Thyme has strong antioxidant properties and, therefore, can reduce the intensity of oxidative processes taking place in the body. The study aimed to assess whether the addition of thyme to diets for fattening pigs containing extruded flaxseeds, a source of n-3 PUFAs, which are particularly susceptible to oxidation, would have a positive effect on redox status and lipid metabolism. The experiment was conducted using 120 weaners (WBP × Neckar crosses) of about 30 kg BW, which were kept until the end of fattening (about 110 kg BW) and divided into three groups of 40 pigs. The control group received a diet with 4% extruded flaxseed. In groups T1 and T3, 1% or 3% of thyme was added to the basal diet. The introduction of 3% thyme resulted in a decrease in the total cholesterol level in the blood and the loin muscle. Moreover, an increase in SOD and CAT activity and a decrease in FRAP and LOOH was noted. Following supplementation with 3% thyme, the n-3 PUFA content and n-3/n-6 ratio increased, while the SFA content was significantly reduced. The results of the studies indicate that thyme has a positive effect on the redox status and lipid profile of the blood and muscles. Due to health concerns, consumers are increasingly paying attention not only to the nutritional value of food products but to their health benefits as well. For this reason, components are added to diets for livestock in order to obtain products with health-promoting properties, e.g., with high content of polyunsaturated fatty acids (PUFA), especially n-3 acids. One ingredient used for this purpose is flaxseed, which improves meat quality due to increased content of PUFAs, such as α-linolenic (ALA), eicosatrienoic, eicosapentaenoic acid (EPA), and docosapentaenoic (DPA) acids . This isHerbs of the family Labiatae are particularly rich in antioxidants. These include many medicinal and seasoning herbs, such as rosemary, thyme, oregano, sage, and mint . Among hPhenolic compounds and other active plant substances can have beneficial effects on numerous biochemical processes in the body, including lipid metabolism, which leads to a decrease in the level of cholesterol and its atherogenic LDL fraction. Herbs with this type of activity include basil, ginseng, dandelion, and thyme ,12. The The aim of the study was to determine the effect of the administration of compound feed with 4% inclusion of extruded flaxseed in combination with different levels of thyme herb (1% or 3%) on lipid parameters and redox processes in fattening pigs, as well as on the oxidative stability and fatty acid profile of the tissues.The material for the research was WBP × Neckar crossbred weaners, from a body weight of about 30.32 ± 0.24 kg (about 84 days of age) until the end of the fattening period . The animals were individually tagged, weighed, and assigned to three feeding groups matched for sex and body weight. Each group comprised 40 animals, kept in 5 pens with 8 pigs in each (4 gilts and 4 barrows). The animals received complete Grower and Finisher diets ad libitum. The content of nutrients, amino acids, phosphorus, and calcium in all groups met the nutritional requirements laid out in NCR for pigs  Table 1Table 1.All experimental procedures used in the study were approved by the Second Local Ethics Committee on Animal Experimentation of the University of Life Sciences in Lublin, Poland.The pigs in the control group received a diet containing extruded flaxseed in the amount of 4%. This composition was chosen on the basis of previous research ,16, whicThe choice of thyme was influenced by a pilot experiment involving observations of the food preferences of piglets, which were given diets containing herbs with antioxidant properties: thyme, caraway, coriander, and purple coneflower. The observations showed that the diet containing thyme was most preferred by the piglets. Dried thyme herb was purchased from the herb shop Zakład Zielarski KAWON-HURT Nowak  and added to the diets when they were being prepared in the feed mixer.The feed was sampled three times during the experiment and analysed for the content of basic nutrients  and minerals  according to AOAC .The thyme herb was analysed as well.The samples were homogenized using a BUCHI mixer B-400 with ceramic blades.The homogenates were analysed for total phenolic content, total antiradical activity using DPPH and ABTS radicals, and flavonoid content.Thyme was extracted by a modification of the method described by Bakowska-Barczak and Kolodziejczyk . Dry thyThe ABTS•+ assay was carried out according to Re et al. . DPPH• fBlood was sampled for analysis from 6 barrows from each group at a body weight of about 70 and 100 kg. The animals had no access to feed for 12 h before blood sampling. Blood was drawn from the jugular vein into 10 mL heparinized tubes. The pigs chosen for analysis were matched for body weight. The same animals from which blood was sampled were slaughtered at about 110 kg BW, and the material collected from them was used for further analysis.Plasma was obtained by centrifuging whole blood at 3500 rpm for 15 min in a laboratory centrifuge  and stored in Eppendorf tubes at −80 °C until analysis.Tests from Cormay  were used to analyse the plasma for selected biochemical parameters, i.e., total protein , uric acid , urea , creatinine , phosphorus ; total cholesterol , triacylglycerols , and HDL cholesterol . LDL cholesterol (LDL-C) was calculated using the formula given by Friedewald :LDL-C  were used to determine the activity of alkaline phosphatase , alanine aminotransferase , and aspartate aminotransferase .Ca, Mg, Zn, Cu, and Fe content was determined by atomic absorption spectrometry (AAS) with a Varian model 720-ES ICP-OES spectrophotometer .The activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and the concentrations of lipid peroxidation products, i.e., peroxides (LOOH) and malondialdehyde (MDA), were determined in the plasma and tissues and parameters of the antioxidant system were measured in the plasma, i.e., ferric reducing antioxidant power (FRAP) and content of vitamin C, according to methods described by Czech et al. .The contents of fatty acids and cholesterol were analysed following fat extraction with a chloroform/methanol mixture, according to Folch et al. . The anaThe percentages of fatty acid methyl esters were estimated by gas chromatography on a Varian CP-3800 chromatograph. The operating conditions for fatty acid separation were as follows: CP WAX 52CB DF 0.25 mm capillary column 60 m in length, gas carrier–helium, flow rate 1.4 mL/min, column temperature 120 °C gradually increased by 2 °C/min to 210 °C, determination time 127 min, feeder temperature 160 °C, detector temperature 160 °C, other gases—hydrogen and oxygen.The content of fatty acids in the longissimus dorsi muscle was calculated according to Weihrauch et al. . Lipid qAll data are expressed as means and SEM (standard error of the mean).Data were analysed by ANOVA with treatment as the fixed effect and pig  as the experimental unit.The normality of the data distribution was tested using the Shapiro–Wilk test and equality of variance was tested by Levene’s test.Treatment means were compared using Tukey’s HSD (honest significant difference) test in Statistica 13 .p < 0.05 was used to determine statistical significance.For all tests, a criterion α level of The content of chemical components, including the antioxidant parameters of thyme herb, is presented in p = 0.003) and in the finisher period compared to the control group and T1 group (p = 0.001). The plasma of fatteners in the grower period receiving a diet with 1% thyme had a significantly lower level of TG than in the control (p = 0.015). In the grower period, the plasma of pigs receiving a diet with 1% and 3% thyme had a significantly higher content of HDL-C than in the control (p = 0.046). In the finisher period, the HDL-C content in the plasma of pigs receiving a diet with 3% thyme (T3) was significantly higher than in the control (p = 0.012). The plasma of group T3 fatteners had a significantly lower content of LDL-C (p = 0.044 grower and p = 0.003 finisher) compared to the other groups. The level of thyme significantly influenced the content of CHOL (p = 0.006 finisher), HDL-C (p = 0.013 finisher), TG (p = 0.013 finisher), and LDL-C (p = 0.016 grower and p = 0.005 finisher)  .p = 0.044 and p = 0.009, respectively). The level of inclusion of thyme also significantly influenced UA and UREA in the plasma of finisher pigs  (p < 0.001 grower and p = 0.005 finisher) relative to the control and was highest in the group receiving a diet with 3% thyme (p = 0.039 grower and p = 0.009 finisher)  . Alkalininisher) .p < 0.001, Vitamin C-p = 0.001; finisher period: SOD–p = 0.047, Vitamin C-p = 0.033). Ferric-reducing antioxidant power (FRAP) in the plasma of grower pigs from groups C and T3 was higher than in group T1 (p = 0.001), while in the finisher period, the FRAP value was significantly higher in the plasma of pigs receiving a diet with thyme (T1 and T3) than in the control (p = 0.004). LOOH content in the plasma of finisher pigs (p = 0.020) and MDA in grower (p = 0.001) and finisher (p = 0.009) pigs were significantly lower than in the control . The level of inclusion of thyme significantly influenced CAT activity (p = 0.026). The content of LOOH and MDA in the longissimus dorsi muscle of pigs from group T3 was significantly lower than in the control . The level of thyme significantly influenced the content of both LOOH (p = 0.041) and MDA (p = 0.049) (SOD and CAT activity was significantly higher in the longissimus dorsi muscle of pigs from the group receiving a diet with 3% thyme compared to the control (= 0.049) .p = 0.016) in the longissimus dorsi muscle relative to the control. The longissimus dorsi muscle of group T3 fatteners had significantly lower content of 18:0 (p = 0.050) and higher content of 20:1 (p = 0.049), 18:2 (p = 0.023), 18:3 (p = 0.039), and 20:3 (p = 0.045) compared to the other groups. The level of thyme significantly influenced the levels of 18:0 , 20:1 , 18:2 , 18:3 , and 20:3   .p = 0.021), while the PUFA content in the longissimus dorsi muscle of pigs from group T3 was significantly higher than in group C (p = 0.029). Significantly higher content of n-3 acids (p = 0.028) and a higher n-3/n-6 ratio (p = 0.046) were noted in the muscle of T3 pigs compared to the other experimental groups (C and T1 groups). The 3% inclusion of thyme in diets for pigs caused a significant reduction in the TI index (p = 0.043) and the cholesterol level (p = 0.013) compared to the control. The level of thyme inclusion significantly influenced the content of SFAs , the content of n-3 acids , and the n-3/n-6 ratio   .Formulation of diets for pigs presents many challenges associated with the choice of feed ingredients that will provide adequate amounts of essential nutrients and, at the same time, ensure that the animal products will meet consumer expectations. Consumers prefer products with the highest possible levels of substances with health-promoting effects, such as valuable protein, minerals, and polyunsaturated fatty acids (PUFAs). A valuable source of PUFAs for animals, especially n-3 PUFAs, is flaxseed, which is increasingly used as a feed ingredient for various livestock animals. This strategy results in beneficial modifications of the fatty acid composition of the meat or other products, such as milk or eggs. Our earlier research  and studIn the present study, the diet containing thyme herb in the amount of 3% had a beneficial effect on the lipid metabolism of fattening pigs, resulting in a reduction in the level of cholesterol and its LDL fraction, as well as that of TG even in the case of 1% inclusion of the phytobiotic. Shao et al.  used a bLipid metabolism is regulated mainly by the active substances in thyme oil, i.e., thymol and carvacrol. These substances have been shown to exert an inhibitory effect on the activity of hydroxymethylglutaryl–coenzyme A reductase, a key enzyme regulating endogenous biosynthesis of cholesterol . The capThere are indications that thyme has hepatoprotective effects in the case of damage to the liver caused by various factors . The benThe results of the present study, in which the effect of thyme herb on redox parameters of the blood was evaluated, confirmed the antioxidant effects of the plant. The plasma of pigs receiving a diet with 3% thyme herb showed increased activity of antioxidant enzymes SOD and CAT, which protect tissues against oxidation, as well as an increased FRAP value (ferric reducing antioxidant power). The value of this parameter is determined by the presence of vitamin C (15%), uric acid 60%), bilirubin (5%), and protein (10%) . The lev0%, bilirThe inclusion of thyme herb in the diet of pigs also modified the fatty acid profile of the loin meat. Both levels of the additive reduced the content of acids 14:0 and 18:0 and the total amount of saturated fatty acids. The use of 3% thyme herb increased the content of acids 20:1, 18:2, 18:3, and 20:3, the total PUFA content, and the n-3/n-6 ratio. In addition, there was a decrease in the thrombogenicity index TI and the cholesterol content. These changes should be regarded as highly favourable for consumers of pork, as a lower intake of saturated fatty acids and cholesterol can minimize the risk of atherosclerosis and other cardiovascular diseases . The incThis is also evidenced by the lower levels of lipid peroxidation markers in this group of pigs. The literature, unfortunately, lacks data on the fatty acid composition of pork from pigs fed with thyme or thyme oil. Chickens receiving an aqueous extract of thyme (50 and 100 mg/kg of feed) and thyme powder (150 and 250 mg/kg of feed) had reduced levels of some saturated fatty acids in the breast muscle, accompanied by an increase in health-promoting unsaturated fatty acids . ResultsThe presence of thyme herb in diets with extruded flaxseed has a beneficial effect by influencing the antioxidant status of the blood and muscle and improving the lipid profile of tissues.The study showed that a 3% inclusion of thyme in the diet significantly slows down oxidant processes, which may be a successful means of protecting meat against spoilage during storage. Furthermore, the 3% inclusion of thyme herb had a clear beneficial effect on lipid metabolism, so the levels of cholesterol and HDL cholesterol were reduced in both the blood and the longissimus dorsi muscle. In addition, the level of n-3 PUFAs in the muscles increased while the level of SFAs significantly decreased. The results of this study must be confirmed by further research.


Granulocyte colony-stimulating factor (G-CSF) administration increased ovarian preantral follicles and anti-Müllerian hormone (AMH) in animal models with diminished ovarian reserve. We investigated whether G-CSF priming before treatment with assisted reproductive technology (ART) improved embryo development and pregnancy rate while increasing serum AMH in patients with poor ovarian reserve.In this prospective randomized open-label controlled trial, 100 patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of in vitro fertilization/intracytoplasmic sperm injection and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming.Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming  than in controls . With priming, significantly more patients achieved cryopreservation of redundant blastocysts. The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls . Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables , logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. No adverse effects of priming were observed.G-CSF priming improved embryonic development and pregnancy rate during ART treatment and increased AMH in patients with poor ovarian reserve. Enhanced preantral follicle growth likely was responsible.https://www.umin.ac.jp/ctr/index.htm.UMIN registration in Japan (UMIN000013956) on May 14, 2014.  Ten years ago, we treated repeated implantation failure in 10 women with diminished ovarian reserve by administering granulocyte colony-stimulating factor (G-CSF) in association with embryo transfers (ET), based upon previous reports . No shorIn diabetic rats, administration of G-CSF consistently decreased ovarian follicular degeneration as well as degeneration and fibrosis of ovarian stroma, while increasing serum anti-Müllerian hormone (AMH) concentrations . G-CSF aConsidering our experience with the 3 patients and these G-CSF effects in animal models, we designed the present prospective randomized clinical trial examining whether G-CSF administration in the early luteal phases of each of 2 cycles (G-CSF priming) preceding the cycle involving assisted reproductive technology (ART) improved embryonic development and pregnancy rate following ART in patients with poor ovarian reserve, as well as whether priming increased serum AMH concentrations. We also investigated associations between effects of G-CSF priming and killer-cell immunoglobulin-like receptor (KIR) types, since some types have been associated with improved implantation following G-CSF administration on the day of ET .Our prospective open-label randomized clinical trial investigated whether G-CSF priming preceding ART enhanced preantral follicle growth, thus increasing the ART pregnancy rate in patients with poor ovarian reserve. Between May 19, 2014 and November 26, 2018, a total of 465 patients sought ART treatment at Women’s Clinic Jinno; 111 met study inclusion criteria. Eleven declined participation, leaving 100 to be enrolled and randomly assigned to groups undergoing or not undergoing G-CSF priming prior to standard ART. Randomization involved patients drawing from a box containing group assignments in sealed envelopes mixed 1:1  During these 2 cycles the G-CSF group, but not the control group, underwent subcutaneous administration of G-CSF  during the early luteal phase, based upon basal body temperature records, vaginal ultrasonographic findings, and, if necessary, serum progesterone determinations  or intracytoplasmic sperm injection (ICSI). Remaining embryos were cryopreserved at the blastocyst stage. When IVF/ICSI and fresh embryo transfer (ET) failed to result in delivery, cryopreserved embryos were thawed and transferred (cryopreserved ET) in subsequent spontaneous cycles. During actual ART treatment using fresh or cryopreserved ET, neither group received G-CSF.Clinical pregnancy and ongoing pregnancy were diagnosed by ultrasonographic detection of a gestational sac and fetal heart movements, respectively. Abortion and delivery were defined as pregnancy loss before 22 weeks and birth after 22 weeks, respectively. Clinical pregnancy rates by IVF/ICSI-fresh ET (per ovarian stimulation) were compared between groups as the primary outcome measure. Cumulative live delivery rates, follicular growth, fertilization, and embryonic development were compared between groups as secondary outcome measures.vs. post-treatment changes in serum AMH concentration were compared between groups. Serum samples were obtained within 4 months preceding study enrollment and just before initiating ovarian stimulation for ART .Follicular development was stimulated with the long protocol as described previously . Briefly6 per mL and/or motility < 20%). Oocytes were considered fertilized when 2 pronuclei were observed 17 to 19 h after insemination or ICSI. At 2, 3, or 5 days after oocyte retrieval, embryos were transferred to the uterus according to number and quality of developing embryos for each patient. Progesterone (25 mg i.m.) was administered daily after ET.Oocytes were collected transvaginally 36 h after hCG administration and inseminated as described previously . ICSI waRedundant embryos were cultured for 5 to 6 days after IVF/ICSI to the blastocyst stage and cryopreserved using a vitrification method. Thawed cryopreserved blastocysts were transferred to uteri on luteal day 5 of a spontaneous natural cycle, as described previously . For lut2), prolactin (PRL), testosterone (T), thyroid-stimulating hormone (TSH), free thyronine (FT3), free thyroxine (FT4), and dehydroepiandrosterone sulfate (DHEA-S) were measured by enzyme chemiluminescent immunoassays on cycle day 3 within 3 months before study enrollment.Serum concentrations of AMH, follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol  for PRL, 0.03 ng/mL  for T, and 2 μg/dL  for DHEA-S.Sensitivities and intra- and interassay coefficients of variation were 0.01 ng/mL  for AMH, 0.06 IU/L  for FSH, 0.11 IU/L  for LH, 5.0 pg/mL  for EDNA was genotyped for 16 KIR genes using PCR-SSOP (sequence-specific oligonucleotide probe) using a commercial kit  and Luminex 100 technology  as previously described .U test or the Wilcoxon matched-pairs signed rank test as appropriate. If data were normally distributed, unpaired t tests or paired t tests were performed as appropriate. Data also were analyzed using the chi-squared test, Fisher’s exact test, or multiple logistic regression analysis as appropriate. P values below 0.05 were considered to indicate significance. Whenever appropriate, results are presented as the mean ± standard deviation (SD).IBM SPSS Statistics Version 27  was used for statistical analyses. Normality was tested by the Shapiro–Wilk test. If data were not normally distributed, analysis was performed using the Mann–Whitney 3 nor FT4 differed between groups, making the TSH difference unlikely to be clinically meaningful. Ranges of AMH values were 0.00 to 1.93 ng/mL in the G-CSF group and 0.00 to 1.77 in the control group. Among the 50 G-CSF patients, 10, 50, 4, and 29 respectively had tubal infertility, ovarian dysfunction, endometriosis, and male infertility, as did 9, 49, 4, and 33 of the 49 control patients, showing no significant differences in prevalence of infertility causes (chi-squared test or Fisher’s exact test).Except for TSH, no significant differences concerning baseline characteristics were evident between the 2 groups completing the study Table . AlthougOne hundred ART patients were enrolled and randomized to G-CSF or control groups (50 patients each). All G-CSF patients completed the study; 1 control dropped out for unknown reasons, leaving 49 Fig. . All 3 pForty-seven G-CSF and forty-nine control patients underwent ovarian stimulation, resulting in successful oocyte retrievals in all G-CSF patients and 48 controls (no follicular growth was induced in one control). No transferable embryos were obtained in 2 G-CSF patients and 4 controls, leaving 45 and 44 fresh ET.P = 0.31, Mann–Whitney U test). Moreover, significantly more G-CSF patients achieved cryopreservation of redundant blastocysts than controls.G-CSF and control groups respectively achieved 14 and 6 clinical pregnancies, 12 and 5 ongoing pregnancies, and 9 and 5 live deliveries. Rates of clinical and ongoing pregnancy per stimulated patient were significantly higher in the G-CSF group (30% and 26%) than in controls , as were numbers of transferred embryos per cryopreserved ET .Subsequently, 21 and 14 cycles of cryopreserved ET were carried out in the G-CSF and control groups, resulting in 6 and 4 clinical pregnancies with 4 and 2 live deliveries. Rates of clinical pregnancy per cryopreserved ET were similar between G-CSF and control groups , although statistical significance was not attained . However, the rate of cumulative live delivery per patient was 32% in the G-CSF group, significantly higher than 14% in controls .Associations of 4 major fertility-related factors  with achievement of cumulative live delivery were analyzed by logistic regression analysis. A backward stepwise method based on the likelihood ratio test was used for selection of variables. Only age and G-CSF priming significantly correlated with cumulative live delivery , gestational age at delivery  or Apgar scores at 1 and 5 min .Sixteen G-CSF and seven control patients respectively delivered 20  and 10  normal live infants, including 2 and 3 sets of twins and 1 and 0 set of triplets. Considering the 13 and 4 singleton newborns in G-CSF and control groups, no significant difference was evident in body weight  were evident in endometrial thickness (11.4 ± 2.2 mm vs. 11.2 ± 2.6) or total amounts of hMG administered (2800 ± 660 IU vs. 2700 ± 810).On the day of hCG administration, number of follicles larger than 16 mm and serum Evs. 4.2 ± 2.8; day-2 embryos, 5.3 ± 4.1 vs. 3.7 ± 2.9; Fig. For all patients’retrieved oocytes, developmental outcomes were monitored during the first 2 days of culture. Comparing 47 G-CSF and 48 control patients with successfully retrieved oocytes, numbers of retrieved oocytes did not differ significantly but numbers of fertilized oocytes and day-2 embryos per retrieval were significantly higher in the G-CSF group than in controls . Oocytes from significantly more G-CSF patients proved suitable for embryo culture until 5 days after oocyte retrieval than did oocytes from control patients  into peripheral blood , potentiClinical safety and tolerance of G-CSF treatment have been established in healthy bone marrow donors treated for 3 to 5 days , patientPlatelet-rich plasma (PRP) injection into human ovaries has been reported to improve ovarian reserve markers and clinical pregnancy rates . HoweverIn patients with poor ovarian reserve, G-CSF priming in 2 consecutive cycles preceding ART significantly improved fertilization and embryonic development in ART therapy. Consequently, rates of implantation and clinical and ongoing pregnancy by fresh ET were significantly increased. The cumulative live birth rate was significantly higher in the G-CSF group than controls. G-CSF priming also significantly increased serum AMH, consistent with enhancement of preantral follicle growth–a mechanism differing from those previously suggested for implantation improvement by G-CSF. We observed no effects of G-CSF priming on miscarriage rates or any association of its efficacy with KIR genotypes. G-CSF priming showed no adverse events in our subjects or their fetuses. All infants born to subjects receiving G-CSF were free of congenital anomalies and had weights, birth weeks, and Apgar scores similar to those born to controls. This study proposes a novel, simple, and safe treatment for poor ovarian reserve.


Several morphological abnormalities may occur during tooth development and may be a predisposing factor for periodontal destruction. Palatogingival groove (PGG) is a developmental deformity that may cause localized periodontitis and endodontic complexities. The groove usually originates as a root indentation in the central fossa of the palatal root of maxillary lateral incisors.Cone beam computed tomography (CBCT) is an excellent radiographic imaging technique capable of identifying PGGs and provides details about the exact site, extent, and depth characteristics of this deformity. Early diagnosis and management of PGGs are of utmost importance, particularly due to their diagnostic intricacies that can pose both clinical and therapeutic challenges.This article aims to report a rare case of palatogingival groove associated with an on-and-off discharge from the maxillary left lateral incisor tooth (#22). Nonsurgical endodontic treatment was carried out with #22, and the palatogingival groove was sealed with composite restoration after a deep curettage. Excellent radiographic healing was observed after a six-month follow-up. Palatogingival grooves (PGGs) are developmental anomalies that typically originate as root indentations in the central fossa of the palatal roots of maxillary incisors. These grooves often extend from the cingulum and progress apically along the root surface, varying in length -9. On raBlack, in 1908, was the first to recognize and refer to this developmental deformity as a radicular groove ,5,8-10. The maxillary lateral incisor is frequently affected tooth due to its embryological challenge of being positioned between the tooth germs of the central incisor and canine ,12.Published literature has documented that the prevalence of PGGs ranged from 0.90% to 44.6%. This disparity in the prevalence rates can be ascribed to variations in diagnostic criteria, research methods, ethnic backgrounds, and geographical locations -9,12-16.The exact cause of PGGs remains obscure. However, the prevailing recommendations for the development of PGGs encompass the following four possibilities: (a) an anomaly in embryonic development, potentially involving the folding of Hertwig's epithelial root sheath, (b) a variant of dens invaginatus, (c) genetic factors, and (d) an attempt to form another root -12,17-19Conventional radiography serves as a non-invasive radiographic imaging technique for acquiring a two-dimensional depiction of the root canal structure. It is practical and cost-effective, making it the primary choice for such purposes. Nevertheless, these imaging techniques provide less detailed, overlapping images with inherent geometric distortions and noise, potentially affecting the accuracy and analysis of these structures. Cone beam computed tomography (CBCT) provides three-dimensional images with higher resolution and detailed accuracy, thus enabling precise evaluation, diagnosis, treatment planning, and post-treatment follow-up ,9,10,20.PGGs offer an excellent harbor for bacterial plaque and calculus aggregation, thus ensuring the onset and progression of periodontal diseases. The clinical implication of PGGs is linked to the occurrence of localized periodontitis, either with or without pulpal involvement, contingent upon the groove extent, depth, and complexity. Deeper grooves may establish a connection with the pulp chamber, leading to pulpal infection. Nevertheless, the primary link between the pulp and the periodontium with a groove is facilitated by accessory canals running along the groove, thus resulting in combined endodontic-periodontic lesions ,18,19,21In the past, teeth with PGGs were typically extracted due to endodontic-periodontal complications and hopeless prognosis . Over thThis article aims to report a rare case of palatogingival groove associated with an on-and-off discharge from the maxillary left lateral incisor tooth (#22). Nonsurgical endodontic treatment was carried out with #22, and the palatogingival groove was sealed with composite restoration after a deep curettage. Excellent radiographic healing was observed after a six-month follow-up.A 16-year-old male patient in good general health reported to our outpatient department with a chief complaint of on-and-off discharge from the palatal aspect of the left maxillary anterior tooth region for the past three months. The patient consulted a private practitioner for the same complaint and was prescribed a course of antibiotics and analgesics for a week (Cap Amox 500 mg thrice daily and Ketorol DT tablet 10 mg twice daily). There was no associated history of trauma. The past medical, dental, and family history were non-significant. No gross facial asymmetry was noticed on extraoral examination. Intraoral examination revealed palatally erupted #12 and #22. The crown of #22 was intact, non-carious, non-tender on percussion, and exhibited a negative response to thermal and electric pulp testing. There were no draining sinus tracts observed on the labial/palatal gingival surfaces. However, a palatogingival groove (PGG) was observed with a mesiopalatal aspect of #22, originating from the cingulum and extending to the gingival sulcus and continuing disto-apically down to the palatal root aspect. Oral hygiene was fair with a mild pocket (4 mm) on the palatal mucosa of #22  radiograph revealed an intact crown of #22 with an ill-defined, hazy radiolucency in the periapical region and dilacerated apical one-third of the root  at 120 KV, 5 mA, an exposure time of 7 s, with voxel size of 0.25 mm × 0.25 mm × 0.25 mm and image matrix of 640 × 640.The axial view revealed a shallow palatogingival groove at the level of the cementoenamel junction with a normal root canal configuration (type I groove) Figure .The sagittal section revealed an ill-defined (3.39 mm x 4.66 mm) periapical hypodensity with intact cortical plates of #22 . Canal patency was achieved using a #10 K-file followed by coronal flaring with G.G. drill numbers 1, 2, and 3 in a step-back manner. The working length was found to be 21 mm using a radiographic technique and confirmed with an electronic apex locator (Root Zx Mini). Biomechanical preparation was done in a step-back manner up to master apical file #40 with continuous use of 3% sodium hypochlorite as an irrigant and intermittent use of saline and ethylenediaminetetraacetic acid (EDTA). Calcium hydroxide was used as an intracanal medicament, and a closed dressing was given. The patient was recalled after three days, and the canals were irrigated with 3% sodium hypochlorite, saline, and EDTA as per standard protocol. Canals were dried, and a long-term dressing of calcium hydroxide and Iodoform mixture (METAPEX) was placed in the canals. There was no pain on percussion with #22 after a week of follow-up.The patient was recalled after one month for obturation of the canal. The obturation of the canal was completed using the lateral condensation technique. Deep curettage of the palatogingival groove was done, and it was sealed with composite restoration Figure .Follow-up was done every month. Complete radiographic healing was observed periapically with #22 at the six-month follow-up Figure .The PGG serves as an excellent gateway for oral microorganisms to penetrate periodontal tissues, resulting in periodontal destruction, along with concurrent pulp necrosis and/or apical periodontitis, thus posing a threat to diagnosis and treatment plans .Numerous classification systems have been proposed to classify PGGs due to notable discrepancies observed in their location, extent, depth, and termination ,14,24,25A thorough clinical assessment reveals a funnel-shaped hollow groove with plaque and calculus accumulation, coupled with bleeding upon probing, pocket formation, and clinical attachment loss .Patients with a PGG pathology may experience various symptoms, including occasional dull pain , acute pOccasionally, a few patients may be completely asymptomatic. In the majority of cases, the patient exhibits a negative history of dental caries, tooth discoloration, and trauma. Depending on the pulpal involvement, the pulp may be vital or non-vital. Advanced lesions with deeper grooves often exhibit no response to electric or thermal pulp testing .Published literature has reported that the presence of PGGs correlates with narrow/deep periodontal pockets and overall poorer periodontal health ,5,12. HoIn the presented case, a shallow pocket measuring 4 mm was observed clinically, whereas there was no evidence of bone loss radiographically. Our case findings were in coherence with previously published reports ,16.Radiographically, teardrop-shaped radiolucency may be seen in cases with primary pulpal infection, whereas pear-shaped radiolucency with widened periodontal ligament space may be observed in primary periodontal lesions . In a feMicrocomputed tomographic evaluation may provide a three-dimensional representation of morphological anomalies. Gu  elucidatOver the last decade, CBCT has emerged as a precise diagnostic tool, offering reconstructed three-dimensional images of teeth and adjacent tissues . RegardiBased on cross-sectional configurations observed in CBCT images, the morphology of extracted teeth, and preoperative radiography, Tan et al.  categoriTan et al.  suggesteIn the presented case, the axial CBCT section revealed a type I palatogingival groove at the level of cementoenamel junction corresponding to a normal, single root canal. The sagittal CBCT section showed a hypodense lesion at the periapex of #22 with intact cortical plates.Published literature has utilized diverse methodologies to evaluate the prevalence of PGGs. These methods include microscopic examination and in vitro microcomputed tomography. For in vivo evaluations, clinical examination, radiographic examination, and CBCT were employed. The findings, however, demonstrate variability ,18,30-34The treatment objectives for a tooth with a PGG are achieving complete elimination of microorganisms, ensuring a thorough and permanent seal of the groove, and achieving complete healing of the periodontium . The recCases with shallow grooves without any periodontal damage may be treated with superficial odontoplasty and the removal of granulation tissue through periodontal procedures. Conversely, when grooves are moderate to complex, a combined approach involving both endodontic and periodontal interventions is required .Various materials have been employed to seal PGGs. Due to their superior properties, mineral trioxide aggregate (MTA) and iRoot BP Plus have emerged as excellent substitutes for previously used materials such as amalgam or glass ionomer ,20.In a recent study, Tan et al.  advocateThe prognosis for a tooth with PGG is contingent on multiple factors, such as the groove location, access to the anomaly, accompanying periodontal disease severity, and the characteristics of the groove  ,3,14.The groove's depth plays a significant role in shaping the configuration of the root canals, potentially posing challenges for achieving effective endodontic sealing. Teeth featuring shallower grooves with less depth extending along the root generally exhibit a more favorable prognosis . MoreoveRecommendationsPGG serves as a “plaque trap” resulting in periodontal diseases with or without endodontic implications. Detailed history, clinical evaluation, and CBCT facilitates an early and accurate diagnosis. Treatment options vary and encompass saucerization and sealing the defect, endodontic therapy, and surgical periodontal procedures. Type I grooves are usually treated with saucerization, curettage, and sealing of the groove and may require endodontic treatment where pulpal involvement occurs. Type II and III grooves necessitate complex endodontic and periodontal interventions.Our patient presented with a complaint of discharge from the maxillary anterior tooth region. After a detailed history and clinical and radiographic examination, a diagnosis of chronic periapical abscess secondary to a palatogingival groove with #22 was made. Nonsurgical endodontic treatment was carried out with deep curettage and sealing of the groove. A six-month follow-up showed favorable radiographic healing with no recurrence episodes.


Histomonas meleagridis is a protozoan parasite that causes histomonosis in gallinaceous birds. This study identified α-actinin 1 of H. meleagridis. α-actinin 1 was predicted as a virulence factor involved in cell adhesion in our previous differential proteomic analysis. It localized in different cellular regions as detected by an indirect immunofluorescent assay between virulent histomonads and attenuated histomonads JSYZ. Administration of recombinant expressed rHma-actinin 1 reduced lesion scores in the cecum and liver, improved weight gain, and induced humoral and cellular immune responses against H. meleagridis in chickens.Histomonas meleagridis is a protozoan parasite that causes histomonosis in gallinaceous birds such as turkeys and chickens. Since the banning and restricted usage of effective drugs such as nitarsone, 80–100% morbidity and mortality occur in turkeys and 20–30% mortality in chickens. New ideas are needed to resolve the re-emergence of histomonosis in poultry. In this study, the α-actinin encoding gene from H. meleagridis was cloned. The 1839-bp gene encoding 612 amnio acids showed close phylogenetic relationships with Trichomonas vaginalis and Tritrichomonas foetus. It was then inserted into the prokaryotic expression vector pET28a(+) and induced with isopropyl-β-D-thiogalactopyranoside. A 73 kDa recombinant protein rHmα-actinin 1 was obtained and purified with a Ni-NTA chromatography column. rHmα-actinin 1 was recognized by mouse anti-rHmα-actinin 1 polyclonal antibody, mouse anti-rHmα-actinin 1 monoclonal antibody, and rehabilitation sera from H. meleagridis infected chickens. Native α-actinin 1 in the total proteins of H. meleagridis can also be detected with mouse anti-rHmα-actinin monoclonal antibody. Immunolocalization assays showed that Hmα-actinin 1 was mainly distributed in the cytoplasm of virulent histomonads JSYZ-D9 and in the peripheral regions (near the plasma membrane) of attenuated histomonads JSYZ-D195. Based on in vivo experiment, when chickens were subcutaneously immunized with rHmα-actinin 1 at 5 and 12 days old and then challenged with H. meleagridis at 19 days old, rHmα-actinin 1 reduced the lesion scores 12 days after infection (31 days old) and increased the body weight gain during the challenged period (19–31 days old). Furthermore, it also strengthened the cellular and humoral immune responses 7 days after the second immunization (19 days old). In conclusion, Hmα-actinin 1 could be used as a candidate antigen to develop vaccines against chicken histomonosis. Histomonas meleagridis is a protozoan parasite belonging to the phylum Parabasalia, class Tritrichomonadea, order Tritrichomonadida, and family Dientamoebidae  × 100. On day 31 , chickens were euthanized, and the liver and caecum were taken for lesion score evaluation, which was graded from 0–4 following a previous study [p < 0.05.The data were statistically analyzed using one-way ANOVA by SPSS 22.0  with Duncan’s multiple range test. Data were expressed as mean ± S.D. value. Differences were considered significant at H. meleagridis and submitted to GenBank with the accession number MW197428 for nucleotides and QZM06938 for amino acids. It was a full open reading frame encoding 612 amino acids with no signal peptide. Its nucleotide had 99.8%, 53.7%, and 61.8% homology to α-actinin 1 (GenBank accession number FM200068), α-actinin 2 (GenBank accession number FM200071), and α-actinin 3 (GenBank accession number FM200072), respectively, from H. meleagridis in Austria [T. vaginalis, from 52.2% to 53.9%. Phylogenetic analysis of the amino acid sequences showed that it was in the same clade as α-actinins from H. meleagridis and Tritrichomonas foetus, and in a sister clade with T. vaginalis  for further analysis. The final concentration was 4 mg/100 mL BL21(DE3).The 1839-bp α-actinin 1 gene was inserted into pET28a(+) (Invitrogen) and successfully expressed xpressed . It was 450 nm.The titer of mouse anti-rHmα-actinin 1 polyclonal antibody was determined to be 102,400, which was the highest dilution when the positive serum was 2.1 times greater than the negative serum at ODH. meleagridis using western blot and indirect immunofluorescence.Two stable hybridoma cells lines against rHmα-actinin 1, 4C2 and 2F1, were obtained and isotyped IgG1. The supernatant of the cell culture of 2F1 was collected to assess the native proteins of Hmα-actinin 1 in H. meleagridis infected chickens  and attenuated (JSYZ-D195) histomonads. Mouse anti-rHmα-actinin 1 polyclonal antibody successfully recognized the native Hmα-actinin 1 of eagridis . This sheagridis A–F but ieagridis G–L. Mouseagridis .p < 0.05), especially in the 150 µg group (p < 0.05).The serum antibodies of chickens in response to immunization with rHmα-actinin 1 are shown in p < 0.05), whereas IL-4 and IL-10 were significantly lower in the 200 µg and 150 µg groups (p < 0.05). At 7 days following secondary immunization (19 days of age), IFN-γ and IL-2 were still significantly higher in the 200 µg, 150 µg, and 100 µg groups than in the other groups (p < 0.05), whereas IL-4 and IL-10 were still lower in immunized groups (p < 0.05).IFN-γ, IL-2, IL-4, and IL-10 responses to rHmα-actinin 1 immunization are shown in Overall, there was no difference in the four serum cytokines the day before immunization (5 days of age). The group immunized with 200 μg rHmα-actinin 1 had the best cellular immunity, followed by the 150 μg group.p > 0.05) (p < 0.05) but did not differ significantly from that of chickens in the UU group (negative control) (p > 0.05). The RWG-2 was highest (97.93%) in group 150 µg, followed by group 100 µg (93.34%) (p > 0.05) (p < 0.05), and scores were lowest in the 150 µg group.Seven groups were designed to evaluate the protective effect of rHmα-actinin 1. No chickens died, and the survival rate of every group was 100% . On days > 0.05) . On days(93.34%) . Liver l > 0.05) . HoweverT. vaginalis, which contains α-actinin 2 (110 kDa) [T. vaginalis infection in mice under experimental conditions [T. vaginalis infection.Re-emergence of histomonosis resulted from the mandatory prohibition of chemical drugs to maintain food safety, making it is necessary to find more effective methods to control this disease. It has been reported that α-actinin in 110 kDa)  and α-ac110 kDa) ,37, are nditions ,29. ThusH. meleagridis, α-actinin 1, α-actinin 2, and α-actinin 3 [H. meleagridis and T. foetus, which had close evolutionary relationships to each other [T. vaginalis. Overall, α-actinin showed evolutionary conservation in trichomonas, and the α-actinin 1 encoding gene in H. meleagridis was successfully cloned in this study.There are three α-actinins in ctinin 3 . The α-ach other ,35, and in vitro using the prokaryotic expression vector pET28a(+). The expressed rHmα-actinin 1 was 73 kDa and was specially recognized by mouse anti-rHmα-actinin 1 polyclonal antibodies, mouse anti-rHmα-actinin 1 monoclonal antibodies, and rehabilitation sera from H. meleagridis infected chickens. This showed that rHmα-actinin 1 had suitable immunoreactivity and was suitable for further research. Native α-actinin 1 was also detected with mouse anti-rHmα-actinin 1 monoclonal antibodies in extracted proteins of whole parasites using western blot. Immunolocalization results showed that Hmα-actinin 1 was located in the cytoplasm of virulent histomonads JSYZ-D9 and in the peripheral regions (near the plasma membrane) of attenuated histomonads JSYZ-D195. This result further verified the differential expression of α-actinin 1 reported in our previous differential proteomic analysis, where it was highly expressed in virulent histomonads and expressed at low levels in attenuated histomonads [H. meleagridis and T. vaginalis, which showed that α-actinin was located in the cytoplasm when it is pear-shaped, but when parasites adhered to host cells and become amoeboid shape, it was localized in the periphery [To reveal the localization of Hmα-actinin 1 in the parasite and its role in virulence, it was successfully expressed tomonads . This reeriphery ,25,38. TT. vaginalis and successfully expressed in pET32a system in E. coli BL21 (DE3) cells. α-actinin was primarily localized in the cytoplasm of T. vaginalis by using indirect immunofluorescence with the polyclonal antibodies against α-actinin [T. vaginalis α-actinin, α-actinin 2 (Tvα-actinin 2), was also successfully expressed in pET21b expression plasmid and transferred into E. coli BL21 (DE3) cells. Immunofluorescence localization showed that during trophozoite development, Tvα-actinin 2 was mainly located in the cytoplasm. However, during amoeboid stage development, Tvα-actinin 2 was primarily found near the plasma membrane. It was observed that T. vaginalis undergoes a morphological transition to an amoeboid-like form during the invasive stage. This further confirmed that Tvα-actinin 2 is a virulence factor associated with invasion [Recombinant expression technology can be used as a tool for studying gene function. α-actinin was found in -actinin . Anotherinvasion .Recombinant expression technology enables the mass production of recombinant subunit vaccines, thereby reducing the cost and time required for preparing natural protein vaccines . RecombiIn vivo protective experiments showed that rHmα-actinin 1 immunized groups showed lower cecal and liver lesions and higher BWG than adjuvant and UC groups. No chickens died in the groups. The liver lesion score was slight compared to caecum because the characterization of the immune system in chickens prevents parasites transmitting from caecum to liver [H. meleagridis, there were variations in body weight gain (BWG2) during challenge period (19–31 days of age) among the groups. The 150 µg group showed the highest level of protection compared to the other immunized groups.to liver ,40. LiveH. meleagridis infection stimulates higher Th1 immune responses than Th2 [rHmα-actinin 1 induced better humoral and cellular immune mechanisms mediated by antibodies and cytokines. Serum antibody levels showed no obvious increase 7 days after the first immunization but increased significantly after the second immunization. This growth trend was also reflected in IL-2 levels. IL-2 is considered a T-cell growth factor that can promote the activation and proliferation of lymphocytes and assist B lymphocytes to produce specific antibodies against protozoan infection ,41. In tthan Th2 ,42.in vivo experiment to analyze the peak period of lesions after H. meleagridis infection, in order to determine the timepoint for necropsy.α-actinin 1 was successfully identified in this study, but the molecular mechanism associated with its different expression and localization in virulent versus attenuated strains needs to be further researched. It is recommended to conduct a preliminary experiment before H. meleagridis. It was successfully expressed in the prokaryotic expression vector pET28a(+) with a molecular weight of 73 kDa. rHmα-actinin 1 showed good immunogenicity and was detected by mouse anti-rHmα-actinin 1 polyclonal antibodies, mouse anti-rHmα-actinin 1 monoclonal antibodies, and rehabilitation sera from H. meleagridis-infected chickens. Immunolocalization analysis showed that α-actinin 1 localized to the cytoplasm of virulent histomonads and in the peripheral regions (near the plasma membrane) of attenuated histomonads. Results indicate that Hmα-actinin 1 may be an important virulence infector. The recombinant protein rHmα-actinin 1 can also stimulate humoral and cellular immune responses against H. meleagridis infection in chickens. Overall, Hmα-actinin 1 may be a potential vaccine candidate against chicken H. meleagridis infection.In summary, this study cloned the 1839-bp α-actinin 1-encoding gene of


In response to the article “Ventral Longitudinal Intraspinal Fluid Collection Presenting as Upper Limb Amyotrophy”  we wouldIn 2018, Nicoletti et al. reported on a 63-year-old man with bibrachial amyotrophy due to a spinal cerebrospinal fluid (CSF) leak, which was not located at this time [Dynamic digital subtraction myelography is the preferred imaging modality to locate a ventral CSF leak . As mostBibrachial amyotrophy is a very rare manifestation of spontaneous intracranial hypotension (SIH) and likely caused by stretching of the cervical nerve roots over the extradural CSF collection . Large e


New techniques and technology in the field of burn surgery that facilitate “minimally invasive” donor site reduction include bromelain enzymatic debridement and autologous skin cell sprays (ASCS). Each of these techniques independently have been shown to decrease the need of autografting and reduce the donor site size. However, the use of both methods together was not possible until recently. The authors hypothesize that bromelain enzymatic debridement plus ASCS will result in donor site reduction compared to conventional excision and grafting.The conventional treatment for deep 2nd and 3rd degree burns was performed. The patients were stratified into those that received conventional excision and autografting (CONTROL group), and those that received bromelain enzymatic debridement with autologous skin cell spray with or without a split thickness skin graft (TREATMENT group). Data collection included demographics, injury characteristics, length of stay (LOS)/%TBSA. Total surface area treated with ASCS, autograft, and donor site size was tabulated. A ratio of donor site to total area treated was calculated and compared to determine relative reduction in donor site size. Data was reported in medians+IQR and comparisons were done with a Mann Whitney test.A single institution retrospective review of patients who underwent treatment of deep 22  and 1463cm2  in the CONTROLS and TREATMENT groups, respectively. There were no differences in age and sex. The total area grafted and/or treated with ASCS was 1342 cm2  in the CONTROLS and 1404 cm2  in the TREATMENT group (p=0.4354). The donor site area to total treatment area ratio for CONTROLS was 0.273  while the TREATMENT group was 0.082  (p=0.013). The O/E ratio for LOS was 1.35 days/%TBSA for the CONTROLS and 1.06 for the TREATMENT group (p= 0.2). No significantly differences were detected in comorbidities between groups. Each group had 1 unplanned readmission but no graft loss. There were no mortalities.Twenty-eight study patients were included over 18 months, 15 CONTROLS and 13 TREATMENT patients. Burns size was 1789 cmBromelain based enzymatic debridement can be successfully utilized with ASCS to perform a “minimally invasive” skin graft. These patients received relatively smaller donor sites and showed a trend for lower LOS. Just as with all surgical therapies, appropriate patient selection and burn characteristics are imperative when utilizing minimally invasive burn surgery. Further larger scale studies are needed to adjust for confounders and determine the individual and synergistic effects of the component novel therapies.Donor site reduction improves care paradigms


N ═ 21) received PEG-rhG-CSF, while patients in the control arm (N ═ 19) received short-acting rhG-CSF. The primary endpoint was the incidence of grade 3–4 neutropenia, and the secondary endpoints were the incidence of febrile neutropenia, chemotherapy delay, and radiotherapy interruption. In addition, dynamic changes in absolute neutrophil count during radical chemoradiotherapy and adverse events were compared between the two groups. There were 0 and 4 cycles of grade 3–4 neutropenia in the PEG-rhG-CSF and rhG-CSF groups, respectively. The incidence of neutropenia of all grades was lower in patients on PEG-rhG-CSF than that on rhG-CSF . No patient developed neutropenic fever. The lowest values of neutropenia during chemoradiotherapy cycles were 2.73 ± 1.02 and 1.91 ± 0.79 × 10 9/mL in the PEG-rhG-CSF and rhG-CSF groups, respectively (p < 0.001). In the PEG-rhG-CSF and rhG-CSF groups, 0 and 8 (11.11%) cycles of chemotherapy were delayed due to neutropenia, respectively (p ═ 0.01). There was no delay of radiotherapy by more than one week in either group. Prophylactic use of PEG-rhG-CSF during chemoradiotherapy for cervical cancer can effectively prevent neutropenia and associated adverse events. PEG-rhG-CSF may be an effective strategy to provide uninterrupted radical chemoradiotherapy for cervical cancer.The standard of care for locally advanced cervical cancer is concurrent chemoradiotherapy, which is associated with significant toxicity, especially hematologic toxicity. To evaluate the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in preventing neutropenia during radical chemoradiotherapy for cervical cancer, 40 patients receiving prophylaxis from February 2018 to July 2019 were randomly divided into two arms in a 1:1 ratio. Patients in the study arm ( Cervical cancer is one of the most common gynecologic malignancies, with approximately 130,000 new diagnosed cases in China each year . The staRecombinant human granulocyte colony-stimulating factor (rhG-CSF) is used for supportive care and safe administration of chemotherapy. rhG-CSF mobilizes neutrophil progenitor cells to reduce the severity of neutropenia and the risk of febrile neutropenia (FN) by at least 50% . Both thHowever, the short half-life rhG-CSF requires daily administration . PegylatEscherichia coli. A full list of the inclusion and exclusion criteria is given in the protocol . All patients provided informed consent to participate. The eligible patients were randomly divided at a 1:1 ratio into either the PEG-rhG-CSF group as the study arm or the rhG-CSF group as control. The study design diagram is shown in The trial was a single-center, open-label, randomized study at the First Affiliated Hospital of Air Force Medical University  and was registered in ClinicalTrials.gov (NCT03206684). Patients were eligible for inclusion if they had cervical cancer with the 2009 International Federation of Gynecology and Obstetrics (FIGO) IB–IVA stage and were intended to receive radical chemoradiotherapy from February 2018 to July 2019. Eligible patients were also required to be aged ≥18 years, have a Karnofsky performance score of ≥70, and have consented to receive radical chemoradiotherapy regimen with docetaxel and cisplatin. The key exclusion criteria included the presence of (1) other active malignancies or brain metastases, (2) existing infection that is difficult to control, or (3) allergy to PEG-rhG-CSF and rhG-CSF or other biologic products derived from genetically engineered 9/L or the absolute neutrophil count (ANC) was less than 0.5 × 109/L, rhG-CSF for salvage treatment was administered at a dose of 150–300 µg once a day until a WBC of 10 × 109/L or an ANC of 1.5 × 109/L was reached. Both PEG-rhG-CSF and rhG-CSF were provided for free by the China Shiyao Pharmaceutical Group.In the study group, PEG-rhG-CSF was given subcutaneously at a dose of 6 mg for patients weighing ≥45 kg or 3 mg for those weighing <45 kg after 48 h of completing one chemotherapy cycle. In the control arm, rhG-CSF was injected subcutaneously at 48 h after the end of chemotherapy at a dose of 300 µg for those weighing ≥45 kg or 150 µg for those weighing <45 kg once a day for three consecutive days. In both groups, if the white blood cell count (WBC) was less than 2 × 102 on day 1 and intravenous cisplatin at 20–25 mg/m2 on days 1–3. Chemotherapy and radiotherapy were withheld for grade 4 neutropenia, thrombocytopenia, FN, or persistent  grade 3 or 4 gastrointestinal toxicity.All patients underwent three-dimensional conformal or intensity-modulated radiotherapy of the clinical target at 50 Gy in 25 daily fractions and two cycles of concurrent chemotherapy, followed by an intracavitary brachytherapy boost using a high dose rate technique and two cycles of adjuvant chemotherapy. The metastatic lymph nodes were treated with a simultaneous integrated boost regimen of 62.5 Gy in 25 fractions. Chemotherapy was given once every 21 days and comprised a dual-agent regimen of intravenous docetaxel at 60–75 mg/mBlood test was done once a week or according to physician decision, whereas physical examination and evaluation for toxicity were repeated weekly during treatment. Neutropenia, FN, and safety evaluation were graded and evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE 5.0) of the World Health Organization. Chemotherapy or radiotherapy delay was defined as suspension of the schedule of treatment for more than one week.The trial was a single-center, open-label, randomized study at the First Affiliated Hospital of Air Force Medical University  and was registered in ClinicalTrials.gov (NCT03206684). The study protocol was following the Guidelines of Helsinki, reviewed and approved by the Ethics Committee of the First Affiliated Hospital, Air Force Medical University (KY20172040-1). All patients provided informed consent to participate.α of 0.005 and a β of 0.20, the required sample size was 160 cycles.The present study intended to reduce the incidence of grade 1–4 neutropenia to 30% in all chemotherapy cycles by the prophylactic use of PEG-rhG-CSF, based on a probability of 65% for the event . Allowinp < 0.05 denoted significance.SPSS19.0 statistical software  was used for data analysis. Demographic and baseline data were statistically described and compared between the study and control arms. The incidence of the efficacy measures and safety events was statistically described, and 95% confidence intervals were calculated. Student’s t-test, Chi-square test, and Fisher’s exact test were used to compare the differences between the arms. n ═ 21) or rhG-CSF (n ═ 19) group. The entire cohort of patients had a median age of 50 years . The patients with FIGO stage IIB–IVA accounted for 70% of the whole cohort. The two arms were balanced in terms of neutrophils, leukocytes, hemoglobin, and platelet count at baseline  vs. 4 (5.56%) cycles; ═ 0.046] .9/L in the PEG-rhG-CSF and rhG-CSF arm (p ═ 0.004), respectively. Salvage treatment was needed during 21 (29.17%) chemotherapy cycles in the rhG-CSF group but was not needed in the PEG-rhG-CSF group. The incidence of chemotherapy delay was significantly lower in the PEG-rhG-CSF arm than in the rhG-CSF arm . However, in both groups, chemotherapy dose adjustment was not needed and there were no radiotherapy delay and FN.The lowest value of ANC throughout treatment was 2.73± 1.02 and 1.91 ± 0.79 × 10p ═ 0.93) and at one month after the end of chemoradiotherapy (p ═ 0.05).The mean ANC level was higher in the PEG-rhG-CSF arm than in the rhG-CSF arm during each period of chemoradiotherapy . In the Among the adverse reactions encountered in the PEG-rhG-CSF and rhG-CSF groups, digestive tract events, bone marrow suppression, and skin reaction were the most frequent. Among the 40 patients in this study, there were no severe adverse events related with PEG-rhG-CSF or rhG-CSF .In this study, the prophylactic use of PEG-rhG-CSF after every chemotherapy cycle effectively reduced the incidence of grade 1–4 neutropenia and delay of chemotherapy in patients with cervical cancer treated by cisplatin and docetaxel. The use of PEG-rhG-CSF resulted in high levels and fast recovery of neutrophils and low incidence of grade 1–4 neutropenia. There were no severe adverse effects of PEG-rhG-CSF treatment.Since 1999, CCRT with cisplatin has become the standard of care for patients receiving radical therapy for LACC. However, in patients with poor prognostic factors, such as lymph node metastasis, large primary tumor size, adenocarcinoma pathologic type, and FIGO stage III–IVA, concurrent chemotherapy using single-agent cisplatin cannot significantly improve the survival rate , 5. TherIn the past, rhG-CSF was used to prevent and treat leukopenia and FN caused by chemotherapy. Koensgen et al.  observedp < 0.05).The plasma half-life of PEG-rhG-CSF is extended to 47 h, and several clinical studies have confirmed that one dose per chemotherapy cycle can effectively prevent neutropenia , 23. In In the present study, there was no grade 3–4 neutropenia in patients who received prophylactic PEG-rhG-CSF, and the incidence of grade 1–4 neutropenia was significantly lower in the PEG-rhG-CSF arm  than in the rhG-CSF arm . Moreover, the incidence of delay in chemotherapy was significantly lower in the PEG-rhG-CSF arm  than in the rhG-CSF arm . The results of this study showed that compared with multiple doses of short-acting rhG-CSF, one prophylactic administration of PEG-rhG-CSF per chemotherapy cycle resulted in better bone marrow protection. The consensus of Chinese experts on the use of pegylated granulocyte stimulating factor during CCRT does not recommend the use of PEG-rhG-CSF during the weekly chemotherapy regimen of CCRT . MoreoveIn this study, we recorded the dynamic changes in neutrophils during chemoradiotherapy and found a relatively high level and rapid increase of the ANC after the end of pelvic irradiation in the PEG-rhG-CSF arm. These results were similar to those in the study by Zou et al. , althougSome limitations of this study were the small number of included patients and the single institute design. However, the total number of chemotherapy cycles met the statistical requirements, because each patient received four cycles of chemotherapy. In addition, the efficacy of rhG-CSF use for three times per cycle in the control group could be a little weak for the prevention of neutropenia. In 2003, Green et al.  reportedProphylactic use of PEG-rhG-CSF during chemoradiotherapy for cervical cancer can effectively prevent neutropenia and its related adverse events. PEG-rhG-CSF use may be an effective strategy to guarantee continuous radical chemoradiotherapy for cervical cancer by ensuring the designed time and dose of chemotherapy and an uninterrupted radiotherapy. This result is worth being further proven by a phase III randomized prospective study.


Chlamydia trachomatis can cause the eyelid to scar and turn inwards, resulting in the eyelashes rubbing against the eyeball, known as trachomatous trichiasis (TT). In Guinea, baseline surveys conducted in 2013 reported inflammatory trachoma prevalences below the World Health Organization (WHO) threshold for elimination, but TT prevalences above threshold. Given this epidemiological context and time since baseline survey, TT-only surveys were conducted in selected districts to determine current TT prevalence. The results of this study provide critical data for assessing Guinea’s achievement of trachoma elimination targets.Trachoma is a public health problem in 42 countries. Inflammation associated with repeated ocular infection with Four health districts, consisting of six evaluation units (EU), were surveyed. In each EU, field teams visited 29 clusters with a minimum 30 households included in each. Participants aged≥15 years were examined by certified graders trained to identify TT and determine whether management had been offered.A total of 22,476 people were examined, with 48 TT cases across the six EUs identified. Five of six EUs had an age-and-gender adjusted TT-prevalence unknown to the health system less than 0.2%, whereas one EU, Beyla 2, had an adjusted TT prevalence of 0.24%.These TT-only surveys, along with findings from other trachoma interventions, suggest that Guinea is close to achieving elimination of trachoma as a public health problem. This study demonstrates the value of undertaking TT-only surveys in settings where baseline surveys indicated active trachoma prevalences below WHO elimination threshold, but TT prevalences above it. Chlamydia trachomatis and is a public health problem in 42 countries, primarily in lower- and middle-income countries; many in sub-Saharan Africa. 2 Infection is associated with active (inflammatory) trachoma, most commonly in pre-school children. Factors associated with transmission are unclean faces and limited availability or use of latrines, which can increase the number of C. trachomatis -carrying flies, Musca sorbens.3–10Trachoma is a bacterial eye disease caused by 11 Repeated infection and associated episodes of active trachoma can lead to scarring of the tarsal conjunctiva,13 which can result in the upper eyelid turning inwards, causing the eyelashes to rub against the eyeball. This painful state is called trachomatous trichiasis (TT) and can damage the cornea, leading to opacities that can result in blindness.14 The prevalence of TT increases with age; it is rarely present before the age of 15 years.16 As of June 2022, over 125 million people worldwide were estimated to be at risk of blindness due to trachoma.1 The SAFE strategy, developed by the World Health Organization (WHO) for the elimination of trachoma as a public health problem, includes: TT Surgery (S), Antibiotics (A), Facial Cleanliness (F) and Environmental improvement (E).18 In trachoma-endemic areas, elimination as a public health problem requires evidence of a trachomatous inflammation—follicular (TF) prevalence below 5% in children aged 1–9 years and a prevalence of TT unknown to the health system below 0.2% in adults aged 15 years and above.Trachoma is the leading infectious cause of blindness worldwide.19 There is limited information on the implementation of F and E activities in Guinea; a study on water, sanitation and hygiene conducted by the Guinea health ministry in collaboration with Sightsavers in 2020 demonstrated the existence of small-scale interventions delivered to rural communities throughout the country, however this activity was not related to the trachoma elimination programme nor was it implemented district-wide.20 Since July 2021, Sightsavers has been supporting a water and sanitation behaviour change programme contributing to the reduction of TF, through the Guinean NGO Tinkisso. Surgical outreach campaigns have been implemented across the country since 2013. Individuals identified with TT during the baseline surveys were offered surgery, and in areas with high TT prevalence, house-to-house screening was conducted using local volunteers trained to identify TT cases, which were later confirmed by ophthalmic nurses. Based on the 2011–2016 baseline surveys, the country-wide TT backlog was estimated at 32,737; the most up-to-date estimate (January 2023) is a backlog of 7,774 cases.In Guinea, national baseline mapping of trachoma took place in 31 health districts from 2011–2016; 18 health districts required implementation of SAFE.21 Sixteen districts have benefited from mass drug administration (MDA) of azithromycin (tablets and syrup) and tetracycline eye ointment. Two districts, Koundara and Mali, did not receive MDA, as the original surveys were considered outdated and potentially unreliable. Instead, they underwent re-survey in 2017 and 2018 using a standard trachoma prevelance survey methodology,22 both returning TF and TT prevalences below elimination threshold.23 Impact surveys were carried out in the 16 MDA-treated health districts between 2017–2019, followed by surveillance surveys in 2020. The results of the latest surveys demonstrated a TF prevalence in 1–9-year-olds under 5% in all districts, however TT prevalence was above the WHO elimination threshold in eight districts.Since 2013, antibiotic interventions have been implemented following WHO recommendations.19 TT-only surveys are a tool that countries can use to support validation of elimination of TT as a public health problem.24 TT-only surveys focus specifically on adults aged≥15 years and have larger sample sizes than normal trachoma prevalence surveys, which recruit fewer adults and therefore estimate TT prevalence with less precision. This paper is one of the first reporting on TT-specific re-surveying in order to determine TT intervention needs and provide data for trachoma elimination dossier preparation for submission to WHO.25Given the long duration since baseline surveys, it is likely that the original TT prevalence estimates do not reflect the current status. Furthermore, since baseline TF prevalence was below elimination threshold, further surveys collecting data on TF prevalence in 1–9-year-olds are unnecessary. For these reasons, TT-only surveys were recommended in four health districts  in which TF prevalence was<5% and TT prevalence was≥0.2% in baseline surveys in 2013–2014 to determine if additional TT interventions are recommended.26 including the use of face masks and disposable gloves, which were changed between each individual examination.Ethical approval for this study was provided by the Guinea Ministère de la Santé (033/CNERS/21) and the London School of Hygiene & Tropical Medicine, UK (16105). Every participant was provided with a clear explanation of the purpose and use of their data. Informed consent was required from individuals surveyed and was recorded in Android smartphones. Barrier measures for the prevention of SARS-CoV-2 transmission were implemented from 2020 onwards as per the standard operating procedures developed by Guinea according to WHO guidance,27 Therefore, two health districts with populations larger than 250,000, Labé and Beyla, were each split into two separate EUs  is defined as a local health administrative unit with a population of 100,000–250,000 people.28 In brief, the sample size was determined by the single population proportion for precision formula that includes the expected prevalence (0.2%), standard deviation corresponding to 95% confidence intervals (1.96), the design effect size (1.47) and the margin of error (0.2%), resulting in a sample size of 2,818 people to examine. This value is then multiplied by the estimated non-response inflation factor (1.2), resulting in a final sample size of 3,382 individuals to enumerate per EU. In Guinea, the number of residents aged≥15 years per household is estimated to be 4,29 resulting in 846 households required in total. A survey team can visit~30 households per cluster per day, meaning a total of 29 clusters per EU were required to meet the sample size.All participants were aged≥15 years, had lived for at least a month in the selected village, and belonged to a selected household. Participant selection methods and number of participants required for TT-only surveys have been standardized, as described elsewhere.www.tropicaldata.org).30 In brief, graders were given two days of training on examination techniques including proper handwashing, everting the eyelids, looking for surgical scars or epilation, and identifying the clinical signs of trichiasis . Graders were tested using Objective Structured Clinical Evaluation techniques to quality-assure clinical evaluation. Recorders were trained to enter data into Android smartphones using the Tropical Data software. All consenting adults were seen by a certified grader. The clinical examination was performed with binocular magnifying loupes (2.5×) and under adequate lighting . Each eyelid was examined separately. All clinicals signs were graded according to the WHO simplified trachoma grading system.32Trachoma graders and recorders were trained and certified following the methods developed by Tropical Data  were then asked questions about health management for the trichiasis; an individual was defined as “known to the health system” if the participant answered yes to the question of having had or been offered surgery or epilation, or if the participant answered no to the question of having had surgery, but the grader saw a surgical scar and subsequent discussion confirmed that there was a history of eyelid surgery.33The graders first looked for the presence of eyelashes touching the eyeball or signs of eyelash removal from the upper eyelids (TT) and on the lower eyelids (lower eyelid trichiasis).Participants with TT were offered free TT surgery, the scheduling of which was done in agreement between the participant and the village and health authorities. Participants with TF were treated with 1% tetracycline ophthalmic ointment (to be applied twice daily for 6 weeks) or a single oral azithromycin dose (1 g for adults).32 TT “unknown to the health system” indicates TT for which the participant had not received an offer of epilation or surgery from a healthcare worker, or in a participant who responded “I don’t know” to the questions asked regarding TT management and did not have any evidence of a surgical scar.27TT is defined as at least one eyelash from the upper eyelid touching the eyeball, or evidence of recent epilation of in-turned eyelashes from the upper eyelid.22Data were recorded in the Tropical Data app using Android smartphones. Data were uploaded to a secure central server, enabling the dedicated Tropical Data data team to clean and validate the data in partnership with the Programme National de Lutte contre les Maladies Tropicales Négligées (PNLMTN). Standardized data analyses using automated algorithms were conducted to calculate the age- and gender-adjusted prevalence of TT.34 due to the low numbers of TT cases.Further data association analyses were performed to determine the relationship between age, gender and the prevalence of all TT known or unknown to the health system using the glmer R package . Models including household, cluster and EU as random effects were tested for best fit using maximum likelihood tests. Odds ratio (OR) values for the likelihood of TT prevalence were determined for each age bracket and gender. Individual age values were grouped into age brackets of 15–45, 46–75, and 75+ years; these age brackets were chosen as the models did not run with smaller age bandsA total of 22,514 people were enumerated, with 22,476 (99.8%) examined from January to February 2021. A mean of 58% of participants per EU were women. In each EU, 29 clusters and~883 households were examined .There were 48 cases of TT across all six EUs, with one EU, Dalaba, in Mamou Region, having zero cases . A largep < .001; gender: p < .05). The OR for gender indicated over double the odds of TT in women compared to men , and individuals aged 46–75 or >75 years had greater odds of having TT relative to those aged 15–45 years .Models testing the three random effects of household, cluster and EU were unable to converge with the inclusion of household and EU; therefore, only cluster was used. P-values indicated that both age and gender were strongly associated with TT prevalence  , neither of these districts has had surveys reporting TT as a public health problem.er 2021) .37 in that women were twice as likely to have TT compared to men, and people aged>46 years were significantly more likely to have TT relative to those aged 15–45 years. The higher prevalence of TT in women is thought to be largely due to them spending more time in close proximity with young children, the main reservoir of ocular C. trachomatis infection.37 Increasing age is an expected association of TT due to the pathogenesis of trachoma: it is the repeated infections over time which lead to the development of conjunctival scarring, and in turn to TT. Therefore, older people are more likely to have experienced a sufficient number of C. trachomatis infections to develop TT, relative to younger people.13The association analysis results concur with the outcomes of many other studies,In five of the surveyed EUs, TT management should be integrated within the routine clinical services. To ensure that Guinea reaches elimination, it is recommended that Beyla 2 undergoes TT-specific public health interventions. The current estimated TT backlog in Beyla district is 391. Thirty-one trichiasis cases were managed in 2015 (prior to the TT-only survey), however there were no cases managed after this point. If a public health approach to TT is implemented, Beyla 2 will require documentation of full geographic coverage of case finding and outreach services, or a subsequent TT-only survey to demonstrate that elimination has been achieved.38 which can be a barrier to documenting trachoma elimination. Many of the standard surveys are supported by external partners, and countries may struggle to undertake TT-only surveys without additional funding. The benefit of these surveys is clear however, when there is a likelihood that old baseline data is not an accurate reflection of current TT prevalence. Geospatial analysis may be a useful approach for providing more precise TT prevalence estimates either through more efficient survey design or generating estimates using existing survey data, if appropriate.39The cost of running TT-only surveys is estimated at $9,707 per EU,Guinea is currently working on a transition plan to ensure the availability of incident TT case management in formerly endemic districts in-line with WHO elimination criteria. At the present time, there are seven EUs in Guinea that have a prevalence of TT unknown to the health system≥0.2% requiring interventions in order to achieve elimination of TT as a public health problem. Completion of these interventions and demonstration of below elimination threshold prevalence should provide the final data needed for dossier submission to WHO for trachoma elimination validation.


As a common central nervous system infection in newborns, neonatal bacterial meningitis (NBM) can seriously affect their health and growth. However, although metagenomic approaches are being applied in clinical diagnostic practice, there are some limitations for whole metagenome sequencing and amplicon sequencing in handling low microbial biomass samples. Through a newly developed ultra-sensitive metagenomic sequencing method named 2bRAD-M, we investigated the microbial signatures of central nervous system infections in neonates admitted to the neonatal intensive care unit. Particularly, we recruited a total of 23 neonates suspected of having NBM and collected their blood, cerebrospinal fluid, and skin samples for 2bRAD-M sequencing. Then we developed a novel decontamination method  for 2bRAD-M by which we efficiently denoised the sequencing data and found some potential biomarkers that have significantly different relative abundance between 12 patients that were diagnosed as NBM and 11 Non-NBM based on their cerebrospinal fluid (CSF) examination results. Specifically, we discovered 11 and 8 potential biomarkers for NBM in blood and CSF separately and further identified 16 and 35 microbial species that highly correlated with the physiological indicators in blood and CSF. Our study not only provide microbiological evidence to aid in the diagnosis of NBM but also demonstrated the application of an ultra-sensitive metagenomic sequencing method in pathogenesis study. Neonatal bacterial meningitis (NBM) is a common central nervous system infection that can seriously affect the health and growth of infants . Even thThe importance of early diagnosis and antimicrobial therapy for NBM infants cannot be overstated, but the challenge is still great . For exaAs advanced laboratory assays, metagenomic approaches  are now being used clinically to improve the diagnostic accuracy of pathogens and avoid inappropriate therapies . CompareMethods) from the neonatal intensive care unit (NICU) and leveraging their microbial signatures from multiple body sites decoded by the cutting-edge 2bRAD-M sequencing, our study aims to identify the underlying associative agents and risk factors associated with central nervous system (CNS) infections in neonates, as well as to provide a basis for the early diagnosis and treatment of neonatal bacterial meningitis in the NICU.In previous studies, little effort has been made to study the microbial signatures of CSF in NBM patients, and systematic sampling  has rarely been conducted. By recruiting 23 patients suspected of having NBM  and blood (a median of 7 ml) of the children for regular clinical examination, and the remaining CSF and blood after examination are sent to Qingdao oebiotech for 2bRAD-M sequencing. As for the environment samples, we selected needles from the same batch and model as those used in the operation, opened them at the start of the puncture operation, and placed them within the operating tray to expose them to the same environment. Upon completion of the puncture, we gathered the needles used to collect environmental samples with sterile forceps using sterile tubes. All samples were stored in a -80 °C freezer before sequencing.The diagnosis of neonatal bacterial meningitis is difficult due to the inconspicuous clinical manifestations, thus the diagnosis mainly depends on the vigilance of the disease. We recruited children with suspected central nervous system (CNS) infectious diseases using the following criteria: 1. pathogenic bacteria detected in blood cultures; 2. clinical and laboratory tests both suggest Central nervous system infection; 3. the child did not improve after receiving antibiotic therapy. For the 23 recruited children, we first collect skin samples by using a sterile swab to rub skin surface within 25cmEscherichia coli and non-lactose fermenting Streptococcus agalactiae. This further highlights the issue of a low positivity rate associated with traditional culturing methods.Our study followed the diagnostic criteria for NBM as outlined in the review “Bacterial meningitis in the neonate: pathogeny, diagnosis and diagnosis” published in the Chinese Journal of Perinatal Medicine, 2016. Specifically, the inclusion criteria were indicated by at least two of the following abnormal clinical observations in CSF: (1) increased white blood cell count (>21x109/L), (2) elevated total protein , (3) decreased glucose , or cerebrospinal fluid bacteria culture positive will be directly diagnosed. After blood and CSF examination, patients with relevant indicators meeting the diagnostic criteria for bacterial meningitis were included in NBM group. Notably, all the enrolled children were patients with elevated blood infection indexes, and clinical manifestations such as fever and poor feeding. These children are at risk for central nervous system infections. The Non-NBM children’s finally diagnosis was sepsis in 6 cases (54.5%), intrauterine infection in 2 cases (18.2%), and pneumonia in 2 cases (18.2%). Moreover, in the NBM children, positive culture results were found in only two cases, which were In this study, cerebrospinal fluid and blood samples were collected from children after routine clinical examination, which did not increase the burden of additional cerebrospinal fluid and blood collection. This study was approved by the Ethics Committee of Qingdao Women and Children’s Hospital (QFELL-YJ-2022-15). A written statement that formal consent was obtained from the parent/guardian.The TIANamp Micro DNA Kit (Tiangen) was used to extract the genomic DNA. 2btags libraries were prepared according to previous studies . First, https://github.com/sunzhengCDNM/MAP2B), was used for taxonomic profiling  and visualized through principal coordinate analysis (PCoA). The Wilcoxon test was used to determine significance, e.g., the Wilcoxon Rank-sum test was used to compare samples between the two groups using Parallel-Meta 3.5 .p-value < 0.05 and |r| > 0.6 were considered statistically significant. Data visualization in this study was performed using R, for example, the “ggplot2”, “ggpubr” package were used for boxplot, scatter plot, and line chart, and the “pheatmap” was employed for the heatmaps.Here, we used MaAsLin2 (Microbiome Multivariable Associations with Linear Models) to identify multivariable associations between the microbiome and physical indexes . MaAsLinTo construct the Random Forest Model, the top-ranking NBM-discriminatory taxa that led to reasonably good fit were identified based on the “rfcv” function in the randomForest package . Random To investigate the prevalence and characteristics of CNS infections in neonates admitted to the NICU of Qingdao Women and Children’s Hospital. A total of 23 neonates were enrolled in the study between January 1, 2021 and March 1, 2022, and suspected of having CNS infectious diseases. Based on their cerebrospinal fluid (CSF) examination results, the patients were divided into two groups: those with neonatal bacterial meningitis  and those without CNS infection .Our study collected a range of clinical data in addition to the diagnosis. This included demographic information such as gestation days, neonates’ weight, gender, and age (days after birth), as well as other clinical details such as antibiotic and probiotic usage and feeding formula . Then, we compared routine physiological indicators of the neonates, such as birth weight, Premature rupture of membranes (PROM), and antibiotic usage (if any), and found no differences . Moreover, we further compared the biochemical test indicators in blood and CSF between the two groups and identified no significant differences in blood regarding the White Blood Cell (WBC), Hemoglobin (Hb), Platelet count (PLT), Neutrophil proportion (NEUT%), Monocyte proportion (MONO%), Lymphocyte proportion (LYM%), C-reactive protein (CRP), Procalcitonin (PCT), Albumin, Lactate dehydrogenase (LDH), and neither the level of Pandy and Glucose in CSF (p > 0.05). As previously mentioned, the CSF WBC and CSF total protein levels are significantly higher in the NBM group than non-NBM group .The mothers of the recruited infants are ethnically identical (Asian). Comparison of the mothers’ age, gestational age and delivery mode between the NBM and non-NBM revealed no significant differences  and negative controls in sequencing Figure 1Although the source tracking results revealed the existence of blood and CSF-specific taxa, there is an obvious overlapped proportion between blood/CSF and negative controls, which may impact downstream analysis e.g., leading to false positives in differential analysis. We then sought to deplete such noise information from blood and CSF samples by a novel decontamination method using negative controls  to removp< 0.05); (2) However, for CSF samples, an opposite comparison results were found, with the NBM group having lower alpha diversity than the non-NBM group ; (3) And there were no significant differences in alpha diversity between the NBM and non-NBM group in skin samples (p > 0.05). This indicates that NBM patients are characterized with higher microbial alpha diversity in CSF but lower microbial alpha diversity in the blood compared to relatively healthy neonates.Previous studies have shown that higher alpha diversity in the microbiome is associated with healthy status of host . To invep = 0.001; Conventional decontamination method: F = 6.833, p = 0.001; RLD: F = 7.635, p = 0.001). However, there was no obvious separation in the taxonomic structure of NBM and non-NBM groups in blood, CSF, and skin ;. In addition, here we see RLD outperformed the conventional decontamination method in differentiating microbiota from different body sites. All the above suggest that the decontamination method used in this study can effectively correct beta diversity analysis results without affecting alpha diversity analysis.As for the beta diversity, we found the discriminating power of the microbiota for NBM is enlarged after decontamination, e.g., the separation trend of microorganisms from different body sites was more significant  models to diagnose NBM from non-NBM neonates. The robustness of the RF models was evaluated by the Area Under the Receiver Operating Characteristic Curve (AUC). We then found that, RF models based on raw data  In CSF  There were no significant differences in skin samples between the NBM and non-NBM groups.After decontamination, we next sought to explore the microbial biomarkers that can distinguish NBM from non-NBM samples. To perform the differential analysis and rule out the influence from the confounders , we employed MaAsLin2 to identify the biomarkers for NBM. We found that: (1) In blood . In CSF samples . In summary, we identified 11 and 8 potential biomarkers for NBM in blood and CSF separately and discovered 16 and 35 microbial species that highly correlated with the physiological indicators in blood and CSF.In order to further investigate the correlation between differential species and physiological indicators, a spearman correlation analysis was conducted. We found that in blood samples . Some of them have been reported as pathogenic bacteria, e.g., P. putida can cause various infections in newborns (Comamonas testosterone (previously known as Pseudomonas testosterone) is a common pathogen in its genus. However, many of these species, including Anoxybacillus rupiensis, Brevundimonas vesicularis, Pseudonocardia ammonioxydans, and Kocuria palustris have rarely been reported as pathogens and are commonly found in soil, rivers, hot springs, and marine organisms  with our method (RLD), and we found that both methods performed better in determining the microbiome from different body sites and disease status, with RLD slightly outperforming the conventional method. In addition, there are currently many useful tools for eliminating contamination introduced in the wet lab using negative and positive controls . HoweverThere are still some limitations regarding the sample size. Thus, in future work, we plan to validate the identified disease-associated biomarkers through a larger cohort with qPCR and digital PCR, and incorporate metabolomic data to better understand the pathogenesis of NBM. In summary, our study identified NBM biomarkers from multiple body sites through the 2bRAD-M sequencing method and an innovative decontamination method, pioneering in providing microbial signatures for the study of the occurrence, prevention, and diagnosis of NBM.https://ngdc.cncb.ac.cn/search/?dbId=gsa&q=CRA009684, CRA009684.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The requirement of ethical approval was waived by the Ethics Committee of Qingdao Women and Children’s Hospital for the studies involving humans because We did not waive the ethical approval. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin because We filled out a written informed consent form. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.The authors confirm contribution to the paper as follows: study conception and design: ZS and XL. data collection: YH. Analysis and interpretation of results: MZ and JL. Draft manuscript preparation: YH, ZS, and MZ. All authors contributed to the article and approved the submitted version.