Inclusion of intake from supplements reduced the proportion with inadequate intake for all micronutrients except calcium among the Wahls group but increased the proportion with excessive intake for vitamin D and niacin among both groups and magnesium among the Swank group. Both diets, especially when including intake from supplements, are associated with reduced inadequate intake compared to the normal diet of people with relapsing remitting MS.The low-saturated fat (Swank) and modified Paleolithic elimination (Wahls) diets have shown promise for MS symptoms; however, due to their restriction of specific foods, inadequate intake of micronutrients is concerning. Therefore, as part of a randomized trial, weighed food records were collected on three consecutive days and were used to evaluate the intake of micronutrients among people with relapsing remitting MS adapting these diets. After randomization to either the Swank or Wahls diets, diet education and support was provided by registered dietitians at baseline and throughout the first 12 weeks of the intervention. Usual intake of each micronutrient was estimated and then evaluated with the EAR-cut point method. At 12 weeks, the Swank group had significant reductions in the proportion with inadequate intake from food for vitamins C, D, and E, while the Wahls group had significant reductions for magnesium and vitamins A, C, D, and E. However, the proportion with inadequate intake significantly increased for calcium, thiamin, and vitamin B Multiple sclerosis (MS) is a chronic neurodegenerative disease that affects nearly 1 million people in the United States ,3 despitPreliminary evidence suggests the low-saturated fat diet developed by Dr. Swank and the modified Paleolithic diet developed by Dr. Wahls are potential adjunct approaches for symptom management . On the Due to the restriction of specific foods in both the low-saturated fat and the modified Paleolithic diets, risk of micronutrient deficiencies is of concern. Inadequate intake of several micronutrients, such as folate and magnesium, is associated with more severe MS symptoms ,21; thusAs part of a 36-week, randomized, parallel-group, single-blinded trial comparing the effect of the low-saturated fat and modified Paleolithic diets on fatigue and quality of life among people with relapsing-remitting MS (RRMS) ; 3-day w2; (3) severe mental impairment; (4) self-reported adverse reactions to gluten-containing foods; (5) diagnosed conditions including eating disorders, severe psychiatric disorders, celiac disease, kidney stones, heart failure, angina, or liver cirrhosis; and (6) insulin, warfarin, radiation, or chemotherapy use. A complete list of inclusion and exclusion criteria can be found in the published protocol [Participants were recruited from within 500 miles of Iowa City, Iowa, and were eligible for enrollment in the study if they (1) had definitive RRMS in accordance with the 2010 McDonald criteria , (2) hadprotocol .n = 87) were randomized at baseline to either the modified Paleolithic elimination  or the low-saturated fat  diets for 24 weeks. During the first 12 weeks of the intervention, participants received two in-person and five telephone-based nutrition counseling sessions from a registered dietitian (RD). Participants also received personalized emails with feedback on their diet checklists every 4 weeks. After 12 weeks of the intervention period, in-person and telephone counseling sessions were discontinued, but participants were allowed to contact the intervention RD for additional support. In addition, the Wahls group participants were instructed to begin reintroducing nightshade vegetables and spices during weeks 13 through 24. The data presented in this article are from the baseline and first 12-week post-randomization period only to avoid variation in diet caused by the reintroduction of the nightshade vegetables and spices among the Wahls group and to limit bias introduced due to dropouts. Data on participants who completed the first 12 weeks of the intervention (n = 39 Wahls and n = 38 Swank) was included in the present secondary analysis  unsaturated fat and four servings each of grains (whole grains preferred) and fruits and vegetables (FV) per day . The Wah80 ng/mL . Adheren80 ng/mL .Three weighed food records were collected consecutively on two weekdays and one weekend day at baseline and again at 12 weeks. Food records obtained from included participants were analyzed using Nutrition Data System for Research software (University of Minnesota Nutrition Coordinating Center). To account for the differing bioavailability of folate and folic acid, we used dietary folate equivalents (DFEs) for all analyses. Because tryptophan can partially meet niacin requirements, niacin equivalents (NEs) were calculated as 60 mg tryptophan is equal to 1 mg niacin. Bioconversion of provitamin A carotenoids were accounted for by using retinol activity equivalents (RAEs) for vitamin A, and bioconversion of tocopherols was accounted for by using alpha-tocopherol equivalents for all vitamin E analyses. Mean intake of each micronutrient from food was calculated for each participant and adjusted for age, gender, and corresponding 2007 Harvard Food Frequency Questionnaires (FFQs) values using the National Cancer Institute (NCI) method to estimate usual intake . Usual iz-tests. The 12-week difference between groups in inadequate micronutrient intake was compared with two-sample proportion z-tests. All analyses were conducted using two-tailed tests, and significance was concluded when alpha ≤ 0.05.Fisher’s exact test was used to compare categorical variables between groups at baseline , and generalized linear models were used to compare continuous variables  between groups at baseline. The within-group baseline to 12-week changes in inadequate micronutrient intake were compared using one-sample proportion 6, 21.6% for folate, 20.7% for vitamin A, 17.4% for zinc, 14.0% for vitamin B12, 12.6% for iron, 11.5% for thiamin, 8.7% for copper, 5.3% for riboflavin, 3.5% for phosphorus, 2.8% for niacin, and 2.7% for selenium (p < 0.01), E , and D . At 24 weeks, the proportion with inadequate intake from food was significantly lower than baseline for vitamins C , D , and B6 . The proportion with inadequate intake of vitamin A from food was significantly higher at 24 weeks compared to baseline intake . Inclusion of intake from supplements lead to a further reduced proportion with inadequate intake for copper; magnesium; zinc, and the vitamins A, C, D, E, B6, and thiamin at 12 and 24 weeks , vitamin C , vitamin D , vitamin E , and magnesium . The Wahls group also had significant increases in the proportion with inadequate intake from food for calcium , thiamin , and vitamin B12  at 12 weeks. All differences remained significant at 24 weeks compared to baseline; however, the proportion with inadequate intake from food at 24 weeks increased for vitamin C  and copper  compared to week 12 values. Inclusion of intake from supplements lead to a further reduced proportion with inadequate intake for magnesium; zinc; and the vitamins A, C, D, E, B6, riboflavin, thiamin, and folate at 12 and 24 weeks  and copper (p = 0.05) at 12 weeks and for vitamin A (p < 0.01) and zinc (p = 0.04) at 24 weeks (p < 0.01) at 12 weeks and thiamin at 24 weeks compared to the Wahls group. After inclusion of intake from supplements, the Swank group had a higher proportion with inadequate intake of vitamin B12 (p = 0.02) and lower proportion with inadequate intake of calcium (p = 0.02) compared to the Wahls group at both 12 and 24 weeks , but no other micronutrient was above 10% for either group . The pro12 among the Wahls group, for which the proportion with inadequate intake from food increased significantly; however, only calcium remained increased after the inclusion of supplements.The findings from this analysis of 3-day weighed food records show that both the low-saturated fat diet proposed by Swank and the modified Paleolithic elimination diet proposed by Wahls are associated with reduced proportion of or no change in micronutrient inadequate intake compared to the usual diet of people with RRMS. This was with the exceptions of calcium, thiamin, and vitamin BThe usual diet of both groups in this study at baseline were severely inadequate (>20% of the group) in terms of intake of calcium; magnesium; and the vitamins A, C, D, E, and folate. The same micronutrients are also severely inadequately consumed among the general U.S. adult population . Similar6, C, D, and E compared to their usual diet at baseline. The reduction in inadequate vitamin C intake is likely due to the recommendation for 2+ servings each of fruits and vegetables per day that are rich sources of vitamin C. The reduction in the proportion with inadequate vitamin D intake from food observed among the Swank group is likely due to consumption of vitamin D-fortified foods such as orange juice and non-fat dairy products. However, despite the decreases in the proportion with inadequate intake for vitamins C, D, and E, intake from food for all are still considered severely inadequate at 12 weeks. Similarly, the proportion with inadequate intake from food for folate, calcium, magnesium, and zinc remained above 20% at 12 weeks. However, the proportion with inadequate intake was reduced to below the 20% threshold for all micronutrients except calcium with the inclusion of intake from supplements. These observations are consistent with previous analyses of experimental menus [In this study, the Swank group had significant reductions in the proportion with inadequate intake from food for vitamins Bal menus  and an oal menus . 12, which likely explains the significant increase in the proportion with inadequate intake for calcium and vitamin B12 at 12 weeks. Similarly, the Wahls elimination diet recommends strict avoidance of grains, which are a rich source of thiamin and likely explain the significant increase in the proportion with inadequate intake of thiamin at 12 weeks. The proportion with inadequate intake for calcium, vitamin D, vitamin E, iron, and magnesium all remained above 20% at 12 weeks among the Wahls group, but despite the increased proportion with inadequate intake for thiamin and vitamin B12, proportion with inadequate intake from food for these nutrients was above the 20% threshold for severe inadequate intake at 12 weeks, and only calcium and iron were still above 20% after inclusion of intake from supplements. These observations are consistent with analysis of 24-hour dietary recalls from a preliminary trial [The Wahls group had significant reductions in the proportion with inadequate intake from food for vitamin A, vitamin C, vitamin D, vitamin E, and magnesium at 12 and 24 weeks compared to their usual diet at baseline. The Wahls elimination diet recommends 6–9+ servings per day total fruits and vegetables, 12 ounces per week organ meats, and 4 ounces per day nuts and seeds, which are rich sources of the micronutrients that improved with the diet intervention. However, the Wahls elimination diet recommends strict avoidance of dairy, a rich source of calcium and vitamin Bry trial , our prery trial , and an ry trial . The severe inadequate intake from food of vitamin D, vitamin E, calcium, and magnesium was present among both groups during the intervention, highlighting the importance of supplementation of select micronutrients among people with MS following either the Swank or Wahls diets. Inclusion of intake from supplements reduced the proportion with inadequate intake for most micronutrient shortfalls. In addition to supplementation, pwMS following either diet may benefit from selecting specific micronutrient-dense foods such as sunflower seeds for vitamin E or pumpkin seeds for magnesium. Because both diets restrict or partially restrict dairy, recommendations for the consumption of calcium-fortified foods such as fortified juices or milk alternatives or calcium supplementation may be beneficial. For micronutrients with few rich food sources such as vitamin D, supplementation is recommended for pwMS , especiaWith combined intake from food and supplements, excessive intakes of several micronutrients are of concern. In both groups, a high proportion with excessive intake was observed for vitamin D and niacin, and for magnesium in the Swank group. As part of the intervention of this study, vitamin D doses were adjusted on the basis of serum laboratory values, and therefore it is uncertain how generalizable the excessive vitamin D intake from food and supplements is in this study. In the healthy population, prolonged excessive intake may increase the risk for micronutrient toxicity; however, it is possible that the chronic inflammation and immunologic activity associated with MS increases micronutrient needs of people with MS. Future studies are needed to evaluate micronutrient status with micronutrient biomarkers to determine the specific micronutrient requirements of pwMS.n = 50) to adjust usual intake; however, dietary data were collected on more days than necessary for the NCI method to help reduce variation caused by the lack of sample size. Finally, laboratory evaluation of micronutrient status with biomarkers was not conducted for nutrients included in this analysis other than vitamin D, and therefore we were unable to determine if inadequate or excessive intake of micronutrients in this study was associated with deficiency or toxicity among pwMS following the Swank or Wahls diets. This study is strengthened by the use of 3-day weighed food records, robust analytical methods, and high diet adherence reported previously as 87% and 80% at 12 weeks for the Swank and Wahls diets, respectively . HoweverThere is great interest among the people with MS and researchers alike for the use of specialized diets as adjunct therapy to improve quality of life and reduce fatigue; however, the restrictive nature of several of these specialized diets causes concern for risk of micronutrient deficiencies. This study is one of the first major attempts to evaluate the intake of micronutrients of diets specialized for MS. These results indicate that compared to the usual diet of people with RRMS, the Swank and Wahls diets are associated with reductions in the proportion with inadequate intake for several micronutrients, especially with intake from supplements, for which deficiency may be associated with symptom burden including magnesium and the vitamins A, C, D, and E.


E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of  Collagenases are essential components of the matrix metalloproteinases family of proteins ; their mN-termini based on fluorescamine [The range of research questions and applications that stem from various functional aspects of collagenases requires an efficient activity assay. Such a setup must be cost-effective, robust, and amenable to further adaptation to automated high throughput screening platforms. However, the majority of currently available assays for monitoring enzymatic activity of collagenases are based on a fluorescently labeled peptide mimicking a single collagen unit, or on the detection of hydroxyproline ,26,27,28escamine . These tN-terminus Gly [Herein, we adapted a method developed for fluorescence-based detection of peptides containing an inus Gly . Based oClostridium histolyticum such as collagenase G, H and T were highly characterized both structurally and biochemically [E. coli BL21 (DE3). Following transformation of the plasmid containing the ColG gene, the bacterial culture was grown at 37 °C. At OD = 0.8, induction was executed by the addition of 1 mM IPTG. Human collagenases have relatively high specificity and often cleave collagen at a single site. In contrast, bacterial collagenases were shown to have broad substrate specificity and are capable of digesting collagen at multiple sites. Among these bacterial enzymes, collagenases from emically ,8,34,35.M value of ColG towards collagen in its purified form and in the lysate  = 1.19 × Vmax (lysate). Enzymatic reaction was performed at 25 °C as collagen type I is thermally instable above 30 °C [After setting an established expression and purification protocol for ColG, and considering the need for an activity assay within the bacterial lysate, we tested activity using the fluorogenic reagent 3,4-dihydroxyphenylacetic acid (DHPAA) assay  at a rane lysate A. In addlagenase . Figure ve 30 °C .E. coli BL21 cells expressing ColG in 2 mL-deep 96-well plates. Following bacterial lysis and centrifugation, 16 µL aliquots were transferred into a new 96-well plate and incubated with 1 mg/mL collagen for 1 h at 25 °C with shaking, followed by the addition of the reaction reagents. E. coli BL21 that was not transformed with ColG plasmid (4th bar from left). On the other hand, activity of purified ColG with collagen and 3,4-DHPAA resulted in a similar intensity to that of lysates of bacteria harboring ColG plasmid. This showed that the florescence signal observed in the assay was the result of collagen’s degradation by the inducible, recombinant expressed ColG in the bacterial lysate.We then investigated if the activity and readout were indeed specific to the degradation of collagen by ColG. To this end, we cultured 500 µL Since we aimed to establish a bacterial-based assay that was amenable to screening and that could be exploited for the discovery of optimized variants of collagenases with improved rates of kinetics, identifying the optimal lysate volume and sampling time that would enable us to follow the initial velocity rate in its linear phase was important. This would ensure that optimized enzymes showed higher fluorescence intensity, which is correlated with higher activity. Thus, in our next step, we characterized the reaction kinetics in the 96-well-plate format.E. coli BL21 cells resulted in distinct colonies that represented the genetic library, each harboring specific mutations of the variant ColG. Following transformation and plating, selected colonies from the agar plate were cultured, lysed, and tested for ColG activity in the 96-well plate.Once we established a robust protocol to detect collagenase activity in the bacterial lysate as a result of inducing ColG recombinant expression, we constructed a focused genetic library of ColG. This aimed at simultaneous screening of hundreds of variants for identifying improved collagenase activity. Considering the high theoretical diversity of such a library and the currently limited capacity to screen it, the mutation rate was calibrated to incorporate about eight nonsynonymous mutations per ColG gene. The library was generated via the application of an error-prone PCR, thus resulting in random and noncontrollable mutational positions. Transformation of the library into A question may arise if structural changes appear due to the mutation. Given the assay specificity, size of the protein, and location of the mutation, we reasoned that no extreme structural change occurred, and the overall fold was maintained, primarily as activity was maintained. However, to explore a possible mechanism for the improved activity of the ColG variant, we modeled this variant following 1ns MD. E. coli BL21 cells were transformed with plasmid containing the mutated gene. Induction of protein expression and subsequent purification were executed in a similar way to that of the wild-type enzyme. In our next step, we evaluated the collagen-degradation kinetics of the wild-type and ColG-F782S variant in their purified form. To this end, E. coli within the bacterial lysate, without the need for further purification. The latter is of immense importance for screening genetic libraries, to identify optimized variants when each colony harbors a different variant of the target enzyme. An additional important factor is the use of bacteria as the host organism. The use of more advanced systems, such as yeast or even mammalian cells, has several advantages in the field of protein-directed evolution [E. coli is an attractive choice as a host organism due to its large selection of cloning vectors and strains, as well as its rapid and well-regulated growth rate. A major disadvantage of the proposed strategy is its relatively low throughput compared to other display-based methods. However, computational predictions and the semirational design of the mutational space can compensate the screening space by generating a focused library based on in silico filtering, thus enabling an efficient screening strategy. The current work employed the screening of 96-well plates, in which few colonies showed slight improved activity, but the activity of most of the samples was reduced. It is likely that the exact number of mutations per gene should be calibrated for each collagenase enzyme, and that the insertion of eight mutations per gene was too high. Indeed, the selected colony had a single mutation in the amino acid level. Thus, thorough calibration of the mutational number should be considered.Degradation of the triple-helix collagen is an important step in the range of cellular paths, as well as in biotechnology applications. The process is most-efficiently executed by bacterial collagenases. Regardless of the degradation mechanism, a robust assay for evaluating the rate of degradation is essential for identifying inhibitors and optimized variants. To this end, and based on a previously reported assay , we genevolution . HoweverAn additional factor to note is the molecular-level origin of the improved activity. Given the relatively large molecular weight of the target protein, mutations could arise in regions not directly related to the enzymatic activity. However, such mutations may lead to enhanced stability or higher expression levels of the variant. In the current protocol, all the mutations translated to a higher fluorescent readout. Thus, the structure-function aspects must be further deciphered in order to integrate successful substitutions into a potent protein variant. N-terminus 6xHIS tag, followed by a cleavable TEV site. The plasmid was transformed to E. coli BL21 and cells; at OD = 0.8, protein expression was induced by the addition of 1 mM IPTG. Following overnight culturing at 25 °C, the cells were harvested. The harvested cells were resuspended in 40 mL of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 30 mM imidazole. The suspended cells were then disrupted by sonication, and the insoluble fraction was removed by centrifugation for 20 min at 15,000× g. The supernatant was applied to a 5 mL column of nickel beads. After washing the resin, the protein was eluted with the addition of 300 mM imidazole. For the execution of the enzymatic assay in the 96-well format, bacterial cells were disrupted by the addition of ‘Bug-Buster’ reagent according to the manufacturer’s instructions, with the addition of 0.4 mg/mL of lysozyme and 250 units/10 mL benzonase.The plasmid expressing ColG was a gift from Hans Brandstetter . The genAn enzymatic assay was repurposed based on a previously described protocol . PurifieE. coli BL21 cells and plated on an LB plate supplemented with 100 mg/mL ampicillin. Individual colonies were randomly selected and grown overnight in 96-well deep-well plates containing 500 mL LB supplemented with 100 mg/mL ampicillin at 37 °C, with shaking. The overnight culture was used to inoculate (at 1:20 dilution) fresh 500 mL LB supplemented with 200 mg/mL ampicillin in 96-well deep-well plates. The cells were grown at 30 °C, with shaking, for about 4 h, to an OD600 = 0.8, and induced with 1 mM IPTG to induce expression of the ColG variants. Following overnight incubation at 25 °C, the cells were pelleted and resuspended in Bug-Buster lysis buffer for further enzymatic activity assay.A genetic library was constructed from the ColG gene with the GeneMorph II Random Mutagenesis Kit , adjusted to produce an average of 6 nonsynonymous mutations per gene. Following mutagenesis PCR, libraries were cloned back into the original vector. The cloned vectors were transformed into − and Na+) were inserted to achieve a neutral simulation cell. Energy minimization was carried out using the steepest descent algorithm, followed by the conjugate gradient algorithm. The system was then equilibrated with a 100 ps MD simulation in the canonical (NVT) ensemble, using the modified Berendsen thermostat [All simulations were performed using the GROMACS software version 2020.1 ,39 and termostat  for fixiermostat . These wermostat .4 was purchased from Thermo Fisher Scientific, Waltham, MA, USA. The Bug-Buster used for bacterial lysis was purchased from Merck (Millipore). The lysozyme was purchased from Merck, Darmstadt, Germany, and the benzonase was purchased from Santa Cruz, Dallas, TX, USA. All other chemicals and reagents were purchased from Merck.The collagen-I at a concentration of 1 mg/mL (Cat#92695), 3,4-DHPAA, and boric acid were purchased from Merck (Sigma), Darmstadt, Germany. The NaIO


Some earlier studies have found indications of significant changes in cardiometabolic risk factors in children born after assisted reproductive technology (ART). Most of these studies are based on small cohorts with high risk of selection bias. In this study, we compared the risk of cardiovascular disease, obesity, and type 2 diabetes between singleton children born after ART and singleton children born after spontaneous conception (SC).p = 0.02), obesity , and type 2 diabetes . After adjustment, there was no significant difference between children born after ART and children born after SC for any cardiovascular disease  or type 2 diabetes . For any cardiovascular disease, the 95% CI was reasonably narrow, excluding effects of a substantial magnitude, while the 95% CI for type 2 diabetes was wide, not excluding clinically meaningful effects. For obesity, there was a small but significant increased risk among children born after ART . Important limitations of the study were the relatively short follow-up time, the limited number of events for some outcomes, and that the outcome obesity is often not considered as a disease and therefore not caught by registers, likely leading to an underestimation of obesity in both children born after ART and children born after SC.This was a large population-based cohort study of individuals born in Norway, Sweden, Finland, and Denmark between 1984 and 2015. Data were obtained from national ART and medical birth registers and cross-linked with data from national patient registers and other population-based registers in the respective countries. In total, 122,429 children born after ART and 7,574,685 children born after SC were included. Mean (SD) maternal age was 33.9 (4.3) years for ART and 29.7 (5.2) for SC, 67.7% versus 41.8% were primiparous, and 45.2% versus 32.1% had more than 12 years of education. Preterm birth (<37 weeks 0 days) occurred in 7.9% of children born after ART and 4.8% in children born after SC, and 5.7% versus 3.3% had a low birth weight . Mean (SD) follow-up time was 8.6 (6.2) years for children born after ART and 14.0 (8.6) years for children born after SC. In total, 135 (0.11%), 645 (0.65%), and 18 (0.01%) children born after ART were diagnosed with cardiovascular disease , obesity or type 2 diabetes, respectively. The corresponding values were 10,702 (0.14%), 30,308 (0.74%), and 2,919 (0.04%) for children born after SC. In the unadjusted analysis, children born after ART had a significantly higher risk of any cardiovascular disease .Most earlier studies are based on small cohorts with high risk of selection bias. Furthermore, most earlier studies have measured markers and risk factors for disease instead of clinical diseases.In this study we wanted to compare the risk of cardiovascular disease, obesity, and type 2 diabetes between singleton children born after ART and singleton children born after spontaneous conception (SC).n = 122,429) and SC  between 1984 and 2015 in Norway, Sweden, Finland, and Denmark.By combining high-quality Nordic registers, we included singleton children born after ART (The findings showed that children born after ART had no increased risk of cardiovascular disease or type 2 diabetes after adjustment for measured confounders. For any cardiovascular disease, the 95% CI was reasonably narrow, excluding effects of a substantial magnitude, while the 95% CI for type 2 diabetes was wide, not excluding clinically meaningful effects.We observed a slightly increased risk of obesity in children born after ART.Since type 2 diabetes and most of the cardiovascular diseases had a limited number of events, the ART population is relatively young, and there was a slightly increased risk of obesity in children born after ART, there is a need for further studies with longer follow-up in this area. The use of assisted reproductive technology (ART) has increased significantly during the last decades, with more than 390,000 children born worldwide annually  and the A major problem is that most published studies are based on small cohorts  aThis was a population-based cohort study carried out in Norway, Sweden, Finland, and Denmark. Data were obtained from the CoNARTaS cohort (Committee of Nordic Assisted Reproductive Technology and Safety) and then cross-linked with data from national patient registers (NPRs), national cause of death registers (CDRs), SWEDIABKIDS (Swedish childhood diabetes register), the Swedish National Diabetes Register (NDR), the Swedish Prescribed Drug Register (SPDR), and BORIS (Swedish childhood obesity treatment register). Socioeconomic data were retrieved from the statistic bureaus in each country. Major birth defects, registered during the first year of birth, were defined according to the EUROCAT classification system . CongeniThe study is reported as per the Reporting of studies Conducted using Observational Routinely-collected health Data (RECORD) guideline . The anaThe CoNARTaS cohort consists of all children born after ART or SC between 1984 and 2015 in Norway, 1985 and 2015 in Sweden, 1990 and 2014 in Finland, and 1994 and 2014 in Denmark. The data were obtained from the national ART registers and medical birth registers (MBRs), all with high quality . Some ofThe MBRs have covered nearly all births in the Nordic countries over several decades. Reporting to the registers is mandatory . In thisIn Norway public and private ART clinics report detailed information to the MBR on all ART cycles that result in ongoing pregnancies verified by ultrasound in gestational week 6−7 . In SwedThe NPRs include diagnoses for all inpatient care since 2008 in Norway, 1987 in Sweden, 1967 in Finland, and 1977 in Denmark. Outpatient visits in public hospitals and specialized healthcare in private clinics have been included since 2008 in Norway, 2001 in Sweden, and 1998 in Finland. In Denmark, outpatient visits in public hospitals have been registered since 1995, and information about specialized healthcare in private clinics since 2003 . The regThe NDR started in 1996. All individuals in Sweden with diabetes mellitus, except for gestational diabetes, from the age of 18 years are included. Today, the register covers 98% of specialized inpatient care and 92% of outpatient visits .SWEDIABKIDS started in year 2000 and is incorporated as a part of the NDR. The register includes children and adolescents in Sweden with a diagnosis of diabetes mellitus under the age of 18 years .The SPDR linked with personal identity number started in July 2005. The SPDR contains all filled prescriptions in Sweden for drugs and includes data about name of drug, Anatomic Therapeutic Chemical (ATC) code, date of prescription, date when the product was collected, and prescription amount .BORIS was established in 2005. The register includes children under 18 years of age who are treated for obesity at pediatric clinics or in hospital care in Sweden. According to the annual report of BORIS, the coverage ratio increased rapidly from 2008, and from 2013 approximately 80% of children treated for obesity are included in the register .The CDRs include all individuals who have died, either in the country or abroad, and who were registered in the country at the time of death. The register started 1951 in Norway , 1952 inThe statistical bureaus are the central authorities for recording statistics on all individuals in the respective countries and contain information such as educational level, country of birth, and emigration. In Norway, information about emigration comes from the National Population Register, administered by the Norwegian Tax Administration.During the time period from 1 January 1984 to 31 December 2015 in Norway, 1 January 1985 to 31 December 2015 in Sweden, 1 October 1990 to 31 December 2014 in Finland, and 1 January 1994 to 31 December 2014 in Denmark, 7,946,009 children were born (stillbirths excluded). We excluded 243,995 multiple births, 2,585 children born after oocyte donation (OD) or missing information about OD , 2,255 children born after sperm donation (only from Sweden and Denmark since there was no available information from Norway and Finland), and 60 children with unknown sex. Consequently, 248,895 children were excluded and 7,697,114 children were included in the study .The primary outcomes were any cardiovascular disease, obesity, and type 2 diabetes.“Any cardiovascular disease” was defined if the child had a discharge diagnosis from hospital (inpatient register) with a code for any specific cardiovascular disease in ICD-8, ICD-9, or ICD-10  or a codObesity was defined if the child had a discharge diagnosis from hospital or outpatient visits with a specific code for obesity in ICD-8, ICD-9, or ICD-10 . Since oType 2 diabetes in the children was, in Norway, Finland, and Denmark, classified exclusively from data in the NPRs. In these countries, type 2 diabetes was defined if the child had a discharge diagnosis from hospital or outpatient visits with the ICD-10 code for type 2 diabetes . Before All children who were diagnosed with both type 1 and type 2 diabetes were considered as having type 1 diabetes and excluded as events, due to their young age and therefore much greater probability of having type 1 diabetes. Maternal diabetes was defined as any diabetes (type 1 or 2 diabetes). In Norway, Finland, and Denmark this was defined if the mother had a discharge diagnosis from hospital or outpatient visits with the ICD-10 code for type 1 or 2 diabetes . In SwedA fixed set of covariates was selected a priori. Child characteristics were sex, calendar year of birth (continuous variable), and country of birth. Maternal characteristics included age at birth of child (continuous variable), smoking at first antenatal visit (yes or no), highest educational level , any cardiovascular disease, obesity, and any diabetes before or at birth of child.Approval for data retrieval was obtained in each country. In Norway, ethical approval was given by the Regional Committee for Medical and Health Research Ethics . In Sweden, the Regional Ethical Committee at the University of Gothenburg approved the study . In Denmark and Finland, ethical approval is not required for register-based studies.n) and percent for categorical variables and as mean and standard deviation (SD) and/or median and range for continuous variables. Cox proportional hazards models were used to calculate the risk of any cardiovascular disease, obesity, and type 2 diabetes. We used age as the time scale and included each child’s time at risk computed from the date of birth until whichever event occurred first: diagnosis of any cardiovascular disease, obesity, or type 2 diabetes; emigration (not available for Finland); death; or end of the follow-up period .Descriptive statistics are presented as frequency , any maternal obesity before or at birth (for the outcome obesity), and any maternal diabetes before or at birth (for the outcome type 2 diabetes). For the outcome type 2 diabetes, no adjustment for maternal age or child’s country of birth was made, due to there being few events.Cardiovascular disease, obesity, and type 2 diabetes were analyzed separately, i.e., if a child was diagnosed with obesity, the child continued to be followed up in the study for the outcomes type 2 diabetes and cardiovascular disease. The same child could occur in several subgroups of cardiovascular disease but only 1 time in “any cardiovascular disease.” We estimated unadjusted and adjusted HRs (aHRs) with 95% confidence intervals (CIs) using Cox regression (maximum likelihood). Corresponding Three sensitivity analyses were performed. Since the NPR in Norway first started in 2008 and since there was no information about educational level from Norway and a substantial proportion with missing data on smoking in Norway, we performed a sensitivity analysis in which data from Norway were excluded. In this analysis, adjustments were made for the same variables as in the main analysis and also for maternal highest educational level and maternal smoking at first antenatal visit. Missing data for smoking were imputed as no smoking in the regression analyses. To evaluate what impact this approach had on the results, we also performed analyses in which missing data for smoking were not imputed. In another sensitivity analysis, children born between 1996 and 2005 were analyzed for the outcome any cardiovascular disease (too few events for type 2 diabetes). This analysis was performed because the follow-up time in the total cohort differed considerably between children born after ART and children born after SC, and a potential underestimation of the true association between ART conception and cardiovascular diseases and type 2 diabetes could exist. In this analysis, the same adjustment was made as in the main analysis for any cardiovascular disease. In a third sensitivity analysis, for type 2 diabetes we further investigated the opposite directions of associations from the overall incidence rates and from the Cox regression. To account for the differences in mean follow-up time after ART and SC, we drew a random sample of 12 children born after SC for every child born after ART, matched on year of birth and country.Finally, 2 subgroup analyses, including data from Norway, Sweden, and Denmark, were performed in which children born after FET were compared to children born after fresh embryo transfer, and children born after ICSI were compared to children born after IVF. In these analyses, adjustment was made for child sex, calendar year of birth, maternal age, maternal any cardiovascular disease (for the outcome any cardiovascular disease), and maternal any obesity before or at birth (for the outcome obesity). The same subgroup analyses were also performed but with data only from Sweden and Denmark. In these analyses, adjustments were made for the same variables as in the subgroup analyses with data from Norway, Sweden, and Denmark and also for maternal highest educational level and maternal smoking at first antenatal visit. We did not adjust for gestational age, birth weight, or birth defects in any of the analyses since these are potential causal pathway characteristics. The analyses were performed in Stata version 15.1.In total 7,697,114 children were included in the analysis. Of these, 122,429 children were born after ART and 7,574,685 were born after SC ; Table 1p = 0.02). Children born after ART also had a significantly higher risk of being registered with an obesity diagnosis than children born after SC, with 96.8 compared to 93.9 per 100,000 person-years at risk , and type 2 diabetes, with 1.7 compared to 2.7 per 100,000 person-years at risk  (p = 0.80) or type 2 diabetes . For obesity, however, there was still a significant difference between the 2 groups   . After a= 0.001) . HRs forp = 0.96; obesity: aHR 1.21; 95% CI 1.11–1.31; p < 0.001; type 2 diabetes: aHR 1.45; 95% CI 0.87–2.41; p = 0.16) . Another sensitivity analysis was performed in which children born between 1996 and 2005 were compared for the outcome any cardiovascular disease. The mean follow-up time in this cohort was 13.7  years for children born after ART and 14.4  years for children born after SC. The analysis did not find any significant difference between children born after ART and children born after SC, neither in the unadjusted  nor in the adjusted analysis   . In this = 0.44) .p = 0.36).In the sensitivity analysis with a matched sample for type 2 diabetes, the overall incidence rates were consistent with the results from the Cox regression: 1.7/100,000 person-years at risk for children born after ART compared to 1.3/100,000 person-years at risk for children born after SC  or obesity  in the adjusted analyses  children born after FET, 93 (0.12%) children born after fresh embryo transfer, 51 (0.13%) children born after ICSI, and 69 (0.12%) children born after IVF developed cardiovascular disease during the study period. The corresponding values for obesity were 81 (0.50%) for children born after FET, 433 (0.68%) for children born after fresh embryo transfer , 219 0.. Neither% childreThis large, Nordic population-based cohort study included 122,429 singletons born after ART and 7,574,685 singletons born after SC and explored whether there was any difference in the risk of developing cardiovascular disease, obesity, or type 2 diabetes between the 2 groups. The main findings were that children born after ART had no increased risk of cardiovascular disease or type 2 diabetes after adjustment for measured confounders, while for obesity a small but significant increased risk was noticed. For the outcome any cardiovascular disease, the 95% CI was reasonably narrow, excluding effects of a substantial magnitude, while the 95% CI for type 2 diabetes was wide, not excluding clinically meaningful effects.Overall, the findings from this study give no reason for immediate worries, in contrast to earlier systematic reviews and meta-analyses ,20, in w2), compared to controls [The finding that children born after ART had an increased risk of cardiovascular disease in the unadjusted analysis but no increased risk after adjustment for confounders such as maternal age and maternal cardiovascular disease suggests that the ART technique itself does not cause the differences between the groups. Instead, observed differences could be an effect of parental characteristics. However, there is an increased risk of hypertensive disorders in women pregnant after ART, and the risk is higher after FET than after fresh embryo transfer, indicating that the technique also may have an effect –48. Thercontrols .Cardiovascular disease in children has different etiology than cardiovascular disease in adults. Whereas risk factors for ischemic heart disease, heart failure, and stroke in adults are hypertension, smoking, atrial fibrillation, and diabetes, congenital heart disease is an important risk factor for these diseases in children –52. EarlThe finding in the present study that there was no significant difference in the risk of developing type 2 diabetes between children born after ART and children born after SC, after adjustment, was based on few events (18 children born after ART with type 2 diabetes) and should therefore be interpreted with caution. In the meta-analysis from 2017 —of 380 cThe observation of an increased risk of obesity among children born after ART may have several explanations. Since obesity is a diagnosis not causing severe symptoms, the willingness to seek treatment may vary. There is a possibility that parents of children born after ART seek treatment to a greater extent than other parents do, or that children born after ART have other conditions that make them see the doctor more often, such as sequelae from preterm birth. Another complicating factor is the rather late start of the outpatient registers, since obesity mainly is an outpatient diagnosis. Still, several mechanisms may explain why children born after ART could have an increased risk of obesity. Epigenetic and genetic changes associated with ART have been observed in different studies –58. EpigA major strength of this study is the large size and population-based design, making it possible to study diseases that are rare in the ART population, where most individuals are still young. Moreover, the study is based on national registers with almost complete coverage rates and high validity, minimizing missing data and the risk of selection bias. Finally, it was possible to adjust for several relevant confounders, such as maternal cardiovascular disease, obesity, and diabetes.An important limitation of the study was the relatively short follow-up time, which also was considerably shorter in children born after ART compared to children born after SC. Thus, there might be an underestimation of the true association between ART conception and cardiovascular diseases and type 2 diabetes. Yet, a sensitivity analysis of children born after ART and SC born between 1996 and 2005 did not find any difference in the risk of developing any cardiovascular disease between children born after ART and SC, suggesting no underestimation of the risk of cardiovascular disease in children born after ART. Still, since cardiovascular disease and type 2 diabetes have a much higher incidence and prevalence in older individuals, the results are only generalizable in a young population. Further limitations were differences between countries in the time periods for data collection, ART registration processes, NPR coverage rates, and dataset sources, which may all create variability. An analysis with a 2-level model with country as the higher level might have been appropriate. However, due to the small number of clusters (4 countries), the large number of observations, and the complex frailty models needed to handle multiple levels in survival analysis, we were not able to come up with a model giving stable and reasonable estimates. We only added country as a covariate in the survival analysis, and it should be noted that we might have underestimated the standard errors. Data on outpatient visits were missing in the beginning of the study period, which resulted in the decision to exclude children who were born before the start of the outpatient registers of the NPRs in the respective countries. This exclusion probably resulted in a substantial loss of obesity data. The obesity data also do not capture obese individuals who have not attended inpatient or outpatient clinics, or in whom obesity was not recorded as a diagnosis. Consequently, the definition of obesity cannot be expected to reflect the true rate of obesity in the general population. Furthermore, the large proportion of missing data on maternal BMI made it impossible to adjust for in the analyses. Instead, we used the ICD code for obesity in the adjustments. Paternal characteristics were also missing, and data on ethnicity were not available due to the European General Data Protection Regulation. Lastly, there was also a risk of unmeasured and unknown confounders.We conclude that children born after ART had no increased risk of cardiovascular disease and type 2 diabetes after adjustment for known confounders, while a small but significant increase of obesity was noticed. For any cardiovascular disease, the 95% CI was reasonably narrow, excluding effects of a substantial magnitude, while the 95% CI for type 2 diabetes was wide, not excluding clinically meaningful effects. The fact that type 2 diabetes and most of the cardiovascular diseases had a limited number of events, and that there was a slightly increased risk of obesity in children born after ART, suggests a need for further studies with longer follow-up. Small differences in a young population could potentially have major consequences in the future.S1 FigART, assisted reproductive technology; CVD, cardiovascular disease; SC, spontaneous conception.(TIFF)Click here for additional data file.S2 FigART, assisted reproductive technology; SC, spontaneous conception.(TIFF)Click here for additional data file.S3 FigART, assisted reproductive technology; SC, spontaneous conception.(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.


Dietary fibers are widely considered to be beneficial to health as they produce nutrients through gut microbial fermentation while facilitating weight management and boosting gut health. To date, the gene expression profiles of the carbohydrate active enzymes (CAZymes) that respond to different types of fibers  in the gut microbes of pigs are not well understood. Therefore, we investigated the functional response of colonic microbiota to different dietary fibers in pigs through metatranscriptomic analysis.Parabacteroides, Ruminococcus, Faecalibacterium and Alloprevotella but decreased Sutterella; INU increased the relative abundance of Fusobacterium and Rhodococcus but decreased Bacillus; and PEC increased the relative abundance of the Streptococcus and Bacteroidetes groups but decreased Clostridium, Clostridioides, Intestinibacter, Gemmiger, Muribaculum and Vibrio. The gene expression of CAZymes GH8, GH14, GH24, GH38, GT14, GT31, GT77 and GT91 downregulated but that of GH77, GH97, GT3, GT10 and GT27 upregulated in the RPS diet group; the gene expression of AA4, AA7, GH14, GH15, GH24, GH26, GH27, GH38, GH101, GT26, GT27 and GT38 downregulated in the INU group; and the gene expression of PL4, AA1, GT32, GH18, GH37, GH101 and GH112 downregulated but that of CE14, AA3, AA12, GH5, GH102 and GH103 upregulated in the PEC group. Compared with the RPS and INU groups, the composition of colonic microbiota in the PEC group exhibited more diverse changes with the variation of CAZymes and Streptococcus as the main contributor to CBM61, which greatly promoted the digestion of pectin.The results showed that the microbial composition and CAZyme structure of the three experimental groups changed significantly compared with the control group (CON). Based on a comparative analysis with the control diet, RPS increased the abundance of The results of this exploratory study provided a comprehensive overview of the effects of different fibers on nutrient digestibility, gut microbiota and CAZymes in pig colon, which will furnish new insights into the impacts of the use of dietary fibers on animal and human health.The online version contains supplementary material available at 10.1186/s42523-021-00108-1. Dietary fibers are defined as the oligosaccharides, polysaccharides and derivatives that cannot be digested by the digestive enzymes into absorbable components in the small intestine but can be partly fermented by bacteria in the large intestine . Common Bifidobacteria but limits the growth of potential pathogenic bacteria, such as E. coli and Salmonella /total abundance}*1,000,000, and the total abundance of each sample was 1,000,000. The expression of each unigene was evaluated as parts per million (PPM)  of > 0.5 or < − 0.5 and n 3.2.1)  was utilThe distributions of microbiota to the CAZymes were visualised via Circos. The contributions of different bacteria to certain enzymes were evaluated by analysing the origin of the enzymes. Then, the microbiota were ordered according to the distribution. The top 10 and 15 genera were respectively selected. The influence of the bacteria on the digestion of dietary fibers will be analysed in follow-up studies.Additional file 1. The changed phyla in the dietary fiber groups compared to the CON group. CON, a control diet; RPS, a raw potato starch enriched diet; INU, an inulin enriched diet; PEC, a pectin enriched diet.Additional file 2. The CAZymes changes in the dietary fiber groups compared to the CON group. CON, a control diet; RPS, a raw potato starch enriched diet; INU, an inulin enriched diet; PEC, a pectin enriched diet.Additional file 3. The network of correlations between bacteria and CAZyme classes changed significantly by the dietary fiber. Green lines represent positive correlations and red lines represent negative correlationsAdditional file 4. Composition and analyzed nutrient contents of experimental diets (as-fed basis).


People living with HIV (PLWH) are particularly vulnerable to worsened outcomes of COVID-19. Therefore, the purpose of this work was to provide a scoping review of the literature to assess the risk factors for COVID-19 mortality among PLWH. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR), searches were conducted in PubMed, Scopus, Global Health, and WHO Coronavirus Database. Articles were eligible for inclusion if they were in English, included PLWH who died after COVID-19 infection, and described risk factors for mortality. Results were descriptively synthesized and pooled thereafter. Study quality was assessed using the Joanna Brigg Institute’s critical appraisal tools. 20 studies were eligible for inclusion, with the pooled death rate being 11.7%. Age was a major risk factor, especially after 50 (23.2%) and after 70 (41.8%), and males had a death rate nearly double that of females. As total comorbidities increased, the death rate also greatly increased; among those with comorbidities, the highest fatality rates were those with cardiovascular disease (30.2%), chronic kidney disease (23.5%), obesity (22.4%), and diabetes (18.4%). Other risk factors for mortality among PLWH included having a Black racial background, being an injection drug user, being a smoker, and having a CD4 cell count below 200. There is a need to better study confounding factors, and to understand how vaccination influences mortality risk. Overall, the findings highlight a need to ensure that focus is placed on the varying demographics of PLWH amidst COVID-19 control efforts.The online version contains supplementary material available at 10.1007/s10461-022-03578-9. The COVID-19 pandemic has immensely impacted the health status of diverse populations. As of December 23rd, 2021, there have been 276,436,619 confirmed cases of COVID-19 worldwide, and 5,374,744 deaths [As of 2020, there were 37,600,000 PLWH worldwide and 690,000 people who died due to HIV-related causes . DespiteThe presence of comorbidities has consistently shown to be a risk factor for worsened outcomes among COVID-19 patients. Such comorbidities include hypertension, diabetes, CVD, and cerebrovascular disease . HoweverIn consideration of the uncertainty of previous findings, there is a need to better understand the relationship between COVID-19 among PLWH, and the overall risks for poor outcomes. While Mirzaei et al. (2021)  have comThis scoping review followed the Preferred Reporting Items for Systematic Review and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) . The steOn July 20, 2021, searches were conducted in four different databases: PubMed, Scopus, WHO Coronavirus Database, and Global Health. As our population of interest was PLWH who died of COVID-19, searches included relevant terms on HIV, COVID-19, and mortality. No restrictions were placed based on date or language in these searches. Full search terms, with the respective database, are listed in Supplementary Table 1.Original research studies eligible for inclusion were required to have a minimum of five PLWH who died while infected with COVID-19 and provided stratified data with characteristics of the patients who died. Studies were also required to be in English. To keep the inclusion criteria as broad as possible, peer-reviewed articles and pre-print papers were eligible, and there were no restrictions placed on study type, or date of publication.After completion of the initial searches, two researchers (KV and PG) independently screened articles. Duplicates were first removed, and articles were thereafter screened by title and abstract. The remaining articles underwent full-text analysis and were excluded if they did not meet the criteria for inclusion. Discrepancies of selected articles among the two researchers were evaluated until consensus was reached about the final articles for inclusion.3), as well as additional features relevant to patient outcomes that were discussed in the study. The additional features included (but were not limited to) the following: a history of an AIDS diagnosis, men who have sex with men (MSM), injection drug use (IDU), smoking status, and having had the influenza vaccination. Thereafter, a pooled analysis was conducted to determine the overall and stratified case-fatality rate. Data was synthesized and analysed descriptively, with the use of MS excel sheets.Study characteristics, as well as patient data, were next extracted from the final articles. The characteristics of the studies considered were city and country of the study, source of data, study design, and proportion of deaths among PLWH infected with COVID-19. Patient data that was extracted included patient total, sex, age, race, comorbidities, HIV viral load, CD4 cell count  critical appraisal tools . As has Searches from all databases produced a total of 1192 articles. 938 articles remained after removal of duplicates, and after screening by title and abstract, 63 articles remained. A total of 20 articles –36 met tCharacteristics of the included studies are listed in Table Quality assessment scores for the cohort studies ranged from 6/11 to 10/11 (mean = 8.1), 5/8 for the cross-sectional study, and 6/10 for the case series. Figure Case-fatality rates among studies ranged from 2.9  to 32.5%Amongst racial groups, it was found that Black individuals had the highest fatality rate (12.9%), followed by Hispanic/Latino individuals (10.3%), then White individuals (10.0%), with people of other races having the lowest rate (8.3%). Total COVID-19 case rates were also highest amongst Blacks, followed by Hispanics/Latinos.Males were found to have a mortality rate nearly double that of females (10.1% and 5.4% respectively). In terms of age, there was a consistent trend showing that an increase in age corresponds to a markedly higher risk of mortality. These differences were largest amongst those 70 years and above, who had a case-fatality rate of 41.8%, compared to those under 40 years of age, who had a fatality rate of 1.2%. There was also found to be a near five times increase in death rates for those above the age of 50, with those of age 40–49 having a rate of 2.7%, and those 50–59 having a rate of 10.1%.In the pooled analysis, death rates among those with and without viral suppression were relatively similar, at 14.2% amongst those virally suppressed, and 14.7% for those who were virally unsuppressed. A lower CD4 cell count did have an impact on fatality rates; those with a CD4 count under 200 had a death rate of 34.2%, compared to 22.9% for those with a CD4 count above 200. A single study, which compared fatality rates amongst patients, showed that those with a viral load above 1000 copies/mL or a CD4 count below 200 had an adjusted hazards ratio of 3.80  .The presence of comorbidities also led to a higher death rate among patients. Those with one or more comorbidities had a fatality rate of 22.3%, and this increased to 47.8% for patients with two or more, and 61.3% for those with three or more. CVD (other than hypertension) patients had an elevated case-fatality rate of 30.2%, as well as diabetes patients (18.4%), obese patients (22.4%), and CKD patients (23.5%). Patients with hypertension had lower death rates (7.9%), along with patients with chronic obstructive pulmonary disease (COPD) (6.7%) and current or past tuberculosis (5.4%).There were varying trends for the additional factors. Patients with a history of an AIDS diagnosis had a fatality rate of 33.6%, though this factor was only evaluated in a single study ; in thatThe findings of this scoping review provide further evidence indicating that PLWH are at a high risk of COVID-19 mortality, with the case-fatality rate among PLWH patients included in this review being 11.7%. Based on these high mortality rates, it is evident that extra support and services are needed during the pandemic in order improve outcomes for this population. One way this can happen is with the expansion of telemedicine services for this demographic. Considering that telemedicine services have been shown in previous studies to be effective in supporting PLWH both prior to , 38, andOur review has shown that a substantial proportion of PLWH were either virally unsuppressed or had a low CD4 cell count; those with a lower CD4 count had a particularly high risk of death. A possible explanation for this is an interruption of adherence to ARTs due to social distancing measures during the COVID-19 pandemic, as this is a problem that has been described in numerous different contexts , 42. AloWhen considering race, it is evident that PLWH who are Black are at a particularly elevated risk of contracting, and dying from, COVID-19 compared to those of other racial groups. These findings have important implications, and further highlight the consequences of racial inequities during the COVID-19 pandemic. Our work hence shows that there is a need to create more programs that are specifically directed towards helping Black individuals amidst the pandemic, particularly across the United States. Therefore, targeted vaccination campaigns, along with resource allocation programs, have the potential to have a sizeable impact. Furthermore, prior research has indicated that medical mistrust regarding COVID-19 among HIV-positive Black individuals may be a contributor to health inequities . AddressThere is a striking difference in death rates among people of different sexes, and for people of different age groups. Males were nearly twice as likely to die from COVID-19 than females, and PLWH above the age of 70 had a death rate nearly 35 times higher than those under 40. It is worthwhile denoting that comparable trends have also been seen among COVID-19 patients not living with HIV . StudyinBased on the findings, it is unclear if MSM are at a higher risk of death. The findings do however show that IDU are at a higher risk of death, and risk of fatality is especially high amongst IDU who are also MSM. Previous research has provided an indication that, during the COVID-19 pandemic, IDU have had lower ART adherence rates and increased rates of illicit substance use overall, and this may be attributed to increased social-distancing measures . There iThe presence of comorbidities was shown to correspond to high death rates, with those living with multiple comorbidities having especially high rates of death. CVD, obesity, CKD, and diabetes are all conditions which appear to elevate death rates, along with smoking. However, an opposite pattern was observed for those with hypertension, COPD, and tuberculosis. Furthermore, there was limited patient data for those with cancer, neuropsychiatric disease, organ transplants, hyperlipidemia, and bacterial superinfection. This emphasizes a need to better understand the risk due to specific comorbidities for this demographic. Similarly, it would be beneficial to better understand the effects of influenza vaccinations, and whether they may be beneficial, or even detrimental for this group.Notably, of the studies included in this review, there were no case–control studies included. This hence restricted the capability to compare outcomes for COVID-19 patients who did, and did not, have HIV. Additionally, while there were a small number of high-quality studies, many of the included studies were either of moderate or low quality and did not appropriately account for confounding factors. It is hence recommended that more high-quality studies, with controls, be conducted to better understand the factors that place PLWH at risk of COVID-19 mortality.There are several important limitations to consider. First, none of the studies provided data on the effects of COVID-19 vaccination, and the extent to which this impacts risk. Secondly, as half of the studies are from USA, it is not entirely clear how much findings from these settings can be generalized to other nations. Furthermore, the findings do not provide a clear indication as to whether having an elevated viral load influences overall fatality rates; considering that many of the social distancing measures during the COVID-19 pandemic have previously shown to impact medication adherence , 42, thePLWH are at a high risk for death by COVID-19. Risk factors for death were found to be having a Black racial background, living with comorbidities, being an IDU, being older, having a low CD4 cell count, and being male. Targeted interventions and pandemic control efforts towards individuals with these risk factors will be imperative to effectively save lives of PLWH during the COVID-19 pandemic.Supplementary file1 (DOCX 38 kb)Below is the link to the electronic supplementary material.


Perturbation of tryptophan (TRP) metabolism contributes to the immune escape of cancer; however, the explored TRP metabolites are limited, and their efficacy in clarifying the susceptibility and progression of esophageal cancer (EC) remains ambiguous. Our study sought to evaluate the effects of the TRP metabolic profile on the clinical outcomes of EC using a Chinese population cohort; and to develop a risk prediction model targeting TRP metabolism.A total of 456 healthy individuals as control subjects and 393 patients with EC who were followed up for one year as case subjects were enrolled. Quantification of the plasma concentrations of TRP and its metabolites was performed using HPLC-MS/MS. The logistic regression model was applied to evaluate the effects of the clinical characteristics and plasma metabolites of the subjects on susceptibility and tumor metastasis events, whereas Cox regression analysis was performed to assess the overall survival (OS) of the patients.p = 0.0114). Furthermore, it was figured out that OS was significantly shorter for patients with elevated XA/KYN ratio , in which medium and high levels of XA/KYN versus low level had a significantly lower OS .Levels of creatinine and liver enzymes were substantially correlated with multiple metabolites/metabolite ratios in TRP metabolism, suggesting that hepatic and renal function would exert effects on TRP metabolism. Age- and sex-matched case–control subjects were selected using propensity score matching. Plasma exposure to 5-HT was found to be elevated 3.94-fold in case subjects (N = 166) compared to control subjects (N = 203), achieving an AUC of 0.811 for predicting susceptibility event. Subsequent correlation analysis indicated that a higher plasma exposure to 5-HIAA significantly increased the risk of lymph node metastasis (OR: 2.16, This study provides a pivotal basis for targeting endogenous TRP metabolism as a potential therapeutic intervention. Esophageal cancer (EC) refers to a malignant digestive tract cancer that develops from the aberrant proliferation of the esophageal squamous epithelium or glandular epithelium. Owing to its insidious onset and scarce early detection methods, EC is usually diagnosed as advanced or metastatic cancer, with a five-year survival rate of less than 16.8% . IncreasCatalyzed by specific enzyme activities, TRP serves as a substrate for three different branches , 5-hydroxytryptamine (5-HT) pathway, and indole pathway) , 9, therGiven the rather complex network of TRP metabolic reactions, the regulation of vital immunosuppressive metabolites has become an attractive target for cancer therapy. Nevertheless, pathological shifts in the TRP metabolic profile have shown inconsistent trends in patients with different cancers. Previously, a nested case–control study was conducted which found that serum levels of 3-HAA and 3-HAA/3-HK were negatively correlated with the risk of pancreatic cancer, while KYN/TRP was not significantly correlated with the risk of pancreatic cancer . AnotherHerein, along the metabolite–clinical phenotype–study endpoint axis, we performed this study to investigate the impact of the TRP metabolic profile on the clinical primary outcomes of EC  of patients) using a Chinese population cohort.A prospective study of the Chinese population was performed. First, clinical indexes were investigated in all subjects  to explain individual differences in the TRP metabolic profile. Later, the effects of plasma exposure to TRP and its metabolites on clinical endpoint events were assessed, namely, susceptibility, lymph node metastasis, distant metastasis, and OS.From August 2018 to November 2018, the study population, namely, 456 healthy individuals (control subjects) and 393 patients with histologically confirmed EC (case subjects), were sequentially recruited at the Cancer Hospital of Shantou University Medical College and the First Affiliated Hospital of Shantou University Medical College. Consistent baseline information was obtained for both groups from the hospital database, namely, demographics, blood routine examination, and biochemical measurements.The exclusion criteria for the selected subjects were as follows: (1) age <18 years; (2) renal insufficiency ; (3) liver insufficiency ; (4) pregnancy or lactation; (5) poor compliance or inability to complete the test; (6) patients who underwent anti-cancer treatments during this period; and (7) a history of other malignancies.An additional exclusion criterion was required for control subjects: metabolic diseases such as diabetes, cancer, cardiovascular disease, severe infections, gout, and other end-stage diseases.The study was approved by the Ethics Committee of Guangzhou Red Cross Hospital (2020-109-02) and conducted in accordance with the Declaration of Helsinki principles. All subjects signed informed consent documentation. The workflow of sample selection is depicted in g for 10 min at 4°C within 2 h. Plasma and blood cells were separated and stored at −80°C for future analysis.Fasting blood collection in the early morning for both groups of subjects was required to minimize the influence of nutrition on the plasma levels of TRP metabolites. The patients pathologically diagnosed with EC did not receive radiotherapy or chemotherapy at this stage. Each blood sample was placed in EDTA anticoagulation tubes and centrifuged at 2,095×Clinical endpoints included susceptibility event, tumor metastasis event, and OS of patients.p >0.05).A case–control study was conducted on susceptibility event. The initial sample population included 393 patients with EC and 456 healthy individuals. According to the propensity score matching (PSM) principle, 166 patients with EC and 203 healthy individuals were selected  of patients at the Cancer Hospital of Shantou University Medical College.Tumor metastasis refers to the continued growth of cancer cells from the primary site  to other sites OS was defined as the time from the start of randomization to the death of any cause and was mainly evaluated based on in-hospital outpatient follow-up and out-of-hospital telephone follow-up. Follow-up information for each patient was obtained from the hospital database and telephone interviews. The telephone follow-up period was as follows: advanced-stage patients were followed-up every 3 months, and early stage patients were followed-up every 6 months. The last follow-up period of this study was December 2019, and the patients were followed up for 1 year.A sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) assay was established for the simultaneous quantification of TRP, KYN, KYNA, 3-HAA, XA, 5-HT, 5-HIAA, IPA, and IAA in human plasma. Analysis was performed using an HPLC system  coupled with an API 4000 triple-quadrupole mass spectrometer .d5 and KYN-d4) were isolated from human plasma by liquid–liquid extraction with prechilled acetonitrile and then separated on an Acquity XSelect HSS T3 column  at a flow rate of 0.30 ml/min by the gradient of the mobile phase consisting of 0.01% (v/v) formic acid in water (A) and acetonitrile (B). The following gradient program was used: 0–0.1 min 5% B; 0.1–0.5 min 5→60% B; 0.5–2.8 min 60% B; 2.8–2.9 min 60→5% B; 2.9–5.0 min 5% B.Nine analytes and internal standards (TRP-Mass detection was performed using an API 4000 triple-quadrupole mass spectrometer under the positive electrospray ionization mode. Electrospray voltage (IS) was set at 5,500 V, desolvation gas temperature was set at 550°C, and ion source gas 1 (CS1) and ion source gas 2 (CS2) were set at 50 psi. The air curtain gas (CUR) was 25 psi. Declustering potential (DP) and collision energy (CE) were optimized for each analyte and internal standard. Ion transitions and optimized multiple reaction monitoring (MRM) parameters are shown in p <0.05, metabolite ratios were also included in the list of dependent variables.Demographic and clinical characteristics were summarized using counts (percentages) for categorical variables and medians (interquartile ranges [IQR]) for continuous variables. Considering that the concentration ranges of all metabolites were skewed, logarithmic transformation was performed prior to analysis. Spearman correlation analysis was applied to study the association between plasma concentrations of upstream and downstream metabolites . If p <0.05 after univariate linear regression were employed into the multivariate regression model, in which p <0.05 was considered as the independent factor of metabolites/metabolite ratios. The R2 was used to evaluate the interpretability of the model.Linear regression analysis was performed to assess the effects of baseline demographic and clinical characteristics on the plasma metabolites/metabolite ratios. Factors with p <0.05 were entered into the multivariate model, and only variables with p <0.05 considered as independent impact factors, were retained in the model. Furthermore, prognostic models of clinical events were constructed for each predictive variable by receiver-operating characteristic (ROC) curves using the area under the curve (AUC) to measure the diagnostic effectiveness. The optimal cutoffs were calculated by selecting the data point that maximized the true-positive rate and minimized the false-positive rate. The Kaplan–Meier method was conducted to assess the effects of every independent variable associated with OS, in which patients were further stratified into three groups: low, moderate, and high in terms of the quartiles of their plasma levels, and the p-value was analyzed with the log-rank test.Based on the propensity index, the appropriate PSM method (“MatchIt” package) was employed to correct the imbalance of baseline data between the study population of case subjects and control subjects for inclusion of the population in the susceptibility event study calculated using the nearest neighbor method. Age and sex were used as covariates to condense a comprehensive score, with a paired caliper value of 0.02. A logistic regression model was applied to evaluate the effects of clinical characteristics and plasma metabolites of the subjects on susceptibility and tumor metastasis events, and Cox regression analysis was performed to assess the OS of patients, while the odds ratio (OR), hazard ratio (HR), and 95% confidence interval (95%CI) were calculated. Variables with p <0.05. All data were analyzed using SAS 9.4 , R , and GraphPad Prism 6. A flowchart of the experimental design and sample selection process is depicted in The criterion for statistical significance was set at p <0.05), whereas a significant negative correlation was found between TRP and IPA in control subjects . Therefore, the corresponding ratios were included in the list of dependent variables for further correlation analyses.Based on these criteria, we ultimately included 456 healthy individuals and 393 patients with EC. The demographic characteristics and clinical data of the enrolled subjects are shown in Univariate linear regression analysis revealed the following: (1) high CREA levels were associated with the plasma levels of KYN, KYNA, 5-HIAA, IPA, and IAA; (2) coagulation index of patients, namely, platelet (PLT), was correlated with plasma exposure to multiple metabolites, namely, TRP, KYNA, 5-HT, and IPA; (3) male patients showed higher plasma exposure to TRP, XA, and 3-HAA; (4) other clinical indexes for hepatic function , nutrition , coagulation , and immune  exhibited certain effects on plasma exposure of metabolites , nutrition , and cardiac function  exhibited certain effects on plasma exposure to metabolites . Hepatic and renal function indexes  and neutrophil ratio (NE) were correlated with plasma exposure to TRP, KYN, KYNA, XA, 3-HAA, 5-HIAA, and IAA. The effects of the remaining factors on the metabolites in the control subjects are shown in Similarly, univariate linear regression analysis showed that age significantly affected the metabolite ratios in the control subjects except for 3-HAA/TRP. In accordance with the above results, the plasma levels of multiple metabolite ratios were still significantly affected by sex and the levels of ALB, GLB, and ALB/GLB; the effects of the remaining factors on the metabolite ratios are shown in These factors were incorporated into a multivariate linear regression model of individual metabolites/metabolite ratios. Multivariate linear regression identified that the CREA levels in the control subjects independently affected several metabolites/metabolite ratios in the TRP metabolic profile, namely, IAA, KYN/TRP, KYNA/TRP, IAA/TRP, and 3-HAA/KYN. Hepatic function indexes  were considered as secondary factors involving metabolites/metabolite ratios, namely, TRP, KYN, KYNA, 3-HAA, 5-HT, KYN/TRP, and KYNA/KYN. Consistent with the case cohort, hepatic and renal function were similarly involved in the dynamics of the TRP metabolic profile in control subjects, and platelets in the control cohort were similarly independent influencers of plasma exposure to 5-HT. According to previous studies, platelets and mast cells were proposed to be the vital reservoirs of 5-HT, and secretion of 5-HT would lead to increased uptake of 5-HT by circulating platelets and mast cells , 18.Given the significant differences in age and sex in the original two cohorts, confounding bias could alter the veracity of the findings on cancer susceptibility event; therefore, matching the two cohorts was required to increase the statistical power of the present case–control study.The MatchIt package of R was used to match the age and sex of the case and control subjects. Before matching, the age of the control subjects was 48.00  years, accounting for 53.73% of males and 46.27% of females, while the age of case subjects was 63.00  years, accounting for 77.35% of males and 22.65% of females. The PSM scores of the control and case subjects were 0.28 ± 0.27 and 0.67 ± 0.27, respectively.Via the nearest neighbor method of PSM, the age of control subjects was 57.00  years old, of which 63.55% were male and 36.45% were female, while the age of case subjects was 58.00  years old, of which 68.67% were male, and 31.33% were female. There was no statistically significant difference between the two groups (p >0.05). The PSM scores of the two groups were 0.55 ± 0.22 in control subjects and 0.55 ± 0.22 in case subjects, which proved that the age and sex of both groups were balanced and comparable , and ten other biochemical indicators were associated with susceptibility to EC . A similar trend was observed between patients with low and high plasma levels of XA/KYN . However, there was no significant difference between patients with medium and high plasma XA/KYN levels , as depicted in Follow-up for this study was completed in December 2019, with survival data available for 358 patients. Of the enrolled patients with EC, 111 died, 247 survived, and 35 were lost to follow-up. Univariate Cox regression analysis showed that plasma levels of XA/TRP, 3-HAA/TRP, XA/KYN, and 3-HAA/KYN, male sex, comorbid hypertension, several blood routine indexes, and several biochemical indexes were significantly associated with the OS of patients  TRP metabolism was significantly imbalanced in patients with EC. Three major metabolic pathways were severely dysregulated in both groups, in which the population with high plasma exposure to 5-HT significantly increased the risk of EC; (2) 5-HIAA was independently associated with increased lymph node metastasis risk; and (3) high plasma levels of XA/KYN conferred a risk effect on the occurrence of death, since KYN metabolism towards XA was significantly increased in the high-risk group of death events. The above results characterized their roles in carcinogenesis and potency as risk markers for EC metastasis and prognosis.in vivo and in vitro studies have shown that 5-HT serves as an effective mitogen in various cell types , and the presence of non-endocrine malignant cells in the digestive tract might lead to an increase in these metabolites in body fluid and urine . To datep = 0.2348), thus revealing that this metabolite could not serve as a biomarker to aid in determining the prognosis of patients with EC yielded insufficient evidence. Previously, Cheng et al. found that the serum 5-HIAA/TRP level in EC patients with metastasis was higher than that non-metastatic patients, which is consistent with our results , speculating that the metabolism of KYN in the direction of XA increased the risk of death. Moreover, it is significant to point out that this metabolite ratio was the first discovery of an association between the TRP metabolic profile and the prognosis of cancer patients.Interestingly, after exploring the association between metabolites and clinical endpoint events, this study suggested that the plasma level of XA/KYN significantly affected OS . Remarkably, activation of AHR drives the transformation of naive CD4 cancers . In addi cancers . Further cancers , 37. Add cancers –40. Seve cancers . Moreove cancers . Hence, However, a limitation of this study is that the sample size was relatively small. Nonetheless, we conducted a strict screening and quality control process on the original sample population, and conducted sample screening and correlation discussions for different clinical endpoint events. Most of our findings are in line with those of previous studies, proving the reliability of our results. There is no denying that a more extensive study cohort would be more convincing to clarify the association with clinical endpoint events. In the future, broader and more comprehensive inclusion of the study population would address this limitation.A comprehensive evaluation of the clinical predictive value of tryptophan metabolism in carcinogenesis and its potential as biomarkers for metastasis and prognosis of EC emerged from the present study. Our results demonstrated that elevated levels of 5-HT, 5-HIAA, and XA/KYN were observed in EC and might be part of the mechanism underlying the susceptibility, lymph node metastasis, and poor prognosis of EC, respectively. This study provides an essential theoretical basis for targeting endogenous TRP metabolism as a potential therapeutic intervention.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Guangzhou Red Cross Hospital. The patients/participants provided their written informed consent to participate in this study.YC, JC, DG, and PY performed the experiment, performed data analysis, and wrote the manuscript. SC participated in the experiment. CZ, CX, and QZ participated in patient recruitment. CL revised the manuscript; ShiZ and ShoZ designed the study and revised manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the 2020 Guangdong Science and Technology Special Fund  item (No: 200113165875501).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Europe has about 10,000 imported cases of malaria each year, or around 80 cases per 100,000 trips to endemic areas. Non-use of chemoprophylaxis in travellers remains the main reason for this. The proliferation of online travel blogs as a source of advice (sometimes the only one used) for preparing a trip to an endemic area may play a role in the decision to use chemoprophylaxis. The aim of this study was to analyse the information offered on malaria in the main travel blogs in English and Spanish.Five hundred travel blogs in English and 100 in Spanish, considered highly relevant were analysed. The relevance were according to different metrics: (1) Alexa Rank; (2) social networks (RRSS) measuring the total followers of Facebook, Twitter, Instagram and YouTube; (3) number of monthly visits using the SEMrush tool; (4) domain authority; and (5) number of backlinks or incoming links using the SEMrush tool.Of the included travel blogs, 57% of those in English and 64% of those in Spanish offered information on malaria, and 79 and 75%, respectively, featured a discussion on malaria written as a blog post or in forum comments. Information on chemoprophylaxis was available in 56.1% of English-language blogs and 10.7% of Spanish-speaking blogs, while its side effects were discussed in 38.6 and 68.8%, respectively (p < 0.001). Content analysis revealed that the information was usually insufficient, incomplete or, more seriously, inaccurate. In many cases, this could discourage users from taking appropriate preventive measures.Travel blogs in English and Spanish provide low-quality information on malaria. The so-called “travel influencers” must communicate reliable, verified and quality information on malaria on their channels in a way that could contribute to reducing the burden of the disease in travellers.The online version contains supplementary material available at 10.1186/s12936-021-03864-2. About 10,000 imported cases of malaria are reported in Europe each year, or about 80 for every 100,000 trips to endemic areas. The global incidence of the disease was estimated at 229,000,000 cases in 2019, of which more than 50% occurred in Africa .Despite the existence of official databases, the real burden of imported malaria in non-endemic countries is probably underestimated . PrevaleThe risk of imported malaria is known to be higher in travellers to Africa, especially migrants and people visiting friends and relatives (VFRs). There are many studies that indicate that chemoprophylaxis use is still insufficient in people with imported malaria cases, both in these high-risk groups as well as young people and travellers with a high educational level , 9.The first source of information that the traveller can access is the official, “classic” route, that is, specific, timely advice offered by health professionals through specialized clinics . Howeverhttps://www.alexa.com/siteinfo); (2) social networks (RRSS) measuring the total followers of Facebook, Twitter, Instagram and YouTube), using Influencer Marketing Hub (https://influencermarketinghub.com/), Fanpage Karma (https://www.fanpagekarma.com/) and Metricool (https://metricool.com); (3) number of monthly visits using the SEMrush tool (https://es.semrush.com/); (4) domain authority, using the MOZ tool (https://moz.com/free-seo-tools); and (5) the number of backlinks or incoming links using the SEMrush tool. Moreover, the monthly and annual visits/users for all the English and Spanish blogs selected using the tools available on the Alexa website were estimated.Six hundred travel blogs (500 in English and 100 in Spanish) with general content for travellers and accounts of trips to malaria-endemic countries were analysed. The blogs were selected based on different metrics assessing their relevance  that included the word “The differences between English- and Spanish-language blogs were determined using Pearson’s chi-square test for categorical variables. Values were considered to be statistically significant when the p value was less than 0.05. Statistical data analysis was performed using IBM SPSS Statistics for Windows, Version 25.0 .Web traffic measurement tools show that collectively, the 600 blogs analysed have a reach of 92,400,000 unique visitors/month, for an incredible 1,108,800,000 visitors per year. No information on malaria was available in 43% of the 500 English-language or in 36% of the 100 Spanish-language blogs, even though they were pages that offered recommendations for visiting endemic countries. On the other hand, about 95% of Spanish blogs did offer advertising or advertising content for travel health insurance.Of the blogs that contained information on malaria (English n = 285 and Spanish n = 64), 79 and 75%, respectively, mentioned malaria in the forum or comments section. Information on chemoprophylaxis was available in 56.1% of English-language blogs and 10.7% of Spanish-speaking blogs, while its side effects were discussed in 38.6 and 68.8%, respectively (p < 0.001). Only 21.1 and 34.4% (p = 0.023) of the blog posts presented up-to-date information (reviewed in the previous year) . A minority  advised uncertified preventive products against malaria or products of dubious effectiveness, such as garlic, onion, vitamin B, bracelets, nitrous oxide, various cosmetics, anti-mosquito candles, or electrical/ultrasonic devices to repel mosquitoes. Few blogs  included references for the information presented. Only 7.0 and 3.1% of the blogs, respectively, provided correct information about self-treatment, while healthcare professionals generated or reviewed the information in just 3.5 and 3.1% of the blogs . Certain “filter criteria” could also be applied, allowing the user to evaluate the information obtained for themselves. However, in other, so-called “mainstream” media, such as newspapers, radio or television, the information presented may also be erroneous, incomplete or biased, but that is not cause for it to be withdrawn. Rather, the user must apply their own personal judgment.The adoption of codes of conduct and ethics, such as HONcode , could bIntroducing a code of conduct for sensitive information provided by these sites is further justified by the fact that malaria prevention is just a small drop in the ocean of popular medical communication. Indeed, the larger problem is so huge that it is really hard addressing this single aspect alone, or even medical travel advice in general. Good initiatives exist, including the HONcode, but most people are not aware of them . MoreoveThe main strength of the study resides in the fact that it opens the door to innovative interventions to improve malaria prophylaxis. It also touches on a much wider topic of interest than the specific focus of the study, namely the ability to refer blog readers to quality information to improve the training of travellers.However, the results of this study should be interpreted in light of the study’s limitations. First of all, the study was performed in only English and Spanish blogs, which may be different from blogs in other languages. Secondly, the study did not differentiate between the country where the bloggers were based. Third, the criteria used for measuring quality was based on researchers’ subjective experience.In conclusion, travel blogs written in English and Spanish often provide low-quality information on malaria. The contents offered were generally insufficient, incomplete or, worse, inaccurate. Further qualitative studies are necessary to analyse the quality of information on malaria in online media.Additional file 1: Table S1. 500 English travel blogs analyzed by alphabetic order. Table S2. 100 Spanish travel blogs analyzed by alphabetic order.