We redefined Robert’s prototypical cytoprotection model, namely the intragastric administration of 96% alcohol in order to generate a general peripheral and central syndrome similar to that which occurs when major central or peripheral veins are occluded in animal models. With this redefinition, we used Robert’s model to examine the cytoprotective effects of the stable gastric pentadecapeptide BPC 157. The intragastric administration of alcohol induced gastric lesions, intracranial  hypertension, severe brain swelling and lesions, portal and vena caval hypertension, aortal hypotension, severe thrombosis, inferior vena cava and superior mesenteric vein congestion, azygos vein failure , electrocardiogram disturbances, and heart, lung, liver and kidney lesions. The use of BPC 157 therapy  counteracted these deficits rapidly. Specifically, BPC 157 reversed brain swelling and superior mesenteric vein and inferior vena caval congestion, and helped the azygos vein to recover, which improved the collateral blood flow pathway. Microscopically, BPC 157 counteracted brain , heart , lung , liver (congestion), kidney (congestion) and gastrointestinal  lesions. In addition, this may have taken place along with the activation of specific molecular pathways. In conclusion, these findings clarify and extend the theory of cytoprotection, offer an approach to its practical application, and establish BPC 157 as a prospective cytoprotective treatment. This study attempts to clarify and extend the theory of cytoprotection, offer an approach to its practical application, and establish a prospective cytoprotective treatment. Cytoprotection is one of the most important concepts in gastroenterology and pharmacology ,2,3. TheOur conceptual reconstruction ,5,6,7 toHowever, this eventuality has not been investigated, nor appropriately combined with Robert’s cytoprotection ,2,3,8, iConceptually, screening the ability of compounds to modulate multiorgan pathology is consistent with Robert’s original proposal, which examined direct injury to the epithelial cells in the stomach by various noxious agents  ,2,3 and The exposure of the stomach mucosa to 100% ethanol for as little as 30 s leads to endothelial failure, generalized thrombosis and vessel occlusion in a multiorgan pathology screening model, with abundant thrombi in superficial capillaries, diffuse vascular stasis and endothelial lesions . CytoproFor the reliable counteraction of intragastric absolute alcohol application leading to a full syndrome like that described in rats, with the occlusion of major vessels ,15,16,17BPC 157 was originally described as an anti-ulcer peptide that is stable in human gastric juice for more than 24 h . It is aeNOS, mTOR and VGFEa, known to interact with BPC 157 and the administration of alcohol [The present study is the first to examine the treatment options for the full occlusive syndrome, including peripheral and central deficits similar to those described in rats with major vessel occlusion, that occurs following the intragastric administration of absolute alcohol , as in Robert’s prototypical cytoprotective model ,15,16,17 alcohol ,58,59.Finally, our findings could demonstrate the benefits of this model for screening potentially cytoprotective compounds.ad libitum. The animals’ care was in compliance with the standard operating procedures (SOPs) of the Pharmacology Animal Facility and the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123).This study was conducted with 12-week-old, 200 g male albino Wistar rats, randomly assigned at six rats/group/interval. The rats were bred in-house at the Pharmacology Animal Facility, School of Medicine, Zagreb, Croatia. The animal facility was registered by the Directorate of Veterinary (Reg. No: HR-POK-007). The laboratory rats were acclimated for 5 days and then randomly assigned to their respective treatment groups. They were housed in polycarbonate cages under conventional laboratory conditions at 20–24 °C, with a relative humidity of 40–70% and a noise level of 60 dB. Each cage was identified by dates, the study number, the group, the dose and the number of the animal. Fluorescent lighting provided illumination 12 h per day. A standard good laboratory practice (GLP) diet and fresh water were provided This study was approved by the local Ethics Committee. The ethical principles of the study complied with the European Directive 010/63/E, the Law on Amendments to the Animal Protection Act , the Animal Protection Act , the Ordinance on the protection of animals used for scientific purposes , the Federation of European Laboratory Animal Science Association (FELASA)’s recommendations and the recommendations of the Ethics Committee of the School of Medicine, University of Zagreb. The experiments were assessed by observers blinded to the treatment.The medication was administered as described in previous studies ,15,16,17The gross lesions were recorded in deeply anaesthetized laparatomized rats, with a camera attached to a VMS-004 Discovery Deluxe USB microscope . Hemorrhagic lesions in the stomach were assessed as the percentage of the total area of the glandular stomach at 1 min after alcohol administration and following medication, at 1, 5, 15 and 30 min; the rats were then euthanized. The other investigated features included the gross presentation of the brain, the superior mesenteric vein, the inferior vena cava and the heart, as well as a thrombus assessment.l2/l1) between two observed brains, veins or hearts and as an inter-factor (and therefore not measured) for deriving the final expression (6). The procedure was as follows:1,2), after dividing both sides by V1 andAs described in previous studies ,15,16,17This measuring procedure, followed by the calculation of the volume ratios, was performed separately for the brain, veins and heart.Brain swelling was recorded in separate rats 15 min after complete calvariectomy. Briefly, 6 burr holes were drilled in three horizontal lines, all of them medial to the superior temporal lines and temporalis muscle attachments. The two rostral burr holes were placed just basally from the posterior interocular line, the two basal burr holes were placed just rostrally to the lambdoid suture (and the transverse sinuses) on both sides, respectively, and the middle two burr holes were placed in the line between the basal and rostral burr holes.After euthanasia, the superior sagittal sinus, the portal vein, the inferior vena cava, the superior mesenteric vein, the superior mesenteric artery, the hepatic artery and the abdominal aorta were removed from the rats, and the clots were weighed ,15,16,17Recordings were made in the deeply anaesthetized rats with a cannula , connected to a pressure transducer , which was inserted into the superior sagittal sinus, the portal vein, the inferior vena cava and the abdominal aorta at the level of the bifurcation before the administration of alcohol 1 min after alcohol application and 1, 5, 15 and 30 min after therapy . Each recording lasted 1 min. For the superior sagittal sinus pressure recording, we made a single burr hole in the rostral part of the sagittal suture, above the superior sagittal sinus, and cannulated the anterior portion of the superior sagittal sinus using Braun intravenous cannulas. We then laparatomized the rats in order to record portal vein, inferior vena caval and abdominal aortal pressure.Of note, the normal rats exhibited a superior sagittal sinus pressure of −24 to −27 mmHg and a portal pressure of 3–5 mmHg, which was similar to that of the inferior vena cava, although it was at least 1 mmHg higher in the portal vein. By contrast, the abdominal aorta blood pressure was 100–120 mm Hg at the level of the bifurcation ,15,16,17ECGs were recorded continuously in the deeply anaesthetized rats for all three main leads by positioning stainless steel electrodes on all four limbs using an ECG monitor with a 2090 programmer  connected to a Waverunner LT342 digital oscilloscope  at 15 min, 24 h or 48 h after ligation. This arrangement enabled precise recordings, measurements and analysis of the ECG parameters at the level of the bifurcation ,15,16,17The stomach (from grossly intact tissue), brain, lungs, liver, kidneys and heart were rapidly removed and fixed in 10% neutral buffered formalin at room temperature for 24 h. Tissue blocks were embedded in paraffin, sectioned at 4 μm, stained with hematoxylin and eosin (H&E) and evaluated by light microscopy using semiquantitative scoring.2, with 80 measurements for each sample of cortex and hippocampus. The dark neurons were subjected to hypoxia; they featured eosinophilic cytoplasm, pyknotic nuclei and loss of Nissl substance.As described ,15,16,17We used a scoring system to grade the degree of lung injury, including observations of focal thickening of the alveolar membranes, congestion, pulmonary edema, intra-alveolar hemorrhage, interstitial neutrophil infiltration and intra-alveolar neutrophil infiltration. Each feature was assigned a score from 0 to 3, based on its absence (0), or presence to a mild (1), moderate (2) or severe (3) degree. A final histology score was determined ,16,17,62The assessment of renal injury was based on the degeneration of Bowman’s space, glomeruli and proximal and distal tubules, vascular congestion, and interstitial edema. The criteria for liver injury were the vacuolization of hepatocytes and pyknotic hepatocyte nuclei, the activation of Kupffer cells and the enlargement of sinusoids. Each specimen was scored using a scale from 0 to 3  for each criterion ,16,17,63To illustrate the possible involvement of pathways, gene expression analysis assessment  was perfThe total RNA was extracted from different tissues, rapidly dissected, snap-frozen in liquid nitrogen and stored at −80 °C. The tissues were homogenized using a Bio-Gen PRO200 homogenizer  in 1000 μL of TRIzol , and RNA extraction was performed using a TRIzol-based reagent method according to the manufacturer’s instructions.After RNA extraction, quantification was performed with DeNovix DS-11 Spectrophotometer .A High Capacity cDNA Reverse Transcription Kit  was used to perform reverse transcription following the manufacturer’s instructions and using a ProFlex PCR System machine .TaqMan Gene Expression Assays  with a TaqMan Gene Expression Master Mix were used for the gene expression analysis of selected genes . QuantitActb was chosen as a reference gene for the normalization of the Nos3, Mtor and Vegfa gene expression data.−ΔΔCt, where ΔΔCt is the difference between the ΔCt of the treated sample and the ΔCt of the non-treated sample. The results were expressed as fold change and as percentages. Fold change values lower then 70% indicated decreased gene expression in BPC-157-treated animals (downregulation), fold change values between 70% and 130% were considered as biological variability (no change in gene expression), and fold change values higher then 130% indicated increased gene expression in BPC-157-treated animals (upregulation).The difference in gene expression between the treated and non-treated samples was analyzed using the formula 2U test to compare the groups. The values were presented as the mean ± standard deviation (SD) or as the minimum, median and maximum. To compare the frequency difference between the groups, the chi-square test or Fischer’s exact test was used. A value of p < 0.05 was considered statistically significant.The statistical analysis was performed by using the parametric one-way analysis of variance (ANOVA) with the post hoc Newman–Keuls test or the non-parametric Kruskal–Wallis test, followed by the Mann–Whitney We found that an assault on the stomach—96% alcohol administered intragastrically—produced gastric lesions and rapidly escalated to a generalized syndrome with peripheral and central deficits similar to those that have been observed when major vessels are occluded in rats ,15,16,17Alcohol insult to the stomach produced large gross hemorrhagic lesions and severe pathology in the stomach  a–d. In tWithin 1 min of intragastric alcohol administration, there was moderate congestion in the liver a,b. AfteAlcohol insult to the stomach led to kidney lesions within 1 min, moderate congestion and progression after 5 min and prominent congestion after 15 and 30 min a–c. In tIn the control rats, there was moderate congestion in the lungs with hemorrhage in the lung parenchyma 1 min after intragastric alcohol administration a,b. AfteIntragastric alcohol rapidly produced heart dilatation and lesions and ECG disturbances a–k. In tp ˂ 0.05 compared with saline-treated rats). The only abnormality was peaked T waves in the third limb lead at all time points.One minute after the introduction of intragastric, ECG recordings showed marked tachycardia with prolonged PQ and QTc intervals e–g. FurtA proportional change in the vessel was used to assess the development of peripheral vessel failure after intragastric alcohol administration a–g. TherThe intragastric administration of 96% alcohol led to severe portal and vena caval hypertension, with the former more pronounced than the latter, and aortal hypotension that persisted throughout the entire experimental period. In addition, the normal (negative) pressure in the superior sagittal sinus was increased  blood pressure in the superior sagittal sinus . The shuAs has been described in previous studies ,15,16,17Alcohol affects thrombocyte function : the acuIn conclusion, in the intragastric alcohol-treated rats, alcohol caused an initial endothelial injury that quickly spread and mimicked the symptoms observed when major vessels are permanently occluded in animal models ,15,16,17eNOS, mTOR and VGFRa in the stomach, brain, heart, lung, liver and kidneys) [In addition, the study of such beneficial effects as the particular activation of the collateral pathways reliant on the injurious occlusion ,15,16,17kidneys) ,52,53,54kidneys) . There wkidneys) . Of notekidneys) ,56,57 ockidneys) ,37,38,39Egr, Nos, Srf, Vegfr, Akt1, Plcɣ and Kras gene expression in the vessel that provided an alternative operating pathway  [Egr1, Akt1, Kras, Src, Foxo, Srf, Vegfr2, Nos3 and Nos1 expression and decreased Nos2 and Nfkb expression; these changes may indicate how BPC 157 exerts its effects [eNOS, mTOR and VGFRa expression may illustrate particular points at which BPC 157 therapy worked simultaneously in each of the organs. An illustrative example may be the simultaneous increase  and decrease (mTOR expression) in the lung. Interestingly, we noted that eNOS, mTOR and VGFRa expression occurred together in the liver (decrease), and in the kidneys (no change). Overall, BPC 157 can induce and interact with a host of molecules to exert beneficial effects. The extent of the benefits of BPC 157, and the mechanisms by which it exerts these benefits, remain to be fully explored.In one study, BPC 157 affected in rats) . In the  effects . Therefoper se, especially cytoprotection studies, must be interpreted with caution [As a final note, it is true that animal studies  caution . In addi caution ,6. Howev caution ,6, in bo caution ,98 and i caution ,99,100,  caution ). An imp caution ,45), a p caution . In this caution ,3,8, ani


Bisphenols are endocrine disruptors that may be associated with altered fetal growth in humans, and they have similar biological functions to mimic hormones. In addition, aggregated chemicals showed an adverse effect although individual concentration was at a low level. However, most studies between bisphenols and birth outcomes have focused on the effect of individual bisphenol. Thus, we explored the associations of urinary bisphenol mixtures with birth outcomes. We conducted a prospective birth cohort study in South Korea. One hundred eighty mother-infant pairs were recruited from 2017 to 2019. Bisphenol A (BPA), bisphenol F (BPF), and bisphenol S (BPS) in one spot urine were analyzed using ultra-performance liquid chromatography–tandem mass spectrometry. We used two statistical approaches to examine potential associations of BPA, BPF, and BPS with birth weight and gestational age: (1) multivariable linear regression; (2) Bayesian kernel machine regression (BKMR). The geometric means of BPA, BPF, and BPS were 2.1, 0.2, and 0.1 μg/L, respectively. In stratified linear analyses by each median value, a higher BPF was positively associated with birth weight (g) . Mixture analyses using BKMR suggested an inverse association between bisphenol mixtures and birth weight. Our findings suggest that in utero bisphenol exposure may influence birth weight and that such relationships may differ considering non-linearity and the combined effect. Bisphenols are a class of endocrine-disrupting chemicals (EDCs) frequently used in manufacturing polycarbonate plastics and epoxy resins, which are found in various consumer products, such as plastic baby bottles, toys, and epoxy food-can linings ,2. DespiBisphenols influence on physiological receptors in different pathways . BPA canConsidering the toxicity of BPA and consumer concerns, manufacturers have been banned from using BPA in their products, with gradual transitions to using bisphenol analogs, such as BPF and BPS. Thus, BPF and BPS have been widely used in manufacturing BPA-free products ,15. SomeAlthough these previous studies found that exposure to environmental chemicals during pregnancy may be associated with adverse fetal growth, most did not consider the aggregate effect of environmental chemical exposure on infant health . Among tn = 8) and no available urinary bisphenol concentration data (n = 56). Therefore, 180 pregnant women were included in the final analysis. This study was approved by the Institutional Review Boards of National Cancer Center, Korea.The MAKE study was a community-based prospective birth cohort study in South Korea to examine the effects of maternal chemical exposure during pregnancy on fetal growth. Pregnant Korean women aged at least 18 years were eligible to enroll. The details of the MAKE study have been described previously . BrieflyEscherichia coli K12; Roche Biomedical, Mannheim, Germany) were added. The samples were enzymatically hydrolyzed for 16 h in a constant temperature bath at 37 °C and then cooled down by adding 100 μg/L of 2 N HCl. After solid phase extraction to remove impurities, the samples were eluted with methanol and then analyzed using ultra-performance liquid chromatography–tandem mass spectrometry .Spot urine samples were collected from each participant while visiting the center and then were frozen at −70 °C. To measure the urinary bisphenol concentrations, a 1-mL sample was transferred to a glass test tube, and then internal standards of 50 μg/L, 1 mL of 2 M sodium acetate, and 50 μg/L of β-glucuronidase  were performed such as linearity, accuracy, precision, and method detection limits. For quality control, seven samples with a concentrated standard 3 to 10 times the detection limit were measured with the same procedures mentioned above. The limit of detection (LOD) was calculated by multiplying the standard deviation of the seven replicates by 3.14 . The precision and accuracy measurements were calculated by multiplying by the relative standard deviation of more than five replicated measurements. The acceptable range of precision and accuracy was 85–115%. The correlation coefficient of the calibration curve was more than 0.999. The LOD for BPA, BPF, and BPS were 0.071, 0.083, and 0.020 μg/L, respectively.For external QC, each analytical laboratory regularly participated in international and national quality control programs. Internationally, all the laboratories participated in the 61st to 63rd programs of the German External Quality Assessment Scheme (G-EQUAS) and also participated in the domestic quality assurance program by the National Institute of Environmental Research (NIER) in South Korea. All the analytical results of the laboratories were qualified within the tolerance ranges by the managers of G-EQUAS and NIER.After delivery, we obtained birth information, including the birth weight (g) and gestational age (week) from medical record. Gestational age at delivery was estimated from the first date of the last menstrual cycle or the first date of ultrasonographic estimation if the date of the last menstrual period was unreliable or if a significant difference occurred between the last menstrual cycle date and ultrasonographic estimation date (>10 days).10-transformed for normality assumption. The urinary bisphenols were corrected for specific gravity (SG) and measured using a urine analyzer  to adjust the urinary dilution [Urinary bisphenol concentrations below the LOD were assigned to LOD/√2 , and alldilution . The SG-t-test to assess the differences in the birth weight and gestational age according to the maternal and infant characteristics. We selected potential confounders, known as influential factors, for bisphenol exposure and birth outcomes from previous studies with a directed acyclic graph (2) [We used Student’s ic graph . All theraph (2)  before pTo estimate the linear associations between each urinary bisphenol and birth outcomes, we used multivariable linear regression models using the SAS 9.4 software . We used three models: unadjusted model, multivariable-adjusted model, and mutually adjusted model.We conducted further analyses using Bayesian kernel machine regression (BKMR) , with thi , C is the concentrations of bisphenols, h denotes the unknown exposure-response function to be estimated, BKMR with a gaussian kernel function is used to estimate the individual and overall joint association of bisphenols with birth outcomes, and the formula is given by:outcomes .As BKMR calculates the high-dimensional estimation of the exposure-response surface, it can visualize these associations by defining the cross-sectional surface. Thus, we present the following in this study: (1) the univariate exposure–response relationship of one bisphenol with birth outcomes, considering that others are fixed at their median value and all the covariates have a constant value; (2) the association of one bisphenol increases by a certain percentile  with birth outcomes at various quantile values of others, considering that all the covariates have a constant value; (3) the bivariate exposure–response relationship of one bisphenol with birth outcomes at various quantile values of another bisphenol, considering that the other bisphenol is fixed at its median value and all the covariates have a constant value; (4) the overall joint association of all bisphenols increase by five percentiles with birth outcomes as referenced to their 50th percentile, considering that all the covariates have a constant value.10-transformed values and centered to handle skewed data, and all the BKMR models were adjusted for potential confounders. BKMR was implemented with the R package bkmr using R software .The BPA, BPF, and BPS concentrations were scaled to logThe detection rates of bisphenols above the LOD in urine samples followed the order of BPA (96.2%), BPF (84.4%), and BPS (66.1%). The SG-corrected geometric means (GM) of the BPA, BPF, and BPS concentrations were 2.1, 0.2, and 0.1 μg/L, respectively . The matp-value = 0.82; BPF: β = 38.5, 95% CI: −18.1 to 95.1, p-value = 0.18) and gestational age . However, BPS was negatively associated with the birth weight  and gestational age ; additionally, all the linear associations were not statistically significant. However, after stratified analyses by the median concentration of each bisphenol, a higher BPF level was significantly associated with increased birth weight   .We conducted BKMR using component-wise variable selection to examine the potential nonlinear and interactive effects and overall effect of bisphenols on the birth outcomes, adjusting for potential confounders. The estimated PIPs on the birth weight for BPA, BPF, and BPS were 0.11, 0.78, and 0.48, respectively, and the PIPs of the gestational age for BPA, BPF, and BPS were 0.21, 0.31, 0.25, respectively. In this study, we found nonlinear associations of BPF and BPS on birth weight. For BPF, an increase in BPF from the 10th to 50th percentile (0.1 to 0.2 μg/L; To examine potential pairwise interactions among the bisphenols, particularly due to the nonlinearity for BPF and BPS, we estimated the predicted change in birth weight and gestational age, considering the other bisphenol patterns at 25th, 50th, or 75th percentiles. Finally, we also examined the overall effect of bisphenol mixtures on birth outcomes. In this prospective birth cohort study, we examined the associations of the gestational concentrations of bisphenols with birth weight and gestational age. We found that higher BPF levels were significantly associated with increased birth weight. We observed a non-linear association of BPF and BPS on birth weight and increasing levels of the bisphenol mixture were associated with decreased birth weight. However, we found no changes in gestational age.In the current study, urinary BPA levels were much higher than those of its analogs. The results of our individual bisphenol analyses were similar to those previously reported in investigations using the MAKE study . ComparePrevious studies have found that maternal bisphenol exposure is associated with birth weight. However, the impact of prenatal exposure to BPA on size at birth is unclear because of the inconsistent findings from current epidemiological studies . Our resSimilar to BPA, two birth cohort studies reported that maternal exposure to BPS and BPF was inversely associated with size at birth ,17, whilPrevious studies have assessed the association between individual chemical biomarkers and birth outcomes. Few have examined the effect of aggregate environmental exposures on birth outcomes ,40,41,42The metabolism of BPS and BPF have not been well studied. However, several experiments have showed that BPF metabolism and distribution are similar to those of BPA . BPA expOur study has several limitations. First, the specificity of the bisphenol mixture exposure assessment has not been evaluated in other studies. Second, our study sample size was small, which may reduce the statistical power to obtain the sufficient precision of our estimates . FinallyHigher exposure to BPF is associated with increased birth weight. However, the overall joint association of bisphenol mixture exposure with birth weight suggests an inverse association with birth weight. Thus, individual chemical exposure and mixture exposure may have different effects on the same health outcome. Further studies are required to substantiate these findings considering non-linearity and the combined effect.


Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene  and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes.The identification of IMPORTANCELegionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields. Legionella spp. have been described as the causative agent of legionellosis in humans. The term refers to two main form of diseases, Legionnaires’ disease, a severe multisystem disease involving pneumonia, and Pontiac fever, a nonpneumonic form, acute, self-limiting influenza-like illness. Additionally, extrapulmonary  and asymptomatic forms are reported , which comprises 16 serogroups; the majority of cases, clusters, and outbreaks are attributable to serogroup 1 (Sg1). Other Legionella non-pneumophila species (non-Lp) are less studied and less commonly associated with human disease; thus, they remain undiagnosed due to limits of current diagnostic methods, which are more specific and sensitive for Lp. Indeed, the commonly used diagnostic method is the detection of a urinary antigen that is more sensitive for Lp Sg1 and does not permit the detection of Lp non-Sg1 or other Legionella species  without l-cysteine (l-cys) is the first step to discriminate Legionella from other bacteria. Serological methods, such as the agglutination test, the direct fluorescent antibody (DFA) test, and indirect immunofluorescence assay (IFA), are mostly used for discrimination between non-Lp species and Lp serogroups  that are represented by a sequence-based typing (SBT) approach for clinical and environmental Lp strains (mip) gene sequencing for non-Lp isolates  can guarantee the genotypic classification of Lp and blue-white autofluorescent species  and Acinetobacter. This marker is not sufficient for Legionella classification, especially for non-Lp, although different studies have already suggested combining rpoB with the mip gene to identify these species more accurately  with 5% sheep blood agar. Moreover, the agglutination for Legionella species antisera test displayed positive results for 34/135 isolates (25.2%) and ambiguous results for 10/135 (7.4%) isolates; in contrast, most isolates (91/135 [67.4%]) showed negative results for the agglutination test. All of them were then submitted for gene amplification as previously described.All 135 isolates showed positive growth on BCYE cysmip and rpoB sequencing analysis at the species level as follows: L. anisa 51/135 (37.7%), L. rubrilucens 26/135 (19.2%), L. taurinensis 22/135 (16.3%), and L. nautarum 15/135 (11.1%). The remaining isolates were represented by L. feeleii 7/135 (5.2%), L. londiniensis 7/135 (5.2%), L. quateirensis 4/135 (3.0%), L. quinlivanii 1/135 (0.7%), and L. steelei 1/135 (0.7%). The positive control was confirmed to belong to L. pneumophila.All isolates (135/135 [100%]) were identified by mip and rpoB results was found in 121/135 (89.6%) isolates; in contrast, discordance was returned for 14/135 (10.4%) isolates. In particular, it is possible to evaluate the number of isolates displaying concordance between the mip and rpoB results, including 49/135 belonging to L. anisa, 26/135 belonging to L. rubrilucens, 15/135 belonging to L. nautarum, 7/135 belonging to L. londiniensis, 1/135 belonging to L. steelei, 22/135 belonging to L. taurinensis, and 1/135 belonging to Lp.Regarding the percentage of genomic identity, complete concordance between mip and rpoB gene sequencing and their ranges of matches compared to the reference strains are shown in mip, rpoB, or both genes are highlighted in bold. Regarding mip gene identification, compared with the respective reference strains, our isolates showed a nucleotide identity interval of 98.2% to 100%, with the exception of two L. anisa isolates and one L. quinlivanii isolate with nucleotide identities of 96.7% and 96.2%, respectively. However, the rpoB gene results showed a nucleotide identity interval of 95.1% to 100%, except for two isolates of L. anisa and four isolates of L. quateirensis, which were identical to each other with nucleotide identity percentages of 92.4% and 94.5%, respectively. Moreover, for the seven isolates identified by the mip gene as L. feeleii (98.2%), a discrepancy with the rpoB gene identity results was found, showing a percentage of identity of 95.4% for six isolates and 95.1% for one isolate.The results obtained by rpoB gene in our isolates, it was important to determine the specific intra- and interspecies variation intervals, as has been done for the mip sequence-based classification scheme created by Ratcliff et al.  did not fall within the intraspecies identity interval (lower cutoff at 95.1%), thus suggesting the possibility that the strains belong to different species. The same considerations can be applied to the four L. quateirensis isolates, which showed a percentage of identity for the rpoB gene of 94.5%.On the basis of the intra- and interspecies intervals calculated from the 329-bp L. feeleii and one isolate of L. quinlivanii, determined according to the mip gene classification scheme, showed borderline results with the rpoB classification scheme based on the observed cutoff values of 95.1% to 95.4% for the presumptive L. feeleii and 95.7% for L. quinlivanii. These findings provide further evidence of their misidentification and the necessity of further investigation.The identity values of seven strains of rpoB gene being characterized as having greater genetic variability (number of DNA mismatches), the deduced amino acid sequences of the mip gene showed a higher number of amino acid substitutions. It is important to emphasize that all 14 isolates focused on in our study showed few amino acid substitutions in the mip gene, from 1 to 3; in contrast, regarding the rpoB gene, only five amino acid substitutions were reported in L. taurinensis.Moreover, mip and rpoB genes, respectively. The dendrogram built using the mip and rpoB gene sequences regrouped all isolates into 10 clades corresponding to a specific Legionella species. In the mip gene dendrogram, no relevant differences were found, with the exception of two isolates of L. anisa (MR 54 and MR 97) that were separated from the corresponding main branch, suggesting a possible misidentification of these isolates. In contrast, the dendrogram built using the rpoB gene showed the same 10 clades but with a higher genetic distance between wild types and the reference strains.mip and rpoB genetic discrepancies between the 14 wild strains and their reference strains used in this study. The rpoB dendrogram showed that in the main clade of L. anisa, two isolates (MR 54 and MR 97) were different from others based on the mip gene dendrogram that was previously described. Moreover, in the L. feeleii clade, one isolate (MR 123) is separated from the main clade, and one isolate belonging to L. quinlivanii (MR 36) and four isolates belonging to L. quateirensis  present differences from the corresponding reference strains ATCC 43830 and ATCC 49507, respectively.Legionella spp. in environmental and clinical samples, and it is well known that the amplification and sequencing of some genes for the direct detection and identification of bacteria can be simple, convenient, and specific in their differentiation of bacterial species. The use of PCR methods in Legionella identification and typing, thanks to their species-specific capability, has increased the power to detect and identify species, reducing the time and cost compared to culture and antibody approaches as well as improving the sensitivity and specificity of identification, especially for clinical approaches.Several studies have compared molecular methods to detect Lp species have been mostly identified by only the mip gene, although several studies have shown that no single system is perfect and that other target genes need to be investigated  distributed in both nosocomial and community environments. The results obtained were compared with the gold standard mip gene classification scheme already developed by Ratcliff et al. (Legionella Infections (ESGLI). Our results confirmed, in agreement with previous studies, that both mip and rpoB are able to discriminate among Legionella species, considering that our isolates (89.6%) showed complete concordance between the two classification schemes.Many of the studies regarding the amplification of ironment –39. Thisf et al.  and stilmip and rpoB results, as there was a low percentage of genomic identity with respect to the reference strains for 14 isolates. In detail, our results suggest that sequencing using only rpoB is able to detect relevant genetic differences between the wild-type and the reference strains, which would otherwise be undetected using only the mip approach . This result is especially interesting given that L. quateirensis and L. anisa showed a variability percentage for the rpoB gene outside the intraspecies interval of variation found here (0 to 4.8%); L. feeleii and L. quinlivanii had values very close to the variation cutoff, suggesting that the identification scheme using only one gene limits the discovery and study of species variation and sometimes limits discrimination between different species. In line with previous results, all the dendrogram representations show that there is lower genetic diversity in the mip gene between and within the clades; in contrast, the diversity in rpoB appears to be greater, leading to the identification of several isolates that showed evident differences from their respective clade or reference strain . The results obtained using the rpoB gene seem to be useful for the identification of non-Lp species, and the results obtained permit the construction of the first rpoB gene classification scheme in the scientific literature.It is important to note that in some cases, there was no concordance between the Lp species appears to be as reliable as that of the entire gene.Thanks to the matrices described above, we built pairwise identity intervals that allowed us to classify our unknown sequences based on comparisons with reference strains. The comparison carried out using the values obtained here seems reliable, and we propose that they be used in a classification scheme. For strains whose similarity percentages are very close to the cutoff values, further in-depth analyses are recommended. Based on the intervals of variation derived from the pairwise identity matrices, the discriminatory power of the 329-bp target region for the non-L. quateirensis described here. The average nucleotide identity (ANI) analysis, performed comparing their entire genome and the L. quateirensis type strain, showed pairwise values below the similarity threshold fixed to 95%, validating the hypothesis that the four strains belong to a presumptive new Legionella species , could contribute to better clarifying the identification of our isolates. This approach has already been applied to the four isolates of  species .mip gene, as all reported isolates showed discrepancies regarding the identification scheme based on the mip and rpoB genes. The role of the mip gene is widely documented; it is involved in the ability of L. pneumophila to replicate in eukaryotic cells and environmental amoebae  and in a few Legionella species , inadequate antibiotic treatments in human infection contest , and disinfection treatment. If a discrepancy is observed in this first step, then a more advanced technology, such as WGS, can be applied. This combined strategy represents an improved screening approach for Legionella isolate identification.Although the non-Legionella environmental surveillance programs of several facilities commonly associated with the risk of Legionella infections, including hospitals, companies, and communities .The isolates involved in this study come from Legionella culture technique was based on ISO 11731:2017  of the untreated, filtered, heated, and acid-treated samples were seeded on plates of the selective medium glycine-vancomycin-polymyxin B-cycloheximide (GVPC)  and incubated at 35 ± 2°C with 2.5% COLegionella genus, suspected colonies were subcultured on buffered charcoal yeast extract (BCYE) agar with (cys) and without (cys) l-cysteine (l-cys) supplementation . Moreover, as a negative control, the same isolates were spread on tryptone soy agar (TSA) with 5% sheep blood agar  and incubated under the same conditions previously described, as Legionella is not able to grow on this medium. Only the colonies that grew on BCYE cys+ agar were considered for the next steps of the study.To confirm the presence of the Legionella colonies were then identified using the Legionella latex agglutination test , which is able to distinguish between Lp and non-Lp. In particular, among Lp, it is possible to identify serogroup 1 (Sg1) from Sg2 to Sg14, while among non-Lp, it is possible to recognize only some non-Lp, such as L. anisa, L. bozemanii 1 and 2, Legionella gormanii, L. longbeachae 1 and 2, L. dumoffii, and L. jordanis. A total of 134 strains of non-Lp and 1 strain of Lp that was previously typed by sequence-based typing (SBT) and included as a positive control were selected for the study.The predicted -Lp isolates was performed to determine the gene sequences of mip and rpoB as described by Ratcliff et al. , and DNA concentrations were determined using a Qubit fluorometer . PCR analysis for all nonf et al.  and Ko ef et al. , respectmip gene amplification was performed using degenerate primers and modified by M13 tailing to avoid noise in the DNA sequence  and 40 pmol of each primer; 100 ng of the DNA extracted from the presumptive colonies was added as the template. The mip amplicons were sequenced using tailed M13 forward and reverse primers (mip-595R-M13R caggaaacagctatgaccCATATGCAAGACCTGAGGGAAC and mip-74F-M13F tgtaaaacgacggccagtGCTGCAACCGATGCCAC) to obtain complete coverage of the region of interest , 1 U of Taq polymerase, and a PCR mixture consisting of PCR buffer 10×, 1.5 mM MgCl2, and 250 μM deoxynucleoside triphosphates (dNTPs). The thermal cycles consisted of 35 cycles, and each cycle consisted of 30 s at 94°C for denaturation, 30 s at 55°C for annealing, and 30 s at 72°C for extension, followed by a final extension at 72°C for 10 min.o et al. . Gene amPCR products were visualized by electrophoresis on a 2% agarose gel and stained with ethidium bromide. Following purification, DNA was sequenced using BigDye chemistry and analyzed on an ABI PRISM 3100 genetic analyzer . Raw sequencing data were assembled using CLC Main Workbench 7.6.4 software.mip sequences were compared to sequences deposited in the Legionella mip gene sequence database using a similarity analysis tool. EWGLI has established an accessible web database (http://bioinformatics.phe.org.uk/cgi-bin/Legionella/mip/mip_id.cgi) that contains sequence data from described species and allows for the identification of non-Lp species. Species-level identification was performed on the basis of a similarity score of 98 to 100% compared to the sequences in the database , National Collection of Type Cultures, Central Public Health Laboratory (NCTC), NITE Biological Research Center, National Institute of Technology and Evaluation (NBRC), and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM). According to Adékambi et al. and Ko et al., the cutoff used for rpoB gene sequence-based identification was fixed at a 94 to 95% similarity percentage using an rpoB gene fragment of 300 to 600 bp  retrieved from the NCBI, were used to determine the ranges of the intra- and interspecies intervals of variation for the rpoB gene, resulting in a pairwise identity matrix for the entire gene with a length from 4,101 to 4,143 bp , retaining the default settings. The developed matrices permitted us to obtain a minimum and a maximum value of variation to establish intra- and interspecies intervals of divergence for the identification of the environmental isolates used in the present study. In detail, our 135 isolates were considered wild strains, and an in-house numbering scheme was used to label them (MR 1 to MR 135) (The matrices were built using the multiple sequence comparison by log-expectation (MUSCLE) program  in Genei MR 135) .Legionella isolates involved in the study, a multiple sequence alignment (MSA) and a phylogenetic tree were performed on the mip and rpoB gene sequences. For each taxon identified as previously described, the reference mip and rpoB gene sequences of the corresponding type strains from several culture collections were retrieved and added to the analysis (https://www.geneious.com), retaining the default settings.To estimate the relationship among the analysis . When reanalysis , a tool analysis . Branch The GenBank accession numbers of sequences generated during this study are listed in


Background and objective:Andrographis paniculata (AP) is a traditionally used herbaceous plant, whose main active constituent is andrographolide. Andrographolide derivative medications and herbal preparations of AP are often used to treat respiratory tract infections. This study aims to systematically evaluate the safety of andrographolide derivative medications and herbal preparations of AP based on clinical studies.Methods: English and Chinese databases were searched for all types of clinical studies that reported adverse drug reactions (ADRs) and adverse events (AEs) of andrographolide derivative medications and herbal preparations of AP. The ADRs and AEs were classified according to manifestations, and graded according to severity. Single-rate meta-analysis was performed for ADR incidence using R software.Results: A total of 262 studies were included, including 125 randomized controlled trials, 23 non-randomized controlled trials, 6 case series, and 108 case reports. In 9490 participants using andrographolide derivative injections, 383 (4.04%) reported ADRs. Meta-analysis showed that the ADR incidence of three most frequently used injections of andrographolide derivatives  were 5.48% [95% CI ], 3.69% [95% CI ] and 5.33% [95% CI ], respectively, which may be slightly higher than the actual ADR incidence, because only studies that reported the occurrence of ADRs or AEs were included, but studies without ADR and AE were not included. The ADRs of andrographolide derivative injections were mainly gastrointestinal, skin and subcutaneous tissue disorders, and anaphylaxis. Fifty-five patients experienced life-threatening anaphylactic shock, three patients died, and the causation attributed to the andrographolide derivative injection. Other ADRs were mild, moderate or medically significant. Nine herbal preparations of AP were tested in 10 studies, and the reported ADRs were mainly mild to moderate gastrointestinal, skin and subcutaneous tissue disorders. Except for five patients using andrographolide derivative injections eventually died, most of the ADRs were alleviated after drug withdrawal and symptomatic treatment.Conclusions: The ADRs of andrographolide derivative medications are few, but can be life-threatening, mainly gastrointestinal, skin and subcutaneous tissue disorders, and anaphylaxis. Injections of andrographolide derivatives should be used with caution. Herbal preparations of AP are essentially safe.Systematic Review Registration: [website], identifier [registration number] Andrographis paniculata (Burm.f.) Nees (AP) is an herbaceous plant in the Acanthaceae family, and has been traditionally used in China and India  is an unfavourable outcome that occurs during or after the use of a drug or other intervention but is not necessarily caused by it. Adverse drug reaction (ADR) is an adverse event for which the causal relationship between the drug and the event is at least a reasonable possibility . AccordiIn the published systematic reviews of andrographolide derivative injections, some reviews reported about safety, but they only briefly stated the ADRs and AEs, and did not systematically review the information of these ADRs and AEs. A few studies systematically reviewed the safety of single andrographolide derivative  . One stuAndrographis (Chuanxinlian)”, “andrographolide”, “adverse drug reaction”, “adverse event” and “safety” were used. The reference lists of studies meeting the inclusion criteria were also searched to identify additional relevant studies. A detailed search strategy for PubMed is attached as We conducted systematic searches in seven electronic databases, including four Chinese databases , Wanfang Database, Chinese Scientific Journal Database (VIP) and SinoMed) and three English databases , from their inception to January 2021. Search terms such as “Four authors in pair  screened the literature independently. Any disagreement was resolved by discussion with a third author (J-pL). Clinical studies that meet all the following criteria will be included: 1) Clinical studies using andrographolide derivative medications or herbal preparations of AP; 2) Clinical studies that reported the occurrence of ADRs or AEs after using these preparations. 3) There are no restrictions on the characteristics of participants, control groups and study types. Studies that meet the following criteria will be excluded: repeated studies, or studies that used other preparations in addition to andrographolide derivative medications, herbal preparations of AP, and symptomatic treatment.Six authors in pair  extracted data and assessed the methodological quality of the included studies independently. Any disagreement was resolved by discussion. The extracted information mainly includes: study type, demographic characteristics of participants, details of interventions  and the information of ADRs and AEs.For different types of studies, corresponding tools were used to evaluate the methodological quality. For randomized controlled trials (RCTs), the risk of bias tool was used . Items iWe evaluated the causal relationship between andrographolide derivative medications/herbal preparations of AP and the adverse events, according to the World Health Organization-Uppsala Monitoring Centre (WHO-UMC) causality assessment criteria . AdverseThe Common Terminology Criteria for Adverse Events (CTCAE) system was used to classify the ADRs and AEs according to manifestations, and grade them according to severity . The CTC2 statistic was less than 50%, and the p-value for the test of heterogeneity was ≥0.10. In these cases, a fixed-effect model was used to compute the pooled estimate of ADR incidence. In all other cases, the studies were considered heterogeneous, and a random-effect model was used to compute the pooled ADR incidence. Other data not suitable for pooling analysis were synthesized qualitatively.The R3.6.1 software was used for data analysis. Single-rate meta-analysis was performed for the ADR incidence of included studies, if the studies were similar in intervention. Results were considered homogenous when the IThe literature search identified 3972 citations, of which 607 were excluded due to duplication. After reviewing the titles and abstracts, 1619 citations were excluded. After scanning the full text, 1484 publications were excluded. Finally, we included 262 studies, including 125 RCTs, 23 non-RCTs, six case series and 108 case reports . Most ofThe sample size of included studies ranged from 20 to 2000, except for case reports. The age of participants ranged from 1 month to 88 years old. The included RCTs, non-RCTs and case series focused on URTIs (43.51%), pneumonia (15.58%), intestinal infections (12.34%), hand-foot-mouth disease (6.50%), bronchitis (5.84%), other RTIs (5.84%) and other diseases (10.39%) such as encephalitis, herpes, multiple sclerosis, hypertriglyceridemia, chronic obstructive pulmonary diseases, knee osteoarthritis, rheumatoid arthritis, type 2 diabetes mellitus and unspecified bacterial infections. The included case reports involved a total of 155 cases, of which 58.71% were URTIs, 14.84% were bronchitis, 5.81% were pneumonia, 7.74% were other RTIs, 2.58% were intestinal infections, 9.03% did not report specific diagnosis, and other cases were lung cancer and arthritis.n = 93) did not report the method of random sequence generation. Among the 125 RCTs, only five trials provided information on allocation concealment (n = 120) did not provide the information of allocation concealment. Among the included RCTs, 51 were assessed with high risk of bias due to insufficient blinding procedures of participants and personnel, because the interventions between treatment group and control group were different in preparation, dosage or frequency. Six trials were placebo-controlled (n = 68) did not provide enough information on blinding of participants and personnel. The majority (n = 87) of the included RCTs did not provide enough information on blinding of outcome assessment. Thirty-two trials were assessed to be at high risk of bias as they assessed subjective outcome measures and the participants or practitioners were not blinded. Six trials assessed subjective outcome measures, and the patients and practitioners did not know which study group they had been assigned to (n = 122) did not provide information on incomplete outcome data. One trial had a protocol that could be obtained, and the outcome measures were consistent with the trial report (n = 124) did not provide enough information on selective reporting  provided the information on clearly stated aim, appropriate endpoints, follow up, contemporary groups and equivalent baseline. Among these included trials, 20 reported follow-up period, 19 reported information on statistical analyses, 15 reported adequate control group, and only two reported the information on prospective data collection (All included non-RCTs (llection . All then = 6) clearly described the aim of the study, but did not report the information on prospective data collection and consecutive recruitment of patients. Among these case series, one was multicentered (n = 5) reported two to four items, and were of lower quality. The most commonly reported items were aim of the study and main findings of the study.All the included case series  investigated injections of andrographolide derivatives, of which 75 (52.82%) investigated andrographolide sulfonate , 54 (38.03%) investigated potassium sodium dehydroandrographolide succinate , and nine (6.34%) used potassium dehydroandrographolide succinate . One study (0.70%) researched andrographolide sodium bisulfite  , and oneAndrographis paniculata for children  for children when administered by intravenous (IV) injection; 50–100 mg/time, 2–3 times/day for intramuscular (IM) injection in adults. For PSDS, the dosage stated in instruction is 0.16–0.4 g/day, IV, for adults. And for PDS, the dosage is 400–800 mg/day, IV, and 100 mg/time, IM, for adults. Among the 75 studies investigating AS, 67 (89.33%) used the injection in accordance with the dosage stated in the instruction. Three studies (4.00%) administered the injection at a dosage lower than the stated dosage . Of theschildren , it is achildren , and the2 = 53%, p < 0.01). A total of 4479 patients were treated with PSDS, of which 146 (3.26%) developed ADRs. Meta-analysis . A total of 645 patients were treated with PDS, and 26 (4.03%) of them developed ADRs. Meta-analysis  experienced a total of 383 ADRs. We performed meta-analysis on the ADR incidence of AS, PSDS, and PDS . A totalanalysis  showed tanalysis  showed tectively , so no mAmong the ADRs of andrographolide derivative injections reported in the included RCTs, non-RCTs and case series, 43.34% were gastrointestinal disorders, and 32.38% were skin and subcutaneous tissue disorders. Other ADRs were anaphylaxis, general disorders and abnormal administration site conditions, nervous system disorders, blood and lymphatic system disorders, abnormal medical examination results, and unspecified anaphylactic reaction. According to the information provided in the articles, the severity of 250 (65.27%) ADRs could not be clearly graded, because only the name and number of ADRs were reported, and no other information was provided. For the remaining 133 ADRs, 107 (27.94%) were graded as CTCAE grade 1, 25 (6.53%) were graded as CTCAE grade 2, and one (0.26%) was graded as CTCAE grade 3. Among the included RCTs, non-RCTs, and case series using injections of andrographolide derivatives, 11 reported that the ADRs were cured without treatment, 36 reported that the ADRs were cured after stopping the drug and receiving symptomatic treatment, and the remaining 95 studies did not report the prognosis of the ADRs.In the included case reports, there were 152 patients receiving injections of andrographolide derivatives, of whom 76 (50.00%) got PDS, 41 (26.97%) AS, 28 (18.42%) PSDS, five (3.29%) ASB , and twoIn the included case reports, 41 patients were treated with AS, 13 (31.71%) of whom were treated at a dosage according to the instruction. Twenty-five children (60.97%) were treated at the dosage of 50–200 mg, IV, However, the weight of these children was not reported, so it is unclear whether they were treated at an off-label dosage. The dosage of the remaining three patients (7.32%) was not reported. Twenty-eight patients received PSDS, 11 of whom were treated at a dosage according to the instruction. Two adults and one child were treated at off-label doses of 800 and 2000 mg, IV, respectively . The dosAccording to the results of causality evaluation, the adverse events of eight patients were certainly related to injections of andrographolide derivatives, 132 were probably related to the injections, 12 were possibly related to the injections. Therefore, adverse events of all these patients were assessed to be ADRs. These 152 patients reported a total of 207 ADRs, of which 97 were anaphylaxis (CTCAE grade 3–5), 41 were skin and subcutaneous tissue disorders (CTCAE grade 1–3), 16 were general disorders and abnormal administration site condition (CTCAE grade 1–3), 15 were gastrointestinal disorders (CTCAE grade 1–3), 13 were abnormal medical examination results (CTCAE grade 2–4). Other ADRs included nervous system disorders (CTCAE grade 1–3), respiratory, thoracic and mediastinal disorders (CTCAE grade 1–3), renal and urinary disorders (CTCAE grade 3), and cardiac disorders (CTCAE grade 2 and 3). Of the anaphylaxis, 55 were life-threatening anaphylactic shock (CTCAE grade 4), and three patients died (CTCAE grade 5)  . Two patAmong these 152 patients, 145 had alleviated ADRs after discontinuation of the drug and symptomatic treatment, and five patients died. Two patients with shock did not recover from shock, and one of them developed multiple organ failure and eventually became vegetative . Three oIn the included studies, two studies  investigIn the included RCTs, non-RCTs, and case series, 10 studies used herbal preparations of AP . Two stuAndrographis paniculata purified extract) was used in two groups with 300 mg/day and 600 mg/day respectively, and the number of adverse events in the 300 mg/day group was more than that in the 600 mg/day group. In one study (Andrographis paniculata ethanol extract) was used in two groups with 1200 mg/day and 1800 mg/day respectively, and the adverse events in the two groups were slightly different in manifestation and quantity. In one study (Andrographis paniculata extract) was used in two groups at three and five capsules per day, respectively, and the adverse events occurred in both groups were nausea.According to the results of causality evaluation, the adverse events reported in nine studies were possibly related to the herbal preparations of AP. In one study , some ofne study , the herne study , the herThe ADRs and AEs of these herbal preparations were mainly gastrointestinal disorders  and skin and subcutaneous tissue disorders . A total of 165 ADRs and AEs of herbal preparations were reported in these studies, of which 43 (26.06%) were graded as CTCAE grade 1, and 4 (2.42%) were graded as CTCAE grade 2. The severity of the remaining 118 (71.52%) ADRs/AEs could not be clearly graded, due to the insufficient information reported in the articles. Since these 10 studies used different preparations, no meta-analysis was performed on the incidence of ADR and AE. The incidence and manifestations of ADRs and AEs were described qualitatively based on what was reported in the original articles. Among these 10 studies, one study reported that the ADRs were cured without treatment , and twoIn the included case reports, three patients took herbal preparation Chuanxinlian tablets at a dosage of 5 tablets/time, 3 times/day . One of This systematic review included 262 clinical studies that reported ADRs and AEs of andrographolide derivative medications and herbal preparations of AP. The included studies mainly investigated three injections of andrographolide derivatives: AS, PSDS and PDS. The results of meta-analysis showed that the ADR incidence of AS, PSDS, and PDS were 5.48, 3.69 and 5.33%, respectively. The ADRs of andrographolide derivative injections are mainly gastrointestinal disorders, skin and subcutaneous tissue disorders, followed by anaphylaxis, general disorders and abnormal administration site conditions, nervous system disorders. The majority of these ADRs are mild to moderate, and a small number of patients may experience severe or life-threatening ADRs. Most of these ADRs caused by andrographolide derivative injections can be alleviated after discontinuation of the drug and symptomatic treatment. In the included case reports, one patient developed anaphylactic shock after using AS, and eventually turned into vegetative state. Five patients died after using injections of andrographolide derivatives . The included studies involved 9 herbal preparations of AP (tablets and capsules). The ADRs and AEs of these herbal preparations are mainly mild to moderate gastrointestinal disorders, skin and subcutaneous tissue disorders.There have been other clinical studies and literature studies on the safety of other drugs used to treat RTIs. A meta-analysis evaluating the effect of clarithromycin on streptococcal pharyngitis included five studies, in which 600 patients were treated with clarithromycin . Among tThere is currently no comprehensive systematic review on the safety of andrographolide derivative medications and herbal preparations of AP. Some published studies have reviewed the ADRs of several andrographolide derivatives. Two studies reviewed the ADRs of four andrographolide derivative injections  . AccordiIn the included case reports, 97 patients developed anaphylaxis after being treated with injections of andrographolide derivatives, of which 55 patients had anaphylaxis that were life-threatening (CTCAE grade 4), and three patients died (CTCAE grade 5). Before prescribing medicine, doctors should inquire the allergic history of patients carefully, and patients who are allergic to the preparation should carefully take the medicine to avoid serious ADRs and AEs. Some of the ADRs caused by andrographolide derivative injections are severe, so they should be used with caution, and possible ADRs should be closely monitored.Treatment dosage is of importance to ADRs. Five injections of andrographolide derivatives were involved in this study, of which only AS specifies the dosage for children in the instruction. Many of the included clinical studies were for children. It is unclear whether the other andrographolide derivative injections were used at an off-label dosage for children in the included studies, because dosage for children is not specified in the instructions. In clinical practice, these preparations may be more likely to overdose when applied to children, because there is no prescribed dosage for children in the instructions. Therefore, if dosages for children are supplemented in the instructions, the safe application of these preparations in children will be promoted.Except for a few studies, the included studies have methodological deficiencies. Moreover, except for case reports, other studies usually report the information on ADRs and AEs very briefly, which will make it more difficult for readers to clearly assess the severity of ADRs and AEs, and to evaluate the causal relationship. More studies with higher methodological quality are needed to provide more high-quality evidence for evaluating the safety of andrographolide derivatives, while herbal preparations of AP can generally be regarded as safe. The CONSORT for Harms checklist is a checklist applicable for reporting information related to harms in RCTs , which iClinical studies that reported the ADRs and AEs of andrographolide derivative medications and herbal preparations of AP were comprehensively searched and included in this study. Moreover, the severity of ADRs and AEs were graded using the CTCAE system. The number, manifestations and severity grading of ADRs and AEs were all reported in this study. Therefore, this systematic review provides comprehensive information on the safety of andrographolide derivative medications and herbal preparations of AP.The limitations of this study are, first of all, most of the included studies have methodological deficiencies, and the overall quality of this study is limited by the quality of original studies. Secondly, we only included studies that reported the occurrence of ADRs and AEs, but did not include studies that clearly reported that patients did not have ADRs or AEs after taking andrographolide derivative medications and herbal preparations of AP. Hence, the ADR incidence of andrographolide derivative injections resulted from meta-analysis may be slightly higher than the actual ADR incidence. Thirdly, due to the large variety and wide application of andrographolide derivative medications and herbal preparations of AP, the number of clinical studies using these preparations is very large. Studies using other drugs in addition to andrographolide derivative medications, herbal preparations of AP, and symptomatic treatment were excluded from this study. In this way, the influence of other drugs on the results of causality evaluation could be avoided, but at the same time, the included studies would not be comprehensive enough. Furthermore, this study only summarized the information on ADRs and AEs obtained from literature. There is a lack of information from ADR monitoring centers . As a result, information on the ADRs of these preparations reported in this study is not comprehensive enough. Moreover, the severity of ADRs and AEs were graded only based on the information reported in the articles. However, except for case reports, other studies only briefly reported the information about ADRs and AEs, and therefore the severity of many ADRs and AEs could not be clearly graded. And that, the severity grading was solely based on the information provided in the articles, so the results should be interpreted and applied with caution.The ADR incidence of three most used injections of andrographolide derivatives  are 5.48, 3.69 and 5.33%, respectively. The ADRs of andrographolide derivative injections are mainly gastrointestinal disorders, skin and subcutaneous tissue disorders, followed by anaphylaxis, general disorders and abnormal administration site conditions, etc. Most of the ADRs are mild, moderate, or medically significant (CTCAE grade 1–3). Based on data from this review, we recommend that AS, PSDS and PDS be used with caution, because a small number of patients can experience life-threatening or lethal anaphylactic shock (CTCAE grade 4 and 5) after using these injections. This is of particular importance in patients with a history of allergy. Possible ADRs should be closely monitored. Herbal preparations of AP are essentially safe. Most of the included clinical studies have limited methodological quality. More post-marketing studies on the safety of andrographolide derivative medications are needed.


COVID-19 can cause damage to the lung, which can result in progressive respiratory failure and potential death. Chest radiography and CT are the imaging tools used to diagnose and monitor patients with COVID-19. Lung ultrasound (LUS) during COVID-19 is being used in some areas to aid decision-making and improve patient care. However, its increased use could help improve existing practice for patients with suspected COVID-19, or other lung disease. A limitation of LUS is that it requires practitioners with sufficient competence to ensure timely, safe, and diagnostic clinical/imaging assessments. This commentary discusses the role and governance of LUS during and beyond the COVID-19 pandemic, and how increased education and training in this discipline can be undertaken given the restrictions in imaging highly infectious patients. The use of simulation, although numerical methods or dedicated scan trainers, and machine learning algorithms could further improve the accuracy of LUS, whilst helping to reduce its learning curve for greater uptake in clinical practice. They should be qualified/certified to perform LUS. To achieve this, research is needed to determine the skill mix and level of expertise required to deliver best practice.During the early phases of COVID-19, LUS is being done by non-radiology practitioners such as emergency medicine physicians, intensivists, nephrologists, obstetricians, paediatricians, and physiotherapists4–7 The locations include: (a) primary care – the family  practitioner’s surgery and a patient’s home/nursing home6; and (b) secondary care – emergency medicine departments, the intensive care unit, paediatric-/adult- medical/surgical wards, and obstetric wards.6,7 This to: (a) diagnose conditions such as consolidation, COVID-19, interstitial lung disease (ILD), pleural effusions and respiratory distress syndrome (RDS)5; b) guide therapeutic intervention,4,5 and (c) monitoring of disease progression or resolution4,6,7 The ideal time to do LUS is when the patient needs it,5–7 and could be done 24 h of the day, seven days a week9 However, research is needed to determine the skill mix and level of expertise required to deliver this practice.Critically ill adult and pediatric patients in resource poor/medium/rich settings with suspected lung disease should have POC LUS.5,6,10,11 The US scanner should:For patients with suspected COVID-19, or other lung disease, POCUS is convenient, due to its: low cost, dynamic examination, non-ionising radiation, and portability.4,11Be handheld, or a cart and easy to clean,4–8At minimum have a curvi-linear, low-frequency  and a li10Potentially have advanced image processing (AIP): compound-, harmonic-, speckle reduction- and color Doppler imaging.4,6,7; (3) using the traditional technique without AIP,   in confined bed spaces with limited access to a power supply; (2) using best practice infection prevention controlout AIP, 4,5 or wiwith AIP . AIP cante-lung) , which a4–8 Practitioners who learn LUS can be undergraduates and postgraduates. The practitioner should not be a complete novice; one with no skills or knowledge of US physics and application techniques. Moving the probe across the regions of the chest5 and interpreting subtle pathology associated with COVID-19,6,7 and other lung disease5 requires skills that cannot be obtained in a short period.4–6,8 LUS, unlike most soft tissue imaging with US, relies on the observation and interpretation of US artifacts.4–8 The practitioner needs to be able to interpret normal and abnormal appearances to detect lung disease. Covid-19, ILD and RDS present on LUS as b-lines.5,6 These are discrete vertical hyperechoic reverberation artifacts, which originate at the pleural line and often extend through to the maximum imaging depth, as illustrated in 4–6 They move with respiration and up to three can be present in healthy lung.5 The observation of three or more is an indication of lung pathology.4–7 In severe cases, coalescent b-lines filling the space between ribs (white lung) are detected5–7 .14 This comprises of seven elements describing essential sub steps of an US examination. It enables assessment of practitioner performance through training until competency rather than relying on a predetermined number of scans. Using the OSAUS generic rating scale, during training for assessment, with structured formative feedback has the potential to improve the practitioner’s LUS skills acquisition. Research is needed to determine the effectiveness of using the OSAUS tool for the LUS setting.Certification of competency. The core competency for all non-radiology and radiology practitioners doing LUS could be assessed using the international multispecialty consensus tool: the 4 The images and reports need regular peer-review, and discussion at local education meetings, to ensure quality assurance and the practitioner’s continuing professional development.4POCUS in acute and critical care settings undertaken by non-radiology and radiology practitioners needs the same governance. The indication for LUS needs to be justified; and the images, and a typed report, should be stored in a secure location such as a hospital’s or national healthcare system’s picture archiving and communication system (PACS). This to allow the LUS examination to be part of the patient’s record, which can be reviewed at any time by secondary care, non-radiology, and radiology practitioners, to aid rapid and future patient management.15 to allow for experimentation in as wide as possible parameter space is needed. This to provide the opportunity for practitioners and medical physicists to refine image settings and probe choice without patient exposure.10 Furthermore, as the use of machine learning to aid in the diagnosis of LUS patients increases, it will help reduce the learning curve for the effective use of this modality.16A need for simulation of US imaging, in freely available US software packages, such as k-Wave,The logistics of setting up a LUS service during and beyond the COVID-19 pandemic will be challenging. To become best care, evidence-based practice, this needs collaboration between the non-radiology and radiology departments, Higher Education Institutions, and US scanner and LUS phantom manufacturers.8 Education using simulation and hands-on will enhance learning and improve patient care.12 The aim is with good governance, LUS will be best practice in the patient pathway for COVID-19 and beyond.For patients with suspected COVID-19, or other lung disease, the role of POC LUS is developing. It requires practitioners with relevant education to ensure timely, safe, and diagnostic clinical/imaging assessments for rapid decision-making.


Aim: Medication-related osteonecrosis of the jaw (MRONJ) occurs after exposure to medication (antiresorptive or antiangiogenic agents) for bone-related complications. It is more common in the mandible than in the maxilla. The present study investigated maxillary MRONJ in elderly patients through a meta-analysis. Methods: Keywords, including “MRONJ”, “maxilla”, and “surgery”, were entered into databases, including Embase, PubMed/MEDLINE, Cochrane Library, and ProQuest, which were searched systematically. Results: Investigating 77 studies, we found that 18 (2 case reports and 16 case series) papers conformed to the standards. The results revealed a 2.6:1 female-to-male ratio of disease occurrence. The average age of patients was 70.6 ± 5.5 years, and most patients were in the third stage (43.6%). The average time of medication usage was 50.0 ± 20.1 months. The pooled proportion of clinical efficacy of surgery was 86%. Conclusion: To prevent and manage MRONJ, all elderly patients should maintain proper oral hygiene and receive dental examinations regularly. Risk assessment and safety management of MRONJ should be performed by medical teams. Most elderly people have at least one chronic disease and are at higher risk than others for developing osteoporosis ,2. Osteofracture ,4. ThereHowever, studies have found that intravenous or oral administration of BPs may cause bisphosphonate-related osteonecrosis of the jaw (BRONJ) ,6. The i®; Novartis Pharmaceuticals, East Hanover, NJ, USA) and zoledronate  experienced painful bone exposure in the mandible, maxilla, or both. An increasing number of studies have determined that osteonecrosis of the jaw is induced by other antiresorptive agents, including denosumab, a receptor activator of nuclear factor kappa-B ligand inhibitor; antiangiogenic agents, such as bevacizumab; and tyrosine kinase inhibitors, such as sunitinib and sorafenib [Osteonecrosis of the jaw induced by drugs was first reported by Marx , who notorafenib ,9,10,11.orafenib .The ratio occurring MRONJ in the maxilla and mandible were between 1:2 and 1:3, possibly with the reason that mandible has the higher hardness and bone density. Most relevant research has focused on identifying the causes of MRONJ and determining the prognosis of its treatment; studies have typically focused on the mandible ,14,15,16The study methods were based on the meta-analysis statistical evaluation and review guide of the Joanna Briggs Institute . In 2003, Marx  reported(1)2000–2021 English-language reference literature.(2)Case report or case series.(3)Patients with maxillary MRONJ or combined with mandibular MRONJ.(4)An intervention of surgical treatment.(5)Prognosis for the intervention outcome of wound healing, recurrence, or complication.(6)Elderly people (age ≥ 60).The inclusion criteria for this study’s literature review were as follows:(1)Patients only with mandibular MRONJ.(2)Osteonecrosis of the mandible unrelated to medication.(3)Surgery not used as the major treatment but rather a conservative therapy, such as the use of antibacterial mouth rinse and full-body antibiotics.(4)Animal experimentation.(5)“Krokodil” drug or nucleoside reverse transcriptase inhibitor-related osteonecrosis.(6)Reporting language other than English.The exclusion criteria were as follows:After excluding duplicate studies (5 papers) and those not conforming to the conditions , we identified 72 papers. After title and abstract review, 31 references were excluded, leaving 41 potentially relevant publications. Their full text was assessed for eligibility, and 23 papers that did not conform to the main criteria were excluded. Finally, surgery was performed in the severe MRONJ in a total of 18 papers that were for the literature review ,33,34,35Two independent reviewers screened the titles identified in the literature search, extracted the data, and analyzed the results. The use of a standard practice was proposed for extracting information regarding the studies, such as the author(s), year of publication, country, study design, and number of participants; patient data, such as gender  and mean age of patients; and disease data, such as osteonecrosis location, AAOMS stage, treatment type , disease history, class of drugs (bisphosphonates/nonbisphosphonates), duration of drug exposure, follow-up time after treatment, and surgery outcomes of interest and effectiveness. One must be alert to the problem of bias in case series studies arising from the absence of a control group and the diversity of clinical and methodological approaches.Two reviewers independently reviewed and extracted the original data from the 18 studies. After performing verification and reaching a consensus, MedCalc (version 19.6.4), a freely available Windows program, was used to conduct the meta-analysis and create the diagrams. Before combining the meta-analysis results, we conducted a heterogeneity test, which yielded an I2 between the studies. If I2 < 50%, then the different studies would have homogeneity and a fixed-effects model should be adopted. However, if the study designs of the combined studies had differences , then a random-effects model is recommended to avoid underestimating treatment variance. If I2 ≥ 50%, this would mean that the combined studies had heterogeneity and a random-effects model should be adopted. Sensitivity analysis can be further conducted, and unsuitable studies excluded before reanalysis. Furthermore, the use of subpopulation analysis is recommended to evaluate the effects of different subject populations. The estimation of combined effects considered the sample size of surgery efficacy and total sample of the studies. When different scales are used for the measurement indexes, the mean continuous variables and weighted sample number are adopted to estimate the combined effect. Furthermore, the fail-safe number test was used to determine publication bias. When the fail-safe number is larger than the tolerance level, this means that the possibility of publication bias is extremely low, and the results of the meta-analysis are more credible.This article was written according to the PRISMA statement .The major clinical prognoses were compared after patients with maxillary MRONJ received surgical treatment or surgical treatment combined with other treatments. The definition of successful treatment used in this study was as follows: (1) wound healing, (2) no recurrence, or (3) no complications. If one of these three outcomes was documented, then the surgical treatment was deemed effective. In the meta-analysis, the effect of clinical treatment was defined by the percentage of successful treatments and the 95% confidence interval (CI). The uncontrolled variables included in this study exhibited no significant differences. Accordingly, using the fixed-effects model was recommended. Other statistical analyses were performed using SPSS version 20 . p-value of <0.05 to be statistically significant for the calculation of heterogeneity. Egger’s tests were used to assess the possibility of publication bias because they are useful adjuncts to meta-analysis; funnel plots were created therefrom. All proportional meta-analyses of case series studies were performed using MedCalc .Forest plots are presented to summarize the data. Each horizontal line on a forest plot represents a case series included in the meta-analysis. The length of the line corresponds to a 95% CI of the corresponding case series’ effect estimate. The effect estimate is marked with a solid black square. The size of the square represents the weight of the corresponding study in the meta-analysis. The pooled estimate is marked with an unfilled diamond at the bottom of the forest plot. CIs of pooled estimates are displayed as a horizontal line through the diamond, where the line might be contained within the diamond if the CI is narrow. A statistically significant difference between both interventions studied was defined if their combined 95% CIs did not overlap. We considered a p < 0.001). This indicates that the scores of the two reviewers had extremely high consistency and were significantly correlated.A total of 18 studies were included in this study, which comprised 2 case reports and 16 case series. Details of the case reports and case series are presented in the systematic literature review table. The kappa value of consistency between the two reviewers was 9.11  revealed no significant difference in the consistency (homogeneity) of clinical and methodological aspects between the studies included in the meta-analysis (p = 0.4141 > 0.05), which indicated no relationship between treatment effect and study size. These results suggest publication bias was not a factor in this meta-analysis.The test of heterogeneity , denosumab, and antiangiogenic agents), and it involves chronic osteonecrosis in the maxilla or mandible. In this meta-analysis, 79.1% of patients were taking BPs during the treatment period. Other treatment drugs were non-BPs, such as denosumab, which accounted for 11.4%. The average time of medication usage was 50.0 ± 20.1 months. Regarding prevalence of comorbidities, osteoporosis (31.5%) was predominant, followed by breast cancer (20.1%) and multiple myeloma (15%). The pathological mechanism of MRONJ is not entirely clear and can be multifactorial. The prevalence of MRONJ has exhibited an increase among elderly patients. As population aging becomes more pronounced, the oral hygiene and health of elderly people are crucial concerns. For consolidated analysis, data were extracted from the collected studies, and our results revealed that there were more elderly female patients with maxillary MRONJ. In both men and women, the average age was 70.6 years. Typical events that may precede MRONJ include acute periodontitis, denture misfit, wounds from invasive surgery, and other dentoalveolar surgeries. MRONJ is most frequently triggered by tooth extraction. In this meta-analysis, 8 of 18 articles presented that the trigger event of MRONJ was tooth extraction. Notably, approximately one-third of MRONJ cases are idiopathic without apparent cause. Such cases may possibly be attributable to subclinical traumas. Therefore, the risk factors of MRONJ include systemic factors  and local factors  ,22,25. MRONJ is a potentially serious adverse event associated with high cumulative doses of antiresorptive or antiangiogenic drugs. A cessation of medication, such as the aforementioned drugs, is termed a “drug holiday” before invasive treatment. It is unclear whether a drug holiday before surgical treatments such as a tooth extraction is appropriate for BPs drug use. In an advisory statement, the American Dental Association in 2011 opined that a drug holiday is not required for patients receiving a relatively low cumulative dose of BPs (<2 years) or denosumab who may continue antiresorptive therapy during invasive dental treatment . Wilde eThe denosumab does not accumulate in bone tissue and has a considerably shorter half-life than the bisphosphonates (28 days vs. 10–12 years) . HasegawSurgical treatments for maxillary MRONJ include sequestrectomy, debridement, resection, and immediate repair with/without flap reconstruction . NicolatAdjuvant therapies are currently recommended for maxillary MRONJ surgery. In a report by Park et al. , endoscoMoreover, several methods may be employed as alternative treatment options for enhancing MRONJ bone healing. Low-level laser therapy is a promising supplementary treatment for MRONJ because it facilitates biological effects, such as inflammation and angiogenesis, and simultaneously increases the inorganic substrate and facilitates wound healing . The recPeriosteal reaction is a nonspecific radiographic finding that indicates periosteal irritation. To clarify the clinical significance of the periosteal reaction in wound healing, Kojima et al.  investigIt is desirable to analyze existing data so that physicians and health professionals may have a reference for the current state of knowledge. For this reason, conducting a proportional meta-analysis of case series studies with a comprehensive systematic search of uncontrolled studies  is suggested when there are no clinical trials in the literature that answer questions about clinical interventions. However, investigators and policymakers should be extremely cautious in interpreting such results because many flaws may be evident in the internal validity of this type of study, specifically, bias in case series studies arising from the absence of a control group and the diversity of clinical and methodological approaches. Nevertheless, the evidence provided from a proportional meta-analysis of case series studies should only be used until appropriate clinical trials are conducted. Okuyama et al.  reportedIn this study, we used a proportional meta-analysis of case studies, specifically case report or case series studies of maxillary MRONJ, in order to evaluate the effectiveness of surgery as treatment. Our evidentiary level was relatively low, which affected the ability to determine the efficacy and safety of interventions, surgical procedures, and prevention programs. Nonetheless, this alternative analysis can help surgeons, physicians, and health professionals to make provisional decisions in conjunction with their clinical expertise and the patient’s wishes and circumstances. We recommend that healthcare professionals weigh the benefits and risks of surgery under this approach and take account of the patient’s values and preferences. Moreover, we recommend further research involving high-quality primary studies and ethical and methodologically sound clinical trials.In this study, the average age of patients was 70.6 years and with a 2.6:1 female-to-male ratio. Most patients were in the third stage (43.6%). Clinical efficacy of surgery was up to 86%. In order for the MRONJ to be prevented and managed, all elderly patients were suggested to maintain a proper oral hygiene routine and receive routine dental examination. In this study, tooth extraction is considered to be a trigger event. Therefore, a detailed inquiry with risk factors for MRONJ is critical before tooth extraction or other dentoalveolar surgery. Patients’ medical histories  and past medication usage (BPs or non-BPs) should be fully understood, and they should be evaluated by a medical team .