Pyrus communis L.) cultivars ‘Regal Red’ and ‘Sweet Sensation’ (red sports of ‘Doyenné de Comice’) after flower inoculation with the typical nectar-inhabiting microorganisms Metschnikowia reukaufii and Acinetobacter nectaris, and a combination of both. Pollination success was monitored by measuring the number of flower visits, fruit set and seed set in two consecutive years, 2019 and 2020. Results revealed that application of a mixture of M. reukaufii and A. nectaris resulted in significantly higher visitation rates of honeybees and hoverflies. By contrast, no effects on flower visits were found when yeasts and bacteria were applied separately. Fruit set and seed set were not significantly affected by any of the inoculation treatments. The only factors affecting fruit set were initial number of flower clusters on the trees and the year. The absence of treatment effects can most likely be attributed to the fact that pollination was not a limiting factor for fruit set in our experiments. Altogether, our results show that inoculation of flowers with nectar microbes can modify pollinator foraging patterns, but did not lead to increased pollination success under the conditions tested.Currently, one of the most important challenges is to provide sufficient and affordable food and energy for a fast-growing world population, alongside preserving natural habitats and maintaining biodiversity. About 35% of the global food production depends on animals for pollination. In recent years, an alarming worldwide decline in pollinators has been reported, putting our food production under additional pressure. Therefore, there is an urgent need to find sustainable ways to ensure this crucial ecosystem service. Recent studies have shown that floral nectar is generally colonized by microorganisms, specifically yeasts and bacteria, which may alter nectar chemistry and enhance attraction of pollinators. In this study, we investigated changes in pollinator foraging behavior and pollination success in European pear ( One of the biggest challenges today is to ensure the production of sufficient and affordable food and energy for a fast-growing world population, alongside preserving natural habitats and biodiversity ,2. AboutPlants use different strategies to attract pollinators, including flower color, size, shape and scent, and they often reward visiting insects with nectar and pollen . Floral Helleborus foetidus and different larkspurs [Given its high sugar content, nectar also provides an ideal habitat for microbes. In fact, although nectar is assumed to be initially sterile following flower opening , it veryarkspurs –29. Espearkspurs ,30,31. Iarkspurs ,33.Because nectar-inhabiting microbes have the potential to improve plant pollination by altering nectar properties, nectar manipulation through inoculation of microbes shows great potential to increase pollination and crop yield. This is especially the case in crops that produce nectar with low sugar concentrations, which decreases attractiveness to pollinators, such as pear ,35. MostPyrus communis L.) cultivars ‘Regal Red’ and ‘Sweet Sensation’ (red sports of ‘Doyenné de Comice) after inoculation of typical nectar microbes, including Metschnikowia reukaufii and Acinetobacter nectaris. Specifically, newly opened pear flowers were inoculated with each microbial species separately, or a combination of both species by hand spraying the inoculum into the flowers. Subsequently, flower visits by bees and hoverflies were monitored during flowering, and pollination success was measured by quantifying fruit and seed set. The experiment was performed in two consecutive years, 2019 and 2020.In this study, we aimed to investigate changes in pollination behavior in European pear  and Acinetobacter nectaris . Metschnikowia reukaufii is one of the most common yeast species in floral nectar [M. reukaufii to non-inoculated nectar [M. reukaufii produces distinct volatile organic compounds that are attractive to flower-visiting insects such as bees [Acinetobacter nectaris also commonly occurs in floral nectar [M. reukaufii [M. reukaufii CdV Mar 33.1 [A. nectaris LMG 26958, were isolated from floral nectar of Gladiolus italicus—communis  and Muscari comosum , respectively. Both strains were stored in 37.5% glycerol at −80°C.Two common nectar-inhabiting microbial species were used in this study, l nectar ,38 and hl nectar –41. Behad nectar ,27,29. F as bees ,26 and p as bees ,43. Acinl nectar –47, ofteeukaufii . The strMar 33.1  and A. nM. reukaufii) and trypticase soy agar (TSA)  supplemented with 5% fructose (A. nectaris). Next, plates were incubated at 25°C and a subculture was made on the same medium after two days. Subsequently, following an incubation period of four days at 25°C, three 250 ml-Erlenmeyers filled with 150 ml yeast extract peptone dextrose  and 150 ml trypticase soy broth  were inoculated with the yeast and bacterial strain, respectively. After an incubation period of three days at 25°C on a shaking platform (120 rpm), cells were transferred into fresh growth medium. Specifically, for each strain 50 ml of homogenized culture medium was added to eight 1 l-Erlenmeyers with 750 ml YPD (M. reukaufii) or 750 ml TSB (A. nectaris), and again incubated under the same conditions. After five days cell suspensions were centrifuged for 9 min at 3,500 g. Pellets were then combined and suspended in tap water to a total volume of 7 l yeast suspension and 7 l bacterial suspension, and immediately used for flower inoculation.In order to prepare the inocula, strains were first plated on yeast extract peptone dextrose agar (YPDA)  of the nesting blocks was between 40 and 50% . In each orchard, four treatments were applied. These included a treatment in which yeast cells were applied, a treatment with bacterial cells, and a combination of both. A treatment in which tap water was applied was included as a control. Each treatment was repeated in each orchard in four plots of five pear trees per plot. Plots were separated by a distance of at least 15 m along the row and 10 m across rows . For the combined treatment of yeasts and bacteria, a combination of 37.5 ml of each suspension was applied. In 2019 the OD600 values for the yeast and the bacterial suspensions were 0.7 and 0.35 (corresponding with 1.1 x 107 and 4.2 x 107 CFU (colony forming units)/ml, respectively; in 2020 they were 0.9 and 0.5 (corresponding with 1.5 x 107 and 7.1 x 107 CFU/ml), respectively.The field studies in this research were performed in pear orchards with consent of the owners. Experiments were performed in Belgium in 2019 and 2020 in three commercial pear orchards, including two orchards with cultivar ‘Sweet Sensation’ and one orchard with cultivar ‘Regal Red Comice’. Both cultivars are red sports of the self-incompatible, late-flowering ‘Comice’ pear cultivar with a short effective pollination period of a few days depending on temperature and flower quality ,50. In 2oss rows . In GutsIn order to assess potential differences in insect attraction among treatments, transect walks were carried out in each orchard at five time points between 10 am and 5 pm in the first three days after microbial inoculation. At each time point, each plot of five trees was observed for 2 min at the sunny side of the trees, and only effective flower visits were noted, i.e. when the anthers or stigmas were touched by an insect. In each orchard, for each treatment and each time point, the sum of effective flower visits of the four plots was then made, representing the number of effective flower visits in a total of 8 min for each transect walk. This value was recalculated to visits per tree per hour (dividing by 20 and multiplying by 60/8). A distinction between taxonomic groups of pollinators was made as follows: honeybees, bumblebees, horned mason bees, other bees, and hoverflies.nd of September and the 23rd of August, respectively. Two fruits per tree (40 fruits per treatment) in each orchard were randomly sampled to quantify seed set. Seed set was defined as the number of well-developed seeds present in the fruit.In March 2019 and 2020, the initial number of flower clusters on the 20 trees per treatment per orchard was counted to estimate the initial number of flowers, i.e. 7.5 times the number of clusters per tree (based on flower counts of previous years). Fruit set, i.e. the percentage of flowers developing into mature fruits, was monitored on individual trees after the June drop for all 20 trees per treatment per orchard. The June drop refers to the natural tendency of fruit trees to drop immature fruit after flowering, usually around the beginning of June, to avoid over-bearing of the tree. In 2019 and 2020 fruits were harvested on the 2M. reukaufii and A. nectaris DNA in the samples. Reactions were carried out in an ABI StepOnePlus real-time PCR system  in a total volume of 20 μl containing 1 μl DNA, 0.2 μl of each primer (20 μM stock), 10 μl 1x SsoAdvanced Universal SYBR Green Supermix and 8.6 μl nuclease free water. Amplification was performed using the primers Mr_LSU_F1_2 (5’-TGC AAG CAG ACA CAA CCT CG-3’) and Mr_LSU_R1_2 (5’-CGC CAG CAT CCT TGA AGA AT-3’) and An_rpoB_F1_1 (5’-AAA CGT GGT GAT CAG CTG ACT-3’) and An_rpoB_R1_1 (5’-GTA AGA CGT TCA GCG ATA C-3’), targeting the large subunit of the ribosomal RNA (rRNA) gene and the gene encoding the RNA polymerase subunit B (rpoB) of M. reukaufii and A. nectaris, respectively. Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, followed by 40 cycles of a denaturation step at 95°C for 15 s, an annealing at 57°C for 60 s and an elongation step at 72°C for 30 s. Fluorescence (520 nm) was detected at the end of the elongation phase for each cycle. The threshold cycle (CT), or the PCR cycle where fluorescence was first detected, was determined automatically using the Applied Biosystems software. The baseline was set automatically, while the threshold was manually set at 0.52 (which was above any background). At the end of each run, a melting curve analysis was performed to confirm amplification specificity. In each analysis, a negative control (DNA replaced by DNA-free water), and a standard ten-fold dilution series of genomic DNA of M. reukaufii CdV Mar 33.1 and A. nectaris LMG 26958 was included. All reactions were performed in duplicate. The amount of target DNA in each sample was calculated from the standard curve and expressed as ng target DNA per gram flower material.To confirm the presence of the applied microbes after inoculation of the flowers, in each orchard six samples of ten random flowers each were collected for every treatment 30 h after inoculation and analyzed by qPCR. For each sample, genomic DNA was extracted from 0.5 g homogenized flower material using QIAGEN DNeasy PowerSoil Kit  according to the manufacturer’s instructions with one modification: in the third and fourth step of the protocol the use of a vortex adapter was replaced by two cycles of 30 s in the Precellys 24 homogenizer  at 4000 rpm. Subsequently, DNA was subjected to a qPCR analysis to quantify All statistical analyses were performed in R version 4.0.2 . The effects of the different inoculation treatments on pollinator visits, fruit set and seed set were analyzed using generalized linear mixed models (GLMMs) with a Gauss, beta and negative binomial distribution, respectively, using the glmmTMB package . InoculaM. reukaufii and A. nectaris was studied using GLMM’s with a Gauss distribution (glmmTMB package [The effect of inoculation treatment (fixed factor) on the presence of  package ), in whi package  to perfoHoneybees were the most abundant flower visitors recorded across the different treatments, followed by hoverflies and mason bees . In 2019In both years, honeybees were significantly more observed in the yeast + bacteria treatment compared to the control  and the bacteria treatment  . Only inFruit set was significantly higher (P < 0.0001) in 2019 (11.2 ± 6.4%) compared to 2020 (8.5 ± 5.4%) and was significantly affected by the initial number of flower clusters per tree (P < 0.0001): a lower number of initial clusters resulted in a higher fruit set . Both inA. nectaris was inoculated at half of the dose in the yeast + bacteria treatment, the bacterium was significantly more abundant on flowers of the yeast + bacteria treatment compared to the flowers where only the bacteria were inoculated (P = 0.003)  .M. reukaufii and/or A. nectaris. Previous experiments under both controlled greenhouse conditions and field conditions have shown that bees prefer nectar inoculated with M. reukaufii over non-inoculated nectar [M. reukaufii and A. nectaris resulted in higher insect visitation rates, although differences were only significant for honeybees (in both 2019 and 2020) and hoverflies (2019). In contrast, no effects were seen when yeasts and bacteria were applied separately. So far, the mechanisms underlying these synergistic effects remain unclear, but it is reasonable to assume that a more attractive mixture of volatile compounds was obtained when combining both microbes [Metschnikowia reukaufii and Acinetobacter species, including A. nectaris, are commonly found together in floral nectar [A. nectaris prefers fructose and does not grow well on sucrose and glucose, Metschnikowia is able to hydrolyze sucrose and consume the resulting glucose, enriching the nectar in fructose [A. nectaris was abundantly present in the yeast + bacteria treatment, while it was less abundant when only the bacteria were applied, even though at a double dose was used. While several studies have focused on the consequences of either nectar yeasts or bacteria for pollination [In this study, we investigated changes in pollinator foraging behavior and pollination success in European pear after flower inoculation with the nectar microorganisms d nectar ,27,29, md nectar  or they d nectar . Our resmicrobes ,54. Howemicrobes . Metschnl nectar . A potenl nectar . Whereasfructose ,45. Likefructose . Such inlination ,27,28,31lination ,33. AlthM. reukaufii and A. nectaris, the total number of insect visits was still low (on average 2.0 insect visits per hour per tree) compared to what is generally considered sufficient for adequate pollination. The recommendations by Mayer et al. [M. reukaufii and A. nectaris. The maximum number of honeybees monitored in a single plot was eight per 8 min, which equals 12 honeybees per tree per hour. On that day the temperature reached 16°C. On time points when the temperature only reached 10°C no honeybees were seen. Honeybees are known to only start foraging at temperatures of 12–13°C [Although highest insect visitation rates were recorded for the combined treatment of r et al.  for adeqr et al. . Moreover et al. ,58, whicr et al.  reported 12–13°C , which m 12–13°C , they onIn 2019 hoverflies were also more abundantly present on trees with the yeast + bacteria treatment. Hoverflies are important pollinators of wild flowers but also visit several crops . SeveralM. reukaufii and A. nectaris did not result in an increased fruit set or seed set, nor did the other treatments. The only studied factors significantly influencing fruit set were year and initial number of flower clusters. In 2019, fruit set was overall significantly higher compared to 2020. This can be explained by the fact that circumstances for setting fruit were better in 2019. Indeed, in 2020 there was a severe cold period in the night of March 31st. At that point clusters were in the white bud stage having a critical temperature of about -3°C, below which crucial flower damage occurs [Despite the higher number of insect visits, inoculation of pear flowers with a combination of e occurs . During e occurs . In Horpe occurs ,67. Thise occurs ,69. In oe occurs ,71. TherIn general, seed set was relatively high in our study, especially in 2019, with average values between 2.9 and 3.6 seeds per pear. A seed set of two to three seeds per fruit is generally considered a good indicator for high quality ‘Comice’ pears, having a better quality compared to fruits without developed seeds ,36. MicrM. reukaufii on plant reproductive success showed different results in different plants, but no positive effects on fruit and seed set were reported so far. Herrera et al. [H. foetidus after inoculation of the nectar with M. reukauffi due to longer pollinator visits resulting in an increase of self-pollen. On the other hand, Vannette et al. [M. reukauffi on fruit-and seed set in Mimulus aurantiacus and Delphinium nuttallianum flowers respectively.The limited research on the effect of a et al.  reportede et al.  and Schae et al.  found noM. reukaufii and A. nectaris resulted in a higher visitation rate of honeybees and hoverflies, while this was not the case when the microbes were inoculated separately. Nevertheless, despite increased insect visitation rates, fruit set and seed set were not increased, most probably because trees were at maximum production capacity. We expect that under poor pollination conditions  and when trees have a limited number of flowers, microbial inoculation may lead to more pronounced effects. Further research is needed to confirm this. Then, nectar inoculation with microbes may be an effective way to enhance pollination and crop yield, which will be especially beneficial for crops that produce nectar with low sugar concentrations like pear.Altogether, we have shown that inoculation of pear flowers with a combination of the nectar microbes S1 FigMetschnikowia reukaufii (yeast), Acinetobacter nectaris (bacteria), and a combination of both (yeast + bacteria). As a control, flowers were sprayed with water. Tree rows are indicated by a number and were separated at least 4 m from each other. In each orchard pollinizer trees were planted to ensure cross-pollination. Furthermore, in each orchard two honeybee hives and ten nesting blocks for mason bees per hectare were provided to support pollination.Experiments were performed in a pear orchard in Tielt-Winge (a), Herk-de-Stad (b), Horpmaal (c) and Gutshoven (d), and included four plots of five trees that were treated. Treatments included inoculation of flowers with (PDF)Click here for additional data file.S2 FigMetschnikowia reukaufii (yeast), Acinetobacter nectaris (bacteria), and a combination of both (yeast + bacteria). As a control, flowers were sprayed with water. No significant differences occurred between treatments within one orchard (p>0.05). Error bars represent standard errors of the mean.Treatments included inoculation of flowers with (PDF)Click here for additional data file.S3 FigMetschnikowia reukaufii (yeast), Acinetobacter nectaris (bacteria) and a combination of both (yeast + bacteria). As a control, flowers were sprayed with water.Treatments included inoculation of flowers with (PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.


For cardiovascular disease prevention, statins alone or combined with ezetimibe have been recommended to achieve low-density lipoprotein cholesterol targets, but their effects on other lipids are less reported. This study was designed to examine lipid changes in subjects with ST-segment elevation myocardial infarction (STEMI) after two highly effective lipid-lowering therapies. Twenty patients with STEMI were randomized to be treated with rosuvastatin 20 mg QD or simvastatin 40 mg combined with ezetimibe 10 mg QD for 30 days. Fasting blood samples were collected on the first day (D1) and after 30 days (D30). Lipidomic analysis was performed using the Lipidyzer platform. Similar classic lipid profile was obtained in both groups of lipid-lowering therapies. However, differences with the lipidomic analysis were observed between D30 and D1 for most of the analyzed classes. Differences were noted with lipid-lowering therapies for lipids such as FA, LPC, PC, PE, CE, Cer, and SM, notably in patients treated with rosuvastatin. Correlation studies between classic lipid profiles and lipidomic results showed different information. These findings seem relevant, due to the involvement of these lipid classes in crucial mechanisms of atherosclerosis, and may account for residual cardiovascular risk.Randomized clinical trial: ClinicalTrials.gov, NCT02428374, registered on 28/09/2014. However, despite the achievement of very low levels of LDL-C, recurrent events after acute coronary syndromes are still observed, suggesting that other lipid components including fatty acids (FA) may contribute to the atherothrombotic disease4. In addition, decreased plasma levels of plasmalogens were reported in subjects with CHD5. In fact, their role in atherosclerosis is still poorly understood, involving possibly antioxidant properties that impact the movement of molecules in and out of the cells, as plasmalogens are components of cell membranes6. High free FA concentrations can induce activation of NLRP3 inflammasome, triggering a pro-inflammatory response related to atherosclerosis, and lipid-lowering therapies, such as statin combined with the inhibitor of intestinal cholesterol absorption (simvastatin + ezetimibe), can partially revert many inflammatory biomarkers9. Following rosuvastatin treatment, a lipidomic study revealed significant decrease in sphingomyelin (SM), triglycerides (TG), phosphatidylinositol (PI) and phosphatidylethanolamines (PE) levels, but for lysophosphatidylcholines (LPC) and phosphatidylcholines (PC), no significant changes were reported10. Despite effective achievement of LDL-C and non HDL-C targets by the use of less potent statin combined with ezetimibe, their effects in other lipids are less reported. Therefore, this study aimed to compare lipid composition in the plasma of subjects with very high cardiovascular risk with STEMI at baseline (first day) and after 30 days of exposure to two highly effective lipid-lowering therapies .Therapies aiming to reduce LDL-C changed substantially the natural history of cardiovascular disease (CVD), especially coronary heart disease (CHD)q-values from Post Hoc test with Bonferroni correction for multiple comparisons of the repeated measures ANOVA .Prior to the lipidomic study, a classic lipid profile was obtained. Figure VA Table . It was q-values from post hoc test with Bonferroni correction for multiple comparisons of the repeated measures ANOVA , Cer , FA , PC , PE  and SM . LPC was the only lipid class that increased  in G2. Following simvastatin plus ezetimibe therapy at D30, there was a decrease in SM . Although not significant , simvastatin treatment after D30 also showed a trend for decrease in PE  and PC . FA, CE, Cer, LPC, TG and LPE were almost unaltered in G4 . Comparing the two arms of treatment at D1 (G1 and G3) lower levels of PC  and PE  were found for simvastatin plus ezetimibe group. Regarding the effects of treatments at D30 (G2 and G4), only the sum of LPC were 14% decreased  in G4 compared to rosuvastatin arm (G2).Considering rosuvastatin administration at D30, there was a decrease for CE  for all studied patient groups Fig. . More co2 = 0.637 and Q2 = 0.246, was validated by CV-ANOVA  and permutation test (100 permutations) of selected Y variables. The poor predictability of the model, indicated by Q2 < 0.5, is provided by the low numbers of observations (n = 10) and the properties of the dataset. Thus, this model must be validated by permutation test and by cross-validation (pcv-ANOVA)11. The Q2VY values for each y variables, from the permutation test, had shown that the Y variable G4 was responsible for the poor predictability of this model (Q2 = 0.246) as shown in the Supplementary Table 2 value calculated is stable regarding this permutation11. In addition, the stable Q2 with low error  obtained towards a permutation test with five randomized dataset, indicating that the model is trustful  for variables considered statistically significant are shown in Supplementary Table Discriminant variables were selected by the combination of multivariate analysis (VIP > 1), and univariate evaluation by post hoc test with Bonferroni correction for multiple comparisons of the repeated measures ANOVA  as shown in Supplementary Table Figure Considering the temporal treatment evaluation (G1 vs G2 and G3 vs G4), Fig. 17. These changes were more pronounced by rosuvastatin therapy, and the significant decrease in Cer observed only with this lipid-lowering drug seems important, as Cer has been considered a robust predictor for cardiovascular events, independently of LDL-C as well as for cardiovascular mortality19. It should be pointed out that, although in the lipid class evaluation, the sum of individual lipids from the same class presented significant changes only for SM class with simvastatin plus ezetimibe therapy, the statistical analysis of individual lipid species has not shown that the treatment altered lipids from many other classes . These findings highlight the importance of evaluating lipid species individually. Lipids with different fatty acid chains, from the same class, may present opposite effects in CVD risk. In fact, lipidomic studies have shown that PC containing polyunsaturated fatty acyl chains were negatively associated with CVD risk, while saturated and monounsaturated fatty acyl chains were deleterious for CVD risk20.This study revealed differences in many lipid concentrations in subjects with STEMI despite comparable classic lipid profiles. After highly effective lipid-lowering therapies (rosuvastatin or simvastatin + ezetimibe), similar trend in the lipid composition were observed for both therapies. However, differences were noted between the lipid-lowering therapies for many lipids such as FA, LPC, PC, PE, CE, Cer and SM and these findings seem relevant, due to the involvement of these lipid classes in crucial mechanisms of atherosclerosis 23. Although FA and their esters constitute the major sources of energy for the heart muscle, their excess has profound effects on the heart causing an enhanced susceptibility to inflammation, oxidative stress and ischemic damage (fibrosis and hypertrophy)24. Different FA can be metabolized into pro- and anti-inflammatory signalling molecules. In particular, some n-6 FA (first and foremost arachidonic acid) are precursors to pro-inflammatory molecules (primarily prostaglandins)24, while saturated fatty acids (SAFAs) act as major inducers of inflammation through several mechanisms, such as the activation of toll-like receptor-4 (TLR4), which promotes the production of inflammatory cytokines 25. Endothelial dysfunction due to the NF-kappa B activation is also induced by SAFA, resulting in increased superoxide production, while NLRP3 inflammation activation increases endothelial permeability27. In our study, in the two arms of therapy, lower concentrations of FA  were observed after 1 month of treatment. These results suggest a potential beneficial effect of therapies on CVD. Interesting to note, both therapies promoted similar reduction in FA, but for FA 16:0, it was significantly reduced at D30 only in the rosuvastatin arm. Our results are in line with a meta-analysis of clinical studies involving atorvastatin and simvastatin published by Sahebkar et al. showing reduction in free fatty acids levels independently of treatment duration, dose and magnitude of reduction in LDL-C levels28.Inflammation and oxidative stress have been considered as a part of the pathophysiology of acute coronary syndromes and their recurrences30. However, findings from recent clinical studies have suggested potential biomarkers of positive prognostic making those studies controversial20. A key issue is the complexity of the enzyme cascade involved in LPC metabolism, which shows that a long way must be traveled until the understanding of the results reported in literature31.LPC is increasingly recognized as a key factor positively associated with atherosclerosis development and cardiovascular diseases.. Fernandez, et al.studying plasma lipid composition and risk of developing cardiovascular disease also observed a decrease in LPCs associated with CVD32. Other interesting study on lipid profile of rosuvastatin in humans has also highlighted changes in LPC and PC compound classes, suggesting an effect of rosuvastatin in LPC and PC metabolism10. Although no significant differences between therapies were observed in the D1 (G1 vs. G3) and D30 (G2 vs. G4) for LPC compounds class, temporal % changes in the rosuvastatin arm were higher than simvastatin/ezetimibe.In our study, LPCs were significantly increased with rosuvastatin treatment at D30, of which five were responsible for this increase , while only three of them had their levels altered after simvastatin + ezetimibe at D30 Table . Interes33. Furthermore, Khaw et al. have associated odd chain phospholipids with lower cardiovascular risk34. Also, in the study of Ward-Caviness et al. a standard deviation increase in log-transformed concentration of LPC 17:0 negatively correlated with the risk of incident myocardial infarction in three cohorts35. Interestingly, our study shows that rosuvastatin therapy increased LPC 17:0 in more than 50% at D30, which could contribute to a cardiovascular protective effect.Some lipid composed of odd chain fatty acids were significantly altered by both treatments. Aforetime, odd chain fatty acids were associated solely with diet, however nowadays they are recognized as products of catabolism of branched-chain amino acids. Branched-chain amino acids were positively associated with CVD risk and type 2 diabetesPE and PC were reduced after treatment with both lipid-lowering therapies. High levels were found in rosuvastatin group for most of the compounds.36. Higher decrease with rosuvastatin compared with atorvastatin treatments was found in the SM/SM + PC ratio37. The same was found here, for rosuvastatin and simvastatin + ezetimibe comparison. These results corroborate Choi et al. findings, that reported a significant decrease in SM, TG and PE, especially in PE 18:0_18:2 levels, and an increase in LPC 20:4 and LPC 18:110. In 2018, Lee et al. hypothesized that these increases may be due to pleiotropic effects of statins, since phospholipase A2, which is the main enzyme involved in LPC metabolism, is inhibited by rosuvastatin38.In addition, our study showed that both lipid-lowering therapies decreased SM concentrations at D30. Sphingomyelin was measured in the large Multi-Ethnic Study of Atherosclerosis (MESA), and the authors reported a modest negative association with incident CVDs, after adjustment for lipoproteins and full adjustment for other risk factorsBesides the superiority in changing lipid concentration for rosuvastatin in the altered classes compared to simvastatin + ezetimibe, only rosuvastatin promoted significant reduction in CE and Cer compounds.. studying the dose-dependent effects on plasma sphingolipidome and phospholipidome in the metabolic syndrome found that rosuvastatin at both 10 and 40 mg/d significantly reduced the concentrations of total and individual plasma sphingolipids and phospholipids with evidence of dose-dependent effects39. For CE, as the results found for LPC, controversial findings have been reported, but Stegemann et al. applying mass spectrometry-based lipidomic profiling, reported an association of CE with cardiovascular disease over a 10-year observation period40.Ng et alConsidering the correlations between lipids and classic clinical parameters, it is interesting to note the number of correlations found at G1 compared to other groups . The lack of correlations for those groups indicates that lipidomic measurements and classic lipid profile addresses different information. In this work, PC, SM and TG were positively correlated to LDL-C for both lipid-lowering therapies at D1, but a negative or no correlation between HDL-C and TG was found, mainly when rosuvastatin was administrated at D30. There is an expected inverse association between HDL-C and TG in diabetic and overweight patients. Patients with insulin resistance have delayed hydrolysis of TG-rich lipoproteins due to reduced lipoprotein lipase activity. As a result, fewer phospholipids and surface components are transferred to HDL, decreasing its effectiveness for the reverse cholesterol transport. Therefore, lower concentrations of cholesterol from HDL are found.20. Studies examining CVDs with lipidomics found that after adjusting for HDL-C and LDL-C levels, only PC remained associated with cardiovascular events45.A previous study reported that LPC are predominantly found in HDL, Cer in LDL, and PC are present in both lipoproteins (HDL and LDL)46.In this work, LDL-C was reduced with both lipid-lowering therapies at similar % changes. Otherwise, Cer was reduced significantly, only with rosuvastatin at D30. As a reduction of LDL-C and Cer is expected to contribute to lower residual cardiovascular risk, rosuvastatin presented better results than simvastatin + ezetimibe. Considering classic lipid parameters, rosuvastatin treatment decreased in 17% HDL-C level after one month. Another study showed that patients on rosuvastatin therapy may have either increased or decreased the HDL-c after 4 weeks of treatment. However, these changes in HDL-C did not affect the incidence of major cardiovascular events in one year follow-up50. Interestingly, the sum of our findings corroborates with a review article recently published by Rai & Bhatnagar showing decreased LPC 16:0 in hyperlipidemia causative disorders, such as high-fat diet, obesity and diabetes. LPC decrease was accompanied by increase in FA and Cer. The authors also highlighted the association of hyperlipidemia with an increase in small-chain fatty acids, SAFA content of DG, TG and PC lipid classes, factors associated with CVD risk51. No direct comparisons between rosuvastatin and simvastatin + ezetimibe in cardiovascular outcomes have been reported.In summary, regarding to the changes in the classic lipid profile, our results are in agreement with previous studies50. In addition, the myocardial injury estimated by troponins was similar in both arms of lipid-lowering treatment. Our sample size is relatively small, but the patients included in the study were all submitted to same treatment strategy (pharmacoinvasive) with similar characteristics at baseline.This study compared two highly effective lipid-lowering therapies in the acute phase of myocardial infarction. However, we are unable to estimate the effects of the acute myocardial infarction per se on lipid composition. It is expected some decrease in lipids due to the healing process involving the necrotic and ischemic myocardium, but these changes have been reported as insignificant in the following days after the acute coronary eventIn spite of comparable classic lipid profile at baseline and after the exposure to treatments, significant differences in lipid composition were found between the two highly effective lipid-lowering therapies. To note, higher % changes in the rosuvastatin arm of therapy compared to simvastatin + ezetimibe were identified, and significant changes for CE and Cer were observed only in the rosuvastatin group. Regarding the correlation studies, we found that lipidomic analysis and classic clinical exams account for different information in both lipid-lowering therapies. In summary, our results indicate important differences in lipid composition that cannot be identified by the classic lipid profile between the studied lipid-lowering therapies. These differences may account for residual cardiovascular risk.Methanol, 1-propanol and dichloromethane in HPLC grade were purchased from JTBAKER . Water was purified by the Milli Q system . Ammonium acetate was obtained from Sigma-Aldrich . The Lipidyzer isotope labeled internal standards mixture kit consisting of 54 isotopes from 13 lipid classes , PC, PE, Sphingomyelin (SM), diacylglycerols (DG), TG, FA, Cholesterol ester (CE), Ceramide (Cer); dihydroceramides (DhCer), hexosylceramides (HexCer), lactosylceramides (LacCer) was purchased from Sciex .This prospective, randomized, open label study was delineated to evaluate differences in the composition of lipids in patients with STEMI at D1 and at D30 after implementing the two lipid-lowering therapies . Plasma of patients were categorized in four groups: patients in the rosuvastatin group at D1 (G1) and at D30 (G2); patients treated with simvastatin + ezetimibe at D1 (G3) and at D30 (G4). These two lipid-lowering therapies were chosen to promote similar changes in the classic lipid profile, allowing the comparison of the more effective inhibition of cholesterol synthesis (rosuvastatin) with the combined mechanisms of LDL-C lowering . Patients were randomized 1:1 for the lipid-lowering therapy using a central computerized system (battle-ami.huhsp.org.br). All patients followed similar protocol, receiving dual antiplatelet therapies, betablockers, and renin-angiotensin system blockers and they were referred to coronary angiogram, and percutaneous coronary intervention, when needed, in the first 24 h of STEMI.52. They had no prior MI and were naive for lipid-lowering treatment. All patients were submitted to pharmacological thrombolysis with tenecteplase in the first 6 h of STEMI, followed by coronary angiogram and percutaneous intervention when needed in the first 24 h of STEMI (pharmacoinvasive strategy). Key exclusion criteria included hemodynamic instability, autoimmune disease, known malignancy, pregnancy and signs of active infections. All patients received the study medications from the hospitalization as well as at hospital discharge. These patients were monitored by phone and followed up in our outpatient clinic . At each visit, the patients brought the boxes of their medications to check their adherence.The study included mainly middle aged males, approximately half of them with type 2 diabetes. From the 25 patients consecutively screened for the trial, three were not eligible due to inclusion/exclusion criteria and two did not complete trial . Table g for 15 min, at room temperature and storage at − 80 °C before analysis. All samples for general biochemical tests, including the classic lipid profile were performed in the Central Laboratory of the University Hospital and the LDL-C was estimated by the Friedewald equation. Biochemical determination of TC, HDL-c and TG* was performed by enzymatic colorimetric assays with commercial kits from Roche in Cobas C 501 module.Fasting blood samples were collected, in the morning at D1 and at D30 after lipid-lowering therapy, in tubes containing EDTA, followed by centrifugation at 130053. Briefly, 100 μL of plasma were transferred to a borosilicate glass culture tube (16 × 100 mm)53. Next, 900 μL water, 2 mL methanol, and 900 μL dichloromethane were added to all samples and the mixture was vortexed for 5 s. Samples were left to incubate at room temperature for 30 min. Next, another 1 mL water and 900 μL dichloromethane were added to the tube, followed by gentle vortexing for 5 s, and centrifugation at 2500g at 15 °C for 10 min. The bottom organic layer was transferred to a new tube and 1.8 mL dichloromethane were added to the original tube for a second extraction. The combined extracts were concentrated under nitrogen. Exactly 100 μL of the isotope labeled internal standards mixture were added to the dried extract and another 30 min incubation was allowed until equilibrium is reached. Finally, 250 μL mobile phase solution (10 mmol L−1 ammonium acetate in 50:50 methanol:dichloromethane) were added. IS mixture is composed by 54 labeled lipid species that covers 10 main lipid classes found in human plasma with different final concentrations reflecting their physiological concentrations54.Lipid extraction was carried out by a modified Blight-Dyer protocol as described elsewhere−1. Each sample was injected twice, with the DMS on (PC/PE/LPC/LPE/SM) and off (CE/Cer/DG/FA/TG). Over 1100 lipid species and 54 labeled internal standards were monitored by selected reaction monitoring (SRM) in positive/negative polarity switching. Positive ion mode was used to detect the lipid classes SM/DG/CE/Cer/TG and the negative ion mode to detect the lipid classes LPE/LPC/PC/PE/FA. Lipid annotation is achieved by measuring specific SRM transitions, where the monitored fragment is related to the fatty acid composition. Also, lipid class is determined by ramping the compensation voltages in the differential mobility unit of the Lipidyzer platform. DMS parameters used were: temperature = low; separation voltage = 3.5 kV and differential mobility spectrometric resolution = low. Electrospray ion source parameters were as follow: voltage (ESI +): 4.1 kV, voltage (ESI -) = − 2.5 kV, curtain gas = 17, CAD gas = Medium, Temperature = 200 °C, Nebulizing gas = 17 and heater gas − 25.Quantitative lipidomics was performed with the Sciex Lipidyzer platform configured by an ExionLC AD instrument (Sciex) coupled to a QTRAP 5500 mass spectrometer (Sciex) equipped with SelexION for differential mobility spectrometry (DMS) and electrospray ionization (ESI) source. The solvent 1-propanol was used as the chemical modifier for the DMS. Samples were introduced to the mass spectrometer by flow injection at 8 µL min59. Quality control (QC) consisted of a standard plasma sample obtained from the Lipydizer kit. The reconstituted lyophilized plasma was extracted following the procedure described previously. The QC sample was injected five times at the beginning of the randomized sample batch, every 10 injections and, at the end of the sample batch.All data obtained from the Lipidyzer Platform were automatically processed in the Lipidomics Workflow Manager (LWM). Signals of all lipids obtained for each sample were quantified using the intensity of internal standard applying the Lipidyzer platform. The software calculates concentration as average intensity of the analyte MRM/average intensity of the most structurally similar IS MRM multiplied by its concentration in nmol/mL. Lipidyzer platform allowed for automated data acquisition, data processing, and reporting. A detailed description of the method can be found in previous studieshttps://www.minitab.com/pt-br/) and Microsoft Excel 2013. Univariate analysis (Repeated Measures ANOVA) was performed in MATLAB  using an in-house script.Box-plot was performed using Minitab 17.0 . Correlation graphics were performed in R 3.6.3 . From the original generated table compiling the lipids identified  for peak areas; in this work, < 20% CV was used as a criterion to retain that particular component in the dataset for further evaluation, which was in agreement with the recomendationThe study protocol was approved by the local ethics committee , which follows the Declaration of Helsinki, and written informed consent was provided by all subjects before their inclusion in the study.Supplementary Information.


Coronary artery (CA) aneurysms in children are a rare but potentially life-threatening finding and are highly associated with Kawasaki disease (KD).We describe a four-year-old female with a vasculitis and CA aneurysms. She had a prolonged course with recurrent fever and systemic inflammation several times upon discontinuation of steroid treatment. Due in part to the CA aneurysms, she initially was diagnosed with KD but due to the unusual clinical course, further evaluation was performed. Abdominal and chest MRI/A revealed diffuse aortitis suggestive of a large vessel vasculitis, specifically Takayasu arteritis. With treatment targeted for Takayasu arteritis, there was resolution of fever and inflammation and the CA aneurysms improved.This case demonstrates the utility in broadening the differential diagnosis in cases of presumed KD with CA involvement in which the clinical course is atypical for KD. Kawasaki disease (KD), the leading cause of acquired pediatric heart disease in the developed world, is a vasculitis that preferentially affects medium-sized arteries, particularly the coronary arteries (CA) . CoronarA 4-year-old female patient presented to her pediatrician with 2 days of fever and emesis. She was initially treated with amoxicillin for presumptive strep throat. Fever continued despite antibiotic treatment and she represented to an outside hospital’s emergency department (Day 5 of illness). By this time, she had developed a rash, hand puffiness and bilateral non-exudative conjunctivitis. She was given a 3 day course of corticosteroids. She defervesced and her symptoms resolved.3 cells/uL, hemoglobin (Hgb) 11.9 g/dL, platelets 393 × 103 cells/uL, sodium 132 mEq/L, and multiple negative SARS-CoV-2 antibody and PCR tests. Echocardiogram was performed due to concern for KD and was normal, including normal CA size and appearance. She was treated on Day 11 with 2 g/kg of intravenous immunoglobulin (IVIG) and high dose aspirin for presumed KD. Her fever resolved and she was discharged home on Day 13. Three days later she had recurrent low-grade fevers and elevated inflammatory markers which prompted readmission and retreatment with IVIG (Day 16). Her physical exam at that time was notable for bilateral conjunctival injection, normal tongue and oropharynx without erythema or lesions, no lymphadenopathy, pulses intact throughout with capillary refill less than 2 s, “reluctant but full” joint range of motion, mild edema of hands and feet, no fingertip or toe peeling, and no rash. Labs at this admission suggesting incomplete KD were hypoalbuminemia 2.7 mg/dL, elevated CRP to 14.4 mg/dL, elevated ESR 130 mm/hr., normocytic anemia with Hgb 10.8 g/dL, leukocytosis to17.4 Th/uL, and mild thrombocytosis to 486 Th/uL Her echocardiogram showed a new, small, saccular aneurysm in the left anterior descending (LAD) CA with maximal dimension of 2.9 mm, z-score 3.27 (Day 17). She met criteria for incomplete KD based on the American Heart Association incomplete KD algorithm with 3/5 clinical criteria, elevated inflammatory markers and abnormal coronary arteries. A second dose of IVIG (2 g/kg) was given but fever persisted and oral corticosteroids were reinitiated at 1 mg/kg BID. She subsequently defervesced and was discharged on oral steroids and low dose aspirin.The fever recurred 10 days after initial symptom onset and she was admitted. Labs were notable for an erythrocyte sedimentation rate (ESR) of 53 mm/hr., c-reactive protein (CRP) 3.9 mg/dL, white blood count (WBC) 23.4 × 10Over serial outpatient follow-up, she had interval increases in CA measurements on echocardiography , with LAD dimension progressing to 4.5 mm, z-score 9.0 and RCA dimension to 3.5 mm, z-score 4.2. She was weaned off steroids over 3 weeks, but developed recurrent fever and increase in CRP (Day 37), prompting readmission. Over this course she was noted to have mild systemic hypertension. Echocardiogram and CT were notable for an interval increase of the previously noted LAD aneurysm, now measuring 5–6 mm (z-score ~ 11) as well as RCA aneurysm with max dimension ~ 4 mm  she developed recurrent fever and elevated inflammatory markers prompting repeat admission and treatment with a second dose of cyclophosphamide (10 mg/kg) and re-initiation of steroids. Given her prolonged and atypical course of presumed KD, with recurrence of inflammation and fever with steroid weans she was evaluated for alternative forms of vasculitis. This included an MRI/A abdomen that showed diffuse wall thickening and diffuse abnormal enhancement of the thoracic and abdominal aorta with suspected involvement of the right brachiocephalic and bilateral common carotid arteries, indicating a diffuse aortitis Fig. . KD doesWe describe a case which illustrates that although KD is the most likely cause of CA aneurysm in children, there are other causes. Our patient was initially diagnosed with incomplete KD and progressive CA aneurysm enlargement over the first 6 weeks of illness. Her clinical course was atypical for KD, in that she had recurrent fever and inflammation over > 2 months with inability to wean from steroids. Even in the absence of treatment, fever is self-limited in KD and rarely lasts beyond 3–4 weeks . The unuTakayasu arteritis is an idiopathic large vessel vasculitis that primarily affects the aorta and its main branches . Though Coronary artery involvement, particularly CA stenosis, is fairly common in TA. Yet TA is rarely considered in infants and young children with CA aneurysm due to the low incidence of TA in this age range. The prevalence of CA involvement ranges from 3 to 30% in prior reports –11. CoroIn addition to KD and TA, there are several other reported etiologies of CA dilation/aneurysm in children. As these diseases are rare causes of CA aneurysm, the literature mainly consists of case series and reports. Multiple autoimmune diseases have been implicated as potential causes of CA dilation/aneurysm. Polyarteritis nodosa has been associated with CA aneurysms in several case reports, with acute myocardial infarction as the presenting symptom of in several reports –18. In aInterest in etiologies of CA enlargement in children increased recently with the description of Multisystem Inflammatory Syndrome in Children (MIS-C), a newly characterized syndrome associated with SARS-CoV-2. In the case of our patient, multiple negative tests for SARs-CoV2 antibodies makes this diagnosis less likely. However, MIS-C has overlapping features with KD, including potential for CA dilation/aneurysm but also clear differences, including an older patient population, different ethnic distribution, more severe inflammation and myocardial involvement, and more frequent presentation in hypotensive shock –38. CoroIn summary, CA dilation or aneurysm, though most commonly associated with KD in children, can also be a manifestation of several other rheumatologic and infectious diseases. This case demonstrates the utility in broadening the differential diagnosis in cases of presumed KD with CA aneurysm but with a clinical course that is inconsistent with KD.


The influence of lymph node dissection (LND) on survival in patients with head and neck neurogenic tumors remains unclear. We aimed to determine the effect of LND on the outcomes of patients with head and neck neurogenic tumors.Data of patients with surgically treated head and neck neurogenic tumors were identified from the Surveillance, Epidemiology, and End Results (SEER) database (1975–2016) to investigate the relationship between LND and clinical outcomes by survival analysis. Subgroup analysis was performed in IVa and IVb group.P < .001), advanced stage (P = .037), African American race (P = .002), diagnosis before 2004 (P < .001), and chemotherapy administration (P < .001) to be independent negative predictors of overall survival. Kaplan-Meier analysis demonstrated that LND was not a predictor of clinical nodal negativity (cN0) in either IVa or IVb patients.In total, 662 head and neck neurogenic tumor patients (median age: 49.0 [0–91.0] years) met the inclusion criteria, of whom 13.1% were in the IVa group and 86.9% were in the IVb group. The median follow-up time was 76.0 months (range: 6.0–336.0 months), and the 5-year and 10-year overall survival was 82.4%  and 69.0% . Cox regression analysis revealed older age (In head and neck neurogenic patients, LND may not impact the outcome of cN0 in either IVa or IVb group. These data can be recommended in guiding surgical plan and future studies. Neurogenic tumors that originate from neural crest derivatives occur in the head and neck in 25–45% of cases –3. Head As known for most head and neck cancers, the regional and distant metastases have been identified as one of the major challenges affecting long-term survival . ThorougThis retrospective study used data from the National Cancer Institute’s SEER project database from 1975 to 2016. The study protocol was designed according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement . InstituThe SEER 18 database was searched to identify all patients diagnosed with neuroblastoma and peripheral nervous cell tumors using the 3rd edition of the ICCC site recode extended classification and the IARC code IV. Site codes for tumors originating from the peripheral nervous system and soft tissue of the head, face, neck, nasal cavity, or paranasal sinuses were searched. The following categories were obtained from the data: age at diagnosis, sex, ethnicity, year of diagnosis, SEER historic stage, reason for not undergoing cancer-directed surgery, survival months, vital status recode, SEER classification for other causes of death, therapy, examination of regional nodes and their respective positivity status. Detailed information on all variables mentioned in the SEER database is available in Appendix C of the SEER manual.The inclusion criteria were primary diagnosis of malignant neurogenic tumor and definitive surgery performed at the primary site. Only patients followed for 6 months were included in this study. The most standard classification of neuroblastoma, called the International Neuroblastoma Staging System (INSS), was not included in the SEER database, and the SEER historic stage was used to obtain the tumor stage. Therefore, disease classification included localized, regional, and distant subgroups. Data on age, sex, ethnicity, tumor size, survival months, and therapy were obtained directly from the original data without further conversion. Overall survival (OS) data for patients in the study cohort were based on vital status, in which patients were coded as alive or dead. Disease-specific survival (DSS) data were obtained according to other causes of death. The clinical nodal stage of clinical nodal negative (cN0) patients and clinical nodal positive (cN+) patients was based on criteria taken from the “SEER historic stage A”, “SEER Combined Summary Stage 2000”, and “Derived AJCC Stage Group, 7th edition”. The diagnosis was determined on imaging and physical examination. According to the number of examined lymph nodes, we divided the patients into two groups: the non-LND and the LND group. The pathology finding of nodes was obtained on “Regional nodes positive”. Regarding the years of diagnosis, 3 time periods were considered as follows: before 1998, 1998–2004 and after 2004. This was done because the study period was too long to ensure that the treatment protocols would remain consistent throughout its entirety.2 test. The correlation between the classification variables was determined using Spearman’s correlation test or the Mann-Whitney U test. The kappa index (kappa) was used to assess the agreement between two clustering results. Kaplan-Meier survival analysis was performed to estimate OS and DSS. The log-rank test or Breslow test were used to determine statistically significant predictors of survival on univariate analysis. Covariates with P < 0.05 in the univariate analysis or important covariates were included in the multivariate model. Multivariate Cox analysis was performed to estimate the independent effects of age, treatment and lymphadenectomy to obtain hazard ratios (HR) and 95% confidence intervals (CI). A P-value of < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS version 25.0 and Stata software version 12.0. A normally distributed test was performed on quantitative data. Normally distributed measurement data are expressed as X ± S, while the non-normally distributed measurement data are expressed as medians (interquartile range). Categorical variables were compared using the χA total of 662 patients diagnosed with head and neck neurogenic tumors between 1975 and 2016 were included. A total of 544 cases were excluded in which 99 cases were patients with non-primary tumor, 219 were patients who did not received surgery, 109 were patients with unclear LND information, 63 were patients with unclear clinical nodal stage, and 54 were patients with incomplete information . Overall, 72.9% (70/96) patients with LND had pathologically positive nodes. After Kappa analyses showed that preoperative lymph node staging and postoperative lymph node staging observer agreements were excellent .In addition, 63.4% (420/662) patients were cN0 and 36.6% (242/662) were cN+. The rate of LND was 14.5% (96/662), the LND rate for IVa group was 56.3% (49/87) and 8.2% (47/575) in IVb group . Similarly, women had a higher OS and DSS rates than men . In addition, SEER historic stage and clinical nodal status were both significant predictors of outcomes in our cohort . Patients diagnosed between 1998 and 2004 and after 2004 had better outcomes than those diagnosed before 1998 . The OS and DSS rates did not differ between patients who received LND and patients who did not . In the IVa disease group, the OS and DSS rates were better in patients with LND than patients with non-LND . However, the IVb group showed opposite results: OS and DSS rates were better in the patients without LND  . Comparative analysis found that patients who received chemotherapy or radiation had more advanced stages of the disease . Multivariable Cox regression analysis (Table P < .001), advanced stage (P = .037), African American race (P = .002), diagnosis before 2004 (P < .001), and chemotherapy administration (P < .001) to be independent negative predictors of OS. The risk of outcomes increased 1.04-fold per year with increasing age (95% CI = 1.030–1.053).Overall, the median follow-up time was 76.0 months (range: 6.0–336.0 months), and 633 (95.6%) patients were followed up over 1 year and 387 (58.5%) patients were followed up over 5 years. The 5-year and 10-year OSs were 82.4%  and 69.0% , respectively. The 5-year and 10-year DSSs were 87.5%  and 79.1% , respectively. The OS and DSS were significant better in children than in adults  but not in cN0 patients . Univariable analysis revealed that children (P = .007), SEER stage (P < .001), diagnosis after 1998 (P < 0.023), LND (P = .018), and radiation administration (P < .001) were predictors of OS and DSS. Multivariable analysis revealed that cN+ (P = .005), and LND (P = .024) were independent predictors of OS and DSS .For stage IVa patients, Kaplan-Meier analysis demonstrated that LND was a positive predictor of OS and DSS in the whole cohort or in cN+ patients  or cN0 patients . Multivariable analysis revealed SEER stage (P = .017), diagnosis year (P = .001), and received chemotherapy (P < .001) to be independent predictors of OS and DSS.For patients with IVb, Kaplan-Meier analysis demonstrated LND was negative predictor of overall survival and DSS in whole patients but not patients with cN+  and 8.2% (47/575), respectively. The overall LND rate in the cohort was relatively low, regardless of whether the clinical stage was positive or negative. Our study also showed that LND did not improve OS or DSS in cN0 patients in stages IVa or IVb. This finding was consistent with other head and neck cancers reported in the literature, including cervical adenoid cystic carcinoma, ENB, and cervical unknown primary site of squamous cell carcinoma –21. AddiHowever, it is odd that most (68.6%) cN+ patients did not get neck dissection in our cohort. And 79.8% 158/198) IVb patients with cN+ but only 18.1% (8/44) IVa patients with cN+ did not received LND by further analysis. This is related to the heterogeneity of two diseases, including the preferred treatment method and sensitivity to chemotherapy or radiotherapy. Except for individuals who refused to receive complete or elective neck dissection, some ENB patients may have received radiation therapy of the neck [8/198 IVbP < .001). The age at diagnosis of neuroblastoma is a standard indicator of prognosis [The clinical features and outcomes varied greatly according to the histology of neurogenic tumors . Our serrognosis . While yrognosis . A retrorognosis . In addirognosis –26. Morerognosis .Advanced-stage neurogenic tumors have a high metastasis rate and poor prognoses . The SEERegarding multimodal treatments, patients who received chemotherapy or radiation had a worse prognosis. This might be related to the advanced disease stage of patients in the chemo/radiation group. In addition, patient prognosis improved from 1975 to 2017 due to the significant evolution in the management of oncologic treatment and dramatic escalations in the intensity of therapy during these years .The major limitation of this study is that no further analysis was performed in the subgroup limited to the small number of cases, especially the IVa group. The two most common diseases in the cohort were neuroblastoma and ENB and they are very different in terms of clinical features and outcomes. In addition, the reliability of our results could be improved by verifying our own data further. Moreover, we were also unable to report on other important variables like the extent of neck dissection and surgical approach, which were not recorded in the SEER database.Our study demonstrated that older age, advanced SEER stage, Asian American race, diagnosis before 2004, and chemotherapy administration significantly influenced the outcomes of head and neck malignant neurogenic tumors. Moreover, LND was not a predictor of OS and DSS in cN0 patients. These data can be recommended in guiding surgical plan and future studies. Larger prospective cohort studies are required to confirm our findings and determine the true prognostic impact of neck dissection. Further studies on different lymphadenectomy methods for cN+ patients are also needed.


Tumour hypoxia promotes an aggressive tumour phenotype and enhances resistance to anticancer treatments. Following the recent observation that the mitochondrial inhibitor atovaquone increases tumour oxygenation in NSCLC, we sought to assess whether atovaquone affects tumour subregions differently depending on their level of hypoxia.n = 15) received atovaquone treatment, whilst cohort 2 (n = 15) did not. Hypoxia-related metrics, including change in mean tumour-to-blood ratio, tumour hypoxic volume, and fraction of hypoxic voxels, were assessed using hypoxia PET imaging. Tumours were divided into four subregions or distance categories: edge, outer, inner, and centre, using MATLAB.Patients with resectable NSCLC participated in the ATOM trial (NCT02628080). Cohort 1 (Atovaquone-induced reduction in tumour hypoxia mostly occurred in the inner and outer tumour subregions, and to a lesser extent in the centre subregion. Atovaquone did not seem to act in the edge subregion, which was the only tumour subregion that was non-hypoxic at baseline. Notably, the most intensely hypoxic tumour voxels, and therefore the most radiobiologically resistant areas, were subject to the most pronounced decrease in hypoxia in the different subregions.This study provides insights into the action of atovaquone in tumour subregions that help to better understand its role as a novel tumour radiosensitiser.Trial registration: ClinicalTrials.gov, NCT0262808. Registered 11th December 2015, https://clinicaltrials.gov/ct2/show/NCT02628080The online version contains supplementary material available at 10.1186/s13550-021-00871-x. Lung cancer is the leading cause of cancer-related death in the UK with a five-year overall survival rate only in the region of 16%  and is aOxygen deprivation or hypoxia is defined as an oxygen tension under physiological normoxia  . HypoxiaTo reduce tumour hypoxia and therefore enhance radiosensitivity, decreasing cellular oxygen consumption rate (OCR) via metabolic reprogramming has arisen as a promising approach . Mathemabc1 complex) of the electron transport chain (ETC)  and outer [1.37 (95% CI 1.2–1.5)] subregions exceeded baseline mean TBRmean in the centre subregion [1.19 (95% CI 1.0–1.3)]. Change in mean TBRmean in the non-hypoxic edge subregion following treatment was negligible . In contrast, no significant change was observed in TBRmean in untreated patients, neither in tumours overall nor in any tumour subregion . TBRmean increased from baseline in the edge region in both cohorts similarly, by + 8.0% in atovaquone-treated and by + 4.6% in untreated patients. Thus, atovaquone caused a decrease in TBRmean in tumours overall and in all tumour subregions except the edge region. Interestingly, the reduction in TBRmean in the centre subregion was lower than in the inner and outer subregions. TBRmax values showed a similar trend with no significant changes from baseline in each distance category and overall for untreated patients  atovaquone-treated patients had an overall and meaningful  decrease in HV from baseline, and the median change was − 28% (95% CI − 58.2 to − 4.4). Meanwhile, only two (13.3%) untreated patients had an overall reduction in HV equal to or greater than 10%, and the median change was + 15.5% (95% CI − 6.5 to 35.5), as previously reported [In both cohorts, the magnitude of tumour HV at baseline was not in itself predictive of hypoxia reduction to atovaquone. For instance, the two untreated patients that displayed a meaningful reduction in HV in tumours overall had either medium (44.8 mL) or very small (3.5 mL) baseline HV. Similarly, no meaningful decrease in HV was observed in four atovaquone-treated patients, who had large (366.9 mL), medium (45.9 and 59.3 mL), and small (24.6 mL) baseline HVs.As median HV decreased from baseline in the centre, inner, and outer tumour subregions, analysis of more intense tumour hypoxia was performed. TBR greater than 1.6 and 1.8 were used to define tumour hypoxic voxels. Only tumours that had sufficient HV (predefined as ≥ 1.5 mL) at these TBR thresholds to accurately assess change were included. Using TBR greater than 1.6 and 1.8, atovaquone-induced reduction in HV was even more pronounced in all tumour subregions except in the edge subregion where no median change in HV was consistently observed Table .Table 2SGiven that the treatment duration differed between patients, its influence on tumour HV changes was evaluated  contrary to untreated patients where no significant change was observed (P = 0.63)  and outer (P = 0.01) subregions. The reduction was non-significant in the centre (P = 0.05) and negligible in the edge subregion (P = 0.86)  Table a. In unt6) Table b.P = 0.96) nor in centre (P = 0.31), inner (P = 0.95), outer (P = 0.91), and edge (P = 0.21) subregions (Table P = 0.01) (Table P < 0.001) and in the inner (P < 0.01) and outer (P = 0.01) subregions  Table b. In thens Table c. In untThus, it seems that atovaquone did decrease the global fraction of hypoxic voxels significantly in tumours overall and in all subregions except the edge subregion. The fraction of the most intensely hypoxic voxels also showed the greatest reduction among tumour hypoxic voxels and in most tumour subregions.mean in this subregion was markedly lower than the hypoxia threshold set at 1.4, and although TBRmean did increase during treatment, it was insufficient to classify these voxels as hypoxic, thus explaining the absence of change in median HV in the edge tumour subregion.Following the recent demonstration that the mitochondrial inhibitor atovaquone decreases tumour hypoxia in patients with NSCLC, this study shows that the reduction in TBR and HV occurred in the outer, inner, and centre tumour subregions. The centre subregion displayed a lower TBR at baseline which is likely to be due to lack of hypoxic PET tracer uptake due to a lack of viable tumour cells (e.g. necrotic regions) and may explain the lesser reduction observed in these regions. Besides, atovaquone did not seem to act at the edge subregion of the tumour. Baseline TBRmean from baseline in tumours of untreated patients, signifying that tumour hypoxia intensified over time, reinforced the reduction observed in atovaquone-treated patients. Moreover, as the nitroimidazole-based tracers FMISO and FAZA used accumulate exclusively in viable cells at oxygen levels responsible for hypoxia-related radioresistance [The increase in median HV and TBRsistance , any decsistance , 22. NotAs hypoxia appeared to decrease to a greater extent in the inner and outer tumour subregions than in the centre subregion in atovaquone-treated patients, we hypothesise that blood perfusion may impact the reported treatment outcome. TBR indices at 4 hours post-injection seemed to fall towards tumour centres, and it is possible that TBR could underestimate the extent of hypoxia, especially at tumour centres. These voxels could be poorly perfused , leading to very low rates of FMISO influx which would cause TBR values to remain relatively low despite generating hypoxic conditions which would cause long-term tracer accumulation. Thus, it would be interesting to assess the relationship between tumour blood perfusion and TBR using baseline perfusion CT and hypoxia PET scans.Fixed distance categories were used to assign voxels to a tumour subregion, in accordance with a previous study on FMISO PET kinetics in NSCLC . Yet, tumean in a voxel. As shown by Grimes et al , one woOur analysis of hypoxia PET scans shows differences in hypoxia in different NSCLC tumour regions at baseline, which could be useful for RT dose guidance. Hypoxia-based RT dose painting is an appealing approach for cancer therapy , 27. AddTBRmean between the two scans for the overall tumour or individual tumour regions.Hypoxia PET image repeatability has been demonstrated to be high with modern PET/CT systems using FMISO head and neck cancer (HNC)  and NSCLIn this study, we provide insights into the action of atovaquone in different subregions of NSCLC tumours. Following the demonstration that atovaquone reduces tumour hypoxia in patients with NSCLC, we show that this reduction mostly occurs in the inner and outer tumour subregions, and to a lesser extent in the centre subregion. Importantly, the most intensely hypoxic tumour voxels, and therefore the most radiobiologically resistant areas, are subject to the most pronounced decrease in hypoxia. This enables a better understanding of the role of atovaquone as a novel tumour radiosensitiser. Further investigation is required to assess whether the PET radiotracer may underestimate the level of hypoxia in the innermost tumour subregion and whether perfusion is perhaps responsible, at least in part, for this observation.Additional file 1: Supplementary Figure 1. Change in tumour HV measured by hypoxia PET-CT. Supplementary Figure 2. Influence of treatment time on tumour hypoxic volume changes. Supplementary Figure 3. Relationship between baseline tumour hypoxic volume and total baseline tumour volume.


Tick-borne encephalitis (TBE) virus is a major cause of central nervous system infections in endemic countries. Here, we present clinical and laboratory characteristics of a large international cohort of patients with confirmed TBE using a uniform clinical protocol. Patients were recruited in eight centers from six European countries between 2010 and 2017. A detailed description of clinical signs and symptoms was recorded. The obtained information enabled a reliable classification in 553 of 555 patients: 207 (37.3%) had meningitis, 273 (49.2%) meningoencephalitis, 15 (2.7%) meningomyelitis, and 58 (10.5%) meningoencephalomyelitis; 41 (7.4%) patients had a peripheral paresis of extremities, 13 (2.3%) a central paresis of extremities, and 25 (4.5%) had single or multiple cranial nerve palsies. Five (0.9%) patients died during acute illness. Outcome at discharge was recorded in 298 patients. Of 176 (59.1%) patients with incomplete recovery, 80 (27%) displayed persisting symptoms or signs without recovery expectation. This study provides further evidence that TBE is a severe disease with a large proportion of patients with incomplete recovery. We suggest monitoring TBE in endemic European countries using a uniform protocol to record the full clinical spectrum of the disease. Tick-borne encephalitis (TBE) is an infection of the central nervous system (CNS) caused by the tick-borne encephalitis virus (TBEV) being transmitted by ticks in several central, eastern, and northern European countries ,2. The sIn 2013, the international network “European genetics study of tick-borne encephalitis” (EU-TICK-BO) was established to investigate host genetic associations with the susceptibility and severity of TBE. To provide a consistent description of TBE patients across all centers, a uniform patient case record form (CRF) was established . Paper CData integrity and plausibility were reviewed by two clinicians from the lead study center. A list of missing items and unclear issues was sent out to each participating center. If the centers were not able to provide a complete basic dataset , their patients were excluded.The findings of each patient in the neurological phase were reviewed, and the following diagnoses were assigned  : meningiPareses of extremities were categorized as peripheral paresis  or central paresis . At discharge from hospital, patients were categorized into “complete recovery” if symptoms or signs of TBE completely vanished or “incomplete recovery at discharge” when TBE symptoms or signs were still present. Patients with “incomplete recovery at discharge” were further categorized into “complete recovery expected” and “incomplete recovery expected” based on the assessment of the treating physician. Persisting symptoms or signs at discharge were categorized into “mild subjective” if patients had one or two subjective symptoms, “severe subjective” with three or more subjective symptoms, and “objective” if at least one objective sign with or without subjective symptoms was observed. The main foci of this work were “findings in acute disease”, “outcome at discharge”, and “blood and CSF findings”. Only “findings in acute disease” were recorded in all included patients.p-values (p), together with odds ratios (OR) and their confidence intervals (CI), were reported. Statistical analyses were conducted in R version 3.6.0  and GraphPad Prism software version 8 .Differences in patient characteristics in acute disease and outcome at discharge were analyzed with the Kruskal–Wallis test with Dunn’s multiple comparisons test for continuous variables and Fisher’s exact or Pearson’s chi-squared test for categorical variables. The Kruskal–Wallis test, with Dunn’s multiple comparisons test, was performed to examine the differences in blood and CSF levels according to diagnostic groups. For binary data, To set our findings in context with previously published TBE cohort descriptions, a literature search was conducted on PubMed using the search term “”. Titles were screened for cohort descriptions using original patient data. Information about frequency of diagnosis , paresis, and fatality rates were extracted from full articles from selected abstracts. We included original articles published between 1975 and 2020, with cohort sizes of at least 20 TBE patients presumably infected by the European subtype of TBEV.The study was approved by the local ethics committee of each center, and appropriate informed consent was obtained from all participating patients.A total of 1045 patients with TBE were recruited for genetic investigations (EU-TICK-BO); 430 of a single center were excluded as they only participated in the genetic analysis; 17 patients were excluded due to their date of admission before 2010 and 43 because of an incomplete basic data set after quality control. Finally, 555 (420 prospectively and 135 retrospectively recruited) cases were eligible for clinical description , with agSixteen (2.9%) patients had TBE despite being vaccinated. Out of these, seven were vaccinated as recommended, six had no complete basic vaccination, and three had their last booster vaccination not according to the recommendation. Most patients had a biphasic course of disease (65.1%), with various symptoms or signs, including fever, fatigue, malaise, headache, body pain, pharyngitis, and gastrointestinal symptoms .A clinical diagnosis was assigned to 553 of 555 patients; 207 (37.3%) had meningitis (M), 241 (43.4%) moderate meningoencephalitis (ME mod.), 32 (5.8%) severe meningoencephalitis (ME sev.), 15 (2.7%) meningomyelitis (MM), 46 (8.3%) meningoencephalomyelitis (MEM) with moderate encephalitis, and 13 (2.3%) meningoencephalomyelitis with severe encephalitis. In two patients, a differentiation of peripheral from central paresis was not possible, and these patients were classified as “unknown”. The distribution of diagnoses according to age is shown in In total, 56 (10.1%) patients had pareses of extremities, of whom 41 (7.4%) had peripheral pareses of extremities, 13 (2.3%) central pareses of extremities, and 2 had unknown peripheral or central pareses of extremities . A detain = 20), bladder dysfunction (n = 20), pain in extremities (n = 17), respiratory paresis (n = 9), and rectal dysfunction (n = 9).Spinal involvement was observed, including paresis of extremities (as reported), disturbance of sensibility (p < 0.0001), and patients with MEM (p = 0.002) had lower rates of comorbidities  and were less often admitted to the intensive care unit (ICU)  (p = 0.0006) and patients with MEM . Further, patients with ME had higher rates of recovery compared to patients with MEM  (p = 0.0006) and patients with MEM   . At disc 0.0006) . Accordi 0.0001) . FurtherWe recorded the outcome at the discharge of 298 patients; 117 (39%) had a complete recovery, 176 (59%) an incomplete recovery, and 5 (2%) died. Based on the treating physician’s assessment, 96 (32%) patients were discharged with “complete recovery expected”, while 80 (27%) were discharged with “no recovery expected” .Symptoms and signs of 121 patients discharged with incomplete recovery were recorded ; 36 30%.p = 0.227), deceased patients were admitted more often to the ICU than patients with compete recovery  and patients with incomplete recovery . Further, deceased patients had higher rates of central paresis than patients with complete recovery . Patients with incomplete recovery were diagnosed less often with meningitis than patients with complete recovery  and more often with meningoencephalomyelitis , central paresis , peripheral paresis , cranial palsy , and pain in extremities . (While there was no significant difference in ICU admission between patients with complete and incomplete recovery ( 0.002). .Patient 1 was a 39-year-old male with a kidney transplant and immunosuppression. He developed a severe diffuse CNS dysfunction (GCS = 3), needed mechanical ventilation, and died 4 weeks after the onset of TBE. The cause of death was described as extensive brain inflammatory damage and destabilization of brain functions. Patient 2 was a 47-year-old male with arterial hypertension and hyperuricemia. He had severe diffuse CNS dysfunction (GCS = 3) and died from elevated intracranial pressure 4 weeks after the onset of disease. Patient 3 was a 48-year-old female with arterial hypertension. She had severe diffuse CNS dysfunction (GCS = 3) and a central paresis. She died 4 weeks after the onset of TBE. The cause of death was described as extensive brain inflammatory damage and destabilization of brain functions. Patient 4 was a 66-year-old male with arterial hypertension. He had a disturbance of sensibility, a paresis of the left arm, and neck muscle dysfunction. He died within 4 weeks of TBE onset, caused by respiratory insufficiency. Patient 5 was a 79-year-old female with arterial hypertension who died after 4 weeks of TBE onset. The cause of death was not recorded.p < 0.001; M lower than MEM, p < 0.001) and lactate concentration . There were no significant differences in leukocyte, neutrophil, and lymphocyte counts and glucose concentration (No differences in blood parameters were observed when patients were grouped according to diagnosis . In CSF,ntration .In total, 37 clinical studies published between 1975 and 2020 were included ,44,45,46This is the first multicenter report on clinical findings of TBE patients from six highly endemic European countries. We confirm the high rates of patients with encephalitic disease caused by TBEV in European countries, as previously described by single-center studies or national studies ,16,20,34Our cohort included 16 patients with a history of previous TBE vaccination; 7 patients were fully vaccinated while 9 were incompletely vaccinated or missed receiving a booster vaccination according to recommendations. In contrast to previous studies, we observed a high proportion of children and adolescents with vaccination breakthrough infections ,56. In tLiterature about the severity of TBE breakthrough infection is inconsistent. Previous case reports and case series studies have described a more severe course of disease in patients with vaccination breakthrough infection, while a recent publication investigating a large cohort in Germany did not substantiate this finding ,58. The Although this analysis was carried out conscientiously, there may be potential limitations. According to viral epidemiological data, in all patients with TBE infections, the European TBEV subtype was presumed, though a differentiation of European, Siberian, and Far Eastern subtypes was not made due to non-feasibility in clinical routine. Possible coinfections with borrelia were excluded at each individual center according to clinical routine. Borrelia-specific diagnostic results were neither recorded nor reviewed by the lead study center. In this study, the true incidence of sequelae of our patients remains unknown. We only recorded the outcome at discharge since standardized follow-up investigations in all patients are of limited feasibility in routine patient care. Further investigation, including a detailed follow-up protocol, will be needed to study this subject. Further, the categorization of patients with incomplete outcomes at discharge for expected recovery is subjective to the corresponding investigator and is influenced by personal experience.This is the first international multicenter study of patients with TBE from different European countries. We observed high rates of patients with encephalitis and high rates of patients with lasting signs and symptoms at discharge. The comparison with previously published cohorts showed a likely underreporting of patients with meningomyelitis and patients with central paresis, which might be caused by different case record forms. Therefore, we suggest the use of a uniform case record form to monitor the full spectrum of disease and to raise awareness for disease prevention, particularly in countries with low vaccination rates.


Pretreatment drug resistance (PDR) can limit the effectiveness of HIV antiretroviral therapy (ART). The aim of this study was to assess the prevalence of PDR among HIV-positive individuals that initiated antiretroviral therapy in 2014–2020 in southwestern China.Consecutive cross-sectional surveys were conducted in Qinzhou, Guangxi. We obtained blood samples from individuals who were newly diagnosed with HIV in 2014–2020. PDR and genetic networks analyses were performed by HIV-1 pol sequences using the Stanford HIV-database algorithm and HIV-TRACE, respectively. Univariate and multivariate logistic regression models were used to explore the potential factors associated with PDR.In total, 3236 eligible HIV-positive individuals were included. The overall prevalence of PDR was 6.0% (194/3236). The PDR frequency to NNRTI (3.3%) was much higher than that of NRTI  and PI . A multivariate logistic regression analysis revealed that PDR was significantly higher among individuals aged 18–29 (adjusted odds ratio (aOR): 1.79, 95% CI 1.28–2.50) or 30–49 , and harboring CRF08_BC . A total of 1429 (43.8%) sequences were linked forming transmission clusters ranging in size from 2 to 119 individuals. Twenty-two individuals in 10 clusters had the same drug resistant mutations (DRMs), mostly to NNRTIs .The overall prevalence of PDR was medium, numerous cases of the same DRMs among genetically linked individuals in networks further illustrated the importance of surveillance studies for mitigating PDR.The online version contains supplementary material available at 10.1186/s12879-021-06847-5. Since the establishment of China’s national free HIV antiviral therapy in 2003, the case fatality rate of HIV infection has been effectively reduced and the life span of people living with HIV/AIDS has been prolonged , 2. HoweTo address the challenges posed by drug-resistant viruses, WHO formulated a global strategy for the prevention of HIV drug resistance in 2012 . SubsequGuangxi in southwestern China, is one of the regions with the most severe HIV epidemic in China –17. HoweThis was a cross-sectional study to estimate the prevalence of PDR in HIV-positive individuals that initiated ART in the Prefecture of Qinzhou, one of the regions with the most severe HIV epidemic in Guangxi. The study design was done according to the 2014 WHO protocol note for PDR . EligibiBasic sociodemographic data , behavioral characteristics , and baseline CD4+ cell count before ART were collected from the National HIV/AIDS Comprehensive Response Information Management System .https://hivdb.stanford.edu/). Drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) was defined as the detection of at least one antiretroviral virus drug in any drug class according to the WHO surveillance drug resistance guideline list [Whole blood was sampled before the initiation of ART. For each person, CD4 cells were determined by the Alere PimaTM Analyzer . Frozen plasma (obtained by centrifuging whole blood) was isolated and sent on dry ice to the laboratory at National Center for AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease Control and Prevention (CDC). RNA was extracted from 200 μl of plasma, amplified, and used to sequence the HIV pol region using an in-house Sanger sequencing protocol , 19. Theine list . Drug suThe HIV genetic transmission network was inferred with HIV-TRACE , establi2 test or Fisher’s exact test, while continuous variables are expressed as the mean ± standard deviation (SD) and compared using the non-parametric Mann–Whitney U-test and Kruskal–Wallis test, where appropriate. Univariate and multivariate logistic regression models were used to explore associations between PDR with demographic and clinical variables applied to each subregion. Specifically, all the variables that were statistically significant in the univariate analysis were included in the multivariate logistic regression model, then select the variables independently associated with PDR step by step based on goodness-of-fit tests . p < 0.05 was considered statistically significant. The 95% CI of the overall PDR prevalence is estimated using the distribution of the sample rate (p) approximating a normal distribution. The calculation formula is:Statistical analyses were done in SAS V9.4 . Categorical variables are presented as number of cases and percentages, while continuous variables are expressed as the mean ± standard deviation (SD). Categorical variables which are presented as the number of cases and percentages were compared using the Pearson’s χ3 before ART and 669 persons (20.5%) had a CD4+ cell count ≥ 350 cells/mm3. The proportions of the subtypes CRF01_AE, CRF07_BC, CRF08_BC, CRF55_01B, B, and others were 56.7% (1849/3262), 10.2% (331/3262), 27.4% (895/3262), 0.9% (30/3262), 0.4% (12/3262), and 4.5% (145/3262), respectively.Table 2 = 8.68, p = 0.003) and EFV  , but the highest proportion of high-level resistance in resistant strains was NVP , followed by EFV . The proportion of PDR of ETR  and DOR  was similar   . The PDR frequency to NNRTI  was much higher than that of NRTI  and PI . The most frequent NNRTI associated mutation was E138A/G/K, which was observed in 1.9% (60/3236) of HIV-positive individuals followed by K103N  and V179D/E . K70E/T  was the most prevalent NRTI associated mutation, followed by T215S/D/A  and V75M/A  (Table Factors associated with HIV PDR are listed in Table Genetic networks with different genetic thresholds (range 0.1–1.5%) were established, it is found that the molecular network with 0.5% gene threshold had a high number of clusters to get a high-resolution molecular network . According to the WHO definition of the prevalence of HIVDR, it is classified as low (< 5%), moderate (5–15%) and high (> 15%) . The surPDR may be influenced by many complex factors. This exploratory study found that age is an influential factor for PDR. Compared with people aged 50 and above, young people aged 18–29 are more likely to have pretreatment drug resistance, in line with studies in Jiangsu Province, Shandong Province, Guangxi, and Vietnam , 33. AdhIn this study, the overall proportion belonging to a genetic cluster was not associated with PDR, as was observed in 13 provinces or cities in China  , LiangshOur study has limitations. The sample transmission categories were based on self-reported information, which could not be validated. In addition, Sanger sequencing can only detect minority drug-resistant strains at a 15–20% prevalence in HIV viral populations of HIV-positive individuals and PDR could have been underestimated in this study . In the In summary, large-scale drug resistance surveillance was carried out on HIV-positive individuals that initiated ART in Qinzhou, which has provided insights into the PDR prevalence, influencing factors, and potential transmission relationships of drug-resistant strains in the region. These findings indicate that there is an urgent need for surveillance programs of HIVDR and routine drug resistance testing in the clinical management of HIV-positive individuals. For ART-naïve persons, the results of drug resistance monitoring could guide clinicians in determining more effective, first-line therapeutic regimens. Also, interventions to improve ART adherence and investigations including more specific information on potential transmission could help reduce or even block continued transmission of drug-resistant strains. For national institutions, large-scale studies can help develop national guidelines for ART.In conclusion, large-scale drug resistance surveillance showed that the overall prevalence of PDR is moderate in Qinzhou. Numerous cases of same DRMs among genetically linked individuals in networks further illustrate the importance of surveillance research in the development of future strategies for mitigating PDR.Additional file 1. Changes in pretreatment HIV drug resistance among HIV-positive individuals in southwestern China, 2014–2020.Additional file 2. Number of genetic transmission clusters and links, as a function of the TN93 distance threshold. Additional file 3. Difference in proportion of HIV-positive individuals belonging to genetic transmission networks who did and did not harbor PDR.Additional file 4. Links difference between HIV-positive individuals with and without PDR in the genetic transmission networks.