Vitamin supplementation reduces pemetrexed toxicity. Raised plasma homocysteine reflects deficiency in vitamin B12 and folate, and is suppressed by supplementation. This observational study of 112 patients receiving pemetrexed-based chemotherapy assessed homocysteine levels after 3 weeks of vitamin supplementation, hypothesising high levels would correlate with ongoing deficiency, thus increased toxicity.Primary endpoint was the composite of proportion of patients with treatment delay/ dose reduction/ drug change or hospitalisation during the first six weeks of chemotherapy, comparing those with normal plasma homocysteine  and those with high homocysteine . Secondary endpoints included toxicity and analyses for depression. Post-hoc analysis examined correlation between interval of vitamin and folate supplementation and pemetrexed on primary endpoint and grade 3–4 toxicities.p = 0.09). Twelve percent of patients gave a past history of depression however 66% of patients had an on study Hospital Anxiety and Depression (HAD) score of >7. Supplementation status was not associated with depression. The median overall survival (OS) was 11.8 months (95% CI 8.6–16.5) in the SS group and 8.8 months (95% CI 6.6–16.2) in the US group (p = 0.5). The number of days (<7 or ≥ 7 days) between vitamin B12 and folate initiation and pemetrexed administration, had no effect on the primary endpoint and grade 3–4 toxicities.Eighty-four patients (84%) were successfully supplemented (SS group). The proportion of patients undergoing a treatment delay/ dose reduction/ drug change or hospitalisation in SS group was 44.0%  and in USS group was 18.8% (95% CI 4.0%–45.6%) (On-treatment homocysteine levels were not a biomarker of toxicity or depression. Standard vitamin supplementation is adequate in the majority of patients receiving pemetrexed. High HAD score were noted in this population giving an opportunity for mental health intervention. The lead-in time for vitamin supplementation can be short. Globally 1.8 million new lung cancer diagnoses occurred in 2012. It is the leading cause of cancer death worldwide in developed countries, and prognosis for advanced disease remains poor. Chemotherapy is important to control symptoms and prolong survival in patients without a targetable mutation or with resistance to targeted therapies, thus predictors of toxicity and prognosis are useful.Pemetrexed is a multitargeted antifolate agent, and is a current standard in treating advanced adeno-NSCLC and malignant mesothelioma. Pemetrexed causes grade 3–4 neutropenia and dose delay in a significant proportion of patients. VitaminPlasma homocysteine levels correlate with folic acid and vitamin B12 deficiency and folic acid supplementation reduces plasma homocysteine . StudiesN-methyl-D-aspartate receptor agonist, causes decreased neurogenesis in the dentate gyrus of the hippocampus [The Hospital Anxiety and Depression Score (HADS) is a validated questionnaire used for screening for anxiety and depression. Depression, being observed in a number of lung cancer patients, could have a biological basis and relate to treatment. In particular, homocysteine, being an pocampus . HoweverThis prospective, observational study of patients receiving vitamin B12 and folic acid supplementation with pemetrexed-based chemotherapy aimed to assess homocysteine levels after 21 days of supplementation. We hypothesised that those with a high level of homocysteine would have more toxicity. Thus the 3-week homocysteine level would serve as a predictor of increased chemotherapy-induced toxicity. We also aimed to provide exploratory data on the correlation of homocysteine levels with depression, and investigate whether the interval between vitamin B12 and folate supplementation and pemetrexed administration affects toxicities.th February 2014 to 25th September 2015. Follow-up was carried out until one year after the entry of the last patient (i.e. 25th September 2016). The study was approved by the Wales Research Ethics Committee 4 under the approval number 13/WA/0356. All patients gave full written consent.This observational single arm study recruited patients with advanced NSCLC and mesothelioma suitable for palliative, pemetrexed-based chemotherapy. Patient recruitment spanned from 25st cycle; vitamin B12 was repeated every 9 weeks during treatment, as per local guidelines.Oral folic acid 400 mcg daily and vitamin B12 1000 mcg intramuscular were commenced at least 1 week before the 1A homocysteine sample was taken on cycle 2 day 1 of chemotherapy, i.e. after at least 3 weeks of supplementation. The HADS questionnaire was introduced following a protocol amendment after 56 patients had been recruited.2 or carboplatin AUC 5/6) or single agent pemetrexed. Patients had to be able to take vitamin B12 and folic acid supplementation and have an estimated life expectancy of at least 12 weeks.Inclusion criteria included age greater than 18 years, advanced stage disease (UICC version 7 stage IIIB/IV), receiving combination pemetrexed-based treatment  were compared to patients that showed abnormal plasma levels .Secondary endpoints include identifying differences in number of patients with grade 3–4 toxicities  and OS between the SS and USS groups. There were exploratory analyses for depression, and post-hoc analyses to examine whether the interval between vitamin B12 and folate supplementation and pemetrexed administration will affect the primary endpoint and grade 3–4 toxicities.We predicted 50% of the study population would be in the USS group, and 40% of USS group and 15% of the SS group would show a treatment delay/ dose reduction/ drug change or hospitalisation rate. To detect a difference between the two groups with 80% power and 2-sided alpha of 5%, 98 patients were required.For the secondary endpoints we predicted that 30% of the USS group and 15% of the SS group would show a grade 3/4 toxicity rate. To detect a difference between the two groups with 80% power and 2-sided alpha of 5%, a total of 242 patients were required.p-value was significant, recruitment would continue to 242 patients, if not the trial would be closed early and the secondary endpoints reported.It was planned that after the first 98 patients had finished 6 weeks of treatment an interim analysis of the primary end-point would be performed. If the reported All tests were two-sided assessed at 5% significance level. Differences were compared using a Chi-square or Fisher’s exact test. Statistical analysis was performed using SPSS version 24.At the time of the interim analysis 115 patients had been recruited to the trial. Three patients were ineligible having received AUC4 carboplatin. 100 patients who received at least one chemotherapy cycle and had a homocysteine level documented were included in further analysis .Fifty-eight percent of patients were female, with a median age of 66 years (range 41–87 years). The performance status (PS) was ECOG 0 in 22.3%, ECOG 1 in 67.9%, and ECOG 2 in 9.8%. A history of depression was reported in 11.7% of patients. There were 17.9% with malignant pleural mesothelioma, the remainder had adeno-NSCLC. The baseline characteristics were well-balanced between USS and SS groups apart from sex (45% men in SS group and 63% men in USS group) and distribution of PS (PS 0:1:2 of 21.3: 67.9: 10.7 in SS group and 25.0: 75.0: 0 in USS group). Only two patients (2%) received single agent pemetrexed, 35% receiving cisplatin-pemetrexed combination and 63% receiving carboplatin-pemetrexed combination.Among the 100 patients, 84% patients had successful supplementation with a normal homocysteine level (SS group).p = 0.09) , while that in the USS group was 18.8%  ( = 0.09) . The trip = 0.999). In the SS group, the proportion of patients with a grade 3–4 toxicity was 14.5% , while that in the USS group was 18.8%  (p = 0.70).There was no statistically significant difference in grade 3–4 toxicities between patients with a PS of ECOG 0/1 vs. those with ECOG 2 .OS was measured from cycle 1 day 1 of chemotherapy. Median OS of SS group was 11.8 months (95% CI 8.6–16.5 months) and 8.8 months in the USS group (95% CI 6.6–16.2 months) (p = 0.70).In the population with HAD score recorded (44 patients), 66% (29/44 patients) had HAD score >7. In SS group, the proportion of patients with a HAD score >7 was 63.9%  vs 75%  in the USS group (p = 0.10). We further found that the number of days (<7 or ≥ 7 days) between vitamin B12 and folate and pemetrexed administration had no effect on the primary endpoint as well as G3-4 toxicities . The duration of vitamin B12 and folate before pemetrexed, defined as <7 days (53 patients) and ≥7 days (47 patients), had no correlation with SS and USS grouping  of patients. We conclude that standard vitamin supplementation is adequate in the majority of patients receiving pemetrexed. This high rate of appropriate supplementation confounded results as the USS group contained a small number of patients. With that caveat, high levels of homocysteine did not correlate with increased toxicity and therefore in this patient group is unlikely to be a useful biomarker to identify patients at an increased risk of toxicity. We did not include baseline homocysteine measurement in this study given this correlation has been previously well explored in studies , 9, our While 15% of the study population experienced grade 3/4 toxicity, 40% underwent a delay in their chemotherapy, change in chemotherapy dose or regimen or hospitalisation during the first 6 weeks of their chemotherapy. This composite end-point reflects those experiencing a toxicity that impaired their ability to continue with the planned schedule or resulted in hospitalisation. We would propose that the measurement of grade of toxicity alone may not adequately reflect how patients are coping with chemotherapy.There was no difference in the rates of grade 3/4 toxicities between patients with PS of ECOG 0/1 patients and those with ECOG 2. Though original trials of pemetrexed in lung cancer excluded those of PS 2 , a subseAt present, the standard supplementation regimen prior to pemetrexed includes vitamin B12 1,000 mcg given intramuscularly and oral folic acid 400 mcg, administered at least 5–7 days prior to the first dose of pemetrexed. In the current study, we found that the interval between supplementation of vitamin B12 and folate and first dose of pemetrexed  did not significantly alter the primary end-point and grade 3–4 toxicities. Multiple prospective and retrospective studies , 14 haveIn our cohort, only 11.7% of patients reported depression at baseline. The HAD score was introduced following a protocol amendment midway through the trial but, of those who had a HAD recorded (n = 44), 66% had a HAD score > 7, indicative of depression. Thus the HAD score appears to substantially increases the pick-up of depression in this patient group, potentially allowing earlier intervention. Within the small numbers, no relationship on trend of HAD score to homocysteine level could be identified.In conclusion, on-treatment homocysteine levels were not shown to be a biomarker of toxicity in this study. Standard vitamin supplementation is probably adequate in the majority of patients. Despite vitamin supplementation there continues to be issues with the deliverability of pemetrexed-based chemotherapy in this patient group. The lead-in time of vitamin supplementation can be shortened. The HAD score suggest a high rate of subclinical or undiagnosed depression, affording an opportunity for potential mental health intervention.S1 Study Protocol(DOC)Click here for additional data file.


Bacillus, Porphyrobacter, Pseudomonas, Salinivibrio, and Halomonas were isolated. When cultivated in freshwater, urea, and calcium chloride, all species are able to biomineralize calcium carbonate in different concentrations. In seawater, the strains that biomineralize the highest concentration of calcium carbonate correspond to Bacillus subtilis and Halomonas sp. SEM–EDX and XRD analyses determined that both bacteria induce the formation of 9–33% halite (NaCl), 31–66% monohydrocalcite (CaCO3 × H2O), and 24–27% struvite (MgNH4PO4 × 6H2O). Additionally, B.subtilis induces the formation of 7% anhydrite (CaSO4). In seawater, B.subtilis and Halomonas sp. were able to precipitate both calcium (96–97%) and magnesium (63–67%) ions over 14 days of testing. Ion removal assays with B.subtilis immobilized in beads indicate a direct relationship between the urea concentration and a greater removal of ions with similar rates to free cells. These results demonstrate that the biomineralization mediated by bacterial urea hydrolysis is feasible in both freshwater and seawater, and we propose its application as a new technology in improving water quality for industrial uses.The processes of biomineralization, mediated by ureolytic bacteria, possess a wide range of technological applications, such as the formation of biocements and remediation of water and soil environments. For this reason, the bioprospecting of new ureolytic bacteria is interesting for its application to these technologies, particularly for water treatment. This study demonstrates the isolation, selection, and identification of halotolerant ureolytic bacteria from Laguna Salada (inland from Atacama Desert) and the evaluation of their ability to precipitate calcium carbonate crystals in freshwater in the presence of calcium ions, as well as the ability to induce the precipitation of crystals from different ions present in seawater. Twenty-four halotolerant ureolytic bacteria whose molecular identification gives between 99 and 100% identity with species of the genus  The geographical location, altitude, and physicochemical parameters of the points sampled in this site are shown in Table Environmental samples were taken in sterile plastic tubes and stored at −20°C for further analysis. Five grams of samples were enriched in 50 mL of the following four culture media: TN medium  dissolved in freshwater and supplemented with 35 g/L NaCl), TM medium (TSB dissolved in seawater), LN culture medium  dissolved in freshwater and supplemented with 35 g/L NaCl), and LM medium (LB dissolved in seawater). For the isolation of cultured halophilic bacteria, the described culture media were prepared on plates with 15 g/L agar (Oxoid); 10 μL of each enriched sample was added and incubated at 30°C until growth was observed. The colony forming units (CFU) were selected according to their morphology and sub-cultured to identify the presence of urease activity.Proteus mirabilis was used as a positive control and Escherichia coli JM109 as a negative control.In order to determine the presence of urease activity, the bacterial isolates were cultured in tubes with Christensen's Urea Agar, modified with 20 g/L NaCl Atlas, . The bacwww.geospiza.com). The sequences were then assembled using ChromasPro version 1.6 (2012) and analyzed using the BLASTN database (www.ncbi.nlm.nih.gov) using the non-redundant GenBank nucleotide collection. The identified bacterial strain sequences were then deposited in GenBank .Genomic DNA was extracted using the Power Soil kit (Mobio Cat. No. 1288-100), according to the manufacturer's instructions. PCR amplification of the rRNA gene was performed using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′TACGGYTACCTTGTTACGACTT-3′)  under access numbers indicated in brackets at the tree in Figure Evolutionary analyses were conducted in MEGA7.0 using Maximum Likelihood with a bootstrap of 1,000. The model used was General Time Reversible, while the branch swap filter analysis setting used was weak. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The nucleotide sequences from this study are available in GenBank  and alginic acid (Sigma-Aldrich), respectively, to a final concentration of 4.5 × 109 cells/ml. The mixture was dripped via a peristaltic pump into a sterile solution of 4% CaCl2 × H2O (Merck) and 4% H3BO3 (Merck) for 2 h to form immobilized cell spheres of 0.4 cm diameter. After 2 h, the beads were washed 3 times with sterile distilled water and stored at 4°C until use.Concentrated cells of B.subtilis strain LN8B were performed in 250 mL Erlenmeyer flasks at an incubation temperature of 30°C and constant shaking at 120 rpm for 2 weeks. The concentrations of urea evaluated were 5, 10, 20, and 30 g/L, and the ratio of SW:spheres were of 1:1.The removal studies with immobilized biomass of Calcium and magnesium ions were analyzed by atomic absorption spectrophotometry , with direct suction. Total carbonates and bicarbonates were measured through acid–base titration  coupled to an X-ray detector (Oxford Inca). The samples were prepared on a carbon tape and covered with gold via sputtering (Denton Desk II). The secondary electron current used was between 10 and 20 kV. The magnification was between 250 × and 5,000 ×. The working distance used was 10 nm, with a spot size between 40 and 55.The XRD analyses were obtained using a diffractometer (Siemens D5000) with a graphite secondary monochromator and CuKα radiation. Data were collected for a 1.0 s integration time in 0.02° 2θ steps at 40 kV and 30 mA. Scanning was from 3° to 70° 2θ. The components of the sample were identified by comparing them with standards established by the International Center for Diffraction Data.3 production between isolated bacteria were conducted to identify statistical significance. This was done using Minitab software version 17.0 (www.minitab.com) through unidirectional ANOVA and Dunnett tests or Student's t-test and Tukey test a posteriori. In all cases, comparisons for which p < 0.05 were considered significant.Unless otherwise indicated, the presented data are the means of three independent experiments. The standard deviations of each set of experiments are represented in the corresponding figure (as bars). The comparisons of the mean CaCOBacillus, Salinivibrio, Halomonas, Pseudomonas, and Porphyrobacter genera  with different appearances, colorations, and morphologies. Twenty-four bacterial isolates demonstrated the ability to hydrolyze urea in the Christensen's Urea Agar assay . All isolates with ureolytic activity were phylogenetically identified based on their 16S rRNA sequences by BLASTN. 98–100% identity was obtained with species of the Bacillus species, 20.83% are of the Salinivibrio species, and 20% are of the Halomonas species. The TN2A isolate, making up 4.17% of the isolates, is closely related to the Porphyrobacter species, and the TM5DR strain, making up another 4.17%, is Pseudomonas gessardii. These last two had 99% identity. A phylogenetic tree was generated based on the 16S rRNA. The relatedness of the sequences is shown in Figure From the isolates with urease activity, 50% are of the Bacillus genus was produced by Bacillus subtilis strain LN8B . This was followed, in descending order, by B.subtilis strain LN13DC , Bacillus sp. strain LN16CC , and B.subtilis TN1B . In the other genera, highlights include Halomonas sp. strain LM12ABN , Salinivibrio sp. strain LM12ABA , B.subtilis strain, and Porphyrobacter sp. strain TN2A . No biomineralization signals or precipitate formations were observed in the controls without bacteria.The freshwater biomineralization test was performed with all the isolates, evaluating the total produced carbonates and the pH evolution at the end of the seventh day Figure . All ideB.subtilis strain LN8B. The pH of the negative control remained constant throughout the test at 6.34 ± 0.01.Regarding the pH evolution on the seventh day of the bioassay, in most cultures, a direct relationship was observed between higher concentrations of total precipitated carbonate and higher final pH levels reached by Salinivibrio sp. strain LM12ABA grew to the highest cell density . The pH of the medium increased over time for all strains, with B. subtilis LN8B . When evaluating ammonium production as an indirect measure of urease activity, the same trend was observed in these three strains  were selected to perform the seawater biomineralization test Figure . Of the y Figure  . B.subtilis strain TN1B, Porphyrobacter sp. strain TN2A, Salinivibrio sp. strain LM12ABA, B.subtilis strain LN13DC, and Bacillus sp. strain LN16CC biomineralize Ca2+, decreasing its concentration, on average, by 27.92% after 7 days of bioassay. B.subtilis strain LN8B showed decreases of 98%, and Halomonas sp. strain LM12ABN showed decreases of 92.77%. Magnesium biomineralization—evidenced by decreases in the concentration of Mg2+ in solution in the presence of B.subtilis strain TN1B, Porphyrobacter sp. strain TN2A, Salinivibrio sp. strain LM12ABA, B.subtilis strain LN13DC, and Bacillus sp. strain LN16CC—was only reduced 2.75% compared to the control (concentration of Mg2+ in control was 1300.67±2.31 mg/L). In contrast, the B.subtilis strain LN8B diminished Mg2+ concentrations by 49.32% compared to the control, reaching a final concentration of 659.2 ± 37.5 mg/L, while Halomonas sp. strain LM12ABN decreased Mg2+ concentrations by 7.86%. Concentrations of total carbonates and bicarbonates at the end of the bioassay generated by B.subtilis strain LN8B were 9773.1 ± 386.7 mg/L and 6767.2 ± 512.7 mg/L, respectively. In comparison, Halomonas sp. strain LM12ABN produced 2387.22 ± 130.69 and 4030.12 ± 164.37, respectively . These results were corroborated by XRD , 31.35% monohydrocalcite (CaCO3 × H2O), 27.73% struvite (MgNH4PO4 × 6H2O), and 7.84% anhydrite (CaSO4)  Figure . The come Figure .2+ and Mg2+ from SW with immobilized biomass in a PVA-Al matrix of B. sutilis strain LN8B. It is observed that the urea concentration has no great influence on the pH increase in SW  and magnesium (63–67%) ions over 14 days of testing. SEM–EDX and XRD analyses determined that both bacteria induce the formation of 9–33% halite (NaCl), 31–66% monohydrocalcite (CaCO3 × H2O), and 24–27% struvite (MgNH4PO4 × 6H2O). Additionally, B.subtilis induces the formation of 7% anhydrite (CaSO4).All bacteria with urease activity described in this study can precipitate calcium crystals from ions present in seawater in different percentages and times. In biomineralization tests in seawater, 2+:NHPseudomonas, Flavobacterium, Arthrobacter  urease was immobilized in polyurethane (PU) foam to compare the efficacy of calcite precipitation between the free and immobilized enzymes. The immobilized urease showed higher Km and lower Vmax values, which were reflected by slower overall calcite precipitation  and magnesium biominerals (such as struvite) from the ions present in SW. Additionally, Halomonas sp. LN12ABN formed 9% halite and B.subtilis formed 33%.Through the exploration of the bacterial capacity to precipitate calcium and magnesium ions from SW, it has been demonstrated that Despite the success in the search for bacteria able to precipitate calcium and magnesium from SW, the components needed to optimize the precipitation of these ions, the design of a bioreactor to pretreat the SW, and the means of immobilizing bacteria in a matrix that increases their survival and efficiency in ion removal are currently still under study. However, the positive results of these preliminary experiments certainly justify further development of the process.Finally, this study reinforces the application of biomineralization mediated by bacteria as a biotechnological tool to improve the quality of SW for industrial processes and, alternatively, as reverse osmosis pretreatment.DA performed all the experiments and the analysis of the results. CM performed all the statistical analyzes and contributed to the writing of the manuscript. DA also participated in writing this article. LAC participated in writing this article. MR participated in the design of the trials, analysis of results, and in writing this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine immunophenotypic pattern in newly diagnosed cases of acute myeloid leukaemia by flow cytometry and its correlation with morphological findings.This study was conducted at Haematology (Pathology) department, Army Medical College, in collaboration with Immunology Department Armed Forces Institute of Pathology, Rawalpindi from 16 November 2016 to 16 November 2017. One hundred and six patients of both genders and all age groups diagnosed as acute myeloid leukaemia were included in the study. Demographic data was noted. Complete blood counts, bone marrow examination and cytochemical stains were carried out and evaluated microscopically for blast percentage and morphology. Immunophenotyping was performed by flow cytometry using standard panel on peripheral blood or bone marrow samples. The surface and cytoplasmic antigens of interest were analysed and correlated with morphological findings.The most commonly expressed antigens were CD13, CD33, CD45 and HLA-DR. Almost all blasts expressed CD45 with no remarkable difference among the subtypes of AML. The mean positivity for CD13 among all AML subtypes was 57% and for CD33 was 67%. Aberrant expression of CD7 and CD19 were expressed in 26.4% and 1.1% of all cases respectively. There was concordance rate of 90% between morphology and FCM in our study.Flow cytometric analysis of acute leukaemia done by a combination of patterns and intensity of antigen expression improves diagnostic yield in AML. CD13, CD33 and CD45 are the most frequently expressed antigens in AML. Our findings suggest a 90% concordance between morphology and flow cytometry. It is pertinent to conclude that flow cytometry results interpreted with morphology are complementary. Acute Myeloid Leukemia (AML) is a clonal disorder of haemopoietic stem cells. It is characterised by inhibition of differentiation resulting in accumulation of cells at various stages of incomplete maturation. There is a decreased production of mature haemopoietic elements.3Diagnostic methods for acute leukemias include immature cell count, morphology, cytochemistry, immunophenotyping, cytogenetics and histochemistry in correlation with clinical features. All these diagnostic methods are complementary. Cell morphology remains the basic diagnostic tool to assess the number and morphology of blasts. Immunophenotyping by flow cytometry is a powerful adjuvant tool in delineating cell surface and cytoplasmic markers in AML. The expression of characteristic myeloid lineage markers CD13, CD33 and CD117 allows the distinction of AML from other types of leukaemias.4In the diagnosis of acute leukemia concordance between experienced observers increases from 70 to 99% when morphologic criteria are supplemented by cytochemical and immunophenotypic information.7Immunophenotypic markers shown in various studies implicating adverse outcomes are CD7, CD9, CD11b, CD13, CD14, CD33, CD34, CD56 and terminal deoxynucleotidyl transferase.9Aberrant phenotype is expression of lymphoid-associated markers in myeloblasts or that of myeloid associated markers in lymphoblasts.This descriptive cross sectional study was carried out in Haematology Department Army Medical College in collaboration with Haematology and Immunology department of Armed forces institute of pathology, Rawalpindi from 16 November 2016 to 16 November 2017. The sampling technique used was non probability convenient sampling Approval was taken from ethical review board and institutional review board. A total of 106 patients of all ages and both genders who were newly diagnosed with AML were included. Relapsed cases of AML and those evolving from myelodysplasia (MDS) or receiving treatment for AML were excluded.The samples were analysed for blast percentage and morphology on leishman stained peripheral blood and bone marrow smears followed by cytochemicalstains (Sudan Black B). Immunophenotyping was done using three colour flow cytometer. This included samples of peripheral blood or bone marrow aspirate from patients of De novo AML.Two and a half ml of venous blood in Ethylene Diamine Tetra Acetic Acid (EDTA) tube was collected for Complete Blood Count (CBC) and peripheral blood film examination, under aseptic conditions. Bone marrow aspiration was done after written informed consent, following standard guidelines. Complete blood counts were generated using automated haematology analyzer Sysmex KX-21. Leishman stained smears from peripheral blood and bone marrow aspirates were examined under microscope for morphology and percentage of blasts.11Labelling of each test tube  was done properly and placed in sequence. Ten µ| of monoclonal antibody was added into each tube. 50µl of whole blood / diluted bone marrow in each tube was added, thoroughly mixed and incubated in dark for 30 minutes at room temperature. Fluorescent activated cell sorter lysing solution (FACSLyse) in distilled water was prepared in 1:10 dilution. In each test tube 2 ml of diluted FACSLyse was added and incubated in dark for five minutes at room temperature. It was centrifuged at 300 g for five minutes at room temperature. The supernatant was discarded and the remaining 50µ| fluid was shaken for resuspension of cells. In each tube 2ml of phosphate buffered saline (PBS) was added. Centrifuge at 300 g for five minutes and supernatant was discarded. The remaining fluid was shaken and 0.5 ml of 3.3% formalin was added to each test tube. The samples were kept at 4ºC till analysis on flow cytometer.0 and where results of primary panel were inconclusive. The populations were considered positive if at least 20% of cells within gate showed the expression of a particular antigen. For cytoplasmic antigen expression, the threshold was 10%.Flow cytometric analysis was made by a top FACScan flow cytometer (Becton & Dickenson).The flourochromes FITC, PE and PerCP were used. The primary panel of monoclonal antibodies (mAbs) used were CD45 (for blast identification), CD13, CD33, CD34, HLA-DR and for AML M4/M5 CD14, for AML M6 anti-glycophorin A and for AML-M7, CD61, CD41 and CD42 were used. The extended panel included CD117 and Anti-MPO was used in cases where the morphology suggested AML-MIsotype control (antimouse IgG1FITC/Ig2αPE) is used as negative control (should not react with human blood/bone marrow cells). If it happens, the test/reagent should be rechecked.Data was analyzed using SPSS version 21. Frequencies and percentages were calculated for variables like gender. For quantitative data analysis, mean along with standard deviation were used to assess the values for monoclonal antibodies Correlation tests were applied using Pearson correlation coefficient. The age of the patients at the time of diagnosis ranged from one year to 80 years (mean age = 31.81±19.972 years). Males constituted 58.5% and females were 41.50%. The male to female ratio was 1.4:1.9 /L and highest being 456.77x109/L both extremes seen in AML-M1.The mean of platelet count was 58.23±81.29 × 109 /L. The highest count was 1000 ×109 /L seen in AML-M2 and lowest being 659 × 109 seen in AML-M1.The mean haemoglobin levels were 8.25±2.16 g/dl. The lowest haemoglobin level was 3g/dl seen in AML-M2 and highest levels in AML-M3 of 13.50 g/dl. The mean TLC was 57.46±79.39, the lowest being 0.99 x 10O (75%±33.42) and weakest positivity in AML-M3 (12%±23.84). CD34 showed mean positivity of 38% ± 30.84 among all AML subtypes, strongest being in AML-MO (74% ± 21.79) and weakest seen in AML-M6 (1.33% ± 2.31). In our study there were 18 cases (n =106) of AML-M3 of which only 1 case of AML-M3 showed positivity for HLA-DR and 3 cases (n=18) were positive for CD34, however none of them were positive for both CD34 and HLA-DR. CD13 was studied among the blast population with mean positivity of 57% ±27.53 among all subtypes with strongest positivity in AML-M4 (72% ± 20.01) and weakest in AML-M6 (22% ± 1.73).The least number of blasts were 22(22%) seen in AML-M2 and AML-M6 and highest abnormal promyelocytes with reniform nuclei were 98 %, found in AML-M3. Out of 106 cases analyzed, AML-M2 was the most frequent subtype constituting 49 (47.2%) cases followed by AML-M3 . The least common being AML-M6  as shown in 0 (r=0.717).CD33 showed mean positivity of 67% ± 27.56, strongest being in AML-M4 (87% ±9.02) weakest in AML-M6 (30% ±11.14).CD13 and CD33 were also studied for correlation using Pearson correlation and was found to be statistically significant among all subtypes. The strongest correlation being in AML-M6 (r= 0.962) and weakest being in AML-MThe concordance rate between morphology and FCM is seen in 102 (90%) cases with confidence interval of 95% in our study. While partial discordance was seen in 4(3.77%) cases where lineage was correctly identified with difference in defining subtypes only.r =-.321, .360 .297 and -.585 respectively) and insignificant correlation with CD33, CD117 and CD19 .The aberrant expression of CD7 showed positivity in 28 (26.4%) cases, most frequently expressed in AML-M2 and CD19 was expressed in 1(1.1%) case. The results of correlational analysis showed that CD 13, CD 34, CD7 and CD14 had a significant correlation with immunophenotyping .19In our study AML-M2 (47.2%) was the most frequent subtype similar to Gosh study AML-M2=34%).4%.3,5,14CD7, a T-cell antigen known to show aberrant expression was most commonly expressed in our study 26.4%) followed by CD19 (1.2%). The same trend was observed in most of the international studies where CD7 was most commonly expressed aberrant antigen followed by CD19.6.4% foll21The combined use of CD34 and HLA-DR was more helpful in distinguishing Acute Promyelocytic Leukemia (APL) from non-Acute Promyelocytic Leukemia (non-APL) none of the eighteen cases of APL were positive for both CD34 and HLA-DR whereas 64.77% of non-APL (83%) were positive for both CD34 and HLA-DR. While out of eighty-eight non-APL, two (2.27%) cases were negative for both HLA-DR and CD34 as compared to 77.87% of APL. Thus the negativity for these two antigens doesn’t refer to APL diagnosis exclusively.For more than 20 years, the FAB classification for acute leukemia has been the major system of classification. This classification enabled the diagnosis of a variety of morphologic and cytochemical subtypes of acute leukemia through a structured criterion. However, studies are indicative of failure of most of categories of FAB system in delineating significant disease groups based on morphology and cytochemistry in terms of survival of patients.In case 1, morphological diagnosis was that of AML-M3 while on FCM; HLA-DR and CD34 were positive and was reported as AML-M2. In case 2, morphology suggested AML-M5 but FCM showed negative expression for HLA-DR, CD34, and CD14, reported as AML-M3. Morphological diagnosis of case 3 was AML-M6 but FCM showed negativity for anti-glycophorin A and was reported as AML-M2. Morphology in case 4 was suggestive of AML-M2. However, on FCM positivity of CD14 suggested AML-M4. This discordance was resolved on cytogenetic and molecular analysis. Morphological analysis along with cytochemical stain (SBB) rendered the diagnosis in >80% of our AML cases. Similar study was carried out by Berlukar et al.24One of the limitations was that this study was conducted on a relatively small sample size. the specific markers for AML were used but there was limited use of markers like MPO and CD117. Both these markers are one of the most sensitive markers of myeloid lineage. These were used in only a few cases. Moreover this study would improve if parallel use of molecular analysis was done. However, there were only very few cases with molecular analysis.It is concluded that immunophenotyping is an essential tool in diagnosis and classification of AML. Our findings suggest a 90% concordance between morphology and flow cytometry However, it depends upon the availability of facility for broad panel of primary and secondary markers, which is available only in specialized centres. Interpretation of immunophenotyping should be done in close correlation with morphological findings as both these complement each other. It is need of the hour to extend facility of immunophenotyping at regional levels in the country so that access to these sophisticated facilities is available to patients of leukemias to avoid long distance travel by members of this diseased community.Our findings suggest complementary use of immunophenotyping with morphology improves outcome in the diagnosis of acute leukemias. Further research with use of extended panel of monoclonal antibodies is recommended. Additionally, development of complementary diagnostic techniques such as cytogenetics and molecular analysis should be developed for more precise diagnosis of AML.MB: Did data collection, statistical analysis & and manuscript writing.SAK: Conceived, designed, review and final approval of manuscript.ND: Did statistical analysis, review & editing of manuscript.DA: Did statistical analysis & review of manuscript.


Lymphocytic choriomeningitis virus (LCMV), hantaviruses, Rickettsia spp. and Borrelia burgdorferi among forestry workers from a province in central Spain  was examined by serological screening. This is the first such study in this rural area, where people often live and work in proximity to domestic and wild animals. Immunofluorescent analyses of the serum of 100 forestry workers detected IgG antibodies to LCMV in 2% (CL 95% 0.55%–7.0%) of this population, to hantaviruses in 4% (CL 95% 1.6%–8.3%) for the serum amyloid A (SAA) serotype, and 2% (CL 95% 0.55%–7.0%) for the Seoul virus (SEO) serotype , to Rickettsia in 8% (CL 95% 4.1%–15%) (3% (CL 95% 1.0%–8.5%) for R. typhi and 5% (CL 95% 2.2%–11.2%) for R. slovaca, and to B. burgdorferi in 7% (CL 95% 3.4%–13.8%). The number of people who have been exposed to these organisms is commonly underestimated since most infections are asymptomatic. Greater epidemiological surveillance may therefore be recommended. Exposure to  Lymphocytic choriomeningitis virus  or hantaviruses that are transmitted to humans mainly through inhalation of aerosolized rodent excreta (feces and urine) and bacteria such as Borrelia spp. or Rickettsia spp., which are transmitted to humans through the bites of ticks or flees. People living in rural areas, especially those whose work brings them into contact with animals (like farmers or forestry workers) may be at increased risk of infection [Wild and domestic animals act as reservoirs for many infectious agents that can be transmitted to humans. These agents include viruses, such as nfection . LCMV can cause viral meningitis in humans . Rodent-Bunyaviridae) are the etiological agents of different clinical syndromes, like hemorrhagic fever [Hantaviruses , have been detected, including members of the spotted fever group (SFGR) such as R. slovaca, an agent of Tick-borne lymphadenopathy (TIBOLA) disease, and R. felis [R. typhi is also a reemerging threat [R. typhi, and two transmission cycles have been described: the classic rat-flea-rat cycle and the peridomestic animal cycle in which the reservoir may be cats or dogs [R. slovaca appears to have two transmission cycles in which the reservoirs are either ungulates and wild boars or domestic ruminants such as sheep, goats, and cattle [R. typhi and R. slovaca in Spanish red foxes might provide a useful indicator of the presence of these pathogens [In Spain, emerging species of rickettsia —the cause of Lyme disease, a wildlife-involving zoonotic disease of the Northern Hemisphere [B. burgdorferi are not uncommon in Spain, but most are asymptomatic. Indeed, a study covering the period 2006–2016 in the healthcare area of Santiago de Compostela  [Finally, misphere —has beenmisphere ,18, centmisphere , and soumisphere . Dogs armisphere , althougmisphere . People , Spain)  and a se, Spain) , found LThe present paper examines the seroprevalence of a number of potential disease-causing agents—the transmission under natural conditions of which is poorly understood—among forestry workers in a rural area of Spain. This work is the first step of a broader epidemiological study that will allow us to better understand the epidemiology of the studied infections and their importance in the study area.This study was performed in Guadalajara province  and stored at −20 ºC (celsius) until analysis. The following information was recorded for each person: age, sex, contact with domestic and wild animals, and history of bite by arthropods. All subjects lived in rural areas.The number of subjects included in the study accounted was for 95% of the province’s total forestry workers (the other 5% did not want to participate in the study). All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki of 1975 (as revised in 2013), and the protocol was approved by the Ethics Committee of the Universidad de Alcalá (Protocol number CEI 2011034). Rickettsia were detected using Vero E6 cells (ATCC 1586) infected with R. typhi (Wilmington strain) or R. slovaca (strain 246 CDC) [B. burgdorferi antibodies were detected employing B. burgdorferi sensu stricto (strain B31 ATCC 35210), propagated in Barbour Kelly medium and fixed on spot slides, as an antigen source [LCMV antibodies were detected using spot slides of L-929 cells  and infected with the Armstrong strain to provide antigens . Antibod246 CDC) . B. burgn source . Rickettsia, and ≥1/256 for B. burgdorferi were deemed positive. All positive samples were serially diluted to determine the endpoint titer . Fluorescein-labeled rabbit anti-human IgG antibodies were used to reveal the binding antibodies . Positive and negative control sera were also examined. Sera showing a typical pattern of fluorescence and an IgG titer of ≥1/16 for LCMV, ≥1/32 for hantaviruses, ≥1/64 for 2 test was calculated (and the Fisher’s exact test as needed). To compare mean differences, t-student parametric test or the Mann–Whitney nonparametric test were used. Significance was set at p < 0.05. Proportions and 95% confidence intervals were used using Wilson’s exact test. Odds ratios and their confidence intervals were estimated as a measure of risk. For the comparison of proportions, the χTwenty (CL 95% 13.3%–28.9%) of the women’s samples  and 16/70 (CL 95% 14.6–34%) of the men’s samples returned a positive result for at least one of the pathogens. The average age of the seropositive subjects was 41 years (IQR 31–48.25). IgG antibodies against LCMV were found in two serum samples , men 2.9% (CL 95% 0.8%–9.8%)), one from a man aged 45 and the other from a man aged 54 years.IgG antibodies against hantaviruses were found in four serum samples. The overall seroprevalence was 4% , men 4.3% CL 95% 1.5%–11.9% (3/70)). The average age of the seropositive subjects was 29 years (IQR 28.5–43).The total seroprevalence for the SAA serotype was 4% (CL 95% 1.6%–8.3%). Of them, 3.33% of women tested positively  and 4.3% of men (CL 95% 1.5%–11.9% (3/70)). For the SEO serotype it was 2% seroprevalence (CL 95% 0.55%–7.0%) with women tested at 3.33% (CL 95% 0.6%–16.7% (1/30)) and men at 1.42% (CL 95% 0.3%–7.7% (1/70)). Two samples had antibodies against both the hantaviruses antigens used; in both cases, the highest titer was for the SAA virus.Rickettsia were found in eight serum samples : 3.3% of the women (CL 95% 0.6%–16.7% (1/30)) and 10% of the men (CL 95% 4.9%–19.2% (7/70)). The median age of the seropositive subjects was 29 years (IQR 28.5–43). The total seroprevalence for the R. typhi serotype was 3% (CL 95% 1.0%–8.5%), with 0% of women (CL 95% 0.0%–11.35% (0/30)) and 4.3% of men (CL 95% 1.5%–11.9% (3/70)). For R. slovaca the seropervalence was 5% (CL 95% 2.2%–11.2%) which included 3.33% of women (CL 95% 0.6%–16.7% (1/30)) and 5.71% of men (CL 95% 2.2%–13.8% (4/70)). One male sample was positive for antibodies against both R. typhi and R. slovaca. IgG antibodies against B. burgdorferi were found in seven samples . 6.7% of women (CL 95% 1.8–21.3% (2/30)) and 7.14% men (CL 95% 3.1–15.6% (5/70)). The median age of the seropositive subjects was 40.5 years (IQR 32.5–48.5). One sample from a man aged 40 years was positive for B. burgdorferi and R. slovaca. IgG antibodies against p = 0.414). Seropositive people were older than seronegative ). The difference in seropositivity between the men and women and the mean age of individuals positive for the different infectious agents are shown in p = 0.165). Sixteen men (22.9%) had some seropositive serology compared to four (13%) of women . All subjects, seropositive and seronegative subjects, referred a history of arthropod bites.R. slovaca and B. burgdorferi.Lymphocytic choriomeningitis virus, hantavirus, Rickettsia spp., and Borrelia burgdorferi in Guadalajara province. The present results should therefore be considered to be only preliminary. The antigen used in the IFAT was not species-specific, since antibodies to other members of the families studied can cross-react with antigens used. In future studies, more antigens will be used in an attempt to see exactly which members of each of the genera and species studied are circulating and we will expand the study to more groups of the population of Guadalajara.The present study is only the first stage of a project to determine the distribution and seroprevalence of human infection with The data obtained about antibodies seroprevalence for LCMV are similar to the values that have been reported for the general population in the center of Spain (1.7%)  and fromThe seroprevalence for LCMV reported here is similar to that recorded in other seroepidemiological studies in other areas of the world: 2.38% in Argentina , 5.1% inWhile LCMV infection is known to be widespread among rodents in Spain , its traThe overall seroprevalence for hantavirus infection was 4% (CL95% 1.6%–8.3%). We used two different hantavirus antigens in two IFA tests—an assay which is sensitive and highly cross-reactive for various hantavirus antibodies—and the risk of missing any hantavirus-reactive samples was low. The present results confirm that there is more than one serotype circulating; the overall seroprevalence for SAA was 4%, and for the Seoul strain, it was 2% . Similar values were obtained by Sanfeliú et al. in Catalonia [In a study involving persons whose occupations entailed close physical contact with rodents in North America , the autR. slovaca and R. typhi) in two IFA tests, to cross-react with other Rickettsia (spotted fevers-group and typhus-group). Chai et al. [R. typhi (2.07%) in different rural areas of Zhejiang province, China and reported it to increase with subject age. Other authors have reported seroprevalence for Rickettsia spp. to be high in people working on farms in Brazil [R. conorii among forest workers. In Catalonia (Spain), a seroprevalence of 5.5% has been reported for R. slovaca [R. typhi [R. typhi of 7.5%, although higher values (>12%) were detected in rural areas, with farmers and stock-breeders being the populations most at risk [R. typhi in the Canary Islands, with no differences in terms of age and sex. The seroprevalence of 5% recorded for R. slovaca in the present work is similar to the 5.5% reported by Antón et al. [Also to test the rickettsial study prevalence, we used two different antigens  and the low power of the study may be the reason that some of the differences found are not statistically significant. In addition, few variables  were analyzed and it would be very important to increase information of other risk factors at home related to the diseases.This study is the first to detect antibodies against the studied infectious agents in people—workers with relations with animals and arthropods—from this area of Spain. Although transmission of the present infectious agents to humans is low, probably, the prevalence of infection is underestimated given that most people remain asymptomatic. In summary, it is important to increase awareness for the studied infection, and better surveillance should be implemented. Further epidemiological studies are planned , to increase the knowledge of the true significance of these infections in public health, because maintaining epidemiological vigilance is essential.


We conducted a cross-sectional survey including 23869 participants and aimed to measure the prevalences of and risk factors for chronic kidney disease (CKD) and diabetic kidney disease (DKD) in a Chinese rural population. CKD and DKD status was defined according to the combination of estimated glomerular filtration rate (eGFR) and presence of albuminuria Participant completed a questionnaire involving life-style and relevant medical history, and the blood and urinary specimen were taken. The age- and gender- adjusted prevalences of CKD and DKD were calculated and risk factors associated with the presence of CKD and DKD were analyzed by logistic regression. The overall prevalence of CKD was 16.4% (15.9–16.8%) and of DKD was 2.9% (2.7–3.1%). In participants with diabetes, the overall prevalence of CKD was 35.5% (95% CI = 33.7–37.3%). Factors independently associated with renal damage were age, gender, education, personal income, alcohol consumption, overweight, obesity, diabetes, hypertension and dyslipidemia. Our study shows current prevalences of CKD and DKD in Chinese rural residents. Further researches could identify potential factors explaining the observed differences and implement the interventions to relieve the high burden of CKD and DKD in rural population. Unhealthy diet, unbefitting physical activity and obesity are also reported to be associated with the increasing risk of CKD2. The burden of CKD is not only restricted to the requirement of renal replacement therapy for end-stage renal disease (ESRD), but also its other serious outcomes, such as cardiovascular events and mortality, are strongly influenced by kidney involvement4. In 2010, the mortality caused by CKD almost doubled comparing with which in 1990 and it was ranked as the 18th risk factor in the mortality list5.Chronic kidney disease (CKD) has been a global public health issue in the past decades and affects more than 10% population worldwide. People with diabetes and hypertension are exposed to 50% risk of developing CKD9. The inadequate awareness and control of diabetes and hypertension exacerbate the socioeconomic and health burden of CKD in Chinese population13. In 2010, the prevalences of total of diabetes and prediabetes were 9.7% and 15.5%, respectively, which accounted for about 92.4 million adults with diabetes and 148.2 million adults with prediabetes14. Diabetes has replaced glomerulonephritis as the major cause of CKD in hospitalized population since 2011 and contributed to 1.23% diabetes-related CKD in general population15. Diabetic kidney disease accounted for 43.2% and 46.2% of ESRD patients in Taiwan and Hong Kong, respectively17. In 2012, the overall prevalence of CKD was 10.8%, estimating about 119.5 million Chinese adults with CKD18.In China, CKD is highly prevalent since the rapid increasing prevalences of relevant risk factors, like diabetes, hypertension, unhealthy diet, unbefitting physical activity and metabolic syndrome12. Liu et al. reported that prevalence of diabetes was 6.6% in 2016, accounting for almost 90 million Chinese with diabetes8. Previous studies also have demonstrated that the prevalence of CKD varies substantially among geographical regions and time frames22. This diversity might be associated with the variability in diet pattern, lifestyle, education and economic condition. Therefore, we conducted a cross-sectional study to provide current data on the prevalences of chronic kidney disease, diabetic kidney disease and associated risk factors in the adult population in China.Nevertheless, the prevalence of hypertension has varied from 29.6% to 19.5% in the past five yearsA total of 23869 people were involved in this study. Generally, participants with lower eGFR, albuminuria and DKD performed older, lower educated and personal income, inadequate physical activities and consumption of fruits and vegetables, and higher prevalences of hypertension, dyslipidemia, diabetes hyperuricemia, than did participants without renal damage Table .Table 1G2 and 3947 individuals performed albuminuria. Totally, 4347 participants were suffering from CKD, in which 962 subjects were DKD patients. The adjusted prevalence of eGFR less than 60 mL/min/1.73 m2 was 2.7% (95% CI = 2.5–2.9%) and that of albuminuria was 15.0% (95% CI = 14.6–15.5%). The overall prevalence of CKD and DKD was 16.4% (95% CI = 15.9–16.8%) and 2.9% (95% CI = 2.7–3.1%), respectively , comparing with those without renal damage, subjects with reduced eGFR were older, more likely to be men, lower educated, unbefitting physical activity, insufficient consumption of fat and awareness of diabetes, higher proportion of hypertension and hyperuricemia, while those with albuminuria performed diet rich in fruits and vegetables, lacking control of diabetes and higher proportion of dyslipidemia Table . One hun2 and albuminuria (Table Ptrend < 0.001). The prevalences of hypertension, diabetes and DKD were positively associated with education and economic condition. All of them performed lowest in subjects with the upper tertile of education and personal income. The adjusted prevalences of DKD were 4.5% (95% CI = 4.0–5.0%), 2.3% (95% CI = 2.0–2.6%) and 1.5% (95% CI = 1.2–1.7%) in tertile 1–3 education (Ptrend < 0.001), and 3.1% (95% CI = 2.6–3.6%), 2.6% (95% CI = 2.2–3.0) and 2.5% (95% CI = 2.1–2.8%), in tertile 1–3 economic condition (Ptrend = 0.03), respectively.Comparing with subjects with upper and lower tertiles of education and personal income, those with middle levels of education and personal income performed lower prevalences of eGFR less than 60 mL/min/1.73 mia Table . The adj2 with an OR of 1.98 (95% CI = 1.75–2.23), 1.32 (95% CI = 1.05–1.65), 1.78 (95% CI = 1.35–2.36), 2.30 (95% CI = 1.61–3.30), 1.40 (95% CI = 1.08–1.82), 1.30 (95% CI = 1.05–1.60) and 5.45 (95% CI = 4.29–6.93), respectively. Risk factors associated with albuminuria were diet rich in fruits and vegetables, unhealthy BMIs, diabetes, hypertension, dyslipidemia and hyperuricemia with an OR of 1.12 (95% CI = 1.04–1.22), 1.49 , 1.13 , 1.49 , 2.39 (95% CI = 2.16–2.64), 2.26 (95% CI = 2.08–2.45), 1.29 (95% CI = 1.19–1.40) and 1.43 (95% CI = 1.26–1.64), respectively. The lower tertile of education and upper tertile personal income were associated with higher risk of reduced eGFR and albuminuria , habitual alcohol consumption, diabetes, hypertension and hyperuricemia were associated with higher risk of eGFR less than 60 mL/min/1.73 mIn participants with diabetes, after adjusting for multiple confounders, age , gender , top tertile of personal income  and hyperuricemia  were associated with increased risk of reduced eGFR. Factors associated with presence of albuminuria included gender , diet rich in fruits and vegetables , hypertension  and dyslipidemia . Moderate physical activity performed statistically preventive effects on reduced eGFR and albuminuria, with an OR of 0.23 (95% CI = 0.13–0.39) and 0.78 (95% CI = 0.65–0.93), respectively  indicated that the prevalence of diabetes had increased to 6.4% in 200434. Even the rate of growth slowed down in the past decade, results of 2016 Global Burden of Disease study suggested that diabetes still affected 6.6% of all-age Chinese population8. In our current study, the prevalences of hypertension and diabetes were much higher than results from previous studies: 28.9% versus 23.2% and 11.4% versus 6.6%, respectively. Results of logistic regression also showed that hypertension and diabetes were associated with higher risk of reduced eGFR with ORs of 1.30 and 1.40, and of albuminuria with ORs of 2.26 and 2.39, respectively. Therefore, the higher prevalence of CKD in Henan rural population could be caused by the higher prevalences of hypertension and diabetes.Hypertension and diabetes are also major risk factors of CKD35. A previous community-based cohort study of American population indicated that every 1 mg/dL increase of serum uric acid from baseline was associated with a 7% higher risk for decreased renal function, defined as an eGFR less than 60 mL/min/1.73 m2 36. Another prospective cohort study conducted in Chinese population demonstrated that the increased level of uric serum acid was associated with both declination of eGFR and new-onset albuminuria37. A meta-analysis, combining 13 studies with over 190000 subjects with normal renal function at the baseline, suggested that hyperuricemia was contributed twofold increased risk of new-onset chronic kidney disease38. According to results of present study, comparing with subjects without renal damage, the prevalence of hyperuricemia was much higher in subjects with reduced eGFR and albuminuria: 27.9% and 11.6% versus 6.5%, respectively. The result of logistic regression further supported hyperuricemia was robustly connected with higher risk of reduced eGFR with an odds ratio of 5.45 (95% CI = 4.29–6.93) after adjusted for relevant covariates.Hyperuricemia induced renal injury by its crystal-independent mechanisms, such as the activation of renin-angiotensin system, endothelial dysfunction and induction of oxidative stress39. According to result of present study, the prevalence of dyslipidemia was 27.8%, similar to the latest national survey which reported a prevalence of 26.3% in Chinese rural residents40. Generally, dyslipidemia performed as a risk factor for albuminuria and diabetic kidney disease, with adjusted ORs of 1.29 and 2.51, respectively. However, in participants with diabetes, dyslipidemia was statistically associated with lower risk of reduced eGFR. For this special phenomenon, there were two potential explanations. Firstly, in all participants with diabetes, the proportion of dyslipidemia in subjects with reduced eGFR was 22.2%, while that was 49.1% and 45.0% in subjects with albuminuria and non-renal damage, respectively. The different statistical distributions might be partly contributed to the result of logistic regression. Secondly, in present study, the high fat diet was statistically associated with the lower risk of development of reduced eGFR and this dietary pattern is a main contributor to dyslipidemia. Nevertheless, it was still a risk factor for albuminuria with an OR of 1.20 in subjects with diabetes and deserved highly attention since its notable prevalence.Of other factors related to renal damage, dyslipidemia tends to progress along with the declination of renal function in patients with CKD, even at the early stages of renal dysfunction and it also contributes to the association between CKD and cardiovascular disease, which is the main cause of death in patients with CKD and ESRD41. Chronic kidney disease patients are at high risk of premature death because of cardiovascular disease, which is partly contributed by their sedentary behavior. Previous studies demonstrated that increased physical activities could slow down the progress of declination of estimated glomerular filtration rate in patients with stage 3–4 CKD and improve their physical function, which could potentially contribute to lower risk of cardiovascular disease44. According to the results of the National Health and Nutrition Examination Survey III, comparing with the participants whose physical activity was defined as inactive, the hazard ratios of mortality for physically active participants was 0.59 in the non-CKD subpopulation and 0.44 in the CKD subpopulation45. Present study also supported that increased physical activity was associated with the lower risk of developing renal damage. Results of logistic regression indicated the odds ratios of reduced eGFR, albuminuria and DKD for moderate physical activity were 0.35, 0.83 and 0.78, respectively. However, in 2012, Hallal et al. reported that 31.1% of adult global population were physically inactive46. In 2015, it was estimated that 41% of the adults were defined as low total physical activity in France47. In present study, there were almost 40% of subjects performing insufficient physical activities. Hence, a public education program aiming at providing information about the benefits of moderate physical exercises and the recommendations of physical activity in general population should be promoted.Sedentary lifestyle enhances the risk of development of many unhealthy conditions, such as hypertension, type 2 diabetes mellitus, osteoporosis and depressionThe present study reported the epidemiologic characteristics and influencing factors of chronic kidney disease and diabetic kidney disease based on a representative rural population in China. The standardized survey tools, training programs and the quality-control procedures ensured the reliability of the results. Nevertheless, several limitations should be addressed. First, the renal indicators of CKD and DKD were acquired from single measurements. The estimated prevalences of CKD and DKD might be overestimated. Second, the recognition of diabetes and hypertension was partly based on the usage of medicine and self-reported history. It might be affected by insufficient awareness rate. Finally, the cross-sectional study is incapable of evaluating causal relationships between indicators of renal damage and relevant influencing factors.Results of present study demonstrate that chronic kidney disease and diabetic kidney disease revealed to a major burden of public health in Chinese rural population. The rapid increase prevalences of hypertension and diabetes will persistently affect the overall prevalence of chronic kidney disease in the future. It’s an urgent to develop specific strategies aiming at reduce the burden of CKD.The subjects were recruited in 4 rural districts of Henan province in China from July 2015 to December 2017: Xuchang, Zhumadian, Luoyang and Zhengzhou. A multistage, stratified cluster sampling method was employed to select participants aged over 18 years old in general population. In the first stage, based on the consideration of local economic and medical condition and the quantity of population, 8 candidate regions were selected from 4 different geographical areas  in Henan province. In the second stage, 1–3 representative rural districts in each region were selected by local Center for Disease Control and Prevention. In the final stage, all permanent residents who satisfied the inclusion criteria and agreed to sign the informed consent were recruited in this study. Altogether, a total of 25393 subjects aged 18 years or older were selected from 4 rural districts and 23869 subjects completed the survey and examination, with a response rate of 94.0%. This study was approved by the Zhengzhou University Life Science Ethics Committee (Code: [2015] MEC (S128)) and the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No. KY-2018-LW-66). All participants gave written informed consent before data collection. The present study was performed in accordance with the Declaration of Helsinki.2) was calculated subsequently. According to the Chinese “Criteria of weight for adults ”, BMI was divided into four levels: underweight (<18.5), healthy weight (18.5–23.9), overweight (24–27.9) and obesity (≥28). Blood pressure was measured using electronic sphygmomanometer  three times at one minute intervals. The mean value of three BP readings was used for statistical analysis, unless the difference between the readings was higher than 10 mm Hg, in which case the mean value of other two closest results was employed. Besides participants’ self-reported use of anti-hypertension medications in the past 2 weeks, hypertension was defined as participants whose average systolic blood pressure (SBP) ≥ 140 mm Hg, and/or average diastolic blood pressure (DBP) ≥ 90 mm Hg48.Data were collected by face-to-face interview in examination centers at local health stations. All subjects completed a questionnaire recording their sociodemographic status , personal and family health history , awareness and control of chronic non-communicable disease , lifestyle  and dietary pattern with assistance of medical students, trained practitioners, doctors and nurses. Anthropometric measurements, such as height, weight and blood pressure (BP), were obtained. Height and weight were measured in light clothing without shoes and body mass index . Fasting plasma glucose (FPG) level was estimated by glucose oxidative method (GOD-PAP). Urinary albumin and creatinine were measure from a fresh morning spot urine. Albuminuria was measured with immune-turbidimetric test. Urinary creatinine was evaluated by Jaffe’s kinetic method. The urinary albumin to creatinine ratio  was calculated automatically.49. Albuminuria was defined as participants with an ACR > 30 mg/g. Indicators of renal damage were presence of an eGFR less than 60 mL/min/1.73 m2 or albuminuria. Chronic kidney disease defined as participants with presence of one or two indicators of renal damage. Diabetes was defined according to the American Diabetes Association (ADA) 2009: 1. FPG ≥ 7.0 mmol/L; 2. Self-reported use of insulin or anti-diabetic medications in the past 2 weeks; 3. Self-reported a previous diagnosis of diabetes by physicians. Control of diabetes was defined as participants with diabetes kept their FPG less than 7.0 mmol/L. Diabetic subjects with albuminuria and/or eGFR less than 60 mL/min/1.73 m2 were classified as diabetic kidney disease. Dyslipidemia was presence of abnormal serum lipid concentrations, according to the Chinese guidelines on prevention and treatment of dyslipidemia in adults, or use of anti-dyslipidemia medications during last 2 weeks50. Hyperuricemia was defined as plasma uric acid concentration >422 µmol/L for man and >363 µmol/L for woman.Estimated glomerular filtration rate (eGFR) was calculated by the 2009 CKD-EPI creatinine equation51. According to the international physical activity questionnaire (IPAQ 2001), physical activity was classified as low, moderate and high degree52.Awareness of those with hypertension and diabetes was considered if subjects reported having a previous diagnosis by physicians. Education were divided into 3 levels: 1. Primary school or lower; 2. Junior middle school; 3. Senior high school or above. Economic condition were also classified into 3 levels according to per capita monthly income (RMB): 1. ≤ 500 Yuan; 2. 500–999 Yuan; 3. ≥ 1000 Yuan. Data of personal income were not applicable in 3007 participants since they couldn’t properly evaluated their economic condition. Diet rich in fruits and vegetables was considered as daily average consumption more than 500 g. High fat diet was defined as daily average meat consumption of livestock and poultry more than 75 gChi-square test for categorical variables and Student’s t-test, Mann-Whitney U-test or Wilcoxon test for continuous variables, as appropriate. Standard population of this study was using the data from the China Population Sampling Census in 2009 (data available on http://www.stats.gov.cn/).Epidata software (version 3.1) was used for data entry and management. All statistical analyses were performed with SAS 9.1  and GraphPad Prism 6  for Windows. A p-value < 0.05 was considered statistically significant. Data was expressed as mean ± SD., median with rang or frequency with percentage, as appropriate. Intergroup comparisons were made using Pearson 2), albuminuria, diabetic kidney disease and chronic kidney disease were reported. Staging of CKD based on the Kidney Disease Outcome Quality Initiative guideline and the stage 3 CKD was further divided into stage 3 A and stage 3B with an eGFR of 45 mL/min/1.73 m2 as the cut-off value53. We further stratified the prevalences of two renal damage indicators and DKD by economic and education condition. Prevalences of hypertension, diabetes, awareness and control of diabetes were reported.The crude and adjusted prevalences of reduced eGFR  with 95% confidential intervals (CIs) were reported. Covariates involved in our multivariable logistic regression model were age , gender, education (≤primary school versus junior high school [reference] versus ≥ senior high school), economic condition (≤500 versus 500 - [reference] versus ≥ 1000), alcohol consumption (yes versus no), BMI , diabetes (yes versus no), hypertension (yes versus no), dyslipidemia (yes versus no), hyperuricemia (yes versus no). Multicollinearity between covariates was calculated by using multi linear regression method.Supplementary Materials


The primary purpose of the present retrospective study was to evaluate the short-term association between ambient PM2.5 and hospitalization for CHF in Beijing, China. A total of 15,256 hospital admissions for CHF from January 2010 to June 2012 were identified from Beijing Medical Claim Data for Employees and a time-series design with generalized additive Poisson model was used to assess the obtained data. We found a clear significant exposure response association between PM2.5 and the number of hospitalizations for CHF. Increasing PM2.5 daily concentrations by 10 μg/m3 caused a 0.35%  increase in the number of CHF admissions on the same day. We also found that female and older patients were more susceptible to PM2.5. These associations remained significant in sensitivity analyses involving changing the degrees of freedom of calendar time, temperature, and relative humidity. PM2.5 was associated with significantly increased risk of hospitalization for CHF in this citywide study. These findings may contribute to the limited scientific evidence about the acute impacts of PM2.5 on CHF in China.There is little evidence that acute exposure to fine particulate matter (PM In the USA, reduction of average concentrations of daily PM2.5 by 3.9 μg/m3 would reportedly decrease the number of hospitalizations for CHF by almost 8000 and save around 300 million dollars in related healthcare costs annually [Congestive heart failure (CHF) is a serious public concern because of its poor prognosis  and consfrom CHF ,4,5, espfrom CHF  and hypefrom CHF . A meta-2.5 [2.5 on hospitalization for CHF in developing countries because there is poor environmental monitoring and disease surveillance data in these countries [2.5 and hospitalization for CHF includes only one relevant study from developing countries [2.5 and hospitalization for CHF in China [2.5 on cardiovascular risk in the setting of severe air pollution is still unclear [2.5 and hospitalization for CHF in Beijing from 2010 to 2012 by using a time-series design. Our findings may have implications for relevant policy formulation, clinical decision making, and prevention of and interventions for CHF.Air pollution is known to be more severe in developing and low- and middle-income countries, and geographical and temporal differences are known to exist in the effects of PM2.5 . Therefoountries . The metin China ,11. More unclear . Thus, tWe obtained data on CHF hospitalization from Beijing Medical Claim Data for Employees, which covers all working and retired employees. Sex, date of birth, dates of hospital visits, medication use, discharge diagnoses in Chinese, and corresponding International Classification of Diseases 10th version (ICD-10) codes are all contained in this database, the details of which have been described previously . In the 2.5  data from the air pollution reports issued by the US Embassy (https://china.usembassy-china.org.cn/zh/), which established an air quality monitoring station on the rooftop of the embassy building located in Chaoyang District, Beijing. Daily meteorological data on temperature (°C) and relative humidity (%) from the Chinese Meteorological Bureau (http://data.cma.cn/) were also collected. A previous study [2.5 levels obtained from the US embassy’s monitor were roughly comparable with city-wide PM2.5 levels. In addition, other studies [2), 97.8% (44/45) of the tertiary hospitals and 79.3% (69/87) of the secondary hospitals in Beijing are located within a 40-km radius of the monitor. Additionally, the application and reliability of the US embassy’s monitoring point data has been discussed in previous studies [2.5 in the national air quality monitoring network and publicized real-time monitoring data. Therefore, data from the US embassy was the only publicly available source for daily PM2.5 measurements during the study period. It is important to note that daily (24-h) mean concentrations of PM2.5 were intended as a proxy for population exposure levels in the current study.We collected relevant daily PMus study  reported studies ,16 repor studies , in all  studies ,16. Unti2.5 concentrations, meteorological data, and CHF hospitalizations linked by date, we performed a time-series analysis in combination with a generalized additive Poisson model to assess the short-term association between PM2.5 and CHF hospitalization in the present study. Confounding covariates incorporated in the model included temperature, relative humidity, public holiday, and day of the week, which is predefined according to previous published studies. We used the following formula in this study:With PM2.5 concentrations;  α is the intercept term.Here t denotes the day of the observation, E(Yt) refers to the expected daily case counts of hospitalization for CHF on day t; ps  denotes the smoother based on the penalized spline function; public holiday is categorized as a dummy variables , and the day of week on day t is adjusted as a categorical variable; β represents log-relative risk of CHF morbidity in relation to unit increase in PMWe controlled for seasonality and time trends using a penalized spline with 7 degrees of freedom (df) per year to exclude unmeasured time trends longer than 2 months in hospital admissions for heart failure. The selection of 7 df per year for calendar time was based on the parameter used in several recent large national studies in China ,18,19. W2.5 concentration with three degrees of freedom, because of the assumption that linear association between PM2.5 level and number of hospitalizations may be not justified [2.5 and hospitalization for CHF by setting up models with a single-day lag from the current day (lag 0) up to previous 3 days , and with 2-day (lag 0–1), 3-day (lag 0–2), and 4-day (lag 0–3) moving average concentrations. Subgroup analyses were conducted stratified by age group (18–64 years and ≥65 years) and sex. In line with previous studies, we explored non-linear exposure-response association using a penalized cubic regression spline of PMustified . We also3 increase in the daily PM2.5 concentration. All analyses were conducted using the R Programming Language  with the “mgcv” and “nlme” package.The characteristics were presented with as mean ± standard deviation (SD) for continuous variables and percentages (%) for categorical variables. The estimated effects were expressed as the percentage changes and 95% confidence intervals (CIs) in daily CHF visits associated with 10 μg/m2.5 concentrations, and meteorological conditions. During the study period, there were 16.7 ± 12.1 hospitalizations per day and the average daily concentration of PM2.5 was 99.5 ± 75.3 μg/m3 (ranging from 7.2 μg/m3 to 492.8 μg/m3). The target in the WHO Air Quality Guidelines (24-h average concentration ≤ 25 μg/m3) was achieved in 124 days (13.6%).2.5 and hospitalization for CHF. We noted a broadly linear association between PM2.5 and hospital admissions for CHF though the association was relatively flat at PM2.5 <100 mg/m3, whereas 2.5 and hospitalization for CHF except for on lag three days. Specially, a 10 μg/m3 increase in PM2.5 concentration on the same day corresponded to 0.35%  increase in the number of hospitalizations for CHF.2.5 on admission for CHF. The associations between PM2.5 and admission for CHF were marginally significant for women  and individuals aged ≥65 years . 2.5 with CHF hospitalization was robust.2.5 with hospital admissions for CHF between January 2010 and June 2012 in Beijing, China. In the study we noted a broadly linear association between PM2.5 and hospital admissions for CHF though the association was relatively flat at PM2.5 <100 mg/m3. Similarly, a time-stratified case-crossover study for 26 large cities in China also reported the similar trend in association of short-term exposure to PM2.5 and CHF hospitalizations [2.5 and asthma and ischemic stroke observed in our previous studies [2.5 and hospitalizations for CHF with clear exposure response. We estimated that increasing PM2.5 daily concentrations by 10 μg/m3 would cause a 0.35%  increase in CHF admissions on the same day. Women patients and individuals aged ≥65 years were more vulnerable to PM2.5. These associations remained significant in sensitivity analyses involving changing the degrees of freedom of calendar time, temperature, and relative humidity.In this retrospective study, we examined short-term associations between PMizations . This is studies ,16. We f2.5 on hospitalization for CHF [2.5 on hospitalization for CHF  in Rome. Similarly, another study in an Australian state-wide setting reported that PM2.5 concentration was detrimentally associated with the incidence of CHF  and of readmission  on warm days and 3%  on cool days in Taipei, Taiwan. Recently, a time-stratified case-crossover study for 26 large cities in China with 105,501 CHF hospital admissions for CHF also reported a similar trend in association of short-term exposure to PM2.5 and hospitalizations for CHF [Because there are geographical and temporal differences in the effects of PMoutcomes , it is mg et al.  provided for CHF .2.5 on CHF hospitalizations were less pronounced, but were in line with some relevant studies in China, such as one that investigated the relationship between PM2.5 and daily mortality [2.5.Compared to what has been reported by previous studies in developed countries, we found the estimated effects of PMortality . Possiblortality ,28 and tortality  and the ortality . In the ortality  found th2.5. Other studies have also shown that patients at higher risk of admission for CHF are more likely to be older, women, and to have multiple coexisting medical conditions [2.5 and onset of exacerbation of symptoms leading to hospital admission, Symons et al. [2.5 was associated with hospital admissions for CHF at 2-days lag on value of OR of 1.09 (95% CI: 0.91–1.30%) after controlling for weather conditions among cases defined by 8-h symptom onset. It should also be noted that admission for CHF rather than the timing of onset of symptom was the primary outcome of interest in the study, as it is in many other published studies [In our study, women patients and individuals aged more than 65 years were more susceptible to ambient PMnditions . To evals et al.  assigned studies ,24,25. L studies  postulat2.5 were collected only from a fixed monitoring station and persons requiring admissions may have had less exposure to PM2.5 than healthy individuals because their physical activity was limited. These factors may have resulted in underestimating the effects of air pollution [2.5 on admission for CHF by using two-pollutant models. Third, as many other studies whose data were from larger admissions databases, the present study also could not take some potential confounders into consideration, such as socioeconomic status, physical activities, and preexisting diseases. Chronic disease burden or lifestyle might also modify the association between PM2.5 and heart failure. Further investigation with more detailed information on individuals’ lifestyle risk factors is required to confirm this finding.The present study had some potential limitations. First, misclassification of exposure may be unavoidable because the concentrations of PMollution . Second,2.5 and hospital admission for CHF in this citywide study. Our findings may contribute to the limited scientific evidence about the acute impacts of PM2.5 on CHF in China.In conclusion, we found significant associations between short-term exposure to PM


Factors associated with lung function were explored in a multiple linear regression model. PM2.5 level in Kampala, Jinja and Buwenge were 177.5 µg/m3, 96.3 µg/m3 and 31.4 µg/m3 respectively (p = 0.0000). Respectively mean forced vital capacity as % of predicted (FVC%), forced expiratory volume in one second as % of predicted (FEV1%) and forced expiratory flow 25–75% as % of predicted (FEF25–75%) of children in high ambient air pollution sites  vs. those in the low ambient air pollution site (Buwenge subcounty) were: FVC% , FEV1%  and FEF25–75% . The proportions of children whose %predicted parameters were less than 80% predicted  were higher among children living in high ambient air pollution than those living in lower low ambient air pollutions areas with the exception of FVC%; high vs. low: FEV1 < 80%, %predicted  and FEF25–75 < 80%, %predicted  Factors associated with lung function were : FVC% urban residence , current cough , underweight , and overweight ; FEV1% underweight  and FEF25–75% urban residence  and exposure to biomass smoke . Children in study sites with high ambient air pollution had lower lung function than those in sites with low ambient air pollution. Urban residence, underweight, exposure to biomass smoke and cough were associated with lower lung function.Air pollution is a major cause of sub-optimal lung function and lung diseases in childhood and adulthood. In this study we compared the lung function (measured by spirometry) of 537 Ugandan children, mean age 11.1 years in sites with high  and low (Buwenge) ambient air pollution levels, based on the concentrations of particulate matter smaller than 2.5 micrometres in diameter (PM Dur5 years ,dle life . Prominedle life ,9,10. Chdle life . Studiesdle life ,13,14. Odle life ,16,17. R) limits . Further3 μg/m3) .We therefore hypothesized that children living in sites with high ambient air pollution  would have lower lung function than those living the site with low ambient air pollution . We also hypothesized that children in Jinja municipality which is a smaller city with lower ambient air pollution would have better lung function than those in Kampala city which has higher ambient air pollution. To test these hypotheses, we studied children at three sites of varying air pollution levels and urbanization in Uganda: Kampala (city), Jinja  and Buwenge subcounty .This was a cross sectional survey of school children in two urban locations:  and one rural site (Buwenge subcounty in Jinja district).Kampala city is the capital city of Uganda. It is characterized by a mixture of residences, industries and commercial areas. The residents are also a mixture of high, middle and low socioeconomic status. The middle and lower class commonly use biomass for cooking, particularly in form of charcoal and wood. Jinja municipality has demographic characteristics similar to those of Kampala city but on a lower scale; the population is smaller, less vehicular traffic and fewer slums. On the hand, Buwenge subcounty, which is found in the Jinja district, is a rural area, with little vehicular traffic and residents mainly use biomass for cooking using the typical three-stone open fire stoves ,19. The The inhabitants of Kampala city and Jinja municipality are mainly Black Africans (99%), and the rest are either Asians or Caucasians. The people from Buwenge subcounty are all Black Africans . The weaThe study was conducted in primary schools that were randomly selected from a list obtained from the district education departments. In Kampala, the schools selected were Buganda Road primary school and Uganda Railways primary school, both in the central division of Kampala. In Jinja municipality, Victoria Nile primary school and Walukuba East primary school were selected. In Buwenge subcounty, we randomly selected Buweera and Kagoma primary schools. All the schools that were selected are day public schools. The children in public schools in both urban and rural areas are usually from the lower socioeconomic class.1 between urban and rural areas. Two hundred participants were needed in each of the three sites  to detect this difference.The sample size calculations were based on power ≥0.80 and two-sided α = 0.05. The values used to calculate the sample size were based on estimates obtained from Asgari et al. study in Tehran . We poweChildren in in primary grades four to seven (commonly 9–12 years old) in the selected schools were all invited to participate in the study Information about the study and consent forms were sent to the parents/guardians through the children, who provided written consent to their children’s participation in the study. The children provided written assent. All participants were screened for any contraindication to spirometry . Those who were found to have any contra-indications were excluded. Children with lung infections other than tuberculosis were later included upon recovery from the infection.A questionnaire was used to collect data on the respiratory symptoms and air pollution exposures. Children completed the questionnaire with the help of their parents or a research assistant. Data on respiratory symptoms such as wheeze, cough and breathlessness at the time of data collection and/or in the 12 months prior to enrolment were also collected. Data on indoor and outdoor air pollution exposures such as exposure to biomass smoke while cooking, burning rubbish, and second-hand tobacco smoke was also collected.® with Spirotrac® V software  [1) and forced expiratory flow at 25–75% of FVC (FEF25–75) and their corresponding percent predicted (%predicted) for age, weight, height, gender and ethnicity were recorded. Predicted parameters were based on NHANES III lung function prediction models [Lung function assessment: Lung function was assessed by Spirometry according to American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines using a Pneumotracham, UK) . Forced n models .2.5 concentrations were measured as an indicator of ambient air pollution. A real-time aerosol monitor, DUSTTRACK II-8530  was used to collect PM2.5 particle levels at each school (two schools per site). Sampling was done over 24 h for one day. Particulate pollutant measurement: PM1), FEV1/FVC (FEV1 ratio) and forced expiratory flow 25–75% (FEF25–75%)) and their percentage predicted values . These parameters were compared between sites using analysis of variance (ANOVA) and by urban status using the independent t-test. Urban residence was defined as residing in either Kampala city or Jinja municipality while rural residence was defined as residing in Buwenge subcounty.Descriptive statistics were used to summarize the participants’ socio-demographic characteristics and lung function parameters , forced expiratory volume in the first second . First, we performed simple linear regression. Factors that showed an association with the parameters with a p-value of ≤0.20 were all included in a multiple linear regression model. The factors assessed for association were; (1) urban status (2) Air pollution:- ambient air pollution  and exposure to biomass smoke , (3) tobacco smoke exposure defined as living with someone who smokes or children smoking themselves, (4) malnutrition (underweight and overweight) assessed by body mass index (BMI) for age. BMI for age was calculated using United States of America Centers for Disease Control and prevention software (children BMI tools for school) [p-value of less than 0.05 was considered to represent significant association. All analyses were performed using Stata version 14 .We analyzed for factors associated with lung function, that is, factors associated with each of percentage predicted values  , (5) soc school) . The varThe study protocol was approved by the Mulago Hospital Research and Ethics Committee and the Uganda National Council for Science and Technology Administrative clearance was obtained from the departments of Education of Kampala city and Jinja district . Parents/guardians provided informed written consent and children gave assent.p = 0.0000). Urban children weighed on average 36.2 kg  compared to rural children who weighed 32.1 kg, p = 0.0000. Underweight rates were 10.9% for Kampala, 17.3% for Jinja and 31.5% for Buwenge. Overweight rates were 8.2% for Kampala, 2.7% for Jinja and 5.6% for Buwenge. Obesity rates were low 0.5%, 3.3% and 0.5% for Kampala, Jinja and Buwenge respectively. Having one or more of the respiratory symptoms  occurred in 54.1% of the children in Kampala, 62.9% of the children in Jinja municipality and 66.7% of the children in Buwenge subcounty (p = 0.035).Of the 537 participants, 185 were from Kampala, 151 from Jinja Municipality and 201 from Buwenge subcounty. The survey in Jinja and Buwenge was conducted in the months of March–April 2015 (beginning of rainy season) while that in Kampala was conducted in the months of June–July 2015 (end of rainy season). Children characteristics are presented in p = 0.011). Exposure to second hand tobacco smoke was higher among rural than urban children . The mean particulate matter pollution (PM2.5) levels by site are shown in 3, 96.3 µg/m3 and 31.4 µg/m3 respectively (p = 0.0000).Air pollution exposures (indoor and outdoor) are shown in 1, FEF25–75% in liters for children in Kampala, Jinja and Buwenge were as follows: FVC 2.2, 2.1, 2.0, (p = 0.0001), FEV1 2.0, 1.9, 1.8, (p = 0.0002) and FEF25–75% 2.6, 2.5, 2.5, (p = 0.3704) respectively. In addition, FEV1 ratios for Kampala, Jinja and Buwenge were 0.90, 0.91 and 0.91 respectively. The percentage predicted parameters, for Kampala, Jinja and Buwenge were: FVC% 103.7%, 98.6%, 104.0%; (p = 0.034), FEV1% 95.6%, 91.8%, 99.2% (p = 0.000), FEF25–75% 88.1%, 87.4%, 94.6% (p = 0.032) as highlighted in 25–75% where the differences where not statistically different (p = 0.85), FEV1 < 80%, %predicted  and FEF25–75 < 80%, %predicted  (Lung function data are presented in ifferent . The pro= 0.052) .25–75%) in a multivariate linear model are presented in p = 0.004), underweight , overweight  and cough ; FEV1% underweight  and FEF25–75% rural residence  and exposure to biomass smoke .Factors associated with each of the lung function parameter , by 65.5 mL per decrease of 8.7 μg per cubic meter in PM10 level (p < 0.001), and by 65.5 mL per decrease of 12.6 μg per cubic meter in PM2.5 level (p = 0.008)) [The finding of lower lung function in children exposed to high levels of air pollution is similar to findings from previous studies in other settings such as Greece, India and Iran ,13,14. Aon areas . In Caliollution . A follo 0.008)) . These f 0.008)) ,28. Air  0.008)) .1% was 5.3% lower and FEF25–75% was 6.8% lower compared to the ones reported by Asgari et al. and other studies. We postulate that these smaller differences could be due to higher exposure of rural children to indoor air pollution due to more frequent use of wood for indoor cooking. Indoor air pollution exposure has also been linked to decrement in lung function. In China Roy et al. found a mean FEV1 of 1427 mL SD 303 mL among children exposed to biomass smoke and 1598 mL SD 325 mL among those not exposed to biomass smoke, a difference of 171 mL (10.7%) [It must be noted that the differences in the percentage predicted parameters in our study between urban and rural children though statistically significant are much lower than observed in other studies. We found that FVC% was only 2.6% lower, FEV2.5) in this study did not reach statistical significance, the lower lung function in urban children in study is probably due to exposure to ambient air pollution. The average PM2.5 levels measured at the urban site of 141.0 µg/m3 far exceeds World Health Organization (WHO) defined allowable limits of 25 µg/m3. This is not the first study demonstrating high pollution levels in the study urban sites. In our study conducted in 2014 we demonstrated average levels of PM2.5 particles of 132.1 µg/m3 in Kampala and Jinja [Although the association between lung function and air pollution (PMnd Jinja . 3) and also had higher rates of biomass smoke exposure than Kampala city (92.7% vs. 83.6%) as well as higher rates of underweight (17.3% vs. 10.9%) and cough (51.7% vs. 46.0%).The study found that children in Jinja municipality had lower lung function than those in Kampala city which has higher ambient air pollution. We expected that lung function would be lowest in Kampala children followed by Jinja municipality and Buwenge. The reasons for this observation are not clear from the data that we have from the survey. However, we think that it probably due to the fact that Jinja municipality’s air pollution was much higher than recommended   [This study did not assess physical activity which is known to affect lung function ,46. Jie  < 0.05) . In anot < 0.05) ,48,49.We found that respiratory symptoms were very common among children irrespective of whether they were from high pollution (urban) or low pollution . Rural children had more cough while urban children had more wheeze. The observation is consistent with that observed in other studies ,50. AsgaRural children were significantly shorter than urban children (145.2 cm vs. 139.5 cm). The finding that rural children are shorter has been reported in Uganda National demographic and health survey (UDHS) and the Uganda National panel surveys (UNPS) ,56. In t2.5 measurements were conducted. There could have been temporal changes in air pollution levels that could affect the outcomes. Children in schools were included. Children in schools may be different from their counterparts out of school in terms of socio-economic status. School going may also increase person to person transmission of respiratory infections hence the high symptom rates. Objective assessment of indoor air pollution was not done. Therefore, the true exposure status of the children to indoor pollutants cannot be ascertained. We also did not perform a detailed nutritional assessment. Nutritional is known to affect lung function and health. The study is a cross sectional survey which limits ability to study factors that could be responsible for the lower lung function.This survey had limitations. Firstly, the survey was not conducted simultaneously at the different sites and only one day PMChildren in study sites with high ambient air pollution had lower lung function than those in sites with low ambient air pollution. Urban residence, underweight, exposure to biomass smoke and cough were associated with lower lung function.