Background: HIV-infected children and adolescents (CA-HIV) face significant mental health challenges related to a broad range of biological and psychosocial factors. Data are scarce on the agreement and discrepancy between caregivers and CA-HIV regarding emotional and behavioral problems (EBPs) in CA-HIV.Objectives: We determined agreement between self- versus caregiver- reported EBPs and describe factors associated with informant discrepancy among caregiver–youth dyads who participated in the “Mental health among HIV-infected CHildren and Adolescents in KAmpala and Masaka, Uganda” (CHAKA) study.Methods: In a cross-sectional sample, caregiver-reported EBPs were assessed with the Child and Adolescent Symptom Inventory-5 (CASI-5), and self-reported problems were evaluated with the Youth Inventory-4 (YI-4) in 469 adolescents aged 12–17 years and the Child Inventory-4 (CI-4) in 493 children aged 8–11 years. Adolescents were questioned about experiences of HIV stigma. Caregiver psychological distress was assessed with the Self-Reporting Questionnaire (SRQ-20). Linear regression models were applied to identify variables associated with discrepancy scores.Results: Self-reported emotional problems (EPs) were present in 28.8% of adolescents and 36.9% of children, and 14.5% of adolescents self-reported behavioral problems (BPs). There was only a modest correlation (r ≤ 0.29) between caregiver- and CA-HIV-reported EBPs, with caregivers reporting more EPs whereas adolescents reported more BPs. Informant discrepancy between adolescents and caregivers for BPs was associated with adolescent age and caregiver’s employment and HIV status. Among adolescents, EP discrepancy scores were associated with adolescent’s WHO HIV clinical stage, caregiver level of education, and caregivers caring for other children. Among children, EP discrepancy scores were associated with child and caregiver age, caregiver level of education, and caregiver self-rated health status. HIV stigma and caregiver psychological distress were also associated with discrepancy, such that adolescents who experienced HIV stigma rated their EPs as more severe than their caregivers did and caregivers with increased psychological distress rated EBPs as more severe than CA-HIV self-rated.Conclusions: EBPs are frequently endorsed by CA-HIV, and agreement between informants is modest. Informant discrepancy is related to unique psychosocial and HIV-related factors. Multi-informant reports enhance the evaluation of CA-HIV and informant discrepancies can provide additional insights into the mental health of CA-HIV. Diagnostic and Statistical Manual of Mental Disorders (DSM) nosology reported an average prevalence across studies of 28.6% for attention deficit hyperactivity disorder (ADHD), 24.3% for anxiety disorders, and 25.0% for depression  were residing in Eastern and Southern Africa  study. Specifically, we describe relations between informant discrepancy and a range of sociodemographic and HIV-related factors for the symptoms of a number of common child and adolescent EBPs. Additionally, we investigate the association of HIV stigma and caregiver psychological distress with informant discrepancy. We also report on the prevalence of self-rated EBPs as compared to caregiver-rated EBPs and assessed the level of agreement. Based on existing research  and two in the urban Kampala City Council . Eligible participants were recruited from each study site consecutively until the required sample size was attained. An equal number of 268 dyads was planned for recruitment at each site. Interviews were conducted in partitioned tents that were erected at each of the study sites to ensure privacy and limit distraction.CA-HIV between 5 and 17 years of age with caregivers older than 17 years of age were included. Additionally, participants were included if both caregivers and CA-HIV could speak English or Luganda , and they remained in the study’s geographical area for the subsequent 12 months. Participants were excluded if they were concurrently enrolled in another study, if they were unwell and in need of immediate medical attention, and if they did not understand the study instruments for any reason. Furthermore, to be able to address the objectives of this study, we only included CA-HIV who had completed the self-report measures for EBPs. Eligible study participants provided written informed consent (caregiver) and assent (CA-HIV) after explanation of the study objectives and procedures. No CA-HIV were enrolled without their assent, and all participants were informed that they could withdraw without prejudice at any time. In the majority of cases, the parents provided informed consent for participation of the CA-HIV, but in cases where the primary caregivers were not parents, the guardians of the CA-HIV provided the informed consent. Approximately 2% of participants assessed for eligibility were not included due to factors such as caregiver refusal, CA-HIV refusal, inability to contact the caregiver to obtain consent, and ongoing participation in another study.The assessment battery comprised structured, standardized, and locally translated instruments. Measures not previously used in Uganda were forward and back translated and locally adapted and piloted before use , 55. AssChild & Adolescent Symptom Inventory-5 (CASI-5)  with an impairment rating  for each disorder. The CASI-5 can be utilized to obtain a symptom cutoff score (number of symptoms required for a DSM-5 diagnosis rated “2” or higher), an impairment cutoff score , a clinical cutoff score (has to fulfill both symptoms cutoff and impairment cutoff scores), and a symptom severity score . The symptom severity scores are calculated by adding the individual ratings of each of the symptoms for each disorder. We evaluated for the presence of EBPs utilizing the symptom cutoff score (see r > 0.65), and convergent, divergent, and discriminant validity in various settings (Caregivers completed the parent version of the (CASI-5) , which icore see Table 1 core see , includicore see , and hassettings –61. The settings  and intesettings .Youth’s Inventory-4 (YI-4)  and test–retest reliability (r between 0.54 and 0.92) and aligns well with other self-report measures and clinical diagnoses (Adolescents between 12 and 18 years old completed the 4 (YI-4) , which iiagnoses . Internaiagnoses .The Child Self-Report Inventory-4 (CI-4) (4 (CI-4)  is a par4 (CI-4) , 64. In The Self-Reporting Questionnaire (SRQ-20) ((SRQ-20)  is a bri(SRQ-20) . The SRQTo assess for HIV-related stigma, adolescents were asked five questions (yes/no) pertaining to stigma experienced in the prior year. The questions asked about i) being teased at home because of HIV status, ii) being teased at school/work because of HIV status, iii) being discriminated at home because of HIV status, iv) being discriminated at school/work because of HIV status, and v) having lost friends because of HIV status. Adolescents responding “yes” to any of the questions were regarded as having experienced HIV stigma in the prior year (yes/no).Child/adolescent characteristics: Gender , age (continuous in years), and whether the child missed any days of school in the last term (yes/no).Household characteristics: Study site , who the child lives with , food security , and socioeconomic index based on common household items  constructed for use in Uganda (n Uganda  (includiHIV characteristics: Whether the child was born with HIV (yes/no), nadir CD4 cell count , current CD4 cell count , reported WHO HIV clinical stage , currently on antiretroviral treatment , possible virological treatment failure , whether the adolescent missed any ARV doses in the past 3 days (yes/no), and whether HIV status has been disclosed to the child (yes/no).Caregiver characteristics: Gender , age (continuous in years), caregiver status , employment (yes/no), HLOE , marital status , caregiver also caring for other children (yes/no), caregiver HIV positive (yes/no), and caregiver health status .EBPs: Rates of individual EBPs  of self-rated and caregiver-rated EBPs and sociodemographic and clinical variables (as specified above). We compared the rates reported by the caregivers and CA-HIV by conducting chi-square or Fisher’s exact tests as indicated.z scores. We subtracted the z score obtained for the CA-HIV from the z score obtained for the caregivers to obtain the standardized difference (discrepancy) score for EPs and BPs. Positive scores indicate caregivers rating problems as more severe and negative scores indicate CA-HIV rating problems as more severe. We repeated the same process using averaged severity scores to compute the discrepancy scores, which were used in sensitivity analyses.Agreement between caregiver- and youth self-rated disorders was evaluated with the kappa statistic based on symptom cutoff scores  and Pearson correlation coefficients for severity scores . To measure the discrepancy between caregivers and CA-HIV, we used a recommended approach by calculating standardized difference scores . We trant tests and analyses of variance (ANOVAs) were conducted for categorical variables and Pearson correlation coefficients for continuous variables. Variables with a p value of less than 0.1 were entered into multiple linear regression models to assess factors that each best predicted the discrepancy between caregivers and CA-HIV of EPs and BPs. CA-HIV age and gender were included in each of the models, even if their p values were not significant on univariate analyses. To assess for the effects of HIV stigma and caregiver psychological distress on discrepancy scores, we added the variables “experienced HIV stigma” and “SRQ-20 total score” as an additional step to the final model. We conducted sensitivity analyses by repeating the regression models, but with discrepancy scores based on averaged severity scores as the dependent variables. We did not correct for multiple comparisons due to the exploratory nature of the analyses. Analyses were conducted using SPSS version 25 software package , and all tests were two-tailed with the alpha (α) set at 0.05.To identify clinical and sociodemographic factors that were associated with discrepancy between caregivers and CA-HIV on EPs or BPs, n = 33, 7.0%) were older age , urban study site , a higher socioeconomic index , caregivers interviewed not the parents or grandparents , and caregivers being employed . Factors associated with children who were excluded  were urban study site , WHO clinical stage 1 or 2 , caregiver younger , and caregiver not HIV positive .The overall sample of 1,339 included 351 children under the age of 8 years who did not qualify for inclusion in this study, as they were too young to complete the CI-4. We excluded one adolescent aged 18 years and three CA-HIV for whom age data were missing. We excluded a further 18 children who had not completed the CI-4 and four adolescents who had not completed the YI-4. Our final sample included 493 children who had completed the CI-4 and 469 adolescents who had completed the YI-4. CASI-5 data were missing for 28 (5.9%) adolescents, and five (1.0%) children were excluded from the analysis as the caregivers who completed the CASI-5 were not above the age of 17 years. Factors associated with adolescents who were excluded (The mean age of CA-HIV was 11.9 (SD = 2.6) years, and 52.9% of CA-HIV were female. The majority of CA-HIV (94.2%) were perinatally infected and were receiving ART (95.4%). Eighty-seven (19.7%) of the adolescents reported experiencing HIV-related stigma in the prior year, with 31 (7.0%) answering “yes” to two or more questions. Of the caregivers assessed, 177 (19.1%) scored ≥6 on the SRQ-20 , and the median score on the SRQ-20 was 4.0 . For detailed descriptive data, see p = 0.029], particularly conduct disorder  and substance use problems .Based on symptom count cutoff scores, caregiver- and self- reported BPs were present among 9.2% and 14.5% of adolescents, respectively. Adolescents self-reported a mean number of 0.21 (SD = 0.58) BPs, and of those reporting BPs, 30.9% reported having at least two BPs. Adolescents self-reported more BPs than their caregivers , specifically higher rates of specific phobia , panic attacks , posttraumatic stress disorder (PTSD) , somatic symptoms , and separation anxiety disorder . Adolescents self-reported higher rates of social anxiety disorder .Caregivers reported that 55.7% of adolescents met symptom count criteria for an EP compared with 28.8% according to adolescent self-report. The mean number of self-reported EPs was 0.54 (SD = 1.12), and of those adolescents reporting EPs, 43.7% met criteria for at least two, and 21.5% met criteria for at least three EPs. Caregivers reported more EPs than adolescents , specifically specific phobia , PTSD , and somatic symptoms . Children self-reported higher rates of SAD .Caregivers indicated that 54.8% of the children met criteria for an EP whereas 36.9% of the children self-reported EPs. The mean number of self-reported EPs was 0.64 (1.07), and of the children self-reporting EPs, 42.9% reported at least two EPs and 18.1% reported three or more. Caregivers reported more EPs than children  and caregiver HIV status . These variables were added to the linear regression model along with caregiver caring for other children , caregiver gender , adolescent gender , and adolescent age  , caregiver employment status , and caregiver HIV status . Discrepancy scores decreased with advancing adolescent age and caregivers rated BPs as less severe than adolescents if caregivers were HIV positive and unemployed.Discrepancy scores for BPs were significantly different by caregiver employment status . When these variables were added to the model, the model was significantly improved  and discrepancy scores were significantly associated with caregiver SRQ-20 scores , such that caregivers with higher levels of psychological distress rated BPs as being more severe than adolescents self-rated.Discrepancy scores were significantly associated with caregiver SRQ-20 scores , CA-HIV WHO HIV clinical stage , caregiver age , caregiver HLOE , and caregiver caring for other children . These variables were added to the linear regression model along with adolescent gender  and study site  , caregivers caring for other children , and a tertiary HLOE in caregivers . Caregivers rated EPs as being more severe than adolescents if the adolescents had WHO HIV stage 4 compared to stage 1, if the caregiver had a tertiary HLOE as compared to no formal education, and if the caregiver was also caring for other children.Discrepancy scores for EPs were significantly associated with adolescent age  and with HIV stigma experienced by adolescent . The model was significantly improved  when these variables were added and discrepancy scores were significantly associated with HIV stigma experienced by adolescents  and with caregiver SRQ-20 scores . Caring for other children was no longer significantly associated with discrepancy scores . Adolescents who had experienced stigma in the prior year rated EPs as more severe than caregivers and caregivers with increased psychological distress rated EPs more severe than adolescents self-rated.Discrepancy scores were significantly associated with caregiver SRQ-20 scores , but not outside the home only  or in both settings , self-rated their EPs as significantly more severe than their caregivers rated, compared with adolescents who did not endorse experiencing HIV-related stigma.To assess for possible effects related to the context in which adolescents had experienced HIV-related stigma, we performed r = 0.10, p = 0.011), CA-HIV WHO HIV clinical stage , caregiver age , caregiver HLOE , and caregiver health status . These factors were added to the linear regression model along with study site  and child gender  , caregiver age , caregiver health status , and caregivers having a primary , secondary , and tertiary HLOE . Discrepancy scores decreased with advancing child and caregiver age. Caregivers rated EPs as more severe than children rated EPs if caregivers rated their own health status as “average” or “poor” as compared to “good” or “very good” or if caregivers had any level of education as compared to no formal education.Discrepancy scores for EPs were associated with child age , and the model was significantly improved when SRQ-20 scores were added to the model . Discrepancy scores were significantly associated with caregiver SRQ-20 scores . Caregiver age  and caregiver health status  were no longer significantly associated with discrepancy scores, with a pronounced change for caregiver health status. Similar to adolescents, caregivers with increased psychological distress rated EPs as more severe than children self-rated.Discrepancy scores were significantly associated with caregiver SRQ-20 scores  from the main results are reported.r = 0.986, p < 0.001). Adolescent age  was only trend significantly associated with discrepancy scores.Discrepancy scores based on averaged total severity scores were significantly correlated with discrepancy scores using total severity scores . Discrepancy scores for EPs were significantly associated with adolescent age  and WHO stage 2 , such that discrepancy in reporting EPs decreased with advancing adolescent age and caregivers rated EPs as more severe than adolescents if adolescents were in WHO HIV stage 2 as compared to stage 1.Discrepancy scores based on averaged total severity scores were significantly correlated with discrepancy scores using total severity scores . Child age  and caregivers having a primary  level of education were only trend significantly associated with discrepancy scores.Discrepancy scores based on averaged total severity scores were significantly correlated with discrepancy scores using total severity scores , largely, were associated with caregiver-rated EBPs, but not with youth self-rated EBPs . In cont3) were more likely to have caregiver-reported conduct problems  class C diagnosis was associated with having at least one prior psychiatric disorder diagnosis and having received prior mental health treatment . Anotherproblems . In a Maproblems .Similar to our research, other studies have also found that discrepancies were not related to who the caregiver was , 77, altAdolescents who reported experiencing HIV-related stigma in the prior year rated EPs, but not BPs, as more severe than their caregivers did. Of note, when the setting where stigma had been experienced was evaluated, this association was significant for stigma occurring only within the home environment. Other studies in Ugandan adolescents have also demonstrated that HIV-related stigma was associated with increased self-rated mental health problems , 29. AdoIncreased severity of caregiver psychological distress was associated with caregivers rating EPs and BPs as more severe than adolescents self-rated. The association was stronger for EPs than BPs and psychological distress ratings suggestive of possible depression in caregivers was also associated with increased discrepancy of EPs, but not BPs, on bivariate analysis. Our findings are consistent with the majority of the literature , 40, 41,One of the strengths of the study is the relatively large sample of CA-HIV from a region with a high HIV prevalence. It also includes youth self-report, which is often not assessed in CA-HIV studies, providing unique information on context and how CA-HIV view their own EBPs. This study also considered a broader range of sociodemographic variables than has typically been evaluated in studies examining discrepancy. The assessment instruments adopted for this study were previously used in a large-scale study of CA-HIV from the US , thus faThis study also has some notable limitations. We utilized a cross-sectional research design and as such could not determine cause-and-effect relationships. The EBPs reported by caregivers and CA-HIV were not confirmed with a clinical interview, and thus we cannot comment on specificity and sensitivity in this sample. The sample included differed from the sample excluded, and this may have influenced results. Adolescents who were older, living in urban areas, who had a higher socioeconomic index, with caregivers interviewed who were employed and who were not their parents or grandparents were more likely to be excluded. The main reasons these participants were excluded was that caregivers did not complete the CASI-5, and within this context, the differences could be explained by caregivers who were likely unable to complete the assessment as they were working, and that older adolescents were more likely to attend appointments unaccompanied. Children who were residing in urban areas, had a WHO clinical stage 1 or 2, with caregivers interviewed who were younger and HIV negative were more likely to be excluded. The age discrepancy with caregivers is likely due to excluding caregivers who were not older than 17 years; the reasons for the other differences are less apparent. We grouped EBPs together into broad constructs when assessing discrepancy and thus cannot comment on factors associated with discrepancy for individual disorders. The approach we used to determine overall severity of BP and EPs may also have implications. We used total severity scores obtained for disorders to compute discrepancy scores, which may have weighted total severity towards disorders with more symptoms. However, sensitivity analysis with discrepancy scores determined with averaged severity scores mostly provided the same results, with some differences noted. The differences were of a small magnitude, such that significant associations became trend significant and vice versa. The strength of association for the factors that differed according to approach used to determine severity scores was thus likely small. Future studies can perform more in-depth analysis evaluating agreement and discrepancy for individual disorders as it is beyond the scope of this manuscript. Although we noted receipt of ART, data were not collected on specific ARV regimens. Lastly, we did not correct for multiple comparisons (and type I error) and thus our results should be considered exploratory.Similar to other studies, we demonstrate that CA-HIV commonly experience EBPs, whether based on caregiver or youth self-report. Furthermore, CA-HIV may be prone to develop more severe psychopathology as reflected by higher rates of psychiatric hospitalizations and increased rates of prior psychotropic and behavioral treatments , 83. MenTo our knowledge, this is the first study to evaluate informant discrepancy of EBPs in CA-HIV. Discrepancy between caregivers and CA-HIV was greater if caregivers were unemployed and had no formal education. These results suggest that socioeconomic status may influence the discordance between caregivers and CA-HIV regarding the presence of EBPs. Although many studies have not found an association between socioeconomic status and informant discrepancies, a meta-analysis demonstrated that agreement between mothers and fathers on internalizing and externalizing problems was lower for children of low socioeconomic status , 86. Of Informant discrepancies have been linked to poorer youth outcomes , 43. FewThe study was conducted in accordance with the Declaration of Helsinki and ethical approval was obtained from the Uganda Virus Research Institute’s Research and Ethics Committee, the Ethics Committee of the London School of Hygiene and Tropical Medicine, and the Uganda National Council of Science and Technology. Eligible study participants provided written informed consent (caregiver) and assent (CA-HIV) after explanation of the study objectives and procedures. No CA-HIV were enrolled without their assent and all participants were informed that they could withdraw without prejudice at any time. In the majority of cases, the parents provided informed consent for participation of the CA-HIV, but in cases where the primary caregivers were not parents, the guardians of the CA-HIV provided the informed consent.EK, JL, KG, and VP contributed to the concept and design of the study. Data collection was done by EK, JL, and RM. LH performed statistical data analysis and wrote the paper. All authors contributed to revising the manuscript and have read and approved the submitted version.This work was supported by an MRC/DFID African Leadership Award to Eugene Kinyanda, Number MR/L004623/1. Leigh van den Heuvel was supported in this work by a NIH-Fogarty Project Titled: “Pittsburgh-Stellenbosch University AIDS-comorbidities Training Research Programme ” and her work reported herein was made possible through funding by the South African Medical Research Council through its Division of Research Capacity Development under the SAMRC CLINICIAN RESEARCHER (M.D PHD) SCHOLARSHIP PROGRAMME from funding received from the South African National Treasury. The content hereof is the sole responsibility of the authors and do not necessarily represent the official views of the SAMRC or the funders. Soraya Seedat is supported by the South African Research Chairs Initiative in PTSD funded by the Department of Science and Technology and the National Research Foundation.Funders have played no role in the study design, data collection, analysis, and interpretation and in writing the manuscript.KG is a shareholder in Checkmate Plus and publisher of the CASI-5, YI-4, and CI-4.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer JA declared a shared affiliation, with no collaboration, with one of the authors JN to the handling editor.


Under the condition of this study, heat-treated nickel–titanium (NiTi) files with smaller cross-sectional area, fewer contact points, and reciprocating movements resulted in a lower screw-in effect. This study compared the maximum screw-in forces of various instruments during their movements. Forty simulated canals in resin blocks were randomly divided into four groups (n = 10): ProTaper Universal F2, ProTaper Gold F2, WaveOne Primary, and WaveOne Gold Primary. To standardize a lumen size, all artificial canals were prepared with ProTaper Universal F1. The rotation speed was set at 350 rpm with an automated 4 mm pecking motion at a speed of 1 mm/s. The pecking depth was increased by 1 mm for each pecking motion until the file reached the working length. During instrumentation, screw-in forces were automatically recorded by customized software. Maximum screw-in forces were analyzed by one-way ANOVA and Tukey’s post hoc comparison with the significance level at 0.05. WaveOne Gold files generated the lowest maximum screw-in forces, followed by ProTaper Gold, WaveOne, and ProTaper Universal ( Because of superior flexibility and higher torsional fracture resistance of nickel–titanium (NiTi) instruments compared to those of stainless-steel (SS) instruments, NiTi instruments are widely chosen for the root canal treatment . ShapingGeometric features and heat treatment could influence the mechanical properties ,5,6 and This screw-in phenomenon has been reported to occur frequently when the cutting tip of the rotary instruments are active and have sharp fluted edges ,14. OverVarious kinds of engine-driven NiTi instrument systems made from different alloys and geometric designs are marketed in contemporary endodontics. Few studies have examined the relationship between the features of NiTi files and the screw-in tendency. Previous studies have demonstrated that the screw-in tendency is affected by cross-sectional design and pitch length rather than shaping motion ,20. The Therefore, the aim of this study was to compare the screw-in forces of various instruments with different heat treatments and kinetics during their movements. The null hypothesis was that the heat treatment, geometric properties, and kinetics of endodontic files will not influence the screw-in force during simulated root canal preparation.Four file systems made of different alloys and with different kinetic movements were tested: Two continuous rotation systems  and ProTaper Gold F2  and two reciprocating systems  and WaveOne Gold Primary ). PTU is made of conventional NiTi alloy; WOP is made by using M-wire; PTG and WOG are made with “Gold-Wire” . All instruments have a tip size of ISO #25 with a different apical taper: PTU, PTG, and WOP have 8% apical taper; WOG has 7% apical taper. Their cross-sectional area at D4, helical angle, and pitch length were measured using 30× magnification under a stereomicroscope  a–d.Forty simulated J-shaped root canals in resin blocks (Dentsply Sirona) were used in this study (n = 10). The curvature of root canal was determined to 35° according to the Schneider method , and theA custom-made test device   was fabrp < 0.05. All statistical analyses were performed using SPSS version 22 for Windows .The maximum screw-in forces during the instrumentation procedures were checked for the normality of distribution using the Kolmogorov–Smirnov test. Then, the data were analyzed using the one-way analysis of variance and Tukey’s post hoc comparison test. The level of significance was set at p < 0.05).The mean and standard deviation values of screw-in forces for different tested endodontic files are shown in Previous studies have reported that the geometric features and metallurgical properties of the NiTi file systems have an effect on their mechanical performances ,25,26,27The resin blocks with simulated root canals were used for the standardization of experimental condition and might have a methodological limitation. Even though experimental conditions were standardized by the use of simulated resin canals, the properties of resin material are different from those of natural dentin. Although extracted natural teeth could be used to simulate a clinical condition, the screw-in forces generated during root canal shaping may be highly affected by the anatomic deviations such as canal curvature and lumen size shown in natural teeth . A previ2 at a 4 mm level) and a unique cross-sectional geometry  similar to a slender rectangular and one- or two-point contacts with the dentinal wall during root canal preparation. These geometric features of WOG might have contributed to generating the lowest screw-in forces during instrumentation.Both the geometric design and manufacturing process (heat treatment) of instruments may affect the flexibility and the reaction forces as well as the screw-in effect . In the In contrast, PTU showed the highest screw-in forces. PTU is made of conventional NiTi alloy and has a triangular cross-sectional area designed to cut root dentin with three-point symmetrical contacts. PTU also has a larger taper (0.08 taper for F2) compared with WOG (0.07 taper for Primary). These characteristics increase its rigidity and may produce a higher screw-in effect.In the present study, PTG showed the lower maximum screw-in force compared with PTU, despite these two file systems having similar geometric features. This might be explained by the characteristics of Gold-Wire, which might share thermomechanical properties with CM-wire, and cause the least amount of canal transportation due to its higher flexibility and minimized reaction force ,31. The Manufacturers have claimed that reciprocating movements could reduce the screw-in effect . A transAlthough the screw-in tendency is harmful to root canal preparation, to date few studies have reported the screw-in effect related to NiTi instruments ,19,20. CUnder the limitations of this study, heat-treated NiTi files (WOG and PTG) generated lower screw-in forces compared to M-wire (WOP) and conventional NiTi files (PTU). WOG, which has a smaller cross-sectional area with fewer contact points, generated lower screw-in forces. Reciprocating movement adopted by WOP and WOG may also have effect on the decreased screw-in effect.


While the value of Ki-67 has been recognized in breast cancer, controversy also exists. The goal of this study is to show the prognostic value of Ki-67 according to progesterone receptor (PgR) expression in patients who have estrogen receptor- (ER-) positive, human epidermal growth factor receptor 2- (HER2-) negative early breast cancer.  The records of nonmetastatic invasive breast cancer patients who underwent surgery at a single institution between 2009 and 2012 were reviewed. Primary end point was recurrence-free survival (RFS), and secondary end point was overall survival (OS). Ki-67 and PgR were assessed with immunohistochemistry for the tumor after surgery. p < 0.001 and 0.005, respectively, for RFS and OS). There was no significant difference in RFS and OS according to Ki-67 when the PgR was high (p=0.120 and 0.076). RFS of four groups according to high/low Ki-67 and PgR expression was compared. The low PgR and high Ki-67 expression group showed worst outcome among them (p < 0.001). In a multivariate analysis, high Ki-67 was an independent prognostic factor when the PgR was low .  A total of 1848 patients were enrolled in this study. 223 (12%) patients had high (≥10%) Ki-67, and 1625 (88%) had low Ki-67 expression. Significantly worse RFS and OS were observed in the high vs. low Ki-67 expression only when the PgR was low (<20%) ( Ki-67 had a value as a prognostic factor only under low PgR expression level in early breast cancer. PgR should be considered in evaluating the prognosis of breast cancer patients using Ki-67. The prognosis of breast cancer patients is highly variable and depends on several characteristics. Breast cancers represent a heterogeneous group of tumors with histopathological, immunohistochemical (IHC), and genetic differences –4. CliniIncreased proliferation is a hallmark of malignant tumors and is an essential parameter for the prediction of therapy response . Ki-67 iThe Saint Gallen Consensus recognized a distinction between “luminal A-like” and “luminal B-like” tumor and supported the value of Ki-67 for the robust prognostic information it conveys . The devThe goal of this study was to clarify the independent prognostic value of determining Ki-67 expression. To this end, we investigated the relationship between Ki-67 and PgR expression levels in clinical practice and correlated the expression of these markers with clinicopathologic variables.th edition) pathologic staging system. Recurrence was divided into locoregional and distant. Contralateral recurrence was not included among recurrent categories in this study. The primary end point was recurrence-free survival (RFS) in relation to Ki-67 and PgR expression status. The follow-up period corresponded to the interval from surgery to the last date of a hospital visit, regardless of the visited department. The secondary end point was overall survival (OS). For follow-ups, electronic medical records of patients were reviewed up to November 2015; deaths were recorded based on reports as of December 2013. RFS was classified into four groups based on correlations with Ki-67 and PgR expression.The records of 1848 patients with pathologically confirmed invasive breast cancer who underwent surgery at the Department of Surgery, Seoul National University Hospital , between July 2009 and December 2012 were retrospectively collected. Patients with ER-positive and HER2-negative breast cancer were included, irrespective of PgR status. Patients diagnosed with in-situ carcinoma or distant metastasis at initial diagnosis or who previously had surgery for breast cancer were excluded, as were those for whom data on PgR or Ki-67 were unavailable. We did not exclude patients who underwent neoadjuvant systemic therapy. IHC analysis was performed from the tissue using core biopsy in a diagnosis. If information from initial tissues was insufficient, we used permanent sections in the patients who were not received neoadjuvant systemic treatment. Biopsy tissue was obtained from patients who underwent neoadjuvant systemic therapy prior to treatment. The study population comprised patients with a diagnosis of stage I to IIIC according to the AJCC , PgR , HER2 , and Ki-67  at the indicated dilutions. Sections were then incubated with the biotinylated anti-mouse secondary antibody and stained using streptavidin horseradish peroxidase . The sections were counterstained with Mayer's hematoxylin, dehydrated, cleared, and then mounted for examination. IHC samples were analyzed by one experienced pathologist at SNUH. The cutoff value used to define low versus high Ki-67 expression was the presence of Ki-67 immunoreactivity in more than 10% of stained nuclei in tumor tissues. The 10% cutoff for Ki-67 was found to have the best predictive value for prognosis at SNUH [ER, PgR, HER2, and Ki-67 expression were determined by IHC in formalin-fixed, paraffin-embedded tissue blocks. Expression levels of hormone receptors, HER2, and Ki-67 were assessed using the avidin-biotin complex technique . Tissues at SNUH . Patientt-test was used for comparing continuous variables between two groups. RFS was defined as the interval from the date of operation to the date of the first observation of a recurrence or the last follow-up date without evidence of recurrence. OS was defined as the interval from the date of operation to the date of death or last follow-up. Survival rates were estimated using the Kaplan–Meier method, and differences between two groups were compared using the log-rank test. In univariate and multivariate analyses of survival rates, Cox proportional hazard regression was used with adjustment for various factors. Cox regression analyses were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). Values were two-sided, and statistical significance was defined as a p value <0.05. All statistical analyses were performed using SPSS software, version 21 for Windows .Patients were divided into low and high Ki-67 groups and low and high PgR groups. Clinicopathologic characteristics were assessed using all pairwise comparisons of groups. Categorical variables were compared using chi-square or Fisher's exact test. Student's p < 0.001) and low PgR (p < 0.001). Histologic grade (HG) dichotomized samples into low and high subsets, with grade 3 are being classified as high and grade 1 + 2 is classified as low. Node-positive and high-HG samples were also identified in high Ki-67 and low PgR subsets. Significantly, more cases with low PgR underwent neoadjuvant chemotherapy, regardless of Ki-67 status. In addition, more patients with high Ki-67 underwent adjuvant chemotherapy, regardless of PgR status. We also considered tumor characteristics according to the operation method. A total of 719 patients underwent mastectomy, with a larger number of these patients having low PgR expression than high PgR expression. However, there was no significant association between Ki-67 subsets and the frequency of mastectomy. Only PgR subsets were significantly different among axilla surgery types. Recurrence, whether local or distant, was observed in 52 patients. Recurrence was local in 10 cases and distant in 42 cases. Mortality findings are based on public data from the Ministry of the Interior; as of 2013 , 11 patients had died  were more commonly associated with high Ki-67  was worse than that in the group with low Ki-67 expression and low PgR expression  [Here, we evaluated the value of Ki-67 as an independent prognostic factor for recurrence and survival in nonmetastatic breast cancer patients with ER-positive and HER2-negative tumors. Consistent with previous reports, our study showed that Ki-67 expression exhibited significant prognostic value, but we further demonstrated that Ki-67 is not always an independent prognostic factor. Specifically, Ki-67 had value as a prognostic factor only in the low PgR expression group. In our study, a comparison of RFS among the four expression subgroups revealed the poorest prognosis in the low PgR high Ki-67 subgroup. And the subset was 120 out of 1848 patients. Therefore, active treatment may be considered in about 6% of patients. In terms of predicting prognosis, our findings suggest that combining the Ki-67 expression level with the PgR expression level improves predictive value. Allison et al. reported a strong correlation between high Oncotype DX Recurrence Scores with grade 3 and low-to-absent PR expression and Ki-67 > 10% . In addi< 0.001) . If a gep=0.416; p=0.664; p=0.526; Our paper raises several additional issues. Although Ki-67 has been studied as a prognostic marker in breast cancer for more than two decades, there are controversies surrounding the methods used for determining its expression and the overall analytical validity of published results. Analytical validity refers to the ability of a test to produce reproducible and accurate results. For a marker to have prognostic and predictive value, an evidence-based “optimal” cutoff point is essential. Thus, one reason for controversy surrounding the use of Ki-67 as a marker is the absence of a universally accepted standard cutoff value, which has resulted in the use of different specific threshold values by different laboratories . Our insTo our knowledge, this is one of the largest retrospective studies to analyze data from a high-quality clinical cancer registry on the routine use and prognostic significance of Ki-67. Although retrospective, it does have the advantage of comprising an unselected, nonmetastatic breast cancer population without selection bias. Notably, pathology and biomarker analyses were prospectively performed in a single, accredited laboratory, and thus represent a ‘real' assessment of the value of IHC in clinical practice.Despite the various limitations of Ki-67 as a marker, its clinical use in the breast cancer field has been adopted for several reasons. It is used to distinguish luminal tumors and is considered a prognostic factor. Evaluating Ki-67 by IHC is inexpensive and easy to implement without investments in sophisticated equipment, leading to the attractive concept of Ki-67 as a low-cost biomarker. The importance of clinical flexibility is fundamental, given the uncertainties surrounding the tailoring of different chemotherapy options to each case, highlighting the importance of establishing a more accurate role for Ki-67.In this study, we found that Ki-67 is an effective prognostic marker only in the context of low PgR status. We further found that patients with low PgR and high Ki-67 expression had the worst prognosis in terms of RFS. From a global perspective, genomic signatures will remain difficult to access in the foreseeable future for most patients. Active treatment can be considered as the default in cases of early ER-positive and HER2-negative breast cancer that satisfy conditions of low PgR and high Ki-67.Our results show that Ki-67 has prognostic value for recurrence and survival in patients with ER-positive and HER2-negative early breast cancer only in the context of low PgR expression level. Thus, PgR expression should also be considered in evaluating the prognosis of breast cancer patients using Ki-67.


Pseudomonas aeruginosa. Additionally, resistance to chlorhexidine resulted in elevated expression levels of proteins associated with chaperones, efflux pumps, flagella and cell metabolism. This study provides a comprehensive overview of the evolutionary proteomic changes in P. aeruginosa following exposure to chlorhexidine and colistin. These results have important clinical implications considering the continuous application of chlorhexidine in hospitals that could influence the emergence of colistin-resistant strains.Use of chlorhexidine in clinical settings has led to concerns that repeated exposure of bacteria to sub-lethal doses of chlorhexidine might result in chlorhexidine resistance and cross resistance with other cationic antimicrobials including colistin, endogenous antimicrobial peptides (AMPs) and their mimics, ceragenins. We have previously shown that colistin-resistant Gram-negative bacteria remain susceptible to AMPs and ceragenins. Here, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins by serial exposure of standard strains of Gram-negative bacteria to chlorhexidine to generate resistant populations of organisms. Furthermore, we performed a proteomics study on the chlorhexidine-resistant strains and compared them to the wild-type strains to find the pathways by which bacteria develop resistance to chlorhexidine. Serial exposure of Gram-negative bacteria to chlorhexidine resulted in four- to eight-fold increases in minimum inhibitory concentrations (MICs). Chlorhexidine-resistant organisms showed decreased susceptibility to colistin (8- to 32-fold increases in MICs) despite not being exposed to colistin. In contrast, chlorhexidine-resistant organisms had the same MICs as the original strains when tested with representative AMPs (LL-37 and magainin I) and ceragenins (CSA-44 and CSA-131). These results imply that there may be a connection between the emergence of highly colistin-resistant Gram-negative pathogens and the prevalence of chlorhexidine usage. Yet, use of chlorhexidine may not impact innate immune defenses  and their mimics . Here, we also show that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in  Staphylococcus aureus (MRSA) and multi-drug resistant (MDR) Pseudomonas aeruginosa biofilms grown on hospital grade-surfaces, these pathogens were shown to maintain up to 11 and 80% of their cell viability respectively  for colistin increased by a factor between 16 and 32 with five of the six strains tested. However, when colistin-resistant K. pneumoniae were tested for chlorhexidine susceptibility, Salmonella, there are multiple mutations leading to chlorhexidine resistance that are not necessarily associated with colistin resistance.Due to the prevalent use of chlorhexidine, especially in clinical settings, there has been increasing concern about whether repeated exposures of bacteria to sub-lethal doses of chlorhexidine is a factor in the emergence of MDR bacteria . For exaptomycin . Of partptomycin , which iptomycin . Wand etCross resistance of bacteria to chlorhexidine and colistin may be due to common features of these antimicrobials. For example, both are cationic (positively charged) with hydrophobic functionality. Notably, most endogenous antimicrobial peptides , Acinetobacter baumannii (ATCC 19606), and P. aeruginosa (ATCC 27853) were obtained from the American Type Culture Collection . Ceragenins CSA-131 and CSA-44 were synthesized from a cholic acid scaffolding as previously described (escribed . LL-37 (P. aeruginosa or Mueller Hinton (MH) for K. pneumoniae and A. baumannii were dispensed in separate wells of a 96-well plate. Aliquots (100 μL) of a prepared inoculum (106 cfu/mL) were added, and plates were incubated at 37°C for 18–20 h. Negative and positive controls were included for each set of MIC measurements. The lowest concentrations of antibacterial agents that inhibited visible growth of bacteria were recorded as the MICs.Minimum inhibitory concentrations were determined using a broth microdilution method in a 96-well plate according to the Serial passaging of bacteria was performed according to a protocol described previously . BrieflyMHA plates were made in 60 mm × 15 mm Petri dishes with the addition of Congo red (40 mg/L) and Coomassie brilliant blue (20 mg/L) dyes. Bacterial cultures were grown overnight in MH broth at 37°C. An aliquot of bacterial culture (1 μL) was then spotted onto the MHA dyed plates and incubated at 37°C for 24 h . Images P. aeruginosa were cultured in TSB (25 mL) to an OD600 nm of 0.5. Cells were pelleted at 3750 × g for 10 min at room temperature followed by three washes with ice-cold PBS. Cell pellets were lysed in 1% sodium dodecyl sulfate (1 mL) in Tris buffer . Cells were briefly sonicated to further lyse the cells. Protein concentration was determined by bicinchoninic acid (BCA) protein assay . Protein digestion was carried out using filter-assisted digestion. Aliquots of 50 μg of protein were transferred to centrifugal filters (30 kD molecular weight cutoff), denatured with guanidine , and centrifuged for 15 min at 14000 × g. This step was repeated three times to remove SDS entirely. Cell lysate was reduced by DTT (20 mM in 6 M guanidinium hydrochloride) at 65°C for 5 min. After cooling for 5 min, alkylation was performed with iodoacetamide . The mixture was then washed two times with ammonium bicarbonate buffer . The washed, alkylated protein sample was re-suspended in ammonium bicarbonate  followed by overnight digestion at 37°C with trypsin . After trypsin digestion, peptides were isolated by spinning down the mixture  with one rinse of the filter using ABC . The combined filtrate was vacuum dried using a speedvac . The dried sample was immediately re-dissolved in 50 μL of buffer (0.1% formic acid in Optima-grade water) for mass spectrometry analysis  and drug-resistant analysis .® RSLC, C18, 2 μm, 100 Å, 75 μm × 25 cm) was used for separation of peptides. The mobile phase was comprised of buffer A (0.1% formic acid in Optima water) and buffer B (Optima water and 0.1% formic acid in 80% acetonitrile).Mass spectrometry data were collected using an Orbitrap Fusion Lumos mass spectrometer  coupled to an EASY-nLC 1200 liquid chromatography (LC) pump . A capillary RSLC column  with a reverse sequence decoy database concatenated and included during the search. After the removal of contaminants, low scoring peptides and reverse matches, the LFQ values for the top three peptides were summed and transformed by a logarithm of 2. The proteins were then separated into wild-type and mutant groups, and only those with at least three correct LFQ values were used for quantification. Accordingly, two-tailed heteroscedastic t-tests were conducted to compare wild-type versus mutant groups. The acquisition of gene ontology terms and functional clusters, as well as the identification of molecular functions and biological process, was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID) and UniProt. Information about protein-protein interaction networks was retrieved from STRING (v10).Label-free quantification (LFQ) values were obtained using PEAKS (version 8.5) and the Swiss-Prot validated Previous work has shown that Gram-negative bacteria become resistant to colistin following exposure to a sublethal concentration of colistin; however, colistin resistance does not correlate with higher MIC levels for AMPs or ceragenins . To deteA. baumannii and K. pneumoniae, however, formed colonies of irregular shape and had rough surfaces  were differentially expressed at a significant level , following exposure to chlorhexidine. Complete data analysis is provided as P. aeruginosa are listed in We used label-free quantitation to compare the relative concentrations of proteins within the proteome of wild-type and chlorhexidine-resistant We searched for relationships among the most upregulated  and the most downregulated  proteins and assigned them to functional clusters. The numbers of differentially expressed proteins for each cluster are shown in Due to the positive charge on chlorhexidine, its initial interactions with Gram-negative bacteria are thought to occur at negatively-charged regions of LPS. Further, reduced susceptibility to chlorhexidine has been previously reported in association with modifications in the cell membrane, including modifications to LPS . LPS modP. aeruginosa  LPS and less lipid A-core oligosaccharide. Its absence is also correlated with attenuated growth rate and dramatic morphological changes  was also observed. Upregulation of MurD may result in a protective modification by increasing the thickness or cross-linking the peptidoglycan layer, which subsequently reduces the efficacy of chlorhexidine by affecting its transportation. Mur enzymes have been an attractive target for the development of antibacterial inhibitors owing to the fact that these enzymes are highly conserved molecules specific to bacteria with a known structure and function. However, no Mur inhibitors are clinically used as antimicrobials .phoP/phoQ two-component system, a regulatory system controlling genes for virulence within Salmonella  was also shown to have an associated role . InteresP. aeruginosa  involved in fimbriae formation and motility. Formation of pili, or fimbriae, has been correlated with virulence in pathogens such as ormation . It has udomonas . Twitchiormation . The uprbacteria . OverallK. pneumoniae to colistin (We have demonstrated that prolonged exposure of Gram-negative bacteria to chlorhexidine results in significantly reduced susceptibility to colistin but not to AMPs and ceragenins. This suggests that there may be a specific mechanism for resistance of Gram-negative bacteria to chlorhexidine that causes even higher resistance to colistin but does not affect susceptibility to AMPs and ceragenins. These observations are consistent with the reported decrease in susceptibility of chlorhexidine-resistant colistin . The facP. aeruginosa and its chlorhexidine-resistant progeny. It is likely that proteins involved in resistance to chlorhexidine function similarly in other Gram-negative bacteria. Our study suggests that increased expression of membrane proteins such as OprF, LptD, and the Tol-Pal system is responsible for chlorhexidine resistance, but this is not the only pathway contributing to resistance. For example, chlorhexidine resistance is also associated with upregulation of PagL, which is affected by the two-component regulatory system phoP/phoQ. Additionally, upregulation of flagella proteins, chaperones and proteins associated with energy metabolism were also observed in the chlorhexidine-resistance pathway. Upregulation of some of these proteins also correlates with colistin resistance. Co-resistance with colistin suggests that LPS modification along with changes in expression of associated membrane proteins could be involved in the primary mechanism of chlorhexidine resistance. The fact that exposure of Gram-negative bacteria to chlorhexidine may lead to colistin resistance and possible drug resistance among Gram-positive organisms suggests that wide-spread use of chlorhexidine should be reconsidered. In order to decrease the speculated effect of chlorhexidine on the development of colistin-resistant strains, application of other antimicrobials such as ceragenins could be a safe choice considering its lower risk of cross-resistance.To understand the changes to Gram-negative bacteria that lead to chlorhexidine and colistin resistance but do not impact AMPs and ceragenins, an in-depth quantitative proteomic study was performed with a parent strain of MH and PS designed the experiments and wrote the manuscript. BSH, JC, MT, SW, BH, AZ, CM, RC, and SD performed the experiments. MH, JA, and JP performed data analysis.PS is a paid consultant for N8 Medical and CSA Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The study shows a low prevalence of primary or active CMV infection among the pregnant women, but the IgG seroprevalence suggests high previous CMV exposure. Importantly, CMV seroprevalence was not associated with the HIV status of the women, perhaps due to the ubiquitous exposure of the population to CMV.This study aimed to investigate the seroprevalence of cytomegalovirus (CMV) infection and risk factors associated with CMV acquisition among pregnant women in Zimbabwe. In a cross-sectional study, pregnant women were recruited in late gestation, seeking antenatal care at council clinics in three high-density suburbs in Harare, Zimbabwe. Anti-CMV IgM and IgG antibodies were quantified in serum using an enzyme-linked immunosorbent assay. Antibody avidity tests were used to distinguish active infection from viral reactivation in anti-CMV IgM-positive cases. Five hundred and twenty four women were recruited: 278 HIV infected and 246 HIV uninfected. Current or active CMV infection defined as IgM positive+low avidity was detected in 4.6% (24/524), 95% confidence interval (CI): 3–6.9 in all women, 5.8% (16/278) in the HIV infected and 3.3% (8/246), 95% CI: 1.4–6.3 in the HIV uninfected. IgG seroprevalence was 99.6% (522/524), 95% CI: 98.6–99.9 in all women. Notably, the difference in the prevalence of active CMV infection between the HIV-infected and HIV-uninfected women was not statistically significant ( Cytomegalovirus (CMV) infection is endemic worldwide, with a 30–61% seroprevalence in developed countries  and 60–1in utero, peripartum, and postpartum exposed infants  infection . Unlike  infants . The con infants ,25.Previous studies have informed that the risk of vertical transmission of CMV is greater in primary CMV infection (30–50% of cases) than in latent CMV reactivation or reinfection (0.2–3% of cases) ,31. HoweDespite the potentially disabling consequences of CMV infection during pregnancy and the unclear role of reactivated versus primary CMV infection in cCMV, there is limited information on the prevalence of CMV infection and its associated risk factors particularly among African populations. This is despite the high burden of CMV reported in the isolated studies performed in African settings. Determining CMV infection prevalence, especially among women of childbearing age, is important in estimating the risk of cCMV infection, magnitude of burden of maternal infection, as well as identifying risk groups that could be targeted for intervention . The curr = 1 and 80% power is 499 mother–infant pairs.In a cross-sectional study design, pregnant women in third trimester, presenting for routine antenatal care at three council polyclinics in the high-density suburbs of Harare, were recruited from February 2016 to August 2016. Only participants who provided written informed consent for both their participation and that of their to-be-born infants were recruited. The study was granted ethical clearance by the Medical Research Council of Zimbabwe (MRCZ/A/2177) and the University of Cape Town Ethics Review Board (628/2017). All human and health research procedures were in accordance with the ethical standards of the committees responsible for human research , as well as with the Helsinki Declaration of 1975, as revised in 2008. Assuming a difference of 13% in the prevalence of CMV  between This study was nested into the University of Zimbabwe-College of Health Sciences Birth Cohort, which aims to recruit at least 1000 well-characterized mother–infant pairs from Harare polyclinics. Participants of the birth cohort were followed up for 2 years. The Birth Cohort study aims to examine the impact on pregnancy outcomes, infant growth, immunity, and neurodevelopment of a range of factors, including coinfection with persistent viruses, maternal nutritional status, and breastfeeding, together with maternal HIV status, levels of immune suppression and immune activation, and antiretroviral therapy (ART) regimens in the HIV-infected women. This sub-study from the Birth Cohort recruited accessed a total of 527 pregnant women, 280 were HIV infected and 247 were HIV uninfected.+ T lymphocyte count or HIV viral load) to prevent mother-to-child transmission of HIV, which usually consists of TENOLAM-E . The Harare polyclinics provide routine antenatal through to postnatal care for mothers and their infants residing in and around the suburbs. The setup of these polyclinics is the same as all the other public polyclinics throughout Zimbabwe.HIV-infected and HIV-uninfected pregnant women ≥28 weeks of gestational age were successfully enrolled under the University of Zimbabwe-College of Health Sciences Birth Cohort exploring HIV exposure, disease acquisition, and progression among children.The current standard care for all HIV-infected pregnant women is Option B+  kit (CE 0197) according to the manufacturer's instructions . Participant specimens, standards, and procedural controls, provided by the manufacturer, were run simultaneously in duplicate. Extinction coefficients were determined at 450 nm. Specimens with an extinction ratio <0.8 were considered negative, extinction ratios of ≥0.8 to <1.1 were considered equivocal while extinction ratios ≥1.1 reflected positive tests for both anti-CMV IgG and IgM antibodies. Specimens with indeterminate test results were repeated and reassigned to a positive or negative group. IgG antibody titer was concurrently determined to estimate the strength or magnitude of an immune response to the CMV antigen.M urea added to detach low-avid antibodies (Participant specimens testing positive for anti-CMV IgM were subsequently tested for IgG avidity by ELISA according to the manufacturer's instructions (EUROIMMUN). The EUROIMMUN CMV IgG avidity assay comprises two parallel assays, one with and the other without 6 tibodies . This entibodies .p-value (p < 0.05) was considered statistically significant. Univariate logistic regression analysis was performed to investigate the association of CMV anti-IgM seropositivity with demographic and clinical factors.Study data were collected and managed using Research Electronic Data Capture (REDCap) . Data wep < 0.001)  compared with that of HIV-uninfected women . The median age of gestation among the pregnant women was 33 weeks (25th–75th percentile: 29–36) and was significantly higher (p < 0.001) in the HIV-uninfected group  compared with the HIV infected . The earlier presentation of HIV-infected women to clinic (measured by lower gestation age) could be due to improved health-seeking behavior induced by HIV treatment programs.Of the 527 participants enrolled for the main birth cohort, 524 participants' (278 HIV infected and 246 HIV uninfected) samples were accessed for this study. The demographic and clinical characteristics of the participants are shown in p < 0.001). The median gravidity in the study population was 2.5 (25th–75th percentile: 2–4). In line with the higher parity, the HIV-infected women also had significantly higher gravidity  compared with the HIV-uninfected women  (p < 0.001). The HIV-infected women were significantly more likely to be divorced or cohabiting than the HIV-uninfected women. Body mass index, level of education, and monthly income were comparable between the HIV-infected and HIV-uninfected women.The median parity in the study population was one child (25th–75th percentile: 0–2), but the HIV-infected women had significantly higher parity  compared with the HIV-uninfected women  (A summary of the anti-CMV antibody frequencies for this study is presented in n = 24) showed low avidity for anti-CMV IgG antibodies while the rest showed high avidity. Interestingly, anti-CMV IgG titers were significantly higher in the HIV-infected women  than in the HIV-uninfected women  (p < 0.001).We performed CMV IgG avidity test on the 39 participants who had positive results for both anti-CMV IgG and anti-CMV IgM, and 62%  was significantly associated with the risk of being seropositive for either anti-CMV IgG or IgM antibodies .We report a seroprevalence of 99.6% for anti-CMV IgG and 7.4% for IgM antibodies, in pregnant Zimbabwean women, with no significant differences in seroprevalence observed between the HIV-infected and HIV-uninfected groups. Thus, anti-CMV seropositivity was not significantly associated with HIV status in the study population. However, high anti-CMV Ig antibody titer was significantly associated with HIV positivity. There were no significant associations between anti-CMV seropositivity and demographic characteristics, such as age, parity, gravidity, level of education, and socioeconomic status. Our findings conflict with previous reports where demographic characteristics were significantly associated with either higher or lower risk of CMV infection ,30. HoweUnderstanding the epidemiology of CMV infection during pregnancy is essential for exploring control measures since cCMV infection is associated with potentially fatal and disabling effects. The high prevalence of anti-CMV IgG antibodies (99.6%) confirms reports in other studies on Egyptian, Ghanaian, Kenyan, Malawian populations and other non-African low-income countries ,19,22,30Markers of lower socioeconomic class have been previously reported as risk factors for CMV infection . EthniciIn previous studies carried out among infants in Zimbabwe, the prevalence of CMV infection at 6 weeks of age was greater than 70%, regardless of HIV exposure, emphasizing the early acquisition of CMV infection ,16. HoweIn contrast to the nearly 100% anti-CMV IgG seroprevalence, we found a seroprevalence of 7.4% for anti-CMV IgM antibodies. Presence of anti-CMV IgM antibodies is indicative of primary infection, reactivation of previous infection, or reinfection with a different viral strain , but witContrary to previous reports and the established coactivation between CMV infection and HIV infection ,13, antiAnti-CMV IgG antibody avidity was performed on all anti-CMV IgM positive participants to confirm current infection. Antibody avidity test is the most accessible test of choice to differentiate between current and past infection especially in the resource-limited settings where polymerase chain reaction (PCR) for CMV DNA detection may not be available . In our Our findings deviate from the study previously carried out among Egyptian pregnant women where all the participants who were positive for anti-CMV IgM antibodies had either high or intermediate avidity IgG antibodies . The disInterestingly, anti-CMV IgG antibody titer was significantly associated with HIV status, with the HIV-infected individuals more likely to have higher anti-CMV IgG antibody titer. We suspect that since there is a massive production of IgG antibodies during a secondary immune response to a pathogen, there could be reactivation of CMV resulting in the surge of anti-CMV IgG production . HoweverWith such alarming levels of CMV exposure in pregnancy, understanding the factors associated with CMV infection will go a long way in ameliorating the burden of cCMV. However, our study did not find any significant association between the risk of being either anti-CMV IgG or anti-CMV IgM positive with either income or education. Education and income as markers of socioeconomic status have been linked to CMV infection, with lower income and lower level of education being associated with a higher risk of being anti-CMV seropositive ,32. ZimbIn conclusion, we report a high prevalence of previous CMV exposure among women of childbearing age. Given that secondary CMV infection has been equally associated with higher cCMV to primary CMV infection in the developing countries, the finding calls for interventions to prevent vertical transmission of CMV to their offspring. CMV infection is independent of HIV status in the study population. The limitation of the study could have been the sample size where we had more HIV-infected participants than HIV-uninfected participants (278 HIV infected and 246 HIV uninfected).


Prostate cancer has become the most common form of non-cutaneous  malignancy in men, accounting for 26% of all new male visceral cancer cases in the UK. The aetiology and pathogenesis of prostate cancer are not understood, but given the age-adjusted geographical variations in prostate cancer incidence quoted in epidemiological studies, there is increasing interest in nutrition as a relevant factor. In particular, foods rich in phytochemicals have been proposed to reduce the risk of prostate cancer. Epidemiological studies have reported evidence that plant-based foods including cruciferous vegetables, garlic, tomatoes, pomegranate and green tea are associated with a significant reduction in the progression of prostate cancer. However, while there is well-documented mechanistic evidence at a cellular level of the manner by which individual dietary components may reduce the risk of prostate cancer or its progression, evidence from intervention studies is limited. Moreover, clinical trials investigating the link between the dietary bioactives found in these foods and prostate cancer have reported varied conclusions. Herein, we review the plant bioactives for which there is substantial evidence from epidemiological and human intervention studies. The aim of this review is to provide important insights into how particular plant bioactives  present in commonly consumed food groups may influence the development and progression of prostate cancer. The intimate relationship between the physical and chemical structure of individual foods, dietary patterns and human health is complex. Diets high in saturated fats have been found to be associated with an increased risk of type 2 diabetes, cardiovascular disease (CVD) and cancer , whilst As a result of prostate-specific antigen (PSA) testing and an ageing population, prostate cancer (PCa) has become the most commonly diagnosed form of non-cutaneous  cancer in men, accounting for 26% of all new male visceral cancer cases in the UK, with over 47,000 new diagnoses every year . PCa incThe highest age-standardised incidences of PCa worldwide are seen in Westernised countries, including Australia, New Zealand, Europe and the Americas, compared to the lowest in South Central Asia ,8. This p = 0.0008) ..24].Briefly, when GRN has been hydrolysed into SFN, it is passively absorbed into the enterocyte of the small or large intestine, where it is conjugated (either passively or via the activity of glutathione-S-transferases (GSTs)) with glutathione, for transportation into the systemic circulation. The conjugate is then either metabolised via the mercapturic acid pathway to produce thiol conjugates which are excreted in the urine, or dissociates or is cleaved by GST, to become free SFN  25,26].,26.25,26SFN has been shown to have multi-modal influences on a wide variety of metabolic and cell-signalling pathways involved in cancer including the induction of antioxidant pathways, induction of apoptosis in cancer cells, inhibition of inflammation and angiogenesis and detoxification of carcinogens . The mosResearch suggests that SFN also plays a significant role in the metabolic regulation of the prostate, thus leading to decreased PCa incidence and progression ,33. PrevAlterations in the acetylation patterns of histones are a characteristic feature in PCa progression . SFN hasp = 0.003) [Consistent with cell and animal models, epidemiological studies have demonstrated a link between the intake of cruciferous vegetables (particularly the active compound SFN) and a reduction in the incidence or progression of PCa ,41,42,43= 0.003) . HoweverIn one such randomised control trial (RCT) involving 98 patients scheduled for a prostate biopsy as part of their routine clinical care (who had not previously received any PCa-related treatment procedures), patients consumed either capsules containing myrosinase-treated broccoli seed extract (BSE) containing 100 µmol of SFN twice daily, or a matched placebo control for 4–6 weeks prior to their biopsy procedure . This stA further human intervention trial undertaken in Norfolk (UK) has demonstrated that the several hundred changes in gene expression and potentially oncogenic pathways  are suppressed in a dose-dependent manner by ingesting broccoli soups with increasing concentrations of GRN . This thAllium vegetables are responsible for their characteristic odour and flavour and comprise approximately 1% dry weight of garlic and 0.5% dry weight of onions. The S-alk(en)yl-L-cysteine sulfoxides (SACSOs) are the main sulfur-containing constituents within allium vegetables, and are precursors to the bioactive components. Alliin  is the major SACSO found in garlic, and isoalliin (trans-(+)-S-(propen-1-yl)-L-cysteine sulfoxide) is the predominant SACSO in onions [Alliaceous vegetables, including garlic, onions, leeks and shallots, are rich in a variety of bioactive compounds, such as flavonols, oligosaccharides, selenium, arginine and organosulfur compounds (OSCs), the latter being of most interest with regards to health benefits, especially in relation to cancer. The OSCs within n onions ,38,39. Wn onions .Multiple cell and animal models have suggested a protective role of alliaceous vegetables in cancer risk, with several reported mechanisms, each targeting most of the cancer hallmarks defined by Hanahan and Weinberg , such asp < 0.001), with the effect being most pronounced for garlic and scallions, and for the risk of developing localised PCa [Large-data-set epidemiological studies have supported this suggested negative correlation between the intake of alliaceous vegetables and the incidence of PCa ,54. Resuised PCa . Researcised PCa . A furthThere are currently no published human intervention studies analysing the effect of alliaceous vegetables on PCa. As part of a recently commenced randomised double-blinded clinical trial in Norwich (UK)  patients will consume dietary supplements of SACSO for 4–6 weeks prior to a TPB. This study aims to assess whether SACSO or its metabolites accumulate within the urine, plasma or prostate tissue, as well as modify the gene expression, when compared to a placebo control. This trial will contribute to our understanding of how these bioactives influence PCa progression. In addition to glucosinolates, cruciferous vegetables including broccoli contain other sulfur-containing metabolites, such as S-methyl-L-cysteine sulfoxide . SMCSO is found in higher concentrations (dry weight of 1%–4%) in comparison to glucosinolate concentration (0.1%–0.6%) . SMCSO iIn vitro studies on the effect of SMCSO and its breakdown products MMTSI and MMTSO on human PCa cells lines are well documented , but theAlong with the S-alk(en)yl cysteine sulfoxides, SMCSO is also known to accumulate within alliaceous vegetables such as garlic. As described above, SMCSO has been shown to accumulate within the prostate and urinary system . This cocis–trans configurations [trans-isomer . Isomerisation from trans-isomer to various other forms can occur secondary to heat, light, and chemical reactions or secondary to in vivo mechanisms [Lycopene is the most abundant carotenoid found in tomatoes, and accounts for >85% of all dietary sources . It is aurations . Despitechanisms ,62. trans isomers being deep red, compared with tetra-cis-lycopenes which are more orange in appearance [The lycopene concentration in fresh fruit varies greatly; up to 15 mg of lycopene has been found per 100 g of deep-red tomato varieties, compared with only 0.5 mg per 100 g of yellow tomatoes ,63. Procpearance .Lycopene is passively absorbed in the small intestinal mucosa via integration into dietary lipid micelles, along with other lipids and bile acids. Micelles are transported from the enterocyte to the liver in the form of chylomicrons in lymphatic fluid, prior to transportation in the plasma to target organs via low-density and very-low-density lipoproteins . The absThe multiple conjugated double bonds are believed to be responsible for lycopene’s various protective (anti-oxidant) effects. Its active singlet oxygen  quencher is believed to assist DNA repair and may react with oxygen free radicals by transfer of the unpaired electron, leaving the carotenoid in an excited triplet state. Indeed, lycopene has been shown to exhibit a singlet quenching ability twice as powerful as beta-carotene and tenfold greater than alpha-tocopherol (vitamin E) .Considerable in vivo work has demonstrated a potential antiproliferative effect of lycopene on PCa cells, with postulated mechanisms linked to cell cycle arrest and apoptosis. Lycopene has been shown to exert antiproliferative effects via its ability to induce G2/M-phase cell cycle arrest and apoptotic cell death on LNCaP cells, especially in unsynchronised cells . These fp = 0.017) and circulating concentrations  of lycopene were associated with reduced  PCa risk [p = 0.026), and PCa risk decreased by 3.5% to 3.6% for each additional 10 μg dL−1 of circulating lycopene. However, this association was not shown to be protective against the incidence of advanced PCa. Similarly, a systematic review and dose–response meta-analysis of dietary intake of lycopenes in relation to PCa risk also showed a dose-dependent associated reduced risk of PCa incidence , with no protection to the risk of advanced PCa [Most of the data linking the association between lycopenes and PCa originate from epidemiological or prospective studies. The literature surrounding the association with lycopene and PCa is mixed and remains controversial, particularly with reference to aggressive and/or advanced PCa. A systematic review and meta-analysis of 42 case–control studies  published up to 2016 showed that dietary intake  of PCa risk when comparing the highest versus lowest tertile of lycopene intake . These ip = 0.032) [In contrast to the above studies, a pooled analysis of 15 studies failed to show any association between the intake of seven major carotenoids and overall risk of PCa. However, high lycopene intake was shown to be inversely associated with risk of advanced stage PCa and aggressive disease  .p = 0.03) for high versus low quintiles, with the greatest risk reduction attributed to high tomato sauce intake  especially for extra-prostatic cancers . In addition to highly concentrated sources of carotenoids found in tomato sauces, the thermal processing involved in the cooking of tomato sauces acts to increase the bioavailability of lycopene; heat causes the binding matrices to disrupt, and an oily base improves uptake into micelles and subsequent absorption in the intestine [As is true for all nutrients, estimation of dietary lycopene intake through the use of questionnaires often creates bias . Furtherntestine .p = 0.0003) and degree of apoptotic cell death  in resected tumour areas when compared to the non-intervention controls [These findings are supported by the results of a clinical study in which 32 patients with biopsy-confirmed PCa were randomised into either a treatment arm (consuming 30 mg lycopene/day in the form of a tomato sauce) or a control arm, for 3 weeks prior to prostatectomy. Intervention with a tomato sauce resulted in an increased abundance of apoptotic cells  did not provide any evidence for lycopene supplementation in terms of protection from PCa incidence. Supplementation in this trial also included selenium. Larger human studies are required to elucidate the potential anti-PCa effects of lycopene .trans-3,5,4’-trihydroxystilbene) is a stilbenoid polyphenol belonging to the phytoestrogens. Two isomeric forms of resveratrol exist; the trans- isomer is the most stable and biologically active form, whereas the cis- isomer is formed following the breakdown of the trans- form secondary to the action of UV light or high-pH conditions during fermentation . Oral absorption of resveratrol is documented as being as high as 75%. Following metabolism, the major metabolites occurring in plasma and urine are sulfates and glucuronides (via deconjugation enzymes such as B-glucuronidase), reduced conjugates of which can accumulate to varying concentrations in tissue. Although intake of 5 g resveratrol daily is considered to be safe, evidence suggests that a daily intake of 0.5–1 g can elicit can reduce tumour cell proliferation in the GI tract , with miResveratrol appears to have a significant multi-modal anti-tumoral activity on PCa cells via increasing apoptosis, cell cycle arrest and downregulation of signal transduction pathways. Resveratrol has been shown to have proapoptotic effects on multiple PCa cell lines, including LNCaP, DU–145 and PC–3; and in the athymic xenograft LNCaP and TRAMP mouse models . In addiResveratrol has also been shown to down-regulate AR expression in TRAMP mouse models , and altp = 0.57) [p = 0.001), whereas moderate red wine consumption demonstrated a protective role, with a risk reduction of 12% . However, the exclusion of one large prospective study with a 16-year follow-up demonstrated no significant association between wine type and risk of PCa [Much of the clinical data supporting this relationship is derived from epidemiological studies. Alcohol intake has previously been associated with a significant dose–response increase in risk of PCa incidence and progression , but the = 0.57) . The defk of PCa . Howeverk of PCa .p = 0.052, 41% p ≤ 0.01 and 50% p < 0.001, respectively), although no effect on PSA, prostate size, testosterone or dihydrotestosterone levels was seen [A randomised placebo-controlled clinical study undertaken in middle-aged men with metabolic syndrome has shown that high-dose dietary supplementation of resveratrol for 4 months (1000 mg/day) resulted in significantly lower serum levels of androgen precursors than a control. Androstenedione, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate were all shown to be lowered by up to 50% , independently of mitogen-activated protein kinase kinase (MEK) signalling [EGCG has been shown to be one of the most potent modulators of the molecular pathways involved in prostatic carcinogenesis ,104. Pregnalling . Additiognalling , suggestKB cyclooxygenase-2 (COX-2), and the targeting of insulin-like growth factor (IGF) [EGCG influences other anti-cancer mechanisms through cell cycle arrest and apoptosis, including protein kinase C pathways, inflammatory pathways such as NF-or (IGF) .p = 0.08) for each cup of green tea consumed per day.However, meta-analyses of epidemiological studies have reported conflicting results. Three meta-analyses have all demonstrated no significant inverse association between green tea intake and risk of PCa  [In the first placebo-controlled clinical trial undertaken analysing the effect of green tea on PCa incidence, 60 patients with high-grade prostatic intraepithelial neoplasia (HGPIN) were randomised into either a placebo group or intervention, receiving 600 mg of green tea catechins per day. Over a 12-month follow up period, the incidence of progression to PCa (as diagnosed by prostate biopsies) was significantly reduced in the treatment arm  . However = 0.02) . However = 0.02) .Further human studies are required to investigate the effects of green tea on PCa incidence and progression, either alone or in combination with other treatment modalities.Curcuma longa (commonly known as Turmeric), an herbaceous perennial plant of the ginger family  and B cell lymphoma extra-large (Bcl-xL), and leading to an activation of caspase-3 and -8. Studies have confirmed the dose-dependent inhibition of PC3 cell viability, associated with a significant downregulation of mRNA and protein expression of inhibitor of DNA binding 1 (Id1) . Daily iThe increasing activity of the AR and the upregulation of AR-related co-factors such as cAMP response element-binding protein (CBP) and coactivator protein p300 are salient features of aggressive and hormone-resistant PCas. Curcumin has been shown to interact with these factors in LNCaP and PC-3 cell lines by inhibiting the expression of the AR , as wellp  =  0.0259) [Multiple clinical trials have been undertaken to support these findings. However, most of the trials have described the use of curcumin as an adjunctive treatment to either radiotherapy, hormonal or chemotherapeutic interventions. A recently published RCT was undertaken in patients managed with intermittent androgen deprivation (IAD) for the treatment of biochemical recurrence of PCa following localised treatment or metastatic disease at diagnosis. Patients received curcuminoid powder in capsule form at a total dose of 1440 mg/day for 6 months, commencing at the time of androgen deprivation treatment (ADT) withdrawal. Although there was no significant difference in the overall “off-treatment” duration of IAD, curcumin treatment resulted in a significantly lower PSA progression in comparison to the control group  .p < 0.001), as was a reduction in the activity of superoxide dismutase (SOD) (p = 0.026). PSA, however, was shown to be reduced to <0.2 ng/mL in all patients, with no significant difference between treatment arms [Due to the antioxidant, radiosensitising and radioprotective properties of curcumin, interest in the adjuvant treatment of curcumin with radiotherapy is gaining increasing interest. In a further RCT analysing oxidation status, patients received either adjuvant treatment of 3 g/day curcumin or placebo during external-beam radiotherapy (up to 74 Gy). Although plasma total antioxidant capacity (TAC) was seen to increase in all patients, this was significantly higher in the curcumin arm (ent arms .Punica granatum) have been heavily researched with regards to their bioactive compounds, which are deemed to be anti-microbial, anti-inflammatory and anti-cancerous [Both the seeds and juice of the pomegranate fruit , suggesting a potential anticancer activity of the bioactives within pomegranate . UrolithIn addition, pomegranate extract has been shown to exhibit an inhibitory effect on the NF-κB inflammatory pathway, thus enabling the extract to demonstrate its pro-apoptotic effect . Increasn = 104) which demonstrated that the consumption of either 1 or 3 g pomegranate extract for up to 18 months resulted in an increase in PSA doubling time  [n = 102) concluded that daily pomegranate intake has no impact of levels of PSA in patients with advanced PCa at one month [n = 70) demonstrated that daily pomegranate supplementation for up to four weeks prior to radical prostatectomy resulted in urolithin A accumulation in prostate tissue (p = 0.031), but had no effect on prostatic oxidative stress, cell proliferation, tumour progression or PSA levels [Much of the clinical research on pomegranate has focussed on its effects on CVD , but the< 0.001) . Howeverne month . A furthA levels .Whilst there is some evidence of pomegranate reducing PCa growth through murine studies and short-term intervention studies, the long-term impact of pomegranate consumption is not fully understood. Current clinical trials are ongoing and include: “Pomegranate Juice in Treating Patients with Recurrent PCa, ClinicalTrials.gov Identifier: NCT00060086” (data pending) which will contribute to our understanding of how these bioactives influence PCa progression. This study, in which up to 40 patients who have undergone radical prostatectomy or radiotherapy for PCa will consume oral pomegranate juice daily for 18 months, aims to determine whether pomegranate juice consumption can decrease or slow the rate of rising PSA levels. From the evidence reviewed, it is clear that certain dietary bioactives may be associated with either a reduction in the risk and/or progression of PCa . StudiesEvidence of the reflux of urine into the prostatic ducts was first published in 1982, when, following intra-vesical instillation of carbon microspheres, 70% of prostate tissue removed by transurethral resection was found to contain microspheres . AdditioDue to its ductal anatomy, the peripheral zone is the most likely part of the prostate to be exposed to urinary components. The ducts draining the peripheral glands enter the urethra as a double lateral line along the whole of the distal urethral segment. The ducts are narrow but enter the urethra at a less obtuse angle than those from other zones, thus potentially making them more prone to the reflux of urine, perhaps with a contribution from turbulence . This anThere is also increasing evidence for a microbiological community associated with both the urinary tract and the prostate itself ,161. WhiHealth claims pertaining to nutrition are highly regulated in order to inform as well as protect the public from false health declarations. In order to substantiate scientific findings, human intervention studies, although challenging, are therefore essential in supporting the role of plant-derived bioactives in preventing PCa ,167. CenOne option for determining the effects of individual plant-derived bioactives is to deliver pure compounds through dietary supplements. However, isolating pure compounds is sometimes challenging due to issues with stability, but also the potential to miss, in some cases, unknown compounds derived from the same plant of interest that may also exhibit bioactivity either additively or synergistically . AnotherAn alternative would be to utilise knowledge on levels of individual plant-derived bioactives across different plants/foods to select appropriate interventions. There are a handful of comprehensive and searchable databases with up-to-date coherent and validated scientific information on the composition of food-derived bioactives. The three core databases which provide extensive data on the composition of bioactive compounds in foods are: USDA Database for the Flavonoid Content of Foods Release 3.3 , Phenol-The eBASIS database is a collation of data relating to bioactive compounds in commonly consumed plant-based foods such as fruits as well as cruciferous and alliaceous vegetables. The database is divided into two principal sections: composition data and bioeffects data. The entire database includes data from over 1300 peer-reviewed publications, forming 44,000 data points, covering 800 compounds in over 270 food plants, with the data linked to authoritative plant and plant-part lists ,175,176.Many plant bioactives have been shown to exhibit multi-modal effects on PCa cells , and foo