The independent risk factors identified were then incorporated in a nomogram model. The predictive accuracy and discriminative ability of the nomogram were evaluated by receiver operating characteristic curve (ROC) and calibration curve. LN metastasis occurred in 59 (20.7%) EGC patients. And most of these patients were submucosal cancers (48/59). Chi-square test indicated lymphovascular emboli, carbohydrate antigen 19-9 (CA19-9), ulcer, tumor size, tumor infiltration and histological grade were the risk factors, and multivariate logistics analyses confirmed all these six factors were independent risk factors of LN metastasis, which were selected to construct the nomogram. The nomogram proved well calibrated and had good discriminative ability . The proposed nomogram could result in more-accurate risk prediction for EGC patients.Lymph node (LN) metastasis is known as one of the most important prognostic factors for early gastric cancer (EGC) patients. Patients without LNM normally have better prognosis. However, there is no evaluation criteria to accurately assess the possibility of LN metastasis. Therefore, this study aims to establish an effective nomogram for prognosis prediction. In this study, 285 EGC patients from January 2010 to December 2015 were enrolled. Pearson’s Chi-Square ( Early gastric cancer (EGC) is defined as invasive gastric cancer that invades no more deeper than the submucosa, regardless of lymph node (LN) metastasis4. An overall survival rate (OS) in EGC was considered to be greater than 90% after standardized D2 lymphadenectomy treatment5, with LN metastasis being the most significant prognostic factor7. However, just about 20% of patients exhibited LN metastasis, most of the patients received excessive surgical treatment8.Gastric Cancer (GC) is known as one of the most frequently diagnosed cancer and leading cause of cancer deaths in China10. ER was composed of endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD), with a comparable long-term outcome to surgery11. However, ER can’t supply treatment of lymph node dissection, so it’s essential to evaluate the status of LN metastasis before and after surgery. According to the Japanese guidelines for ESD and EMR for EGC12, the absolute indication of ESD/EMR is as follows: differentiated-type adenocarcinoma without ulcerative findings, of which the depth of invasion is clinically diagnosed as T1a and the diameter is ≤ 20 mm. Only the patients who fulfill the absolute indication are recommended for ER treatment. However, because the absolute indication is excessively strict, the indication for ESD was expanded for the patients with no or lower risks of lymph node metastasis13. Therefore, a quantitative prediction model of LN metastasis based on individual information is urgently needed, which is crucial to weigh the benefits and risks of treatment.Nowadays, endoscopic resection (ER) has been considered to be an alternative option besides surgery, which could improve the quality of life for patients16. However, there are few reports on nomogram of LN metastasis in patients with EGC. In the present study, we aim to establish a nomogram for predicting lymph node metastasis in EGC patients.A nomogram, also called a nomograph, is a graphical calculating tool, which has been developed in most types of cancer at present285 EGC patients in total who underwent surgical treatment in Sun Yat-sen University Cancer Center from January 2010 to December 2015 were selected into this retrospective study. The inclusion criteria were as follows: (i) diagnosed as early gastric cancer and (ii) underwent gastrectomy and D2 lymphadenectomy, (iii) achieved radical (R0) resection. The exclusion criteria were as follows: (i) distant metastasis, (ii) multiple cancers, (iii) stump cancer or recurrent cancer, (iv) accepted preoperative treatment, and (v) died in the perioperative period. In addition, those patients with indications of postoperative chemotherapy have accepted chemotherapy, either S-1 or XELOX regimen.Follow-up assessments including clinical and laboratory examinations, for the first 2 years were conducted every quarterly. During the 3rd to 5th years, assessments were conducted twice a year, and every year after that until death. The primary endpoint was 5-year OS. OS was calculated from the date of surgery until death or to the last follow-up contact, which was used as a measure of prognosis.17. The maximum diameter of the lesions was recorded as the tumor size. The surgery type includes distal, proximal and total resection. The tumor location was separated into three parts . Tumor histological grade was divided into two groups: the undifferentiated group, which includes undifferentiated and poorly adenocarcinomas, and the differentiated group, which includes moderately and well differentiated adenocarcinomas18.We further collected the clinic-pathological data of all included patients. The clinical information included age, gender, surgery type, carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9) and carbohydrate antigen 72-4 (CA72-4) before surgery. The pathological information included depth of tumor infiltration, tumor size, tumor location, Lauren type, ulcer, lymphovascular emboli and histological grade. The depth was measured at the deepest point of penetrated carcinoma cells and tumor size was defined as the maximum diameterχ2) test (including continuity correction when appropriate). The independent risk factors of LN metastasis were assessed by multivariate logistic regression analyses. Moreover, the OS were calculated by the Kaplan–Meier method and compared using the log-rank test. Multivariate analyses were used Cox proportional hazards regression models. The primary end point was 5-year OS.Statistical analyses to identify risk factors were conducted by the SPSS (version 26.0). The continuous variables were shown as the mean ± standard deviation (SD). The categorical variables were analyzed by Pearson’s Chi-Square (rms19 in R studio (version 1.3.10), based on the results of the multivariate logistic regression model. The performance of the nomogram was assessed by calibration curve as indicator of internal calibration, and ROC as a measure of discriminative ability20. Meanwhile, the Hosmer–Lemeshow test was used to evaluate goodness of fit of the nomogram.Then, a nomogram was formulated using the package of P values (two sided) < 0.05 were considered statistical significance in all statistical analyses.All procedures performed in studies involving human participants were in accordance with the ethical standards of our institutional research committee and with the 1964 Helsinki declaration and its later amendments. This article does not contain any studies with animals performed by any of the authors. Due to the retrospective nature of the study, this research was approved by the Ethics Committee of the Sun Yat-sen University Cancer Center, and inform consent was granted a waiver.P < 0.05). Patients with lymphovascular emboli, larger size > 2 cm, or ulcer have a higher possibility of LN metastasis . Tumors with submucosal invasion was associated with higher LNM metastasis than those with intra-mucosa invasion (p = 0.001). Undifferentiated carcinomas were related to lower lymph node metastases (p = 0.005). Patients with CA19-9 ≤ 35U/ml have a lower probability of LN metastasis than those with CA19-9 > 35U/ml (p = 0.028). But LN metastasis was not correlated with gender, age, CA72-4, CEA, tumor location, surgery type, or Lauren type  of those 285 cases. The average number of retrieved lymph nodes was 32.6 ± 14.5(range 2–98). The median age of those EGC patients was 57 ± 11.5 years. Non-parametric test indicated that LN metastasis was associated with lymphovascular emboli, CA19-9, ulcer, tumor size, tumor infiltration and histological grade , CA19-9 , ulcer , tumor size , tumor infiltration  and histological grade  were the risk factors, indicating that all these six factors were independent risk factors of LN metastasis. Therefore, these six clinical-pathological factors were selected to construct a predictive nomogram for LN metastasis , indicating a good discriminative ability  mouths. In our study, the 1-year, 3-year and 5-year OS in EGC were 97.9%, 92.6% and 90.6%, respectively, which were calculated by Kaplan–Meier method. Additionally, the 5-year OS of those with LNM and without LNM were 93.4% and 79.5%. The primary end point of this study is 5-year OS. Kaplan–Meier method (Log-rank test) indicated that OS was significantly associated with age (p = 0.003), CEA (p < 0.001), CA19-9 (p = 0.033), CA72-4 (p = 0.009), LN metastasis (p < 0.001), lymphovascular emboli (p = 0.008), surgery type (p = 0.013), pN stage (p < 0.001) and TNM stage (p < 0.001). But it was no relationship with gender (p = 0.177), tumor size (p = 0.959), tumor location (0.246), tumor infiltration (p = 0.324), histological grade (p = 0.168), ulcer (p = 0.302), or Lauren type (p = 0.556). Kaplan–Meier plots are shown in Fig. 23. The rate of LN metastasis in patients with EGC ranges from 5 to 22%, which means that approximately 70% to 80% of EGC patients undergo overtreatment with D2 lymphadenectomy25. The development of less invasive treatments, including endoscopic mucosa resection (EMR) and endoscopic submucosa dissection (ESD), have had an important impact on the treatment strategies revolution in the last few decades26. The EGC patients meeting suitable conditions (low probability of LN metastasis), were recommended to receive less invasive treatments. Therefore, we retrospectively analyzed 285 EGC patients based on clinical-pathological features to identify the risk factors for LN metastasis and construct a nomogram to guide treatment.Since long, gastrectomy with D2 lymphadenectomy was considered as the standard and optimal treatment for EGC patients27. And therefore, lymphovascular emboli is significantly associated with LN metastasis28. CA19-9 > 35U/ml has been reported as a reliable risk factor for predicting LN metastasis previously29. Larger size (≥ 20 mm), presence of ulcer, tumor submucosal infiltration and undifferentiated type were related to regional lymph node metastasis36. The results of our study were consistent with the studies mentioned above.In this retrospective study, the incidence rate of LN metastasis was 20.7% in EGC patients. The incidence rate of LN metastasis for the patients with one or more risk features, such as lymphovascular emboli, presence of ulcer, tumor size > 2 cm, submucosa infiltration, undifferentiated type, or CA19-9 > 35 U/ml, was higher than those patients without these clinical-pathological features. Multivariate analyses showed that these six features were independent risk factors for LN metastasis, and the presence of lymphovascular emboli was considered the most important factor. Previous studies have confirmed these features were independent risk factors. Lymphovascular emboli is the characteristic of lymphovascular invasion, which is considered a rate-limiting step in the lymph node metastatic processAnd then, we established a nomogram with these six variables related to LN metastasis, which results in more intuitive and accurate in predicting the risk of LN metastasis. The high AUC value (o.842) and calibration curve indicates a good discriminative ability and universal clinical applicability of our nomogram.37. For those EGC patients with low probability of LN metastasis, ESD or EMR was the optimal treatment38. However, there is still a lack of criteria to evaluate the probability of lymph node metastasis. Our study provides a helpful method to solve this problem. For example, a differentiated mucosal patient without lymphovascular or other features, has a low risk of LN metastasis (less than 5%), and is suitable for EMR. On the contrary, an undifferentiated submucosal patient with lymphovascular emboli, larger tumor size (> 2 cm) and ulcer, has a probability of more than 90% for LN metastasis regardless of other features. This patient is recommended to undergo gastrectomy with D2 lymphadenectomy instead of endoscopic treatment. Thus, we think this quantified and visual predictive model allows clinicians to make more objective decisions on treatment options based on the possibility of LN metastasis.LN metastasis has been well considered as one of the most important prognostic factors in both early gastric cancer and advanced gastric cancerThis study had some potential limitations. Firstly, the final selected sample size is small, so we consider improving the nomogram by expanding the sample size in the future. Secondly, this was a single-center retrospective study. In order to further improve the clinical application environment of the nomogram, additional external validation on different populations is required. Finally, P53, HER-2 or other genetic information was not included in the nomogram, which may provide more individualized evidence of our nomogram. In the future, we intend to incorporate more variables into the nomogram to improve its accuracy, including the information of imaging examination and molecular detection. In spite of the above limitations, we believe that our rosette map can be helpful for clinical work. A strength of our nomogram is that it was built from the variables which could be easily obtained before ESD or EMR treatment. It therefore means that this nomogram can be used to preliminarily determine the risk of lymph node metastasis of a patient before surgery, and hence to prescribe a preliminary treatment.In conclusion, we established a nomogram to help clinicians predict the risk of lymph node metastasis in the patients with early gastric cancer. This tool could help clinicians and patients quantify the potential incidence of lymph node metastasis. Surgery plus lymph node dissection is recommended for certain patients with a high risk of LN metastasis, and endoscopic dissection is suitable for those with a low risk. In the future, we will conduct further research to improve clinical application value of the nomogram.


Oxford unicompartmental knee arthroplasty (UKA) is widely used for treating patients with unicompartmental knee diseases. However, the factors affecting the outcomes of and patients’ satisfaction with Oxford UKA remain controversial. The aims of this study were to evaluate the clinical and radiological outcomes, including patient satisfaction, after Oxford UKA and identify the influencing factors related to patients’ satisfaction.We retrospectively analyzed the data of patients who underwent UKA in our medical institution from 2013 to 2018. Demographic information, clinical scores, patient satisfaction and imaging findings were recorded. The patients were followed up for at least 1 year. Multivariate Logistic regression analysis was performed to identify influencing factors related to clinical outcomes including patient satisfaction.P < 0.001; 113.0 ± 27.2 vs. 167.2 ± 23.9, P < 0.001), and the varus deformity was corrected . Six patients (7.5%) developed postoperative complications, and no case suffered from prosthesis loosening. The time since operation (TSO) and angle E were found to be predictors of KSS improvement . The postoperative KSS and angle E were found to be predictors of patient satisfaction .A total of 80 patients (involving 87 knees) were included. The Numerical Rating Scale (NRS) scores and Knee Society Scores (KSS) were significantly improved after operation as compared to preoperative scores (6.0 vs. 1.0, Oxford UKA can improve the NRS score and KSS and correct varus deformities. A shorter TSO and smaller angle E are indicators of a greater KSS improvement. A higher KSS and smaller angle E are indicative of higher patient satisfaction. Knee osteoarthritis (OA) is the most common cause of knee pain and dysfunction in elderly people. Due to population aging, the incidence of knee OA has been on a substantial rise. The incidence of knee OA is 28% in people over 45 years old  and is aCurrently, it is generally accepted that UKA is an alternative treatment for unicompartmental OA of the knee. More than 60 years have passed since Dr. McKeever designed the first unicompartmental knee prosthesis and performed the first UKA in the early 1950s . Since tIn recent years, the value of UKA has been increasingly recognized by orthopedic surgeons. The advantages of UKA over total knee arthroplasty (TKA) include less trauma, shorter operative time, less intraoperative bleeding, faster recovery and better proprioception of the patient , 6. In tWe retrospectively analyzed the data from patients who underwent UKA in our medical institution between November 2013 and December 2018.The inclusion criteria included: (1) UKA due to medial compartment knee OA or medial femoral condyle SONK, (2) preoperative imaging results indicative of a lesion that did not involve the lateral compartment, and (3) preoperative evaluation showing the anterior and posterior cruciate ligaments and the collateral ligaments were intact. The exclusion criteria were: (1) the presence of other inflammatory knee diseases, meniscus injury or traumatic arthritis, (2) incomplete medical records, and (3) a follow-up period less than 1 year.All patients received an Oxford unicompartmental knee prosthesis , and all the operations were performed by the same experienced orthopedic surgeon. The standard procedure for Oxford UKAs was done. Mixed analgesics were injected into the soft tissues around the knee before the incision was closed. Prophylactic antibiotics were administered 30 min before the operation. The drainage tubes were removed within 24 h after the operation. Routine anticoagulant and analgesic therapy was given. Knee flexion and extension exercises were started on the first day after the operation, and patients were instructed to practice partial weight-bearing walking with walking aids appropriate for their specific conditions.The time interval between the date of operation and January 2020 was defined as the time since operation (TSO). The longer the TSO, the more the time that elapsed since the operation and vice versa.Demographic data such as age, sex, BMI and operated side were recorded. The Numerical Rating Scale (NRS) score , 14 and A 6-level Likert scale  was usedX-rays were taken before the operation and at each follow-up. The positioning and alignment of the implants were evaluated according to the recommendations of the implant manufacturer (Zimmer Biomet) . The indt-test was used to compare the differences between the two groups for the continuous variables with normal distribution, and the Mann-Whitney U test was employed for those with non-normal distribution. The categorical data were compared by using the McNemar X2 test or Fisher’s exact test. The significant predictors emerging from the univariate analysis were selected, and a forward stepwise procedure in a multivariate logistic regression model was performed. The results of the regression analyses were presented as the odds ratios (ORs) with 95% CIs. Statistical analyses were conducted by using SPSS for Windows . Differences were considered to be statistically significant at P < 0.05.The Kolmogorov-Smirnov test was used to assess the normality of the data for continuous variables. Continuous variables with normal distribution were expressed as the means ± standard deviations (SDs) and 95% confidence intervals (CIs). The remaining variables were expressed as the medians and quartiles. The categorical variables were expressed as the sums and percentages. The 2 , with female patients being significantly more than male ones (P < 0.001). Thirty-seven patients had preoperative complications, including hypertension, diabetes, cardiovascular diseases, and hepatic and renal dysfunction. All patients were followed up for an average of 28.9 ± 15.0 months . Postoperative complications occurred in 6 patients (7.5%), including 4 cases (5%) of lower limb vein thrombosis, 1 case (1.3%) of surgical site infection, and 1 case (1.3%) of acute periprosthetic joint infection, which were treated with debridement, antibiotics and implant retention (DAIR). No prosthesis loosening was found at the end of the follow-up period. Both the NRS score and KSS improved after the operation (P < 0.001)  underwent UKA in our medical institution from November 2013 to December 2018. Eighty of them (87 knees), including 22 males (25 knees) and 58 females (62 knees) were included and17 patients were excluded because they were lost to follow-up. On average, 80 patients were aged 64.5 ± 7.4 years  and had a BMI of 26.1 ± 3.9 kg/m04 knees P = 0.000). The two groups were compared in terms of 16 variables, including age, sex, BMI, operated side, SONK, preoperative complications, preoperative NRS, preoperative FTA, operative duration, angle A, angle B, angle E, angle F, prosthesis overhang, prosthesis loosening and TSO. The preoperative NRS, angle E and TSO were found to be significantly different between the two groups (Table P = 0.024)] and TSO [OR = 1.044 ] were found to be predictors of KSS improvement (Table The 87 cases treated by UKA were ranked in terms of postoperative improvement in KSS and divided into a less-KSS-improvement group 43 cases) and a more-KSS-improvement group (44 cases). The average KSS improvement was 28.7 ± 20.2  in the less-KSS-improvement group and 79.2 ± 20.1  in the more-KSS-improvement group. There were significant differences in the KSS improvement between the two groups ( cases anP = 0.001)] and angle E [OR = 7.723 ] were predictors of patient satisfaction  (Table P = 0.017) 131313.At the last follow-up, the level of satisfaction among the 87 cases was as follows: patient satisfaction was excellent in 23 cases 26.4%), very good in 34 (39.1%) cases, good in 16 (18.4%) cases, fair in 10 11.5%) cases, poor in 3 (3.4%) cases and terrible in 1 (1.1%) case. There were 73 cases (83.9%) in the satisfaction group and 14 cases (16.1%) in the dissatisfaction group , indicating that UKA can correct varus deformities to a certain extent. However, excessive correction of a varus deformity may lead to an excessive load on the lateral compartment. It can also lead to accelerated deterioration of OA in the lateral compartment, which is one of the main reasons for UKA revision [Due to wear of the articular cartilage of the medial compartment, the medial joint space of the knee becomes narrow, and the FTA increases in patients with medial knee OA. In this study, the preoperative FTA and the postoperative FTA were significantly different . Previous studies [In our study, the positioning of the implants was evaluated according to the recommendations of the implant manufacturer . Our stu studies  have poi studies . Chatell studies  pointed P = 0.001; P = 0.032). OR showed that for every 10-point reduction in the postoperative KSS, the risk of patient dissatisfaction increased by 2.59 times, and for cases with excessive deviation in angle E, the risk of patient dissatisfaction rose by 6.723 times. Therefore, it is reasonable to believe that the correct prosthesis position can improve the postoperative KSS and thus improve patient satisfaction.Previous studies reported that the satisfaction rate of UKA ranged from 73 to 96.4% –28, and This study had some limitations. First, the total number of cases included in this study was small (87 cases), resulting in a small sample size in each group after grouping. Second, all the data in this study were from a single institution, and all UKAs were performed by the same surgeon and his assistants. However, our institution is a regional medical center with a large operation volume, and the results can be, to certain degree, generalized to other population. A multicenter study is warranted to increase the generality of the conclusions. Third, some of the patients in this study were followed up for a short time, which may affect assessment of the long-term efficacy, but all patients completed a follow-up after at least 1 year (12–72 months), and the average follow-up time was 28.9 ± 15.0 months. Finally, all patients in this study were treated with an Oxford unicompartmental knee prosthesis, which has a typical mobile-bearing design. Therefore, the conclusions of this study might not applicable to other types of prostheses. Additional studies are needed to compare the differences between the fixed- and mobile-bearing designs.The Oxford UKA can significantly improve both the NRS score and KSS, and it can also correct varus deformities. A shorter TSO and smaller angle E are predictors of greater KSS improvement. In addition, a higher KSS and smaller angle E are predictors of higher patient satisfaction.


Pesticides are a safety issue globally and cause serious concerns for the environment, wildlife and human health. The handheld detection of four pesticide residues widely used in Basmati rice production using surface-enhanced Raman spectroscopy (SERS) is reported. Different SERS substrates were synthesised and their plasmonic and Raman scattering properties evaluated. Using this approach, detection limits for pesticide residues were achieved within the range of 5 ppb-75 ppb, in solvent. Various extraction techniques were assessed to recover pesticide residues from spiked Basmati rice. Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERs) acetate extraction was applied and characteristic spectral data for each pesticide was obtained from the spiked matrix and analysed using handheld-SERS. This approach allowed detection limits within the matrix conditions to be markedly improved, due to the rapid aggregation of nanogold caused by the extraction medium. Thus, detection limits for three out of four pesticides were detectable below the Maximum Residue Limits (MRLs) of 10 ppb in Basmati rice. Furthermore, the multiplexing performance of handheld-SERS was assessed in solvent and matrix conditions. This study highlights the great potential of handheld-SERS for the rapid on-site detection of pesticide residues in rice and other commodities. Oryza sativa, “Queen of Grains”) is a staple food for more than 60% of the world’s population and its supply must more than double by 2050 to adequately feed growing populations2. Challenges for global rice production include agricultural water scarcity, urbanisation of farming land and climate change3. In addition, the COVID-19 pandemic has added a fourth dimension by disrupting the global food supply chain. Producers now face immense pressures from labour shortages and crop losses because of the pandemic. Rice is also continually under threat from pests  which can consume between 5% and 20% of the crop4 and fungal diseases; rice blast (Magnaporthe grisea) and sheath blight (Rhizoctonia solani), which cause major production constraints in Asia5. Under favourable conditions fungal blight can cause up to a 50% decrease in rice crop yield annually around the world6.Rice  and contaminate the food chain, causing serious harm to wildlife and human health. Extremely toxic, carcinogenic and mutagenic residues can be adsorbed into the body through the skin, eyes, respiratory system and digestive system. Studies have shown a strong correlation between the accumulation of pesticide residues in the body and damage to the reproductive, nervous and immune systems, development of liver and cerebrovascular diseases, and cancers of the liver, gall bladder and breast9.Modern agricultural practices rely on synthetic chemicals, pesticides, fungicides and insecticides to improve rice yield by 20–50%10 high levels of acephate (organophosphate), carbendazim (benzimidazole fungicide), thiamethoxam (neonicotinoid) and tricyclazole (fungicide) were frequently found above the Maximum Residue Limits (MRLs) in Basmati rice during the period between 2011 and 2020 11, which are highly accurate but can also require complex extractions, long analysis times and expensive instrumentation12. Therefore, it is difficult for these techniques to meet the requirements for on-site analysis thus, there is a clear need to develop rapid, cost-effective and portable techniques as screening tools. Surface-Enhanced Raman Spectroscopy (SERS) is a surface-sensitive technique in which inelastic light scattering of molecules is greatly enhanced , through the attachment or adsorption of target analytes close to, onto the surface or in between nano-roughened metallic surfaces or metallic nanoparticles (NPs) 13. SERS has been successfully employed previously for the detection of food and environmental contaminants such as, mycotoxins14, antibiotic residues15, mercury16 and tropane alkaloids17.Conventional methods for pesticides in rice rely on liquid chromatography (LC) or gas chromatography (GC)-coupled with mass spectrometry (MS)et al. detected carbendazim residues in Oolong tea using gold nanoparticles (AuNPs) as substrates18. Liu et al. used a shell thickness-dependent Raman enhancement technique using silver-coated AuNPs (Au@Ag NPs), for the rapid detection of pesticides on various fruit peels19. Fu et al. developed a gold nanorod (GNR) array as a SERS substrate for the detection of thiabendazole in apple20. Li et al. used a SERS imaging technique to determine trace levels of thiophanate and its metabolite carbendazim in red bell pepper21. Wang et al. developed a gecko-inspired nanotentacle SERS (G-SERS) platform for the simultaneous detection of three pesticides in fruit and vegetables22. Whilst vital works they all focus on developing lab-based techniques with Raman spectrometer systems or microscopes. The on-site analysis of pesticide residues currently relies on enzymatic or colorimetric screening techniques which lack sensitivity, reliability and multiplexing capabilities. Consequently, producers rely on costly and lengthy confirmatory techniques, resulting in slow product turnaround.At present, developments have been made to successfully detect pesticide residues within various food matrices using SERS-based techniques. Chen 23 original Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERs) extraction24 and buffered QuEChERs25 (to help with the recovery of problematic acid- and base-sensitive pesticides). QuEChERs was originally designed for extraction and cleanup of pesticide residues from matrices with a high moisture content (>75%) and low-fat content26. Rice is considered a complex food matrix containing fatty acids, amino acids, dietary fibre, vitamins and other essential micronutrients27. Consequently, numerous non-targeted compounds are likely to be removed during solvent extractions such as, QuEChERs. Some works have successfully adapted the technique for grains and rice, however analysis mainly consists of LC-MS and GC-MS28, with very few focusing on SERS. Herein, several extraction procedures were examined to determine the most suitable method for pesticide recovery from Basmati rice when exploiting handheld-SERS for analysis.Many extraction procedures have been previously developed for the analysis of pesticide residues in fruit and vegetables including swab techniquesOverall, this study presents vital improvements to the research area by developing a handheld-SERS-based platform to detect acephate, carbendazim, thiamethoxam and tricyclazole in Basmati rice using AuNP substrates. The novelty of the work lies in the combination of a handheld AuNP SERS-based technique with a QuEChERs extraction (more commonly applied to spectrometry techniques). As a result, this combination could facilitate SERS enhancement and improve sensitivities in matrix conditions. With future developments the technique could easily be adopted in-field thus providing a rapid, affordable and sensitive SERS-based platform. Overall, there is the potential to improve on-site applications for pesticide analysis in rice and with minor adaptions could also be applied to monitor other toxic contaminants , a range of food matrices  or environmental samples .−1, 1270 to 1271 cm−1, 1415 to 1416 cm−1 and 590 to 592 cm−1 for acephate (I), carbendazim (II), thiamethoxam (III) and tricyclazole (IV), respectively. In addition, some vibrational bands disappeared from the spectrum obtained using the handheld device however, slight changes are to be expected due to its reduced size and power output. A detailed description of each Raman band is highlighted in Supplementary Table Raman spectral data was acquired on solid pesticide powder using a benchtop microscope and a handheld instrument, to characterise and ascertain their unique fingerprint spectra and main vibrational bands. Initially, both Raman techniques were used to determine the performance and accuracy of the handheld device, in comparison to the conventional Raman microscope. As expected, the spectral data from the Raman microscope clearly showed the main vibrational bands for each of the pesticides analysed Fig. . The resmax) at approx. 520 nm are produced using the Turkevich method29 whilst, the Frens method30 modifies the stoichiometric ratio between sodium citrate and Au, allowing a wider range of diameters to be produced (~10–100 nm). During synthesis the surface of colloidal AuNPs is passivated via chemisorption and/or physisorption of stabilising citrate ions31. The citrate ion shell remains loose on the surface of the particles and can be easily displaced by interactions such as gold–sulfur (Au–S) and gold–nitrogen (Au–N) bonding32. Due to the distance-dependence of electromagnetic field SERS enhancements , substrates were synthesised using the Frens method to optimise the performance of handheld-SERS. Figure max shifting from 519 nm (black line) to 587 nm (pink line), when the concentration of sodium citrate is decreased from 0.1% to 0.01%, respectively. Additionally, the results obtained through Dynamic Light Scattering (DLS) could confirm an increase in particle size from 25.4 nm to 81.8 nm by decreasing the concentration of sodium citrate  the main vibrational bands could clearly be observed at the same concentration  the SERS intensity slows, due to complete AuNP aggregation and saturation of ‘hot spots’ by the adsorbed dye  for Au substrate (λmax = 519 nm) using handheld-SERS could be calculated as 8.04 × 103, using the main characteristic peak of R6G at 1362 cm−1, in the absence and presence of AuNPs , onto the surface of the AuNPs prior to analysis. The main vibrational bands of RG6 were observed at 1312 cmm−1 Fig.  and are max at 528 nm displayed the greatest increase in SERS intensity . Experiment parameters were optimised including, Au substrate concentration, the ratio of pesticide to Au, the reagent (and volume) required for ‘hot spot’ formation and the required incubation time  at 100 ppm was prepared by dissolving in ethanol:dH36. Furthermore, the addition of HCl lowers the pH of the surrounding medium, reducing the repulsive forces further. High concentrations of HCl can promote a greater degree of particle–particle interactions, leading to ‘fast’ aggregation. In this case, particles stick together during their first collision and form large aggregates rapidly, which can provide a strong increase in SERS intensity37. This degree of aggregation could be observed by the extreme broadening of bands after the addition of TRI , the inter-particle distance is reduced and the peak absorbance redshifts, resulting in a broadening of the absorption spectra (~650 nm). This can be explained by the displacement of citrate ions from the surface, shielding the repulsive forces between particlesmax = 528 nm) and HCl (2 M) could provide sufficient electromagnetic field enhancements and ‘hot spot’ formation, allowing the detection of all four pesticide residues. The major SERS bands for all pesticides can also be observed clearly in the reference spectrum , limit of quantification (LOQ), linear fitting and R2 values for each pesticide are summarised in Supplementary Table 38. The results confirmed a linear relationship between zero and 1 ppm for ACE . The LOD was calculated as 62 ppb, 47 ppb, 75 ppb and 5 ppb for ACE, CBM, THI and TRI, respectively. Therefore, these results confirmed that handheld-SERS could only detect below the recommended EU MRLs for TRI. One issue with the proposed mechanism is selectivity. As the chemical structure of pesticides differs greatly, interactions with the nanomaterial will also vary. For example, TRI is a sulfur containing organic molecule known to bind strongly to Au39, thus, it is not unexpected that the detection limits for this pesticide standard are lower. The results from handheld-SERS were validated against a benchtop Raman microscope  and compared to handheld-SERS. As expected, all pesticides were detected using the Raman microscope below the EU MRLs for Basmati rice, which are set at 10 ppb for ACE, CBM, THI and TRI by the European Commission40. Overall, the results confirmed the possibility to measure and quantify parts per billion (ppb) levels of pesticides using handheld-SERS. However, only TRI could be detected below the recommended MRLs thus, improvements to sensitivity are essential.Pesticide standards were analysed using handheld-SERS to obtain the working range Fig. . The resACE Fig. , CBM , a solvent extraction (E2), QuEChERs acetate (E3) and original QuEChERs (E4) Fig. . The reshod Fig. . The res42. The results can confirm that ACE, CBM and THI are unstable or degrade during the original QuEChERs extraction. Although these pesticides are more suited to an acidic medium the same was not true for TRI, which preferred the conditions of the original QuEChERs. As it was important to use an extraction suited to multiple pesticide residues, QuEChERs acetate could successfully extract all four pesticide residues from basmati rice and took less than 15 min to conduct. Therefore, QuEChERs acetate was selected for further analysis of pesticide recoveries using handheld-SERS.When the same optimised conditions were applied to recover ACE . Additionally, the detection limits were compared to those obtained using the benchtop Raman microscope .Basmati rice was spiked with pesticide residues at concentrations ranging from 0.5 ppm to 10 ppm for ACE Fig.  and 1 pprds Fig. . However43 and acidic conditions (<pH 7.4)44. In these conditions, citrate is protonated thus, the negative charges on the particle surface are greatly reduced, resulting in aggregation and increased clustering. Therefore, the conditions of the extraction are favourable for accelerating aggregation and ‘hot spot’ formation. As a result, lower concentrations of pesticide can become trapped within adjacent nano-gaps and are therefore detectable using handheld-SERS. In the case of ACE, it is often difficult to extract as it degrades easily and requires different conditions compared to other compounds such as, the use of low temperatures, problems which have also become apparent during GC-MS analysis of the compound42. To confirm that the analytical performance of our method is comparable to, or an improvement on previous techniques reported in the literature, several parameters have been compared for the detection of ACE, CBM, THI and TRI in rice  and low pH (5.0) of the extraction medium. As reported previously, bare-AuNPs lack stability in high electrolyte environments (>0.35%)4%, w/v aaccuracy (%) was calculated to be in the range of −17% to 4.5%, therefore, the results could confirm that some of the residues were not completely released during the extraction (Supplementary Table accuracy = −16.6% to 19%). Finally, the REprecision can be used to directly compare the performance of the two techniques; handheld-SERS and the Raman microscope. As the results were all close to zero , ahead of confirmatory laboratory techniques such as, LC-MS or GC-MS. Developments to these methodologies will help to improve food safety by enhancing the on-site analysis of toxic contaminants within key food groups.To evaluate the repeatability and reliability of the QuEChERs acetate extraction the % recovery and RSD (%) were obtained by spiking basmati rice with individual pesticides at three different concentrations. The average recovery and RSD of the extractions were within the range 83.4% to 115.0% and 3.6% to 23.8%, respectively , CBM (730 cm−1 and 735 cm−1), THI (638 cm−1 and 640 cm−1) and TRI (1318 cm−1 and 1372 cm−1) could all be visually identified at concentrations 0.25 ppm and 2.5 ppm as a standard mixture  and hexachlorobenzene) contain persistent organic pollutants (POPs) which can resist degradation thus, remain in the environment for many years and have the ability to bioaccumulate and biomagnify within the food chainure Fig.  and the ure Fig. . All fouTRI Fig. , clubs, TRI Fig. , spades,TRI Fig.  and spikTRI Fig.  conditio47 or convolutional neural networks (CNN)48. These will be important steps forward to improve the rapid on-site analysis of multiple contaminants within food and environmental samples.The results highlight that the detection of TRI in a complex rice sample using this approach can be achieved however, the presence of other pesticides may be difficult to quantify. SERS signals for analytes within a complex matrix can be suppressed by other analytes which are highly Raman active, have higher binding affinity to the substrate or which saturate SERS ‘hot spot’ positions. A known problematic area for SERS is spectral overlapping which will need crucial attention if multiplex analyte analysis is to improve. Areas for improvement may include: 1) enhancing the specificity of the Au substrate, which will ultimately help to reduce matrix effects, the detection of non-targeted compounds and interactions between those analytes which cannot bind as strongly to the substrate; 2) reduce spectral overlapping and interference between multiple residues in a sample, through the use of artificial intelligence (AI) and/or machine learning algorithms e.g. self-modelling mixture analysis (SMA)A rapid, handheld technique for the extraction of four pesticide residues from basmati rice has been developed. Selection of suitable AuNP substrates allowed maximum handheld-SERS enhancement. Sensitivities were improved by incorporation of a modified QuEChERs extraction, not commonly employed for SERS. The approach could detect concentrations extracted from spiked basmati rice within the range 0.6–800 ppb. Three out of four pesticides in matrix conditions could be detected below the MRL of 10 ppb in rice, set by the EU Commission. The average recoveries and RSD values were calculated within the range of 83.4% to 115.0% and 3.6% to 23.8%, respectively. Additionally, the results were validated against a benchtop Raman microscope. Multiplex analysis using handheld-SERS allowed pesticide concentrations in solvent and basmati rice to be detected at 0.25 ppm and 2.5 ppm, respectively. Overall, the results confirmed that there is potential for AuNP substrates, combined with QuEChERs extraction and handheld-SERS to successfully detect pesticide residues in basmati rice. Future developments to substrate design and the incorporation of machine learning algorithms may help to improve sensitivities, spectral overlapping and multiplexing performance in the future. Thus, there is substantial merit in the approach as a tier one screening tool, for the on-site analysis of pesticide residues in rice, agricultural crops  and environmental samples (i.e. groundwater and soil).−1. Spectral data was averaged (n = 3), smoothed using Savitzky-Golay filtering and fitted with OriginPro 8.5 software. AuNP size measurements were conducted using a Zetasizer NanoZS .Ultraviolet-visible spectroscopy (UV–vis) measurements were performed using a Cary 60 spectrophotometer . Handheld-SERS measurements were performed using a HRS-30 spectrometer equipped with a 785 nm laser and Raman fibre optic probe operated at 400 mW (80% laser power) at an integration time of 5 s . Benchtop SERS measurements were also carried out using a DXR2 Raman Microscope  operated with an excitation laser light at 785 nm, laser power of 24 mW, integration time of 5 s, ×10 objective lens within the spectral range of 400–1600 cm2COONa)2·aq), gold (III) chloride trihydrate , acephate , carbendazim (Methyl benzimidazol-2-ylcarbamate), thiamethoxam (3-(2-Chloro-5-thiazolylmethyl)tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadiazin-4-imin), tricyclazole , rhodamine 6G (R6G), hydrochloric acid 37% (HCl), acetic acid ≥99% (HAc), acetonitrile ≥99.9% (MeCN), absolute ethanol ≥99.8%, magnesium sulphate (MgSO4), sodium chloride (NaCl), sodium acetate (NaOAc), potassium chloride (KCl), 96-well flat bottom ELISA plates and polyester foam tipped sterile swabs were all purchased from Sigma Aldrich (UK). Indian basmati rice samples were produced and supplied by Green Saffron .Sodium citrate tribasic dihydrate (HOC(COONa)  and heated until rapidly boiling. Upon reflux 10 mL, 5 mL, 2.5 mL or 1.75 mL of 1% sodium citrate in dH2O was quickly added to the boiling solution, under vigorous stirring. The solution was removed from the heat after the colour changed from yellow to wine-red/purple-red/purple-brown/murky-brown which indicates the citrate reduction of gold ions, followed by DLS analysis to confirm particle size.Spherical nanogold was synthesised using a previous method2O) and stored at 4 °C. All further dilutions of the stock solution were prepared from 0.001 ppm to 10 ppm in dH2O. In a typical experiment, minor adjustments were made to a previous method49 and optimised conditions for analysis were as follows; 1 mL of AuNPs  was added to a clear glass vial followed by 50 µL of pesticide at varying concentrations. Finally, 5 µL of HCl (2 M) was added to the mixture, inverted several times and incubated at room temperature for 2 min. For comparison, the same experimental conditions were applied to a 96-well ELISA plate for analysis with a Raman microscope.Stock solutions of pesticide residues were first prepared by dissolving in a relative solvent (ethanol or dH2O and air dried. For extraction 1 (E1) and extraction 2 (E2) basmati rice samples were spiked by weighing 5 g of dried basmati rice into a clean plastic weigh boat and mixing with 1 mL of pesticide solution at concentrations ranging from 1-100 ppm. After mixing several times with a spatula over the course of 30 min, the samples were left to completely dry at room temperature overnight (~12 h). For extraction 3 (E3) and extraction 4 (E4), 5 g of dried basmati rice was weighed into a 50 mL centrifuge tube and spiked with 2 mL of pesticide solution at concentrations ranging from 10 ppb-100 ppm. The samples were placed on a roller for 30 min and left to completely dry at room temperature overnight (~12 h). The four extraction procedures were conducted as follows:Prior to spiking, basmati rice samples were cleaned thoroughly by rinsing in tap water, dHSwab extraction (E1): Swab sticks pre-soaked in ethanol (EtOH) were drawn through the rice samples evenly for ~90 s and immersed in 1 mL of the extraction solvent (EtOH). The swab tip was removed and vortexed in EtOH for ~30 s to release pesticide residues. Subsequently, 50 µL of the extracted residues were removed for SERS analysis.Solvent extraction (E2): 1 g of spiked basmati rice was placed directly into a 2 mL plastic Eppendorf tube with 1 mL of EtOH. The sample was vortexed for ~30 s to release pesticide residues and 50 µL was removed for SERS analysis.50. Due to the low water content of rice, water was first added  to hydrate the sample, followed by 15 mL of extraction solvent  for single-phase extraction of the sample and vortexed for 1 min. Liquid-liquid partitioning was performed with the addition of salts; 7 g of MgSO4 and 1.8 g NaOAc (E3) or 4 g of MgSO4 and 1 g of NaCl (E4) and vortexed for 1 min followed by a 5 min centrifugation at 1970 xg (RCF). Subsequently, 10 mL of the supernatant was removed and analysed using the handheld spectrometer and benchtop microscope.QuEChERs acetate (E3) and original QuEChERs (E4): QuEChERs extractions were conducted with minor adaptions to a previous reportFor all extractions, SERS analysis was conducted using the method described in the section ‘analysis of pesticide standard solutions using handheld-SERS’. Due to the hazardous nature of the chemicals used during the procedures, all waste was collected and disposed of accordingly.CR is the concentration of pesticide recovered, CSS is the concentration of spiking solution, VSS is the volume of spiking solution (mL) and VES is the volume of extraction solvent including dH2O (mL) . Using Eq.  was calculated for each pesticide using a prominent SERS peak typical to each pesticide and was between the range 55% and 79%. Therefore, from our calculations 21–45% was either not released in the extraction or not detected using handheld-SERS due to matrix interferences.For QuEChERs, initially the concentration of pesticide extracted was calculated using Eq. ;1\documeentclass1pt{minimaFurther information on research design is available in the Supplementary Information (Clean)REPORTING SUMMARY


Acinetobacter baumannii (A. baumannii) from wound infections, determine their resistance and virulence profile, and assess the impact of Silver nanoparticles (AgNPs) on the bacterial growth, virulence and biofilm-related gene expression. AgNPs were synthesized and characterized using TEM, XRD and FTIR spectroscopy. A. baumannii (n = 200) were isolated and identified. Resistance pattern was determined and virulence genes  were screened using PCR. Biofilm formation was evaluated using Microtiter plate method. Then, the antimicrobial activity of AgNPs was evaluated by the well-diffusion method, growth kinetics and MIC determination. Inhibition of biofilm formation and the ability to disperse biofilms in exposure to AgNPs were evaluated. The effect of AgNPs on the expression of virulence and biofilm-related genes  were estimated using QRT-PCR. In vitro infection model for analyzing the antibacterial activity of AgNPs was done using a co-culture infection model of A. baumannii with human fibroblast skin cell line HFF-1 or Vero cell lines. A. baumannii had high level of resistance to antibiotics. Most of the isolates harbored the fimH, afa/draBC, cnf1, csgA and cnf2, and the majority of A. baumannii produced strong biofilms. AgNPs inhibited the growth of A. baumannii efficiently with MIC ranging from 4 to 25 µg/ml. A. baumannii showed a reduced growth rate in the presence of AgNPs. The inhibitory activity and the anti-biofilm activity of AgNPs were more pronounced against the weak biofilm producers. Moreover, AgNPs decreased the expression of kpsMII , afa/draBC,bap, OmpA, and csuA/B genes. The in vitro infection model revealed a significant antibacterial activity of AgNPs against extracellular and intracellular A. baumannii. AgNPs highly interrupted bacterial multiplication and biofilm formation. AgNPs downregulated the transcription level of important virulence and biofilm-related genes. Our findings provide an additional step towards understanding the mechanisms by which sliver nanoparticles interfere with the microbial spread and persistence.We aimed to isolate  Acinetobacter species are commonly found in the environment, however, some species within this genus are generally associated with various habitats such as soil, water, sewage, human, foods and animals. Acinetobacter baumannii (A. baumannii) has an inevitable ability to sustain harsh conditions and spread as a serious pathogen4. A. baumannii infects moist tissues such as mucous membranes or areas of the skin that are exposed to this pathogen, either through wound or injury6.FimH) and pilli structures for attachment to the host cells8. Furthermore, numerous bacteria secrete toxins and extracellular enzymes which play a crucial role in the apoptosis or necrosis of epithelial cells or immunocytes. Various virulence factors of A. baumannii such as adhesins genes like kpsMII (group 2 capsule synthesis) and fimH, tratT (serum resistance associated), fyuA (yersiniabactin receptor) and iutA (aerobactin receptor) have been investigated previously10. An important polysaccharide for biofilm formation is encoded by pgaABCD locus11. Biofilm production is a strategy to escape from harsh conditions and immune responses, hence play as reservoirs for drug-resistant systemic infections. Biofilm-producing A. baumannii has been isolated from several infectious origins such as pneumonia and devise-associated infections. Bacterial within biofilm can resist significantly more against antibiotics compared to planktonic mode of growth12. Hence, biofilm-mediated infections are in relapse more frequently13.The adhesion and colonization or biofilm formation include primary stage in bacterial infections. Major adhesion virulence factors in this step include type I fimbriae (21. Silver nanoparticles (AgNPs), which have low toxicity in ecosystems and have high rate of surface capacity, can inhibit accumulation of biofilm materials responsible for evasion and protection24.Therefore, there is an urgent need to enhance the effects of antimicrobials against pathogenic bacteria. In recent years, interest has enhanced towards application of nanoparticles as therapeutic regimensA. baumannii from wound infections, determine their resistance and virulence profile, and assess the impact of AgNPs on the bacterial growth, virulence and biofilm-related gene expressions in the isolated strains.The aim of this study was to isolate The study was approved by the Institutional Review Board and ethics committee of Assiut University, Egypt and Mustansiriyah University, Baghdad, Iraq. A written informed consent was obtained from participants. All experiments were performed in accordance with relevant guidelines and regulations.25. The obtained samples were stirred, forming AgNPs. AgNPs formation was proved by Ultraviolet–visible spectral analysis. The absorbance spectra were determined using UV spectroscopy at a wavelength of 300–700 nm. The obtained NPs were characterized using transmission electron microscopy (TEM), X-ray diffraction methods (XRD) and Fourier transform infrared (FTIR) spectroscopy27. In TEM, a drop of NPs dispersion was placed on a carbon-coated copper grid and dried at room temperature and the micrograph of the preparation was taken.AgNPs (1000 µg/ml) were prepared via chemical reduction of silver nitrate in presence of polyvinylpyrrolidone (PVP) as a stabilizer according to−1.In case of XRD, crystalline structure of AgNPs powder was identified using the X-ray diffractometer with CuKα radiation (λ = 1.5405 Å) in the 2θ range of 10°–80°. For FTIR, samples were prepared by KBr-discs technique and the spectra were recorded in the range of 400–4000 cmA. baumannii were identified from wound infections. Phenotypic identification included culture onto the MacConkey agar , Leed Acinetobacter agar , and CHROMagar Acinetobacter/MDR medium, and incubated under aerobic conditions at 37 C for 48 h. Gram staining, motility, oxidase, peroxidase, and oxidative-fermentation reactions were performed according to standard techniques. API 20NE bacterial identification system  was used for species identification. Molecular identification of isolates was also conducted using PCR for detection of recA specific gene and bla-oxa51 gene as previously described28. The sequence of the primers are listed in supplemental Table Two hundred isolates of 29, bacterial cultures were plated onto the surface of Muller Hinton agar plates  and susceptibility was measured by the disc-diffusion methods. The following antibiotics were tested: cephalothin (30 μg), cotrimoxazole (23.75/1.25 μg), nitrofurantoin (300 μg), trimethoprim (5 µg), ceftazidime (30 µg), tobramycin (10 µg), amikacin (30 µg), tetracycline (30 μg), gentamicin (10 µg), streptomycin (10 µg), erythromycin (15 µg), levofloxacin (5 µg), rifampicin (5 μg), chloramphenicol (30 μg), azithromycin (15 µg), imipenem (10 μg) and ciprofloxacin (5 μg). Staphylococcus aureus ATCC25923, A. baumannii ATCC 19,606 and Escherichia coli ATCC25922 were used as quality control organisms in the test29.Following Clinical and Laboratory Standards Institute guidelinesafa/draBC, cnf1, cnf2, csgA, cvaC, fimH, fyuA, ibeA, iutA, kpsMT II, PAI, papC, PapG II, III, sfa/focDE and traT were screened using PCR. The primer sequence and product size are listed in Supplemental Table Virulence-related genes including A. baumannii cultures (containing about 105 CFU/ml) were plated on the surface of Mueller Hinton agar plates . Then, wells (5 mm is diameter) were made using a borer and 50 μg/ml of the AgNPs dispersions were added into the wells. Plates were then incubated at 37 °C for 24 h and the inhibition zone diameters were measured30.The antimicrobial activity of the AgNPs was evaluated by the well-diffusion method. A. baumannii was determined using the broth micro-dilution method according to the guidelines of the Clinical and Laboratory Standards Institute31. Briefly, AgNPs formulation was serially diluted in 100 μl Mueller–Hinton broth in wells of 96-well plate. Bacterial suspensions were added and plates were incubated for 24 h at 37 °C. MIC was determined visually by determining the wells with no visible bacterial growth.MIC of the formulated AgNPs against clinically isolated 32. Briefly, isolates were cultivated in Muller Hinton Broth  containing 2% w/v sucrose  and incubated for 24 h at 37 °C in 96 well microtiter plates . After incubation, wells were gently washed twice with PBS solution. Biofilm formed by adherent organisms was stained with crystal violet (0.2% w/v) and the excess dye was washed with distilled water. After drying, 95% ethanol was added to the wells and the optical density (OD) was measured at 570 nm using a microplate reader. Average OD values were determined for all tested isolates and the negative controls. Cut-off value was defined as the value of mean OD of the negative control plus 3X standard deviation of the negative control reads. Isolates were considered weak biofilm producers if they have a mean OD values ≤ two times the cut-off value, moderate biofilm producer if OD ≤ four times the cut-off value and strong biofilm producer if OD > four times the cut-off value.The biofilm-forming abilities of the tested strains were carried out using the microtiter plate method as previously describedIn some experiments, the effect of AgNPs on biofilm formation was tested. To do that, the isolates were treated with 100 µl of 25 µg/ml AgNPs in BHI medium and analyzed as mentioned before. The ability of AgNPs to reduce biofilm formation was assessed by comparing the OD of the AgNPs treated wells versus control untreated wells. To test the ability of AgNPs to disperse the pre-formed biofilm layers, bacterial suspensions were allowed to grow overnight in the 96-well plates, then different aliquots of AgNPs were added to the overnight bacterial cultures. Plates were incubated for additional 24 h, and finally, the strength of the biofilms exposed to the AgNPs was re-evaluated. The experiment was performed in triplicate for each isolate.A. baumannii strong, moderate and weak biofilm producers. Bacterial suspensions at OD595 in Muller Hinton broth were incubated at 37 °C in the presence of 5 μg/ml AgNPs (sub-MIC concentration) and OD595 was measured at 6, 24, 48 and 72 h using a microplate reader . Then, growth curves were plotted using the mean of three replicates for each strain ± SD33.Using the 96-well plate, we compared the effect of AgNPs on the growth of 3 representative kpsMII and afa/draBC genes) and biofilm-related genes  was evaluated using RT-PCR. RNA was extracted from bacteria treated with AgNPs using TRIzol reagent  and untreated bacteria were used as controls. cDNA was synthesized subsequently by Superscript III kit  according to the manufacturer protocol. PCR reactions were carried out using ePower SYBR Green PCR Master Mix  in a 7500 Sequence Detection System . Samples were normalized to the expression of the 16S rRNA gene and the fold change in the expression of the target genes was calculated using 2−ΔΔCT method34. Expression level of each gene in AgNps treated cells was compared to the expression of untreated bacteria as a control.The effect of AgNPs (25 µg/ml) on the expression of the virulence-related genes  were added in a total volume of 0.5 mL. Control samples treated with PBS were included. Kinetic studies were conducted at different time points to evaluate the extracellular killing activity at different contact times . Post-treatment samples were serially diluted in sterile PBS, plated on Mueller–Hinton agar plates, incubated for 24 h at 37C and CFUs were counted. The extracellular killing activity of AgNPs was evaluated by determining the percentage killing at the different time points, which was calculated by the following formula: (CFU of the control untreated samples − CFU of the AgNPs treated samples/CFU of the untreated control samples) × 100.First, the extracellular killing activity was evaluated as previously describedA. baumannii was carried out using the same co-culture infection model of A. baumannii and human fibroblast skin cell line HFF-1or Vero cells35. Briefly, cells were challenged with 1 × 108 CFU/mL log-phase A. baumannii and incubated for 2 h. Extracellular bacteria were removed by 2 h treatment with gentamycin then AgNPs (25 µg/ml ) or sterile PBS (plain control) were added and incubated for different time pointes . Following incubation, cells were lysed by Triton X-100 (0.1%) and samples were diluted in PBS, plated on Mueller–Hinton agar plates, incubated for 24 h at 37C and CFUs were counted. The Intracellular killing activity of AgNPs was evaluated by determining the percentage killing at different time points relative to the control untreated cells as described previously. All experiments were performed in triplicate and the mean ± standard deviation was presented.Second, the ability of AgNPs to kill the intracellular 33. About 1 × 104 cells/200 ul were cultured in 96-well-plate at 37 C and 5% CO2 in DMEM  supplemented with 10% Fetal Bovine Serum. After incubation of the cells overnight, AgNPs were added at different concentrations and incubated for 24 h. The viability of the fibroblast cells was measured by the MTT assay protocol  according to manufacturer instructions. Experiments were performed in triplicates and absorbance was measured at 570 nm using Epoch microplate reader .The cytocompatibility of AgNPs was evaluated using two cell lines; the human fibroblast skin cell line HFF-1 and Vero cell line  as previously describedp value < 0.05 was considered statistically significant.All the obtained results were statistically analysed with the GraphPad Prism  and the statistical software package for the Social Sciences software . A The study was approved by the Institutional Review Board and ethics committee of Assiut University, Egypt and Mustansiriyah University, Baghdad, Iraq. A written informed consent was obtained from participants. All experiments were performed in accordance with relevant guidelines and regulations.−1, 1670 cm−1 and 2900 cm−1, which correspond to CN bond, carbonyl group and C–H stretching, respectively38. Slight shifts in the spectrum of AgNPs indicate the formation of coordination bonds between oxygen or nitrogen of PVP with Ag atoms.Shape and size of AgNPs, which were prepared via chemical reduction of Ag salts, were characterized using TEM. AgNPs had a spherical shape with particle size in the range of 10–50 nm . Only azithromycin, nitrofurantoin, rifampicin, imipenem and gentamycin displayed limited degrees of resistance. The resistance profile of the 200 A. baumannii isolates and distribution of the virulence-related genes are shown in supplemental file Antibacterial susceptibility of the isolated A. baumannii isolates (n = 200) carried the fimH , afa/draBC , cnf1 , csgA  and cnf2 , followed by ibeA , cvaC , iutA , papC , traT , PAI , fyuA , kpsMII , PapGII  and papGIII . The majority of A. baumannii  produced strong biofilms, 22 (11%) isolates produced moderate and 13 (6.5%) isolates produced weak biofilms.Most of the A. baumannii biofilm producers . However, the biofilm formation seems to interfere with the formulation’s inhibitory activity. The inhibitory activity was more pronounced on weak biofilm producers, where AgNPs induced a mean inhibition zone diameter of 22 ± 5 mm, compared to 17 ± 4 mm and 10 ± 4 mm in moderate and strong A. baumannii strains was determined in the presence of silver nanoparticles or its absence as a control. The growth rate was estimated by measuring the OD595 at different time points . Normal cells showed a marked increase in OD595 after 6 h and reached a stationary phase over the duration of 24–72 h. Generally, A. baumannii showed a reduced growth rate in the presence of AgNPs , compared to untreated cells. Consistent with the resistance phenotype of the strong biofilm producers, the effect was more obvious with moderate and weak biofilm producing bacteria . Significant difference was observed between strong and moderate biofilm producers vs the weak group as shown in Fig. A similar effect was observed when MIC values of the formulated AgNPs against the isolated Furthermore, exposure to AgNPs inhibited the strong, moderate and weak biofilm production significantly and biofilms reached 63 ± 15%, 58 ± 16% and 42 ± 9% of controls. AgNPs were also able to disperse the already formed strong, moderate and weak biofilm. Percentage of biofilms treated with AgNPs were 85 ± 12%, 77 ± 15% and 51 ± 11%, respectively  and 3.4-folds (p = 0.001), respectively. RT-PCR analysis of total RNA isolated from strong biofilm producers treated with AgNPs showed that the transcription of bap, OmpA, and csuA/B was significantly decreased by 4.5-, 3.1- and 3.1-folds, respectively, when compared with the untrerated bacteria  and Vero cell lines. Cell viability test showed that exposure of the human fibroblasts to different AgNPs concentrations, starting from 0.25 to 25 µg/mL , did not affect the viability of the cells compared to untreated control cells, indicating that the nanoparticles are not harmful and biocompatible Fig. .Figure 444. The ability of bacteria to form biofilms is one of the main virulence factors that interferes with antibiotic activity and immune defense response mechanisms48. A. baumannii has developed many virulence factors and is responsible for severe life threatening infections53. The bacterium affects different sites, including wound infections, pneumonia, UTI. Biofilm formation abilities and various adhesins participate in the pathogenesis of their infections and resistance to antimicrobial drugs54. Herein, we observed that the majority of A. baumannii were resistant to most of antibiotics and could produce strong biofilms (82.5%). Interestingly, all the isolates which contained fimH and afa/draBC could produce strong biofilms. A. baumannii from wound infections mostly harbored the fimH, afa/draBC, cnf1, csgA and cnf256. It is worth considering that all of these strains were also MDR. The most effective antibiotic against MDR isolates included nitrofurantoin (12%) and imipenem (23%). Our results are in line with other studies which reported a high rate of biofilm formation among MDR-A. baumannii57.Severe infections associated with the multidrug-resistant bacteria, have increased significantly in the last decades60. Recently, several reports have described the improved antimicrobial activity of AgNPs to be used for topical administration against multidrug-resistant bacteria61.In the last years new approaches have been developed to enhance the antimicrobial treatment such as the use antibiotic combinations, bacteriophage therapy, usage of antimicrobial NP-based formulations of old antimicrobial agent62. The inhibitory activity of AgNPs on Gram-negative bacteria is more efficient than their activity on the Gram-positive bacteria64. AgNPs exerts potent antibacterial activity through different mechanisms. They have been reported to adsorb in the form of ionic silver (Ag+) onto the cytoplasmic membranes of Gram-negative bacteria which leads to destruction of the cell membrane, leakage of bacterial contents and death65. In addition, AgNPs also inhibit synthesis of bacterial cell wall66. They cause bacterial death through interaction with sulfur groups in essential proteins, generation of reactive oxygen species leading to bacterial death67. The high anti-Acinetobacter activity of the AgNPs could be attributed to the small size of the particles and the larger surface area, which allows more contact with the bacterial surface68.The anti-Acinetobater activity of the formulated AgNPs was shown by producing marked zones of inhibition and delay in microbial growth curves. Similar results were reported by previous studies using 14–27 µg/mL AgNPsA. baumannii. Also, overnight incubation of sliver to well-formed biofilms lead to their partial removal. This allows the potential use of AgNPs as a biofilm-disrupting agent69. Consistent with our results, previous studies have shown efficient effect of AgNPs in inhibiting biofilms produced by different microorganisms71. Different investigators have evaluated the ability of silver nanoparticles to inhibit biofilm formation by A. baumannii, Pseudomonas aeruginosa (synergy with tobramycin), Staphylococcus aureus (such as synergy with vancomycin), Escherichia coli and Klebsiella pneumonia at similar concentrations78.Our results showed a potent ability of silver in inhibiting the strong biofilms produced by kpsMII and afa/draBC) and biofilm-related genes , which adds to the known mechanism by which silver interferes with microbial growth. We observed that 25 µg/mL of AgNPs significantly decreased the expression of virulence and biofilm-related genes.Importantly, this is the first report that shows that ability of AgNPs to inhibit the expression of different virulence-related genes (Csu gene enables A. baumannii strains to attach and form strong biofilms through formation of pili79. When Csu is inactivated, the ability of pili production and subsequently the ability of bacteria to format biofilms is inhibited80. Finally, we concluded that decreasing the expression of bap, OmpA, and csuA/B by AgNPs is a main approach for decreasing biofilm activity of A. baumannii that will help in reducing antibiotic resistance mechanisms81. The limitations of our study included lack of an in vivo study, which we plan to study in the future work.Studies have shown that the presence of A. baumannii isolates. The analysis of the isolates revealed that most isolates showed high level of resistance to antibiotics, carry bundle of virulence-related genes and different abilities to produce biofilms. Furthermore, treating the bacteria with AgNPs significantly interrupted bacterial growth and multiplication. The activity of AgNPs on A. baumannii growth kinetics, biofilm inhibition and dispersion was affected markedly by the strength of the produced biofilms. AgNPs downregulated the transcription level of important virulence and biofilm-related genes. Our findings provide an additional step towards understanding the mechanisms by which AgNPs interfere with the microbial spread and persistence.This study has enhanced our understanding of the characteristics of clinical Supplementary Information 1.Supplementary Information 2.


To identify predictive factors for visual outcomes of patients presenting with a posterior segment intraocular foreign body (IOFB).  A retrospective chart review was performed for all consecutive patients operated for posterior segment IOFB removal between January 2009 and December 2018. Data were collected for patient demographics, clinical characteristics at presentation, IOFB characteristics, surgical procedures, and postoperative outcomes. A multiple logistic regression model was built for poor final visual acuity (VA) as an outcome . p = 0.009) and number of additional complications (p = 0.01) were independent risk factors for a poor final VA.  Fifty-four patients were included in our study. Ninety-three percent of patients were men, with a mean age of 40.4 ± 12.6 years. Metallic IOFB comprised 88% of cases with a mean ± standard deviation (SD) size of 5.31 ± 4.62 mm. VA improved in 70% of patients after IOFB removal. Predictive factors for poor VA outcome included poor baseline VA, larger IOFB size, high number of additional diagnoses, an anterior chamber extraction, a second intervention, the use of C3F8 or silicone tamponade, and the presence of vitreous hemorrhage, hyphema, and iris damage. Predictive factors for a better visual outcome included first intention intraocular lens (IOL) implantation and the use of air tamponade. In the multiple logistic regression model, both baseline VA ( A high number of concomitant complications and poor baseline VA following posterior segment IOFB were significant predictive factors of poor visual outcome. Despite growing prevention efforts, ocular injuries remain a major cause of blindness with an estimated incidence of 2.4 million cases in the United States . Among tThe aim of this study is to identify predictive factors for visual outcomes of patients presenting with a posterior segment IOFB treated in a tertiary university hospital in Quebec, Canada, and to review the surrounding literature.This retrospective study was approved by the Institutional Review Board of the Centre Hospitalier Universitaire de Quebec–Université Laval and adheres to the tenets of the Declaration of Helsinki.All consecutive patients treated, hospitalized, or operated for posterior segment IOFB at the CHU de Quebec–Université Laval, a university tertiary hospital, between January 1, 2009, and December 31, 2018, were included in the study. Exclusion criteria included nonposterior segment IOFB and patients who had immediate exenteration, enucleation, or evisceration.Data collection included age, gender, VA preoperatively and at least one month after the last intervention, final diagnosis, IOFB characteristics , and delay between the injury and the surgery in days. The type and date of interventions, extraction type (pars plana or anterior chamber), surgical procedure ), intraocular lens implantation , the number and the type of additional diagnoses and complications at presentation , the need for a second intervention (excluding phacoemulsification and intraocular lens implantation (phaco-IOL)), and an evisceration or enucleation were also collected. The context and mechanism of the injury as well as the presence of a relative afferent pupillary defect (RAPD) were not collected because of incomplete data related to these variables.The surgical technique used to remove IOFB depended on clinical characteristics but usually involved a pars plana vitrectomy with IOFB extraction using forceps or magnets, followed by the use of a tamponade. Primary repair also varied depending on clinical characteristics but was generally performed using permanent suture of varied size depending on the localization of the entry wound.t-test or Mann-Whitney U test as appropriate for continuous variables and chi-square analysis for categorical variables. Shapiro-Wilk test and Q-Q plots with 95% confidence intervals were used to test for normal distribution of continuous variables.Data are presented as mean ± standard deviation (SD) for continuous variables and as frequencies (percentages) for categorical variables. Best corrected visual acuity (BCVA) was converted to an Early Treatment Diabetic Retinopathy Study (ETDRS) letter score from the metric Snellen scale for analysis. For the purposes of analyses, missing data for delay before presentation was imputed using the median value for the 5 (9%) missing cases. Patients were separated into two groups based on final VA: the good VA group with vision greater than 50 ETDRS letters  and the poor VA group with vision less than or equal to 50 ETDRS letters. Comparisons of both groups were performed to identify characteristics associated with a poor final VA. The following prognostic factors were analyzed for possible associations with final VA: age, time of injury to intervention, entry site, IOFB characteristics , extraction site, clinical features on initial presentation , IOL implantation , the need for a secondary intervention (excluding phaco-IOL), and the need for a scleral buckle. Any damage to the iris that could reasonably be associated with the trauma was considered as an iris damage, regardless of size or severity. Characteristics and variables were compared between the two groups using independent Student's p > 0.2 removed. Given the small sample size, variables were included in the model while respecting a target of 5–9 events per predictor variable in building the models  of 1  days. Seventy-two percent of patients (n = 39/54) were operated ≤48 hours from injury.A total of 54 patients were included in the study . There wn = 39, 72%), vitreous hemorrhages , hyphemas , retinal detachments , retinal tears , iris damage , choroidal detachment , and endophthalmitis .The most common additional diagnoses and complications seen with IOFB were cataracts (p = 0.013). In four cases (7%), the VA remained stable, while in 12 cases (22%), the VA worsened.Thirty-three percent of patients presented with an initial VA of 6/18 or better and 15% of patients had an initial VA of 6/9 or better. Forty-four percent of patients presented with an initial VA of no light perception (NLP), light perception (LP), or hand motion (HM). On final presentation, 50% of patients had a VA of 6/18 or better and 33% of patients had a final VA of 6/9 or better. Moreover, only 19% of patients had a final VA of NLP, LP, or HM . Accordin = 11) had a first intention IOL implantation. 14 patients (26%) underwent a second intervention (excluding phacoemulsification with intraocular lens (IOL) implantation). Second interventions performed mainly included vitrectomy (with or without combined buckle) used in case of complications following the primary intervention, such as retinal detachments with or without vitreoretinal proliferation. The intraocular tamponade used during the surgery was air (43%), SF6 (28%), C3F8 (13%), and silicone oil (17%).Among the 54 patients, 21 (39%) received surgical intervention within 24 hours of the incident. IOFB removal was achieved via the pars plana in 48 cases (89%) or via the anterior chamber in 5 cases (9%). All patients underwent a vitrectomy. Nineteen patients (35%) had a scleral buckle (combined to the vitrectomy) and 5 (9%) had a corneal graft. 43 patients (80%) underwent phacoemulsification. Thirty-two eyes (59%) had phacoemulsification with secondary intraocular lens (IOL) implantation while only 20% (p < 0.001), a larger IOFB size (p < 0.001), a higher number of additional diagnoses (p < 0.001), the presence of vitreous hemorrhage (p = 0.002), hyphema (p < 0.001), iris damage (p = 0.042), an anterior chamber extraction (p = 0.009), a second intervention (p = 0.018), and the use of C3F8 (p = 0.002) or silicone tamponade (p = 0.027) (p = 0.026) and air tamponade (p = 0.019).The results revealed that poor final BCVA was associated with the following factors: a poor baseline VA (= 0.027) . Likewisp = 0.009) and a higher number of additional diagnoses/complications at presentation (p = 0.01).For further clarification of independent risk factors for a poor visual outcome, a multiple logistic regression model was built . IndepenDespite growing awareness for the use of protective eyewear, IOFBs continue to be a common cause of blindness in both developed and undeveloped countries . We descAs in previous studies, patients are predominantly male with an average age of 40 years , 12, 13.n = 1, 2%) though this patient did not develop endophthalmitis.The majority 88%) of IOFBs in our study were inorganic and metallic in origin. In general, inorganic, metal IOFBs are better tolerated than organic matter. Most metals are inert and, if small and deeply lodged, may be left in place . Hammeri8% of IOFp < 0.001). Smaller IOFBs , but the extent of retinal detachment could differ between outcome groups. On the contrary, cataract development does not seem to influence final visual outcomes in our study and in others [Complications identified as poor prognostic factors in our study were consistent with findings in the literature, including vitreous hemorrhage , 16 and n others , 18, givn others , 22, RAPn others , 16, retn others , 13, PVRn others , 22, andn others . We wereThere are many conflicting studies regarding the management of IOFB in terms of the optimal time of removal. Recent studies suggest that immediate IOFB removal may not be as critical to vision preservation as previously thought , 23. A lOur study found no association between the time before removal and the final visual outcome. These results are consistent with Wickham et al. , Liu et Only one-third of the patients in our study presented with an initial VA of 6/18 or better and 15% of patients had an initial VA of 6/9 or better. We found that better initial VA was associated with better final VA. This correlation has previously been found in numerous studies , 12, 13.The requirement for repeated surgery is associated with a poorer visual outcome , 25, 26.The use of C3F8 gas or silicone oil tamponades compared to air tamponade was associated with poor visual outcome in this study. Similar results were reported by Liu et al. with C3F8  and AkesThis is a retrospective study with a relatively small sample size that provides epidemiological data for IOFB in our region. Our results are comparable to previous studies in other regions, including a study in North Carolina in which similar factors of poor visual outcomes  were identified among a cohort of 59 predominantly young male patients . StrengtIn conclusion, this study identified multiple risk factors for poor VA following posterior segment IOFB over the span of a decade. Independent risk factors included worse baseline VA and a high number of concomitant complications . These factors will allow ophthalmologists to more accurately assess the trauma severity and visual prognosis of patients with a posterior segment IOFB. Future directions include assessing the impact on long-term visual acuity and the impact of the type of intervention on long-term visual prognosis.


Retinitis pigmentosa (RP) is a heterogeneous group of inherited ocular diseases that result in progressive retinal degeneration. This study aims to describe different Swept-source Optical Coherence Tomographic (SS-OCT) changes in Palestinian RP patients and to explore possible correlations with Visual Acuity (VA).A cross-sectional observational study was conducted on Retinitis Pigmentosa patients diagnosed with RP in a tertiary eye hospital. Full history and ocular examination were made. SS-OCT imaging was done for all eyes assessing the presence of cystoid macular edema, epiretinal membrane, macular holes, and external limiting membrane, ellipsoid zone status. Also, central macular thickness and choroidal vascular thickness were measured.p = 0.001), ellipsoid zone(p = 0.001), and external limiting membrane (p = 0.001).The study was run on 161 eyes of 81 patients; 53 males and 28 females. The average age at examination was 26.1 (6–78) years. Twenty-six eyes (16.1%) were of syndromic RP patients, mostly Usher syndrome; 20 eyes (12.4%). The mean Logaritmic minimal angle of resolution (LogMAR) of Best Corrected Visual Acuity (BCVA)of the study sample was 0.66 ± 0.7. The most prevalent change was cystoid macular edema , followed by epiretinal membrane . A macular hole was noted only in one eye (0.6%). Ellipsoid zone and external limiting membrane were absent in 55 eyes (35.0%) and 60 eyes 37.5%. Vitreous hyperreflective foci were found in 35 eyes (43.8%). LogMAR of BCVA was associated significantly with cystoid macular edema  of ellipsoid zone +/− external limiting membrane and choroidal vascular thickness, which should be evaluated serially using high-resolution OCT. Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of hereditary retinal disorders , comprisMore than 100 genes have been associated with RP . DiseaseAssessment of patients with RP includes a detailed history, full standard ophthalmological exam, imaging modalities, and functional testing, with molecular genetic testing increasingly being undertaken .Over the last two decades, the study of retinal anatomy and pathology has been revolutionized by the advent of optical coherence tomography (OCT) . This beSwept-source (SS) OCT is a variation of OCT that offers improvements in visualizing the vitreous, retina, and choroid at one image. The increased scan speeds, decreased signal attenuation, more comprehensive imaging, and deeper tissue penetration make SS-OCT ideal for capturing a wide range of views and studying structures below the retinal pigmented epithelium (RPE), especially the choroid .OCT can show progressive degeneration of the photoreceptor layers through the change in the integrity of the outer retinal hyperreflective bands . OCT alsThe Palestinian community has a relatively high consanguinity rate, which significantly increases the prevalence of recessively inherited disorders such as RP , 14. A gA cross-sectional observational study was conducted on all patients diagnosed with RP at St. John Eye Hospital between October 2016 and December 2018. Informed consent was obtained from all participants and from parents and/or legal guardians of participants under 18 years of age. Examinations were carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) and to ensure the privacy and confidentiality of the participants. Approval of the St John Eye Hospital institutional ethics committee was obtained.The study included 161 eyes of 81 patients diagnosed with RP. Diagnosis of RP was based on typical clinical history, fundoscopic appearance, fundus autofluorescence, and full-field ERG. The study included patients of all age groups, all modes of inheritance, and both syndromic and non-syndromic RP. We excluded patients with any other retinal pathology or systemic disease that might cause macular changes on OCT. Excluded comorbidities included diabetic retinopathy, central serous Chorioretinopathy, age related macular degeneration, retinal vein occlusion, Stargardt disease and cone-rod dystrophy. Additionally, patients with media opacity precluding OCT image acquisition and patients with poor image quality were excluded.A total of 100 patients with RP registered in the hospital’s database were recruited for a single visit, of them, 19 patients were excluded according to the above- mentioned criteria . Full history and standard ophthalmological examination were performed. BCVA was measured with the logarithm of the minimal angle resolution (logMAR) vision chart.OCT imaging was done using the DRI Triton, Swept Source OCT  device by a dedicated imaging technician. SS-OCT imaging was performed using horizontal 9 mm wide raster of 128 scans and 16-line radial 6 mm wide scans. Three readers did SS-OCT image assessment; two retina specialists , and an OCT specialist (ES). The agreement of two readers, out of three, decided the final decision. Image assessment and measurements were done with the aid of IMAGE net 6 OCT Topcon software.The OCT variables assessed included; (i) macular changes: presence or absence of CME, macular hole, and automated measurement of Central macular thickness (CMT) in μm; (ii) the presence or absence of other retinal changes: ERM, choroidal neovascularization (CNV), vitreomacular interface abnormalities  includinP-value of less than 0.05 was considered to be statistically significant. All statistical analyses were done using SPSS version 20.0.Descriptive statistics were first applied to assess the patients’ baseline characteristics . A Chi-square test was used to compare categorical variables. Independent t-test and Mann-Whitney test were used to compare means in normally distributed data and nonparametric data. Kruskal Wallis test was used for comparing nonparametric values in more than two groups. Possible correlations between continuous variables were explored using the Spearman’s ranked order correlation test. A This study recruited 81 RP patients and assessed 161 eyes. It included 53 males (65.2% of eyes) and 28 females (34.8% of eyes). The average age at examination was 26.1 (6–78) years. Twenty-six eyes (16.1%) were from patients with syndromic RP, including Usher syndrome , Bardet-Biedl syndrome , and RP with unspecified syndromic features . This sample’s mean LogMAR BCVA was 0.66 ± 0.73 (0.00–3.00), and the mean CMT was 230 ± 85.7 (86–577) μm. The subfoveal CVT thickness could be measured in 72 eyes, with an average subfoveal CVT being 289.3 ± 103.3 (69–612) μm (Table p = 0.161). The second most common finding was ERM . A macular hole was noted only in one eye (0.6%) of the studied sample. EZ was reported to be absent in 55 eyes (35.0%) and abnormal in 63 eyes (40.1%). ELM was found to be missing in 60 eyes (37.5%) and abnormal in 45 eyes (28.1%). However, no cases of VMT/VMA or CNV were observed ; of the Syndromic RP patients,  had CME, compared to  of non-syndromic patients; however, this was not statistically significant (p = 0.260), and 17.1, 60% had an absent, abnormal EZ respectively in a non-significant relationship (p = 0.197).Vitreous Hyper-reflective foci (HRF) were examined in 72 eyes and were found in 35 eyes (43.8%) of the cases assessed. Of these (with foci present), 26% were associated with CME in a non-significant relationship (p = 0.007), where females had worse VA than males. CME had a highly significant relationship with BCVA (p < 0.001); interestingly, eyes without CME had a much worse VA in terms of BCVA. Both the EZ and ELM status shows a highly significant relationship with BCVA (p < 0.001), with worse VA associated with EZ and/or ELM being absent. At the same time, the presence of ERM does not show a significant relationship with BCVA. Similarly, the presence of vitreous HRF had no significant association with BCVA.Table p = 0.005) and between subfoveal CVT and BCVA . Additionally, subfoveal CVT was found to be negatively correlated with age . We repeated the analysis using data of one eye that was randomly selected from patients with two eyes. The results showed a non-significant week negative correlation between BCVA and CMT , sub foveal CVT , and age .Spearman’s ranked order correlation test was used to assess the correlation between BCVA and CMT, CVA and age. We observed a weak significant negative correlation between CMT and BCVA ; younger than those reported in other studies; 33.4–49.3 years , 18. Thi. A wide range of CME prevalence in RP patients has been reported in previous studies, where some reported as small as 5.5% by Hagiwara , AdackapMacular holes were found less frequently, with only one eye (0.6%) displaying a lamellar hole, similar to those recorded by Hagiwara et al. (0.5%) . PreviouP < 0.001). Indeed, Battaglia et al. showed that ELM remains significantly associated with BCVA after multivariate regression, whereas EZ did not [This study identified a higher percentage of eyes having an absent EZ (35%) compared to other studies , 25–27. p = 0.007). This may be related to the cultural beliefs of the studied population, and females are less likely to seek medical attention at an earlier stage [p < 0.001). This contrasts with the non-significant relationship reported by others [In this study, BCVA was found to be significantly correlated with several variables. The first is gender; females, in this study, had a worse BCVA than males.  [In this study, the average CMT for RP patients was marginally above the healthy population (230.0 ± 85.649 μm) and correlated with decreased BCVA (= 0.005) .The aver= 0.005) . A signi= 0.005) . This sup = 0.039), as expected, and negatively with BCVA (p = 0.015). Similar significant relationships were noted by Aknin et al. [It has been hypothesized that the factor involved in photoreceptor degeneration is choroidal thinning caused by decreased blood flow . Our stun et al.  and Ayton et al.  but incon et al.  using Enp = 0.551). Interestingly, eyes without vitreous HRF had a worse BCVA than eyes with HRF (LogMAR 0.684 vs. LogMAR 0.590). This may indicate that their presence reflects an early or intermediate stage of the disease, rather than an advanced stage. This is also supported by the fact that they were more likely to be found when the EZ was shortened and abnormal (60%) rather than when absent. To date, HRF has been studied in other retinal layers, but not in the vitreous. And vitreous HRF was only studied in diabetic retinopathy and uveitis.HRF has been identified in other retinal diseases composed of macrophages, migrating RPE cells, and extravasated lipoprotein. This study is the first to describe HRF in the vitreous, thereby increasing our limited current understanding of their role in the disease process. Although vitreous HRF was present in 43.8% of studied eyes, this was non-significantly correlated with BCVA  of EZ +/− ELM and CVT, which should be evaluated using high-resolution OCT better to identify candidates for gene therapy trials/treatments; as well as identify CME.