Atypical femoral fractures (AFF) have low union rates. The use of teriparatide has been advocated for the post-operative healing of AFF, but the evidence is limited to case reports and some series due to its low incidence.We present a case series of four female patients to support the use of teriparatide after the surgical fixation of their AFF. Three of the patients had a complete AFF and one had an incomplete fracture. Their mean age was 70 (52 - 87) years, mean body mass index 24.6 (18.3 - 29.3), mean bone mineral density T-score of -2.3 (-4.8/-1.0), with a prior history of anti-resorptive therapy with bisphosphonates and denosumab.Teriparatide was started at an average of 8 (2-18) days post-fixation, with 20mcg daily for six months. Immediate full weight-bearing was permitted in three patients, while one was non-weight bearing for two months. The mean time to union was 12 (10 - 14) weeks. No side effects were observed over a mean follow-up of 58 (50 – 72) weeks.The use of teriparatide facilitated the quick union of AFF after surgical fixation. It appeared to be safe and promoted fracture healing in AFF. The use of teriparatide has been advocated to improve the healing of surgically fixed AFF, but the evidence is limitedWe present a case series of four patients with surgically fixed AFF, which united rapidly after being treated with teriparatide. This case series adds to the evidence for the post-operative use of teriparatide. We also discuss the optimal treatment dosage and duration.3.Four patients who received teriparatide after surgical fixation of AFF were retrospectively identified. All patients met the American Society for Bone and Mineral Research (ASBMR) Task Force 2013 revised case definition for AFFPatient 1 sustained a midshaft fracture following a fall from a standing height. Patient 2 sustained a subtrochanteric fracture following a fall from a chair. Patient 3 sustained a midshaft fracture as she sat down due to “weakness”. Patient 4 had no trauma but had prodromal pain in the preceding three weeks before presentation. Although the fracture was incomplete, there were radiolucent lines laterally on the anteroposterior radiograph and anteriorly on the lateral radiograph.No peri-surgical complications or adverse effects in relation to the use of teriparatide were noted.2.Bisphosphonates are commonly used in the treatment of osteoporosis. It reduces the rate of bone turnover. The bone undergoes more mineralisation and is not substituted by younger, less mineralised bone, rendering it more intolerant to deforming forces and hence more brittle. AFF may occur, and this is a concern with the increasing use of bisphosphonates in the ageing population1.With decreased bone turnover, a longer time to union, up to an average of 23 weeks, was observed after surgical fixation of AFF, despite halting any further use of bisphosphonates. A significant portion, up to 12%, would require revision within 48 weeks. For these, the union would occur only later, at an average of 44 weekset al found that the union time was significantly reduced with teriparatide. Two other series reviewed only had 10 and 15 AFF2.The use of teriparatide to aid union has been described. Teriparatide is a recombinant form of parathyroid hormone (PTH) that causes increased bisphosphonates turnover and increases bone turnover. With its anabolic effect, the time to union and union rate after AFF have been reduced, in the few case reports and series. In Im and Lee's review, the largest series had 44 AFF in which Miyakoshi 2.There is also no established dosage, frequency, and duration of treatment. In Im and Lee’s review, the interval from surgery to initiation of teriparatide ranged from 1 to 11 months, while the dose ranged from 20mcg daily to 56.5 mcg weekly, for a period of three months to two years3. It should be commenced as soon as possible after the fixation as it has been shown to provide better radiographic healing as compared to a six months delay in therapy4. It is not definite when the therapy should cease, although the shortest reported duration is three months2.The use of teriparatide to aid union after surgical fixation of AFF is off the Food and Drug Administration (FDA) list of approved indications. Two dosing regimes have been described, either a daily subcutaneous injection of 20mcg daily or a weekly injection of 56.5mcg. Daily injection has been shown to achieve union in a significantly shorter time with an average of 26 weeks as compared to 44 weekset al Not all the patients received teriparatide in their study, and there was insufficient information on the treatment regime for those treated with it. These might be the reasons for the difference in our results1. Tsuchie et al's study showed a mean union time of 26 weeks. However, they included those treated non-surgically, which may increase the overall time to union3.All our cases united within 14 weeks. This was sooner than the mean 22 weeks reported by Bogdan We also noted that Patient 4 achieved union within ten weeks, earlier than the other cases. A possible explanation for this is that teriparatide indeed has the potential to cause early union in all cases, and it became apparent when Patient 4 was coincidentally reviewed at an earlier date. A shorter interval for review for future cases may be required to determine this.5.None of our patients developed adverse effects from the treatment. Nevertheless, physicians need to be cognisant of the potential adverse effect of hypercalcaemia which may occur in up to 11% of patients. Non-uraemic calciphylaxis has been reported, albeit extremely rare. There is also a theoretical risk of osteosarcoma, though it was only noted in rat studies and may be dose and duration-dependent. Hence, it might be desirable to stop the treatment once the fracture is united. Nonetheless, this needs to be balanced against the risk of future fracture, as the bone density undergoes accelerated deterioration upon cessation of teriparatide. Ordinarily, in osteoporosis, this may be mitigated by taking other anti-osteoporotic medications such as bisphosphonates or denosumab, but this may not be suitable for patients who already had AFFIn conclusion, current literature provides encouraging results for the use of teriparatide after the surgical fixation of AFF. It is difficult to perform a study with a high level of evidence due to its rare occurrence. Nevertheless, each series adds to our understanding of the utility of teriparatide in improving the union in surgical fixation of AFF. We have described in detail our treatment regime and future studies should also include this to guide clinical management.


To explore the reported variability in the surgical management of ileocolonic Crohn’ s disease and identify areas of standard practice, we present this study which aims to assess how different colorectal surgeons with a subspecialty interest in inflammatory bowel disease (IBD) surgery may act in different clinical scenarios of ileocolonic Crohn’s disease.Anonymous videos demonstrating the small bowel walkthrough and anonymised patients’ clinical data, imaging and pathological findings were distributed to the surgeons using an electronic tool. Surgeons answered on operative strategy, bowel resections, management of small bowel mesentery, type of anastomosis and use of stomas.Eight small bowel walkthrough videos were registered and 12 assessors completed the survey with a questionnaire completion rate of 87.5%. There was 87.7% agreement in the need to perform an ileocolonic resection. However, the agreement for the need to perform associated surgical procedures such as strictureplasties or further bowel resections was only 57.4%. When an anastomosis was fashioned, the side to side configuration was the most commonly used. The preferred management of the mesentery was dissection close to the bowel.The decision on the main procedure to be performed had a high agreement amongst the different assessors, but the treatment of multifocal disease was highly controversial, with low agreement on the need for associated procedures to treat internal fistulae and the use of strictureplasties. At the same time, there was significant heterogeneity in the decision on when to anastomose and when to fashion an ileostomy.The online version contains supplementary material available at 10.1007/s00384-021-03892-z. Most patients with ileocolonic Crohn’s disease (CD) require surgical intervention during their lifetime, and in view of the relapsing course of the disease, a multidisciplinary team (MDT) approach  is mandaTo explore this variability in the surgical management of ileocolonic CD and identify areas of standard practice, we present this study which aims to assess how different colorectal surgeons with a subspecialty interest in inflammatory bowel disease (IBD) surgery may act in different clinical scenarios of ileocolonic CD, with the primary objective to evaluate the inter-observer variability in the operative strategy in complex ileocolonic CD cases, with particular reference to surgical approach, extent and site of resections, use of bowel sparing techniques and diverting stomas.According to a previous study protocol , anonymoColorectal surgeons with expertise in minimally invasive surgery and IBD were selected as committee members to develop a survey on operative strategy for treatment of complex ileocolonic CD. Inclusion criteria for the committee members included evidence of previously published experience in CD surgery guideline development , distancThe committee identified items for inclusion in the survey by discussion through e-mails and face-to-face meetings. The final survey Appendix  includedwww.enalyzer.com). Anonymised patients’ clinical data, imaging and pathological findings were also shared with the committee members.The anonymous videos demonstrating the small bowel walkthrough were distributed to the committee members together with the anonymous survey using an electronic tool  [ĸ values from 0.21 to 0.4 were considered as indicating fair agreement, while values from 0.41 to 0.6 and >0.61 were considered as indicating moderate and substantial agreement, respectively.Categorical variables are presented as frequency or percentage and were compared with the use of the chi-squared test or Fisher’s exact test, as appropriate. Continuous variables are presented as mean (±standard deviation) or median (range) and were compared with the use of Student’s appa (ĸ)  along wiEight small bowel walkthrough videos Appendix  were disk= 0.82; 95% CI 0.64, 0.99). However, the agreement for the need to perform associated surgical procedures such as strictureplasties, further bowel resections or repair of internal fistulae was quite low, with an agreement of 57.4% . Amongst all the assessors, only 1 suggested conversion to open surgery in one of the procedures (estimated conversion rate of 1.04%). The full results of the survey are shown in Table There was an agreement of 87.7% amongst the assessors in the need to perform an ileocolic resection . When the anastomosis was not fashioned, there were reported differences in the management of the distal colonic end; however, a double-barrelled stoma (76.4%) was the preferred option over a closed intra-abdominal stump (11.8%) or an ileocolic anastomosis protected by a loop ileostomy (11.8%). When an anastomosis was fashioned, the side to side anastomosis was the most commonly used as detailed in Appendix There was significant heterogeneity in the decision to fashion or not an anastomosis, with an agreement of 70.4%  with a slight preference for resection of the mesentery close to the bowel (33.3 to 77.8% of the cases) rather than radical cancer-like resection (0 to 41.7%) or resection of macroscopically affected mesentery only ( 0 to 25%).There was significant heterogeneity in the management of the mesentery, with an overall agreement of 45.6% ESM 2Assessors’ agreement for the need to perform anastomosis and/or defunctioning (DOCX 15 kb)ESM 3Assessors’ agreement on the anastomosis configuration (DOCX 16 kb)


COVID-19 has been declared a pandemic since March 2020 and it has been responsible for millions of deaths worldwide. The SARS-CoV-2 causes a spectrum of diseases mainly affecting the respiratory system. It can also complicate other systems causing thromboembolic phenomena and myocardial ischaemia. An entity of hypoxia has been described in these patients which show no clinical signs and symptoms of respiratory distress despite being extremely hypoxic. This is called silent or happy hypoxia. The exact mechanism for this is not known. We report 4 cases which had similar presentations of silent hypoxia but had different course of illness and different outcomes. All 4 patients did not show any signs of respiratory distress, but had oxygen saturation less than 82%. 3 of them needed intensive care unit support for oxygen therapy and subsequently needed noninvasive ventilation. Only one required invasive ventilation. The fourth patient did not require intensive care support. The patient who required invasive ventilation succumbed due to severe COVID pneumonia whereas the other 3 patients were discharged from the hospital. Silent hypoxemia can go undetected in COVID-19 patients particularly in the time of a pandemic. This case series highlights the importance of meticulous clinical examination including oxygen saturation measurements in suspected or confirmed patients with COVID-19. The course of illness can be different in different populations, and this needs further clinical evidence. SARS-CoV-2 virus is responsible for the spectrum of conditions in COVID-19. It is predominantly a respiratory infection that can complicate other systems. It may present with a wide spectrum of symptoms ranging from mild self-limiting upper respiratory tract infection like illness to severe pneumonia and death. The SARS-CoV-2 infection leads to severe respiratory failure in some of the patients who require advanced respiratory support. Often deterioration to this level is evident by clinically detectable respiratory distress as dyspnoea or tachypnoea. “Silent” or “apathetic” hypoxia is an entity described in patients suffering from COVID-19 pneumonia which is described as objective hypoxia in the absence of apparent respiratory distress [There is a certain group of patients who presents with silent hypoxia. Those patients lack proportionate clinical signs of respiratory distress, even though their chest X-rays show diffuse pneumonia and have evidence of hypoxia detectable in pulse oximetry or arterial blood gas analysis. However, sudden and rapid deterioration may occur in this subset of patients. To assess the severity of pulmonary involvement, a clinical examination should be meticulous, and adequate emphasis should be given to pulse oximetry reading and blood gas analysis. The cause of silent hypoxaemia is controversial. The outcome of these patients can also be variable, and no predictors have been identified to comment on the prognosis. We report 4 cases with silent hypoxaemia with the same presentation, but a different course of illness. The purpose of this report is to highlight the importance of “silent hypoxia” in COVID-19 patients.A 53-year-old manual labourer with a history of fever, productive cough, and sore throat for 3 days presented to a local hospital. He is a current smoker and did not have any comorbid diseases. He was diagnosed with a COVID-19 infection by a rapid antigen test and was transferred to a specialised COVID-19 treatment centre. He was not in acute distress and conversant with unlabored speech. His respiratory rate was 16/min without any dyspnoea. He had a blood pressure of 134/77 mmHg with a heart rate of 88/min. On pulse oximetry measurement, his saturation was found to be 66% on room air, and arterial oxygen partial pressure was 56 mmHg. His CXR showed bilateral patchy consolidations on admissions . He was A 51-year-old lady with a background history of diabetes mellitus and hypertension presented to a local hospital with cough and fever for 7 days duration. She had a BMI of 39. She was transferred to a specialised COVID treatment centre after tested positive for SARS-CoV-2 rapid antigen. She denied any dyspnoea or chest pain on admission. Her respiratory rate was 20/min with a blood pressure of 155/80 mmHg and a pulse rate of 77/min. Despite the absence of signs of respiratory distress, her saturation on admission was 60% on air. Arterial blood gas revealed an arterial oxygen saturation of 51 mmHg with 26 mmHg of CO2 on room air. Her chest X-ray showed bilateral patchy consolidation . She was2 of 54 mmHg. He had a respiratory rate of 16/min and initial CXR showed bilateral patchy consolidations  of 82% on air with PaO2 of 56 mmHg with normal respiratory rate and effort. His blood pressure was 160/80, and his pulse rate was 90/min. His oxygen saturation improved to 98% on FiO2 of 40%. His CXR showed bilateral air space opacification . A chestSARS-CoV-2 is a droplet borne virus responsible for the global pandemic of COVID-19 infection. Millions of cases have been reported worldwide. The disease spectrum ranges from asymptomatic infection to acute respiratory distress syndrome causing death in a considerable number of patients. The course of infection can also complicate with thromboembolic phenomena, myocardial ischaemia, and secondary infections , 4.Hypoxaemia is defined as arterial oxygen partial pressure less than 80 mmHg, and it becomes severe when it falls below 60 mmHg . HypoxaeThe exact mechanism for silent hypoxaemia is not clear. But there are several hypotheses to explain the occurrence of this entity. Most of the hypothesis is related to the preservation of the ability to remove CO2 from the body during COVID-19 infection. Pneumonia analysis of COVID-19 shows the virus causes the alveoli to collapse, reducing the oxygen level and causing hypoxia, but the reaction still enhances the normal lung ability to expel carbon dioxide. Since carbon dioxide removal is still effective, patients do not feel shortness of breath . It is aThere are not enough data available to predict the clinical course or outcome in these patients with silent hypoxaemia. Chandra et al. reported a case of a 56-year-old patient who presented with silent hypoxemia who made complete recovery only with supplemental oxygen up to 15 L/min via a nonrebreather mask. He did not require ventilator support . Lari et2 level and diffuse pneumonia in CXR. Once the patients were diagnosed to have silent ischaemia, they were started on oxygen therapy. Case 1, 2, and 3 patients showed an increment in their oxygen requirement with disease progression following the diagnosis of silent hypoxaemia. In contrast the 4th patient remained on the same requirement and subsequently weaned off without the need for the ICU care. Therefore the course of illness is not similar in all patients with silent hypoxaemia. The outcome of patients was also different. Case 1 had prolonged hospital stay, and the recovery was complicated with several episodes of silent hypoxaemia while 2nd patient died after getting ARDS. The 4th patient was discharged without any complications.We have reported 4 cases that we encountered who had the clinical entity of silent hypoxaemia. None of them showed apparent signs and symptoms of respiratory distress despite having alarmingly low SpOAll 4 patients received IV dexamethasone as a part of their treatment regimen. The third patients received IV tocilizumab when he was deteriorating rapidly. Use of tocilizumab can be commenced in hospitalized patients with COVID-19 in whom there are no signs of infection . The eviSilent hypoxemia is an important entity which needs to be detected early in patients with SARS-CoV-2 infection. However, the course of illness could be unpredictable. The exact cause of the silent hypoxaemia is also not known. In our experience, further clinical evidence is required to interpret the associations and outcome of silent hypoxemia.Silent hypoxemia can go undetected in COVID-19 patients. This highlights the importance of meticulous clinical examination including oxygen saturation measurements in suspected or confirmed patients with COVID-19. The course of illness can be different in different populations, and this needs further clinical evidence.


Medication-Related Osteonecrosis of the Jaws (MRONJ) is a difficult-to-treat complication of the therapy of osteoporosis and some malignancies cured with bisphosphonates and antiresorptive drugs. The pathomechanism is unclear, but there is increasing observation that Actinomyces infection may play a role in its development and progression. The aim of our study was to demonstrate that histological examination using a validated triple staining procedure for Actinomyces bacteria strains can detect a high rate of Actinomyces infection in patient's samples with MRONJ. 112 previously hematoxylin-eosin (HE) stained samples submitted with the clinical diagnosis of MRONJ were re-evaluated histologically using an appropriate triple special staining validated for the identification of Actinomyces infection. During the first evaluation, when pathologists did not specifically look for Actinomyces, only 8.93% of the samples were reported as positive. In contrast, re-evaluation with triple staining provided a yield of 93.7% positive samples, therefore, we suggest the triple special staining to be standard in MRONJ histology evaluation. These results show that if the clinician suspects Actinomyces infection and brings this to the attention of the pathologist, it could significantly increase the number of correct diagnoses. It serves as an aid for clinicians in therapeutic success of MRONJ by selecting a long-term adequate antibiotic medication which is suitable for the elimination of actinomyces infection. MRONJ is a condition characterized by a bone lesion that does not heal for at least 8 weeks, the patient has previously received or is receiving antiresorptive (bisphosphonate) agents or angiogenesis-inhibitor, and has no history of malignancy or radiation of the jawbones5.Medication-Related Osteonecrosis of the Jaws (MRONJ) was first described in 2003 as a complication associated with the treatment of osteoporosis and malignant diseases of the bone6. The altered remodelling and reduced blood supply impair the immune defense of the bone10. This mechanism is suggested to facilitate the development of osteomyelitis as a result of mechanical trauma and infection14, which may progress to osteonecrosis due to the poor regenerative potential caused by these factors15.Although it is well established that MRONJ is a complication of the above-mentioned therapies, the exact pathogenesis is unknown and seems to be multifactorial18, the severity of the underlying disease, the patient's immune status, the comorbidities  and the concomitant use of other therapies . According to a retrospective analysis, about 0.5% of the Hungarian population receives bisphosphonate treatment for some reason. The incidence of MRONJ was found to be 0.9% in patients taking bisphosphonates for oncological indications and 0.1% in patients with non-malignant diseases19.A distinction is made between low and high-risk groups for MRONJ. The severity of risk depends on how long the antiresorptive treatment has been applied, the route and dose of administration—the risk of developing MRONJ is 100–1000 times higher after intravenous administration20 which are present as normal inhabitants in the oral cavity. In cases where it was detected, the average treatment time for MRONJ was longer22.There is consent that one of the key factors in the development of MRONJ is bacterial infection, the source of which is the oral flora, including Actinomyces strains25. They are characterized by low virulence and lack of hyaluronidase enzymes, so they do not penetrate the intact mucosa. Disease development requires a damage to the integrity of the mucosa, as well as the presence of co-pathogenic bacteria , creating an anaerobic environment suitable for Actinomyces strains27. The most relevant pathogenic species are Actinomyces israelii, Actinomyces viscosus, Actinomyces meyeri, Actinomyces naeslundii and Actinomyces gerencseriae29.Actinomyces species are microaerophilic or anaerobic, opportunistic, Gram-positive non-spore-forming bacteria. They form radially arranged branched filamentous colonies—hence the name ‘ray fungus’ given to them upon discovery—and play a key role in biofilm formation and thus in the formation of dental plaque35. The majority of these cases develop following an injury disrupting mucosal integrity, and Actinomyces colonies are usually found in the necrotic area of the bone. Poor healing propensity, and a high relapse rate are also characteristic. However, it is unclear whether Actinomyces strains are causal factors in the development of MRONJ, osteomyelitis, osteoradionecrosis and COD associated bone necrosis, or colonize the necrotic area later causing further damage to surrounding tissues37.There are many similarities in the clinical features and morphology of jaw necrosis related to various etiologies including MRONJ, osteoradionecrosis, osteomyelitis, cemento-osseous dysplasia (COD) induced osteomyelitis and chronic diffuse sclerotizing osteomyelitis38. However, many of them did not mention Actinomyces among the detected bacterial strains at all. It is assumable that the results were highly dependent on whether Actinomyces were specifically searched for using appropriate special stains, or they were recognized as a random finding. When choosing the test method, it is important to consider that microbiological culture is limited in its ability to detect Actinomyces strains and often gives false negative results39. This may explain why some authors have found a much lower rate of Actinomyces infection in MRONJ25.The reported proportions of MRONJ samples with a histological confirmation of the presence of Actinomyces is markedly variable in the literature, ranging from almost 100% to around 12%We aimed to re-evaluate hematoxylin–eosin (HE) stained histological samples from patients diagnosed with MRONJ for the presence of Actinomyces.5.Samples were obtained from patients diagnosed with MRONJ between 2011 and 2020 at the Department of Oro-Maxillofacial Surgery and Stomatology and Oral Diagnostics Department, Faculty of Dentistry, Semmelweis University, without any selection. The involved patients belonged to stage 2 and 3 according to American Association of Oral and Maxillofacial Surgeons (AAOMS) classification40.In accordance with the recent guidelines, surgical removal of sequesters was completed with antibacterial treatment as follows: patients started antibiotic therapy 3 days before surgery and continued for 10 days in the postoperative period. In cases when appropriate microbiological data were available, definitive antibiotic therapy was used based on the sensitivity. In other cases, the empirical therapy included amoxicillin-clavulanic acid  as the first choice, or clindamycin (300 mg 4 × 1) or doxycycline  in patients with penicillin allergy. Cooling of the area and the use of 0.2% chlorhexidine mouth rinse at least 3 times a day for one week after surgery was recommendedAltogether 117 samples of 83 patients from the archives of the Department of Pathology and Experimental Cancer Research, Semmelweis University from 2011 to 2020 were re-evaluated. The study design was approved by the local Ethics Committee (SE RKEB 137/2020). The data of all patients were anonymized and procedures were in line with the requirements of the Helsinki Declaration. Due to the retrospective study design there was no need for ethical approval of any informed consent. Hereafter we use the term “evaluation” for the original hematoxylin-eosin (HE) study and “re-evaluation” for the second examination with specific stains for Actinomyces. A total of 117 archived samples were compared with the original histological findings during the re-evaluation. 3 samples were excluded due to the lack of unambiguous osteonecrosis and 2 other due to the insufficient bone content for further analysis. Finally, 112 archived samples were involved in the study.All of the involved patients were treated with bisphosphonates, 101 cases with oncological indication, and 11 cases with osteoporosis. The latter group received bisphosphonate therapy only. Multiple (2–4) specimens were available from 33 patients due to persistent or recurrent lesions.We compared the histological data with available microbiological results of the patients included in this study. Microbiological examination was performed in 39 cases within 3 months before or after the operation. For the calculation of sensitivity and specificity we made a 2 × 2 table with groups of subjects divided according to the triple staining as reference method in columns, and categories according to microbiological results in rows.All the included samples were obtained from the surgical treatment of osteonecrosis following bisphosphonate treatment and the clinical diagnosis was MRONJ. Sections from the decalcified, formalin-fixed paraffin-embedded biopsy were stained with HE. Histological criteria included the presence of completely necrotic bone trabeculae and bacterial aggregates along with variable inflammatory infiltrates as it was previously described. During the re-evaluation, beside the HE histomorphology, the presence of Actinomyces was confirmed using Gram , periodic acid—Schiff , and Grocott’s methenamine silver  staining. Slides were evaluated using a Nicone Eclipse E600 POL microscope first, then the most representative samples were digitalized using a Pannoramic digital slide scanner . Image acquisition was performed using the Case Viewer software .Samples containing the characteristic filamentous bacterial colonies (“sulphur granules”) showing positivity with all three stainings were regarded as „positive” to Actinomyces.Original routine histological evaluation had been performed by 14 different pathologists in the described time period and the clinical diagnosis of osteonecrosis was confirmed in all cases. Re-evaluation of the samples was performed by 1 trained pathologist focusing on the presence of Actinomyces infection. During the re-evaluation, the examiner was blinded regarding the previous pathology report of presence or absence of Actinomyces or any microbiological data. To test the examiners reliability, he performed a repeated re-evaluation of the same samples, one and a half year following the first re-evaluation (the examiner was masked to the previous results). The intraobserver agreement was calculated with Cohen’s kappa; value is 0.93 (95% truncated CI 0.79–1). That indicates good agreement.In the samples containing both viable and non-viable bone tissues the two areas were also compared from the perspective of Actinomyces content. Bone was considered vital when viable osteocytes were visible in the lacunae.SPSS Statistics 27  was used for statistical analysis. Group comparisons were performed using the Related-Samples McNemar Change Test with 95% confidence interval.dncs_data1.xlsx, that was used as input data. The confidence interval limits was truncated to 0 or 1 if the limits are below 0 or over 1. The calculations were made in R  using the psych (Revelle 2021.2.1.9) package.The intraobserver agreement was assessed by using Cohen’s kappa with its 95% confidence interval was determined; we included the data to a useable structure to Semmelweis University Regional and Institutional Comitte of Science and Research Ethics 137/2020 (Hungary). Due to the retrospective study design there was no need for ethical approval of any informed consent: Semmelweis University Regional and Institutional Comitte of Science and Research Ethics 155/2012 (Hungary). The study was done according to the declaration of Helsinki.The study design was approved by the The evaluated samples contained necrotic, acellular bone trabeculae with bacterial aggregates between them Fig. ., usuallAmong the 112 archived samples 102 (91.07%) were reported as Actinomyces negative during the first routine histological evaluation with HE stainings, of which 95 were found to be positive during the re-evaluation with triple specific stains. Compared with the original report 7 samples (6.25%) were found to be negative during the re-evaluation. In the evaluation, the presence of Actinomyces was detected in 10 samples (8.93%) and this result was confirmed in all cases in the re-evaluation, so in the end a total of 105 (93.75%) samples were found to be positive. The result proved highly significant (p = 0.0000) , all of which were evaluated histologically as “positive” during the re-evaluation. No samples were histologically negative and microbiologically positive. 2 of 39 samples were negative with both histological and microbiological examinations. Comparison of triple staining histology and microbiology results resulted in high specificity (1) and very low sensitivity (0.054). Negative predictive value of routine microbiological test was 0.054.Beside the two Actinomyces positive culture we found Fusobacteria, Prevotella, Eikenella, and Enterobacteria strains. In the remaining 35 histologically positive cases, the following bacteria were found in the microbiological culture Prevotella (12), Fusobacteria (5), Parvimonas (5), Veillonella (4), Eikenella (2), Streptococcus (7) Klebsiella (6) Staphylococcus (4), Enterobacter (2), Enterococcus (1) Haemophylus (2) and Citrobacter (1) strains.43. A highly variable proportion of Actinomyces positivity can be found in the literature, however, in the studies with lower prevalence the methods of Actinomyces detection, including microbiology or HE staining of histological samples, were probably inadequate regarding sensitivity25. Cerrato's analysis of 30 publications showed that Actinomyces positivity occurred in 96.4% to 25.4% of the samples7. These variances are presumably related not only to methodological differences but whether the study design was focused on the presence of Actinomyces or not.MRONJ is a multifactorial, not a life-threatening disease, but significantly impairing the quality of life. There are still many questions about the pathogenesis, but most authors agree that infections may play a major role in its development. Many of them have raised the possibility that Actinomyces strains are potentially involved in this process, in addition to other bacteriaIn our study, we re-evaluated the histopathologycal samples in our database that had been received with a MRONJ diagnosis over a 10-year period to examine the frequency of Actinomyces. To the best of our knowledge there has been no study to date that has re-stained and re-evaluated previously tested samples. The re-evaluation of archived samples with a special focus on and using appropriate stains for Actinomyces, showed positivity in 93.75% compared to the 8.93% of the original routine evaluation. There were no cases where the sample initially found to be positive was found as negative in the re-evaluation. Only 7 out of the 112 samples proved to be negative in both the routine and the re-evaluation.These results suggest that one of the reasons for the significant discrepancy in the literature reports on the prevalence of Actinomyces may be a methodological problem. The routinely used HE staining resulted in a sparse detection of Actinomyces, which therefore seems more likely to be an accidental finding on MRONJ samples.52. Microbiological results were available in case of 39 samples in the patient’s records. Actinomyces could only be detected in two samples, representing a prevalence of 5.13% versus the 93.75% result of histology. All two samples were evaluated histologically as “positive” during the re-evaluation. These data confirm the well-known fact that microbiology is not a proper method to detect Actinomyces and underlie the importance of adequate histological assessment.The sensitivity of microbiological cultures is very poor regarding the detection of Actinomyces species44. During the review of the microbiological data, we found that Prevotella, Fusobacteria, Parvimonas, Veillonella, Eikenella, Staphylococcus, Streptococcus and Klebsiella species were most frequently found in the samples.In dental plaques, Actinomyces species create co-aggregates with other bacteria, mainly Fusobacteria, Prevotella, Eikenella and Veillonella strains, during the formation of the biofilm. The role of this cooperation seems to be basically important in the pathogenesis of osteomyelitis as well, in which these species adhere to collagen fibers and promote the development of osteonecrosis37, however, and increasing number of authors suggest a causal role of Actinomycetes in the development of MRONJ based on the higher prevalence detected by histological evaluation44. Russmueller et al. found a high (89%) prevalence and concluded that Actinomyces strains play a prominent role in MRONJ and will change our understanding of it. In a systematic review published in 2020, Cerrato concluded that osteomyelitis caused by Actinomyces and MRONJ may have a common origin with the notion that the lower prevalence reported in earlier series is at least partially related to methodological problems. Our study supports Cerrato's latter view.The potential causal role of Actinomyces in the pathogenesis of MRONJ is still an unresolved issueThe therapy of MRONJ is generally based on the recommendation of AAOMS . According to the AAOMS principles, systemic antibacterial therapy is recommended from stage 3, but the above mentioned position paper does not mention the length of antibiotic therapy.12. In contrast, the recommended antibiotic treatment protocols for actinomycosis of the jawbone start with intravenous broad-spectrum antibiotics  for 3–4 weeks and continue with oral penicillin for 9–12 months or longer48, whereas Actinomyces strains are sensitive to beta-lactam compounds, but these compounds show very poor potential to penetrate fibrotic, necrotic and inflamed tissues and large bacterial aggregates49. Therefore, following a few weeks of intravenous therapy penicillin V can be used for the long term, significantly decreasing the incidence of gastrointestinal side effects compared to penicillins with beta-lactamase inhibitors52.The MRONJ protocols of different countries usually recommend a few weeks of antibiotic treatment after surgeryRusmueller et al., or even lower than others, Anavi-Lev, Lee, Naik, Hansen, Franco-Pretto53. However, the antibiotic regime used in our patients is unlikely to affect the proportion of Actinomyces positive cases, since it is started only three days before the sampling. It is important to note that these short preoperative treatment is insufficient to eliminate Actinomyces.The proportion of Actinomyces positive samples in our study were undoubtedly higher in comparison with some previous reports, although not far from the result of Summarized, it would be important for the clinician to consider the possibility of Actinomyces infection in all MRONJ samples and bring this to the attention of the pathologist, who will then search for it using validated methods. This will presumably increase the frequency of detected Actinomyces infections and allow for the selection of an appropriate therapy. The focused histological examination, including triple stains and appropriate clinical issues, can be significant, and result in much higher detection rate of Actinomyces in comparison with isolated HE staining. This is highlighted by the fact that the microbiological detection of Actinomyces using conventional techniques shows poor sensitivity.Additionally, long-term antibiotic treatment protocols should be considered in the postoperative pharmacotherapy of MRONJ, in order to eradicate Actinomyces species. The currently applied too short antibiotic therapy may contribute to the large number of therapeutic failures.


This study aimed to analyze the serum melatonin levels and changes in sleep patterns in pediatric patients with coronavirus disease 2019 (COVID-19). This study was designed as a descriptive, cross-sectional study. Serum melatonin levels and sleep parameters of children with the diagnosis of COVID-19 who had mild and moderate disease  were compared with those of children admitted with non-COVID-19 nonspecific upper respiratory tract infection . The sleep disturbance scale for children (SDSC) questionnaire was applied to the participants› primary caregivers to analyze their sleep patterns at present and six months before symptom onset and to investigate the impact of COVID-19 on sleep patterns.  The entire study cohort consisted of 106 patients. The COVID-19 group included 80 patients, while the control group consisted of 26 patients. The mean serum melatonin levels were 136.72 pg/mL and 172.63 pg/mL in the COVID-19 and control groups, respectively (p = 0.16). There was no significant difference between the groups in terms of 6 subcategories of the SDSC questionnaire regarding the present time and 6 months before symptom onset. The total SDSC scores were also similar in two different evaluation time points described above (p = 0.99).We conclude that COVID-19 did not impact the sleep parameters of children. Serum melatonin levels of all patients were higher than the reference range; however, they were higher in the non-COVID-19 patient group than the COVID-19 group. Since serum melatonin levels were higher than the reference values in children with COVID-19, and this disease is significantly less morbid in children, melatonin may have protective effects against COVID-19.  Although it is known that melatonin has antiinflammatory, antioxidant, and immunomodulatory functions during infection with bacteria and viruses, its specific role in the novel coronavirus disease 2019 (COVID-19) has not been widely investigated [1]. To date, data obtained regarding COVID-19 infection showed significantly lower morbidity and mortality rates in the young patient population and children [1–3]. This finding implies that patient age may have a critical role in this disease’s pathogenesis. Since the serum concentration of melatonin is inversely correlated with age, it would not be unreasonable to investigate the potential role of melatonin in the pathogenesis and progress of COVID-19 . Melatonin is a hormone secreted by the pineal gland, and it is the primary regulator of sleepPhotosensitive retinal-containing receptors mediate the release of melatonin from the pineal gland [4].Thus, its secretion is inhibited during daylight. It is known that COVID-19 is a zoonotic disease, and bats are the natural carriers of coronavirus [4]. Bats are nocturnal hunters; they avoid daylight and hide in dark caves during the daytime. As a result, they continuously have high levels of melatonin compared to the human. Coronavirus infection is not fatal in bats with minimal or no symptoms. Despite these facts, the alleviating role of melatonin hormone or relatively high serum melatonin concentrations in the young patient population and children with COVID-19 has not been investigated.This study aimed to analyze the protective role of melatonin in children with COVID-19 infection. Also, we aimed to investigate these children’s sleep patterns to interrogate the relationships between melatonin levels and sleep patterns and the impact of COVID-19 on the sleep patterns of these patients.This study was approved by the Ethical Review Committee of Ankara City Hospital (06.06.2020/E1-20-564). Pediatric patients admitted to Ankara City Hospital, Pediatric Health Center after confirmation of COVID-19 infection between June 2020 and August 2020 by at least two positive PCR test results, were included in this prospective study. Children hospitalized at the same center with the clinical suspicion of COVID-19 but had at least two negative PCR test results constituted the control group. These control group patients presented with high fever and other nonspecific upper respiratory tract infection symptoms without an apparent history of contact with known COVID-19 cases. The age range of the target population was 6–16 years since the sleep disturbance scale for children (SDSC) questionnaire assesses children and adolescents in this age group . Patients with a history of previous COVID-19 infection, history of melatonin treatment in the previous 4 weeks, sleep apnea syndrome, chronic neuromuscular disease, and those whose legal guardians did not give consent were excluded. The study participant flowchart is shown in Figure 1. The COVID-19-positive patients were classified according to disease severity based on clinical and laboratory data [7]. Patients with no clinical symptoms or findings and normal chest imaging results were classified as asymptomatic, while those who had a high fever , myalgia, throat pain, coughing, or nasal discharge with normal findings of lung auscultation were classified as a mild disease. On the other hand, patients who had a high fever, coughing, or wheezing accompanied by pneumonia or those who had COVID-19-associated changes in their computerized chest tomography without clinical findings were classified as a moderate disease. Patients who had dyspnea and low oxygen saturation  were classified as severe, and those who had rapidly progressing acute respiratory distress syndrome or respiratory failure with multiple organ dysfunction were classified as critical. Waldhauser et al. analyzed the alterations in serum melatonin levels with growing and aging [2]. The reported values in this study were used as references of serum melatonin values. These authors reported that the mean serum melatonin levels were 160 pg/mL for the 6–7 age group, 110 pg/mL for the 7–12 age group, 85 pg/mL for the 12–15 age group, and 55 pg/mL for patients older than 15. Serum samples were collected from all patients on the day of admission and within three days of their first symptoms for analyzing the melatonin levels. They were collected between 06:00 and 07:00 am in the dim light of less than 30 lux  from the patients after overnight fasting. Postural conditions and environmental lighting were the same for all patients during venipuncture. First, 2 mL of blood was drawn from the antecubital vein and subsequently centrifuged. The serum was kept at –20 °C. All study participants went to bed between 21:30 and 22:30 and woke up between 07:00 and 08:00 am. Serum melatonin levels were analyzed using the ELISA method . The SDSC questionnaire was applied to the primary caregivers by the same researcher (P.G.) on the phone. The primary caregiver was requested first to answer the questions considering the last week, which covered disease onset  and second by considering the six months before infection . The primary reason for the evaluation of sleep at two different time frames was analyzing the impact of COVID-19 infection on the patients’ sleep characteristics and comparison of the patients and controls in this regard. This questionnaire containing 27 Likert-type questions was designed to evaluate sleep disturbances in children and adolescents and was validated for the Turkish language . It evaluates subsets of sleep disturbances, namely, disorders of initiating and maintaining sleep, sleep breathing disorders, disorders of arousal, sleep-wake transition disorders, disorders of excessive somnolence, and sleep hyperhidrosis) in children and adolescents. The cut-off point is 39 on this scale; points higher than this are associated with sleep disturbances [5]. A reliability test has been performed in order to test the data set obtained from the questionnaire forms. Alpha coefficient  has been used for reliability analysis. Sleep disorders in children have been measured using a questionnaire consisting of 24 questions and the reliability of the sleep disorder scale was found to be 0.840 for the last 6 months and 0.825 for the last week. Data analyses were performed using IBM SPSS Statistics v. 23.0 . Whether the distribution of continuous variables was normal or not was determined by the Kolmogorov–Smirnov test. The Levene test was used for the evaluation of the homogeneity of the variances. Unless specified otherwise, continuous variables were presented as means± standard deviations (SD) for normally distributed data, and medians and ranges  for data with skewed distribution. Categorical variables were given as the numbers (n) or percentages (%). The student’s t test was used to compare continuous normally distributed variables between two independent groups, while the Mann–Whitney U test was implemented for comparing the nonnormally distributed data. Categorical variables were compared using the Pearson chi-square test or Fisher exact test. Correlations between variables were evaluated using the Pearson or Spearman correlation analysis. A p value of less than 0.05 was considered significant.The target population of this study included 118 patients. Five patients who refused to give blood samples, 5 patients whose primary caregivers could not provide the authors with reliable data, and 2 patients with inconclusive laboratory data were excluded. Thus, the entire study cohort consisted of 116 patients. Among these patients, 58 were COVID-19 patients, while 26 patients were assigned to the control group for measuring serum melatonin levels, and 65 were COVID-19 patients, while 20 patients were assigned to the control group for applying SDSC . All COVID-19 patients had either mild (85%) or moderate disease (15%) based on the classification. Demographic data of the patients and serum melatonin levels are shown in Table 1. Comparative analysis revealed that serum melatonin levels were relatively lower in the COVID-19 patient group than the control group. However, the difference was not statistically significant .The differences in serum melatonin levels were evaluated by grouping the participants based on their age and corresponding reference levels, as shown in Table 2. There were 5 patients in the 6–7-year-old age group. However, since there were no control group patients in this age group, they were not included in Table 2. The serum melatonin levels were higher than the age-referenced values in both patient groups of our study (Table 3) [2]. Comparison of the serum melatonin levels based on patient age elucidated that the COVID-19 patients had lower serum melatonin levels than the patients in the control group in all age subgroups. Although serum melatonin levels of our COVID-19 patients were lower than control group patients in all age groups, a statistically significant difference was detected only in the 7–11 age group (p = 0.03). All study participants, except for the COVID-19 patients in the 7–12 age group, had significantly higher serum melatonin levels than the reference values (Table 3). Comparison of sleep parameters regarding 6 months before symptom onset and present time between COVID-19 patients and control group patients are displayed in the Appendix. There was no significant difference regarding the six subsets of sleep disturbances evaluated by SDSC performed based on 6 months before symptom onset and current data. The changes in the SDSC subset scores and total scores are displayed in Table 4. The comparison of both total SDSC scores and SDSC subcategory scores of two time points  revealed no statistically significant difference between the two groups (p = 0.99). A review of the SDSC scores of the COVID-19 patients showed that 4 (6.2%) patients had scores higher than 39  in the ‘present time’ evaluation, while 5 (7.7%) patients had scores higher than 39 in the evaluation regarding 6 months before symptom onset. One particular control group patient had SDSC scores higher than 39 in both evaluations regarding the present time and six months before symptom onset.To the best of our knowledge, this study was the first to investigate the serum melatonin levels and sleep patterns in children with COVID-19. The role of melatonin in COVID-19 is still unclear, as data concerning this disease’s pathophysiology is still accumulating [8]. In the adult and elderly population, melatonin is thought to have beneficial effects providing better clinical outcomes for COVID-19 patients admitted to critical care units [1]. Melatonin exerts these positive effects by reducing vessel permeability, decreasing anxiety and sedation need, and improving sleep quality. Therefore, some researchers advocated its use as adjuvant therapy. In our study, serum melatonin levels were lower in children and adolescents with mild or moderate COVID-19 than the control group patients with nonspecific upper respiratory tract infections. However, the difference was not statistically significant. Also, serum melatonin levels of the children and adolescents in both COVID-19 and control groups were significantly higher than the reference values adjusted for age [2]. This finding leads to the hypothesis that serum melatonin levels increase during the acute phase of all viral infections, including the new type of coronavirus infection. The common denominator of studies investigating the relationship of melatonin with viral, bacterial, and parasitic infections is the expectation of using melatonin to treat these infections [9–11] However, none of these studies were conducted with pediatric patients.Serum melatonin concentrations start to decline with age after the neonatal period [2]. Contrary to this information and the reference values obtained from healthy children, average melatonin levels of 12–15 years old children were higher than that of patients who were 15 years old and older [2]. Although conflicting data exist, a manuscript published in 1992 reports a minor increase in melatonin levels during the puberty [12]. We compared the melatonin levels between COVID-19 positive patients and COVID-19 negative patients with an infection and did not observe serum melatonin levels showing a positive correlation with age in either group, contrary to data from healthy children. This observation leads to the question of whether there is an age-related difference between healthy and infected children concerning serum melatonin levels however, there is no study investigating this question in the literature. We were not able to test this hypothesis since healthy controls were not included in our study. Crowley et al. have investigated the relationship between light exposure and melatonin secretion from the pineal gland and have compared pre-mid and late pubertal melatonin levels. The authors stated that pre- to mid-pubertal group showed significantly greater melatonin suppression on light exposure but no significant differences were seen between pre-, mid, and late pubertal groups in morning melatonin suppression [13]. This study shows that phases of puberty affect melatonin secretion patterns. This points out to the notion that melatonin secretion as a pro-inflammatory or antiinflammatory response to infection could be different from healthy children, similar to the different levels of suppression caused by light exposure in different stages of puberty.On the other hand, it is known that COVID-19 usually has a mild course in pediatric patients [14–15]. As such, approximately 90% of pediatric patients who do not have comorbidities usually exhibit mild or moderate symptoms with relatively lower morbidity rates. Detection of high serum melatonin levels in pediatric COVID-19 patients has strengthened the theory that melatonin may have protective effects against the progression of COVID-19. Since there were no severe COVID-19 cases in our study, serum melatonin levels could not be compared between mild or moderate cases and severe cases. Lifestyle changes due to the COVID-19 pandemic significantly increased the risk of sleep disorders both in patients and healthy population [16–17]. Some studies investigated the sleep disorders in patients who stayed in self-isolation or who were hospitalized for long periods or those who had a history of long-term admission to the intensive care unit. In one of these studies, the frequency of insomnia was 17.4% in Italian patients quarantined for 14 days [16]. Another study from Taiwan investigated sleep disorders in social media users and reported that the rate of sleep disturbances was 55.8% in this patient population [17]. Also, a French study presented complicated sleep disturbance cases in the young adult population during the pandemic period [18].It is widely accepted that melatonin prevents sleep deprivation, reduces delirium risk, and shortens the length of stay in patients staying at intensive care units [19]. Our literature did not reveal any studies investigating the impact of COVID-19 on sleep parameters in the pediatric patient population. One of our aims was to investigate the changes in children’s sleep patterns with COVID-19 and compare these changes with the changes detected in a group of children who had nonspecific upper respiratory tract infections by questioning the primary caregivers of the participants. The two groups showed similar results in both evaluations regarding the present time and 6 months before symptom onset. Similarly, no difference was found by comparing the SDSC scores concerning present time and six months before symptom onset. These results demonstrated that COVID-19 did not cause any significant changes in the sleep patterns of children. It was reported that melatonin decreased sleep onset latency and prolonged total sleep time [20]. Although the serum melatonin levels of all study participants were higher than the reference values in our study, no difference was detected in sleep parameters related to initiating and maintaining sleep. We aimed to take serum samples under similar temporal and environmental conditions considering that a difference of even 1 h can result in a significant difference in melatonin levels. It is also known that changes in sleep timing or exposure to light in the evening can result in different melatonin onset times, leading to different melatonin levels in the following morning [21]. Therefore, although we aimed to obtain standardized conditions, the wide range observed in the serum melatonin levels might have been caused by the inevitable differences in the conditions. Our study has some limitations which need to be considered while evaluating its findings. First, the SDSC questionnaires were performed by phone. Although the optimal method of performing a questionnaire is in-person interviews, we had to conduct them on the phone due to contact isolation. Second, the primary caregiver was expected to give sleep-related information regarding the present time and 6 months before symptom onset. The reliability of the latter can be considered doubtful. Third, since there were no severe COVID-19 cases in our cohort, we could not compare the serum melatonin levels of mild or moderate COVID-19 cases with those of severe cases. This limitation is significant because melatonin is used for adjuvant treatment of some patients with severe COVID-19 who are followed in intensive care units [1].Our study showed that COVID-19 did not cause any changes in sleep parameters of children. Although we detected that serum melatonin levels of all patients participating in our study were higher than the age-adjusted reference values, they were higher in the non-COVID-19 patient group than the patients diagnosed with COVID-19. This finding does not support our hypothesis; however, serum melatonin levels higher than the reference range in pediatric patients with COVID-19 can be considered as a sign of melatonin’s protective effect in these patients. Nevertheless, our results need to be confirmed by further prospective studies conducted with more extensive patient series. This study was approved by the Ethical Review Committee of Ankara City Hospital (06.06.2020/E1-20-564). Written informed consent was obtained from all patients’ parents or legal guardians of the children included.


We have previously shown that 67% of patients with newly diagnosed coeliac disease (CD) presenting to gastroenterologists have evidence of neurological dysfunction. This manifested with headache and loss of co-ordination. Furthermore 60% of these patients had abnormal brain imaging. In this follow-up study, we re-examined and re-scanned 30 patients from the original cohort of 100, seven years later. There was significant reduction in the prevalence of headaches (47% to 20%) but an increase in the prevalence of incoordination (27% to 47%). Although those patients with coordination problems at baseline reported improvement on the gluten free diet (GFD), there were 7 patients reporting incoordination not present at baseline. All 7 patients had positive serology for one or more gluten-sensitivity related antibodies at follow-up. In total, 50% of the whole follow-up cohort were positive for one or more gluten-related antibodies. A comparison between the baseline and follow-up brain imaging showed a greater rate of cerebellar grey matter atrophy in the antibody positive group compared to the antibody negative group. Patients with CD who do not adhere to a strict GFD and are serological positive are at risk of developing ataxia, and have a significantly higher rate of cerebellar atrophy when compared to patients with negative serology. This highlights the importance of regular review and close monitoring. Coeliac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten in genetically susceptible individuals and it affects 1% of the population .To what extent do patients with classic CD presentation suffer from neurological problems is an ongoing enquiry. Research has found evidence of cognitive deficits , which mWith almost all of these experiments being single-timepoint, what remains most understudied is the longitudinal outlook of these patients. Importantly, it must be addressed if the GFD is as an effective treatment in stopping these central nervous system injuries from progressing further. Currently, one leading hypothesis for the pathogenesis of these problems focus on similar mechanisms to the gastrointestinal phenotype; which circulating autoantibodies against gluten products exhibit cross-reactivity with key body tissues and cause downstream harm . GliadinIn 2010 we conducted a 3-year prospective study with the primary aim of establishing the prevalence of neurological involvement at the time of diagnosis of CD in patients presenting to gastroenterology clinics with the typical gastroenterological symptoms of CD . We alsoIn this follow-up study we reviewed 30 of the original participating patients 7 years after their baseline assessment. They underwent detailed clinical examination and repeat brain imaging. This investigation aimed to characterise any change in the clinical  phenotype of these patients, and also to investigate for relationships between antibody status and rate of brain atrophy.The study was based at the Departments of Gastroenterology, Academic Department of Neurosciences and Neuroradiology, Sheffield Teaching Hospitals NHS Trust, Sheffield, UK. The study (research number STH20656) was approved by the local ethics committee and informed consent was obtained from all participants. All 100 patients who had participated in the original study were send an invitation to participate in the follow-up study. The ethics approval allowed for only a single invitation letter to be send to each of the 100 original participants. Thirty patients who responded to this invitation were seen and reassessed and underwent further brain imaging as per the original protocol (see below). Reasons for patients non-responding included: patients had moved away from Sheffield , and the COVID pandemic, which disrupted all non-COVID research in the UK for 9 months. Additionally, the terms of ethical approval did not allow for a second invitation letter to be sent at a later date. All responders were seen.As per the original protocol, all patients had been diagnosed with CD following gastroscopy and duodenal biopsy . All pat3 isotropic resolution, TR/TE = 10.5/4.8 ms. The FLAIR sequence was a 3D routine, 0.97 × 0.97 × 0.56 cm resolution, TR/TE = 4800/304 ms, TI = 1650 ms.Patients underwent MR imaging of the brain, using the same scanner  and sequences as in the original study. Acquisitions included volumetric T1-weighted (T1W) and volumetric FLAIR structural imaging, and MR spectroscopy of the cerebellum (vermis). The methodology of the MR spectroscopy acquisition and the cut-offs for abnormal spectroscopic measurements NAA/Cr (N-Acetyl Aspartate to Creatine ratio) have been described previously. The T1W sequence was a 3D “MPRAGE” routine, 0.8 cmhttp://www.diedrichsenlab.org/imaging/suit.htm (accessed on 25 March 2021), The original study had indicated that both cerebellar grey matter (GM) and the thalamus may be at risk of atrophy in this patient cohort. Accordingly, volumetry of these regions was performed on the newer scans and re-performed on the baseline scans, to ensure software/workstation comparability. All scans first underwent bias correction using the “N4ITK” package. For estimated of cerebellar GM the “SUIT” pipeline was used , anti-gliadin (AGA) IgG and IgA , endomysium-EMA , and IgG and IgA transglutaminase 6-TG6  antibodies was undertaken in all patients during their visit for clinical assessment. All tests were performed at the NHS immunology lab as part of normal clinical care. The exact kits used differed in some instances compared to patient’s baseline assessments (both for AGA and TG6 antibodies).Baseline neurological symptoms of the returning sample were compared by chi-square analysis to the original whole sample to determine if they were significantly different as a subgroup in some manner. Clinical characteristics were then summarised descriptively in terms of how the rate of neurological symptoms has persisted over time. As the kits used to test for gluten antibodies changed in some instances between timepoints these were not explored individually. Instead, where relevant patients were classified based on if they were either negative for all tests, or positive for any test at follow-up.For imaging analyses, descriptive statistics were produced to show the rate of brain atrophy over the whole cohort. Statistical analysis then focused on comparing the rate of change (pYBVC) between those patients who still had positivity of any gluten-related antibody at follow-up, compared to those in which all antibodies were within normal levels. Age was first compared between these groups to determine if it was significantly different and therefore should be included as a covariate in subsequent analyses or not. Groupwise testing then compared pYBVC of all cerebellar GM (SUIT), the thalamus (FIRST), and overall brain volume (SIENA) between antibody groups, after visual inspection of histograms was performed to decide if parametric or non-parametric models should be used.The baseline and follow-up clinical and serological characteristics of the 30 patients are summarised in Amongst these 30 patients, at the time of diagnosis of CD (baseline study), clinical history revealed that 14/30 (47%) complained of frequent and intractable headaches, 8/30 (27%) complained of balance problems and 3/30 (10%) of sensory symptoms. At the same time, on examination, 10/30 (33%) had gait ataxia, 3 of which also had nystagmus and one had myoclonic tremor. These figures were not significant (Chi square) to what was observed in the whole cohort of 100 patients from which these 30 patients were re-assessed. Further comparisons of baseline data between the returning cohort and other groupings were therefore not sought.At follow-up (average 7 years later) the clinical history revealed that only 6/30  complained of ongoing headaches and 4 of these 6 said that their headaches were much better since being on gluten-free diet. Fourteen patients (47%) mentioned gait instability. Seven of these patients had not reported any balance problems at the time of their baseline assessments. Of the remaining 7 patients who had reported balance problems at baseline and at follow-up, 3 said that their balance had improved on GFD, the other 4 stated that it was stable. Sensory symptoms were reported by 3/30 (10%) patients.On examination at follow-up 17/30 (57% as opposed to 33% at baseline) of patients had mild gait ataxia . Four patients (13% as opposed to 0% at baseline) had loss of vibration sensation in their feet. Two patients had nystagmus (the same 2 that had nystagmus at baseline) and one had myoclonus (same patient as baseline).All 7 patients who did not have ataxia at baseline assessment but developed mild gait ataxia at follow-up were positive for one or more serological tests for gluten sensitivity. The remaining 10 patients had ataxia from baseline. Of the 10 patients, 3 were found to have a neuropathy at follow-up neurophysiological assessment, 3 showed improvement of their ataxia on GFD, 3 remained the same and in one the ataxia had completely resolved.Neurophysiological assessments in the 4 patients with evidence of reduced vibration sensation in their feet, at follow-up, showed a peripheral neuropathy in 3 . Therefore, the prevalence of neuropathy in this cohort was 10%.Serological tests at follow-up showed that 15 (50%) of patients were positive for one or more tests signifying gluten sensitivity. These included AGA (9 patients) and/or TG6 antibodies (10 patients). Amongst those patients positive for TG6 and/or AGA antibodies there were 6  who were positive for TG2 antibodies and 5  who were positive for EMA. The mean TG2 titre amongst the 30 patients at baseline was 207 U/mL and at follow-up 6 U/mL .Brain imaging was performed in 29 patients; one patient failed to attend their appointment. MR spectroscopy from the vermis (NAA/Cr) significantly improved in 12 patients (40%), remained the same in 10 (33%) and got worse in 7 (23%). Of the 7 patients with worse spectroscopy, 5 had positive serology.Brain volume analysis was performed on 28 patients, as one subject was excluded due to undergoing a clinical scan routine at their follow-up appointment such that the T1W acquisition was not comparable to their baseline. For the remaining 28 patients, average percentage yearly brain volume change (pYBVC) of the different ROIs was as follows: cerebellar GM = −0.53% (±0.42); thalamus = −0.49% (±0.38); whole brain = −0.16% (±0.15).t-test p = 0.612). Independent t-tests revealed the rate of atrophy of cerebellar GM  was significantly greater for the antibody-positive group (mean pYBVC = −0.73% ± 0.45) compared to the antibody-negative group  or the whole brain (p = 0.486).Of these 28 patients, 14 still had positivity of at least one gluten-related antibody at follow-up. Those who were still antibody positive were not significantly different in age (mean = 55.3 ± 10.2 years) to those who had become antibody negative ; the cerebellar grey matter, the thalamus, and the whole brain. While the thalamus and whole brain analyses did not reveal significant findings, a significantly higher rate of atrophy was found in the cerebellum for those patients who still had antibody positivity at follow-up. The cerebellum was included as a ROI given its known relevance in gluten-related disorders. It is, for example, the primary site of damage in gluten ataxia , while tIt is potentially concerning that the two brain ROIs, which were selected because they had been shown to be at risk of atrophy in this cohort—the cerebellum and thalamus—each showed a rate of atrophy almost three times as fast as that of the whole brain. Although those with antibody negativity have some level of protection, a faster rate of atrophy in at-risk ROIs may therefore be persisting even in those on well controlled diets. While it has been shown that different regions of the brain shrink at different rates in heathy ageing , this noFifty percent of the patients assessed in this study had positive serology for one or more serological marker for CD 7 years later. Such an immunological response is associated with slow but definite progression of the ataxia. These observations highlight the importance of regular clinical review with repeat serological testing, not just with EMA and TG2 antibodies but also with AGA and TG6 antibodies. Of interest is the fact that only 37% of these 30 patients were still under active gastroenterology follow-up.Two of the patients who participated in this follow-up study were subsequently followed-up in the gluten neurology clinic because of the presence of ataxia and an abnormal brain scan, in combination with positive serology. After a review by the dietitian, a repeat scan and serological testing approximately one year later showed significant improvement on MR spectroscopy associated with clinical improvement. This suggests that there might still be a potential for neurological improvement even in the context of previous imperfect adherence to GFD, assuming that there is still some cerebellar reserve and no permanent atrophy.Four patients developed symptoms of peripheral neuropathy at follow up and in 3 the neurophysiological assessment showed large fibre neuropathy in 2, and small fibre neuropathy in one. This is in keeping with previous observations that peripheral neuropathy is a late manifestation in patients diagnosed with classic CD .This study has its limitations. Our intention was to try and review as many patients as possible from the original cohort. We only managed to review 30 patients, although we did recruit and assess all patients that contacted us following the invitation letter. The smaller number was a result of the interruption of the study by the COVID-19 pandemic and the fact that many patients had moved out of the Sheffield area. Furthermore, the ethics approval prevented us from attempting to contact the patients for a second time, something that we did consider doing after ease of travelling restrictions due to the pandemic. However, we believe that this cohort of 30 patients is a representative group as indicated by the fact that the neurological symptoms and signs were not significantly different to those observed in the baseline cohort of 100 patients, and age and sex of the cohort at baseline was similar to that of the original group.Future lines of investigation should seek to replicate these findings with larger cohorts, and where possible, more deeply explore the relationship between neurological symptoms/brain imaging findings and relevant clinical markers, such as specific gluten-related antibodies, other markers of dietary adherence, and also indices of cognition and quality of life. Molecular analysis of regulatory factors and of genes relevant to processes of neuroinflammation, neurodegeneration, and autoimmunity would also be desirable to further identify risk factors and better understand the pathogenesis of these findings.In conclusion, we showed here that whilst the prevalence of headache improves significantly amongst patients with CD presenting to gastroenterologists, there is evidence of development of mild gait ataxia in some patients who have positive serology for gluten antibodies 7 years later. A significantly higher rate of atrophy was found in the cerebellum of those patients who still had antibody positivity at follow-up. This highlights the importance of regular review and close monitoring using a battery of serological tests. Those patients who remain positive need further dietetic input and closer monitoring.


Rheumatoid Arthritis (RA) is a chronic disease characterized by severe inflammation that leads to degradation of articular cartilage and the formation of bony erosions. Currently, certain anesthesiologist-led pain management clinics have begun to take on a collaborative role in the treatment of patients with RA, as this progressive disease impairs work capacity due to chronic pain. We present three clinical cases in which platelet-rich plasma (PRP) was used for the treatment of RA in patients seeking a new therapy for pain control and improved range of motion, specifically in certain joints of the hand. The Patient Activity Scale II was employed as a standardized method to assess RA disease severity, recorded on the day of injection, at one month, at three months, and at six months. All of the included patients, ages 49, 60, and 63, had an established diagnosis of RA affecting the proximal interphalangeal and metacarpophalangeal joints of the hand. Over the course of six months, two out of three patients reported a 20% reduction in pain from the initial visit and a 30% improvement in overall well-being. The third patient noted a 50% decrease in pain from the initial visit and a 50% improvement in overall well-being. PRP treatment consistently resulted in functional improvement for each of the three patients treated, while also reducing long term pain and inflammation. Initial clinical and laboratory studies have shown that autologous plasma rich in platelets serves as a source of an abundance of growth factors once activated. The multitude of these growth factors injected into and around the diseased joints improves functionality in patients with RA indicating PRP may be a safe and beneficial therapy in patients with RA primarily affecting the joints of the hand. Rheumatoid Arthritis (RA) is a chronic disease characterized by severe inflammation that leads to degradation of articular cartilage and the formation of bony erosions. A patient with RA primarily presents with significant swelling in the wrists, metacarpophalangeal, metatarsophalangeal, and proximal interphalangeal joints. Unfortunately, this condition affects about five in 1000 individuals worldwide, with a peak incidence in the sixth decade of life. Notably, women are two to three times more likely than men to develop RA over the course of their lifetime [Therapeutic approaches today primarily consist of disease modifying antirheumatic drugs (DMARDs). These agents have been noted to be key components in managing a patient’s RA immediately after the initial diagnosis . HoweverToday, anesthesiologist-led pain management clinics have started to take a more active role in the management of patients with chronic pain as this progressive disease impairs work capacity, leads to disability, and impacts a patient’s psychological state due to chronic pain ,5. The uWe present three clinical cases in which PRP was used for the treatment of RA in patients seeking a new therapy to control their chronic disease. Moving forward we believe that PRP therapy can serve as an additional treatment option, in which the body can use inherent physiologic tools to heal itself after the direct infusion of platelet-rich plasma.MethodsThree patients presenting with RA were treated with activated PRP. At the bedside, peripheral venous whole blood was drawn into 12 mL tube and PRP was created using a Juventix centrifugation systems  protocol by spinning at 3500 revolutions per min for eight minutes, to concentrate the platelets within the tube. Processing allows the visible gel separator within the tube to isolate the platelet-rich plasma clearly above the red blood cells and granulocytes for easy extraction. Next, a 32-gauge meso needle is placed at the top of the vial. An 18-gauge draw needle is then attached to a 10 mL syringe to withdraw the sample, directing the needle directly above the gel separator, thus extracting the entire platelet component.Patients were positioned in a manner that made their injection sites readily accessible for the administration of serum. For injection into the joint, a 7 mL sample of PRP was mixed with 1 mL of lidocaine into a syringe and attached to a 25-gauge needle. Each patient received a standard 0.5 mL dose intra-articularly and a 1.5 mL dose peri-articularly in each affected joint.In addition to clinical examination, the Patient Activity Scale II (PAS-II) was employed as a standardized method to assess RA disease severity. The PAS-II has been studied to be an effective and appropriate generic self-report indicator to assess active rheumatic disease .The survey was recorded on the day of PRP injection, at one month, at three months, and lastly, at six months. The following two questions were primarily used to determine disease severity throughout the course of this report: “How much pain have you had because of your illness in the past week on a scale of 0 to 10?” and “Considering all the ways that your illness affects you, rate how you are doing on the following scale of 0 to 10.” The goal of each of these questions was to serve as an effective assessment tool based on the patient’s mobility and functional capacity to quantify disease activity using patient reported outcomes. The numerical scores were evaluated and compared at each of the four time points.ResultsCase 1A 49-year-old male diagnosed with RA in 2019 presented to the clinic after previously declining initiation of DMARD therapy to treat his condition and subsequently struggled with significantly decreased range of motion, primarily in his hands, for one year before seeking pain management therapy. The patient came to the office in November of 2020 seeking therapeutic treatment of persistent inflammatory RA of the right fourth and fifth metacarpophalangeal joints. At his initial visit, the patient was given a PAS-II survey to complete. He noted “some difficulty” upon standing from a chair, doing outside work, and moving/lifting heavy objects, and reported he could complete the remaining activities “with no difficulty” such as opening car doors and reaching for objects. He reported 7/10 pain with his illness in the previous week. Additionally, he reported feeling at a level of 6/10 when asked about the effect of his illness on his daily functional capacity. On physical exam, the aforementioned joints were visibly swollen and tender to palpation. Per our methods, the patient received 0.5 mL intra-articularly and 1.5 mL peri-articularly.After one month, the patient was re-evaluated and objectively reported increased range of motion and diminished pain in the areas treated with PRP therapy. At three months, using the PAS-II scale, the patient reported only 3/10 pain with his illness and 4/10 overall, in terms of his illness. By six months, the patient felt as though the procedure gave him 100% relief and he rated on PAS-II scale being at 2/10 for pain and at a 1/10 as a whole, in light of the illness. Overall, the patient reported a 50% decrease in pain compared to his initial visit and overall well-being regarding the scope of his RA.Case 2A 60-year-old female diagnosed with RA in 2018, who had declined DMARD therapy with her rheumatologist, was seen in the pain management office in November of 2020 for treatment of her continued pain and inflammation primarily affecting the right third proximal interphalangeal (PIP) joint . At the initial visit, the patient was given a PAS-II survey to complete. She noted “much difficulty” with grasping and moving heavy objects. She reported 6/10 pain from her RA within the past week and again a 6/10 overall rating for her illness. Per our methods, the patient received 0.5 mL intra-articularly and 1.5 mL peri-articularly.After one month, the patient was evaluated again and objectively reported a mild decrease in her pain level, though still with significant restriction in her full range of motion. At three months, using the PAS-II scale, the patient reported 5/10 pain level and 5/10 overall, in terms of her illness. By six months, her PAS-II scale ratings were a 4/10 in terms of pain, and a 3/10 rating for overall daily functional capacity with her disease. Overall, she reported a 20% decrease in pain compared to her initial visit and 30% improvement in overall well-being in the scope of her RA.Case 3A 63-year-old male was diagnosed with RA in 2020 and was referred from his primary care physician to a rheumatologist for management of his condition. He was initially prescribed meloxicam for pain relief and then further sought therapy at our pain management clinic. He was seen in the office in November of 2020 for PRP administration in his afflicted joints - right third metacarpophalangeal (MCP) and right fifth PIP joints. At the initial visit, the patient was given a PAS-II survey to complete. He noted “some difficulty” with opening car doors and doing outside work such as yard work. He reported 4/10 pain from her RA within the past week and again a 4/10 overall illness rating. Per our methods, the patient received 0.5 mL intra-articularly and 1.5 mL peri-articularly into the affected joints.After one month, the patient was re-evaluated and objectively noted an improvement in his range of motion, with only a minor reduction of pain. At three months, using the PAS-II scale, the patient reported 3/10 pain level and 3/10 overall illness rating. By six months, his PAS-II scale ratings were a 2/10 in terms of his pain and a 1/10 regarding the effect the disease had on his overall well-being. Overall the patient reported a 20% decrease in pain compared to his initial visit and 30% improvement in overall daily, functional capacity in terms of his RA.Over the past decade, the application of PRP has substantially grown as a biological treatment for healing and regeneration of damaged tissues. Its effective pathophysiologic response has the potential to mitigate the significant damage caused by RA. Activation of the fibroblast-like synoviocytes (FLS) leads to the formation of “pannus,” a hyperplastic synovial lining that invades the periarticular bone leading to erosions and cartilage degradation . AdditioCurrent treatment options are aimed at the reduction of pain and inflammation to improve the quality of life of these patients, as this disease significantly affects activities of daily living. Since early diagnosis and treatment initiation halts the progression of joint damage in 90% of patients with early RA, a timely diagnosis and effective intervention is critical . PRP hasThe benefits of PRP in the pathogenesis of repair and regeneration in the setting of RA has been largely evaluated in laboratory, preclinical studies. Tong et. al. administered PRP in human rheumatoid FLS cells and found that it effectively altered the production of inflammatory mediators and enhanced neovascularization, which likely accelerates the healing process. PRP also regulates key factors in the P13K/AKT pathway, which is significantly involved in the progressing pathogenesis of the disease . Most noMuch of the clinical data supporting the use of therapeutic PRP has attempted to shed light on the benefits of PRP in treating patients with Osteoarthritis (OA), which shares some similarities in pathogenesis compared to RA, in regards to the pathophysiology of cartilage matrix degradation and progressive joint remodeling , 22. KeyThis report focuses on the use of PRP in RA patients seeking treatment to improve joint pain and inflammation. We present these cases to support the idea that PRP should be considered as an additional therapeutic option in the treatment of both the major biochemical and clinical effects of RA. In our experience, 0.5 mL of intra-articular PRP and 1.5 mL of peri-articular PRP resulted in significant functional improvement six months after injection with promise of sustained improvement. These results were independent of the duration of disease and past or ongoing treatment for these patients. Additionally, no adverse reactions were noted over the six-month follow-up period in the three patients who received PRP treatment. There are very few serious adverse events reported for PRP in the literature, likely due to the autologous nature of the treatment ,26. LimiIn conclusion, the precise mechanism of action of platelet-rich plasma shows promise as an effective agent for the management of RA in patients seeking an adjunct or alternative treatment to DMARD therapy. Initial clinical and laboratory studies have shown that autologous plasma rich in platelets serves as a source of an abundance of growth factors once activated. The multitude of these growth factors directly injected into the diseased joint accelerates healing and improves functionality in patients with RA. PRP is likely to be a safe and beneficial therapy in patients with any stage of RA. Intra-articular and peri-articular administration of PRP in outpatient settings is an easily adoptable treatment strategy, with growing emergence in our Board Certified Pain-Medicine practices. Overall, we believe that PRP should be further evaluated in clinical trials to outline the long-term clinical benefits in the improvement of joint structure and composition, as well as to evaluate the significant improvement expected in quality of life in patients with RA.