Little attention has been paid to chest high resolution computed tomography (HRCT) findings in idiopathic pulmonary arterial hypertension (IPAH) patients so far, while a couple of small studies suggested that presence of centrilobular ground-glass opacifications (GGO) on lung scans could have a significant negative prognostic value. Therefore, the aims of the present study were: to assess frequency and clinical significance of GGO in IPAH, and to verify if it carries an add-on prognostic value in reference to multidimensional risk assessment tool recommended by the 2015 European pulmonary hypertension guidelines.Chest HRCT scans of 110 IPAH patients were retrospectively analysed. Patients were divided into three groups: with panlobular (p)GGO, centrilobular (c)GGO, and normal lung pattern. Association of different GGO patterns with demographic, functional, haemodynamic, and biochemical parameters was tested. Survival analysis was also performed.GGO were found in 46% of the IPAH patients: pGGO in 24% and cGGO in 22%. Independent predictors of pGGO were: positive history of haemoptysis, higher number of low-risk factors, and lower cardiac output. Independent predictors of cGGO were: positive history of haemoptysis, younger age, higher right atrial pressure, and higher mixed venous blood oxygen saturation. CGGO had a negative prognostic value for outcome in a 2-year perspective. This effect was not seen in the longer term, probably due to short survival of cGGO patients.Lung HRCT carries a significant independent prognostic information in IPAH, and in patients with cGGO present on the scans an early referral to lung transplantation centres should be considered. Pulmonary hypertension (PH) is defined as an increase in mean pulmonary artery pressure (PAPm) ≥ 25 mmHg at rest as assessed by right heart catheterisation (RHC) ; recentlGround-glass opacities (GGO) is a radiological term for areas of hazy increased lung parenchyma density not sufficient to obscure bronchial and vascular margins on chest HRCT . They arIPAH remains a life-threatening disease despite constant development of therapeutic options and improved management plans , 18–24. Although well appreciated, the phenomenon of inhomogeneous lung attenuation in IPAH has not been thoroughly investigated and described. Consequently, the objectives of our study were: to assess frequency and clinical significance of GGO in IPAH, and to verify if it carries an add-on prognostic value in reference to ESC/ERS multidimensional risk assessment tool.Patients diagnosed with IPAH in our hospital between 1997 and 2011, and for whom chest HRCT scans were available for review, were retrospectively analysed. Diagnosis of IPAH was based on evidence of PAH on RHC and exclusion of known causes of PH. All included patients had PAPm ≥ 25 mmHg, PAWP ≤ 15 mmHg, and PVR > 3 Wood units [The HRCT examinations were performed between Jan 1997 and Nov 2011, with use of either a single-row helical unit Picker PQ 2000 (Siemens) or a multi-row Somatom Sensation 16 (Siemens). In part of the patients images were obtained with a sequential acquisition technique with 1-mm section thickness at 10 mm intervals, in others high resolution images were reconstructed from contrast enhanced spiral acquisition. All scans were acquired in the supine position, at end-inspiration. There were no differences in the radiological protocols between studies obtained with both CT equipments, and differences in technical specifications of both units made no impact on assessment of elements included in the analysis. The images were reassessed by the radiologist experienced both in PH and interstitial lung diseases, blinded to patients’ clinical details and outcomes. The main focus was on assessment of lung parenchyma homogeneity, and patients were divided into three groups: with cGGO pattern, with pGGO pattern, and with normal pattern. CGGO were further evaluated as distributed evenly or with a zonal predominance, pGGO as patchy or perihilar. Mediastinal lymphadenopathy  and smooth thickening of interlobular septa were also recorded.2), PVR [Data collected for analyses were: sex, age at the time of chest HRCT, cigarette smoking status, history of haemoptysis, presence of patent foramen ovale, World Health Organisation functional class (WHO FC), 6-min walking test results , plasma N-terminal pro-brain natriuretic peptide (NT-proBNP), DLCO percent of predicted value (%pred), haemodynamic parameters: mean right atrial pressure (RAPm), systolic, diastolic and mean pulmonary artery pressures, cardiac output (CO) and cardiac index (CI), mixed venous blood oxygen saturation (Svo2), PVR , result 2), PVR ). Median2, with cut-off values and three levels of risk as proposed in the document [For risk stratification 6 of 13 risk factors recommended in the 2015 ESC/ERS PH guidelines were used: WHO FC, 6-min walking test distance (6MWD), NT-proBNP plasma level, RAPm, CI, and SvOdocument . The Fredocument .The STATISTICA v. 13 (Statsoft) computer software was used for statistical analysis. Continues data were presented as median with interquartile range, and Kruskal–Wallis’ or U Mann–Whitney’s tests were used for comparison between three or two groups, respectively. Categorical variables were shown as actual number with percentage where appropriate, and the Pearson’s chi-square test was used for comparison between groups. Logistic regression was performed to determine factors associated with different HRCT patterns, the normal pattern was a reference class. The Somers’ D was used to built the multivariate regression models. Transplant-free survival was calculated from the date of chest HRCT scanning to the date of death or LTx (complete observation) or the date of last follow-up, with 23rd Apr 2020 terminating the period of observation. Survival analysis was performed using the Kaplan–Meier method. Survival between groups was compared with log-rank test. Univariable and multivariable Cox proportional hazards models were used to evaluate the predictive value of the HRCT patterns as well as the other analysed variables. Parameters with p < 0.2 in the univariate analysis were included in the multivariate analysis. Hazard ratios (HR) with 95% confidence interval (95% CI) were presented. P < 0.05 was considered statistically significant.No ethics committee approval or informed consent were required due to retrospective character of this study. It was conducted in agreement with the Declaration of Helsinki and the European General Data Protection Regulation.The studied group consisted of 110 IPAH patients: 102 (93%) incident, 80 (73%) women, age median value was 44.3 years (range 17.3–78.5 years). Fifty (46%) patients had the abnormal lung pattern on HRCT scans. The pGGO were found in 26 (24%) patients, perihilar localisation was seen in 50% of them. The cGGO were found in 24 (22%) patients, nodules were widespread and with no specific zonal predominance in all cases. The remaining 54% of the patients were classified as normal HRCT pattern. One patient with pGGO had lymphadenopathy and septal thickening. Among patients with cGGO, two had lymphadenopathy, and another one had septal thickening. Three patients with normal pattern had lymphadenopathy. The characteristics of the whole studied group, as well as detailed comparison of the three groups according to the lung HRCT pattern are presented in Tables The abnormal HRCT pattern was more frequent in women  than in man , p = 0.004. Patients with abnormal pattern had lower median age: 37.5 (26.6–49.6) vs 46.8 (37.6–59.0) years, p = 0.029. The cGGO and pGGO groups had higher rates of patients with positive history of haemoptysis compared to the normal pattern group: 29% and 23% vs 9%, p = 0.046. There was no difference in WHO FC between the groups, but patients with the abnormal HRCT pattern had higher median value of 6MWD than patients with the normal pattern: 429 (380–484) vs 379 (298– 450) m, p = 0.018.The distinctive features of the pGGO group were: (i) negative association with cigarette smoking: only 19% of these patients were ever-smokers vs 45% of the remaining patients, p = 0.019, (ii) higher number of low-risk factors: 3.0 (2.0–4.0) vs 2.0 (0.0–3.0) in the remaining patients, p = 0.024. The distinctive feature of the cGGO group was higher median RAPm: 10.0 (8.0–13.0) vs 7.0 (4.0–10.0) mmHg in the remaining patients, p = 0.008.There were no significant differences between the lung HRCT patterns in terms of presence of patent foramen ovale, oxygen saturation, DLCO%pred, NT-proBNP, or haemodynamic parameters other than RAPm.2, and positive history of haemoptysis.The results of logistic regressions are shown in Table The median time of follow-up was 5.0 years, range 0.1–22.1 years. During this time 73 (66%) patients died (65 patients) or underwent LTx (8 patients), 26 24%) patients were still alive at the end of the study, and 11 (10%) were lost to follow-up. The patients’ LTx-free survival depending on the lung HRCT pattern is illustrated in Fig. % patientFor 71% of the patients all six risk factors were documented, further 24% had 5 risk factors available, 5% of patients had 2–4 risk factors documented. 27% of the patients were in the low-risk group, 60% in the intermediate-risk group, and 13% in the high-risk group. The median number of low-risk factors was 2, 23% of the patients had no low-risk factors, 27% had at least 4 low-risk factors. There were no significant differences in distribution of the HRCT patterns in the risk groups, but pGGO pattern was associated with a higher number of low-risk predictors compared to other patterns — Table All three patients with cGGO and either lymphadenopathy or septal thickening had survival shorter than 1 year. The patient with pGGO, lymphadenopathy and septal thickening as well as all patients with normal pattern and lymphadenopathy survived longer than 2 years.Significant independent predictors for negative outcome in the perspective of the whole follow-up were: intermediate/high risk group  and DLCO%pred . Independent predictors in the two-year perspective were: presence of cGGO , DLCO%pred , and decrease in oxygen saturation during 6-min walking test . Results of uni- and multivariate Cox regressions are shown in Tables Chest HRCT plays an essential role in the workup of PH , and it In our study, the groups of patients with centri- and panlobular GGO patterns shared some similarities that distinguished them from the patients with normal pattern: more frequent female gender, younger age, better 6MWD, and positive history of haemoptysis. We failed to find explanation for association of GGO with gender and age. Better 6MWD could be secondary to younger age of these patients. Haemoptysis is a recognised complication of IPAH associated with poor prognosis . The pre2. The pGGO group had the lowest rate of cigarette smokers and the highest median number of low-risk factors. Independent predictors of pGGO were: positive history of haemoptysis, higher number of low-risk factors, and lower CO.We found features distinctive for each GGO pattern group. The cGGO group was characterised by the highest RAPm, and that was in agreement with previous studies on smaller groups , 17. IndWe also demonstrated significant differences in short-term survival rates between the groups. Presence of cGGO turned out to have a negative prognostic value for outcome in a 2–year perspective, with sixfold increase in death/LTx risk. The 1,2,3-year survival rates in the cGGO group were similar to those reported for high-risk group PAH patients in national and international registries , 22, altIn the clinical context of PAH with GGO present on HRCT lung scans, it is of utmost importance to differentiate between IPAH and PAH with overt features of venous/capillaries (PVOD/PCH) involvement . SurvivaOur research has shortcomings related to its retrospective character, possible selection bias, disregard of medical treatment in the analysis, and still small number of included patients. Nevertheless, it gives insights into different phenotypes of IPAH patients depending on lung HRCT pattern and proves independent significance of cGGO in predicting survival in a real-life patients cohort.Further research is needed to elucidate many intriguing issues related to IPAH phenotype associated with GGO on lung HRCT, e.g. the histopathologic background of cGGO and pGGO, mutual dependence of causes and effects between GGO-related structures and PH course, relation between GGO and right ventricle performance assessed with echocardiography and/or cardiac magnetic resonance imaging, or effect of PAH-specific treatment on GGO.GGO are frequent findings on the lung HRCT scans of IPAH patients. Panlobular and centrilobular patterns of GGO are equally prevalent, but have different clinical significance. Presence of cGGO on HRCT scans of the lungs of IPAH patients has an add-on prognostic value for an outcome in a 2-year follow-up in reference to already well-established prognostic factors collected in the ESC/ERS multidimensional risk assessment tool, and could serve as an complementary element in making therapeutic decisions, e.g. the timing of referral for LTx assessment.


Mycoplasma suis (M. suis) belongs to the group of haemotrophic mycoplasmas and is known as the causative agent of infectious anaemia in pigs. In the last few years valuable insights into the mechanism of adhesion and invasion, shedding patterns and cell tropism of M. suis were gained by the use of new molecular techniques. However, details on M. suis induced lesions as well as the distribution of M. suis in different organs are still lacking. Therefore, seven splenectomised pigs were experimentally infected and clinical and laboratory investigations as well as a detailed histopathological examination were performed. Detection and quantification of M. suis DNA in blood and various tissue samples was done using a quantitative real-time PCR.M. suis was found in all investigated tissues with the highest copy numbers found in the kidneys. In Giemsa stained sections M. suis was only detected red blood cell (RBC)-associated.During the course of experimental infection, periodically occurring signs of infectious anaemia of pigs including severe icteroanaemia, fever, apathy and anorexia were observed. In addition, dermatological manifestations such as haemorrhagic diathesis presenting as petechiae occurred. The most important haematological alterations were normochromic, normocytic anaemia, hypoglycaemia as well as increased bilirubin and urea concentrations. Necropsy revealed predominant evidence of haemolysis with consecutive anaemia, as well as disseminated intravascular coagulation. M. suis was detected in organs of experimentally infected pigs. Pathological findings are most likely resulting from haemolysis, consecutive anaemia as well as from disseminated intravascular coagulation and subsequent organ impairments.In the present study, no RBC independent sequestration of  Mycoplasma suis is a so far uncultivable haemotrophic pathogen that causes acute or chronic infectious anaemia in pigs (IAP) worldwide [M. suis via parenteral exposure during zootechnical procedures or through ranking fights have been identified as key epidemiological routes [M. suis are rarely and incompletely described in literature.orldwide –5. Cliniorldwide , 3, 6. Aorldwide , 7–11. Ul routes , 9, 11. M. suisis primary found in the blood on the surface of red blood cells (RBCs), inside RBCs or, to a lesser extent, free in the plasma [M. suis to the endothelium has also been proven so far [e plasma , 6. In an so far , 12.M. suis infected animals reached maximal copy numbers of 105 to 1010 M. suis/mL blood within 6–7 days post infection (dpi) in splenectomised pigs [3 to 109 copies/ mL blood within 23 to 30 dpi in intact animals [M. suis occurs within 1 to 2 days [M. suis bacteraemia [M. suis infections. Possibly, propagation as well as sequestration of M. suis in organs and a subsequent pathogen release under immunosuppressive conditions may be involved in the largely unknown pathogenesis.Establishment of persistent infections as well as fluctuating bacteraemia are hallmarks of haemoplasma infections , 3, 13.  animals . After ao 2 days , 16 but teraemia . SimilarM. suis in different organs as well as tissue alterations due to M. suis in experimentally infected pigs in order to identify potential M. suis target tissues and sequestration sites which could be involved in IAP pathogenesis.Thus, the aim of the present study was to investigate the possible RBC-independent sequestration of M. suis infections such as icterus and haemorrhagic diathesis presenting as petechiae and cyanosis of the ear tips. As those three animals  fulfilled the abortion criteria of the scoring system, they were euthanised on 8 dpi. The four remaining animals  showed mild clinical signs of IAP first on 13–15 dpi which manifested as cyanoses of the ear tips and petechiae generalised over the body. Clinical signs exacerbated in three animals  between 16 and 18 dpi with highly impaired general condition, high fever (up to 41.6 °C), anorexia, dyspnoea, skin pallor and ecchymosis on both ears. Due to fulfilment of the abortion criteria, two animals  were euthanised at 17 and 20 dpi, respectively. Antibiotic treatment with oxytetracycline and oral supplementation of glucose resulted in an improvement of clinical signs in the two remaining animals. However, recurrent occurrence of acute IAP was observed in both animals and they had to be euthanised on 41 (ID 31) and 62 dpi (ID 71), respectively. The course of the score points over time is displayed in Fig. On 7 and 8 dpi, three animals  showed clinical signs of acute IAP including impaired general condition, fever up to 41.4 °C, apathy, anorexia and skin alterations described for All animals developed a normochromic, normocytic anaemia at the time of acute clinical signs. RBC, haemoglobin and packed-cell-volume (PCV) concentrations from 0 dpi until euthanasia of the last animal 62 dpi) are shown in Fig.  dpi are With the onset of first clinical signs related to acute IAP, five  out of the seven animals developed hypoglycaemia. Hypoglycaemia was also observed in the recurrent IAP attacks. The lowest glucose concentration of 0.2 mmol/L was recorded on 8 dpi (ID 76). At the time of acute IAP attacks increased bilirubin concentrations were recorded ranging from 3.25 μmol/L to 160.2 μmol/L. Urea concentrations increased in six out of seven animals  at the onset of clinical signs. At the individual termination point of the experiment, serum iron concentrations decreased in all animals.M. suis was first detected in the blood on 4 dpi in three animals  by qPCR. On day 6 all animals were M. suis positive. M. suis blood loads ranged between 2.2 × 103 M. suis/mL on 4 dpi (ID23) and 9.6 × 109 M. suis/mL on 8 dpi (ID 32).M. suis was found in all investigated tissue samples with the highest bacterial loads in the kidney (range: 1.76 × 1010 M. suis/g tissue (ID 32) - 5.98 × 1011 M. suis/g tissue (ID 31) and the lowest bacterial loads in the liver (range: 1.52 × 104 M. suis/g tissues (ID 74) - 2.32 × 106 M. suis/g (ID 73)).As shown in Table Gross necropsy findings included severe icterus with yellowish discoloration of the skin and mucous membranes in two out of the seven animals ID 73; 74). In addition, accumulation of watery translucent, icteric discoloured fluid was found in the abdominal cavity in animal 74. Signs of anaemia such as pale musculature were found in two animals . Both animals further presented severe swelling of the mediastinal and intestinal lymph nodes which were also reddened by blood resorption. Additionally, moderate to severe yellowish discoloration of the tunica intima of the aorta was observed in three animals  .Macroscopically, changes of the kidneys were noticed in all animals. In addition to brightened hyperaemic tissue , multiple and blurred whitish lesions with a diameter of up to 5 mm were observed, extending either over the entire renal tissue  or only over the renal cortex (31).Macroscopic examination of other investigated organs  revealed no obvious findings in all seven animals.Microscopic examination revealed low-grade nonspecific reactive interstitial hepatitis involving lymphocytes, plasma cells, eosinophilic and basophilic granulocytes as well as macrophages in three animals . Additionally, in four animals ID 73, 74, 31, 71) a multifocal erythrophagocytosis by Kupffer cells was detected and in two animals, haemosiderin was found in these macrophages . A total of three animals showed mild to severe oedema of the gallbladder bed . In four animals  hyaline thrombi representing atypical coagulation products appeared in the hepatic sinusoids and vessels  and non-purulent interstitial nephritis with dilatation of the lymph vessels (ID 31). Microscopic examination of the lymph nodes revealed signs of blood resorption , increased phagocytosis of the RBCs and the occurrence of siderophages . The mesenteric lymph nodes of two animals showed follicular hyperplasia (ID 31) and sinus histiocytosis (ID 23), respectively. In two animals  fibrin was also found in the sinuses of different lymph nodes.V. cava. The animal ID 32 showed a pronounced circular fibrin exudation in the aorta with concomitant infiltration of macrophages and lymphocytes. A largely unchanged and haematopoietically active tissue with dominant erythropoiesis was found in sternal and femoral bone marrow , multifocal low-grade acute bleeding and oedema in the leptomeninx (ID 76), multifocal high-grade perivascular plasma cellular and histiocytic infiltrates in the plexus of the fourth ventricle (ID 32) and endothelial cell swellings (ID 73) were found. Vasal and perivascular neutrophilic granulocytes and monocytes were detected in choroid plexus , leptomeninx (ID 71), cerebral cortex (ID 74) and subependymal in the lateral ventricle (ID 73).M. suis was detected in Giemsa stained slides of all investigated organs. In the Giemsa staining M. suis was only associated with RBCs  were evident in splenectomised pigs of the present study as early as 7 dpi. Although cutaneous manifestations resulting from a so far unknown pathogenesis have formerly thought to occur only in chronic stages of the disease, a more recent study described lesions from 17 dpi onwards in experimentally infected non-splenectomised pigs .M. suis infection [Despite numerous lesions no pathognomonic features for IAP were found in the present study which is in accordance with Dent et al. . The mosnfection . As hyalM. suis induced centrilobular necrosis of the liver was observed in one animal and has been described previously [. In general, the most common cause of centrilobular necrosis is hypoxia of tissues. Hypoxia during IAP likely results either from anaemia or DIC [M. suis infection [eviously , 26. In a or DIC . The hyanfection .To summarise, the macroscopic and histologic lesions are likely to result from haemolysis with consecutive anaemia as well as disseminated intravascular coagulation and resulting organ impairment.M. suis in blood samples throughout the entire investigation period. Sequestration of haemotrophic mycoplasma in tissues has been proven in previous studies [M. suis for endothelial cells was identified [M. suis in tissues we sampled different organs and were able to detect M. suis in different quantities in the respective tissues. In contrast to M. haemofelis infected cats higher tissue loads were observed in the present study. In order to determine haemoplasma copy numbers in tissue other than expected from the blood supply Tasker et al. [Antibiotic treatment did not result in clearance of the pathogen as indicated by permanent detection of  studies –30 and ientified . To inver et al.  have defM. suis was exclusively associated with RBCs in Giemsa stained slides our study indicated no evidence of so far unknown potential host cells. However, further investigations with methods showing a higher sensitivity  are certainly needed.Such calculation cannot be extrapolated to pigs as data on organ perfusion for pigs are lacking. However, if we adopt the equation from cats  a higherM. suis and resulting macroscopic and microscopic lesions were investigated in experimentally infected pigs. M. suis was detected in various organs by qPCR, however no evidences of RBC independent sequestration in different organs were found. Observed gross-necropsy and histopathological findings in M. suis infected pigs are most probably resulting from anaemia caused by haemolysis together with disseminated intravascular coagulation and consequential effects on other organs.In the present study tissue sequestration of M. suis qPCR negative female piglets  [7 M. suis/mL; M. suis field strain K323/13) 1 week after splenectomy. Clinical assessment was performed according to a score system described by Stadler et al. [Seven 28-days old,  74, 76) . Briefly 74, 76) . Pigs wer et al.  with somEDTA-anticoagulated blood and serum samples were collected every 2 days for the first 8 dpi and afterwards once a week until the end of the trial planned on 90 dpi.The following haematological parameters were recorded from EDTA-anticoagulated blood using the Scil Vet™ ABC tool : RBC count (T/L), haemoglobin concentration (mmol/L), packed-cell-volume (PCV) (%), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and leukocyte count (G/L). Glucose (mmol/L), iron (μmol/L), urea (mmol/L) and bilirubin (μmol/L) concentrations were determined by a Hitachi C 311 Chemistry Analyzer  from serum samples.M. triceps); skin; stomach; V. cava. After sampling, tissues used for histopathological examination were immediately transferred to 4% paraformaldehyde and fixed at 4 °C for 24 h. Tissues were embedded in plastic and stained according to Giemsa as well as with haematoxylin-eosin-phloxin (HEP).Necropsy and a macroscopic pathological examination were performed on all animals. Tissues of the following organs were collected during necropsy: aorta; bone marrow; brain (cerebrum and cerebellum); gall-bladder; heart; intestines; kidney; liver; lung; mesenterial lymph nodes; pancreas; skeletal muscle  as described elsewhere [2, 30 mM sucrose, 1% (v/v) Triton-X-100) followed by purification with the GenElute™ Bacterial Genomic DNA Kit  according to the manufacturer’s instructions [M. suis DNA was quantified afterwards by real-time PCR using the StepOne System™ (Applied Biosystems®) and primers specific for the M. suis msg1 [lsewhere , 32. Foruis msg1 .Data were analysed with Excel (Microsoft® 2016) and the statistical software IBM SPSS, Statistics 22.0 . Haematological and biochemical parameters were evaluated by means of descriptive statistics which included the calculation of the mean, standard deviation (SD) as well as standard errors of the mean (SEM). Differences in mean blood parameters between 0 dpi and the consecutive sampling days were investigated by a paired sample t-test.


Streptococcus pyogenes (S. pyogenes) and Haemophilus influenzae. The aim of this study was to investigate the effect of antibacterial agents against cell-invading bacteria.Recurrent tonsillitis is one of the most common otolaryngological disorders caused by cell-invading bacteria, such as Haemophilus influenzae (NTHi) and four strains of S. pyogenes was investigated. The antibacterial agents used were garenoxacin (GRNX), clarithromycin (CAM), amoxicillin (AMPC), cefditoren pivoxil (CDTR-PI), and levofloxacin (LVFX).The intracellular invasion of Detroit 562 cells by five strains of nontypeable S. pyogenes fully invaded Detroit 562 cells in 6 h and were less sensitive to CAM. GRNX, CAM, and LVFX were effective against bacteria invading the cells, but AMPC and CDTR-PI were not effective. GRNX was the most effective.Both NTHi and GRNX was the most effective agent against bacteria invading cells. Streptococcus pyogenes (S. pyogenes), Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pneumoniae [H. influenzae (NTHi) and S. pyogenes invade the cells and escape from the action of antibacterial agents [Recurrent tonsillitis is one of the most common otolaryngological disorders . The moseumoniae . Among tl agents . When toH. influenzae is a leading cause of acute and chronic otitis media, chronic sinusitis, and tonsillitis [H. influenzae lack capsular polysaccharides and are referred to as NTHi and that H. influenzae frequently persist within dense biofilm communities that are thought to provide resistance to host clearance and bactericidal activity of some antibacterial agents [S. pyogenes is an important human pathogen that can cause severe, life-threatening, invasive infections, such as soft tissue infection, sepsis, and streptococcal toxic shock syndrome [S. pyogenes is generally an extracellular pathogen that can survive and persist within the host by the expression of a broad array of virulence functions directed to circumventing the host immune mechanisms [S. pyogenes entering cells and escaping from the action of antibacterial agents.sillitis . It is rl agents . S. pyogsyndrome . S. pyogchanisms . It is bS. pyogenes) [H. influenzae (BLNAR) is particularly common [International guidelines recommend penicillin as the first-choice antibiotic treatment for acute sore throat (suspected to be caused by yogenes) . Howeveryogenes)  and are yogenes) . Therefoy common . TherefoS. pneumoniae, one of the most important causative pathogens in community-acquired pneumonia (CAP). Similarly, garenoxacin (GRNX) is an oral des-fluoro (6)-quinolone with potent antimicrobial activity against common respiratory pathogens [Levofloxacin (LVFX), a broad-spectrum fluoroquinolone with potent activity against Gram-positive bacteria, is currently recommended to treat respiratory tract infections and pneumonia due to athogens . LVFX anathogens . Additioathogens . These dMycoplasma pneumoniae and Chlamydia pneumoniae that do not have cell walls [Legionella and nontuberculous mycobacteria, reflecting its excellent transferability from tissues to cells [Clarithromycin (CAM) exerts its antibacterial activity through its inhibitory effect on protein synthesis and is therefore effective against atypical pathogens such as ll walls . It alsoto cells .S. pyogenes isolated in Japan.The present study investigated the in vitro antibacterial activity of antibacterial agents against clinical strains of NTHi and S. pyogenes, one bacterial strain (NTHi1 or S. pyogenes1) was used to confirm the time of cellular invasion. NTHi invaded the cells 2 h after they were attached to the cells . GRNX had a significantly higher bactericidal effect than CAM and LVFX (p < 0.05). However, no bactericidal effect was observed from treatment with AMPC or CDTR-PI , but treatment with AMPC or CDTR-PI had no bactericidal effect . GRNX had a significantly higher bactericidal effect than CAM and LVFX . However, no bactericidal effect was observed from treatment with AMPC or CDTR-PI , but treatment with AMPC or CDTR-PI had no bactericidal effect .Fibronectin-binding protein (F1 protein) is mentioned as a mechanism by which he cells . In Japahe cells  reportedl agents . Moreovebiofilms . In addienotypes . In thisH. influenzae invades Detroit 562 cells supports the results of the present study [In addition, phosphorylcholine is mentioned as a mechanism of intracellular invasion by NTHi, and the higher the expression level of phosphorylcholine, the more it penetrates into cells . The prent study .S. pneumoniae and H. influenzae have increased exponentially worldwide. Penicillin-resistant S. pneumoniae, such as penicillin intermediately-resistant S. pneumoniae (PISP), penicillin-resistant S. pneumoniae (PRSP), and BLNAR, are particularly common in Japan [S. pyogenes isolates, the MICs of LVFX were eightfold higher than those of GRNX, CDTR-PI, and AMPC. Quinolone disrupts the DNA replication of type II topoisomerase, thereby inhibiting bacterial growth. Moreover, type II topoisomerases include DNA gyrase and topoisomerase IV and consist of two dimers of subunit types A and B. The quinolone resistance-determining regions (QRDRs) within subunits A and B are closely related to resistance [S. pyogenes strains, 12 (85.7%) had two or more mutations in QRDRs. This is considered one of the reasons why S. pyogenes were less sensitive to LVFX.Since the late 1990s, respiratory tract infections caused by antibiotic-resistant strains of in Japan . In NTHisistance . Shoji esistance  reportedH. influenzae that has entered the cells [Invasion of cells by bacteria has been cited as a cause of repeated tonsillitis. In this study, neither AMPC nor CDTR-PI was found to have a bactericidal effect on bacteria invading the cells. It is known that β-lactam antibacterial agents have low intracellular transmissibility, and their antibacterial action is reduced against he cells . TherefoGRNX is highly effective in the treatment of patients with upper and lower respiratory tract infections . Takagi The present study showed that CAM was effective against bacteria invading cells. Patel et al.  reportedOur study has some limitations. First, BLNAS and other resistant strains were not investigated. Since the number of strains of resistant bacteria is increasing, more resistant strains should be included in future studies. The second limitation pertains to the epithelial cells used. Although the use of normal human epithelial cells may be more clinically relevant, we used a pharyngeal cancer-derived cell line. Because these cells were of human origin, we consider that the results of this study were not different from those that would have been obtained with the use of normal cells.GRNX was the most effective agent against cell-invading bacteria. Administration of GRNX should be considered when the efficacy of penicillin and cephem antibiotics and of β-lactam is insufficient in daily medical practice.The following antibacterial agents were used in the study: analytical grade powders of GRNX , CAM , amoxicillin (AMPC) , cefditoren pivoxil (CDTR-PI) , and LVFX .S. pyogenes oropharyngeal isolates from patients with otitis media with effusion and recurrent tonsillitis (aged 21–35 years) and patients with only recurrent tonsillitis (aged 24–42 years), respectively, at the Kagoshima University Hospital between March 2019 and December 2020. All bacteria were stored in skimmed milk with glycerol at − 80 °C until use. An aliquot of each bacterial stock was thawed and cultured overnight at 37 °C in a 5% CO2 incubator on chocolate II agar  or sheep blood agar  plates, as appropriate. After washing in 0.5% bovine serum albumin–phosphate-buffered saline (PBS), the bacteria were used for intracellular invasion assays. The concentrations of NTHi and S. pyogenes were adjusted to 1.0 × 108 colony-forming units (CFU)/mL at an absorbance of 580 nm. The Institutional Review Board of Kagoshima University approved this study.We collected NTHi nasopharyngeal isolates and The susceptibility of bacteria to antibiotics was studied by the broth microdilution method, performed according to Clinical Laboratory Standards Institute guidelines . The tesemm gene encoding the M protein was performed according to the protocol presented by the Center for Disease Control and Prevention (http://www.cdc.gov/ncidod/ biotech/strep/protocols.html), with minor modifications previously described [Genotyping of the escribed .2 incubator as previously described [4 viable cells per well in a 96-well BD Falcon tissue culture plate with a low-evaporation lid . The plates were used when > 90% confluence was observed following overnight incubation.Detroit 562 cells , a human pharyngeal carcinoma epithelial cell line, were grown to confluence in minimal essential medium  supplemented with 1 mM sodium pyruvate , 10% fetal bovine serum , penicillin (100 U/mL), and streptomycin  at 37 °C in a 5% COescribed . The celS. pyogenes strains (1.0 × 108 CFU/mL) were added to Detroit 562 cells cultured in a 96-well plate and allowed to adhere at 37 °C in a 5% CO2 incubator for 6 h. Each well was then treated with gentamicin (200 μg/mL) at 37 °C in a 5% CO2 incubator for 1 h. After washing five times with 200 μL of PBS, the cells were treated with 100 μL of each antibacterial agent at 37 °C in a 5% CO2 incubator for 6 h. Controls were treated with PBS without antibacterial treatment. After washing five times with 200 μL of PBS, the cells were treated with 100 μL of saponin at 37 °C in a 5% CO2 incubator for 15 min. Further, 100 μL of the samples from each well was plated on chocolate II agar plates or sheep blood agar and cultured overnight, and the number of colonies formed was counted as described previously [One hundred microliters each of the NTHi and eviously .p < 0.05 indicated statistical significance.All statistical data were analyzed using SPSS for Windows software , and the values were presented as mean ± standard deviation. Furthermore, we used unpaired one-way analysis of variance with Tukey’s method for statistical data analysis. Differences showing


Montelukast is a selective leukotriene receptor antagonist that is widely used to treat bronchial asthma and nasal allergy. To clarify the association between montelukast and neuropsychiatric adverse events (AEs), we evaluated case reports recorded between January 2004 and December 2018 in the Food and Drug Administration Adverse Event Reporting System (FAERS). Furthermore, we elucidated the potential toxicological mechanisms of montelukast-associated neuropsychiatric AEs through functional enrichment analysis of human genes interacting with montelukast. The reporting odds ratios of suicidal ideation and depression in the system organ class of psychiatric disorders were 21.5 : 20.3–22.9) and 8.2 (95% CI: 7.8–8.7), respectively. We explored 1,144 human genes that directly or indirectly interact with montelukast. The molecular complex detection (MCODE) plug-in of Cytoscape detected 14 clusters. Functional analysis indicated that several genes were significantly enriched in the biological processes of “neuroactive ligand–receptor interaction.” “Mood disorders” and “major depressive disorder” were significant disease terms related to montelukast. Our retrospective analysis based on the FAERS demonstrated a significant association between montelukast and neuropsychiatric AEs. Functional enrichment analysis of montelukast-associated genes related to neuropsychiatric symptoms warrant further research on the underlying pharmacological mechanisms. Montelukast is a selective leukotriene receptor antagonist used to treat bronchial asthma and nasal allergy. Although montelukast is generally well tolerated, several clinical trials and post-marketing studies have reported serious neuropsychiatric adverse events (AEs) . The pothttp://www.fda.gov), and is used in the post-marketing safety assessments of approved drugs. The objective of this study was to evaluate the association between neuropsychiatric AEs using well-established pharmacovigilance indices such as reporting odds ratio (ROR).The FAERS is a spontaneous reporting system (SRS) involving reports of AEs in a real-world setting that are voluntarily submitted by healthcare professionals, pharmaceutical companies, and patients. The FAERS database is publicly available, can be downloaded from the FDA website .Data from April 2004 to December 2018 were extracted from the FAERS database on the FDA website. The informatic structure of the FAERS database is based on the international safety reporting guidelines issued by the International Council on Harmonization (ICH), known as ICH E2B guidelines . We inteFollowing the FDA’s recommendation, we excluded duplicate reports of the same patient from different reporting sources from the analysis and extracted reports. Drugs in FAERS are classified into four categories: primary suspect drug (PS), secondary suspect drug (SS), concomitant (C), and interacting (I), according to the anticipated degree of involvement for AEs. Only reports with the PS drug code were included in this analysis.https://www.meddra.org), which is the dictionary for terminology used in the FAERS database. This study relied on the definitions provided by MedDRA version 21.0. To evaluate montelukast-associated AEs, we utilized the system organ classes (SOCs) of “psychiatric disorders,” “general disorders and administration site conditions,” “nervous system disorders,” “respiratory, thoracic and mediastinal disorders,” and “gastrointestinal disorders” , DSigDB , and STITCH (https://stitch.embl.de). The indirectly associated genes were retrieved from iRefIndex 15.0  (http://cytoscape.org). This plug-in was utilized to choose hub modules of the gene−gene interaction network in Cytoscape with a degree cutoff = 2, node score cutoff = 0.2, k-core = 2, and Max. Depth from seed = 100 as the criteria. Next, we used “clusterProfiler (version 1.4.0),” an R package, to perform functional analysis and visualization of functional profiles for genes and gene clusters. We used the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to explore the biological significance. KEGG enrichment analysis was performed using clusterProfiler with organism = “hsa,” pvalueCutoff = 0.05, pAdjustMethod = “BH,” and qvalueCutoff = 0.1. The thresholds in the KEGG enrichment analysis are pvalueCutoff = 0.05 and qvalueCutoff = 0.1. The default thresholds in the KEGG enrichment analysis are pvalueCutoff = 0.05 and qvalueCutoff = 0.2. Lin et al. applied pvalueCutoff = 0.05 and qvalueCutoff (not listed)  . Molecul.vib.be) . The clu listed) . We coulThe FAERS database contains 11,527,470 reports from January 2004 to December 2018. After excluding duplicates according to the FDA recommendations, 9,702,166 were analyzed. The RORs of suicidal ideation, suicide attempts, and depression in the SOC of psychiatric disorders were 21.5 (95% CI: 20.3–22.9), 9.5 (95% CI: 8.5–10.5), and 8.2 (95% CI: 7.8–8.7), respectively.https://www.dgidb.org), DSigDB , and STITCH (https://stitch.embl.de) and retrieved 26 genes  that interact with montelukast directly. All the genes of “9606. mitab” from iRefIndex 15.0 (https://irefindex.vib.be) were integrated into a network with 20,877 nodes and 429,350 edges. The genes that directly or indirectly interact with the above 26 genes were integrated into a network with 1,144 nodes and 35,384 edges. MCODE plug-in found 14 clusters  and “respiratory syncytial virus infections”  in cluster 1 and “mood disorders”  and “major depressive disorder”  in cluster 2.Furthermore, data retrieved from DisGeNET was used to characterize diseases associated with montelukast. We found significant enrichment in genes involved in the following diseases related to montelukast : “pneumoWe elucidated the AE profile of montelukast using the SOC of psychiatric disorders associated with the drug in the FAERS database. The lower limits of the 95% CIs of RORs related to the SOC of “psychiatric disorders” were more than 1, and the signal was significantly detected. Neuropsychiatric AEs have been found to account for the most important costs associated with comorbidity in asthma and have a negative impact on the patients’ quality of life , althougTo better understand the toxicological mechanisms of montelukast-associated neuropsychiatric AEs, we curated drug−gene interactions from public databases. A total of 1,144 human genes interacting with montelukast were investigated. Some of these genes that were highly enriched in DisGeNET were related to “mood disorders” and “major depressive disorder” . HCRT h. HCRT enThe common AEs caused by montelukast are upper airway infections, anaphylaxis, nausea, vomiting, diarrhea, elevated levels of liver enzymes, agitation, anxiety, depression, sleep disturbance, and eosinophilic granulomatosis with polyangiitis (EGPA), also known as Churg–Strauss syndrome . The preMany studies supporting an association between leukotriene-modifying agents including montelukast and suicidality are primarily based on reviews of individual safety reports in AE databases which are subject to reporting bias and confounding factors. On the contrary, case-control and cohort studies, and clinical trials do not support an association between the two. Ecological studies have demonstrated a lack of positive association between leukotriene-modifying agents and suicidality at the population level . Althougin vitro and in vivo experiments because of our currently limited knowledge about disease-associated proteins and their interactions. Therefore, the association between montelukast and genes should be confirmed experimentally. Furthermore, we identified a list of 26 proteins interacting with montelukast. However, not all genes from databases like DGIdb are direct pharmacological targets; many may be indirectly affected by drugs. Our analysis uses a protein–protein interaction network to map these 26 affected proteins to larger networks and demonstrate that the networks are enriched in genes pertaining to mood disorders. We seized on the genes HCRT, KALRN, and HTR2A to substantiate the connection to mood disorders. As these genes are not direct targets of montelukast, this approach must be validated by showing that a given “hotspot” in such a protein–protein interaction network distinguishes drugs that cause “mood disorders” from drugs that do not. The modular assembly of drug safety subnetworks (MADSS) algorithm may be suitable for solving this problem (Some limitations of our functional enrichment analysis should also be noted. Our results do not offer any hard evidence regarding the potential mechanisms of montelukast-associated neuropsychiatric AEs. For now, the drug–gene interactions investigated have not been validated in any experimental model or  problem .Our retrospective analysis demonstrated a significant association between montelukast and neuropsychiatric AEs. The genes that were thought to be associated with neuropsychiatric symptoms due to their interaction with montelukast were found to be significantly enriched in functional categories of psychiatric disease, which necessitates future pharmacological research.


Introduction: To examine English language YouTube videos that covered both COVID-19 and peripheral artery disease (PAD).Methods: The research was planned from October 1 to 5, 2020. Two cardiologists (CB and ES) executed online searches in which the term COVID-19/coronavirus was paired with common keywords about PAD, including ‘peripheral artery disease + COVID-19,’ ‘leg pain + coronavirus,’ ‘leg vascular disease + COVID-19,’ ‘atherosclerosis + COVID-19,’ and ‘claudication + coronavirus.’ For each video, a record was made of the number of days on YouTube, length, number of views and comments, and the number of ‘likes’ and ‘dislikes’. Videos were also categorized according to content as informative videos , patient experience videos (with patient testimonies), or news update videos . Moreover, DISCERN and Medical Information and Content Index (MICI) were evaluated.Results: Totally, 91 YouTube videos met study inclusion criteria. News update videos were the most-watched when compared with informative and patient experience videos . The DISCERN score was significantly higher in the informative group: 2.8 for informative videos, 1.7 for patients' experience videos, and 1.8 for news update videos (p= 0.001). The most common theme was clinical symptoms in the informative videos (82.4%). The mean MICI score was calculated as 3.7±1.4 points for informative videos.Conclusion: YouTube videos about COVID-19 and PAD are widely-viewed information sources for patients. Our study has demonstrated that YouTube videos about COVID-19 and PAD generally had poor quality content. The newrecorded . As a reDue to the ease of transmission of COVID-19, people all over the world saw public transportation limited, quarantine rules applied, and outpatient polyclinic appointments postponed. As a result, patients and their families increasingly turned to social media for medical information . In thisYouTube has no process for evaluating video quality, resulting in a wide variation of accuracy and utility. In the present study, our purpose was to examine English language YouTube videos that covered both COVID-19 and peripheral artery disease (PAD).The research was planned from October 1 to 5, 2020. Approval from the Institutional Ethics Committee was not necessary because no patient data were used. Two cardiologists (CB and ES) executed online searches in which the term COVID-19/coronavirus was paired with common keywords about PAD, including ‘peripheral artery disease + COVID-19,’ ‘leg pain + coronavirus,’ ‘leg vascular disease + COVID-19,’ ‘atherosclerosis + COVID-19,’ and ‘claudication + coronavirus.’YouTube statics show that the duration of an uploaded video is a critical factor in the video’s popularity; and previous reports claim that, in terms of popularity, the optimum length of a video is between 2 and 15 minutes . Thus, oFor each video, a record was made of its number of days on YouTube, length, number of views, comments, and number of likes and dislikes. All videos were classified as either originating from healthcare providers, nonprofessional individuals, or news agencies. Additionally, videos were classified according to whether the target audience was healthcare workers or patients. Videos were also categorized according to content as informative videos , patient experience videos (with patient testimonies), or news update videos .To analyze the contents of the videos, two cardiologists completed DISCERN and Medical Information and Content Index (MICI) questionnaires. The DISCERN score provides an objective evaluation about video content quality and was calculated using a questionnaire with five yes/no questions. ‘Yes’ reflected a positive aspect and scored one point, while ‘No’ scored zero; thus, DISCERN scored videos in a range from zero to five . MICI waFor the statistical analysis, the Statistical Package for the Social Sciences version 20  program was used. An independent samples t-test was used to compare independent groups and χ2 and Fisher exact tests were used to compare categorical data. A post hoc test was used to compute pairwise comparisons. Inter-rater compatibility was determined by Cohen kappa score. Quantitative data were represented as mean ± standard deviation. Categorical data were noted as frequency (n) and percentages (%). The data were analyzed at a 95% confidence level and considered statistically significant if the P-value was less than 0.05.In total, 91 YouTube videos met our inclusion criteria, while 47 videos were excluded due to inappropriate duration (32), presentation in a language other than English (9), and irrelevant content (6). Regarding the information source, 40 videos were categorized as informative, 29 videos as patient experiences, and 22 videos as news updates. The study's flow chart is found in Figure News update videos were the most-watched when compared with informative and patient experience videos . Similarly, the mean duration of videos was significantly longer in the news update group (p=0.040). In contrast, video length, views per day, likes, dislikes, and comments for each video were comparable between groups . The main source of informative videos was professional healthcare providers (47.5%). All three of the video groups overwhelmingly targeted patients: 90.0% of informative videos, 86.2% of patient experience videos, and 90.9% of news update videos (p=0.838). The DISCERN score was significantly higher in the informative group: 2.8 for informative videos, 1.7 for patient experience videos, and 1.8 for news update videos . Additionally, the number of news update video views was significantly higher when compared to other groups . The duration was significantly longer for the news update group when compared to the patient experience group (p=0.033), but not significantly different when compared to the informative video group . A total of 43 videos (47.2%) and 45 videos (49.4%) included knowledge about disease prevalence and disease transmission, respectively. Additionally, the issues of screening tests and treatment of disease were mentioned in 53 (58.2%) and 54 videos (59.3%), respectively. The mean MICI score was calculated as 3.7±1.4 points for informative videos  are active internet users, and reports revealed that 95% of them watch YouTube videos . At a tiThe DISCERN score was developed to measure the quality of written health information and has been validated in previous studies. Ferhatoglu et al. used DISCERN scores to analyze YouTube videos about obesity surgery, concluding that videos produced by professional healthcare providers had significantly better DISCERN scores than those produced by non-professionals . In anotNagpal et al. used MICI scores to analyze YouTube videos during the Ebola pandemic . Dutta eThe number of views for YouTube videos indicates the approximate number of people reached. In the study by Yuksel and Cakmak on COVID-19 and pregnancy, news update videos had significantly more views per day in comparison to patient experience videos, but not compared to informative videos . The preThere are some limitations of this study that is the first to analyze YouTube videos about COVID-19 and PAD. First, although we evaluated YouTube videos for the whole six-month period beginning with the first COVID-19 cases in early March, it is difficult to take into account the ongoing rapid changes in knowledge. Second, we analyzed YouTube videos in English only, and we acknowledge that video-sharing around the world is occurring in many different languages. Cross-language comparisons of the quality of YouTube videos about COVID-19 and PAD may be the subject of future study. Finally, we identified the five most common keywords to search for videos about COVID-19 and PAD; however, the inclusion of more terms related to COVID-19 and PAD may increase the number of videos.In conclusion, YouTube videos about COVID-19 and PAD are widely-viewed information sources for patients. Our study has demonstrated that YouTube videos about COVID-19 and PAD generally have poor quality content but, with improvement, YouTube videos could potentially become effective and accessible sources of information about COVID-19 and PAD.


Multivariately, HIV-infection was not associated with Agatston-scores , the number of coronary plaques  or SSS . ASCVD risk scores and years of cocaine-use significantly increased all CAD outcomes among HIV-infected individuals, but not among HIV-uninfected. Importantly, none of the HIV-medications were associated with any of the CAD outcomes. HIV-infection is not directly associated with CAD and therefore HIV-infected are not destined to have worse CAD profiles. However, HIV-infection may indirectly promote CAD progression as risk factors may have a more prominent role in the acceleration of CAD in these patients.Our objective was to assess whether human immunodeficiency virus (HIV)-infection directly or indirectly promotes the progression of clinical characteristics of coronary artery disease (CAD). 300 African Americans with asymptomatic CAD  who underwent coronary CT angiography at two time points (mean follow-up: 4.0 ± 2.3 years) were randomly selected from 1429 participants of a prospective epidemiological study between May 2004 and August 2015. We calculated Agatston-scores, number of coronary plaques and segment stenosis score (SSS). Linear mixed models were used to assess the effects of HIV-infection, atherosclerotic cardiovascular disease (ASCVD) risk, years of cocaine use on CAD. There was no significant difference in annual progression rates between HIV-infected and—uninfected regarding Agatston-scores (10.8 ± 25.1/year vs. 7.2 ± 17.8/year,  While data on hard end-points  show clear associations with HIV-infection, information regarding sub-clinical stages of CAD are contradictory6. It is unclear whether discrepancies of previous results are due to the cross-sectional study design, or whether there may be other factors which promote CAD in PLWH, which were less investigated, such as illegal substance use. However, the chronic effects of substance use (such as cocaine) on CAD also prove to be controversial with results showing clear associations7, while other investigations failed to identify any connection9. As nearly half of all PLWH report substance use beyond marihuana11, it is of utmost importance to investigate how conventional (cardiovascular) and unconventional (HIV-infection and substance abuse) risk factors accelerate subclinical CAD in PLWH.Antiretroviral therapy (ART) has transformed the disease course of human immunodeficiency virus (HIV)-infection. While manifestations of acquired immunodeficiency syndrome are now scarce, people living with HIV (PLWH) still have shorter life expectancies as compared to the general population due to the increased risk of noncommunicable diseases such as coronary artery disease (CAD)12. Among reasons for the high prevalence of HIV-infection among African Americans is drug abuse, the second leading cause of HIV-infection in both African Americans men and women12. Thus, it is crutial to understand the interplay of risk factors and CAD in this patient population.Although African Americans constitute only about 13% of the U.S. population, 43% of the estimated new cases of HIV in 2016 were in African Americans13. However, Agatston-score does not detect noncalcified plaques which are strong predictors of later outcomes14. Furthermore, it is not only the number of plaques, but the extent of CAD and the degree of stenosis, which are important and which can be summarized by the segment stenosis score (SSS)15.Coronary CT angiography (CTA) provides a non-invasive method to assess CAD. Coronary atherosclerosis may be characterized by the Agatston-score, an independent predictor of future major adverse cardiac eventsTherefore in our prospective longitudinal observational study, we wished to assess whether HIV-infection had an independent effect on CAD or whether it may facilitate atherosclerosis through promoting the effects of risk factors in African Americans. Furthermore, we wished to assess the potential adverse effects of illicit drugs in this patient population.Detailed study methodologies can be found in supplementary material.7. Inclusion criteria were (1) age ≥ 21 years and (2) self‐designated African American race. Exclusion criteria were: (1) history of serious physical disease or current physical disease which may preclude the follow-up of patients or hinder the image quality of coronary CT angiography, including chronic obstructive pulmonary disease; (2) pregnancy; (3) chronic kidney disease with an estimated glomerular filtration rate of < 60 mL/minute per 1.73 m2; and (4) contraindication to CT angiography scans, including a history of contrast allergy.Between May 2004 and August 2015, 1429 cardiovascularily asymptomatic individuals were prospectively enrolled in an epidemiological observational study investigating the effects of illicit drugs, HIV, ART and inflammation on CAD5. In the current manuscript we aimed to assess the longitudinal effect of illicit drugs on coronary Agatston-score, number of coronary plaques present and SSS. With the intention of later volumetric analysis we deemed the analysis of 300 individuals’ 2 CT scans feasible and comparable to other CAD progression analyses in different patient populations16. Therefore, we randomly selected 300 individuals showing at least > 10% stenosis  on the baseline coronary CTA17.Previously, in this cohort the associations between HIV-infection and clinical characteristics of CAD (presence of CAD and presence of different plaque types) were investigated based-on cross-sectional data5. Atherosclerotic cardiovascular disease (ASCVD) risk scores were calculated as a measure of overall cardiovascular risk based on the ACC/AHA Guidelines at the time of the CTA scans18. All participants were tested for HIV-infection by ELISA and if positive, then confirmed by Western blot test. Furthermore, at the time of the CTA examinations high‐sensitivity C‐reactive protein (hsCRP) was also measured. For HIV‐infected participants, detailed information regarding duration of known HIV-infection, and medications including: nucleoside reverse-transcriptase inhibitor use (NRTI), non-nucleoside reverse-transcriptase inhibitor use (NNRTI) and protease inhibitor use (PI) were collected. A medical chart review was used to confirm the information on the medical history and medications provided by the study participants.Briefly, all participants self-reported years of cocaine use defined as use by any route for at least 6 months, administered at least 4 times a month7. Briefly, coronary Agatston-score was calculated using the Agatston-score method19. Coronary plaque was defined as any discernible structure that could be assigned to the coronary artery wall and could be identified in at least 2 independent planes and caused at least a 10% reduction in lumen caliber17. Overall, the total number of plaques present in the coronaries was counted. Furthermore, the degree of stenosis present in each of 17 coronary segments was classified as: (0) none (0%); (1) minimal (10–24%); (2) mild (25–49%); (3) moderate (50–69%), (4) severe (70–99%) or (5) occluded (100%) and used to quantify the SSS by summing the stenosis score of each segment per patient20. All images were randomly interpreted by a level-3 certified specialist in cardiac CT imaging with 5 years of experience and blinded to all clinical data.Detailed scan protocols have been published22. We calculated univariate models to assess the effect of each predictor. If it was associated with the overall amount or the progression rate of the given outcome at p < 0.10, then we included that predictor into a multivariate model. We always included HIV in the multivariate models as we wished to assess its potential modifying effect. We further did sub-analyses stratifying our data based-on HIV-serostatus to assess whether HIV modifies the effect of cocaine use. For HIV positive cohorts, we also included HIV-associated medications in the analysis. We also stratified our patient population to cocaine-users: individuals who used cocaine at either time point; and cocaine non-users: participants who did not use cocaine at either time point to remove the potential effects of cocaine use on outcomes. All analyses were conducted in the R environment v3.6.123. A 2-sided p value smaller than 0.05 was considered statistically significant.In brief, we used linear mixed models which allow the analysis of repeated observations at non-standardized intervals. It also allows to assess whether a risk factors affects the overall amount of an outcome (i.e. Agatston-score) and/or if it changes the annual progression rate of the outcome (i.e. yearly progression of Agatston-score)The Institutional Review Board at the Johns Hopkins School of Medicine  approved the study protocol. All procedures used in this study were in accordance with HIPAA, local and federal regulations, and the Declaration of Helsinki.All study participants provided written informed consent.3 and 2.4 ± 1.1 copies/mL, respectively. During the follow-up period, none of the HIV-uninfected participants acquired HIV. The reported cocaine use increased from 161 to 174 over the follow-up period (p = 0.29). The average duration of cocaine use among cocaine users increased from 13.7 ± 8.7 years at baseline to 14.9 ± 8.6 years at the end of follow-up (p < 0.0001). The mean ASCVD risk score increased from 7.6% ± 7.2% at baseline to 10.2% ± 7.8% (p < 0.0001) between the two CT scans (4.0 ± 2.3 years).A total of 300 study participants were included in this investigation. The demographic and clinical characteristics of all participants and HIV‐related clinical factors of HIV‐infected participants are presented in Table p = 0.04). The mean Agatston-score changed from: 51.2 ± 174.6 to 93.3 ± 256.8 over the follow-up period (p < 0.0001). At baseline there were 621 plaques, on average 2.1 ± 2.0 per patient, were visible, while at follow-up 861 plaques, on average 2.9 ± 2.1, were identified (p < 0.0001). Overall, the mean annual change in Agatston-score was 9.9 ± 23.6 units. On average, 0.26 ± 0.34 plaques developed each year and SSS increased by 0.54 ± 0.97 annually.At baseline 118 (39.3%), while at follow-up 144 (48.0%) participants had Agatston-scores greater than zero . However, each year of cocaine use increased Agatston-scores by 3.36 units , the number of coronary plaques by 0.03  and SSS by 0.05 . Furthermore, each percentage increase in ASCVD risk score increased the annual progression rate of Agatston-score by 0.88 units/year , and the overall number of coronary plaques by 0.03  and SSS by 0.06 . Also, on average statin users had more disease regarding all three CAD markers as compared to individuals who did not take statins. Detailed univariate and multivariate results are presented in Table In multivariate analysis, HIV-infection was not associated with either CAD parameter , the number of coronary plaques by 0.03  and SSS by 0.07 . Each percentage increase in ASCVD risk scores increased the annual progression rate of Agatston-scores by 0.87 units/year , the overall number of coronary plaques by 0.02  and SSS by 0.05 . Importantly, none of the HIV associated medications had effect on any of the CAD parameters. Detailed results are presented in Table .Among PLWH, in multivariate models, each year of cocaine use increased Agatston-scores by 4.05 units , but not any of the other CAD markers. Interestingly, hsCRP had a more prominent effect among HIV-uninfected, by increasing the number of coronary plaques by 0.05  and SSS by 0.14 . Detailed results are presented in Table However, among HIV-uninfected, cocaine use had no effect on any of the CAD markers Table . Neverthp = 0.017; 0.64 ± 1.08/year vs. 0.39 ± 0.76/year, p = 0.017, respectively), with borderline differences regarding the annual progression are of the number of plaques .Cocaine users tended to have worse CAD parameters as compared to non-users . However, each percentage increase in ASCVD risk scores increased the number of coronary plaques by 0.06  and SSS by 0.12 . Detailed results can be found in supplemental Table Multivariate analysis indicated, that among cocaine users, HIV-infection had no effect on any of the CAD parameters . However, each percentage increase in ASCVD risk scores increased Agatston-scores by 1.21 units , the number of coronary plaques by 0.02  and the annual progression rate of Agatston-scores by 1.07 units/year  and SSS by 0.01/year . Detailed results are in supplemental Table Among the 126 42.0%) of cocaine non-users, in multivariate models, HIV-infection had no effect on any CAD marker (2.0% of cThe findings of this investigation show that (1) overall, HIV-infection was not independently associated with changes in any CAD markers among the total study population, and (2) duration of cocaine use was significantly associated with increased coronary Agatston-scores, number of coronary plaques and SSS. However, these associations were more apparent in PLWH as compared to uninfected individuals. These findings imply that HIV-infection may indirectly promote CAD progression as other risk factors, such as duration of cocaine use may have a more prominent role in the acceleration of CAD in PLWH.2, it is of utmost importance to better understand the possible causes of decreased life expectancy. Furthermore, as African Americans in the United States have been disproportionally affected by HIV-infection and drug abuse, it is crucial to understand whether and how HIV-infection exacerbates cardiovascular comorbidity among this population. Reports of the possible associations of HIV with CAD are inconsistent. While the Multicenter AIDS Cohort Study reported a significant association between HIV and an increased prevalence of noncalcified coronary plaque4, both a Baltimore prospective heart study and a Swedish prospective cohort study failed to identify a significant associated between HIV-infection and the presence of any plaque and noncalcified or mixed plaques6. Interestingly, the Swedish prospective cohort study demonstrated that HIV-infection was negatively associated with the total number of plaques and SSS6.As life expectancy of PLWH irrespective of ART therapy is still shorter than that of the general population24. CAD is a dynamic process where noncalcified plaques progress over time into calcified plaques25. Cross-sectional studies only provide information regarding associations at a specific point in time and do not provide information on how risk factors may truly affect the course of a disease. Also, as PLWH are at an increased risk of using illegal substances11, the possible detrimental effects of drugs, such as cocaine use, potentially have a more prominent impact on the lives of these individuals. Lai et al. has found that in PLWH long-term cocaine use (defined as use for over 15 years) significantly increased the odds of significant stenosis7. Lai and colleges also found that chronic cocaine users had an increased prevalence ratio to have any coronary stenosis or Agatston-score when corrected for multiple risk factors5. However, among acute coronary syndrome patients, Bamberg et al. found no association between plaque markers and cocaine use. However, they were at increased risk of acute coronary syndrome as compared to matched individuals26. Similar results were found by Chang and colleges among acute coronary syndrome patients, where cocaine-use was not associated with coronary stenosis or calcifications9.A possible reason for the conflicting results may be the fact that cross-sectional analyses are inherently limited in nature. There is a scarcity in longitudinal analyses on the effects of HIV-infection on CAD. Tarr et al. found similar results are our current investigation where HIV-infection was not associated with progression of CADAs compared to previous investigations our longitudinal assessment allows a unique opportunity to base our inferences on longitudinal changes in risk factors and CAD outcomes, rather than cross-sectional analyses. It seems that there is a linear relationship between the years of cocaine use and Agatston-score, the number of coronary plaques and also SSS. However this relationship is only apparent in PLWH. Our results may provide an explanation to why PLWH have accelerated CAD progression and why previous investigations are contradictory. It seems that while HIV-infection does not have a direct adverse effect on Agatston-score, the number of plaques or SSS, it may amplify the effects illicit drugs, such as cocaine. Therefore, cocaine use may have a prominent role in the development and acceleration of CAD interpedently from other risk factors in this patient population. While we have effective therapies for cardiovascular risk such as statins, the only way to overcome the negative effects of cocaine use is through abstinence. As PLWH report higher rates of substance use, it is of utmost importance to promote and achieve abstinence from illicit drugs.Our study has several limitations. First, all our study subjects were African Americans. While racial/ethnic homogeneity could be considered a strength, the findings derived from this study population might not be generalizable to other racial and ethnic groups without caution. Furthermore, due to the longitudinal nature of our analysis, we did not assess the effect of risk factors on noncalcified, mixed or calcified plaque proportions. Since CAD is a dynamic process where noncalcified plaque progress into calcified plaques, accurate inferences regarding longitudinal compositional change can only be done using volumetric analysis. Also, only PLWH took statins, therefore we were not able to assess the effects of statin in uninfected individuals. Furthermore, our investigation only looked at cocaine use among potential illicit drugs. As illegal substance use is more common in this population, other illicit drugs may also have potentially affected our observed results.In conclusion, PLWH are not destined to have worse CAD, as HIV-infection is not independently associated with any marker of CAD. However, cardiovascular risk factors and cocaine use may have a more profound effect on CAD progression in those with HIV-infection. Therefore, more aggressive management of cardiovascular risk factors and illicit drug use is needed. Furthermore, more strict normal values and earlier initiation of preventive therapies might be needed in these patients to achieve similar outcomes.Supplementary Information.


Manifestations of hypokalaemia in ectopic adrenocorticotropic hormonesyndrome(EAS) vary from mild muscle weakness to life-threatening arrhythmia. Herein, we present a rare case of EAS with concomitant rhabdomyolysis(RM) as a result of intractable hypokalaemia.68Ga-DOTA-NOC. Mediastinoscopic lymph node biopsy was performed. The pathological diagnosis was small-cell and large-cell neuroendocrine carcinoma with positive ACTH staining. The patient was prescribed mifepristone and received one cycle of chemotherapy. The patient could not tolerate subsequent chemotherapy and died of dyscrasia.A 64-year-old man was admitted for limb weakness and facial hyperpigmentation for 2 weeks. Lab tests revealed intractable hypokalaemia (lowest at 1.8 mmol/L) and metabolic alkalosis. The diagnosis of RM was based on a creatine kinase(CK)level of 5 times the upper limit. The elevated CK and myohemoglobin (Mb) levels returned to within the normal range after the alleviation of hypokalaemia. The patient was diagnosed with ACTH-dependent Cushing’s syndrome (CS) based on unsuppressed serum cortisol after a low-dose dexamethasone suppression test(LDDST) and remarkably elevated ACTH levels. The diagnosis of EAS was made based on the results of a high-dose dexamethasone suppression test(HDDST) and bilateral inferior petrosal sinus sampling(BIPSS). Multiple lymph nodes in the left supraclavicular fossa, right root of neck, mediastinum and bilateral hili of the lung were found with abnormal uptake of RM is a rare complication of EAS with insidious onset and atypical clinical manifestations. Serum potassium levels should be vigilantly monitored to avoid RM in EAS. Cushing’s syndrome (CS) is a set of clinical syndromes caused by excessive production of cortisol. Ectopic adrenocorticotropic hormone syndrome (EAS) accounts for approximately 12-17 % of CS cases in which ACTH is produced by tumours [A 64-year-old man was admitted to a local hospital for limb weakness and facial hyperpigmentation for 2 weeks. He also reported oedema of the lower limbs for 1 week. He had a history of smoking for 40 years(30 cigarettes per day). Lab tests revealed severe hypokalaemia  and metabolic alkalosis. Potassium supplementation  failed to normalize the serum potassium (peaked at 2.3 mmol/L). As the level of serum creatine kinase (CK) was remarkably elevated, the patient was transferred to our hospital. Upon admission, mild moon face and facial hyperpigmentation was noticed when compared with photos taken 2 years before. Oedema of the lower extremities and decreased muscle strength were also found during physical examination. Hyperpigmentation at interphalangeal joints and ecchymosis gradually developed during hospitalization.left/PRLperipheral and PRLright/PRLperipheral were 4.3 ± 0.5 and 2.5 ± 0.3, respectively). The left, right and peripheral ACTH levels were 789.9 ± 5.2 pg/mL, 773.6 ± 18.2 pg/mL and 726.5 ± 26.7 pg/mL (three samples for each), respectively, and the left, right and peripheral ACTH/PRL levels were 12.4 ± 1.4, 21.4 ± 2.4 and 49.1 ± 2.2, respectively. The diagnosis of EAS was made based on the HDDST and BIPSS results. Chest and abdominal CT as well as neck ultrasound were further arranged. Abdominal CT showed diffuse enlargement of the bilateral adrenal glands  level was 7.4 mmol/L, and the glycosylated haemoglobinHbA1c) level was 5.7 % on admission. During hospitalization, the FPG level gradually increased. Diagnosis of diabetes was made, and insulin therapy was initiated. Bone mineral density indicated low bone mass. Blood pressure, lipid profile, and coagulation function were generally normal.No thrombus was found by colour Doppler ultrasound in the arteries or veins of the lower extremities. On admission, remarkably elevated levels of serum CK and myohemoglobin (Mb)were noticed with slightly increased creatine kinase isoenzyme(CKMB) , the urologists and anaesthetists considered it very risky for the patient to undergo bilateral adrenalectomybased on hypokalaemia, infection and general status. Additionally, aggravation of the condition was more related to malignancy than hypercortisolaemia, especially after the application of mifepristone. As the priority was determined to be to treat neuroendocrine carcinoma, which was unresectable, the patient was transferred to the oncology department to receive chemotherapy. In addition, mifepristone was applied at an initiating dosage of 75 mg per day and a maintaining dosage of 150 mg per day. The patient received one cycle of chemotherapy with the EP regimen (etoposide 100 mg iv drip days 1–3 and cisplatin days 1–3) but could not tolerate subsequent chemotherapy. He died of dyscrasia within 1 month.68Ga PET/CT is effective in locating pulmonary neuroendocrine tumours, while 18-F FDP is suitable for malignancies known or speculated to be more aggressive. For 68Ga and 18-F FDP PET/CT-negative cases, somatostatin imaging can be adopted, but persistent hypercorticosteraemia may suppress the expression of the somatostatin subtype 2 receptor and cause false-negative results in some cases [We reported a representative case of EAS caused by a very aggressive malignancy, with atypical manifestation of CS and no opportunity for radical resection at diagnosis. As a rare cause of CS, EAS has always been a clinical challenge. First, EAS often has an insidious onset. Typical signs of CS, such as moon face and central obesity, are often absent from the influence of cachexia, leading to delayed diagnosis. Second, some tumours are highly malignant and progress rapidly, and diagnosis and treatment are urgently needed. The diagnosis from ACTH-dependent CS to the specific causes was roughly divided into two steps. The first step is to distinguish whether the lesion is located in the centre  or the periphery. The amplitude of ACTH and the HDDST, pituitary MR, chest and abdomen CT results can be suggestive. However, as the incidence of incidental pituitary adenomas is as high as 10 %, identification of a pituitary adenoma does not always guarantee the identification of the responsible lesion. Moreover, the ACTH levels and HDDST results of some peripheral carcinoid and pituitary adenomas overlap. Some tumour biomarkers are not specific and increase in chronic inflammation. With the combination of HDDST and BIPSS, 95 % of the cases can be distinguished , 4, 5. HThe ultimate cure for patients with EAS is radical resection of the malignancy, which was not feasible for our patient who had multiple metastases. LCNEC and small-cell carcinoma are both high-grade neuroendocrine carcinomas. Early surgical resection is the first-line therapy for LCNEC, and the 5-year and 10-year survival rates are approximately 30 and 10 %, respectively. Most patients with small-cell carcinoma require chemotherapy or a combination of chemotherapy and radiotherapy, and the 5-year and 10-year survival rates are approximately 10 % and less than 5 %, respectively . For EAS+, we considered hypokalaemia instead of hypophosphatemia to result in RM.In patients with CS, extra cortisol binds to mineralocorticoid receptors and leads to hypokalaemia. Some medications, such as diuretics and mifepristone, contribute to aggravation. Hypokalaemia in patients with EAS or adrenocortical carcinoma(ACC) is often intractable due to the extremely high cortisol level. K+ regulates blood flow in muscle tissue by dilating vessels. As skeletal muscles consume large amounts of oxygen, hypokalaemia impairs blood supply and causes ischaemic damage. Hypokalaemia also increases the permeability of the myolemma by disturbing transmembrane ion transport [+level is below 3.0 mmol/L, elevation of myoenzymes may occur when the K+level is below 2.5 mmol/L, and RM might occur when the K+level is below 2.0 mmol/L [This patient had RM caused by extreme hypokalaemia, a rare complication of EAS. There are diverse causes of rhabdomyolysis. Patients with CS are susceptible to infection due to immunodeficiency caused by hypercortisolaemia, which is a common aetiology for RM. Disturbances of electrolytes such as hyponatraemia, hypokalaemia and hypophosphataemia should also be considered in the aetiological diagnosis of RM . For theransport . The sev0 mmol/L , 12.An interesting issue is that RM is reported far more frequently in primary aldosteronism (PA), although PA and CS are both related to severe hypokalaemia. One explanation is the very low incidence of EAS and ACC. Another possible mechanism is that hypokalaemia-induced RM is caused by the imbalance of oxygen supply and consumption. Patients with EAS and ACC usually have significant protein catabolism and muscle loss due to extreme hypercorticosteraemia. Many of them also have restricted physical activities due to pathological fracture or cachexia. As oxygen consumption is determined by muscle content and activity status (resting or exercising), patients with CS have a lower oxygen requirement. In contrast, muscle content and physical activity are generally normal in patients with PA, making them more vulnerable to impaired blood and oxygen supply caused by hypokalaemia.Clinical manifestations of RM vary greatly. The typical triad of myasthenia, myalgia and coloured urine occurs in less than 10 % of patients, while more than 50 % of patients have no symptoms or signs –15. EndoIn summary, RM is a rare complication of EAS with atypical clinical manifestations. Serum potassium levels should be vigilantly monitored to avoid RM in EAS.


Nocebo effect is prevalent among neurological diseases, resulting in low adherence and treatment outcome. We sought to examine the nocebo effect in randomized controlled trials (RCTs) in multiple system atrophy (MSA).Q and I2.We searched RCTs in MSA from Medline since September, 2021. RCTs for drug treatment conducted in adult MSA patients with more than 5 cases in each treatment arm were included. We assessed the number of dropout due to placebo intolerance. We also did a symptomatic/disease-modifying subgroup analysis based on two different treatment purposes. The STATA software was used for statistical analysis. Overall heterogeneity was assessed using the Cochran Data were extracted from 11 RCTs fulfilling our search criteria. Of 540 placebo-treated patients, 64.2% reported at least one adverse event (AE) and 7.5% reported dropout because of AEs. The chance of dropping out because of an AE and experiencing at least one AE did not differ between placebo and active drug treatment arms. Besides, the pooled nocebo dropout rate in the symptomatic subgroup was similar to that of the disease-modifying subgroup.In MSA RCTs, nocebo dropout rate was not at a low level among neurological disorders. Nocebo effect was an important reason of dropout because of AE in placebo and active drug treatment arms. Different treatment purposes may not influence nocebo effect. The psychosocial context can play an essential role in most medical treatments. Positive physiological or psychological effects induced by the treatment context are referred to as well- known placebo effects. Placebo effect has impact on therapeutic response, adherence, and quality of life , 2. On tNocebo effect is prevalent among neurological diseases and a variability in the magnitude of nocebo effect has been demonstrated in various neurological disorders . To dateThis systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .A computer-based PubMed literature search was conducted on 28th Sep, 2021. We used two Medical Subject Headings (MeSH) terms. Term A was “placebo” and Term B was “multiple system atrophy” or “MSA.” Limitations were set to specify language to be English.We included the studies if they met the following criteria: (1) referred specifically to MSA; (2) referred specifically to humans; (3) RCTs; (4) pharmaceutical studies; (5) there was a placebo arm; (6) each treatment arm had at least 5 patients; (7) adult participants; and (8) detailed data of drop-outs in each treatment arm were available in the report; (9) excluding reviews, letters without original data, editorials; (10) when there was more than one publication from the same studied population, only data from the most comprehensive report was included in our analysis and the remaining were excluded.Two reviewers independently performed the search, reviewing all articles and collecting data. Items were extracted from each study and the extracted information included: information on the article identification; year of publication; evaluation period; total number of subjects; number of placebo-treated subjects; total number of placebo deaths; number of placebo-treated subjects who dropped out because of AEs; number of placebo-treated subjects who dropped out for other reasons; number of male subjects treated with placebo; mean age of placebo-treated subjects; number of active drug-treated subjects; total number of active drug deaths; number of active drug-treated subjects who dropped out because of AEs; number of active drug-treated subjects who dropped out for other reasons; number of male subjects treated with active drug; mean age of active drug-treated subjects; active drug; disease-modifying drug or symptomatic drug; route of drug administration and geographic location. The authors independently evaluated each of the included studies according to modified JADAD scale . RCT witQ test  and I2 value . The fixed-effect model was applied if there was no or low heterogeneity. Data were analyzed using a random effects model if there was high heterogeneity. Pooled ORs for comparison between the two arms were estimated as illustrated in the forest plots. The Egger’s test was used to assess the presence of asymmetry in the funnel plots .The meta-analysis was conducted using STATA 15.0 (Copyright 1985–2017 Stata Corp LP). We calculated the pooled dropout rates because of any AEs for each group and its 95% confidence intervals, as well as the pooled AE rates for each group and its 95% confidence intervals. We choose odds ratios (ORs) and 95% confidence intervals (CIs) as the appropriate parameters to evaluate the dichotomous outcomes, such as the dropout rate. Overall heterogeneity was assessed using the Cochran The process of the article selection is presented in Fig. CI = 3.0–13.7%) and 11.8% (95% CI = 5.1–20.8%) respectively. One study [OR = 0.626, 95% CI = 0.389–1.007, p = 0.053). There was no heterogeneity among the included studies . As shown in the funnel plots , there was no publication bias among the included studies . Besides, 17 deaths occurred in placebo-treated patients of 7 studies and 22 deaths occurred in active drug-treated patients of 8 studies (Table The pooled estimate of dropout rate because of AEs other than death in placebo-treated patients and active drug-treated patients was 7.5% (95% ne study  reportedCI = 43.2–82.6%), compared to 72.4% (95% CI = 53.4–88.0%) for active drug treated patients. As illustrated in the forest plot . There was no heterogeneity among the studies included in the meta-analysis . As shown in the funnel plots .Only eight of the studies , 14–19 rlot Fig. , the chaots Fig. , there wOR = 0.484, 95% CI = 0.173–1.358, p = 0.168; disease-modifying subgroup: OR = 0.672, 95% CI = 0.392–1.149, p = 0.146). There was also no heterogeneity among the studies included in two subgroups . Moreover, the pooled nocebo dropout rate in the symptomatic subgroup  was similar to that of the disease-modifying subgroup .The MSA patients included in 11 studies were classified into two subgroups according to different purposes of experimental treatment. Three of the studies were symptomatic and eight of the studies were disease-modifying  and chronic inflammatory demyelinating polyneuropathy (CIDP)  [CI = 5.3–8.4%, nocebo AE rate 57.8%, 95% CI = 50.1–66.7%) [CI = 6.8–11.5%, nocebo AE rate 64.7%, 95% CI = 53.6–74.4%) [Nocebo varies significantly among neurological diseases. Weighed against other neurological disorders investigated with identical methodology, comparable to those found in fibromyalgia  , the noc1–66.7%)  and Park6–74.4%) . In term6–74.4%) . MSA is 6–74.4%) . Patient6–74.4%) , 26. MSA6–74.4%) , 28. Thu6–74.4%) .A treatment may be investigated with a potential disease-modifying purpose, others as a symptomatic drug. From the conceptual perspective of the nocebo effect, different treatment purposes may influence patients’ expectation towards the experimental drug and the study participation. We did a further subgroup analysis. Our results showed that in the symptomatic subgroup/disease-modifying subgroup, the chance of dropping out because of AEs other than death did not differ between placebo and active-drug treatment arms. Moreover, the pooled nocebo dropout rate in the symptomatic subgroup was similar to that of the disease-modifying subgroup. Our results indicated that nocebo effect did not appear to be affected by different purposes of experimental treatment in MSA RCTs.Our results should be interpreted with caution due to the limitations of the design. Firstly, the trail dropouts did not accurately reflect the nocebo effects and its severity. Because it is difficult to accurately attribute AEs in the placebo group to drug administration or a consequence of disease worsening or non-specific symptoms, dropouts used as measure of nocebo effect exist deficiency. Secondly, this meta-analysis was performed using only eleven RCTs, which involved pharmacotherapy in MSA and met our stringent inclusion criteria. Some MSA-related RCTs, which involved other diseases such as neurogenic orthostatic hypotension and pure autonomic failure, were not included in our analysis. Therefore, our estimates may not fully reflect the extent of nocebo effect in MSA patients.Clinicians should be aware of the nocebo phenomenon and attempt to reduce nocebo effect in clinical trials and clinical practice . A numbeIn RCTs for MSA, 64.2% of the participants reported AEs related to nocebo and 7.5% of the participants reported dropout related to nocebo. With dropout rate as measure of nocebo effect, the total patients participating in those RCTs reported similar dropout independently of the study arm they belonged, which suggests some AEs arise from the anticipated pharmacological effects of the drug other than active drug itself. Nocebo effect was the important reason of dropout because of AE in the placebo and the active drug groups and is an important factor for treatment adherence in MSA patients during clinical trials and in clinical practice. Furthermore, the nocebo effect does not appear to be affected by different purposes of experimental treatment in MSA RCTs.


Bombus longipennis is the species of Bumblebees (Hymenoptera: Apidae), which are important pollinators for wild plants and greenhouses crops. The complete mitochondrial genome (mitogenome) of B. longipennis was determined by next-generation sequencing. The mitogenome was 18,458 bp in size with 87.2% A + T content, containing 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an AT-rich control region (D-loop). Gene arrangement was found to be identical to those of other mitogenomes of bumblebees (e.g. Bombus terrestris and Bombus ignitus). All 13 PCGs initiated with typical ATN codons. Among them, 11 PCGs terminated with TAA or TAG; only cox2 and nad4 have incomplete stop codon T. All 22 tRNAs can be folded into typical cloverleaf structure. Phylogenetic analysis based on the concatenated nucleotide sequences of all 13 PCGs indicated that B. longipennis was more closely related to other species of subgenus Bombus, which clustered into a monophyletic group. Bombus longipennis Friese, 1918 was the species belonging to the subgenus Bombus in Bombus genus, and widely distributed in North China with elevations of 1303–4011 m  are important pollinators for wild plants and greenhouses crops . Specimens were stored in the Institute of Zoology and Ecology, College of Life Science, Northwest Normal University, Lanzhou, China (accession number: TZ2020016). The genomic DNA was extracted from thoracic muscle of a single specimen, and then was sequenced by Illumina NovaSeq 6000 platform with both directions of 150 bp reads. The MITObim v1.9.1 , indicating significant A + T bias. This mitogenome contained 13 protein-coding genes (PCGs), 22 tRNAs genes, two rRNA genes, and an AT-rich control region (D-loop). The identical pattern of gene arrangement was shared by the mitogenome of B. longipennis with other bumblebee species (e.g. Bombus terrestris and Bombus ignitus)  analysis based on the best-fitting substitution model of TIM + F+I + G4 according to BIC with 1000 ultrafast bootstrap replicates. The concatenated nucleotide sequences of 13 PCGs from 20 Apidae species including geographic populations of Bombus terrestris and Bombus hypocrite were used to construct the phylogenetic tree. Two stingless bees Melipona fasciculata (accession number: MH680930.1) and Melipona scutellaris (accession number: NC_026198.1) were used as outgroup for phylogenetic analyses. The result indicated that B. longipennis was more closely related to other species of subgenus Bombus, which clustered into a monophyletic group (B. longipennis reported in this study will provide more essential molecular data for further phylogenetic studies related to bumblebees and other pollinators.So far, there are 17 bumblebee species, whose mitochondrial genomes have been reported. These species including ic group . The phyic group . The mit