To evaluate the performance of contrast-enhanced mammography (CEM) compared to magnetic resonance imaging (MRI) for estimating residual tumor size after neoadjuvant chemotherapy (NAC) in women with newly diagnosed breast cancer. The institutional review board approved this study. This prospective study included women with newly diagnosed breast cancer who underwent breast CEM and MRI at the end of the last cycle of NAC and before definitive surgery. Size of residual malignancy on post-NAC CEM and MRI was compared with surgical pathology. Agreements and correlations of CEM and MRI measurements with histological size were assessed. Thirty-three patients were included with a mean age of 45 years (range 22–76). The sensitivity, specificity, and positive and negative predictive value for detection of residual disease of CEM were 76%, 87.5%, 95%, and 86.4%, and those of MRI were 92%, 75%, 92%, and 75%. Comparing CEM to MRI, the mean difference was −0.8 cm, concordance coefficient was 0.7, and Pearson correlation was 0.7 (p = 0.0003). The concordance coefficient between measurements of each imaging modality and pathologic tumor size was 0.7 for CEM and 0.4 for MRI. Pearson correlation was 0.8 for CEM and 0.5 for MRI. Mean differences between CEM, MRI, and residual histopathological tumor size were 0.8 cm and 1.8 cm, respectively. CEM has good correlation and agreement with histopathology for measuring residual disease after NAC. CEM was comparable to MRI, showing high positive predictive value and specificity for detecting residual disease. Neoadjuvant chemotherapy (NAC) was used as a primary therapeutic strategy for locally advanced and inflammatory breast cancers. It may decrease the extent of the tumor, increasing the chances of successful breast-conservation surgery, and provide prognostic information to evaluate treatment response .Response to NAC is currently assessed by combining clinical examination and conventional imaging techniques such as mammography, breast magnetic resonance (MRI), and ultrasound (US). To date, breast MRI has proven to be superior to mammography and US in the assessment of tumor extent and presence of additional foci and highly sensitive in identifying residual disease following NAC –6. UnforContrast-enhanced mammography (CEM) is a new imaging tool that uses a dual-energy technique to combine the benefits of digital mammography with intravenous contrast utilization. In the diagnostic setting, CEM has a higher sensitivity for breast cancer detection compared with the sensitivities of full-field digital mammography (FFDM) alone and FFDM combined with ultrasound –16. For We aimed to evaluate the accuracy of CEM in assessing residual disease extent and predicting pathologic complete response (pCR) compared to MRI after completion of NAC in patients with a diagnosis of breast cancer.This prospective study was conducted to evaluate the performance of CEM for the diagnosis of residual breast cancer after NAC. This study enrolled patients who underwent NAC followed by surgery with curative intent for locally advanced primary breast cancer between August 2015 and December 2017. The eligibility criterion was pathologically confirmed breast cancer by core biopsy. Patients were excluded if they had (a) a pacemaker, (b) previous allergic reaction to contrast media, or (c) history of breast cancer treated with chemotherapy. The study was approved by the local ethics committee, and informed consent was obtained from all patients.Bilateral craniocaudal (CC) and mediolateral oblique (MLO) views were acquired using a commercially available FFDM system . A pair of low- and high-energy images during single-breast compression was obtained per view. Before image acquisition, 1.5 ml/kg of non-ionic contrast medium  was injected using an automated injector at a flow rate of 3 ml/s. The first image was obtained 1.5–2 min after injection initiation, and all images were obtained within 4–7 min. The complete examination protocol has been previously described and explained .2, TR/TE 12500/78.2 ms, bandwidth 250 kHz, FOV 32 cm × 32 cm, matrix 256 × 192, slice/gap 4/0.4 mm); sagittal 3D T1W fat suppressed volume imaging for breast assessment (VIBRANT)  acquired dynamically (1 pre- and 3 post-gadolinium injection phases) with a 87 s temporal resolution; and high spatial resolution post-contrast T1W fat suppressed 3D VIBRANT . Contrast medium (0.1 mmol/kg of gadodiamide) was injected with a 10-second timing delay into the antecubital vein using an 18 to 20-gauge needle at a flow rate of 2 mL/s followed by a flush of 20 ml of saline solution. The average acquisition time was 30 min.All breast MRI examinations were performed using a 1.5T MRI system  with a dedicated eight-channel breast coil. The following sequences were acquired: axial 2D T1W fast spoiled gradient echo ; axial 2D Short-TI Inversion Recovery ; axial 2D diffusion weighted imaging SE EPI  was used for interpretation of MRI studies. CEM examinations were interpreted on 5-megapixel monitors at full resolution with one breast on each monitor using a dedicated mammography software program .The response evaluation criteria in solid tumors (RECIST), version 1.1  was usedAll patients underwent breast-conserving surgery or mastectomy after completion of NAC, and surgical specimens were evaluated by a pathologist for the size of residual malignancy. Surgical pathology reports were reviewed for the following parameters: histologic tumor subtype, tumor size, estrogen receptor, progesterone receptor, and HER2 statuses. Tumor size was defined as the largest dimension based on macroscopic and histopathological examination. In the case of multifocal breast cancer, a sum of the diameters for all target lesions was calculated. We considered pCR as the absence of invasive cancer in the breast irrespective of ductal carcinoma in situ or nodal involvement (ypT0/is).Tumor sizes measured by CEM, FFDM, and MRI were compared to the tumor size measured in pathology (reference standard). Also, CEM and FFDM results were compared to MRI results. Agreements of FFDM, CEM, and MRI measurements with histological size were assessed using Bland–Altman plots. Lin's concordance and Pearson correlation coefficient were used to assess agreement between diagnostic imaging tools and pathology results. The sensitivity of a test was defined as the proportion of patients with a positive study among those with residual tumors. Specificity was defined as the proportion of patients with a negative study among those without residual tumors. Statistical analyses were performed using MedCalc for Windows, version 14.8.1 . A p value <0.05 was considered significant.A total of 33 women were included with a mean age of 45 years . Regarding menstrual status, 27 (81.8%) of the women were premenopausal and 6 (18.2%) were postmenopausal. A total of 22 (66.7%) had heterogeneously dense breasts or dense breasts. The majority underwent mastectomy . The mean time intervals between studies and surgery were 40 days for CEM and FFDM and 47 days for MRI. The mean time interval between the end of NAC and surgery was 55 days .Histological results comprised invasive ductal carcinoma , mixed invasive carcinoma , and invasive lobular carcinoma . The molecular subtype distribution in descending order was as follows: luminal B , luminal A , triple negative , and HER2-Overexpressing . Detailed information of each women included in the study is described in Pathologic mean tumor size after NAC was 1.6 cm (range 0–7.5 cm) compared with 2.4 cm (range 0–13.1 cm) for CEM, 2.4 cm (range 0-7.2 cm) for FFDM, and 3.6 cm (range 0–12.5 cm) for MRI. CEM accurately showed final tumor size to within 1 cm in 23 patients (69.7%) and overestimated tumor size by more than 1 cm in 8 patients (24.2%). FFDM accurately showed final tumor size to within 1 cm in 13 patients (39.4%) and overestimated tumor size by more than 1 cm in 15 patients (45.4%). MRI accurately showed final tumor size to within 1 cm in 12 patients (36.3%) and overestimated tumor size by more than 1 cm in 19 patients (57.6%).The concordance coefficient between measurements of each imaging modality and pathologic tumor size was 0.7 for CEM, 0.3 for FFDM, and 0.4 for MRI. Pearson correlation was 0.8 for CEM, 0.3 for FFDM, and 0.5 for MRI. Mean differences between CEM, FFDM, MRI, and residual histopathological tumor size were 0.8 cm, 0.7 cm, and 1.8 cm, respectively. Limits of agreement and more detailed information are described in Among the 25 patients who had RD, 19 were positive by CEM and FFDM, and 23 were positive by MRI. Progressive disease on imaging was correctly described on CEM and MRI , in two Pathologic complete response was achieved in 8 cases (24.2%); all of those were IDC, 6 (62.5%) luminal B, and 3 (37.5%) triple negative. Radiologic complete remission rate was 13 women in CEM (39.4%), 11 in FFDM (33.3%), and 6 in MRI (18.2%). Among the eight patients who had pCR, only one was positive by CEM for residual disease, three were positive by FFDM, and two were positive by MRI . The speMean difference between CEM and MRI was -0.8 cm (SD = 2.8). Lin's concordance coefficient was 0.7 (95% CI 0.4–0.8), and Pearson correlation was 0.7 (p = 0.0003). Limits of agreement were -6.3 to 4.7 cm between modalities. In comparison to MRI, CEM had measurements within 1 cm in 54.5% and overestimated size by more than 1 cm in 31.8% of the cases. All patients reported as having a complete radiologic response by MRI had negative CEM studies.The accurate assessment of residual disease at the end of neoadjuvant chemotherapy is imperative for surgical planning. Currently, MRI is accepted as the best imaging method for treatment monitoring , 3, 6. CIn our study, we evaluated CEM for detecting and measuring residual tumor after NAC. All imaging methods overestimated residual tumor size in most patients. Difference between CEM and histopathological results was within 1 cm in almost 70% of the cases. A similar difference was reported in a recent study with patients submitted to neoadjuvant chemotherapy and endocrine therapy . Limit oMRI demonstrated the best sensitivity and area under the receiver operator characteristic curve (AUC) for detecting residual tumor after NAC. CEM showed better specificity and PPV than MRI and FFDM. As well as our results, high specificity and PPV of CEM were also reported in women with locally advanced breast cancer , 22. DesOur results showed that when MRI was used as the reference, CEM showed good correlation and concordance Figures , and 5. This study had potential limitations. It was a single institution trial, and the small study population and uncommon molecular subtype distribution may limit the applicability of our results to the general population. For better comparison to MRI, CEM analysis was based only on recombined images. There is the risk of losing calcifications without enhancement, which may underestimate residual tumor measurements. Nevertheless, in our study, it was not identified. Although the radiologist was blinded to the results of other imaging modality when reviewing CEM and MRI, all studies were reviewed by the same radiologist, thereby introducing inherent bias. We were not able to evaluate the relationship of NAC agents, tumor histology, and molecular subtype with the performance of CEM.CEM has good correlation and agreement with histopathology for measuring residual tumors after NAC. CEM is as reliable as MRI in assessing the presence of residual tumor. Our findings are encouraging since CEM might be useful for patients with contraindications to MRI and patients in regions with limited MRI availability or lack of reimbursement.


Significant regional variations were determined in fertility control behavior of rural women (p < 0.05). Almost all of its predictors explained by Theory of Planned Behavior (TPB) also showed significant regional variations (p < 0.05). Current fertility control policy should be strengthened more not only to improve fertility behavior of rural women but also to establish regional equity in fertility control by improving their reproductive decision-making in a rational way.Women’s fertility decision is quite difficult in male-dominant rural culture due to their poor reproductive autonomy. A cross-sectional survey was conducted in rural community of Bangladesh between November 2017 and February 2018 among 1285 respondents selected by multi-stage stratified sampling to explore regional variations of rural women’s fertility control behavior and its determinants using hierarchical and other inferential statistics. Data collection was done by face-to-face interview using questionnaire. Average parity was 2.5 per woman and 41% respondents had three or more children. About 60% of them used modern contraceptives (MCs) and oral contraceptive pill (OCP) was their first choice. Male participation in contraceptive use was less than 5%. Regional variation, women’s empowerment, fertility control knowledge, family planning (FP) attitude, social influence, perceived behavioral control (PBC) and fertility intention were significant predictors of fertility control behavior ( Fertility control behavior is the planned outcome of fertility decision that helps clients to choose an appropriate contraceptive method in a rational way according to their expressed needs and situation to carry out their reproductive intentions successfully ,2. Use oAccording to global context, total fertility rate (TFR) in 2015 was 2.5 live births per woman with a huge regional difference . The higIn 2016, Bangladesh had an estimated population of more than 160 million, which makes it the 8th most populous country in the world ,17. PopuAlthough decision-making in fertility control is a major concern in reproductive autonomy, most of the rural women are not familiar with rational ways of contraceptive decision-making even among the MC users . FertiliThis cross-sectional survey was conducted in rural areas of three divisions  of Bangladesh.The study was conducted among 1285 married women of reproductive age (15–49 years) having at least one child currently living together with their husbands in the rural area at least for 12 months before the survey.The respondents were selected by using multistage sampling technique . InitialData were collected by the researcher and 4 trained interviewers with previous exposure in health research between November 2017 and February 2018. Face-to-face interview was done through a structured questionnaire having 10 parts. Data collectors asked questions orally and filled out the responses. Socio-demographic and reproductive characteristics of respondents were described in the first part through 18 items such as age, income, number of children, etc. The second part was used to describe fertility control behavior of rural women using 6 items related to birth control and birth spacing. It was mixed-type, including both Likert as well as dichotomous types of answers. The third part described women’s autonomy using 10 items such as decision-making, economic access and mobility. Women’s fertility preference was assessed in the fourth part using 6 items such as preference for larger family and son preference. Part 5 included 8 items regarding information about contraceptive use and birth spacing to assess fertility control knowledge. Spousal relation was assessed in part 6 through 6 items such as spousal communication and male participation in FP. Part 7 was constructed to assess women’s FP attitude using 8 items. Social influence in fertility control was described by 8 items in part 8. Women’s PBC was assessed in part 9 through 6 items such as self-control on contraceptive-decision for birth control and birth spacing. Intention to fertility control was assessed in part 10 using 6 items such as readiness to use contraceptive and readiness for fertility control. Likert scale was followed from part 3 to part 10, except part 5, where dichotomous type was used. Equal numbers of positive and negative questions were included in the scales of fertility preference, fertility control knowledge, FP attitude and fertility control intention scales and only in PBC scale there were 4 positive and 2 negative questions. Cronbach’s Alpha (α) of fertility control behavior scale was 0.72. Cronbach’s α for women’s empowerment and fertility preference were 0.77 and 0.79, respectively. Cronbach’s α of FP attitude scale was 0.80 and for PBC scale it was 0.87. Cronbach’s α for social influence and fertility control intention were 0.72 and 0.78, respectively. Only spousal status and fertility control knowledge scales showed Cronbach’s α score between 0.6 and 0.7.Data processing and analyses were done using Statistical Package for Social Sciences (SPSS) version 20. Frequencies, percentage, mean and standard deviation (SD) were used for descriptive statistics. Hierarchical regression model was used to describe significance of predictors as well as regional effect on fertility control behavior. Kruskal–Wallis H and one-way analysis of variance (ANOVA) tests were used to describe the regional variations of fertility control behavior of rural women with its predictors. Factors related to fertility control behavior of rural women were arranged in groups for hierarchical analysis .Ethical clearances were taken from the Ethical Committee of Mahidol University (COA. No. MUPH 2017-193) and also from the Bangladesh Medical Research Council (BMRC) (Reg. No. 07427092017) to conduct the study.The typical rural picture was found to describe the socioeconomic status of the respondents. Nearly half of the respondents (44.5%) were in the group of 21–35 years. The average age of the respondents was 27.7 years with SD of 7.8 years . Around Among the respondents, about 80% had a history of early marriage and mean age of marriage was 16.6 years with SD of 1.1 years. Among the respondents, 78.6% had adolescent pregnancy and mean age of first pregnancy was 18.2 years with SD of 1.3 years. Around 43% had a history of pregnancy more than two times and 7% respondents had a history of abortion. Around 42% had a history of delivery more than two times and average parity was 2.5 per woman. Women having more than two living children were 41% and among the respondents, almost half of them (48.1%) conceived within two years of marriage. Average initial gap of first pregnancy from their marital age was 1.6 years with SD of 0.7 year .Almost half of them (48.6%) regularly used MCs and 11.3% irregularly used. Among the respondents, around 27% shared about switching in contraceptive use. Only around 8% used natural methods. Among the users of MCs, more than half (54.2%) used oral contraceptive pills (OCPs), 22.5% used injections, 3.6% used implant and 2.7% used Copper-T. Among the users of MCs, condom was used only in 7.5% cases and 9.5% gave a history of using permanent methods of contraceptives, either tubal ligation or vasectomy . p < 0.001) and accounted for 55.6% of the variation in fertility control behavior of rural women (p < 0.001). Finally, the area was added to find out the significant effect of regional variation on fertility control behavior. The inclusion of area to the regression model explained an additional 0.2% of the variation in fertility control behavior. Although this change in R² was small, it was significant  = 4.06, p < 0.05).The three-stage hierarchical multiple regression revealed that background factors at stage one contributed significantly to the regression model  = 199.49, al women . Next, cp < 0.001) (p < 0.001) (p < 0.05) from fertility control behavior of rural women of Chittagong division. Women’s age, family income, empowerment and fertility preference showed significance predicting fertility control behavior in the first model of background issues. Age and women’s empowerment persisted as significant predictors in the second model. Fertility control knowledge from background issues also showed significance in the second model. FP attitude, social influence, PBC and fertility intention were highly significant to predict fertility control behavior among the core items of TPB in the second model. When all independent variables were included in stage three of the regression model, socio-economic variables showed no significance predicting fertility control behavior of rural women. Only women’s empowerment and fertility control knowledge from background issues were significant to predict fertility control behavior in the final stage of the regression model (< 0.001) . FP atti< 0.001) . SpousalThe average score of women’s fertility control behavior for all divisions was 8.9 (±4.6). The average women’s empowerment score and average fertility preference score at the national level were 34.3 (±5.2) and 20.1 (±3.5), respectively. Mean score of fertility control knowledge was 5.6 (±1.7) and average FP attitude score among total respondents was 30.1 (±3.3). The average social influence score and average PBC score at national level were 29.0 (±3.6) and 21.6 (±4.3), respectively. The average fertility control intention score was 20.9 (±3.6) and mean score of spousal status among total respondents was 7.6 (±3.1) .2 (2) = 49.41, p < 0.001, with a mean rank fertility control behavior score of 544.28 for Chittagong, 669.55 for Dhaka and 715.91 for Rajshahi. Chittagong was very highly significantly different from Dhaka (p < 0.001) and Rajshahi (p < 0.001) but Dhaka and Rajshahi were not significantly different (p > 0.05). Significant regional variations of women’s empowerment (χ2 (2) = 64.03, p < 0.001), spousal status (χ2 (2) = 26.72, p < 0.001), PBC (χ2 (2) = 17.42, p < 0.001) and fertility control intention (χ2 (2) = 18.96, p < 0.001) were also confirmed by Kruskal–Wallis H tests. Post hoc tests confirmed the vulnerability of Chittagong from Rajshahi and Dhaka , FP attitude  = 12.58, p < 0.001) and social influence  = 25.10, p < 0.001) in fertility control were confirmed through one-way ANOVA tests. Tukey post hoc tests revealed the vulnerability of Chittagong from other divisions in fertility preference, FP attitude and social influence. In the case of fertility control knowledge, there was no statistically significant difference found among rural women of three divisions as determined by one-way ANOVA  = 2.12, p > 0.05) with a mean fertility knowledge score of 5.5 for Chittagong, 5.6 for Dhaka and 5.8 for Rajshahi. Although there was no significant difference, Rajshahi showed higher score  = 6.09, er score .This study showed that addressing women’s fertility decision remained challenging in a male-dominant rural community. Fertility control behavior as well as its predictors were explained through TPB in different backgrounds. Less than two-thirds (59.9%) of the respondents were MC users. The rate of MC use was still slightly higher in the study area in comparison to a recent Bangladesh Demographic and Health Survey (BDHS) . One of OCP was the most common method in contraceptive use, followed by injection in second choice, which was similar to the national findings of BDHS . Rural wAge, income, religion, family size, parity, etc. sometimes showed similarity with national findings of BDHS, 2014  and in aSES, women’s empowerment, fertility preference and fertility control knowledge were taken as background issues of TPB model of fertility control behavior in this study. Although women’s age and family income were significant in the primary stage of hierarchical regression model, in the final stage these were not significantly related to fertility control behavior of rural women in this study that differed from many other studies on fertility control ,22,23,26Women’s empowerment was in favor of fertility control behavior. Higher empowerment score showed better state in parity and contraceptive use. Importance of women’s empowerment in fertility control was also mentioned in other Bangladeshi papers ,9. WoldeAll the core factors of contraceptive use explained by TPB, except external control, showed significant effects on fertility control behavior of Bangladeshi rural women. Better FP attitude, positive social influence, higher PBC were in favor of better fertility control behavior of rural women that were described similarly in some studies using TPB ,21. HighRespondents were randomly selected only from 3 divisions not covering all divisions to describe regional variations of fertility control behavior of rural women. Multistage stratified sampling based on national TFR at first stage minimized the selection bias and made the sample representative including respondents from all age groups. Rural women might be more positive in a few cases towards the items related to individual issues in MC use. As it was a cross-sectional survey, the study had few inherent limitations to describe the significance of predictors with fertility control behavior of women. Except these, the study adopted TPB model nicely to explain regional variations of fertility control behavior of rural women and its predictors. A mixed-method or qualitative study can be effective to explain more the reasons of regional disparity in MC use in rural Bangladesh based on current study findings.Marked regional variations were observed in fertility control behavior of rural women along with its predictors. Regional disparity was an extra burden in male-dominant rural culture to achieve the national birth control target. Regional disparity should be minimized in contraceptive decision-making of rural women to ensure rational use of contraceptives. Fertility control awareness as well as income-generating programs for rural women could be helpful to improve their reproductive autonomy in fertility decision. The findings of this research will be helpful for planners to make current fertility control programs more region specific. Appropriate strategies should be included in the national fertility control program based on regional disparity in fertility indicators to make it more effective, not only to improve fertility control behavior of rural women but also to establish regional equity in fertility control.


Haemophilus parasuis is a commensal organism of the upper respiratory tract of healthy pigs and causes high morbidity and mortality in piglets. The aim of this study was to investigate the epidemiology of H. parasuis in China from 2014 to 2017.H. parasuis isolates by serotyping, antimicrobial susceptibility, biofilm formation and with enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays.We characterized 143 50) were in the range of 0.25–16 μg/mL and MIC90 were 2–>512 μg/mL. A total of 99 isolates of H. parasuis (69.2%) were able to form biofilms and 59.6% (59/99) performed weak biofilm-forming ability. ERIC-PCR revealed a very heterogeneous pattern with 87 clusters.Serotyping revealed serovar 5 as the most prevalent (26.6%) followed by serovars 4 (22.4%), 7 (9.1 %), 13 (6.3%), 12 (5.6 %), and non-typeable (8.4%). In a panel of 23 antimicrobials, the minimum inhibitory concentration 50% (MICH. parasuis isolates showed a high serovar and genotypic lineage diversity, different abilities to form biofilms and a high degree of genetic diversity. Biofilm formation was related to antimicrobial susceptibility but there were no statistically significant associations between the antimicrobial susceptibility and either the serovars or the ERIC-PCR clusters. This study showed a high prevalence of high-MIC H. parasuis strains and suggests the need for a continuous surveillance of clinical isolates of H. parasuis.These  Haemophilus parasuis is the causative agent of Glässer’s disease in pigs and is one of the most important pathogen in the modern swine industry. The disease is characterized by pneumonia, meningitis, arthritis, polyserositis, and septicemia. Outbreaks of Glässer’s disease have resulted in serious lethality and economic losses in the global swine industry  isolates and these possess wide differences in virulence  is a genotyping method based on conjugated enterobacterial repeat sequences. It is a valuable tool for typing H. parasuis isolates including the serologically NT ones in a rapid and low cost manner  test against 15 reference strain antiserums as previously described  values of antimicrobials for the nstitute  and a prnstitute . The 23  and M07 .50) and 90% (MIC90) of the isolates. The reference strains H. influenzae ATCC 49247 and Escherichia coli ATCC25922 served as quality controls for MIC determinations.Ranges of susceptibility were performed varying from 0.25 to 512 μg/mL of the antibiotic described above and recorded along with the MIC that inhibited growth of 50% (MICH. parasuis isolates from overnight agar cultures were suspended in 5 mL tryptic soy broth (TSB)  containing 10 μg/mL NAD and 5% bovine serum  and 200 μL of this culture suspension was aliquoted in triplicate into flat-bottom 96 well polystyrene plates and incubated for 24 h at 37 °C. The wells were washed three times with 200 μL of sterile phosphate-buffered saline to remove loosely adherent cells. The remaining attached bacteria were fixed with 200 μL of absolute methanol per well for 15 min before the plates were emptied and left to dry at room temperature. Biofilms were stained with 200 μL of 1% (w/v) crystal violet for 5 min. Excess stain was removed with three washes of distilled water and the plates were dried at 37 °C for 15 min. The stain was then released by adding 100 μL of 33% (v/v) glacial acetic acid per well. The amount of released stain was quantified by measuring the absorbance at 570 nm with an automated Elx800 Universal Microplate Reader .Biofilm formation experiments were performed in 96-well microtiter plates as previously described with minor modifications . BrieflyHaemophilus parasuis isolates were grown on tryptic soy agar  plates and several colonies of each isolate were inoculated into 5 mL TSB containing 10 μg/mL NAD and 5% bovine serum and incubated for 24 h at 37 °C. Total DNA was extracted from these cultures using the TIANamp Bacteria DNA Kit . The isolates were characterized by ERIC-PCR following published guidelines (idelines . PCR reaHaemophilus parasuis strains (143) were serotyped using GD and IHA tests and 131 were typeable. The serotype prevalence was serotype 5 (26.6%), 4 (22.4%), 7 (9.1%), NT (8.4%), 13 (6.3%), 12 (5.6 %), 1 (4.2%), 14 (4.2%), and 6 (3.5%). In addition, there were also some serotypes that existed only in one or two strains such as serotype 3 (2.1%), 2 (1.4%), 8 (1.4%), 10 (1.4%), 11 (1.4%), and 9 (0.7%) (9 (0.7%) .Serotypes 4 and 5 were the major serotypes associated with the most recent epidemics in China. In 2104 and 2015, serotypes 12 and 13 were the main serotypes after serotype 4 and 5 but after 2016, the number of isolates of serotype 7 increased rapidly, significantly beyond serotypes 12 and 13. Serotype 7 became one of the major epidemic serotypes ; Fig. 2.H. parasuis  were biofilm positive, and 59.6% (59/99) performed weak biofilm-forming ability. However, 24 isolates were moderate biofilm producers and 16 isolates (11.2 %) were strong biofilm producers. A total of 44 (30.8%) H. parasuis strains were not able to form biofilms on our polystyrene test surfaces  have not been reported until the current work. The high MIC values we found suggested that the use of these drugs should be regulated. Furthermore, these results indicated that there has been a rapid increase in the rate of resistance. This attributable at least in part to the use of antibiotic additives in animal feeds and the extensive use of antimicrobial agents in veterinary medicine. Additionally, there is a limit that there are currently no approved clinical breakpoints available for H. parasuis. Therefore, we should strictly control the use of antimicrobial agents in food animals and need a continuous surveillance of antimicrobial susceptibility to minimize the emergence of resistance in future. In a previous study, we have detected the antimicrobial resistance genes for the 143 H. parasuis (H. parasuis in piglets is a combination of transferable antibiotic resistance genes and multiple target gene mutations.Treatment of infections with crobials . Neverth 2 μg/mL , while Mparasuis , combineH. parasuis forms biofilms in vitro and suggests a causal relationship between colonizers and biofilm growth (H. parasuis isolates differed in their ability to form biofilms as has been reported previously (Biofilm formation is essential for persistent infections . These sm growth . Individeviously . Differeeviously .H. parasuis could be divided into those that were highly virulent, moderately virulent and non-virulent although there may be individual variations (Streptococcus pneumoniae showed that biofilm formation in vivo was associated with reduced invasiveness and a dampened cytokine response (H. parasuis isolates have significantly higher resistance to β-lactams than the non-producers (In general, the 15 different serovars of riations . Howeverriations . It is wresponse . In the roducers . The perH. parasuis. ERIC-PCR has been used to successfully subtype H. parasuis strains isolated from different regions, confirming the high heterogeneity and high genetic variability of H. parasuis (H. parasuis strains and 68 genotypes in the 99 biofilm producers similar to previous results (In the current study, we also utilized ERIC-PCR and found it possessed discriminatory power for parasuis . We obta results . This anH. parasuis strains had high-MIC values to common antimicrobial agents in China. These showed a marked geographic variability indicating that the prudent use of antimicrobials is very important. These H. parasuis isolates were also diverse serologically and genetically and possessed with different levels of biofilm-forming ability in vitro. Biofilm formation correlated with their antimicrobial susceptibility. This study suggests the need for a continuous surveillance of clinical isolates of H. parasuis.In conclusion, this study showed 10.7717/peerj.5040/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5040/supp-2Supplemental Information 2Click here for additional data file.


Infective endocarditis (IE) is a serious mainly bacterial infection associated with high mortality. Epidemiology of fatal IE is however largely unknown. We studied occurrence and trends of fatal IE in a population-based setting.All adults (≥18 years of age) who deceased due to IE in Finland during 2004–2016 were studied. Data was collected from the nationwide, obligatory Cause of Death Registry. Background population consisted of 28,657,870 person-years and 651,556 deaths.P < 0.0001). On average, IE contributed to 1.16 (95% CI: 1.08–1.24) out of 1000 deaths in general adult population. The proportionate amount of deaths with IE was highest in population aged < 40 years followed by gradual decrease with aging. Incidence rate and proportion of deaths caused by IE remained stable during the study period.Infective endocarditis contributed to death in 754 cases and was the underlying cause of death in 352 cases. The standardized incidence rate of deaths associated with IE was 1.42 : 1.32–1.52) per 100,000 person-years. Incidence rate increased progressively with aging from 50 years of age. Men had a two-fold risk of acquiring fatal infective endocarditis compared to women (risk ratio (RR) 1.95; 95% CI: 1.71–2.22; Our study describes for the first time the population-based epidemiology of fatal IE in adults. Men had a two-fold risk of acquiring fatal IE compared to women. Although occurrence of fatal IE increased with aging, the proportion of deaths to which IE contributed was highest in young adult population. Infective endocarditis (IE) is a serious mainly bacterial infection associated with high mortality. Incidence of IE admissions has previously found to be 2–8/100,000 person-years . CurrentAdults (aged ≥18 years) with IE-related death during 2004–2016 were studied. Deceased with IE contributing to death recorded in death certificate were retrospectively identified from the nationwide Cause of Death Registry of Statistics Finland . Issuance of death certificate with determination of the causes of death and collection of death certificates into Statistics Finland database is required by law, and has a complete coverage of population. The physician issuing the death certificate determines the one cause of death he/she deems is the underlying cause of death, and this is recorded as the official cause of death based on World Health Organization (WHO) classification. In addition, other causes contributing to death are recorded. All death certificates are verified by the competent authority in charge of forensic medicine.Infective endocarditis was recognized by International Classification of Diseases, Tenth Revision (ICD-10) codes I33, I38 or I39. Age- and sex-specific population and mortality data of the Finnish population from the years 2004–2016 were obtained from Statistics Finland . The stuP < 0.05. All P-values were two-sided. The SAS system version 9.4 (SAS Institute Inc.) was used for statistical analyses.Age- and sex-specific incidence rates and proportions of death were calculated. Standardization of incidence was performed with direct method and European 2013 standard population. Differences in continuous variables were analyzed by using a t-test. The associations of age, sex and study year with the incidence rate and proportion of deaths were studied by using Poisson regression modeling. Incidence rate and proportionate amount of deaths were modelled with using the logarithm of corresponding population or number of deaths as an offset parameter . Impact n = 306) were older (mean age 73.1 years (SD 15.8)) than men ; P < 0.0001). Infective endocarditis-associated death occurred in hospital or other healthcare facility in 89.5%, at home in 8.6% and elsewhere  in 1.6% of cases, while 0.3% of deaths occurred abroad. There were no monthly (P = 0.642) or seasonal (P = 0.214) differences in occurrence of IE-associated deaths.Infective endocarditis contributed to death in 754 cases during 2004–2016. The mean age of deceased with IE was 68.6 (standard deviation (SD) 16.7 years) . Women (P < 0.0001). Incidence rate was lowest in population aged 18–29 years, remained similar in population aged 30–49, and increased progressively with aging from 50 years of age (Table P < 0.0001). Association was not modified by age (interaction P = 0.102). Overall incidence rate was 1.95 (95% CI: 1.79–2.13)/100,000 person-years in men and 0.99 (95% CI: 0.88–1.12)/100,000 in women. Incidence rate of IE-associated deaths had no significant trend of change (P = 0.994) during the study period  per 100,000 person-years. Occurrence was significantly age-dependent . Association was not modified by age (interaction P = 0.234). Proportion of deaths associated with IE remained stable (P = 0.465) during the study period  out of 1000 deaths in general adult population. The proportionate amount of deaths with IE was highest (4.42 (95% CI: 3.41–5.64)/1000) in adult population aged < 40 years followed by gradual decrease with aging down to 0.39 (95% CI: 0.28–0.53)/1000 in nonagenarians (Staphylococcus aureus. Cardiovascular diseases were the leading non-infectious underlying causes of death (16.3%) with coronary artery disease/myocardial infarction (39.8%) and cerebrovascular disease/stroke (18.7%) as major contributors. Valvular stenosis or insufficiency was the underlying cause of death in 46 cases (6.1%) with aortic valve affected in 60.9% (of which 89.2% were stenosis), mitral valve in 23.9% (stenosis in 63.6%) and tricuspid valve in 6.5%.Infective endocarditis was the underlying cause of death in 352 cases (46.7%). Septicemia was recorded to be the underlying cause of death in 11.5% of cases and other infectious disease in 2.8% (Table P < 0.0001) was also similar to all deaths associated with IE /100,000 in total adult population. Age distribution of deaths with IE as the underlying cause was similar to all deaths associated with IE  among patients with IE –8, 18, b–20% amonIn our study infective endocarditis-associated death occurred in hospital or other healthcare facility in 89.5% and at home in 8.6% of cases. Individuals without IE diagnosis prior to death most likely died outside the hospital or other healthcare facility, e.g. at home. The incidence rate for IE hospital admissions in Finnish adult population is found to be 6.33/100,000 and 30-day mortality after admission to be 11.3% . PreviouWe found that in IE-related deaths septicemia was the underlying cause of death in 11.5% of cases. Patients with IE often have uncontrolled infection and complications of sepsis (e.g. septic shock) might lead to death. In these cases septicemia might be coded as the underlying cause of death and IE as a cause contributing to death.Infective endocarditis, septicemia or other infection was deemed as underlying cause of death in 61% and valvular heart disease in 6.1% of deaths associated with IE. Valvular heart disease is a known risk factor for IE. Although determination of the underlying cause of death may be prone to subjective interpretation of findings, our results suggest that significant valvular disease was the cause of death related to IE in 6% of the cases.Occurrences of infectious diseases including sepsis , pneumonTo our knowledge this is the first population-level study on the occurrence of fatal IE. The current study is based on nationwide, obligatory database with a complete population coverage. There are however limitations to the study. Usage of registry data has certain limitations as we did not have the access to the more detailed personal level data, e.g. microbiological, clinical or autopsy data. The general autopsy rate of all deaths in Finland during the study period was 28.4% compared to mean of 16.6% in all members of the European Union . The diaIn conclusion, our study describes the population-based epidemiology of fatal IE in adults for the first time. Infective endocarditis-associated death occurred in average to 1.4 out of 100,000 person-years and contributed to 1.2 of 1000 deaths in adult population. Men had a two-fold risk of acquiring fatal IE compared to women. Occurrence of fatal IE increased progressively with aging from 50 years of age, but the proportion of deaths to which IE contributed was highest in young adult population. Incidence rate of fatal IE remained stable over time.Additional file 1: Table S1. Incidence Rate of Deaths with Infective Endocarditis as the Underlying Cause in Adult Population of Finland during 2004–2016. Table S2. Proportion of Deaths with Infective Endocarditis as the Underlying Cause in Adult Population of Finland during 2004–2016.


The aim was to determine the prevalence of glue‐sniffing among street children.A descriptive cross‐sectional study design was adopted.Children are the source of hope and could be the major ailments for the development of society, nation and the world but there are large numbers of children on the street sniff glue and struggling with multiple disease and uncertain future.A study was conducted in 52 street children to determine the prevalence of glue‐sniffers and its impact on street children in Butwal, municipality of Nepal. This study was conducted in 52 street children, 5‐ point Likert scale and interview schedule was performed with the help of structured and semi‐structured questionnaire to collect data. Finally, the collected data are analysed by using descriptive statistical methods such as frequency, percentage and mean.The study findings revealed that a large number of children, that is 40.38% was between the age group of 9–12 years and 92.31% were male. The current prevalence of glue‐sniffing among street children is 88.46%. Similarly, 58.7% of respondents had started glue‐sniffing 1 year ago. Out of 46 respondents who sniffed glue, 89.13% were unaware of its effect. Nearly, half of the respondents 45.65% had encountered health issues such as headache, chest pain and stomach ache.They have deprived children and denied not only of their rights as children but also of their normal childhood. Without guidance, education and security, they are heading towards an uncertain future. They can have enough potentiality and talent if they are brought into a better environment and might have real hope for the bright future. Meanwhile, it is also reported about the abuse of other kinds of volatile substances, such as paint thinners, nail varnish remover, gasoline and lighter fluids subsequently appeared. However, the term “glue‐sniffing” has come to be applied, in a general way, to all kinds of solvent abuse by inhalation. Glue‐sniffing is usually taken by the young children as a forerunner to alcohol abuse, and probably, it became substitute because of its low cost, readily availability and provides faster onset of action , there are approximately 5,000 street children over the country among them 800–900 in Kathmandu only. Glue‐sniffing among street children in Nepal can be taken as an initiation to the use of other more teratogenic drugs. Peer pressure and easy availability of glue “dendrite” are two of the prominent reasons for addiction to sniffing. Addiction to glue‐sniffing is making street children more dependent on substances. Such type of drug dependency is resulting in an increase in street children carrying out petty thefts. This trend, in the course of time will lead children to even more violent and unsocial activities  was the biggest problem faced by the street children, mostly caused by police and peers. Adhesive was the most common solvent used by the street children along with thinners and balm. In addition to glue and cannabis, cigarette smoking was also found to be pervasive among the respondents. The reasons stated for glue‐sniffing included peer pressure, curiosity, pleasure, coping with stress and overcoming family issues. Respondents who slept in public places and parks/ streets were more likely to sniff glue—25% of the glue‐sniffers slept in parks/ streets compared with 13% non‐glue‐sniffers; 28% of the glue‐sniffers slept in other public places compared with 14% of the non‐glue‐sniffers. Moreover, street children who sniffed glue were more likely to use other drugs than non‐glue‐sniffers  years, with the children in Peshawar slightly younger (13.0 SD 2.33 years) than the children found in other cities. The maximum proportions of children were between 15–16 years of age. The largest proportions of children interviewed were Pakistani (81%), more than half of whom had migrated from smaller cities. 12.5% of the total children were Afghani followed by Bengalis (5.8%). One third of the children interviewed belonged to single‐parent families with large family size. Almost three quarters of the children were not currently living along with their families. These children prefer staying in groups, had no permanent place to live, had been sleeping in parks (50%), friend's place (12%) and darbars (6.7%) etc. (76%) of the children never attended formal education. The remaining 24% had 3.01 (SD 1.97) years of educational attainment. The average daily income reported was Rs. 79.7 (SD 44.7), which was sourced through cleaning cars, scavenging solid waste garbage and begging  included in the sample. The mean age of children using solvents was 14.3 , followed by fever (19.7%), GIT upsets (18.7%) and skin infections (12.6%). Adhesive glue was the primary drug of choice consumed by 374 (90%) of the interviewed street children. Other solvents abused included petrol (25.5%) and thinners (10.6%). Use of petrol was significantly popular in Quetta (43%) and Peshawar (41%). In Karachi, children were found to be involved with glue (95.2%), thinners (27.6%), petrol (13.3%), as well as tincture (5.7%). An average amount of Rs. 39.7 ± 30.1 was being spent on these solvents with minimal geographical variations. A little more than 60% of the children reported regular use of these substances for more than 2 years via various techniques. On average they inhaled almost 80 g  was the only drug used by the respondents in this study. The most common health problems faced by them were head lice infestation (81.2%), headache (66.7%), cut injury (60.4%), common cold (52.1%), dental caries (52%), burning micturation (47.9%), cough (47.9%), being underweight (43.8%), abdominal pain (39.6%), tinnitus (37.55%), gum bleeding (33.3%), joint pain (31.2%), eye inflammation (25%), leg cramps (25%), palpable lymph nodes (25%), chest pain (18.8%), skin lesions (16.7%) and abnormal vision (8.3%) , most of the respondents (40.38%) were of age group 9–12 years mean (mean age was 10.07) where 92.31% were males whereas only 7.69% females. Participants based on education showed that maximum of the respondents 55.77% were illiterate; however, 65.38% respondents lived on the street  were 9–12 years old. Maximum respondents (92.31%) were boys, 55.77% respondents were illiterate, only 34.62% respondents lived with their family and most of them (67.31%) used to pick rags for their livelihood, 26.92% children begged in different places of Butwal. 51.92% children had been living on street for more than 1 year.Among the 46 glue‐sniffers, maximum (58.7%) started glue‐sniffing since 1 year and 25 (54.35%) of them were sniffing glue more than five times a day. Similarly, most of them (87%) brought glue from shops and most respondents (73.91%) used to sniff glue with their friends. Likewise, most of the respondents (71.5%) came on the street to seek employment and major reason behind the glue‐sniffing was to cope with stress. Among total glue‐sniffers, 89.13% did not know about the effect of glue‐sniffing. Only 10.87% possessed the knowledge regarding effect of glue‐sniffing. Along with glue, street children used to abuse different types of substances such as alcohol, tobacco, cigarette and gutkha (tobacco with betel nut). In the same way, most of them (54.35%) told that they had not encountered any kind of health problems. 23.91% complained about stomach ache, 15.22% and 6.52% of them had suffered from chest pain and headache/nausea respectively. Out of total respondents, 86.54% had faced other various kinds of problems apart from those related to health like arrested by police, beaten and scolded by people, frostbite and rain.Out of 52 respondents, 46 respondents sniffed glue. Therefore, the prevalence rate of glue‐sniffing in ward no. 4, 6 and 13 of Butwal Municipality was found to be 88.46% .On the basis of finding, it is recommended that street children are vulnerable to many things including drug abuse and health complications. Programme with broad overview of street children's issues is needed to prevent from getting into substance addiction and drug abuse. The government of Nepal should prohibit the sale of glue and other volatile solvent agents to children. Also, shopkeepers should be made aware about the ill effects of glue on children and should be punished for selling such substances to children. Additionally, further research is required with large sample size to see the prevalence of glue‐sniffing and its complications among adolescents in various major cities of country.7


Fusarium graminearum is responsible for production of the mycotoxin deoxynivalenol (DON) on maize and wheat in Ontario, Canada. It has been understood since the early 1980s that in most parts of Canada, the predominant chemotype of F. graminearum is 15ADON, and not the 3ADON chemotype mainly found in Europe and Asia. The discovery of F. graminearum strains that did not produce DON but the structurally related 7-α hydroxy, 15-deacetylcalonectrin (3ANX) and its hydrolysis product 7-α hydroxy, 3,15-dideacetylcalonectrin to (NX) demonstrated that we still have a lot to learn about this well studied but complicated fungus. We conducted a survey of maize and wheat samples from Ontario farms. In the 2015 crop year, we isolated 86 strains and tested a representative subset of 20 using the published genetic probes for assessing genotype. We also developed a targeted LC-MS/MS method for the identification and quantitation of known toxins from this species to determine chemotype. The results showed that 80% of our strains produced some 3ANX in addition to 15ADON and one strain produced 3ANX and no 15ADON. Comparison of chemical data with genotyping revealed that in more than 50% of the cases there was no clear agreement. These data demonstrate the importance of chemical analysis for understanding the toxigenic potential of strains, especially using a LC-MS method that is capable of differentiating 3ADON and 15ADON. For this collection, genotyping of isolates did not produce reliable information on the chemotype. This is the first report of 3ANX toxin production concurrently with 15ADON and suggests that the 3ANX producers in North America likely originated from the 15ADON background. Fusarium graminearum and related species that produce the mycotoxin deoxynivalenol (DON) are important pathogens of maize and small grains in temperate areas worldwide [F. graminearum isolated from grain that had resulted in human toxicosis [orldwide ,2. The doxicosis . The Japoxicosis ,5.F. graminearum. This revealed that all strains from southern [via a 15-acetyl-DON (15ADON) [F. graminearum strains worldwide by determining the major monoacetate precursor produced by different populations [F. graminearum strains isolated from wheat across nine provinces in China, 11 were 3ADON producers with two strains producing 15ADON [F. culmorum was more common than F. graminearum in cooler wheat growing areas such as the UK and most of continental Europe [F. graminearum from Portugal and the former Yugoslavia were reported as 15ADON producers [In response to the catastrophic epidemics of FHB in Ontario from 1980, research began on the toxins produced by Canadian strains of southern  and eastsouthern  examined15ADON) . This diulations . Early aulations ,13. Chemg 15ADON . MirochaADON . Thl Europe . These sl Europe –20. Howeroducers .F. graminearum chemotypes  but did not produce 3ADON, 15ADON, DON, fusarenone X (4ANIV), nor nivalenol (NIV) [TRI1 gene. The TRI1 enzyme in F. graminearum is a P450 oxygenase responsible for the addition of hydroxyl groups to the C-7 and C-8 positions of calonectrin [F. sporotrichioides adds a single hydroxyl group to the C-8 position [About a decade ago, Gale and co-workers discovered a population of ol (NIV) . Closer ol (NIV) . This 3-onectrin . The anaposition  and it iposition . After tposition . Other lposition ,48, evenposition . In suchposition  but to u2). Working from ~500 samples of the grain from 94 farms, kernels were plated on a semi-selective medium for Fusarium resulting in a large number of isolates of F. graminearum. A proportionate subsample of the strains collected were subjected to comprehensive chemical analysis specifically designed to distinguish all significant F. graminearum metabolites and the available probes used for genotyping the strains The purpose of this report is to discuss the analytical methods used and the relationship between the genotype and chemotype.Farmers in Ontario produce 50% of maize and most of the soft wheat produced in Canada. Spanning 1000 km, growing conditions vary from 3500 to 2900 corn heat units with average morning June RH from 91 to 81%, respectively in a wheat, maize and soy rotation. Each year, grain samples are taken throughout the cereal producing areas of the Province . The kernels were surface sterilized and plated onto PCNB medium . From the well-mixed grains in each sample, 10 kernels of maize or 14 kernels of wheat were plated. The plates were incubated at 26°C for 7 days in darkness. All fungal colonies that appeared to be Fusarium were then transferred to Potato Dextrose Agar , and incubated at 26°C in darkness. After one week, each F. graminearum isolate was identified based on morphological characteristics and stored at 4°C. From the 2015 season, 77 strains were isolated from samples of wheat from 25 farms and 9 from maize samples from two farms.wt/vol) agarose gels, pre-stained with ethidium bromide to confirm amplification. Gel photographs were taken on MultiImage Light Cabinet . Invitrogen PCR SuperMix  was used for all PCR reactions. PCR primers were synthesized by Integrated DNA Technologies .Aerial mycelium from a fresh plate of each isolate was used to extract genomic DNA with the Macharey-Nagel Nucleospin 96 Plant-II kit (GmbH & Co. KG #740663.4). All PCR reactions were amplified in a T100 Thermal Cycler . 10 μL of amplified PCR products were run in 1.5%  representing differing weather patterns. The attempt was to represent as large a geographic area as possible. The subset included 18 strains isolated from wheat and two from maize. These cultures were deposited in the Canadian Collection of Fungal Cultures (DAOMC 251903–251922) with GenBank (accession numbers MH108123-MH108142). To identify the phylogenetic clade of F. graminearum, a portion of the translation elongation factor (TEF) gene was amplified by PCR primer sets EF-1 and EF-2 [From this collection, 20 strains were selected to represent each location where ,000 km2 . These sand EF-2 ,51 and sTRI3 and TRI12 was used to differentiate between 15ADON and 3ADON producers of F. graminearum according to Ward et al. [TRI8 PCR assay developed by Alexander et al. was also tested [TRI1 allele specific amplification  and differing forward primers for the 3ADON (5’-AATGCTCGCGAACTAATCAC-3’), and 3ANX (AGCGAAATGATCAA-3’5’AATGCT) genotypes. Each 29 μL PCR reaction consisted of 22.5 μL of Invitrogen PCR SuperMix (Thermo Fisher Scientific), 2 μL of 10 μM for each forward and reverse primer, and 2.5 μL of fungal genomic DNA. The thermal cycling conditions were 94°C for 2 min, 40 cycles of 94°C for 30s, 49.5°C for 30s, 68°C for 1 min, and a final extension at 68°C for 5 min. A GeneRuler100-bp DNA size ladder (Thermo Fisher Scientific) was used to score the molecular size of each amplicon.A multiplex PCR assay targeting d et al.  followind et al. . The TRIo tested . In ordeo tested  and a TRication; ). The TRThe toxigenic potential of each of the 20 strains was determined by analysis of extracts obtained from agar plate cultures after Visagie et al. . BrieflyLiquid fermentation culture flasks were also tested; the first stage medium comprised molasses 20 g, glucose 30 g, fish-meal 15 g, Pharmamedia 15 g per L of ultrapure water; 50 mL in 250 Erlenmeyer flasks. A MEA plug from a plate of each strain was macerated in sterile distilled water (27 mL). An aliquot (2.5 mL) was used to inoculate each of two flasks with test strains. The cultures were placed on a rotary shaker (3.81 cm throw) at 220 rpm at 28°C for 72 h in darkness. The resulting mycelium from the starter culture was macerated and 2.5 mL aliquots were added to six 250 mL Erlenmeyer flasks containing 50 mL of GYEP medium . These cultures were incubated at 28°C for 10 days in darkness . The filF. graminearum DAOMC 242077 and a portion was converted to NX using potassium hydroxide [Chemical standards of NIV, 4ANIV, 3ADON, 15ADON were purchased from Romer Labs . 3ANX was isolated from cultures of ydroxide .6 maximum injection time (IT) of 64 ms and isolation width of 1.2 amu. Full MS scans between mass range m/z 100–1000, were acquired concurrently throughout the entirety of the LC run to allow for retrospective analysis and putative detection of other fungal metabolites. The full MS scan settings were AGC target 3 ×106, max IT of 64 ms and resolution of 17, 500. The APCI settings were: spray current, 3.5 kV; capillary temperature, 300°C; sheath gas, 32.00 units; auxiliary gas, 10.00 units; probe heater temperature, 250°C; S-Lens RF level, 50.00.All MS data were collected with a Q-Exactive Quadrupole Orbitrap mass spectrometer  coupled to an Agilent 1290 high-performance liquid chromatography system. Mycotoxins were resolved using a Zorbax Eclipse Plus RRHD C18 column  maintained at 35°C. The mobile phase was comprised of water with 0.1% formic acid (A), and acetonitrile with 0.1% formic acid (B) . The gradient began by holding mobile phase B at 5% for 30 seconds, before increasing to 22% over 30 seconds. In order to resolve 3ADON and 15ADON, mobile phase B was then increased to 37% over 3 minutes before increasing to 100% over 1.5 minutes and held for two minutes before returning to 5% over 45 seconds. Five μL injections were used with a flow rate of 0.3 mL/min. Compounds were analyzed by a combination of full MS, and MS/MS at a resolution of 17, 500, automatic gain control (AGC) of 1 ×10F. graminearum was detected in 6% of maize kernel samples and 7% of maize producing farms. A total of 398 grain samples were collected from 66 wheat-producing farms in 2015. F. graminearum was detected in 19% of grain samples overall and 39% of wheat producing farms, all from counties in southern Ontario (see above). These strains were confirmed as F. graminearum by the TEF gene after O’Donnell et al. [F. graminearum that were recovered came from the most southern counties of Ontario between Wellington and Kent Counties .A total of 141 samples of shelled maize were collected from 28 farms from the 2015 crop year. l et al. . In thisF. graminearum were grown under three conditions  and screened using our trichothecene specific LC-MS/MS method. For the liquid fermentation cultures, the concentration of each analyte was also quantitated  produced 3ANX and no 15ADON. Interestingly, we found that 80% (n = 16) of the isolates examined, co-produced detectable levels of both 15ADON and 3ANX in at least one medium. Some same-strain differences were observed in different growth media. Under the growth conditions where the 16 strains co-produced 15ADON and 3ANX, the amount of 15ADON was greater than the amount of 3ANX in 88%, 100% and 63% for MEA agar, GYEP agar and liquid culture respectively.The 20 representative isolates of ntitated . The 15ATo confirm that 3ANX was present in the same fermentation cultures as 15ADON, the MS/MS spectra of a 3ANX standard was compared to 3ANX detected in strain DAOMC 251912 at two different collision energies . Not onlTRI3 and TRI12 reported by Ward et al. [TRI8 assay reported by Alexander et al. [TRI3, TRI12, and TRI8 assays. As expected, the 3ANX control strain DAOMC 242077 tested as a 3ADON genotype for TRI3, TRI12, and TRI8 assays. The RFLP-PCR assay targeting TRI1 [TRI1 assay developed by Toomajian [TRI1 assay correctly distinguished the 3ADON and 3ANX strains but suggested the 15ADON positive control strain was both an ADON and 3ANX genotype.The results of the genotype analysis are shown in d et al.  and Stard et al.  as well r et al.  indicateing TRI1  and a seoomajian  are purpTRI3, TRI12 and TRI8 assays. The RFLP-PCR assay targeting TRI1 resulted in ADON genotype, whereas the assay developed by Toomajian suggested that DAOMC 252911 was both a ADON and 3ANX genotype, which is consistent with the 15ADON control strain (DAOMC 180378). The three strains producing only 15ADON were DAOMC 251904, 251910 and 251917. The genetic analysis performed in this study obtained a consensus match with the 15ADON positive control strain for DAOMC 251904, 251910 however the RFLP-PCR assay could not identify DAOMC 251917 as an ADON nor a 3ANX genotype. Overall, there was agreement between the genotype and chemotype approximately half the time.In contrast to the agreement observed in the positive control strains, we found that genotypes based on the literature probes from the systematic collection in Ontario generally did not align with all genetic assays for the positive control strains . For 55%TRI3 and TRI12 assay. Although these six strains produced both 15ADON and 3ANX in liquid culture, the PCR results matched those obtained from the 15ADON strain (DAOMC 180378).Strains that could produce both 15ADON and 3ANX in liquid culture were isolated from both wheat (n = 14) and maize (n = 2) including DAOMC 251906, 251907, 251908, 251913, 251916 and 251921. All strains resulted in 15ADON amplification for TRI1 RFLP-PCR assay and ADON genotype for the Toomajian assay. The TRI3, TRI12 and TRI8 assays indicated a15ADON genotype for DAOMC 251909. This strain resulted in ADON genotype for the RFLP-PCR assay, however indicated that it was the 3ANX genotype by the Toomajian TRI1 PCR assay. Tests of strains DAOMC 251912 and 251919 resulted in ADON genotype for TRI1 RFLP-PCR and the Toomajian assays, but did not amplify in the TRI8 assay. DAOMC 251914, 251915 and 251920 did not amplify in the TRI8 assay, but the RFLP-PCR assay and the Toomajian assays suggested the strain was the ADON genotype. The tests of DAOMC 251918 indicated the 15ADON genotype for the TRI8 assay however did not amplify in the RFLP-PCR assay and resulted in 3ANX genotype for the Toomajian assay. For DAOMC 251922, the RFLP-PCR assay resulted in a ADON chemotype and the TRI8 assay indicated 15ADON , which showed superior ionization of both the monoacetylated and non-acetylated target analytes.All of the trichothecenes listed in in 3ADON . For thend 3ADON . This waTRI3 and TRI12 PCR assay. All but five  15ADON and 3ANX producers were determined to be the 15ADON genotype with the TRI8 assay. All but one (DAOMC 251918) were ADON genotype using the RFLP-PCR assay. There was no consistent pattern for 15ADON and 3ANX producers using the Toomajian assay. For this assay; 9/16 (56%) resulted in genotype matching the 15ADON producing control strain (DAOMC 180378), 4/16 (25%) resulted in genotype matching the 3ADON producing control strain (DAOMC 242075), 2/16 (13%) resulted in genotype matching the 3ANX producing control strain (DAOMC 242077). Overall, this confusing pattern suggests that these strains have Tri-cluster genotypes more similar to 15ADON strains than the known 3ANX strains.The chemical screening method developed here allowed for highly confident detection of all trichothecenes needed to determine a strain’s chemotype. This facilitated the unexpected observation of 15ADON and 3ANX in the same culture extracts. The contention has been that production of NX represented strains with a unique genetic background producing only NX . BroadlyF. graminearum resulting from a single ascospore that produced both DON and NIV. Kim et al. [Fusarium trichothecenes are produced along metabolic grids [TRI1 [This phenomenon has been observed by others. Sugiura et al.  reportedm et al.  reportedm et al. . Kulik em et al.  reportedm et al.  also repic grids . Alternaic grids , mutatiods [TRI1 , mutatiods [TRI1 ,38 or evds [TRI1 .F. graminearum strains isolated in a survey of Ontario, 97% of 155 isolates were the 15ADON genotype [The available information at the time of the discovery of the 15ADON chemotype in 1983 through the subsequent decade was that it dominated the populations in the New World from Canada ,61–63, tgenotype .One possible explanation of the ability of some 15ADON producing strains to concomitantly produce 3ANX is a lowered specificity in two key enzymes, TRI1 and TRI8. The TRI1 enzyme of both 15ADON and 3ADON populations will produce 7,8-dihydroxycalonectrin . HoweverF. graminearum strains through the mid 1990s produced were the15ADON chemotype meaning when wheat was analyzed for the two acetates of DON, 15ADON was present in the sample [The present data show that the capacity to produce 3ANX exists in and probably originated from the 15ADON background. In our opinion, this makes sense. As noted, the available data in the USA and Canada on the chemotype of e sample ,62,67,68e sample ,69.F. graminearum called NA1 (15ADON genotype) and NA2 (3ADON genotype). These authors reported that the frequency of recombinant genotypes is increasing rapidly. They further stated that trichothecene genotype is “an increasingly unreliable marker of population identity”. Liang et al. [“much more work was needed to resolve this question”.From collections gathered in 1999 and 2000 from plant material showing FHB symptoms, Gale et al.  reportedg et al.  suggesteg et al.  suggesteF. graminearum ascocarps [F. graminearum populations. Kelly & Ward, [Cropping over a large part of Ontario involves rotations of wheat, maize and soybeans and the region has experienced epidemic years for FHB or GER for over 100 years. These became more serious in the early 1980’s ,73 and rscocarps . This pe & Ward,  reported & Ward, .F. graminearum isolates that are able to produce both 15ADON and 3ANX are common in Ontario. This is a region where the 3ADON strains have never been common. The 15ADON strain used here, DAOMC 180378 did not produce detectable levels of 3ANX. The present data do not shed light on whether the 15ADON/3ANX chemotype was present 40 years ago. Although a pathway has been inferred for the accumulation of 3ANX, experience with other Fusarium species is clear that more than one pathway can exist within a population. More than 80% of our strains produced some 3ANX in addition to 15ADON. Comparison of chemical data with the methods used for genotyping revealed that in more than 50% of the cases there was no clear agreement. The seemingly rapid change in F. graminearum populations evidenced by both these chemical data and the genetic data of Kelly & Ward, [F. graminearum strains. As such we are arguing that chemical analysis is critical for all future studies of this fungus and the use of LC-MS/MS methods such as the one described here that are capable of differentiating all of the known compounds are necessary. Strain data from all three years of the study will be examined for any influence of agronomy on chemotypes recovered in a subsequent report.The present data demonstrate that  & Ward,  explain  & Ward,  this oldS1 Table(PDF)Click here for additional data file.


To measure the knowledge of healthcare professionals about increasingly prevalent Autism Spectrum Disorder (ASD) along with perceptions around its management and prognosis and comparison across various specialties.This Cross sectional survey based comparative analysis took place at Maternity and Children Hospital and King Faisal Hospital Makkah from December 2017 to May 2018. The validated self-administered “Knowledge about childhood autism among health workers” questionnaire was used along with additional questions regarding perceptions about ASD. The mean and mean percent scores were calculated. Chi squared test and ANOVA were applied to find the association between quantitative and qualitative variables respectively.Out of 162 participants, 153 returned the questionnaire and 147 were included in final analysis. Physicians constituted 81.6% (120) of participants. The mean score for participants was 9.80(S.E.M ±0.32) where non-physicians yielded higher mean score (11.2±4.41) as compared to physicians (9.6±3.28) (p=0.113). Psychiatrists had highest score of 16/19 while general physicians had lowest (6/19). Participants with more years of experience had higher mean scores . About 72.10% (106) of participants opted for medication as a treatment option. Nearly 38.1% (56) of participants were skeptical about improvement of ASD with early interventions.There is a lack of knowledge about ASD amongst healthcare professionals in Saudi Arabia. Experienced professionals working with ASD children can be utilized to deliver targeted trainings nationwide. One in every 68 children are estimated to have autism spectrum disorder (ASD), making it one of the most common neurodevelopmental disorders.3There is no known cure for ASD, but it is established that early intervention and intensive behavioral therapy (BT) is concomitant with a better outcome.Ethical approval was obtained from the Ethical committee of the Hospital. Informed consent was taken after study objectives were explained and then the survey was distributed. Respondents were requested to complete and return it there and then, in order to ensure a good response rate and decreased chances of obtaining information from collateral sources.Data were collected by convenience sampling using a survey form encompassing demographic details, a validated questionnaire (English) titled Knowledge about Childhood Autism Among Health Workers (KCAHW)10KCAHW Questionnaire is self-administered and contains 19 items, each having three options to choose from (correct option yields 1 score while incorrect ones get a 0). It has four domains. Domain-1 consists of eight items about difficulties in social interaction. Domain-2 has one item about communication and language development. Domain-3 contains four items about restricted, repetitive and stereotyped behaviors and interests. Domain-4 contains six items about etiology, comorbid conditions and the onset. A maximum score of 19 and minimum score of 0 is achievable.Data was entered in SPSS 21.0 version after scoring the responses. Descriptive analysis was done to calculate means, frequencies and percentages. Mean and SD of the whole sample along with mean percent score were calculated. Comparison of mean scores of physician vs non-physicians was done by applying t-test keeping value of p < 0.05 . Chi squared test was applied to observe association between qualitative variables like gender and nationality etc.The total number of participants was 162. Questionnaires filled and returned totaled 153, yielding a response rate of 94%. Out of these, six had incomplete information and were excluded from the final analysis resulting in an inclusion of 147 questionnaires. Of these, 113 participants completed the KCAHW Questionnaire Males covered 44.90% (66) of the study sample and females constituted 48.30% (71). About 6.8% were unidentified. Mean age of the sample was 28.02 (SD ±13.09). About two-thirds (104) of the participants were Saudi Nationals whereas 6.80% did not reveal their nationality.Physicians constituted 81.6% (120) of the sample. HCPs consisted of pediatricians, psychiatrists, psychologists, physiotherapists, occupational therapists (OTs), general physicians (GPs), surgeons, gynecologists, medical specialists and few medical trainees.Pediatricians were highest in number (23.80% =35) and physiotherapists comprised 14.3% (21) of the total (highest number among non-physicians). Ratio of participants from the two hospitals was 3:2. Less than half (40.8%=60) had a clinical experience of more than 5 years. The total score of the study population was 1108/2147 with the mean score 9.80(S.E.M ±0.32). Mean percent score was found out to be 51.60%.The mean score in Domain-1  was 5.7/8 (S.E.M ±0.14) (71.25%). In Domain-2 (language), it was 0.62/1(S.E.M ±0.04) (62%). In Domain-3 (behaviors) it was 2.2/4(S.E.M ±0.11)) (55%) and in Domain-4 (etiology), it was 0.73/6 (S.E.M ±0.06) (12.16%). Males had mean score of 9.8(S.D ±3.32) vs 9.7(S.D±3.72) amongst females. Saudi nationals scored 9.5(S.D ±3.54) as compared to 10.6(S.D ±3.28) by other nationalities.The mean score for physicians’ and non-physicians was 9.6(S.D ±3.28) and 11.2(S.D ±4.41) respectively. On applying independent sample t test, difference between the two groups was not statistically significant (p=0.225). Compared among specialties, using ANOVA, difference between mean scores was statistically significant (p= 0.012). Psychiatrists had highest score of 16/19 (n=1) while general physicians had lowest score of 6 (n=1) .There was statistically significant difference (p=0.01) in knowledge among the professionals based upon their years of clinical experience. Participants with < 1 year of clinical experience had score of 8.22, with 1-5years experience had a score of 9.5 and > 5 years of experience had 10.9 mean score.Participants were found to have minimum knowledge about symptoms of ASD such as staring purposelessly and preoccupation with parts of objects, along with etiology and comorbidities associated commonly with the condition. They scored better regarding communication and social behavior symptoms. In the question about interventions helpful in the management of ASD, a vast majority of professionals (72.10%=106) opted for medication as a treatment option. There was no statistically significant difference between physicians and non-physicians in choice of medication as a treatment option (P=0.31).Regarding non-pharmacological treatment options, 57.8% (85) thought a referral should be made to psychiatrists, 56.5% (83) thought a referral to psychologists is important, 68.7% (101) believed in referring to the speech therapists (STs) whereas 51.7% (n=76) thought of occupational therapy (OT) as a possible intervention. However, 65.65% (n=21) of medical specialists, 50% (n=16) of gynecologists, 38.14% (n=13) of pediatricians, 33.33% (n=12) of surgeons and 25.00% (n=1) of psychologists did not consider OT as a useful intervention in management. The difference between physicians and non-physicians regarding myths and misconception about ASD was not significant statistically This study aimed at exploring HCPs’ knowledge regarding ASD in Saudi Arabia. Preferred approaches towards management, pharmacological and otherwise were also looked into. A few questions regarding misconceptions and awareness were incorporated in order to make a comprehensive assessment regarding the understanding and concepts of HCPs around this important neurodevelopmental disorder.Mean score of all healthcare professionals was 9.80±3.44. This translates into a mean percent of 51.57 and implies that HCPs in our study had moderately low levels of knowledge regarding childhood ASD overall. This is comparable to the scores in previous studies done in community health workers that yielded scores of 9.6 and 9.8.9However, mean percent improved from 51.57% to 65.53% when only domains 1, 2 and 3 (questions about sign and symptoms of ASD) were taken into account, implying that HCPs had somewhat better understanding of presentation of ASD as compared to the etiology or comorbidities addressed in domain 4 of KCAHW. Similarly, Eseigbe et al found the highest knowledge gap was associated with onset of ASD and its comorbidities and the least was concerning communication impairments.7Regarding individual domains like 1 , 2 (communication) and 3 (repetitive and odd behaviors), mean percent was 71.25%, 62.00% and 55% respectively. Poorest knowledge was about comorbidities and etiology of ASD (domain 4) i.e. 12.16%. Out of HCPs only 28.60% and 44.20% knew that ASD can be a comorbidity associated with epilepsy and intellectual disability (ID) respectively .This has practical implications as more than 50% children with ASD might have a comorbidity with ID, and in some studies prevalence of ID in ASD is reported as high as 72.8%.Our findings are somewhat similar with an African study which found pediatricians and psychiatrists practicing in a tertiary health facility were associated with having higher knowledge about ASD as compared to general practitioners.7Similarly, in our study amongst clinicians, psychiatrists followed by pediatricians had highest knowledge. Another similar study about HCPs in tertiary care centers (psychiatry or pediatrics) found the mean knowledge to be 12.35±4.4, which was higher than in our study.A positive finding was that medical trainees had a high score of 81.58%. This might be in agreement with previous studies that medical curriculum and psychiatric clerkships do equip students with an ample knowledge about psychiatric illnesses however there may be fading away of this knowledge without ongoing training and education.13Interestingly, pediatricians had a mean score of 9.96, higher only by fractions than other specialties like gynecology (9.80) and surgery (9.46). This may have important implications as pediatrics and general practice are the specialties that are the first point of contact for these children and families. Running programs and workshops to sensitize these specialties may prove to have an exponential effect in successful early detection and prompt referral of such children.In addition to the KCAHW questionnaire, we asked a few questions to assess the professionals’ views about modalities of interventions needed to manage ASD, as this would affect referral to appropriate and concerned professionals. Participants had to choose one or more options out of various pharmacological and non-pharmacological measures , OT and BT which they deemed would help in management of ASD.Most of the HCPs i.e. 72.10% (106) opted for medication as a treatment option. Interestingly, there was no statistically significant difference between physicians and non-physicians in this regard (P=0.31). Keeping in view that so far, no medications have been proven to be efficacious in the treatment of the core social communication impairment seen in ASD and current pharmacological management (mostly with risperidone and aripiprazole) is only aimed at targeting symptom of irritability associated with ASD.15Regarding non-pharmacological treatment options, only near half of HCPs believed in referring to speech therapists, psychiatrists, psychologists and occupational therapist as a possible intervention. Alarmingly, many did not recognize the key role played by psychiatrists and psychologists in the diagnosis and management of ASD. This is even higher than previous studies, which showed that more than 25% of HCP’S did not consider the need for ST and special education services in the management of this disorder.16There were many misconceptions regarding etiology, symptomatology and prognosis of ASD. Overall, no statistical difference was found between physicians and non-physicians regarding questions related to myths and misconceptions about ASD. In response to the notion that “ASD is a rare condition as compared to the West”, although 60% disagreed, a large number of professionals were either not sure or thought otherwise regarding the increasing autism prevalence. Without having prior knowledge and awareness we cannot expect HCP’s to be prepared to deal with the rising magnitude of this condition.One popular belief is that vaccinations can cause ASD.While exploring views “Even with early interventions, ASD has poor prognosis”, 38.1% disagreed or weren’t sure that children with ASD can improve. This was the most worrisome finding as early intervention has a proven role in improving functionality and outcome in autism.4Finally, two questions were asked in order to view the participants’ perceptions about their own knowledge and understanding around ASD along with their views about awareness activities and campaigns which have occurred. More than three quarter of HCP’s agreed to the notion that “There is a little awareness about ASD among HCPs in Saudi Arabia”, whereas half of them were not sure or thought otherwise about the statement that “Enough campaigns had been done to raise awareness about autism in the Country”. This could be because of most of the courses and symposiums about autism have been carried out in specialist settings that provide services for people with autism or special needs. Thus, this may limit the abilities of HCPs like pediatricians and surgeons to attend such courses.Our sample was not fully representative, as it was recruited from a tertiary care government hospital and from maternal and children hospital in Makkah. Thus, results cannot be generalized to the whole region or country. In addition, the numbers in each specialty were not equal which can give an uneven perspective.Despite these limitations, our study is the first of its kind in Saudi Arabia, as it does not only provide evidence and comparison about presence of a huge knowledge gap regarding this prevalent disorder amongst various physicians and non-physicians, but also identifies the specific areas of knowledge deficiency. Decreased knowledge about comorbidities associated with ASD, prognosis and possible treatment modalities especially non-pharmacological options, can be improved by stressing around these areas, as well as improving education and training, inclusion in discussions and ward rounds along with use of media and electronics in order to increase awareness and early recognition of autism amongst healthcare professionals countrywide.There is a dearth of knowledge about ASD amongst HCPs in Saudi Arabia. Specific and focused strategies need to be put on in order to bridge this knowledge gap to ensure early detection, timely referral, appropriate diagnosis and evidence based interventions. Experienced HCPs working with children with ASD can be deployed in order to deliver tailored trainings especially regarding comorbidities and appropriate referral modalities to HCPs nationwide.


Cell Death & Disease; 10.1038/cddis.2017.102; published online 4 May 2017.Correction to: The PDF and HTML versions of the article have been updated to include the Creative Commons Attribution 4.0 International License information.


The proportion of at-risk patients for the three hospitals in Kenya and Ghana (66.2%) were significantly higher than that of the three South African hospitals (53.7%) . Discharge risk of malnutrition was 71.2% (n = 394). Mean length of stay (LOS) was 6.46 ± 5.63 days. During hospitalization, 20.6% lost ≥5% body weight, 18.8% were referred for nutrition support, and discharge BMI (23.87 ± 7.38 kg/m2) was significantly lower than admission BMI (24.3 ± 7.3 kg/m2) (p < 0.001). Admission nutrition risk was associated with lower admission and discharge BMI (p < 0.001), longer LOS (p < 0.001), increased 3-month re-admission rates  and increased mortality . Nearly two-thirds of patients were at risk of malnutrition on admission. This was associated with longer LOS and greater hospital mortality. The nutritional status of patients deteriorated during hospitalization. Routine screening practices with appropriate nutrition support action should be implemented as a matter of urgency.This study aims to determine the prevalence of risk of malnutrition on admission and discharge in African hospitals, and to identify the association with selected indicators. In this multi-center prospective cohort study, adult patients from hospitals in South Africa, Kenya, and Ghana were screened on admission and discharge and contacted 3 months post-discharge. Relevant morbidity and mortality outcomes were assessed. At risk of malnutrition was indicated if NRS-2002 score ≥3. Adult patients ( Globally, the prevalence of malnutrition in adults on admission to hospital varies between 11–74% ,13,14. IIn this multi-country, multi-center prospective cohort study, adult patients (≥18 years) admitted to hospital within the preceding 48 h and who gave informed consent were screened for malnutrition on admission and discharge, and contacted 3 months post-discharge. The following hospitals were conveniently selected and participated: three hospitals from South Africa , two hospitals from Kenya , and one hospital from Ghana . The following patient-groups were excluded: pediatric patients (<18 years); pregnant and lactating females, patients admitted to an intensive care unit (ICU), burns or relevant acute care wards; patients admitted to psychiatry or eating disorder units; as well as patients on dialysis.The sample size calculation was based on the primary objective, and to determine the proportion of patients at risk of malnutrition on admission to hospital with a precision of 3.6%, 90% power and a 95% confidence interval. To achieve this, 2100 participants  were required.Facility-coordinators and fieldworkers were responsible for data gathering at each of the facilities. Extensive fieldworker training and adherence to standard operating procedures ensured quality of data. Baseline data were collected within the first 48 h of hospital admission. Patients that were admitted for ≥7 days qualified for inclusion in the discharge group. This data was then obtained at either actual discharge or day 28 of hospitalization for those staying longer.® (NRS-2002) scoring system developed by the European Society for Clinical Nutrition and Metabolism (ESPEN) . However, just under one-fifth  of patients presented with a BMI <18.5 kg/m2. Females had a significantly higher BMI . Significantly more females also presented with obesity than males (p < 0.001) (Median BMI of patients was 23.7 kg/m< 0.001) .p < 0.001). The greatest weight loss was experienced by patients with HIV/TB (14.06%), oncology (10.87%), and endocrine diseases (8.06%).In total, 1153 (54.3%) patients experienced weight loss before hospital admission, with an average percentage weight loss of 6.77 ± 10.81% (−26.32% to 61.54% range). Just under a quarter experienced weight loss exceeding 5% in the period before hospitalization  . SignifiThe average NRS-2002 score was 2.65 ± 1.63. A total of 1714 (80.6%) patients succeeded to the final assessment phase of the NRS-2002. Of these, 75.1% (1288/1714) scored positively for being at risk of malnutrition (NRS ≥ 3), which represents 60.6% (1288/2125) of all patients on admission .n = 172/2126) of the admitted patients were referred for nutritional support within the first 48 h after admission  and percentage weight loss prior to admission . During hospitalization, 58.6% (n = 313/534) of patients lost weight, with 20.6% (110/534) losing more than 5% body weight (n = 295) of the patients reported a reduced food intake  while in hospital and only 18.8% (n = 102/544) of all patients were referred for specialized nutritional support during hospitalization.Mean hospital LOS was 6.46 ± 5.63 days and this was positively, albeit weakly, related to age  of the patients developed complications requiring medical intervention during hospitalization. Patients developed an average of 0.79 ± 1.19 (0–5 range) complications. Significant differences were found between primary diagnosis and the number of complications incurred during hospitalization (p < 0.001), with the highest number of complications found among patients with HIV/TB (1.96), general surgical conditions (1.22), and gastroenterology conditions (1.31).Just less than half   .In total, 452 patients were available for follow-up telephonically 3 months after the hospital discharge. Self-reported weight changes revealed that the majority  had gained weight post-discharge, while the weight of 21.5% (82/381) remained constant, and 26.8% (102/381) had lost weight in the post-discharge period.p = 0.001). BMI on primary admission was negatively related to the number of complications that developed in the follow-up period .Of the group, 73 (36.86%) developed a complication that required medical intervention and 66 (17%) had been readmitted to hospital in that period. Of those readmitted, the percentage weight loss prior to baseline admission was significantly higher (12.35 ± 15.94%) than the weight loss in those not readmitted (6.44 ± 8.78%)  patients died in this 3-month period, which was more than the 36 (1.7%) patients that died during hospitalization.p < 0.001), whereas the admission average BMI (24.31 ± 7.31 kg/m2) was significantly higher than the discharge BMI (23.87 ± 7.38 kg/m2) (p < 0.001). The inverse link between NRS score and BMI was confirmed with a significant negative relationship noted between admission NRS score and a lower BMI . It was also noted that the proportion of patients with a BMI <18.5 kg/m2 at discharge (24.3%) was significantly more than the admission values , indicating a shift from a normal to decreased BMI status that developed during hospitalization.Wilcoxon signed rank testing for non-parametric paired data revealed that the NRS score on admission (2.95 ± 1.15) was significantly lower than the discharge score (3.11 ± 1.46) (p < 0.001), longer LOS (p = 0.001), and increased mortality (p < 0.001). . .2 = 164.6, p < 0.001). When the three hospitals from South Africa  were compared to the three hospitals from outside of South Africa , the proportion of at-risk patients in the former (53.7%) were significantly less than the proportion of at-risk patients in the hospital outside of South Africa (66.2%) . . .p < 0.001). Significant differences were noted between the proportion of patients at risk of malnutrition and the primary diagnosis . The highest prevalence of malnutrition risk was found in patients with HIV/TB (84.3%), hematology (77.7%), respiratory (75.1%), endocrinology (73.8%), and gastroenterology (71.1%) conditions  was at least a decade younger than those reported for similar studies done in Europe, ranging between 57–72 years ,15,20,21 years wa2) was found, but the range was quite varied (8–60 kg/m2). Females presented with a significantly higher BMI than males, a finding that was also reported for another South African study [A normal mean BMI  experienced weight loss prior to admission and in a quarter of cases, the weight loss exceeded 5%. Sorenson et al.  reportedSixty percent of all patients in this study scored positively for being at risk of malnutrition (NRS score ≥ 3) on admission. These results are in line with another study (Switzerland) using the same tool, that identified 62.7%  of patie2, and/or MUAC < 22 cm for women and 23 cm for men) reported 19.3% [The proportion of at-risk patients for the three hospitals in Kenya and Ghana 66.2%) were significantly higher than that of the three South African hospitals 53.7%). Using a different tool (MUST), another study from South Africa reported a risk of malnutrition of 72.3% 6.2% were reported.7%. Usined 19.3% . It is cIn the present study, the largest proportion of at-risk patients were seen in those with chronic diseases, with HIV/TB patients presenting with the highest prevalence at 84%. Bearing in mind the persistent malnutrition (undernutrition) reported on the African continent at 20%  and the 2 (paired data). Alvarez-Hernandez [More than half (59%) of the patients lost further weight during hospitalization and in 20% of cases, the weight loss exceeded 5% of body weight. This resulted in a significantly lower discharge mean BMI, compared to admission values and a greater proportion of patients with BMI values below 18.5 kg/mFactors contributing to the worsening of nutritional status during hospitalization could be inadequate food intake and inappropriately low referrals for nutritional support. Food intake during hospitalization was poor with 54% of patients reporting a reduced intake. This was substantially more than the 32.4% of hospitalized patients with a reduced food intake reported by Sorensen et al. . During Self-reported information was obtained telephonically 3-months post-discharge. Hospital admission BMI showed significant negative correlations with the number of complications requiring medical interventions in the 3-month period. Again, this demonstrates the greater susceptibility of undernourished individuals to develop complications.Hospital readmission was noted in 17% of cases. A greater weight loss prior to initial hospital admission and a lower BMI on admission were found in those readmitted. The positive association between poor nutritional status and hospital re-admission has also been confirmed by Koren-Hakim et al. who reported a 46.5% readmission rate (6-months post-discharge) positively associated with the at-risk group (using MNA-SF tool) ,5.Being at risk of malnutrition on admission to hospital was associated with various parameters including lower BMI on admission and discharge, longer LOS and higher mortality. Although more complications developed during hospitalization and post-discharge in the at-risk group, and the latter patients were readmitted more, this did not differ significantly from the non-at-risk group.As found in the present study, LOS was positively associated with at-risk of malnutrition by various groups ,18,37,38The large economic implications of malnutrition in the hospital setting because of longer LOS, more complications, and readmissions has been displayed in other countries ,11,19,38Patients had to stay in the hospital for seven days or longer to be included in the discharge group. With an average length of stay of 6 days, the study sample for the discharge data and ultimately for the post-discharge data, was smaller than anticipated. This resulted in loss of statistical power to detect significant associations for paired data. Missing data were not imputed resulting in various group sizes. It is possible that selection bias was introduced if the sicker patients did not consent to participate. Because of ethical restriction on data access, it was not possible to obtain information from those that did not consent. Self-reported data were used for some of the variables which could introduce reporting bias.Nearly two-thirds of all patients were found to be at-risk of malnutrition on admission, placing African patients at the top of the range in relation to the global statistics. This was associated with longer length of stay and greater hospital mortality. The nutritional status of patients deteriorated during hospitalization. The reduced food intake and poor referral practices for nutrition support could have contributed to this. Routine screening practices with appropriate specialized nutrition support action should be prioritized and implemented as a matter of urgency in African hospitals. There is a need for more studies in this area across Africa.