Chitosan belongs to the group of biopolymers with increasing range of potential applications therefore searching for new raw materials as well as new techniques of obtaining of this polysaccharide are currently a subject of interest of many scientists. Presented manuscript describes preparation of chitosan from crickets. Obtainment of final product required a number of processes aimed at removal of undesirable substances such as waxes, mineral salts, proteins or pigments from above-mentioned insects. Chemical structure of fractions obtained after each step was compared with the structure of commercial chitosan by means of techniques such as X-ray diffraction and FT-IR spectroscopy. Final product was subsequently used for preparation of polymer capsules that were modified with active substance characterized by antibacterial and anticancer activity—nisin. Next, sorption capacity of obtained materials was evaluated as well as a release profile of active substance in different environments. Based on the conducted research it can be concluded that crickets constitute an alternative for shellfish and other conventional sources of chitosan. Furthermore, obtained capsules on the basis of such prepared chitosan can be considered as drug delivery systems which efficiency of release of active substance is bigger in alkaline environments. Staphylococcus aureus without negative impact on human dermis cells [Application of materials of natural origin in wide variety of areas is one of the most interesting trends nowadays. Compounds such as polysaccharides, proteins or other bioproducts have become more and more popular. Special emphasis is directed onto chitin and its derivates . One of is cells .Application of chitosan in preparation of systems of drug delivery was also well-documented ,15. FurtGrowing possibilities of medical application of chitosan resulted in increased demand for this polysaccharide. Therefore, new sources as well as techniques of acquiring of this biopolymer are a subject of scientists’ interest ,22. IntePresented publication reports preparation of chitosan from crickets. Multistage chemical processing results in obtainment material that subsequently constitutes a raw material for synthesis of polymer capsules. These can be used as carriers of active substances such as nisin. It is a peptide of natural origin that is characterized by strong antibacterial properties. Additionally, currently scientists conducted studies on its cytotoxic effect on cancer cells ,26,27.Ethyl alcohol , hydrochloric acid , sodium hydroxide , hydrogen peroxide (30%), citric acid (anhydrous) and calcium chloride  were bought in Avantor Performance Materials Poland . Chitosan , phosphate buffer , alginate  and nisin were obtained from Sigma Aldrich S.A. .−1  and X-ray diffraction. Additionally, a weight loss of the processed materials was also investigated when selecting parameters of every stage leading to the preparation of the material with the most similar structure to the structure of commercial chitosan.Chitin is one of the components that builds an external skeleton of many insects such as crickets. However, body of these animals consists of many substances including proteins, waxes and pigments therefore an extraction of chitin-chitosan complex from crickets required a multi-stage chemical process aimed at removing all undesirable substances. Here, such multistep process is presented wherein after each stage a chemical structure of the obtained material was compared to the structure of commercial chitosan using techniques such as Fourier transform infrared (FT-IR) spectroscopy via Nicolet iS5 iD7 ATR Thermo Scientific  wherein the spectra were recorded in the range of 4000–500 cmFirst step of the process involved a removal of waxes by Soxhlet extraction wherein an ethyl alcohol was used as a solvent. Waxes belong to the group of hydrophobic substances and their presence in insects’ bodies can substantially hinder further reactions that are carried out using aqueous solutions. Thus, a fraction of dried crickets was introduced into the thimble and subsequently the Soxhlet extraction was conducted for different amounts of cycles. Next, obtained material was dried at room temperature and subjected to the further stages. Parameters of the process were selected considering weight losses of the processed material.In the second step, fraction of crickets devoid of waxes was treated with hydrochloric acid solution to remove mineral salts. Process was carried out at room temperature with constant stirring. Removal of mineral salts was performed for different periods of time and using HCl with different concentrations. Next, obtained material was washed with distilled water and dried at room temperature. Parameters of the process were selected by defining a weight loss of the processed material as well as by comparison of the structure of obtained fraction with the structure of commercial chitosan. For this purpose, X-ray diffraction and FT-IR spectroscopy were employed. In order to remove protein substances, fractions free of waxes and mineral salts were treated with sodium hydroxide. The process was conducted using 5 M NaOH solution (at elevated temperature and with constant stirring). Reaction was carried out under reflux in different periods of time. Obtained material was washed with distilled water, dried at room temperature and subjected to the XRD and FT-IR techniques.2O2 solution at elevated temperature was employed. Process was carried out for 1 h at elevated temperature wherein obtained material was washed using distilled water and dried at room temperature. Below, in Final step of a chemical treatment of crickets involved a removal of natural pigments such as melanin. For this purpose, a HPrepared chitosan was subsequently used for preparation of polymer capsules designed as drug carriers.The deacetylation degree (DD) of chitosan is one of the most important parameters of this biopolymer which determines its physicochemical and biological properties and thus its application potential. The value of DD corresponds to the molar percentage of monomeric glucosamine units wherein it ranges from 0 to 100 .In order to determine the value of DD, the acid-base potentiometric titration was performed. For this purpose, approx. 0.5 g of chitosan extracted from crickets was dissolved in 0.1 M HCl. Then, the titration using 0.1 M NaOH solution was performed with simultaneous pH measurements made using the pH-meter Elmetron CX-701 . The procedure was conducted three times.161—molar mass of monomeric unit of chitosan which is fully deacetylated, g/mol.The deacetylation degree is subsequently calculated using the following equation:Such an equation allows one to determine the number of moles of amino groups titrated in reference to the total number of moles of monomeric units (both deacetylated and acetylated ones).2 were prepared. Next, alginate solution and chitosan solution were mixed in suitable proportions. Then, nisin solution was added and the whole mixture was dropped slowly to the saturated solution of CaCl2 under continuous mixing conditions. Additionally, capsules based on alginate were also prepared and modified with nisin as reference materials for comparison with alginate/chitosan-based capsules with and without active substance. The procedure of their preparation was the same—the only difference was that the reaction mixture did not contain chitosan solution.Obtained chitosan of crickets’ origin was subsequently used for the preparation of capsules containing active substance. For this purpose, 3% solution of chitosan  in 0.05% acetic acid solution, 0.5% aqueous solution of alginate, 0.1% aqueous solution of nisin and saturated solution of CaClIn Next, prepared capsules were subjected to the investigations aimed at determining their chemical structure, sorption properties and a release profile of nisin from such carriers. A presence of functional groups in the structure of prepared materials was verified using FT-IR spectroscopy. The equipment and applied conditions were the same as in the case of FT-IR analysis of fractions obtained after each step of the chemical treatment of crickets.Sorption capacity of prepared capsules is very important in viewpoint of the potential application of such materials as drug carriers. In order to characterize this property, obtained materials were immersed in distilled water. After these periods of time, swelled capsuled were placed on a moisture analyzer  wherein the drying process was conducted at 37 °C. The percentage loss of mass was determined after 15 and 30 min and corresponded to the amount of absorbed solution.Release profiles of nisin from the interior of chitosan/alginate-based capsules and alginate-based capsules were determined in two different environments. First environment simulated conditions occurring in human stomach—study was conducted in 2% citric acid solution (pH ~ 2); second one resembled pH which is characteristic for duodenum—such conditions were simulated by phosphate buffer (pH = 7.4). Study was carried out as follows: a suitable amount of capsules containing the highest amount of nisin solution (5 mL) were introduced into sterile vessels containing 200 mL of 2% citric acid and PBS solution and placed on a shaking incubator (Hanchen ES-60E Temperature Controlled Incubator and Shaker Scientific Incu-Shaker Shaking Incubator). Vessels were tightly closed with parafilm to limit the possible impact of the external environment. The release process was performed at 37 °C so at the temperature simulating conditions occurring in human body and at a shaking speed of 80 rpm. After every 15 min 3 mL of tested solution were taken, introduced into the cuvette and concentration of active substance in tested sample was determined using UV-Vis spectrophotometry  wherein maximum absorbance characteristic for nisin was verified at approx. λ = 230 nm. Importantly, in each case after sampling the medium, in which the release process was performed, was supplemented with 3 mL of 2% citric acid or PBS solution so that the total volume of the release medium remained constant. To sum up, the study was conducted to verify the drug release ability of both chitosan/alginate-based capsules and alginate-based capsules in various environments simulating in pH and temperature conditions occurring in human body. This, in turn, allowed to determine conditions ensuring the most effective release of the active substance.Examples of weight losses resulting from the process aimed at removing waxes including different amount of cycles are shown in Weight losses calculated after extraction conducted for 4 and 5 cycles were very similar and were within the range 33.2–35.5%. Thus, it can be stated that four extraction cycles are enough for removal of waxes from processed raw material. Importantly, this is also favorable from an economical viewpoint of view and is in line with the principles of Green Chemistry because lower amount of cycles required lower energy demand. These are the reasons due to which 4 extraction cycles were chosen as a process aimed at removal of waxes from the starting material.Results of the second step of processing of crickets including time, concentration of HCl solution applied, amount of extraction cycles as well as a difference between a mass of starting material and a mass of fraction obtained after demineralization are summarized in Based on the obtained results it can be stated that weight losses of processed materials were significantly lower compared to those ones calculated after the removal of waxes. Thus, it may be concluded that crickets contained a relatively lower amount of mineral salts than waxes. Next, it can be reported that the lowest differences between mass of the starting material and the mass of the fraction after demineralization were observed in the case of processes conducted for 30 min using 2 M HCl (sample 2 and sample 6). Additionally, it was observed that the amount of cycles during the extraction had no significant impact on the course of the removal of mineral salts, so four extraction cycles were again considered as sufficient.Nonetheless, an important aspect of performed investigations was an impact of the conditions applied on the structure of final material thus X-ray diffraction and FT-IR spectroscopy were performed to compare the chemical structure of obtained fractions with the structure of commercial polysaccharide. Obtained XRD patterns are presented in Obtained diffraction patterns allowed to conclude enabled the statement that chemical structure of the fractions obtained by means of the above-mentioned parameters were very similar and relatively close to the XRD diffraction pattern of commercial chitosan. On all XRD diffractograms pattern peaks at approx. 2θ = 9° and 2θ = 19° were observed which correspond to the XRD pattern of standard chitin. Importantly, XRD peaks characteristic for chitosan are at approx. 2θ = 10° and 2θ = 20° (JCPDS # 039-1894) ,29.Additionally, a pattern peak at approx. 2θ = 26° (very slight) and 2θ = 31° was observed for sample 2. XRD peaks characteristic for chitin are also at approx. 2θ = 23° and 2θ = 26°  thus it XRD diffractogram of this sample and XRD diffractogram of commercial chitosan were presented separately once again in Next, FT-IR analysis of sample 2 was carried out. Based on the obtained spectra it was possible to determine a presence of characteristic functional groups in the structure of obtained material and compare its spectrum with spectrum of commercial compound. In −1 corresponding to the stretching vibrations of -NH2 and -OH groups can be observed. Moreover, bands at 1650 cm−1 can also be noticed for both materials which corresponds to the stretching vibrations of -CO group in the acetylamide. When comparing the intensity of these two peaks, it can be concluded that the material obtained has more acetylamide groups compared (band with higher intensity) to the commercial chitosan. It is probably caused by the fact that intensity of this band decreases with the increase of the degree of N-deacetylation of the chitin and commercial chitosan is deacetylated form of chitin so has a higher N-deacetylation degree. In turn, tested fraction at this stage of the chemical treatment resembles chitin more than chitosan which confirms results of XRD analysis. On the spectrum of commercial chitosan as well as on the spectrum of the tested sample band within the range 1100–1000 cm−1 derived from stretching vibrations of the -C-O-C- group in the glycosidic linkage was also observed. In the case of analyzed fraction this peak has a low intensity compared to this one of commercial polysaccharide. This may be a result of the presence of a number of organic compounds such as proteins or pigments in the tested material (they will be removed in the course of further processing), which probably cause a signal weakening.Considering results of FT-IR analysis it can be reported that spectrum of fraction obtained after removal of waxes and mineral salts is very similar to this one deriving from commercial chitosan. On both spectra, peak characteristics for the same functional groups were observed. In both cases a broad band at 3250 cmResults of deproteination of fractions deprived of waxes and mineral salts are presented below. As in the case of previous stages, a yield of conducted processes as well as a chemical structure of obtained material were considered when selecting the most favorable parameters. Based on the above-presented results it may be concluded that process conducted for 20 h resulted in majority cases in higher yields. However, this may also indicate that a material was not sufficiently purified from proteins. According to the literature reports crickets are characterized by a high amount of proteins in the body therefore such a stage is a very important in viewpoint of their further processing and studies. This was a reason why it was stated that process should be carried out longer so for at least 45 h. Therefore, only fractions obtained after deproteinization performed for 45 h, 55 h and 65 h were subjected to the XRD analysis.In As it may be noticed on all XRD diffractograms peaks at approx. 2θ = 19° and 2θ = 9° may be observed so at values corresponding to peaks characteristic for chitin. Nonetheless, on XRD patterns of samples treated with 5 M NaOH for 55 h and 65 h second peak is slightly closer to 2θ = 20°. Moreover, the first visible peak is much more like a peak at approx. 2θ = 10° visible on XRD pattern of commercial chitosan. Importantly, this peak is relatively wide for commercial chitosan. Additionally, on XRD pattern of sample treated with NaOH for 45 h a small wide peak at approx. 2θ = 23° is also visible which is characteristic for chitin—it was not observed on XRD patterns of samples treated with NaOH for 55 h and 65 h (longer time of treatment with NaOH resulted in more effective deacetylation of chitin to chitosan). Thus, the sample subjected to the deproteinization performed for 55 h and 65 h was considered for further experiments. However, when selecting the most favorable conditions of each process it is significant to consider principles of Green Chemistry. That is, when there is no difference between fraction after removal of waxes for 55 h and 65 h then the shorter time of the reaction should be selected. This, in turn, provides lower energy demand and thus is more ecological and economical. Therefore, the sample after 55 h deproteinization was subsequently analyzed using FT-IR spectroscopy.In It may be observed that FT-IR spectra in −1 which can be attributed to the stretching vibrations of -OH and -NH2 groups was observed. Next, bands at 2870 cm−1 corresponding to the stretching vibrations of the methylene groups assigned to the chitosan pyranose ring were also visible. Bands at 1650 cm−1 corresponding to the stretching vibrations of C=O group as well as deformation vibrations of the -NH group were also observed. Additionally, bands at 1580 cm−1 characteristic for bending vibrations of -NH deriving from the amide group were noticeable. What is more, the band at 1347 cm−1 corresponded to the deformation vibration of C-H group in the methyl group of the N-acetyl group of chitosan. The broad band in the range of 1125–1025 cm−1 resulted from the stretching vibrations of the glycosidic linkage C-O-C. Moreover, bands occurring at 880 cm−1 corresponded to the stretching vibrations of C-N group.On both spectra a wide band at 3350 cm2O2 solution. Reaction was carried out for 1 h at an elevated temperature while a yield was 95.17%. Obtained fraction was analyzed in the same way as previously—by means of FT-IR spectroscopy. In In the last step, the sample deprived of waxes, mineral salts and proteins selected, based on the previously performed processes—the sample after four extraction cycles, demineralization for 30 min using 2 M HCl and 55 h deproteinization—was subjected to the reaction with HIn On both spectra vibrations, characteristics for the same groups were observed. Thus, it may be tentatively concluded that material obtained as a result of multistep processing of crickets can be defined as chitosan and, importantly, the mentioned insects can be a source of this polysaccharide. Differences in the intensity of observed peaks can be a result of a presence of slight residues of other organic substances that were not removed during the processing. Next, XRD analysis was performed for obtained final fraction. In Any significant differences between obtained XRD diffraction patterns of samples before and after depigmentation were not observed. Two slightly shifted peaks in comparison to peaks visible on diffractogram of commercial chitosan were observed. Thus, it can be concluded that processed insects contained slight residues of pigments.In Chitosan derived from crickets was used in the next part of presented studies as a raw material for synthesis of polymer capsules that could be considered as drug carriers.Results of the performed titration are presented in + ions (derived from HCl) by OH− ions (from NaOH) according to the following reaction:In 2 groups deriving from glucosamine residues of chitosan. Firstly, -NH2 groups are protonated (-NH2 + H+ 3+). Next, when NaOH is added, -NH3+ ions are neutralized by OH− ions and therefore in such a manner the concentration of -NH2 groups may be determined.The second equivalent point may be assigned to the protonation of -NHAnalyzing the titration curve obtained and two equivalent points visible on the curve the deacetylation degree (DD) calculated using the Equation (2) was In 2 solution and subjected to the further studies.Prepared capsules were separated from the CaClPrepared chitosan/alginate-based capsules modified with nisin were subjected to the FT-IR analysis. Additionally—for comparison—spectroscopic analysis was also performed for alginate-based capsules without an active substance. In −1. This band in the case of chitosan/alginate-based capsules showed higher intensity compared to the same band visible on FT-IR spectrum of alginate-based capsules. This, in turn, may indicate intense interactions between these two reagents  due to numerous hydrogen bonds and this is a reason why this band is wider for capsules consisting of two reagents compared to the alginate-based capsules [Functional groups characteristic for analyzed materials were identified at specific absorption bands and showed in capsules . Furthercapsules ,32. Simicapsules ,34.Results of studies on sorption properties of capsules with special emphasis on impact of introduced additive on the tested sorption capacity is presented in Obtained capsules were characterized by swelling properties. Such properties grew over time because volume of absorbed solution was higher after 30 min of swelling compared to this one observed after 15 min. Introduction of nisin into analyzed capsules resulted in an increase of their sorption capacity. It was probably caused by the fact that nisin contains in its structure many hydrophilic groups that can form hydrogen bonds with absorbed medium. Groups such as amino or carboxylic dissociate under the influence of water and resulted ions from the same groups repel electrostatically. Then, structure of the tested material becomes less compact and therefore it can absorb more water. Additionally, additive was added in the form of an aqueous solution therefore absorbed liquid can also interact with the solvent.3+ groups (protonated amines) from chitosan and as a result a three-dimensional hydrogel network formed via a physical crosslinking was obtained [It may be reported that alginate-based capsules showed slightly higher swelling ability than chitosan/alginate-based capsules. Nonetheless, the differences were slight and probably due to the higher crosslinking density of capsules consisting of two components, i.e., both chitosan and alginate. It may be concluded that carboxylate groups present in the structure of alginate probably electrostatically interacted with -NHobtained . The intobtained . Consideobtained . FurtherResults of a release of nisin from alginate-based capsules and chitosan/alginate-based capsules are presented below. In − ions present in tested medium enhance electrostatic repulsion occurring between COO− anions . Therefore, a structure of capsules becomes less compact and introduced substance has more free space to get out from the polymer system can be released to the tested environment.Analyzing above-presented results it may be concluded that release of nisin is significantly more effective in slightly alkaline environment both in the case of alginate-based capsules and chitosan/alginate-based capsules. In In the case of release process observed for chitosan/alginate-based capsules at the beginning nisin was released gradually while the release of the largest amount of this substance occurred after 75 min of the research. After 2 h of the measurements a total amount of nisin was released from tested materials. Importantly, it is worth mentioning that during the first 45 min of the research only a little substance was released and its sudden ejection was observed even after 75 min. Compared to alginate-based capsules, a maximum release of nisin from chitosan/alginate-based capsules occurs more than twice as late (is long-term) that constitutes an important information in viewpoint of the application of such systems as drug delivery systems.Thus, it may be concluded that the application of chitosan extends the time of release of nisin from the developed systems. The pKa of the primary amino group of chitosan is approx. 6.5 and exactly this functional group affects the solubility of this polysaccharide in acidic environments. On the other hand, in neutral and alkaline environments a partial neutralization of this group as well as an aggregation of chitosan are observed that limits a release of nisin . Similar2 groups present in chitosan bind with H+ ions forming NH3+ cations. Such ions repel each other and as a result structure of a capsule also becomes less compact. However, the mentioned interactions are significantly less intense compared to the interactions between OH− and COO− that take place in an alkaline environment. It is caused by the fact that during the synthesis applied volume of alginate solution was four times bigger than volume of chitosan solution. Therefore, release of an active substance from the tested capsules was significantly better in an alkaline solution. It is important information in the viewpoint of the potential application of such materials as drug delivery systems. It may be reported that a release of a drug from such prepared materials is more effective in organs such as duodenum  compared e.g., to stomach (acidic conditions).Due to the solubility of chitosan in an acidic environment a nisin release was possible in such medium however a percentage amount of a released active substance was only 19.16%. A slight signal deriving from the analyzed substance was noticed only after 75 min of the research. It was probably caused by the fact that in such conditions NHCrickets can be classified as a good raw material for preparation of chitosan—polysaccharide, due to its properties, is increasingly used for biomedical purposes. However, multistep chemical processing is necessary to remove from the mentioned insect substances such as waxes, mineral salts, proteins and pigments. Obtained material was characterized by properties and structure almost the same as those ones of commercial chitosan that was proved by means of the following techniques: FT-IR spectroscopy and X-ray diffraction.− and COO− ions that resulted in the loosening the structure of polymer capsules. Furthermore, it was proved that a release of nisin from chitosan/alginate-based capsules occurred significantly longer than a release from alginate-based capsules (without chitosan). Importantly, a possibility of achieving an extended release profile of an active substance is undoubtedly a big advantage of the developed systems. Obtained results indicated that a maximum release of nisin from chitosan/alginate-based capsules was observed after approx. two-times longer period of time than in the case of alginate-based capsules.Obtained chitosan of crickets’ origin was used in the next part of the research as a raw material for preparation of polymer capsules modified with nisin. Prepared capsules were characterized by good swelling properties. Introduction of nisin into the capsules resulted in an increase of sorption capacity. Based on conducted studies on release of active substance in different environments from prepared materials it can be concluded that release is significantly more effective in alkaline solutions. It is caused by the electrostatic interactions between OHPolymer capsules based on chitosan of crickets’ origin and containing nisin constitute an interesting material that can be considered for application for biomedical purposes with special emphasis on application as delivery systems of drug with antibacterial or anticancer activity.


In multivariate analysis, the risk of venous thrombosis was independently associated with previous venous events  and NLR values ≥5 . Moreover, the relative risk in both low- and high-standard risk groups was almost doubled in the presence of NLR ≥ 5. These findings were validated in two Italian independent external cohorts  of contemporary PV patients. Our data support recent experimental work that venous thrombosis is controlled by innate immune cells and highlight that NLR is an inexpensive and easily accessible prognostic biomarker of venous thrombosis.We investigated the neutrophil-to-lymphocyte ratio (NLR) as a predictor of thrombosis in polycythemia vera (PV). After a median follow-up of 2.51 years, of 1508 PV patients enrolled in the ECLAP study, 82 and 84 developed arterial and venous thrombosis, respectively. Absolute counts of total leukocytes, neutrophils, lymphocytes, platelets, and the NLR were tested by generalized additive models (GAM) to evaluate their trend in continuous scale of thrombotic risk. Only for venous thrombosis, we showed that baseline absolute neutrophil and lymphocyte counts were on average respectively higher (median: 6.8 × 10 JAK2 genes (JAK2 V617F and exon-12 mutations). Typically, PV displays an elevated red-cell mass, usually along with leukocytosis and thrombocytosis [Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by uncontrolled clonal proliferation of multipotent bone marrow progenitors, sustained by acquired genetic mutations in ocytosis .Thrombosis can be the first presenting sign preceding the diagnosis of MPN in 20% of cases, with a persistent risk during the follow-up, where the incidence is the highest in patients with PV and similar in essential thrombocythemia (ET) and primary myelofibrosis (PMF) , 5. In aThe relationship between leukocytosis and thrombosis has been extensively investigated in several experimental studies , 13 baseThus, no definitive prognostic role could be ascertained as regards leukocytosis, thus preventing it from being included in the thrombotic risk score of PV patients.However, new data are emerging on the role of nonmyeloid inflammatory cells, such as T lymphocytes and monocytes in the process of immune thrombosis; in particular, experimental work has consistently showed that T-reg lymphocytes are involved in the regulation of the prothrombotic action of activated neutrophils in the process of fibrin formation and dissolution . Based oIn the present work, we investigated whether NLR can identify PV patients at higher risk of both venous and arterial incident events by analyzing the ECLAP database that involved PV patients prospectively followed for a median of 3 years and includes a large number of formally validated arterial and venous thrombotic events.The present study included 1508 of the 1638 PV patients enrolled in the ECLAP study, selected on the basis of the availability of differential blood counts at baseline that allowed to calculate the NLR.9/L. Data regarding clinical outcomes, treatments, and laboratory values during the prospective follow-up were recorded at follow-up visits at 12, 24, 36, 48, and 60 months.All patients with new and old PV diagnoses (according to Polycythemia Vera Study Group (PVSG) criteria) were included prospectively with no exclusion criteria with respect to age, therapy, or duration of disease. Treatment strategies had to comply with the recommendation of maintaining the hematocrit value at less than 0.45 and the platelet count at less than 400 × 10The study protocol conformed to good clinical practices and to the Declaration of Helsinki on medical research in humans.The primary endpoint was the incidence rate of arterial and venous thrombosis , and major and minor thrombotic complications as defined above. These events were objectively diagnosed as previously reported .Univariate analysis was performed to evaluate differences in proportions by the chi-square or Fisher exact tests where appropriate. Differences in continuous variables were tested using the nonparametric Wilcoxon rank-sum test.In order to investigate the role of NLR in the time-to-thrombosis prediction, we overcame the traditional Cox proportional-hazard model, which restricts the log hazard ratio (HR) to be linear in the covariates, by applying the generalized additive models (GAM) . GAM proNLR values were dichotomized using the best threshold found according to Liu’s method. Thrombosis-free survival of the resulting two groups was compared using the Kaplan–Meier (KM) estimator and tested with the log-rank test.Cox proportional-hazard models were applied for multivariable analyses. The Harrell’s C-statistic was calculated to measure the incremental discriminatory ability of a new predictor .During a median follow-up of 2.51 years, 166 thromboses  were recorded in 160 of 1508 patients (10.6%). Arterial thromboses were mostly cerebral, while venous thrombosis mostly affected lower extremities (DVT), with or without pulmonary embolism (PE) (Table p = 0.001) and previous thrombosis (p = 0.003), especially if arterial (p < 0.001), and with the presence of at least one cardiovascular risk factor (p = 0.009). No association with baseline leukocytosis or the NLR was found. In patients with venous thrombosis, not age but history of thrombosis (p < 0.001) was also a risk factor for incident events, especially in cases with prior venous events (p < 0.001).Table p = 0.041), and above all, it was greater in categories of patients with higher NLR values (p = 0.002), which resulted from a simultaneous increase of neutrophils (p = 0.002) and a decrease of lymphocytes (p = 0.001).Differently from arterial, venous complications significantly occurred for progressive increase of BMI (p = 0.002) emerged, leading to increase the NLR values (p = 0.005)  Fig. . The hazTherefore, since the NLR integrates these two opposing trends, it was more representative of the association of lymphocyte and neutrophil counts with venous thrombosis than individual counts alone, where confidence intervals resulted wide at higher counts.Conversely, neither absolute values of neutrophils and lymphocytes nor NLR were found associated with arterial events Fig. .Total leukocyte and platelet counts were also tested, but they did not correlate with either venous or arterial thrombosis (data not shown).p < 0.001) and NLR values ≥5, a figure representing the best cutoff resulted applying Liu’s method ), the risk of venous thrombosis was independently associated with previous venous events  and previous arterial thrombosis  retained the prognostic statistical significance for arterial thrombosis, together with an effect of CV risk factors of borderline significance  and one that added to the latter the NLR value above 5, looking for any incremental value of the statistic. The probability that predictions and outcomes are concordant measured by the C-statistic for the first model was 59.24%, while after adding the NLR variable, it increased to 65.09%.Kaplan–Meier curves of time-to-venous thrombosis probability stratified by either conventional risk classification or values of NLR are plotted in Fig. n = 282 and Rome, n = 175) of contemporary PV patients, whose clinical characteristics are presented in Table The correlation between NLR values and venous thrombosis found in our learning cohort Fig.  was valiIn this series of patients with PV, we sought to investigate for the first time whether the baseline ratio of circulating neutrophils to lymphocytes (NLR) could be a marker of thrombosis as reported in many studies in the general population . To this9/L, p = 0.022) and lower  in patients with incident venous thrombosis, overall resulting in a higher NLR ratio. The most evident finding was the linear association between the absolute number of lymphocytes and the risk of events expressed by the log of HRs in the GAM models. We found that the category of patients presenting at baseline with lymphocyte values less than 2 × 109/L had an increased risk of venous thrombosis with HR greater than one, whereas the HRs for venous thrombosis progressively decreased as the lymphocyte counts increased. These changes are consistent with an opposite prognostic tendency of neutrophils, whose increments showed an increasingly higher trend of risk .In univariate analysis, we found that the absolute number of neutrophils and lymphocytes was on average respectively higher  and age ≥65 years.In a multivariate Cox proportional-hazard model using NLR cutoff equal to five, found to most efficiently discriminate against the highest risk categories, we found that this cutoff value was associated with major venous events (HR = 2.18), independently of age and previous venous thrombosis, and this was confirmed in the sensitivity analysis after excluding minor venous thrombosis. On the contrary, NLR did not show any significant association with arterial events that were predicted by previous arterial event (Interestingly, the prognostic discriminatory power of conventional risk factors for venous thrombosis was increased in the presence of an elevated NLR as demonstrated by the values of the C-statistic increasing from 59% to 65%, in either low- or high-risk PV groups. This finding could therefore suggest the value of incorporating this inflammatory biomarker in a scoring system for venous thrombosis in PV.JAK2V617F, which has recently been reported to be correlated with venous thrombosis [It will be interesting to investigate the relationship between NLR values and the allelic burden of rombosis . FurtherWe are fully cognizant of the limitations of the current study considering that this is a post hoc analysis of a clinical trial using data that had already been collected for other purposes and referred to patients diagnosed with PVSG diagnostic criteria . HoweverIn conclusion, these clinical findings support recent biological concepts that innate immune cells contribute to venous thromboembolism . Indeed,Reproducibility checklist


We have observed white turbidity when a midazolam injection is administered from a lateraltube during the administration of a peripheral parenteral nutrition (PPN) solution. The aim ofthe current study was to determine how to avoid compound changes when co-administering amidazolam injection and a PPN solution.Midazolam solutions were prepared by diluting a midazolam injection with a 5%glucose intravenous infusion. We examined the formulation of the midazolam injection and a PPNsolution at the concentrations used in a clinical setting for changes in appearance, pH, andmidazolam content in test tubes and during administration conditions.2=0.9918). The capture rate of midazolam infused with PPN solution was 91.0% ata 1/6 dilution, whereas it decreased to <90% at a 1/4.8 dilution.With a 1/4.8 dilution of midazolam in undiluted solution, clouding occurred. Astrong correlation was revealed between the midazolam content as measured throughhigh-performance liquid chromatography and the mixture’s midazolam concentration(ROur results suggest that the administration of a midazolam injection solutiondiluted by ≥6-fold with glucose solution or saline from a side tube during the administrationof a PPN solution did not cause changes in composition. Guidelines concerning terminal fluid therapy have beenpublished, and peripheral parenteral nutrition (PPN) solutions are now widely used.5 A patientindicated for sedation usually receives a midazolam injection from a side tube during thesimultaneous administration of a PPN solution. However, since a midazolam injection is stableunder acidic conditions, the move toward alkalinity causes precipitating white turbidity orcrystal precipitation. A delay in detecting this turbidity or precipitation may seriously affecta patient’s health. Clinicians must be sensitive to formulation changes because of the injectionroute.6 Although changes in midazolaminjection formulations during total parenteral nutrition infusions have been reported, fewreports comprehensively describe the changes occurring during simultaneous midazolam injectionand PPN solution administration; only visual changes and pH observations were described, notchanges in midazolam content.7 Therefore, weexamined changes in appearance, pH, and midazolam content during administration conditions basedon differences in the composition of a PPN solution and a midazolam injection solution.Midazolam exerts a sedative effect by activating the inhibitory neurotransmitterGABA receptor in the central nervous system, and it introduces and maintains anesthesia as aninjection, as intensive care sedation during artificial respiration, and as sedation duringpalliative care.1. ® injection solution 10 mg; Maruishi Pharm Inc.) is administeredthrough a side line at 2 mL/h. To reproduce this situation, we mixed 8.4 mL of PPNsolution and 0.4 mL of midazolam injection in a test tube at the ratio of 42:2 at roomtemperature and under normal light conditions. We used an amino acid and glucose injectionwith electrolytes and a vitamin B1 infusion  which is a colorless and transparent infusion agent that contains approximately 7.5%glucose, 3% amino acid (18 types), electrolytes, and vitamin B1 components (pH isapproximately 6.7). We prepared midazolam solutions,  assuming clinicaldoses of 10–240 mg by diluting a midazolam injection with a 5% dextrose solution  to create a range that included anundiluted solution, i.e., 1/24 (We often treat patients for whom a PPN solution is administered at1000 mL/day (42 mL/h) through a main duct, and a midazolam injection; column temperature, roomtemperature, flow rate, 0.8 mL/min; detector, UV - 2075 Plus intelligent UV/Vis detector(JASCO Corp.); detection wavelength, 254 nm, mobile phase, 1 mM citrate-phosphatebuffer (pH 5.0): methanol: acetonitrile=1:1:1 (V/V). The measurement was performed usingan absolute calibration curve method using a midazolam injection solution as a standardsolution. The calibration curve showed linearity in the range 3.1–50.0 μg/mL. Wedetermined the relationship between the midazolam content as measured through HPLC and themidazolam concentration of the mixture by fitting the approximate curve. We performed aPearson’s regression analysis to determine the correlation.To observe changes in the appearance and pH and for high-performance liquidchromatography (HPLC), we first slowly added 0.4 mL of each sample dropwise to8.4 mL of PPN solution, and we observed the visual changes (color tone/turbidity)immediately after the dropwise addition. After recording changes in appearance, we stirred themixture for 30 sec using a vortex mixer and measured the pH and midazolam concentrationof each sample. The pH was measured using a pH meter . Themidazolam content was analyzed through HPLC with reference to Kobo’s method,We investigated changes in the combined PPN solution and midazolam solution, and inthe combined saline solution and midazolam solution at room temperature and under normal lightconditions. We prepared four midazolam solution samples by diluting a midazolam injection(Dormicum injection 10 mg) with saline to 1/2.4, 1/4.8, and 1/6, and an undiluted PPNsolution (500 mL) was added dropwise via an infusion pump at 40 mL/h with a planectainfusion set filter . The 0 min time point occurred immediately after thesyringe pump began to push the solution. We collected 3.5 mL of each mixed solution,having passed through the infusion in-line filter with a 0.22 μm aperture, from the tip ofsyringe pump over a 5 min period after 10, 30, 60, and 120 min. For the collectedmixture, the pH was measured under the same conditions as in Experiment (A); the midazolamcontent was measured through HPLC, and the content ratio to the theoretical value derived fromthe standard solution was determined. We added 500 mL of saline  dropwise at40 mL/h under the same conditions as with the PPN solution, and midazolam solution sampleswere injected from the side tube at 2 mL/hr. The dose concentration of the midazolaminjection solution and the measurement conditions of the collected mixture were the same asthose used for the PPN solution.2=0.9918, showing a strong correlation  was 6.72±0.03, and thepH of sample O was 6.66±0.01 . Thecoerelation .The pH values of the collected mixture of PPN solution (with a pH of approximately6.7) at 0, 10, 30, 60, and 120 min after the infusion of midazolam solution diluted to 1/6began were 6.69±0.07, 6.72±0.08, 6.64±0.00, 6.71±0.08, and6.71±0.08, respectively. The pH values of the mixture with saline (with pH 4.5–8.0)at the same time points were 5.47±0.04, 5.56±0.15, 5.14±0.42,4.75±0.11, and 4.71±0.01, respectively. For the midazolam solution diluted to1/4.8, the pH values of the mixture with the PPN solution were 6.82±0.10,6.83±0.11, 6. 76±0.07, 6.83±0.13, and 6.81±0.11, and the pH valuesof the mixture with saline were 5.24±0.12, 5.59±0.08, 4.89±0.00,4.83±0.10, and 4.77±0.10, respectively. The pH values of the mixed solution withPPN solution when injecting the midazolam solution diluted to 1/2.4 were 6.74, 6.75, 6.75,6.73, and 6.72. The pH values of the mixed solution with saline were 5.56, 5.37, 4.55, 4.36,and 4.39. The pH values of the mixture with the PPN solution when injecting undiluted midazolamsolution were 6.64, 6.61, 6.60, 6.57, and 6.60, and the pH values of the mixed solution withsaline were 5.52, 5.04, 4.12, 4.00, and 4.08, respectively .The midazolam content ratios to theoretical values at 0, 10, 30, 60, and120 min of the mixture with the PPN solution when injecting a 1/6 dilution of midazolamwere 3.4%, 3.5%, 0.4%, 83.2%, and 91.0%, respectively. The midazolam content ratio totheoretical values of the mixture with saline were 1.6%, 0.8%, 37.1%, 82.7%, and 100.3% . The mid2=0.999 or more. An appearance change was observed onlyin the mix of PPN solution and midazolam undiluted solution. The mixture was colorless andtransparent under other conditions.The midazolam content ratio to theoretical values of the mixed solution with PPNsolution when injecting midazolam undiluted solution were 0.1%, 33.5%, 33.4%, 54.7%, and 50.2%,and those of the mixed solution with saline were 0.1%, 0.0 %, 69.3%, 99.1%, and 95.0%,respectively . The cal14 The midazolam injection solution used for the sedation of terminal-stagepatients maintains a stable aqueous solution under acidic conditions, and composition changesoccur under alkaline conditions. Mixing injectable drugs is not possible when one or both of thefollowing are observed: (1) a change in the mixture’s physical appearance is recognized, (2)within 24 h post-compounding, one or more of the compounded ingredients is decomposed by≥10%. When changes in a formulation are prevented, it remains necessary to observe the reactionproduct, maintain the content of the active ingredient, and ensure that the medicine isadministered to the patient safely and securely.15Our experiments clarified a method of combined administration for a PPN solution anda midazolam injection solution that avoids changes in the mixed solution characteristics.Guidelines regarding infusion therapy for terminal cancer patients have been issued in severalcountries. The importance of nutrition has also been highlighted in Japan, and guidelines wereissued that consider the use of PPN solution preparations.16 Kuramoto et al. mixed equal volumes of ahigh-calorie infusion preparation and a midazolam injection solution in two-, three-, four-, andfive-fold dilutions; they examined changes in appearance and conducted a filter-resistance testand reported that a five-fold dilution of the injection can be administered from the side tubeof a total parenteral nutrition infusions line without causing composition changes.7Avoiding formulation changes and reaction products and maintaining the content ofthe active ingredient will guarantee drug efficacy and safety. In an examination of thestability of midazolam hydrochloride in a parenteral nutrient solution with intravenousnutrients containing amino acids at 1.5%, 2.5%, and 5% concentrations and midazolam injectionsat 0.1 and 0.5 mg/mL concentrations, the midazolam and amino acid content at 5 h aftermixing did not change, and changes in appearance, pH, and precipitation did not occur.17 The most interesting point revealed in the current study is that althoughvisible white turbidity occurred in the solution with samples E–O, when we stirred the samplesagain, the white turbidity disappeared and 95.27% of the midazolam could be detected throughHPLC (indicating that only 4.73% was lost). These results were contrary to our expectation,because the solutions measured pH 6.6–6.7, which is more basic than the value of thechanging point; i.e., the pH value at which crystals are thought to precipitate. To clarify thepH dependence of midazolam, the pH of the critical point at which no formulation change isobserved could be identified using Azuma’s method or predicting a formulation change.18 The critical-point pH can be determined throughAzuma’s method, but the reason for the value being close to the pH value of the PPN solution isthat the mixed solution pH is included in the beaded infusion. The influence of the pH bufferappears to be large. Moreover, although we observed that the midazolam content of the solutionafter stirring does not fall, in clinical settings there is no guarantee that the mixture underthe same conditions will be stirred. Therefore, midazolam administration at a ≥6-fold dilutionis recommended.In Experiment (A), white turbidity was confirmed at concentrations exceeding a 1/4.8dilution of the midazolam injection solution. Hydrochloric acid as a solubilizing agentinfluences changes in the midazolam formulation, leading to large variation in the clinicalformulation. The pH range of midazolam injections is 2.8–3.8, and crystals precipitate at themore basic region including the change point 4.72.In Experiment (B), the midazolam content was measured through HPLC, and weinvestigated whether a difference occurred in the composition change between a PPN solutiontransfusion and saline. We measured the pH of unbuffered saline immediately after mixing withsaline and after 10 min, and the pH fluctuation after 30 min tended to be acidic dueto the strongly acidic midazolam injection. This is consistent with the detection of a midazolaminjection after 30 min, as shown in Figure. 4. In 19 Our results clarified that thecloudy midazolam observed in Experiment (A) is a formulation that does not reduce the midazolamcontent if it is redissolved. It is not yet known how the redissolved midazolam solution wouldaffect the body. Although the cloudy component can be thought of as midazolam, an analysis ofthe components captured by the in-line filter is necessary because the cause of the reactionbetween the midazolam solution and the PPN solution that generates this white turbidity is notknown.20 Since the injection of a highconcentration of midazolam was captured by the in-line filter, it is considered that the in-linefilter should be used in a clinical setting. In Experiment (B), we performed a formulationchange test by setting the time for a midazolam injection to reach a patient’s blood vessel fromthe side tube at a maximum of 120 min. The compatibility of midazolam with morphine,haloperidol, and similar drugs which are frequently used in the palliative field should bedetermined through compound change tests.23Midazolam was reported to be adsorbed by vinyl chloride, and we therefore used aninfusion route with polybutadiene (non-vinyl chloride) which does not adsorbmidazolam.24 Since the midazolam dosethat could sedate terminal cancer patients was 20–30 mg/day, midazolam administrationstarts at 1–2 mg/h and the dosage is increased until sufficient sedation is achieved.Sedation using midazolam is effective at 80%–90%; at approximately 5% it causes complicationsleading to death, but midazolam is generally considered to be safe.25 Midazolam is also recommended as a palliative care sedative. Safeadministration is desired; i.e., avoiding changes in formulation, complications such aspulmonary embolism, and problems such as catastrophic blockage or clogging of the in-linefilter. It is important to promote the proper use of injectable medicines and to investigate andsolve problems occurring with their use.In palliative care, sedation is administered to reduce patient pain. Sedation isnecessary in approximately 30% of cancer patients, for the relief of delirium, dyspnea, malaise,and pain. In a systematic review of studies of midazolam doses, the median starting dose was0.5–1.7 mg/h, the median maintenance dose was 18–62 mg/day, and the range was2–450 mg.In conclusion, our findings demonstrated that the administration of a midazolaminjection solution diluted by ≥6-fold or more with glucose solution or saline from the side tubeof a PPN solution did not cause any change in the solution composition.


Despite recent rises in fatal overdoses involving multiple substances, there is a paucity of knowledge about stimulant co-use patterns among people who use opioids (PWUO) or people being treated with medications for opioid use disorder (PTMOUD). A better understanding of the timing and patterns in stimulant co-use among PWUO based on mentions of these substances on social media can help inform prevention programs, policy, and future research directions. This study examines stimulant co-mention trends among PWUO/PTMOUD on social media over multiple years.We collected publicly available data from 14 forums on Reddit (subreddits) that focused on prescription and illicit opioids, and medications for opioid use disorder (MOUD). Collected data ranged from 2011 to 2020, and we also collected timelines comprising past posts from a sample of Reddit users (Redditors) on these forums. We applied natural language processing to generate lexical variants of all included prescription and illicit opioids and stimulants and detect mentions of them on the chosen subreddits. Finally, we analyzed and described trends and patterns in co-mentions.Posts collected for 13,812 Redditors showed that 12,306 (89.1%) mentioned at least 1 opioid, opioid-related medication, or stimulant. Analyses revealed that the number and proportion of Redditors mentioning both opioids and/or opioid-related medications and stimulants steadily increased over time. Relative rates of co-mentions by the same Redditor of heroin and methamphetamine, the substances most commonly co-mentioned, decreased in recent years, while co-mentions of both fentanyl and MOUD with methamphetamine increased.Our analyses reflect increasing mentions of stimulants, particularly methamphetamine, among PWUO/PTMOUD, which closely resembles the growth in overdose deaths involving both opioids and stimulants. These findings are consistent with recent reports suggesting increasing stimulant use among people receiving treatment for opioid use disorder. These data offer insights on emerging trends in the overdose epidemic and underscore the importance of scaling efforts to address co-occurring opioid and stimulant use including harm reduction and comprehensive healthcare access spanning mental-health services and substance use disorder treatment.The online version contains supplementary material available at 10.1186/s12954-022-00628-2. Stimulant co-use –4, speciRecent findings also suggest that reasons for co-use, particularly with methamphetamine, are multifaceted—such as ease of access, helping boost the “high,” improving daily function , and helping manage withdrawal symptoms . HoweverEstimate the volumes and short- and long-term trends of opioid-stimulant co-mentionsQuantify estimated co-use rates between pairs of specific opioids and stimulants based on these discussionsIdentify when stimulants are initiated evidenced by mentions among PWUO at an individual level based on timelines of postsThe insights we derive in this study are purely data-driven through natural language processing (NLP) methods, as opposed to hypothesis-driven, and our methods account for growth in the number of Reddit users over time. Being data-driven, we did not start our analyses with any underlying assumptions or hypothesis. Our intuition was that the growing number of overdose deaths due to co-use of opioids and stimulants would be reflected on the Reddit data, but this intuition did not affect our approach. A better understanding of the timing and patterns in stimulant co-use among PWUO as evidenced by mentions of these substances on social media can help inform prevention efforts including harm reduction, as well as future research directions. In the remainder of the article, we refer to people who post on specific subreddits of interest as Redditors, people who use or used prescription and/or illicit opioids, people who used an opioid reversal agent , and people with OUD who are being treated with medications for opioid use disorder (MOUD) as PWUO/PTMOUD, people who use stimulants as PWUS, people who use drugs more broadly as PWUD, and people who co-use opioids and stimulants as PWCU.The overarching objectives of this study are to utilize social media conversation data on Reddit to:subreddits and the moderation of forum-specific content by forum users. Reddit communities have also been found to serve as a means of social support for PWUD [Reddit is particularly popular among PWUO and the broader community of PWUD as it can offer anonymity . Reddit for PWUD , 15, 19.for PWUD , Blueligfor PWUD , and Disfor PWUD  for sevefor PWUD –25. ReddTo find potential PWUO on Reddit, we identified 14 opioid-related subreddits spanning discussions on prescription and illicit opioids and MOUD . We focused the study on a set of common opioids, opioid-related medications, and stimulants, including both prescription and illicit types . Thus, we next included additional filters for the terms stimulant, meth, and oxy  of the substances. Since drug names and related expressions are often misspelled on social media, we generated commonly used lexical variants of the terms using the LexExp tool . We founn = 5 following the mention of a negation trigger. Any opioid, opioid-related medication or stimulant term was considered negated if it appeared in the context window following a detected negation expression and if an end of sentence marker  did not occur between the negation term and the drug term. Negated terms were excluded from the final count of mentions. We also manually reviewed samples of posts detected by our searches to identify keywords with a large amount of noise  and excluded those from our counts and further analysis. Of the 13,812 Redditors sampled, a subset who posted in these subreddits had no mention of either an opioid, opioid-related medication or a stimulant (N = 1506) and was excluded from the analyses. All analyses except for those examining the total numbers of Redditors (including PWCU) over time were based on subsets of the remaining 12,306 Redditors who posted about at least one opioid, opioid-related medication, or stimulant.The third NLP task involved implementing a negation detection strategy, customized for Reddit data, to exclude negated drug mentions. Specifically, we used a subset of negation terms from the NegEx algorithm   over the same time frame; and the annual ratio of PWCU to PWUO/PTMOUD. Due to the growing user base of Reddit over time, we expected numbers of PWUO/PTMOUD and PWCU to both increase annually. Hence, the ratio of PWCU to PWUO/PTMOUD likely serves as the best indicator of social media trends in opioid and stimulant co-use within this population.We first studied trends in opioid and stimulant co-use between 2011 and 2020 through multiple metrics, including, the number of Redditors posting each year; the number of Redditors who mentioned at least one opioid or opioid-related medication and one stimulant  frequency of co-use of specific types of opioid-stimulant pairs among PWUO/PTMOUD aggregated over the entire study period; (2) proportion of specific opioids co-used with any stimulant in a given year among the total number of PWCU in that year, to study how mentions of specific opioids or opioid-related medications co-used with any stimulant changed over time; and (3) proportion of specific stimulants co-used with any opioid or opioid-related medication in a given year among the total number of PWCU in that year, to study how mentions of specific stimulants co-used with any opioid changed over time. Redditors with mentions of more than one type of opioid or opioid-related medication or stimulant within a given year were separately considered for each specific opioid-stimulant pair.We combined the opioids and opioid-related medications listed in our analytic sample into 5 categories: 1) heroin, (2) fentanyl & analogs (including carfentanil), (3) Medications for Opioid Use Disorder , (4) opioid overdose reversal agents , and (5) prescription opioid pain relievers . We combined stimulants into 4 categories: (1) methamphetamine, (2) amphetamine-type stimulants , (3) methylphenidate-type stimulants , and (4) a combination of methamphetamine and heroin . Categories included generic names, trade names, and common misspellings or lexical variants  versus those who mentioned a stimulant first .We collected 13,812 Redditor timelines from the 14 subreddits, 12,306 of whom mentioned at least one of our selected opioid, opioid-related medication, or stimulant keywords. Redditors mentioning any opioid or opioid-related medication, or any stimulant in their timelines were approximately evenly distributed—9329 Redditors mentioned opioids or opioid-related medication at some point in their timeline; 9151 mentioned stimulants  and prescription opioids were most frequently mentioned with prescription amphetamine-type stimulants . This suggests possibly distinct populations within the social media substance use forums we studied with preferences for particular types of substances. Further understanding the distinction between populations focused primarily on prescription drug misuse and those focused on illicit substance use, and the transitions between these preferences, is a future area for exploration of online data that may yield insights for better targeting prevention and harm reduction efforts. Indeed, while there is research on prescription stimulant misuse , less isWe found noticeable increases in the proportion of stimulant mentions among individuals also discussing MOUD over time, consistent with recent reports indicating that stimulant use among people receiving treatment for OUD may be increasing , 3. Co-uWhile relative rates of stimulant co-use among individuals using fentanyl appear to be on the rise, the relative rate for co-use among individuals using heroin showed a gradual decrease. This is perhaps a result of changes in the illicit opioid supply over recent years, as heroin has experienced supply shortages , 35 and Future work can also focus on further utilizing NLP and machine learning methodology to conduct more fine-grained characterization of the data, such as annotation of posts to filter out posts that do not self-report consumption of a substance mentioned –43. AdvaOur study has several limitations. Absent contextual information, the primary limitation is the assumption that an individual mentioning an opioid, opioid-related medication, or stimulant is currently using that substance, which may result in overestimation of the number of PWUO/PTMOUD, PWUS, and PWCU, particularly in analyses where these mentions spanned multiple years. Nonetheless, the number of mentions could be a proxy for use and at minimum, mentions of a substance represent some degree of interest or consideration of a particular substance. Also, since the forums from which the Redditors were initially identified are topic-specific, most posts explicitly or implicitly refer to personal experiences.Second, individuals who post on Reddit may not necessarily be representative of all PWUD and caution when generalizing from these findings is warranted. According to a 2019 PEW Research Center report, Reddit has an overrepresentation of males and younger people . Since tThird, not all PWUO, PWUS, and PWCU necessarily mention all the substances they may use. Additionally, we excluded cocaine from our analyses due to concerns around data quality given challenges associated with accurately identifying mentions. Further, our analyses were limited to opioid-related subreddits and excluded any stimulant-related subreddits given our focus on PWUO/PTMOUDs. These limitations may both lead to an underestimation of stimulant and opioid-stimulant co-use. Fourth, we were only able to conduct our analyses on a sample of Redditors due to the time required for retrieving longitudinal Redditor data. Our concerns about this limitation are mitigated by the finding that statistics derived from smaller samples of Redditors early on in our study remained almost identical to those obtained when larger numbers of individuals were included. The exponentially rising number of Reddit subscribers over time also posed a challenge for us, since we could not quantify how much of the increasing opioid or opioid-related medications and stimulant mentions could be attributed to increases in their use in the community versus the growing Redditor numbers. To address this, we examined the ratio of PWCUs to Redditors, which revealed that the growth rate in substance mentions was higher than the growth rate of Redditors. It is possible that other factors contributed to the rising substance mention rates, but we were unable to include such confounding factors in this analysis. Lastly, methodological limitations inherent to all analyses of unstructured text data apply as not all mentions/expressions are detectable by NLP methods , 47. HowOur study included only publicly available data that were retrievable via the Reddit API. No private information was obtained. Subreddits that require membership before viewing content were not included. Since our study falls under the category of observational studies conducted on publicly available data, the Emory University Institutional Review Board (IRB) deemed the study to be exempt (category 4). It was determined that no additional harm to individuals was introduced by the use of the publicly available data. In clinical settings, when protected clinical data of patients are used for research, the standard practice is to seek informed consent prior to inclusion. With social media based observational studies such as ours, obtaining consent requires contacting the subscribers directly, and this action requires a higher level of IRB approval as the direct communication with the subscriber may be considered to be intervention. Such contact also presents bigger challenges in determining if the study presents any additional risk or unanticipated harms to the individual. The ethical considerations that need to be taken into account when conducting research using social media data are evolving through discussions and collaborations among active researchers in this space. Currently, there is some consensus that it is appropriate, and perhaps necessary, to utilize all available public data for addressing serious public health crises, such as the ongoing opioid overdose epidemic . ExcludiWe examined real-world, longitudinal social media data from online opioid-related forums to explore patterns in opioid and stimulant discussions and found large increases in co-mentions of opioids or opioid-related medications and stimulants. These findings are consistent with those from other health data sources suggesting growing co-use of these substances and resultant harms. Although a multitude of factors may influence substance use trends, many are difficult to elucidate and explore using traditional data sources such as survey records or health care data. The social media data we explored offer important exploratory insights into which opioids or opioid-related medications and stimulants are most frequently co-mentioned and how these patterns have changed over time. These data derived from real-world conversations may help in hypothesis generation and yield early insights to shape prevention activities addressing health harms associated with opioid and stimulant co-use.Additional file 1: Appendix.


Patient experience with community pharmacy services can be informed by human-centered design principles and approaches. Pharmacy services may benefit from detailed evaluations of consumer experience and patient-centered service design.To use an online journey mapping platform to understand the patient experience with selecting a Medicare Part D plan for individuals that did, and did not, use a free, pharmacy-led, Medicare Part D consultation service.U tests. Open-ended survey responses were coded by the research team using an inductive approach.This was a two-group cross-sectional survey study in a single, rural community pharmacy. Surveys consisted of 7 demographic items, 30 Likert-type items, and 7 open-ended response items. The pharmacy used purposeful convenience sampling to distribute a paper survey to individuals 65 years of age and older currently enrolled in Medicare Part D between June and August 2019. Surveys were distributed to 36 patients currently enrolled in a Medicare Part D plan, with 18 surveys distributed to patients who had previously used a pharmacy-led Medicare Part D consultation service and 18 surveys distributed to patients who did not use the service. Surveys were uploaded to an online journey mapping platform, producing data visualizations for each group. Multiple choice survey items were analyzed using descriptive statistics, wth service user and nonuser groups compared using Mann-Whitney Persona, Empathy, and Current Journey outputs, which mapped Good Experiences and Bad Experiences within the Medicare Part D plan selection experience. Personas differed in their median household incomes . Empathy and Current Journey outputs showed that service users had a wider variety of emotions compared to non-users. Mann-Whitney U tests yielded 5 items with statistically significant differences  in the plan-selection experience, with both groups similarly uncertain about their plan decision. Qualitative responses indicate patient trust was universally important to a complex decision-making process.In total, 36 surveys were returned to the community pharmacy for a response rate of 100%. The journey map platform generated An online journey mapping platform provided insight into how patients experience a pharmacy service that extends beyond satisfaction. For community pharmacies providing Medicare Part D plan consultation services, pharmacies should consider how they can improve the service experience through communication style and patient-centered service design. •Design Think practices like journey mapping may aid in patient-centered service design.•The elaboration likelihood model (ELM) may help to explain patient experience with a Medicare Part D consultation service with Medicare•Information processing ability and heuristics may influence patient decisions. Previous studies have identified that while patients frequently report positive experiences and high levels of satisfaction with community pharmacy Medicare Part D consultation services, they inconsistently use the information gathered from consultation services to switch Part D plans and infrequently use the services in successive years despite potential cost-savings.18.The objective of this study was to use a journey mapping methodology to depict and analyze patient experiences with selecting a Medicare Part D plan for two groups: pharmacy patients who used a pharmacy Medicare Part D consultation service and those who did not. This study makes an important contribution to the literature, using an innovative methodology to assess the patient experience with a community pharmacy service designed to assist patients with a challenging Medicare Part D plan-selection decision.22.1This was an exploratory study using a two-group cross-sectional paper survey combined with a third-party online journey mapping analysis platform. Surveys were administered between 6 and 8 months after participation in the pharmacy-led consultation service. The study was reviewed and approved by The University of Iowa The Institutional Review Board.2.2The study pharmacy is independently owned and located in a small rural town in the Midwest United States. The pharmacy offers various services like vaccinations, medication synchronization, and medication therapy management (MTM). Medicare Part D consultations are available at the community pharmacy year-round for individuals 65 years of age and older or for those looking to enroll in a Medicare Part D plan for the first time. The pharmacy provided information and resources to be used for Medicare Part D plan-selection decisions during the open enrollment period: October 15th to December 7th, 2018 All consultations were provided by one of two individuals: the supervising pharmacist or one certified technician with Medicare Part D plan-selection experience and training. Data on who provided the consultation could not be linked to the individual survey response.Patients eligible for Medicare Part D were mailed a letter in early October 2018, notifying them of the upcoming open-enrollment period and offering free Medicare Part D consultations. For interested parties, consultations were scheduled in 30-min blocks at the community pharmacy. If patients were unable or unwilling to receive the consultation in-person, the same information was offered via a telephone consultation. Before the scheduled consultation, the pharmacist completed a comprehensive medication review (CMR), with third-party software  used to compare Medicare Part D plans based on the current medication regimen. The pharmacy provided each patient with a statement disclosing that the consultations were conducted with the patient's best interests in mind.The pharmacy staff member conducting the service verified the patient's current Medicare Part D plan before printing off the plan-comparison output generated by pharmacy software. This software evaluated patients' chronic medications and pharmacy preferences to identify plans that offer improved insurance coverage. The software generated a print-out that can be shared with the patient. This printout contained information on the patient's current plan, with comparisons to the three lowest-cost Medicare Part D plan alternatives. Pharmacy staff informed patients that several plans existed, and the ones shown were associated with the lowest yearly patient expenditure. After explaining the components of benefit design , the patient was informed on how to change plans. Patients were encouraged to make plan-switching decisions on personal devices but were allowed to use pharmacy computers to make same-day plan-switching decisions at the request of the patient.2.3The online journey-mapping platform started technical development during the first quarter of 2017. The software team that built the platform regularly design and develop applications for projects where data sets are both collected and visually displayed via a web interface. The first journey-mapping beta project took place during the summer of 2017 and informed several changes related to the proprietary algorithm as well as updates to the user interface. As the beta program progressed, updates were implemented in collaboration with both the leadership team experienced with journey map deliverables and the development team familiar with valid approaches to data collection and visualization. The journey-mapping platform entered full production in November 2019 after multiple rounds of iterative updates from beta projects.Discover, Search, Assess, Decide, Assist. These domains focus on the entirety of the Medicare Part D selection experience. Using the standardized items, a study author with Medicare Part D consultation used an iterative process to refine items to reflect the Medicare Part D experience. In total, 7 demographic items, 30 Likert-type items, and 7 open-ended response items were initially included in the survey. Community pharmacy personnel providing the Medicare Part D consultation service reviewed the survey for initial validity and reliability. After revisions suggested by community pharmacy personnel, the survey was piloted with two Medicare Part D beneficiaries currently enrolled in a Medicare Part D plan but unaffiliated with the study pharmacy to identify issues with survey clarity. After piloting, modifications were made based on suggestions to improve readability. Journey mapping domains and example items from the specific journey-mapping platform are included in Appendix A.Survey questions were based on standardized items from the online journey mapping platform and tailored to fit the Medicare Part D plan selection experience. Survey items corresponded to predetermined journey mapping domains: Persona, Empathy, and Current Journey outputs. The Persona outputs can be described as an avatar representing generalized characteristics of each survey group based on median responses. The Empathy outputs are a visual depiction of four emotion domains: Think & Feel, Hear & See, Pain, Gains. Multiple choice survey response items were mapped to each emotion domain and validated by the journey mapping platform development team. The Empathy output also procured an overall emotional experience score, quantifying the overall experience based on the range of emotions felt in the Medicare Part D plan selection process. Scores may range in values from 0 to 10, with larger values associated with better experiences. The Current Journey outputs provide a visual depiction of the comprehensive Medicare Part D plan selection process, progression through 5 decision-making domains: Discover, Search, Assess, Decide, Assist. The Current Journey outputs mapped Good Experiences and Bad Experiences on the Medicare Part D plan selection journey. These experiences are often denoted as Touch Points in journey mapping best practices.,Bad Experiences) or depict moments of experience success (Good Experiences). During Good Experience moments, patients and customers may be further engaged with the service or experience and are more receptive to additional experiences.,Data from the returned paper surveys were entered into the online journey-mapping platform. The software used in this project includes algorithms and analytics that produce visual representations of the Medicare Part D journey for pharmacy service users and nonusers. The platform generated 2.4The pharmacy used purposeful convenience sampling to distribute a paper version of the journey mapping survey to patients between June and August 2019. Recent work developing patient journeys with healthcare services has considering data collection from as few as 8 participants sufficient to develop a patient persona.2.5U tests to identify significant differences in responses between service users and nonusers. Open-ended survey responses were coded by the research team using a qualitative content analysis approach.Journey map outputs were reviewed and discussed with the platform developer. Scaled survey items were analyzed using Mann-Whitney 3,Empathize, Define, Ideate, Prototype, Test.Empathy domain to better understand the patient and their needs. As a result, journey mapping may be a useful tool to focus efforts on defining problems patients may experience throughout their experience, exploring potential solutions to these problems, and pragmatically testing these solutions. The Design Thinking framework and practical applications of the framework's domains like journey mapping may be a viable option in future research for assessing patient-experience with pharmacy services and developing interventions and services using a patient-centered approach.Journey mapping was used as a practical application of the Design Thinking framework.4In total, thirty-six journey map surveys were distributed, completed, and returned to the community pharmacy, for a response rate of 100%.4.1Personas were different in their household incomes. Individuals who did not use the service reported a median household income of $50,000–$74,999 compared to $25,000–$39,999 for those who used the service. Individuals who used the pharmacy consultation service were older than those who did not use the pharmacy consultation service. Journey mapping software outputs for Persona are included in The two patient 4.2Think & Feel category. Patients who used the service had a variety of experiences with the service, ranging from Frustration and Uncomfortable to Good Feelings and .Favorable Comparison Nonusers reported only positive experiences, including Good Experience and Good Feelings. Service users had more Pain and Gain responses than those who did not use the service. For individuals who used the pharmacy service, their overall emotional experience score was 5.8, while individuals who did not use the pharmacy service generated an overall emotional experience score of 6.7. Empathy outputs generated by the third-party journey mapping software are included in Individuals who used the pharmacy service experienced more empathy points in each of the four empathy domains, with the largest visual difference between the groups in the 4.3Current Journey outputs, service users had more extreme Good and Bad Experiences. Service user journeys had more frequent and larger fluctuations in their experience compared to service nonusers. Areas with visual differences in the plan-selection journey appeared to involve collecting and assessing information related to Medicare Part D plans, the need for Medicare Part D assistance late in the decision-making process, and plan selection/decision-making processes. Despite different journeys, both groups appear to end up at similar places, with both groups reporting low optimism regarding their decision. Service users appeared to have more Touch Points during their Medicare Part D plan-selection journey based on the visual depiction of their Current Journey depicted in From the 4.4U tests showed that five survey items had responses with statistically significant differences between service users and nonusers. Most notably, individuals who used the service had a greater appreciation for the features or benefit designs of other plans  and felt they had more opportunities to compare Medicare Part D plans . General item categories and outputs are included in Mann-Whitney 4.5After thematic analysis, the authors agreed on two major themes that describe patient plan-selection experiences: “complexity and uncertainty in the decision-making process” and “the value of trust in the plan selection process.” Representative quotes from service nonusers [NU] and service users [U] may be found in Appendix B.4.5.1You must look at every detail to get the best deal for you-company, pharmacies, medications. [NU9].It is hard to decide if you want a higher deductible or cheaper drug cost. [NU1].Just went to Farm Bureau and let them decide. [NU14].Went with AARP suggestions, overwhelmed with doing it on my own. [NU2].Patients who did not use the pharmacy service appreciated how challenging and complex the plan selection process could be. These individuals expressed varying degrees of frustration with the plan-selection process but often used non-pharmacy resources to help make their plan decision. In some instances, the individuals who did not use a pharmacy service seemed more comfortable deciding on their own and appeared more aware of the various components of benefit design and different components of their specific insurance plan, often engaging in independent research and plan comparison. Individuals who did not use the service used a wide variety of information sources to make their plan decisions, including family, friends, and insurance agents. When individuals sought help, they often let the individual make their plan selection for them.There are so many options for plans [U4].Least enjoyable: comparing what they did or did not cover. So much paperwork to go through [U7].Comparison left me frustrated, went back to my original plan because my agent can help me. [U12].The most enjoyable part was having my options presented to me and explained in an understandable form. [P10].For individuals who did use the pharmacy service, they appeared more aware of the number of plans that were available to them. While some patients appreciated the opportunity to compare plans, many found the plan-comparison process to be frustrating, specifically covering details such as medication coverage. In one instance, the comparison process drove an individual to select a plan from a previous year. While some patients felt the pharmacy service presented options in an understandable form, others reported there were aspects of plan information they did not understand.4.5.2Some uncertainty at first, but confidence because my husband talked to his health insurance agent. [NU12].I took the advice of trusted agents. [NU13].Both groups of individuals had a challenging time trusting the Medicare Part D plan information they received but were aware that plan-switching may be beneficial every year. Individuals who did not use the pharmacy Medicare Part D consultation service were often still uncertain of their plan decision but seemed to have high levels of trust with the individual that assisted them with their plan decision and/or the information they acquired. Ultimately, service nonusers readily accepted the recommendations provided by trusted agents.I needed to trust the person selling it. Hopefully she was honest and not just working to make a sale. [U6].Least enjoyable was trusting the decision I made was best for me [U14].Still not confident I have the best plan for me!! [U14].For individuals using the pharmacy service, trust was more challenging to establish. Patients using the pharmacy service had a difficult time trusting the pharmacy staff member providing the consultation and the information they received. In some instances, patients felt like the pharmacy staff member was trying to “sell them” a specific plan. Ultimately, service users were often uncertain if they had selected the best plan.5The journey mapping process highlighted important differences in the plan-selection experience for service users and nonusers. These differences in experience occurred over the entire service process and were highlighted by differences for users and nonusers in the two personas and the open-ended comments.Commonalities between pharmacy service users and nonusers were that all individuals found the Medicare Part D plan-selection process to be challenging, highlighting that collecting information and assessing it for trustworthiness was difficult, especially in such a technical domain. Both groups reported depending on additional assistance with their Medicare Part D plan selection, but their access to, and use of trusted experts to help with decision-making varied. Despite the differences in information acquisition and plan comparison, both groups still had doubts about their ultimate plan decision, suggesting a target for improvement.One important difference between pharmacy service users and nonusers was their average income level. Older individuals with lower fixed incomes appear to have sought additional support from their community pharmacy more often than younger individuals with more financial resources. People with higher incomes may already have access to services like a financial planner or an insurance agent who may be providing advice. Less well-off or older adults may have less access to these resources.,,Another difference was that individuals who used the Medicare Part D consultation service reported more opportunities for plan comparison and greater awareness of other plan features than those who did not use the pharmacy service. This points to the pharmacy conferring their unique expertise – but perhaps doing so at a level that exceeds the patient's desire for and capacity to use complex information. Consistent with other studies, this level of information overload seems to have led to patient's having a more challenging plan-selection experience.Individuals who did not use the service, on the other hand, appeared to already have a better baseline understanding Medicare Part D insurance and the plan-selection process, often highlighting the importance of understanding specific insurance jargon (i.e. deductible) in their open-ended comments and choosing plans based on this understanding. Individuals who did not use the community pharmacy service may not have been as overwhelmed with information if their agent offered a narrower recommendation, but still left their decision up to an individual they had previously trusted with difficult financial decisions. While, it is difficult to disentangle what effect might be due to education and income and what effect is due to the pharmacy service itself, the data point to targets for service improvement focusing on patient-centered information and facilitating trust within the encounter.5.1,,,With patients making their Medicare Part D plan decision based on the extent to which they could trust the information presented by the pharmacy team, it is important to explore how trust influences older individuals and their decision-making processes.5.2Elaboration Likelihood Model (ELM) may be a useful model for conceptualizing the relationship between patient preferences for information and trust during challenging decision-making experiences like selecting a Medicare Part D plan.,elaborate, or the ability to perform issue-relevant thinking. The extent an individual elaborates influences the types of persuasive messaging and information they may be receptive to, specifically central and peripheral persuasive messaging. Central routes to persuasion often are useful when elaboration is high. Individuals who engage in high levels of issue-relevant thinking undergo careful processing of information and message, ultimately using the information to make an informed decision. Peripheral persuasion is less direct, where individuals use simple decision values like communicator credibility or trustworthiness to make their decision. Elaboration for individuals influenced by peripheral persuasion messages is low, with issue-relevant thinking minimally involved in persuasion and the subsequent decision-making process.The In the context of the Medicare Part D consultation service, patients were exposed to a multitude of plans with an emphasis on specific benefit designs. Patients were required to perform high levels of elaboration, processing large amounts of complex information and use it to make informed Part D plan selections. By providing patients with high-level detailed plan information, the pharmacy assumed that patients were willing and able to elaborate. When faced with plan comparison opportunities via the community pharmacy service, patients were consistently frustrated and reported negative experiences, which may be attributed to moments requiring high levels of elaboration such as processing information and comparing plans to make a plan-switching decision. Patients who did not use the service were infrequently required to elaborate, as patients who did not use the pharmacy service were presented with considerably less information and were infrequently required to compare plans, which resulted in a better experiences potentially due to lower elaboration requirements. When patients who used the pharmacy service were unable to elaborate, they defaulted to heuristics, like the trustworthiness of the pharmacy team, to make their decision. Given that the information presented to patients emphasized cost-related features of specific plan information, patients may have defaulted to a trust heuristic which may have worsened the plan-selection experience. Individuals who were able to elaborate, or unable but not required to elaborate, may have had more positive experiences as information and persuasive messaging were aligned with patient ability and preference.5.3Patient-centered design and consumer behavior research methodologies can be used to refine community pharmacy interventions and the associated impacts of these refinements on patient behavior and outcomes. While we used a journey mapping software platform, more traditional ethnography methodology and participatory approaches can be used to evaluate the patient experience and facilitate a better understanding of the patient decision-making process. To corroborate findings, additional methodologies should be employed, such as patient observations, interviews, and focus groups. There may be additional benefit in prototyping other interaction designs for community pharmacy services, specifically focusing on Medicare Part D consultations, to compare across patient-pharmacist interaction evaluations. For example, how should messages about plan cost be framed so the patient does not feel like the pharmacist has a financial conflict of interest? Research should be conducted to explore differences in patient perceptions of services based upon the person providing the service: pharmacist, pharmacy intern, or pharmacy technician. As journey mapping does not formally test patient preferences and needs related to an experience, more work is needed to formally test and evaluate patient preferences for Medicare Part D consultation service delivery, including the types of information patients prefer, how users would like information presented, and the level of information complexity that users need to make informed Medicare Part D plan decisions.5.4There are several study limitations to consider. Data were collected from a single, rural community pharmacy located in rural Midwest USA. Other pharmacies may have alternative ways of delivering Medicare Part D consultation services and more diverse patient populations may have different experiences. The community pharmacy distributed and collected surveys that may have contributed to social desirability bias, however, patients who used the service reported worse Medicare Part D experiences than those who did not. Both a pharmacist and pharmacy technician delivered the service, and as a result, experiences may have differed based on the service provider. Further, there may have been variations in the patient experience based on the method of service delivery (telephonic vs. in-person). These service variations are likely to contribute to variations in the patient experience but were unable to be linked to specific survey responses. As such, patient preference for delivery method and service provider should be explored in future studies.6Using a journey mapping process that included qualitative and quantitative data collection and analysis depicted the entirety of a patient experience, both good and bad, with selecting a Medicare Part D plan, with or without using a pharmacy-provided Part D plan selection service. Quantitative data triangulated these results and identified key differences in experiences and demographics between community pharmacy Medicare Part D consultation service users and nonusers. Qualitative data provided context and themes informing theoretical applications and explanations associated with the patient experience. By integrating these methodologies, consumer trust literature, and ELM, we can identify clear areas of pharmacy service improvement with recommendations for patient-centered refinement informed by existing theory. Based on these findings, community pharmacies offering Medicare Part D consultations would benefit from redesigning interventions to focus on the way information is delivered to patients, what information is provided, and how they can instill trust in the patients they serve.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.