Bloodstream infection (BSI) is one of the major causes of morbidity and mortality for patients undergoing hematopoietic stem cell transplantation (HSCT). The unrelated cord blood transplantation (UCBT) can provided opportunities for patients without suitable donors for bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT), while few studies have addressed BSI after UCBT. The aim of this study was to analyse the incidence and risk factors of BSI, causative organisms, microbial resistance, and its impact on the clinical outcomes and survival of patients.There are 336 patients, were divided into two groups depending on whether developing BSI. Demographic characteristics, laboratory data, and clinical outcome were compared between different groups. The risk factors of BSI was examined using logistic regression and the survival was examined using the Kaplan-Meier method and log-rank test.P = 0.0574), respectively. The 3-year OS of the MDR group was significantly lower than that of the non-BSI group .Ninety-two patients (27.4%) developed early BSI with 101 pathogenic bacteria isolated, and the median day of developing initial BSI was 4.5 d. Gram-negative bacteria were the most common isolate , followed by Gram-positive bacteria  and fungi . Thirty-seven (36.6%) isolates were documented as having multiple drug resistance (MDR). Myeloid malignancies, conditioning regimens including total body irradiation (TBI), and prolonged neutropenia were identified as the independent risk factors for early BSI. The 3-year OS was 59.9% versus 69.2% in the BSI group and no-BSI group (Our data indicate that the incidence of early BSI after UCBT was high, especially in patients with myeloid disease and a conditioning regimen including TBI and prolonged neutropenia. Early BSI with MDR after UCBT had a negative impact on long-term survival. Infection is a common complication after hematopoietic stem cell transplantation (HSCT), and bloodstream infection (BSI) is one of the major causes of morbidity and mortality for patients undergoing HSCT . In the In this paper, we reported the results of our analysis of early BSI in 336 patients undergoing UCBT at a single institution. The aim of this study was to analyse the incidence and risk factors of BSI, causative organisms, microbial resistance, and its impact on the clinical outcomes and survival of patients.Between January 2012 and January 2017, 361 consecutive patients with malignant hematologic disease undergone single-unit UCBT following a myeloablative conditioning regimen in our transplantation centre. These patients did not have suitable related or unrelated bone marrow transplantation donors. Among the 361 patients, 25 suffered from primary engraft failure and were excluded from this analysis. Therefore, the final data analysis included 336 cases.Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC700603, Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619.All patients received a myeloablative conditioning regimen including or not including total body irradiation (TBI). All patients were treated with cyclosporine A (CsA)  and mycophenolate mofetil (MMF)  for the purpose of GVHD prophylaxis. CsA was administered from day − 1 at 3 mg/(kg.d) intravenously, then oral CsA at a dose ratio of 1:2 when oral intake could be tolerated with a trough level of 150–250 ng/mL. CsA was discontinued on day 180 or before if no GVHD occurred. From day + 1 to day + 28, patients took MMF orally three times a day to the total dose of 30 mg/(kg.d). The rapidity of tapering was based on the presence or absence of GVHD, infectious disease, and relapse risk. Infection prophylaxis consisted of oral Itraconazole, oral cefprozil and intravenous acyclovir. Ganciclovir or foscarnet was applied as a pre-emptive CMV therapy. Serum levels of C-reactive protein (CRP) were measured at least three times a week or at times of fever. During the pre-engraftment period, patients who experienced a neutropenic fever would be treated with broad-spectrum antibiotic. In the process of neutropenic fever (with body temperature ≥ 38 °C), patients received an empirical antibiotic regimen consisted of imipenem/cilastatin and aminoglycoside (amikacin), which was later modified in accordance with their clinical condition and the results of microbial culture. In the event that the fever continued for 48 h and/or the clinical symptoms got worse, patients would receive intravenous vancomycin; in the event that the fever failed to respond to these regimens by day 5, patients would receive the empirical antifungal treatment with intravenous liposomal amphotericin B or caspofungin. At the onset of fever or in the presence of any clinical symptom compatible with an infection, 2 sets of blood cultures were drawn from the peripheral vein and central catheter, and identification and an antimicrobial susceptibility test were performed. Mueller. Hinton Agar containing the 50 g/L off-fiber blood of Sheep was an antimicrobial-sensitive medium. Based on the protocol of drug susceptibility test recommended by the Clinical and Laboratory Standards Institute (CLSI), the results were analyzed by the MicroScan Walkaway 96 PLUS (U.S. Siemens) and Vitek 2 compact (French bioMerieux). Quality control strains were provided by the Ministry of Health Clinical Testing Center including: 9/L. Neutropenia and severe neutropenia were defined as an absolute granulocyte count < 0.5 and < 0.1 × 109/L, respectively. Patients who survived for more than 28 days after transplantation but failed to achieve neutrophil recovery and with < 5% cells of donor origin were regarded as having primary engraft failure. BSI was defined as the isolation of bacteria or fungi from any blood culture in the context of fever or other clinical signs consistent with infection. For coagulase-negative Staphylococci, Streptococcus viridans and other common commensal at least 2 blood cultures were required to be positive [9/L) during the UCBT was identified as having early BSI. Pathogens with intermediate susceptibility or resistance were considered resistant. In this research, the prolonged neutropenia was identified by more than the observed median time of neutrophil recovery in all patients. Overall survival (OS) was defined as the time from UCBT to death or to the last observation. Disease-free survival (DFS) was defined as the time from UCBT to relapse, death or the last observation.Neutrophil recovery was the first day of three consecutive days during which the absolute neutrophil count (ANC) exceeded 0.5 × 10positive . BSI occData on the patients’ characteristics, transplantation procedures, and infectious complications were collected once patients entered the laminar flow ward and were then stored in a computerized database. In cases of inconsistent or missing data, clinical charts were reviewed. A follow-up study on each transplant case was performed annually to update the data on relapse, survival, and complications. The final data set used for analysis was established on October 1, 2017.P < 0.05) or marginally significant (P < 0.20) in univariate analysis were included in multivariate analysis using a forward stepwise method to explore the independent effects of different variables on the incidence of BSI.Categorical variables were compared with the chi-squared test or Fisher’s exact test (both 2-sided) and the Mann–Whitney U test for continuous variables. Continuous variables are expressed as the median and range. The cut-off value for continuous variables was selected based on the observed median . Various clinical factors were evaluated as potential risk factors for BSI in univariate and multivariate analyses combined with the logistic regression model. Factors found to be significant  and R version 3.0.1 (The CRAN project).The OS of different groups was determined using the Kaplan-Meier method and log-rank test and the DFS was determined using survival analysis containing competing event. A 7/kg  and 2.32 × 105/kg , respectively.Table In all patients, 92 patients (27.4%) were documented to have early BSI, 5 patients with twice the occurrence of BSI, and 4 patients with two types of pathogenic bacteria in the same blood culture. Finally, 101 pathogenic bacteria were isolated. The median day of initial BSI was 4.5 d . Within + 7 d, the cumulative frequency of BSI was 84 times, accounting for 91.3% of cases.Candida albicans). The predominant Gram-negative bacteria in descending order were as follows: Escherichia coli (39 isolates), Klebsiella pneumoniae (9 isolates), and Pseudomonas aeruginosa (6 isolates). The predominant Gram-positive bacteria were Streptococcus viridans (10 isolates), Staphylococcus epidermidis (9 isolates), Enterococcus faecium (7 isolates), and Streptococcus mitis (7 isolates). In all isolated pathogens, the number of Escherichia coli was the most, while ESBL-producing E. coli represented 89.7%. In the case of Klebsiella pneumoniae, 6 isolates (66.7%) produced ESBL. No MRSA was documented in our study.Overall, there were 101 isolates over 97 episodes of BSI. Table Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa among Gram-negative bacteria and Streptococci, coagulase-negative Staphylococcus (CoNS), and Enterococcus faecium among Gram-positive bacteria to analyse their resistance to common antibacterial drugs. The results are shown in Tables Klebsiella pneumoniae presented a low rate of resistance to imipenem/cilastatin and amikacin and a relatively high rate of resistance to penicillin, third- or fourth-generation cephalosporins and quinolones. 33.3% Klebsiella pneumoniae were resistance to imipenem/cilastatin. Although Gram-positive bacteria possessed a rather large number of streptococci and CoNS, they were mostly opportunistic pathogens with a low resistance rate. Most Enterococcus faecium specimens were resistant to penicillin and quinolones, while they were 100.0% sensitive to vancomycin, linezolid and tigecycline. In addition, we recorded 37 isolates (36.6%) of MDR. MDR were all Gram-negative bacteria, and five of the nine isolates of Klebsiella pneumonia (55.6%) and 32 of the 39 isolates of Escherichia coli (82.1%) were MDR. In this set of data, we found that Escherichia coli presented low resistance rates to imipenem/cilastatin, amikacin, and piperacillin/tazobactam, which were 5.1, 12.8, and 15.4%, respectively. Only 2 strains of carbopenem-resistant Escherichia coli were discovered, but they were sensitive to amikacin.As for antibiotic susceptibility, considering the category and quantity of the isolated bacteria, we selected p = 0.037), disease (p < 0.001), conditioning regimen including or not including TBI (p = 0.001), state of disease (p = 0.016), number of TNC with a best cut-off at 4.17 × 107/kg (p = 0.035), and time of neutrophil recovery with a best cut-off at 17d (p < 0.001) were statistically significant. And Total CD34+ cell dose with a best cut-off at 2.32 × 105/kg (p = 0.079) was marginally significant. Multivariate analysis showed that ALL compared with myeloid disease , a conditioning regimen including TBI , and prolonged neutropenia  were statistically significant for early BSI after UCBT.Table p > 0.05), PES , and III-IV aGVHD , the median length of time that patients stay in laminar flow ward  showed no significant differences in the two groups. Some clinical indexes, such as the CRP maximum value , day of the CRP maximum value  and day of first fever , were significantly different between the two groups.We also compared the differences in developing acute graft-versus-host disease (aGVHD), pre-engraftment syndrome (PES), and some other clinic indexes between the BSI group and non-BSI group. The incidence of acute GVHD . The 3-year OS was 59.9%  versus 69.2%  in patients with BSI and without BSI, respectively (P = 0.0574), and the 3-year DFS was 57.7%  versus 62.4%  in these two groups, respectively (P = 0.257). Considering the influence of BSI with MDR on prognosis, we made a more detailed grouping. Figures p = 0.012). The 3-year OS was 51.1%  versus 69.2%  in the two groups, and the 3-year DFS was 48.4%  versus 62.4% . The OS and DFS in the non-BSI group and non-MDR BSI group were not different. We also analysed the 100-day OS after UCBT between these three groups, and no difference was found. And considering the AML was identified as a independent risk factor for developing BSI in multivariate analysis, we did separate outcome analysis for AML patients, the results showed that there were 42 patients developing BSI in all 110 patients. The short-term and long-term survival were no significant difference in BSI group and non-BSI group. While, in MDR-BSI and non-BSI group, the long-term survival was significantly different. The 3-year OS was 50.0%  versus 79.4% , and the 3-year DFS was 48.2%  versus 77.1%  in these two groups.The median follow-up of surviving patients was 584 d . First, we compared the differences in survival depending on whether they developed BSI after UCBT. The median day of OS in patients with BSI was 1304 d, while in patients without BSI, it was 1503 d  as independent risk factors for early BSI after UCBT. In previous research, the prolonged neutropenia phase was described to be an independent risk factor for infection . Sanz et7d as indp = 0.0574), while the 3-year DFS in these two groups were not different. Moreover, patients developing MDR-BSI usually had a worse prognosis compared with patients without BSI. These findings emphasize the importance of preventing early BSI after UCBT to improve the prognosis of patients. Meanwhile, early BSI-related mortality did not increase in our data, which was inconsistent with other studies [Escherichia coli, was the empiric antibacterial therapy used in our centre. Antibiotics were changed over time before we received the antimicrobial susceptibility test results, and imipenem/cilastatin was not used for more than 1 week to reduce the occurrence of carbopenem-resistant Enterobacteriaceae. These effective strategies for potential infection and support treatment may have contributed to the low mortality in the early phase after UCBT in patients with BSI. And the immune damage caused by MDR bacteria, depletion of the intestinal microbiota and secondary bone marrow suppression caused by the use of broad-spectrum antibiotics may lead to poor long-term survival in patients with MDR infection [Regarding survival, we found that the 3-year OS of patients with BSI (59.9%) was lower than that of the patients without BSI (69.2%) with marginal significance p = 0.057, while t studies . Imipenenfection –27.we found that the incidence of early BSI in patients with malignant hematologic disease after UCBT was common, particularly in patients with myeloid disease and conditioning regimens including TBI. Patients who developed BSI, especially with the MDR, had worse survival. Understanding the epidemiology and antibiotic resistance of pathogens was of guiding significance for the selection of empirical antibiotics. Early diagnosis of BSI and prompt treatment with effective antibiotics are necessary for patients undergoing UCBT.


We aimed to investigate the expression of proteins related with autotaxin (ATX)–lysophosphatidate (LPA) signaling and the clinical implications in primary and metastatic thyroid tumors.We constructed tissue microarrays with 545 primary thyroid tumors . Immunohistochemical stains for proteins related to ATX–LPA signaling  were performed.The expression of ATX was highest in MC, while the LPA1 expression was higher in PDC and AC, and the expression of LPA2 and LPA3 was highest in PTC (p < 0.001). Additionally, the expression of ATX, LPA1, and LPA2 was higher in conventional-type PTC than in follicular-variant PTC (p < 0.05). PTC with BRAF V600E mutation showed higher expression of ATX, LPA1, LPA2, and LPA3 than PTC without BRAF V600E mutation (p < 0.001). In univariate analysis, ATX positivity (p = 0.005) and LPA1 positivity (p = 0.014) were correlated with shorter overall survival in PTC.Proteins related to the ATX–LPA axis showed different levels of expression in primary thyroid tumors according to subtype. RhoA pathways and is involved in various cellular processes [Autotaxin (ATX) is a glycoprotein transcribed by the ENPP2 gene on chromosome 8 . ATX is rocesses . LPA recrocesses . This ATrocesses , 6.Thyroid cancer is a relatively common malignancy, affecting about 1% of the population. The most common histologic type of thyroid cancer is papillary thyroid carcinoma (PTC), followed by follicular carcinoma (FC), medullary carcinoma (MC), poorly differentiated carcinoma (PDC), and anaplastic carcinoma (AC). These histologic subtypes of thyroid cancer are known to have different cell origins, clinical manifestations, metastatic patterns, and clinical prognoses. Previously, ATX has been reported to be highly expressed in undifferentiated carcinoma , and it Patients diagnosed with PTC who underwent surgery between January 2012 and December 2013 at Severance Hospital, Sinchon-dong, South Korea, were included in the study. As for the remaining subtypes, patients who underwent surgery between January 2000 and December 2014 at Severance Hospital were included. Those who had received preoperative chemotherapy were excluded. This study was approved by the Institutional Review Board of Yonsei University Severance Hospital. Hematoxylin-and-eosin-stained slides of all cases were retrospectively reviewed by a thyroid pathologist . Clinicopathologic data were obtained from the patients’ medical records and included age at diagnosis, disease recurrence, metastasis, current status, and length of follow-up. Tumor size, location (right or left lobe), extent , and number of metastatic lymph nodes were also noted from a review of the slides and the surgical pathology reports.Tumor stroma of PTC was classified as follows: desmoplastic type, tumor stroma composed of cellular fibroblast/myofibroblast proliferation; sclerotic type, tumor stroma composed of collagenous component with few cellular components; pauci type, near absence of a stromal reaction; or inflammatory type, tumor stroma composed of inflammatory cells such as lymphocytes.A representative area showing tumor and tumor stroma was selected on a hematoxylin-and-eosin-stained slide, and a corresponding spot was marked on the surface of the paraffin block. Using a biopsy needle, the selected area was punched out, and a 3-mm tissue core was transferred to a 6 × 5 recipient block. Two tissue cores from the invasive tumors were extracted to minimize extraction bias. Each tissue core was assigned a unique tissue microarray location number that was linked to a database containing other clinicopathologic data.The antibodies used for immunohistochemistry in this study are shown in Table Immunohistochemical markers were assessed by light microscopy. All stained slides were semi-quantitatively evaluated . Tumor at test and Fisher’s exact test were used for continuous and categorical variables, respectively. When analyzing data with multiple comparisons, a corrected p-value with application of the Bonferroni multiple comparison procedure was used. Statistical significance was set at p < 0.05. Kaplan–Meier survival curves and log-rank statistics were employed to evaluate the time to tumor recurrence and overall survival. Multivariate regression analysis was performed using the Cox proportional hazards model.Data were analyzed using Statistical Package for the Social Sciences for Windows, version 23.0 . For determination of statistical significance, Student’s All retrieved cases were subject to this study, consisting of 338 cases of PTC, 111 cases of FC, 69 cases of MC, 23 cases of PDC, and four cases of AC. Basal characteristics of PTC are listed in Additional file The expression of proteins related to the ATX–LPA axis in thyroid cancer showed differences in ATX, LPA1, LPA2, and LPA3 (p < 0.001). ATX was most highly expressed in medullary carcinoma, while LPA1 was predominant in PDC and AC, and LPA2 and LPA3 were predominant in PTC , LPA1 (p = 0.014), and LPA2 (p = 0.001) expression was different according to the histologic subtype, and the expression of ATX, LPA1, LPA2 and LPA3 (p < 0.001) was different according to BRAF V600E mutation status. ATX, LPA1, and LPA2 expression was higher in conventional type than in FVPTC, while PTC with BRAF V600E mutation showed higher expression of ATX, LPA1, LPA2, and LPA3 than did PTC without BRAF V600E mutation  in PTC, and LPA1 expression was different according to the vascular invasion status in FC (p < 0.001). In PTC, desmoplastic type showed high expression of LPA2. LPA1 negativity was correlated with vascular invasion in FC Fig. .Fig. 3CoThe impact of expression of proteins related to the ATX–LPA axis on the prognosis of thyroid cancer was analyzed. As a result, ATX positivity p = 0.005) and LPA1 positivity p = 0.014) were two factors that demonstrated correlation with shorter overall survival (Table  and LPA1 were twoWe investigated the expression of proteins related to the ATX–LPA axis in thyroid cancer. First, ATX was more highly expressed in medullary carcinoma than in other subtypes. Previous studies have shown that ATX-related protein expression was higher in thyroid cancer or metastatic thyroid cancer than in normal thyroid tissue or benign thyroid neoplasm , 10, butThe clinical implication of our study is that the ATX–LPA signaling axis may be a possible therapeutic target in thyroid cancer. Ki16425, a nonlipid competitive inhibitor of LPA1 and LPA3, actually suppressed bone metastasis in a mouse model . Debio07The expression of proteins related to the ATX–LPA axis is different according to tumor subtype and is notably higher in metastatic thyroid cancer than in primary tumor. Furthermore, proteins related to the ATX–LPA axis may represent candidates for effective treatment target in thyroid cancer.Additional file 1. Additional tables.


Occupational exposure to organophosphate pesticides, such as chlorpyrifos (CPF), increases the risk of Alzheimer’s disease (AD), though the mechanism is unclear. To investigate this, we subjected 4-month-old male and female wild-type (WT) and TgF344-AD rats, a transgenic AD model, to an occupational CPF exposure paradigm that recapitulates biomarkers and behavioral impairments experienced by agricultural workers. Subsequent cognition and neuropathology were analyzed over the next 20 months. CPF exposure caused chronic microglial dysregulation and accelerated neurodegeneration in both males and females. The effect on neurodegeneration was more severe in males, and was also associated with accelerated cognitive impairment. Females did not exhibit accelerated cognitive impairment after CPF exposure, and amyloid deposition and tauopathy were unchanged in both males and females. Microglial dysregulation may mediate the increased risk of AD associated with occupational organophosphate exposure, and future therapies to preserve or restore normal microglia might help prevent AD in genetically vulnerable individuals exposed to CPF or other disease-accelerating environmental agents. The interaction of genes and environment contributes to Alzheimer’s disease (AD). For example, agricultural workers, military personnel, industrial manufacturers, veterinarians, horticulturists, aircraft maintenance personnel, and pilots are all potentially at risk of occupational exposure to organophosphates (OPs), which are associated with increased risk of AD. We report here that occupational-like exposure of young animals to the OP chlorpyrifos (CPF) accelerates AD-like cognitive deficits and severe neurodegeneration in male, but not female, TgF344-AD rats, a genetic model of AD. CPF exposure also causes chronic dysregulation of brain microglial cells, while amyloid and tau pathology are not affected. Thus, microglial dysregulation after environmental toxin exposure may represent a second hit that advances the disease. Future therapies to preserve or restore normal microglia might help prevent AD in genetically vulnerable individuals exposed to CPF or other disease-accelerating environmental agents. Understanding the underlying mechanisms could help identify new ways to prevent or treat AD in these populations. However, investigation to date has focused narrowly on acute, high-level exposures,9 rather than the more common moderate and prolonged occupational-like exposure.10 To help address this critical gap, we subjected a rat model of AD to a model of occupational chlorpyrifos (CPF) exposure, a commonly applied OP pesticide. We used TgF344-AD rats, which overexpress two mutations that cause AD: APPSW and PS1∆E9. These animals develop amyloid plaques, hyperphosphorylated tau, neuroinflammation, neuronal cell loss, and behavioral deficits akin to human AD patients, rendering them well-suited for preclinical studies.13 For our environmental model, we applied an established human-derived occupational exposure paradigm that recapitulates characteristic biomarkers and behavioral impairments in a cohort of Egyptian agricultural workers occupationally exposed to CPF.17 A physiologically based pharmacokinetic/pharmacodynamic model18 was used to establish the doses, duration of treatment, and route of administration to inhibit blood ChE activity in rats to levels comparable with those reported in the Egyptian agricultural workers. Accordingly, we exposed 4-month-old male and female TgF344-AD rats and wild-type (WT) littermates to CPF in this manner, and then longitudinally assessed behavior and brain pathology for more than 2 years. We studied both males and females, because epidemiological studies indicate that women are more susceptible than men to AD,19 whereas men are more susceptible than women to the deleterious effects of OP exposure.23Synergistic interactions between genes and the environment play a prominent role in major neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease.n = 26-31/group) to the early-life occupational-like CPF exposure paradigm and then assessed behavior and pathology at 6, 9, 12, 15, and 24 months of age , cholinesterase activity returned to baseline in all groups and remained relatively constant throughout the study   Fig. . Althoug98) Fig. . In the 98) Fig. . Further98) Fig.  and perc98) Fig. . Female 98) Fig. .Fig. 1MaAt 9 and 12 months of age, respectively 17 and 31 weeks after CPF exposure, males and females in all groups were indistinguishable from WT vehicle controls in locomotor activity, NOR, and BM Figures  and S4. t test (P = 0.004). This learning deficit in TgF344-AD males was exacerbated by CPF exposure (P = 0.007)   Fig. . CPF-exp03) Fig. . In fema03) Fig. .28 At the earlier time points, we chose not to conduct repeated MWM testing, despite increased sensitivity, because of its effects on stress hormones29 and the role of stress hormones in AD pathology.31 As the 24-month time point was the final stage of testing, however, we proceeded with MWM. CPF-exposed TgF344-AD males exhibited significant learning and memory deficits at 24 months of age, relative to WT littermates, in the MWM reversal learning and memory probe tests, while CPF-exposed WT males did not  staining showed that aging TgF344-AD rats progressively develop cortical and hippocampal vacuoles Fig. . These vOverexpression of mutated APP and mutated PS1 responsible for processing APP leads to amyloid plaque deposition in TgF344-AD rats, a key pathological feature of the brains of AD patients. Initial survey of amyloid plaques in TgF344-AD rats revealed prolific deposition in the prefrontal cortex, cerebral cortex, entorhinal cortex, and hippocampus, with sporadic deposition in the striatum and cerebellum. Plaque deposition progressively increased with age from 12 to 24 months in TgF344-AD groups, but no significant differences at any age in any group were observed as a function of CPF exposure Fig. . Amyloidβ1–40 and Aβ1–42. Soluble forms of these peptides are toxic and disrupt synaptic transmission, with Aβ1–42 fragments being more toxic than Aβ1–40 fragments.32 At the 15-month time point, amyloid peptide deposition soluble in triton  or 5 M guanidine-HCl  was further analyzed by Aβ1–40 and Aβ1–42 peptide ELISAs. Soluble and insoluble Aβ1–40 and Aβ1–42 were increased in TgF344-AD male and female rats relative to sex-matched controls  in TgF344-AD rats relative to WT controls .s Figure . Furtherols Fig. . Total tols Fig. .34 Here, we observed microglial cell population changes in the hippocampus and cortex of male and female TgF344-AD rats  lowly ramified (0–3 processes), (2) highly ramified (≥4 processes) with large soma, (3) highly ramified (≥4 processes) with small soma, and (5) dystrophic/aged. While microglial morphology changes with age, and TgF344-AD rats displayed distinctly different microglial morphologies from age-matched controls throughout their lifetime, we found no significant differences in morphology as a function of CPF exposure in any animals Figure . Thus, C35 whereas BM uses the entorhinal cortex and hippocampus.28 Importantly, the interval of time between behavioral tests was held constant across all animals, in order to increase reliability of the results. Thus, early-life occupational CPF exposure accelerates the onset of broad deficits associated with cortical and hippocampal-dependent tasks of learning and memory in male TgF344-AD rats, but not in WT male littermates or CPF-exposed female rats of either genotype. Notably, the distance traveled in the open field decreased significantly for females in both vehicle- and CPF-exposure groups at all time points after 6 months of age, but this difference was not seen in males. All males and females were handled and aged similarly, and the basis for this interesting sex-specific difference in locomotor activity is not known.The goal of this work was to determine whether occupational-like CPF exposure would impact the trajectory of disease development, defined here as the phenotype induced by overexpression of mutant APP and PSEN1. We combined two preclinical models to investigate the interaction between known genetic mutations in AD expressed in TgF344-AD rats and early-life occupational-like exposure to CPF, a commonly applied OP pesticide. Male and female rats of both genotypes exhibited significant inhibition of acetylcholinesterase enzyme activity during the 3-week CPF-exposure period, with enzymatic activity returning to normal 8 weeks later and remaining in normal range thereafter. Two months after completion of CPF exposure, when rats were 6 months old, male TgF344-AD rats exhibited significantly impaired cognitive functioning in the NOR and BM cognitive tasks. Six-month-old TgF344-AD rats in the vehicle control arm of the environmental exposure, however, retained normal cognition. WT littermates also did not manifest cognitive deficits as a function of CPF exposure at this time point. While both NOR and BM tasks measure learning and memory, they employ a unique interplay of different brain regions. For example, NOR utilizes the perirhinal cortex, prefrontal cortex, and hippocampus,Repeated measures testing in animal behavior can be challenging, and it is important to note that the 9-month-old animals in our study were no longer naive to the BM learning and memory tasks. Thus, it is possible that these animals may have recognized that the escape location had previously rotated, rendering the memory probe less useful. However, learning can still be assessed under these circumstances, because the goal of the task  can be achieved and animals remain motivated to find an escape. Furthermore, the NOR test works well for repeated testing, as objects are changed every time. In NOR, memory deficits observed in 6-month-old male CPF-exposed TgF344-AD rats were absent at 9 and 12 months. At 15 months of age, however, the cognitive deficits resurfaced in male TgF344 AD rats. Unfortunately, in the case of NOR, the performance of vehicle-exposed TgF344-AD rats was already so severe that there was no room for detection of possible additive effect of CPF exposure. However, a significant worsening of BM reversal learning was noted in both WT and TgF344-AD male rats as a function of early-life occupational-like CPF exposure. In females, no deficits in either task for either genotype or treatment group were observed.36 Because repeated surgical mass removal was prohibited by animal care staff, rats were euthanized if they required more than one mass removal. Although the reduced number of animals at the final time point resulted in greater variability, it was clear in the MWM reversal learning phase and memory probe that early-life occupational-like CPF exposure resulted in more greatly impaired cognitive function in TgF344-AD males than in WT male littermates. In general, males appear selectively susceptible to developing cognitive impairment after early-life occupational-like CPF exposure, with exacerbated effect in the presence of genetic susceptibility to AD.At 24 months of age, the terminal point of the study, males continued to show susceptibility to cognitive impairment after early-life occupational-like CPF exposure, while females functioned normally. Initially, 10 animals were designated for analysis at the 24-month time point. However, survival in all groups, regardless of sex, genotype, or exposure, was roughly 50%. At this age, ~95% of Fischer rats develop neoplasms that impede mobility or reduce quality of life.37 Under pathological conditions, however, microglia may become dysregulated such that they do more harm than good, ultimately increasing toxicity and exacerbating neurodegeneration.42 In our model, microglia in rats with familial AD mutations are dysregulated early in disease. This dysregulation persists chronically and can be exacerbated by environmental insult. Although monocyte populations in TgF344-AD males did not increase in response to CPF treatment, there was a significant shift in the activity state of this cell population toward more phagocytosing. Conversely, females had increased monocyte populations, without a shift in the phagocytosing population in response to CPF exposure. One explanation for this may be that female monocytes increase in number yet remain capable of maintaining a healthy balance of activity, while the males experience a shift in monocyte activity that may advance the disease process.Although female TgF344-AD rats never developed cognitive impairment like males, both sexes displayed cortical and hippocampal vacuolization that was exacerbated by early-life occupational-like CPF exposure. Notably, this effect was more severe in male TgF344-AD rats. Amyloid and tau pathology were not affected by CPF exposure in either sex, but we did observe early microglial proliferation and reactivity as a function of CPF exposure in both males and females, with greater severity in males. Throughout life, a balanced microglia response is critical for maintaining homeostasis in the brain. Under healthy conditions, microglia respond to damage, communicate with neuroinflammatory cells, and clear toxic debris.43 Future work focused on the effect of CPF on microglia is needed to fully understand the process in this model. Nonetheless, the data are such that it can be proposed that therapeutic strategies aimed at major regulators of phenotypic change of microglia under pathological conditions, such as the TREM2-APOE pathway,44 could potentially protect genetically vulnerable populations from disease following microglial-activating environmental events.It is interesting that early-life exposure to CPF induces chronic microglial dysregulation. One explanation may be that microglia undergo a form of cellular senescence following continuous free radical damage, as described by Streit et al.10 Although the goal of this study was to determine consequences of occupational-like CPF exposure in the development of AD-associated behavior and pathophysiology, we also observed interesting effects in aging WT rats. For example, we observed that CPF induces chronic alterations in locomotor activity in WT females, as well as learning and memory deficits in WT males. Furthermore, microglial populations were selectively altered to a more phagocytosing phenotype upon aging in WT males as a function of CPF exposure.Previous studies have described an array of behavioral impairments in WT animals following various exposures to CPF, including deficits in learning, memory, activity, attention, motor coordination, and behavior related to affective state.45 have recently reported significant differences in the transcriptome of male and female microglia from an adult mouse brain, and observed that transplantation of female microglia into adult male mice is neuroprotective in cerebral ischemia. However, alternative hypotheses may also be offered for the lack of behavioral phenotypes observed in female AD- and CPF-exposed groups. For example, we do not know whether exposure levels of CPF differed in the brains of males and females, though we do know that males showed the largest deficit and the least robust inhibition of AchE in the plasma following CPF exposure. In addition, relative to males, females have elevated plasma PON1 activity, a key xenobiotic metabolizing enzyme involved in CPF detoxification that might protect females from CPF-induced neurotoxicity.21 It is also possible that the behavior tests employed in this study were not sensitive enough to detect cognitive impairments in females. While numerous measures were taken to reduce confounding factors, including a meticulously designed behavior schedule, separation of sexes throughout behavior testing, and spraying equipment between tests to remove scents, males and females may have reacted differently to the testing environment. Finally, although previous studies have highlighted the effects of ovulation on behavior,44 it was not feasible to control for ovulation cycles in our experiments, and we are therefore unable to conclude whether this impacted outcome measures.Given that male TgF344-AD rats also showed greater susceptibility to neurodegeneration and resulting cognitive deficits, this male-specific susceptibility may lie within the microglial response to early-life occupational-like CPF exposure. Indeed, Villa et al.15 This same study also reported significant transcriptomic changes in the hippocampus of animals treated with 10, but not 3, mg/kg/d CPF, using the same CPF-exposure paradigm.15It is interesting that no significant behavior effects or pathology were observed in male or female rats exposed to 3 mg/kg/d CPF. This exposure inhibited plasma cholinesterase ~50%  and 75% , as compared with the 10 mg/kg/d CPF dose that reduced plasma cholinesterase activity by ~75%  and 90% . An independent study found that 21 days of CPF exposure inhibited brain cholinesterase levels by 50–60% with 3 mg/kg/d and 90% with 10 mg/kg/d, compatible with our observed dose response in neurotoxicity.The experimental design employed in this study may prove useful for determining potential links between an array of environmental insults and genetic vulnerabilities to neuropsychiatric disease. We recognize that while the model of early-life occupational-like CPF exposure here is representative of exposures that occur in many countries worldwide, it is not typical of exposures experienced in the USA agricultural industry. Still, global agriculture workers, military personnel, industrial manufacturers, veterinarians, horticulturists, aircraft maintenance personnel, and pilots are all potentially at risk of occupational exposure to OPs, and therefore may also be at an increased risk for developing AD. Our results suggest a new therapeutic direction for protecting these individuals.t test was used. All analyses were conducted using GraphPad Prism . Alpha levels were set to 0.05, and outliers were determined using ROUT method in Prism and removed with Q = 0.1%. Significance is denoted as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Analyses were blindly conducted. The code was not broken until analyses were completed.Data were normally distributed. For multiple mean comparisons, ANOVA was used, followed by post-hoc comparison (Tukey’s method). For direct comparison, two-tailed Student’s Animal procedures were performed in accordance with University of Iowa Carver College of Medicine Animal Care Committee’s regulations.All animal procedures were performed in accordance with the University of Iowa Office of Animal Resources and Institutional Animal Care and Use Committee (IACUC). We used WT Fischer 344 and heterogeneous TgF344-AD male and female rats on a Fischer 344 background. TgF344-AD rats co-express human APP 695 with mutations  and PS1 with a deletion of exon 9 driven on a mouse prion promoter (MPP). A male TgF344-AD breeder was kindly provided by Dr. Robert Cohen and bred with Fischer 344 females from Charles River. All animals were maintained on a 12-h light/dark cycle (6:00 a.m. to 6:00 p.m.) in temperature-controlled conditions (70–72 °F), and animals were provided normal chow (Envigo 7913) and water ad libitum. Animals were housed in a specific pathogen-free (SPF) facility in standard caging (Thoren brand filtered caging) with cellunest bedding (Shepherd Paper Products). Rats were genotyped and given a 4-digit identification number for blinding purposes. Placement into experimental groups was randomly determined at the beginning of the experiment, and all WT controls were littermates. To account for the effects of environmental enrichment on cognition, animals were housed two or three to a cage throughout the duration of the experiment. Occasionally, aging animals required standard veterinary care, and thus, all animal care procedures deemed necessary were performed by a member of the University of Iowa’s Office of Animal Resources veterinary staff according to IACUC regulations. Behavior analysis was performed at least 2 weeks after surgical procedures were performed to reduce confounding factors of anesthesia on behavioral tasks.Four-month-old male and female WT and TgF344-AD rats were randomly placed into treatment groups. Subcutaneous (S.C.) injections of vehicle, 3 mg/kg/d CPF, or 10 mg/kg/d CPF  were administered between the hours of 8:00–10:00 a.m. daily for 21 days. The vehicle consisted of 10% ethanol and 90% peanut oil. Animals were monitored daily for pain or discomfort. Throughout the exposure period, tail vein blood was collected under isoflurane starting on day one (baseline) and repeated three additional times, 7 days apart, ending on the final day of exposure. Blood was collected in ACD anticoagulant prior to compound administration at each time point, and thus represents circulating levels from the previous day’s administration. Plasma was isolated using a standard practice and samples were stored at −80 °C until further analysis.46 Briefly, 10 μl of collected plasma was assayed using a Quantichrom Acetylcholinesterase Assay Kit (BioAssay Systems), and samples were analyzed on a Spectromax M2 plate reader at 380 nm. Cholinesterase enzyme activity was normalized to protein concentration, which was measured using a Bradford assay.Plasma cholinesterase enzyme activity was analyzed using a modified Ellman’s assay.At 6, 9, 12, 15, and 24 months of age, animals were subjected to a carefully designed behavior paradigm that consisted of open field, novel object recognition, and Barnes maze  with reversal task or MWM (24 months of age) with reversal task. Behavior testing was designed to place the most aversive tests at the end of the testing paradigm to reduce confounding factors of consecutive days of testing  was a black, open topped Plexiglas box, measuring 100 × 100 × 30 cm. Rats were placed in the center of the open-field apparatus and allowed to freely explore for 10 min for each test. ANY-Maze video tracking software (Stoelting Co.) was used to measure the distance traveled by each animal, and analysis was conducted blind to treatment group.47 and the open-field apparatus (previously described) was used during object recognition analysis. Objects were made of plastic, ranged from 3 to 6 cm in size, and were of different shapes in different colors. NOR testing was completed on animals up to five different times, and thus, different objects were used for each round of repeated testing to maintain novelty in this task. A total of 10 different objects were used throughout the duration of this experiment. Dim lighting was provided in all stages of the testing. Open-field analysis (as previously described) was preformed one day prior to familiarization testing to allow the animal to habituate to the testing apparatus. On the second day of NOR testing (the familiarization phase), rats were placed in the open-field box containing two identical objects (A1 and A2) fixed to the floor and allowed to freely explore the apparatus for 10 min. On the third and final day of NOR testing, the choice phase of the task was performed. Two new objects (A3 and B) were fixed to the floor. Object A1, A2, and A3 were identical, but different from object B, the novel object. The A3 object was used to eliminate any potential for smells from the familiarization to influence the choice phase of the task. Videotape recordings of the familiarization and choice phases were analyzed independently by two blinded scorers, who recorded the time each rat explored each of the objects during the familiarization and choice phases. The percentage of time spent exploring the new object (B) during the choice phase, corrected for any location preference during the familiarization phase, was taken as a measure of memory. Exploration of an object was defined as when the rat directed its nose toward the object at a distance of < 5 cm.A novel object recognition test was performed to assess recognition memory.48 The Barnes maze apparatus was a white, circular surface, 120-cm in diameter, and was raised to a height of 40 cm. The maze consisted of 20 holes, 10-cm in diameter, equally spaced around the border of the surface with an escape cup placed under one hole . Four, high-contrast images, placed equal distance around the apparatus and 10 cm from the top of the table, were used as spatial cues. Over the course of 20 months of behavior testing, 12 different high-contrast images were used as spatial cues. Bright lighting above the apparatus was used to motivate the animals to find an escape location. Each test animal was subjected to 4 days of training comprised of four trials per day, spaced a minimum of 20 min apart, and the time spent to find the escape location and the number of errors (nose pokes in incorrect holes) committed prior to finding the escape cup were recorded for each trial and averaged for each day. The trial ended when the rat entered the escape cup or after 80 s. On day 5, a probe trial was conducted, during which the escape cup was removed and the animal’s memory of the escape location based upon spatial cues was analyzed. ANY-Maze video tracking software (Stoelting Co.) was used to measure the percent time spent in the target area and the percent time spent in the target quadrant. The ratio of the number of nose pokes in the target hole to the number of nose pokes in incorrect holes was manually scored. Nose pokes were defined as head deflections into a hole, but successive pokes into the same hole were not counted as multiple nose pokes. An area extending 5 cm from the escape hole in all directions was used as the target area for measurements.Barnes maze testing was conducted to assess learning and memory at 6, 9, 12, and 15 months of age.Reversal testing was used to further assess learning and memory. Following initial Barnes maze analysis, rats were given 2 days of rest and then tested for learning and memory, as well as cognitive flexibility. Accordingly, the escape hole was rotated 180° and associated with a new spatial cue. Again, each animal was given time to explore the Barnes maze platform freely for 80 s or until they located the new escape location. Four trials were conducted each day at a minimum of 20 min apart for 3 days, and the latency to find the escape and the number of errors committed prior to finding the correct escape location were recorded to test the animal’s capacity to learn the escape location. On day 4, the escape cup was removed to test the animal’s memory and the probe assay was scored as previously described.49 The MWM was a silver, circular water tank, measuring 167-cm in diameter, filled with 24–25 °C water to ~60 cm. A clear, plastic platform was placed submerged below the surface of the water in association with one of four high-contrast spatial cues located around the maze. Each test animal was subjected to 4 days of training comprised of four trials per day a minimum of 20 min apart, and the time spent to find the platform or 120 s (if the platform was not located) was recorded for each trial and averaged for each day. On day 5, a probe trial was conducted, during which the platform was removed and the animal’s memory of the escape location based upon spatial cues was analyzed. ANY-Maze video tracking software (Stoelting Co.) was used to measure the latency to and the number of times the animal crossed the previous platform location area.The MWM test was conducted to analyze learning and memory in 24-month-old rats.Reversal testing was used to further assess learning, memory, and cognitive flexibility. Accordingly, the escape platform was rotated 180° and associated with a new spatial cue. Again, each animal was placed in the water tank until they found the location of the platform or for 120 s. Four trials were conducted each day for 3 days, and latency to find the platform was recorded to test the animal’s capacity to learn the escape location. On day 4, the platform was removed to test the animal’s memory and the probe assay was scored as previously described.Chlorpyrifos (CAS no. 2921-88-2) 98% purity was purchased from Chem Service and diluted in 10% ethanol and 90% peanut oil (Sigma). Paraformaldehyde (PFA) was purchased from Electron Microscopy Sciences and diluted to working concentrations in PBS. An antibody against APP  obtained from ThermoFisher Scientific was used at dilutions of 1:1000 for western blot (WB). An antibody against abnormally phosphorylated tau (clone tau-ps199/202) was obtained from Sigma and used at a concentration of 1:1000 for immunohistochemistry (IHC) and 1:4000 for WB. An antibody against Iba1 (1:5000 for IHC) was obtained from Wako Ltd., and an antibody against CD68 (1: 1000 for IHC) was obtained from Abcam. β-Actin (1:1000 for WB) was obtained from Santa Cruz. Secondary antibodies against mouse and rabbit  were obtained from Cell Signaling, and secondary ImmPRESS IgG HRP and ImmPACT Nova Red and DAB kits were obtained from Vector. Congo Red was obtained from Sigma.g for 20 min at 4 °C and the supernatant was set aside. Pellets were rehomogenized in 3 × (tissue/volume) buffer supplemented with 10% sucrose and salts and then centrifuged at 27,000g for 20 min at 4 °C. The supernatant was combined with the first supernatant and the pellet was saved for WB analysis (crude pellet). To isolate sarkosyl-insoluble tau, the combined supernatants were incubated for 1 h at 37 °C in 1% sarkosyl and 1% β-mercaptoethanol. The samples were centrifuged at 150,000g for 35 min at room temperature. The resulting pellet (sarkosyl-insoluble P3) was analyzed via WB. For Aβ analysis, the aliquot was centrifuged at 10,000g for 15 min at 4 °C. The supernatant (soluble fraction) was centrifuged at 16,000g for 20 min at 4 °C and the resulting supernatant was diluted in cell lysis buffer for further analysis. The pellet from the initial centrifugation was rehomogenized in 4 × (tissue/volume) 5 M guanidine-HCl for further analysis.Transcardiac perfusions with ice-cold PBS were performed while animals were deeply anesthetized under isoflurane and brains were quickly collected and separated into hemispheres. The left hemisphere was fixed in 4% PFA for 24 h prior to routine processing and paraffin embedding for histochemical analyses. The right hemisphere was snap frozen in liquid nitrogen and stored at −80 °C until further processing. Upon processing, snap-frozen hemispheres were homogenized in 2 mL of ice-cold lysis buffer  supplemented with 1 mM PMSF (Sigma), phosphatase inhibitors, and protease inhibitors  for biochemical analysis. Briefly, brains were mechanically dissociated using a tissue tearor homogenizer for 10 pulses. Samples were allowed to stand for 15 min at 4 °C and then separated into three equal aliquots and stored at −80 °C until further processing. One aliquot was used for tau analysis, another for Aβ analysis, and the third was used for protein analysis. For tau analysis, the aliquot was centrifuged at 27,0002O2 for 15 min. Sections were blocked in normal horse serum (Vector) for 20 min, hybridized with various primary antibodies for 1 h, and then incubated with appropriate ImmPRESS secondary kits (Vector). Sections were developed with Nova Red or DAB (Vector) and counterstained with hematoxylin. Routine dehydration in a graded series of ethanol and xylene was preformed prior to coverslipping (Surgipath MM24 Leica). For amyloid burden, sections were directly stained with Congo red according to the manufacturer’s recommendations and imaged using Cy5 fluorescent excitation.50 Fluorescent images were obtained using a Zeiss Axiolmager.M2 microscope and quantified using contrast intensity methods in ImageJ. IHC-stained slides were scanned using an Aperio ImageScope and analyzed using Aperio Digital Slide Studio software . Individually optimized NuclearV9 algorithms were used for Iba1+ and CD68+ cell counts. Individually optimized ColorDeconvolutionV9 algorithms were used to assess Iba1+ stained area.Ten-micrometer para-median sagittal sections were sliced at 30-μm intervals using a microtome and mounted on glass slides, beginning approximately at lateral 0.6 mm and extending approximately through lateral 3.25 mm. For histological analysis, three slides containing a total of nine sections beginning at lateral 0.6 mm and spanning 150 μm were analyzed for each subject. The entire cortex, encompassing the angular insular (AI), somatomotor areas (MO), somatosensory areas (SS), posterior parietal association areas (PTLp), and visual areas (Vis), was analyzed. Sections were routinely dewaxed, hydrated in a graded series of ethanol, and placed in a steamer for 25 min in Citra + buffer (R&D) for antigen retrieval. Endogenous peroxidase was quenched with 0.6% HFor morphological analysis, para-median sagittal 10-μm sections, mounted three per slide, spaced 150-μm apart were stained with H&E (ThermoScientific) using routine protocols. Again, sections were imaged using an Aperio ImageScope and analyzed using Aperio Digital Slide Studio software . Representative sections were subsequently assessed by a neuropathologist. Vacuoles were manually counted from every section and normalized to area analyzed for each region of interest.51 Briefly, detergent-soluble Aβ1–40, 42 species were separately detected in rat brain homogenates prepared with cell lysis buffer described above at a 1:25 dilution. Detergent-insoluble Aβ1–40, 42 species were detected by extraction of homogenate pellets in the chaotropic agent, 5 M guanidine-HCl, followed by a 1:12,500 dilution in lysis buffer. Protein levels were normalized by BCA protein assay (Pierce Biotechnology). Aβ species were separately quantified in detergent-soluble and -insoluble (5 M guanidine-HCl-extracted) fractions using Aβ1–40, 42 ELISA kits (Wako Ltd.) in accordance with the manufacturer’s instructions. For tau analysis, pellets were rehomogenized in a 10% salt-sucrose solution to obtain various soluble and insoluble fractions for western blot.52 Crude pellet tau was obtained by re-homogenization of pellets with TBS (pH 7.4), followed by centrifugation at 1000g for 5 min at 4 °C. 5ein were electrophoretically separated using 4–20% tris-glycine gels. Electrophoresed proteins were then transferred to PVDF membranes, blocked in TBS containing 5% (w/v) BSA (Sigma), and subsequently hybridized with various primary antibodies. Membranes were then incubated with the appropriate HRP-conjugated secondary antibody prior to development with chemiluminescent substrates. ImageJ software was used for densitometric analysis of blots. All blots derive from the same experiment and were processed in parallel.Biochemical analysis of Aβ peptides was conducted according to a two-step extraction method.53 (1) lowly ramified (0–3 processes), (2) highly ramified (≥4 processes) with a large soma, (3) highly ramified ( ≥ 4 processes) with a small soma, and (5) very highly ramified (>5 processes) with a large soma and usually described as dystrophic, aged, or bushy microglia.For morphological analysis of microglia, three Iba1-stained sections were imaged using randomized stereological techniques in the hippocampus, using a three-dimensional counting frame of 300 × 200 × 10-μm on a 500 × 500-μm grid. Positively stained cells were characterized from every section, and percentages of characterized cells were calculated from the total number of cells assessed. For the 9-, 12-, and 15-month time points, greater than 75 cells were analyzed per section. For the 6- and 24-month time points, greater than 50 cells were analyzed per section. The morphology of microglia was classified according to four categories, as modified from Diz-Chaves et al.n = 4/group) were analyzed using a BioRad Luminex 200 Flow Cytometer system. Fluorescence intensity was measured and data analyzed using Bio-Plex Data Analysis software.Serum was collected at 6, 9, 12, 15, and 24 months of age using standard protocols, and samples were stored at −80 °C until analysis. Samples underwent a single freeze–thaw cycle for analysis. Analytes in serum were measured using a Th Complete 14-Plex Rat ProcartaPlex™ Panel  according to the manufacture’s recommendations. Neat (undiluted) serum preparations from all treatment groups at all time points (Supplementary Information Figures and Tables


Oral diseases may cause serious health problems, especially in socially disadvantaged populations and in low-income countries. In populations living in the rural areas of Tanzania there is paucity of reports on oral health. The study aim was to estimate the prevalence, severity and socio-demographic distribution of oral diseases/conditions in adolescents living in Maasai population areas of Tanzania and to compare oral diseases/conditions between Maasai and non-Maasai ethnic groups.A total of 23 schools were randomly selected from 66 rural public primary schools in Monduli and Longido districts, Tanzania. All pupils in the selected classes, 6th grade, were invited to participate in the study. A total of 989 were invited and 906 (91.6%) accepted the invitation and completed an interview and a clinical oral examination.p < 0.05). Adolescents from Monduli (OR = 1.7), males (OR = 2.1), being born within Arusha region (OR = 1.9) and Maasai (OR = 1.7) were more likely to present with gingival bleeding (p < 0.05). DMFT> 0 increased by age (OR = 2.0) and was associated with non-Maasai ethnic group (OR = 2.2), (p < 0.05). Adolescents from Monduli district (OR = 10.0) and those born in Arusha region (OR = 3.2) were more likely to present with dental fluorosis (p < 0.05). Dental erosion was more common among non-Maasais (OR = 2.0) as well as having mother with high education (OR = 2.3), (p < 0.05).Out of 906 study participants (age range 12–17 years), 721(79.6%) were from Maasai and 185 (20.4%) from non-Maasai ethnic groups. Prevalence of poor oral hygiene, gingival bleeding, dental caries experience (DMFT> 0), dental fluorosis TF grade 5–9, dental erosion (into dentin), tooth wear (into dentin) and TMD was 65.6, 40.9, 8.8, 48.6, 1.9, 16.5 and 11.8%, respectively. Multiple variable logistic regression analysis revealed that, girls (OR = 2.0) and participants from Longido (OR = 2.6) were more likely to present with good oral hygiene (Oral diseases like dental caries and dental erosion were less common, but gingival bleeding, dental fluorosis, tooth wear and TMD were common findings in adolescents attending primary schools in the Maasai population areas of Tanzania. Notable differences between Maasai and non-Maasai ethnic groups and certain correlations to sociodemographic factors were detected. Our findings can be utilized by policy makers in the planning of oral health programs in public primary schools of Maasai population areas of Tanzania. Oral diseases are among the most common diseases and yet it is not unusual that these type of problems receive little attention especially so in countries with weak health care systems . Most coA relation between oral diseases/conditions and sociodemographic factors such as sex, age, education, ethnicity and wealth/family income have been reported worldwide –10. StudPoor oral hygiene have commonly been reported among both children and adolescents and may lead to a number of oral health problems such as dental caries, gingivitis, periodontitis and tooth loss , 25. ForReports from the Eastern part of sub-Saharan Africa show that dental fluorosis is very common and in certain regions its prevalence has been found to be nearly 100% , 31. TheThe presence of more severe dental erosion in adolescents in many parts of the world has been reported with varying prevalence (3–26%) –38. To oTemporomandibular disorders (TMD) are a significant public health problem worldwide affecting 3 to 11% depending on diagnosis . StudiesTo the best of our knowledge, there is no information on the oral diseases of contemporary adolescents living in Maasai population areas in Arusha region, northern Tanzania. Thus, the aim of this study was to estimate the prevalence, severity and socio-demographic distribution of oral diseases/conditions in adolescents living in Maasai population areas of Tanzania. It also aimed to explore whether the socio-demographic differences in oral diseases/problems varied according to Maasai and non-Maasai ethnicity.The sample size was estimated based on the assumption that the prevalence of dental erosion among adolescents was 50%. The estimated minimum sample size for this study, 845 adolescents, was obtained by assuming a margin error of 5% and, confidence intervals of 95%. Furthermore, the sample size was multiplied by 2 to account for the design effect (D), and increased by 10% to account for contingencies such as non-response or recording errors.A cross-sectional study was carried out in Maasai population areas of Monduli and Longido districts, in the Arusha region, in the northern part of Tanzania from June to November 2016. The study aimed to focus on 12–14-year-old adolescents, attending rural public primary schools. A list of all primary schools comprising public  and private schools  was obtained from both districts. After having excluded urban and private schools, 23 (13 from Monduli and 10 from Longido) out of a total of 66 (38 from Monduli and 28 from Longido) eligible rural public primary schools were randomly selected using a one-stage cluster sample design with school as the primary sampling unit and random number generator software. In each randomly selected school, a class expected to contain adolescents aged 12–14 years was purposively identified (6th grade). All children available in the identified class were invited to participate in the study. Thus, the inclusion criteria were adolescents expected to be in age 12 to 14 year old attending rural public primary schools of Monduli and Longido districts. The exclusion criteria were adolescents attending urban and private primary schools, those absent during the interview/oral examination day and those with learning difficulties.The questionnaire was constructed in English, translated into Swahili and back-translated to English independently by qualified translators from the University of Dar Es Salaam, Tanzania. Closed- and open-ended questions were used to gather information.Pre-testing of the questionnaire took place in fifty primary school children (12–14 year olds) before the actual fieldwork regarding wording, meaning, and content of each item, and appropriateness of format and modified as needed. Two especially trained medical nurses performed face-to-face interviews in Swahili/Maa (Maasai language) with each adolescent. Face-to-face interview was done in a school setting, either under the tree or inside the classroom depending on availability and each child was interviewed privately while others were inside their classes. In this study, the pilot participants were not part of the main study.Socio-demographic factors were assessed in terms of age, ethnicity, sex, place of residence , father’s and mother’s education, house ownership, number of children, household socio-economic status (perceived affluence of my household) and household wealth index . For thewhat is the highest level of school your mother/father has attended? Responses were (0) for none, (1) for she/he started but did not complete primary school, (2) for completed primary school (3) for she/he started but did not complete secondary school, (4) for she/he completed secondary school, (5) for she/he started but did not complete college/university, (6) for completed college/university, (7) for I don’t know. In the statistical analyses, the items were dichotomized as (0) for low education (from options (0), (1), (2), (3) and (7) and (1) for high education (from options (4), (5) and (6).Parents’ education was assessed by asking Do you have pain in your temple, face, jaw or jaw joint once a week or more? Does it hurt once a week or more when you open your mouth or chew? The response was either yes or no and positive answer to any of the two questions is considered affirmative to TMD diagnosis [Durable household assets indicative of family wealth  was recorded as (Yes) “available and in working condition” or (No) “not available and/or not in working condition.” The TMD epidemiological questions were iagnosis .After an interview, clinical oral examinations were performed by the principal investigator (L.S). The child was examined under field conditions outside or inside the classroom sitting on a chair in natural day light, avoiding the direct sun light. When necessary, the teeth were cleaned and dried by sterile gauze and isolated by cotton rolls. Disposable mouth mirrors and sickle probes (No. 23 explorer or Shepherd’s hook) were used.Oral hygiene was assessed using the Simplified Oral Hygiene Index (OHI-S) . The scoGingival health was assessed by Gingival Bleeding Index (GBI) . Dental Data were analyzed using the Statistical Package for Social Sciences (SPSS) for PC, version 24 . STATA 14.2  was used to adjust for the cluster effect of school. Principle component analysis was used to construct a socio-economic index categorized into wealth quintiles  and based on ownership of assets such as furniture and household characteristics including electricity, type of water source roof material and toilet types . DescripA total of 989 grade 6 primary school adolescents were invited to participate in the study. Of those, 930 adolescents accepted to participate. Twenty-four (2.6%) children who attended the study were excluded during analysis due to too high or low age. Thus, the study included 906 children 12–17 years, mean age 13.4 years (SD 1.2) of which 56.1% were females. The final response rate was 91.6%. Among the participants, 52.9% were from Monduli district and the remaining from Longido district. Of the participants 79.6% were from the Maasai ethnic group and the remaining 20.4% from the non-Maasai group.Duplicate clinical examinations , 3 weeks apart, were carried out with 93 randomly selected participants. Analysis performed on duplicate examination records revealed Kappa value of 0.98, 0.87, and 0.69 for DMFT, TF-index, and dental erosion respectively. Higher Kappa values for DMFT and TF-index might have been contributed by the examiners common knowledge about the disease/condition in the population being rated .p < 0.001). All socio-demographic characteristics except sex and age group differed statistically significantly between the two ethnic groups.As depicted in Table p < 0.05). Good oral hygiene was more common among the non-Maasai adolescents (45.9%) than the Maasai adolescents (31.5%). A total of 27.4% of the males and 40.0% of the females had good oral hygiene (p < 0.05). Similarly, gingival bleeding was more common in Maasai adolescents (44.4%) than non-Maasai (27.6%) (p < 0.05).The overall prevalence of poor oral hygiene (OHI-S ≥ 1) was 65.6%  and gingival bleeding was 40.9%. As depicted in Table p < 0.05). Among the 12–14 year old adolescents, a total of 8.2 and 13.3% of the 15–17 year old adolescents had DMT > 0 (p < 0.05).Dental caries prevalence (DMFT> 0) was 8.8%. The overall mean DMFT was 0.13 (SD 0.5). The DMFT was composed of the decayed (D = 90.0%), missing teeth due to caries (M = 5.0%) and filled teeth (F = 0). Adolescents with both, decayed and missing teeth due to caries were 5.0%. The overall mean DMFS was 0.22 (SD 0.9). The mandibular first permanent molar was the most commonly registered tooth with dental caries (60.0%) and the occlusal surface was the most common registered site with caries (71.6%). As shown in Table p < 0.05). About 51% of the adolescents born in Arusha region was registered with severe dental fluorosis TF score 5–9, while only 12.8% of the adolescents born outside Arusha region (p < 0.05) had so.Dental fluorosis, TF grade 1–9, was recorded in 89.7% of the participants and more severe fluorosis TF-grade 5–9 in 48.6%. More severe dental fluorosis (TF grade 5–9) was more common among the Maasai than non-Masaai . Furthermore, dental erosion was registered among 24.0% of the adolescents from Monduli districts and 36.8% of the adolescents from Longido districts (p < 0.05). However, none of them was registered with the most severe grade of dental erosion (grade 4). In general, the pattern of tooth wear was more similar across the ethnic groups and 44.9% of the Maasais and 50.3% of the non-Maasais was registered with some type of tooth wear (p > 0.05).Dental erosion extending into dentine grade 3–4) was registered in 1.9% (grade 1–4 was 30.0%) of the participants and tooth wear extending into dentine (grade 2–4) in 16.5% (grade 1–4 was 44.3%). As shown in Table  was regip > 0.05).TMD pain (TMD-p) was found in 11.8% of the participants. The prevalence of TMD among Maasais and non-Maasais was 11.2 and 14.1% respectively  in our study was low and in agreement with other studies from East Africa –21. As eAs expected, due to the high fluoride content in the drinking water –69, seveIn the present study, dental erosion extending in to dentine was found only in 1.9% of the adolescents. This is lower than that found in other comparable studies from Saudi Arabia and Sweden which reported prevalence of 11.9 to 26.0% , 37. TheThis study revealed that 16.5% of the adolescents had tooth wear extending into dentine. This is contrary to findings from England where a higher prevalence (30.0–53.0%) has been reported , 76 and Comparing studies using a similar diagnostic system , the prevalence of adolescents with TMD pain in this study was 11.8% which is slightly higher than in other previous studies in Sweden and United States of America , 79–81, The cross-sectional method utilized in data collection, has some drawbacks as it is difficult to establish a causal relationship. The information on oral health was collected by interviews/clinical examination conducted by trained medical/dental personnel. Information bias, recall bias and social desirability bias might have been introduced due to the self-report method employed. In addition, fewer male than female adolescents and school non-attenders in our total study sample, might have contributed to a selection bias that could have affected the study findings and generalization. Relatively fewer males than females in our study sample might be due to the fact that, in their setting male adolescents are responsible for taking care of their livestock, as a result some do not attend schools at all and some start attending schools very late age-wise. Although the methods used in this study have been utilized in other studies in East Africa , 87, preThe study showed that oral diseases/conditions like dental caries and dental erosion were less common, but gingival bleeding, dental fluorosis, tooth wear and TMD were common findings in adolescents attending primary schools in the Maasai population areas in Tanzania. Notable differences between Maasai and non-Maasai ethnic groups and certain correlations to sociodemographic factors were detected. Our findings can be utilized by policy makers in the planning of oral health programs in all public primary schools of Maasai population areas of Tanzania in order to address oral diseases/conditions in this area.


Mucormycosis is an acute and invasive fungal infection with a high mortality rate. Mucorales are less sensitive than other types of fungi to most antifungal agents. Amphotericin B (AMB) is one treatment option for this infection, but in recent studies, the antifungal activity of statins against Mucorales was shown. Therefore, therapy that combines AMB with these agents may have better effects in management of patients with mucormycosis. We evaluated the in vitro activity of AMB alone and in combination with statins, against Mucorales.Rhizopus oryzae isolates, obtained between Jan 2009 and Oct 2016 from patients with uncontrolled diabetes mellitus and cancer referred to the Department of Parasitology and Medical Mycology of Tehran University of Medical Sciences, Tehran, Iran. It was performed by microdilution method, based on the Clinical and Laboratory Standard Institute (CLSI) M38-A2 guideline.Susceptibility profiles of AMB alone and in combination with two statins, atorvastatin (ATO) and lovastatin (LOV) determined against clinical (n: 15) and environmental (n: 5) R. oryzae isolates.All clinical and environmental isolates were susceptible to AMB (MIC≤1 μg/mL). The results of the interactions between AMB and the two statins were positive. The AMB-ATO (GM: 0.13 μg/Ml) combination produced greater activity than the AMB-LOV (GM: 0.26 μg/mL) combination. AMB, in combination with ATO and LOV, reacts positively against clinical and environmental This combination strategy may lead to more effective treatment of mucormycosis and fewer side effects using low dose of AMB. Rhizopus spp., whereas the Mucor, Rhizomucor, Absidia, Saksenaea, Apophysomyces and Cunninghamella species make up a smaller percentage of clinical isolates  regions using PCR product sequencing  and environmental (n=5) isolates of quencing . All isoStatins and antifungal agent: Amphotericin B  was purchased as a standard powder and dissolved in dimethylsulphoxide (DMSO). The statins used in this study were lovastatin  and atorvastatin , provided as a standard powder and prepared in methanol at a concentration of 256 mg each in 50ml methanol. Stock solutions of these drugs were stored at −70 °C until use.In vitro antifungal susceptibility testing: The in vitro antifungal susceptibility was determined using Clinical and Laboratory Standard Institute (CLSI) document (M38-A2)  of AMB, LOV and ATO were determined visually using a mirror to compare the growth in wells with drugs and in control wells without drugs. Candida krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were used as quality control. The geometric means (GM) and ranges of the MIC were determined for each drug alone and in combination after 24h. If no growth was observed, the incubation time was extended to 48h. In addition, the MIC50  and MIC90  were calculated for all drugs using the aforementioned MIC determination criteria. All experiments were performed two times for each strain.(M38-A2) . BrieflyR. oryzae by PCR sequencing of ITS regions. The MIC data of AMB and the two statins are summarized in Twenty clinical and environmental strains were identified as R. oryzae isolates than LOV (MIC>16). The results of the interactions between AMB and the two statins (LOV and ATO) were positive. The AMB-ATO (GM: 0.13 μg/Ml) combination exhibited more activity than the AMB-LOV (GM: 0.26 μg/mL) combination. When AMB was combined with ATO, the MIC of 15 isolates of R. oryzae were reduced. The MIC50 and MIC90 of the AMB-ATO combination were 0.25 μg/mL, which was less than with AMB alone.All clinical and environmental isolates were susceptible to AMB (MIC≤1); the MIC range for AMB was 0.031 to 1 μg/mL, GM MIC; 0.38 μg/mL, MIC50; 0.5 μg/mL, MIC90; 1 μg/mL. Between the two statins, ATO (GM0: 9.5 μg/mL) exhibited better activity against Rhizopus, and are by far the most common strains recovered from patients  exhibited more activity against the isolates than the AMB-LOV combination GM: 0.26 μg/mL) or AMB alone (GM: 0.38 μg/mL). AMB is toxic and has side effects ; therefo μg/mL orR. oryzea isolates, the MIC data were high (GM LOV: >16 μg/mL and GM ATO: 9.5 μg/mL). Statins in these concentrations are not appropriate for use alone as antifungal drugs to treat invasive fungal infections because these MICs are higher than the maximum available concentration ranges in human serum. Therefore, in treating invasive fungal infections, the application of statins is only feasible in combination with other antifungal agents  natural statins, which are metabolites of microorganisms, such as LOV, and 2) completely synthetic compounds, such as ATO . SynthetR. oryzae isolates. This combination strategy may help clinicians treat mucormycosis in lower drug concentrations and without serious side effects.AMB in combination with ATO and LOV showed a positive activity against the clinical and environmental Ethical issues  have been completely observed by the authors.


A novel point-of-care ultrasound (PoCUS) training program was developed to train rural healthcare providers in Kenya on the Focused Assessment with Sonography for Trauma (FAST), thoracic ultrasound, basic echocardiography, and focused obstetric ultrasonography. The program includes a multimedia manual, pre-course testing, 1-day hands-on training, post-testing, 3-month post-course evaluation, and scheduled refresher training. This study evaluates the impact of the course on PoCUS knowledge and skills. Competency results were compared based on number of previous training/refresher sessions and time elapsed since prior training.Trainees were evaluated using a computer-based, 30 question, multiple-choice test, a standardized observed structured clinical exam (OSCE), and a survey on their ultrasound use over the previous 3 months.Thirty-three trainees were evaluated at 21 different facilities. All trainees completed the written exam, and 32 completed the OSCE. Nine trainees out of 33 (27.3%) passed the written test. Trainees with two or more prior training sessions had statistically significant increases in their written test scores, while those with only one prior training session maintained their test scores. Time elapsed since last training was not associated with statistically significant differences in mean written test scores. Mean image quality scores  were 2.65 (2.37–2.93) for FAST, 2.41 (2.03–2.78) for thoracic, 2.22 (1.89–2.55) for cardiac, and 2.95 (2.67–3.24) for obstetric exams. There was a trend towards increased mean image quality scores with increases in the number of prior training sessions, and a trend towards decreased image quality with increased time elapsed since previous training. Forty percent of trainees reported performing more than 20 scans in the previous 3 months, while 22% reported less than 10 scans in the previous 3 months. Second and third trimester focused obstetric ultrasound was the most frequently performed scan type. Frequency of scanning was positively correlated with written test scores and image quality scores.This novel training program has the potential to improve PoCUS knowledge and skills amongst rural healthcare providers in Kenya. There is an ongoing need to increase refresher/re-training opportunities and to enhance frequency of scanning in order to improve PoCUS competency.The online version of this article (10.1186/s12913-018-3196-5) contains supplementary material, which is available to authorized users. Low and moderate-income countries (LMICs) face numerous challenges in the provision of healthcare services to their populations. The World Health Organization (WHO) estimates that 60% of the world’s population does not have access to basic x-ray, computed tomography scanners or other means of tissue imaging in their local health centers . UltrasoRecognizing the powerful clinical role of ultrasound, health care leaders have developed point-of-care ultrasound (PoCUS) training programs specifically designed for LMICs . These pThe Kenyan healthcare work force is highly limited, with an estimated 14 doctors per 100,000 patients , 60% of A novel PoCUS training program was started in Kenya in 2013 and has since undergone a number of changes to improve quality and effectiveness . The proFor pre-training, each participant receives an educational manual. The manual originally included a comprehensive amount of information. In December 2013, the manual was revised to include only focused, easily read content with supporting illustrations and diagrams. In April 2014, instructional multimedia videos were embedded into the manual to better demonstrate technique and to provide multiple examples of normal findings and common disease states. The theory behind these changes is that ample knowledge gained prior to a hands-on training would hasten the process of skills acquisition and clinical scanning competency.Since November 2014, trainees are required to achieve a score of 90% on an online test to be enrolled into the program. After pre-training preparation, trainees then participate in a 1-day supervised hands-on training session. Sessions are conducted in small groups led by certified instructors using human volunteers. Emphasis is placed on maximizing hands-on learning. An observed structured clinical exam (OSCE) is conducted at the end of the initial training session to document baseline skills. Trainees are awarded a PoCUS machine at the end of the first session for use at their facilities to gain competence through practice and patient care.Follow-up in-facility testing is scheduled 3–4 months after initial training. Trainees that do not pass both the written and OSCE-based skill tests during the facility assessment are invited for refresher training 3–4 months later. The follow-up refresher sessions serve two purposes. First, participants are required to re-study the manual in order to strengthen and improve knowledge retention. Knowledge is assessed using a standardized, 30-question multiple choice question (MCQ) exam. Second, refresher skill training is provided to advance the participants’ expertise through hands-on practice and instruction. Scanning ability is assessed again using a post-course OSCE.The goal of this study is to assess trainee knowledge and skill after participating in a novel PoCUS training program designed for rural healthcare providers in Kenya and to gather feedback to make improvements to the training program design.This is a descriptive analysis of results from an independent, on-site evaluation of healthcare providers who participated in the novel PoCUS training program in Kenya. The study was carried out in January 2015.Trainees who had previously participated in the novel PoCUS training program were eligible for the study. All eligible trainees were contacted to volunteer for the study. Inclusion criteria were (a) having previously participated in the PoCUS training program and (b) providing informed consent to participate in this study. Exclusion criteria were participants in the PoCUS training program who (a) declined to provide informed consent, (b) had received any form of ultrasound training prior to attending the PoCUS training program , and/or (c) had not done any scanning since attending the PoCUS training program.The study was conducted at the participants’ current healthcare facilities.The following baseline statistics were collected from the participants: (a) clinical designation , (b) date of initial PoCUS training, (c) number and dates of subsequent refresher courses and assessments, and (d) results of all previous assessments. In addition, the participants were asked to fill out a survey  exam. Each trainee’s results were compared to his or her score at the time of initial training. A passing score was defined as 27/30 (90%).A standardized OSCE .To verify consistency of OSCE results between examiners, a test of the inter-rater reliability was performed using four course examiners on 11 trainees for each of the applications. The intra-class correlation coefficient was 0.695  The paired t-test was used to compare test scores, with an alpha of 0.05 or less considered statistically significant. Spearman’s correlation was used to determine the association between frequency of scanning with test scores and image quality scores.Results from this study were shared with the convenors of the PoCUS training program and the participants.This study was approved by the Aga Khan University, Nairobi Research and Ethics Committee (2014/REC-64 (v3)).Thirty-three trainees from 21 rural and under-resourced healthcare facilities participated in the written and skills testing. Of these, 15, 9, 7, and 2 trainees had previously completed initial training, 1 refresher session, 2 refresher sessions, and 3 refresher sessions, respectively. All completed the written exam, and 20 completed all items on the OSCE. The remaining 13 trainees were unable to recruit a female volunteer in her first trimester of pregnancy, and therefore they were not tested on two skills: identifying the gestational sac and sagittal view of the uterus in the first trimester.Trainees were surveyed on their use of PoCUS in clinical practice. Ninety-four percent (31/33 participants) completed the survey. In the 3 months prior to the study, 22% reported performing fewer than 10 scans, 39% reported performing 10–20 scans, and 39% reported performing > 20 scans (Table Obstetric ultrasounds were the most frequently performed scan type Table .Table 2Sn = 15), mean score was 71.1% compared to a prior score of 74.4%, mean difference 3.6% (p = 0.33). For participants with two prior training sessions (n = 8), mean score was 76.3% compared to 61.2%, which was the mean score from two previous testing sessions. The mean difference was 15.1% (p = 0.03). Participants with three or more prior training sessions (n = 9) had a mean score of 77.8% compared to 64.2%, which was the combined mean score from three previous testing sessions. The mean difference for this group was 13.5% (p < 0.01)  of all trainees passed the written test. Group mean score was 73.6% compared to a prior mean score of 68.2%. For participants with one prior training session n = 5, mean sn = 7), scores were 81.4% compared to 72.9% previously, mean difference 8.6% (p = 0.06). For those trained > 6 months prior to the study (n = 17), scores were 64.7% compared to 56.6% previously, mean difference 8.1% (p = 0.08). For trainees that were required to obtain a 90% score for initial training (n = 8), scores averaged 86.2% with a mean difference of 3.8% (p = 0.34)  and image quality scores .Frequency of scanning was positively correlated with written test scores  was second/third trimester obstetrics. This finding corroborates other findings from the region that have shown that the primary use of PoCUS in sub-Saharan Africa is in the management of pregnant women , 3. TraiThe number of training sessions was evaluated as a factor affecting performance. Trainees with two or three prior training sessions were found to have statistically significant increases in their MCQ test scores. We also noted a trend towards increased image quality scores with increase in number of prior training sessions.In addition, we considered the time elapsed since the previous training session. Trainees who had received training more recently (< 3 months) performed better than those who received training > 6 months prior. We also noted a trend towards decreased image quality with increase in time since the previous training. This highlights the need to increase opportunities for re-training, especially for clinicians who are unable to return to the capital city for refresher training. Arranging training sessions either in-facility or in nearby towns will be useful.Limitations of this study include the fact that frequency of ultrasound use was based on a combination of ultrasound logs and self-report. Some clinicians keep an up-to-date log while others do not, and this limited our data. Our data is also limited by a small sample size. Future work will focus on increasing opportunities for hands-on training and feedback. We will also explore interventions aimed at enhancing frequency of scanning, which we believe will increase the quality of ultrasound performance by our trainees.Achieving an effective educational PoCUS program for clinicians in rural, resource-limited settings is a logistically difficult endeavor. Each aspect including course design, development of training resources, availability of expert trainers, knowledge and skill evaluation requires careful implementation and ongoing assessment . Our proAs reported in this paper, we are performing on-site evaluations to offer practical advice to trainees about applying ultrasound to their practice, to assess their ongoing knowledge and proficiency in practical skills, to elicit feedback about the program, and to offer further hands-on training tips. Going forward, we will work to increase opportunities for hands-on skill training and modeling and create more opportunities for quality assessment and feedback. Additionally, we will design and evaluate interventions geared to increasing the frequency and quality of PoCUS performance. Our overall goal is to advocate for the continued development of PoCUS as a tool to assist rural, under-resourced healthcare providers in their clinical decision-making.Additional file 1:Appendix S1. Ultrasound use assessment. Survey of ultrasound use within the last 3 months. Appendix S2. Observed Structured Clinical Exam Assessment form. OSCE evaluation criteria. (DOCX 44 kb)