similar between the brands. Further studies are needed to determine if there is any property in the alloy or in the morphology of the screws that could give rise to this outcome.When analyzing the variable “fracture torque,” although it was verified that the replica screws (Iconekt® and Exaktus®) had a lower maximum torque, 65.11 Ncm and 62.89 Ncm, respectively, compared to the original Mis® brand, 70 Ncm and 69 Ncm, there were no statistically significant differences.In the present study, we conclude that since no statistically significant differences were found, Iconekt® and Exaktus® replica screws do not present different fracture resistance when compared to MIS® brand screws.Although not the central point of the study but a pertinent analysis nonetheless, it was found that the fracture site of Iconekt® brand screws presented a different pattern from the other brands, with statistically significant differences. The MIS® screws fractured in the screw arm region, the Exaktus® screws also fractured in the screw arm region, and the Iconekt® screws fractured in the thread region.This finding merits further study, either with a possible evaluation of the chemical composition of the materials of the screws or a more detailed analysis of the design of the screws and their comparison; a larger sample could permit identification of a statistically expressed difference regarding the fracture torque.


Single-screw extruders are usually operated with the screw fully filled (flood-fed mode) and not partially filled (starve-fed mode). These modes result in completely different processing characteristics, and although starve-fed mode has been shown to have significant advantages, such as improved mixing and melting performance, it is rarely used, and experimental studies are scarce. Here, we present extensive experimental research into starve-fed extrusion at feeding rates as low as 25%. We compared various operating parameters  at various feeding rates and screw speeds. The results show a first insight into the performance of starve-fed extruders compared to flood-fed extruders. We explored starve-fed extrusion of a polyethylene material which contains a Very High Molecular Weight Polyethylene fraction (VHMWPE). VHMWPE offers several advantages in terms of mechanical properties, but its high viscosity renders common continuous melt processes, such as compression molding, ram extrusion and sintering, ineffective. This work shows that operating single-screw extruders in extreme starve-fed mode significantly increases residence time, melt temperature, and improves melting and that-in combination—this results in significant elongation of VHMWPE particles. Single-screw extruders are among the most important machinery in polymer processing. They are usually operated in flood-fed mode, where the screw beneath the hopper is fully filled and takes in as much material as possible. However, single-screw extruders can also be operated in starve-fed mode, where the screw is not fully filled. As its output depends on the feeding rate and not on the screw speed, a partially filled screw exhibits processing characteristics  that differ completely from those of a screw operated in flood-fed mode . The staIn the current work, we aimed to discover the effect of starve-fed extrusion on different processing parameters like melt temperature, residence time, degradation of the material, specific energy input and melting performance. Most notably, we were looking at the influence of starve-fed extrusion on PE blends which contain a VHMWPE fraction. Since blends containing a VHMWPE fraction are rather difficult to process we are showing the possibility to process these blends with starve-fed extrusion. To quantify the influence on starve-fed extrusion on the size and the shape of these particles we developed a program which can quantify both.A black high-viscosity bimodal hexane copolymer polyethylene compound for HDPE pipes from Borealis with high density and an outstanding resistance to slow crack growth. The melt-flow rate (MFR) of this polymer measured according to ISO 1133 was 0.25 g/10 min (190 °C/5 kg). In the residence-time distribution measurements, we used this polymer in its natural (white) color and added 0.2 g of fluorescent dye (lunar yellow). In the melting-performance experiments, we used the same white polymer, to which we added 2% of carbon black masterbatch.1–P6 had an upper limit of 2000 bar and P7 had an upper limit of 500 bar. A pressure valve at the screw tip was used to adjust the backpressure to specified values. A schematic representation of the extrusion line can be seen in A single-screw extruder was used with a diameter of 45 mm and a length of 41 times the diameter that had a grooved-barrel feeding section. The extruder was equipped with seven pressure transducers at various axial positions, as listed in The temperature profile of the extruder  was heldA commercially available barrier screw from KraussMaffei was used. The solids-conveying section, the melting or barrier section and the melt-conveying section were 500 mm, 650 mm and 450 mm long, respectively. The mixing section consisted of a double maddock and a distributive toothed mixing element and was 240 mm long. To control the amount of material fed to the single-screw extruder, we used a volumetric dosing system which feeds directly into the feed hopper of the extruder.The experiments were performed at two different screw speeds and four different feeding rates. All parameters were evaluated for the operating points listed in Output;Melt temperature;Specific energy input (SEI);Degradation after extrusion;Residence-time distribution;Melting performance;VHMWPE particle distribution;Shape distribution of the VHMWPE particles.For each operating point we analyzed:The melt temperature of the extruded samples was measured inside the pressure valve by a temperature-measuring sword with a length of 7.60 mm and a width of 5.15 mm from Graeff GmbH.P of the extruder is calculated by:The drive power A lower specific energy input indicates that less mechanical energy is applied to the material.−1, −1 and −1.An Anton Paar Plate-Plate Rheometer (MCR302) was employed using the frequency-sweep method from 628 rad/s down to 0.01 rad/s. Viscosity, storage module and loss module were determined for this range to investigate degradation effects. Measurements were carried out at 210 °C under nitrogen to avoid degradation during the rheology measurements using a parallel plate geometry with a diameter of 25 mm and a thickness of 0.8 mm. The pellet and all extruded samples were measured using the same method. Deviation of an extruded sample’s curve from that of the pellet is considered to signify degradation. In particular, the deviation at the zero-shear viscosity is a sign of degradation. The ratios of the viscosity of the pellet and the extruded samples at zero shear rate have been used to determine degradation.c(t) is the fraction of molecules that have been in the extruder for time t or longer. The cumulative residence time distributions To investigate the residence-time distribution in the extruder for a given setting, fluorescence spectroscopy was used . An amouTo investigate the influence of various feeding rates on melting performance, screw-pulling tests were conducted. To this end, each operating point  was configured and held for 30 min. Subsequently, the extruder was stopped abruptly and cooled down to room temperature. The screw was then pulled out of the extruder, and the solidified melt removed from the screw, cut into pieces, polished and finally scanned. The images were analyzed by transforming them into a black-and-white representation as shown in To check whether extrusion increased the homogeneity of the materials, VHMWPE particles, which are suspended within—and are immiscible with—were tested in the polymer matrix. For this analysis, the extruded samples were cut by a Leica Rotation Microtome RM2265 into 12 µm-thick slices that were embedded in (Eukitt) resin between glass and cover slide. The resin was left to harden for 10 h, and then an Olympus SZX10 stereomicroscope equipped with a UC90 camera  was used2 or touching the edge of the image. Parameters recorded for each particle were area, circularity, and mean size of the ellipse axis.The images were analyzed by means of the Wavemetrics Igor Pro 8 software tool. In order to keep the analysis as objective as possible, code was written to repeatedly perform the same automated steps. The images were first converted from RGB to a gray-scale representation, and then the built-in procedure for bimodal distributions was used to determine a threshold from the intensity distribution. In cases in which this did not deliver satisfactory results, we used the built-in iterative method. Particles were identified in the binary image as ellipses, using the threshold and excluding particles smaller than 5-pixelTo determine the influence of starve-fed extrusion on the shape of VHMWPE particles, the circularity (roundness) (Ř) parameter was used, which is calculated by Equation (6).The influence of feeding rate on the output measured is illustrated in For comparison, As in the previous case of 100% feeding rate, with increasing backpressure an overall increase in the pressure profile can be observed for both screw speeds investigated. With decreasing feeding rate, the pressure zone in the first few sections of the extruder depressurizes. An increase in backpressure increases the pressure in the extruder. For low feeding rates, the backpressure length increases with increasing backpressure. Due to the low feeding rate and the ensuing depressurization in the feeding section, the grooves of the extruder no longer affect the output-pressure behavior, which results in a transformation from grooved single-screw to smooth single-screw extruder behavior.The relationship between feeding rate and specific energy input for screw speeds of 50 rpm and 200 rpm is shown in The results clearly show that with decreasing feeding rate the specific energy input rises. A high SEI value indicates that more mechanical energy is put into the material, which may result in severe degradation of the material. In contrast to the melt-temperature, the specific energy input for the lower screw speed (50 rpm) also increases significantly with decreasing feeding rate. Hence, this indicates that for the lower screw speed the cooling unit of the single-screw extrusion machinery was able to compensate the increased mechanical energy input, but was unable to do so for the higher screw speed.The frequency-sweep method is a cheap and fast way of determining the degree by which the extruded material has degraded. The influence of screw speed and feeding rate on degradation is illustrated in −1The ratios of the viscosities at a shear rate of 0.01 sWhile the degree of degradation is visible at a screw speed of 200 rpm and 25% feeding rate, it is practically non-existent at 50 rpm, independent of feeding rate and 200 rpm. Degradation can be caused by long residence time of the material inside the extruder  or by high shear stresses or high shear rates .The residence time distribution of the different screw speeds with different feeding rates can be seen in The values of the mean residence time for screw speeds with different feeding rates can be seen in The results of the cumulative residence-time distribution measurements can be seen in At lower screw speeds, the influence of the feeding rate is particularly pronounced. The average residence times for various screw speeds and feeding rates are listed in With decreasing feeding rate the width of the residence timer distribution function increases. An increase in the residence time distribution function results in enhanced elongation mixing. The mean residence time shows a significant increase when applying starve-fed mode for both screw speeds. The cumulative residence time increases significantly with both decreasing screw speed and decreasing feeding rate. Especially for very low feeding rates the residence time increases tremendously.A decrease in feeding rate results in accelerated melting of the material due to higher residence time and increased SEI to the material. For both screw speeds, we consider the optimal feeding rate in terms of melting performance to be 50%. For a feeding rate of 25% the melting performance decreases compared to a feeding rate of 50%. The pressure profile c.f.  reveals In The different analyzed parameters for 50 rpm and 200 rpm with different feeding rates can be seen in The results clearly show that the extrusion process significantly improves the homogeneity of the samples in terms of number of particles, average size of particles and overall area covered. Further, reducing the feeding rate decreases particle size for screw speeds of 50 rpm. With decreasing feeding rate the overall area covered by particles is reduced.2 were ignored, and the remaining ones were classified according to circularity. The circularity parameter was used to analyze the shape of the VHMWPE particles. A value of one indicates a perfect cycle, while lower values reflect elongation of the particle shape. All particles smaller than 45 µmIt can be seen that a reduction in feeding rate results in elongation of the VHMWPE particles. Since using different extrusion equipment, including high-performance screws  and various mixing elements, showed no influence on white-spot formation, we conclude that starve-feeding is a promising approach to dealing with this problem.In summary, the results clearly show that the behavior of extremely starve-fed extruders differs completely from that of flood-fed extruders. A reduction in feeding rate gives rise to a significant increase in melt temperature due to higher shear and significantly longer residence time. In addition, the pressure build-up at constant backpressure is completely different for highly starve-fed extruders. While flood-fed grooved extruders show a constant backpressure build-up, starve-fed extruders exhibit no measurable pressure. This completely different pressure build-up behavior for the starve-fed mode was already observed . The inc