SEPT9 and SDC2 for detection of colorectal cancer (CRC) has yet to be reported.  Combination of multiple biomarkers was an effective strategy to improve sensitivity in cancer diagnosis and screening. However, the performance of the combination of methylated SEPT9 and SDC2 was used. Methylation statuses of SEPT9 and SDC2 were examined in 19 sets of cancer tissues and paired adjacent tissues and further evaluated with 225 serum samples, including 111 CRC patients and 114 no evidence of disease individuals.  A new qPCR-based assay combining the detection of methylated SEPT9 and SDC2 methylation levels were higher in 94.7% and 100.0% of cancer tissues than in their paired adjacent tissues. The sensitivities for detecting CRC by SEPT9 methylation alone and SDC2 methylation alone were 73.0% (95% CI: 63.6–80.8%) and 71.2% (95% CI: 61.8–79.2%), respectively, with the same specificity of 95.6% (95% CI: 89.6–98.4%). However, when SEPT9 methylation was combined with SDC2 methylation to detect CRC, the sensitivity was improved to 86.5% (95% CI: 78.4–92.0%) with a specificity of 92.1% (95% CI: 85.1–96.1%). SEPT9 and SDC2 detection in serum has the potential to be a noninvasive strategy for CRC screening. The combination of methylated  Colorectal cancer (CRC) is among the three most common cancer types worldwide . It is aThe Chinese CRC screening guideline recommends screening adults of 40–74 years old with the guaiac fecal occult blood test (gFOBT) followed up with digital rectal exam and colonoscopy . HoweverSEPT9 methylation, the only blood-based biomarker approved by FDA for CRC screening, has been used clinically for nearly 10 years [SEPT9 methylation for CRC detection was relatively low, especially for early stage cancers [Syndecan-2 (SDC2) gene was also found to be hypermethylated in the feces or blood samples of most CRC patients [DNA methylation, the addition of methyl groups to the cytosine residues of DNA, is a form of epigenetic modification that mainly occurs in CpG islands usually present in promoter regions . Abnorma10 years . It has 10 years . However cancers , 4. Syndpatients , 14.Biochemical tests are the most common medical testing program in clinics; they measure sodium, potassium, chloride, bicarbonate, blood urea nitrogen, and so on. For the majority of biochemical tests, blood is usually obtained from the patient's vein, and serum or plasma is obtained from the blood sample for the next step test. However, most of the serum or plasma is discarded after the biochemical test is completed. Therefore, use of the remaining serum or plasma after the blood biochemical test might be an effective approach to detect CRC.SEPT9 methylation and the fecal immunochemical test (FIT) individually, and the specificities were 81.5% and 97.4%. When test results for SEPT9 methylation and FIT were combined, the CRC detection rate was 88.7% with a specificity of 78.8% [SEPT9 and SDC2 methylation for CRC screening has never been reported. In this study, we evaluated the performance of a new blood-based early CRC screening assay (ColoDefense), which combined the detection of SEPT9 and SDC2 methylations in a single qPCR reaction, with the remaining serum samples after the blood biochemical test. Such an approach also has an added advantage of avoidance of repeated blood draw.Combination of multiple biomarkers and/or methods has become a trend in cancer diagnosis and screening to improve sensitivity . For exaof 78.8% . Howevern = 19) and paired adjacent paracancer tissues (n = 19) from CRC patients were collected at the time of surgery at the Affiliated Hospital of Xuzhou Medical University. Serum specimens were collected from 111 CRC patients undergoing colonoscopy at the same hospital and histologically confirmed by a pathologist. Control serum specimens were collected from 114 subjects with no evidence of diseases (NED). The remaining serum specimens after the blood biochemical test were collected and frozen at -80°C until used. The study was approved by the Institutional Review Board of the Affiliated Hospital of Xuzhou Medical University (Ethics Committee reference number: XYFY2017-KL105-02), and the informed consent was obtained from all participating patients and healthy control subjects.Fresh-frozen cancer tissues (Genomic DNA was isolated from tissue specimens using a DNA mini kit (Qiagen). For serum samples, the volume was adjusted to 3.5 mL with PBS buffer if the serum volume was less than 3.5 mL, and each sample was extracted using a cfDNA extraction kit (Suzhou VersaBio Technologies Co. Ltd.). Subsequently, bisulfite conversion of purified DNA and purification of the converted product were performed with a bisulfite conversion kit (Suzhou VersaBio Technologies Co. Ltd.). All the kits were used according to the manufacturers' instructions.SEPT9, methylated SDC2, and an internal control (ACTB) were performed in the same reaction. Three separate PCR replicates were prepared with purified bisulfite-converted DNA from each serum sample, and single PCR reaction was performed with purified bisulfite-converted DNA from each tissue sample. Real-time PCR was performed on the LC480-II thermal cycler (Roche Diagnostics) using the following cycling conditions: activation at 95°C for 30 minutes, 50 cycles at 95°C for 10 seconds and 56°C for 30 seconds, and final cooling at 40°C for 30 seconds.Purified DNA obtained from the above steps was tested by real-time methylation-specific qPCR using the ColoDefense test (Suzhou VersaBio Technologies Co. Ltd.), a new blood-based methylation assay for colorectal cancer screening. For the ColoDefense assay, methylated SDC2 and SEPT9; ∆Cp was defined as the difference between the Cp values for the target (methylated SDC2 or methylated SEPT9) and the internal control gene (ACTB). The results for serum specimens were “invalid” if the ACTB Cp was greater than 35.0, and methylated SEPT9 and methylated SDC2 were “detected” if their Cp values were less than 50.0. Methylated SEPT9 was analyzed by using a 1/3 rule in which a serum sample was scored positive if one of the three PCR replicates had a valid amplification curve . And methylated SDC2 was analyzed by using a 2/3 rule, whereby to be called positive, two of three PCR replicates of a serum sample must have valid amplification curves . The serum sample would be considered as positive if either methylated SEPT9 or methylated SDC2 was positive. Data were subjected to statistical analysis by using IBM SPSS for Windows version 22.0, and the t test was used for comparison between two samples at the significant level of p < 0.01. Data from sensitivity and specificity were used to plot the receiver operating characteristic (ROC) curve. Because most Cp values from normal individuals were not detected in the qPCR reaction, we had to set the Cp values to 50.0  for those not detected samples to plot the curve [∆Cp was used to determine the tissue methylation status of he curve .SEPT9 and SDC2 genes in 19 colorectal cancer tissues and paired adjacent paracancer tissues. The SEPT9 and SDC2 methylation levels were higher in 94.7% (18/19) and 100.0% (19/19) of cancer tissues than in their paired adjacent paracancer tissues  with a specificity of 95.6% (95% CI: 89.6–98.4%). The sensitivity by methylated SDC2 alone was 71.2% (95% CI: 61.8–79.2%) with the same specificity of 95.6% (95% CI: 89.6–98.4%). However, when methylated SEPT9 was combined with methylated SDC2 to detect the CRC, the overall sensitivity was improved to 86.5% (95% CI: 78.4–92.0%) with a specificity of 92.1% (95% CI: 85.1–96.1%).When the serum samples were subjected to the ColoDefense assay, the positive detection rate for each stage of CRC is shown in SEPT9 alone was positive in 38.5% of stage I (5/13), 81.6% of stage II (40/49), 69.2% of stage III (27/39), and 100% of stage IV (7/7). Methylated SDC2 alone was positive in 53.9% of stage I (7/13), 67.4% of stage II (33/49), 79.5% of stage III (31/39), and 85.7% of stage IV (6/7). However, for the methylated SEPT9 and methylated SDC2 panel (ColoDefense), the sensitivities for different stages of CRC were 69.2% for stage I (9/13), 85.7% for stage II (42/49), 89.7% for stage III (35/39), and 100% for stage IV (7/7)  .SEPT9 alone was 0.854 (95% CI: 0.800–0.907), and the AUC for methylated SDC2 alone was 0.881 (95% CI: 0.835–0.928). However, the methylated SEPT9 and methylated SDC2 panel (ColoDefense) improved the AUC to 0.922 (95% CI: 0.883–0.961), indicating both high sensitivity and high specificity of the assay and a good performance in distinguishing the CRC subjects from NED subjects.The ROC curves for ColoDefense detecting CRC in serum are shown in SEPT9 alone, methylated SDC2 alone, or the methylated SEPT9 and methylated SDC2 panel (ColoDefense), and there seemed to be no significant difference among the positive detection rates of different volume groups .Due to the inconsistent volume of serum samples in this study, serum samples were divided into three groups by volume  for further analysis . There wSEPT9 alone or methylated SDC2 alone or a combination of methylated SEPT9 and methylated SDC2 between different ages, genders, or locations of the tumors .Furthermore, there was no significant difference among the positive detection rates of methylated SEPT9 methylation assay for CRC screening, the Epi proColon 2.0 assay, has been approved by FDA and Chinese FDA (CFDA) [SEPT9 was the only blood-based biomarker approved by FDA for CRC screening, its sensitivity did not show obvious advantage over FIT [SEPT9 with FIT or CEA could significantly enhance the sensitivity of CRC detection compared with any assay alone [CRC is a leading cause of cancer-related deaths worldwide, and late-stage CRC patients have low five-year survival rates. Blood-based screening strategies present the advantage of minimal invasiveness compared to endoscopies and are expected to have higher compliance rates than stool-based tests . At presA (CFDA) . Epi proA (CFDA) . AnotherA (CFDA) . And sevA (CFDA) , 17. Oveover FIT . The comay alone . HoweverSDC2 as a new blood-based biomarker for CRC was noticed in recent years. Two recent studies reported that the sensitivity of methylated SDC2 for CRC screening with serum or plasma samples was 87.0% with a specificity of 95.2% [SDC2 from these studies were improved by nested PCR or calculation of the percentage of methylated SDC2 with ACTB as the reference gene. In this study, we introduced a new blood-based early CRC screening assay, ColoDefense, which combined two methylation biomarkers, SEPT9 and SDC2, in a single PCR reaction. Compared with Epi proColon 2.0, methylated SEPT9 alone from ColoDefense showed similar sensitivity and specificity, and both had relatively low positive detection rates for stages I and III CRC . Taken together, the combination of methylated SEPT9 and methylated SDC2 significantly improved the positive detection rates for stages I and III CRC (SEPT9 alone or methylated SDC2 alone was 73.0% or 71.2%, respectively, with specificity of 95.6% for both. In contrast, the combination of methylated SEPT9 and methylated SDC2 increased sensitivity to 86.5% with a specificity of 92.1%, resulting in 13.5% and 15.3% increases in sensitivity compared to methylated SEPT9 alone and methylated SDC2 alone whereas specificity only decreased 3.5% (Methylated of 95.2%  or 89.4% III CRC . The ovesed 3.5% .For Epi proColon 2.0, real-time PCR reactions are performed with 3.5 mL fresh plasma samples as initial input, which means that approximately 10 mL of blood is needed for each test. As the blood biochemical test is the most common test in the clinics and large amount of serum remains after each test, using the remaining serum samples for CRC screening may avoid repeated blood draw. In this study, we found that the ColoDefense assay on such serum samples did show high sensitivity and specificity for CRC detection. In particular, by classifying serum samples by volume, it was found that serum volume was not the most important factor affecting CRC positive detection rates . TherefoSEPT9 and SDC2. The results demonstrated that the CRC positive detection rates were significantly improved by the methylated SEPT9 and methylated SDC2 panel without significant impact on specificity. It suggested that the methylated SEPT9 and methylated SDC2 panel might be the best combination for early CRC screening with high sensitivity and specificity.In this study, we evaluated a new blood-based CRC early screening assay, ColoDefense, which combines two methylation biomarkers,


Plasmodium falciparum  parasites is a major hurdle to the control of malaria. This study monitored changes in the genetic diversity and the multiplicity of P. falciparum parasite infection in asymptomatic children living in southern Ghana at 3 month intervals between April 2015 and January 2016.Genetic diversity in merozoite surface protein 1 (msp 1) and merozoite surface protein 2 (msp 2) genes.Filter paper blood spots (DBS) were collected quarterly from children living in Obom, a community with perennial malaria transmission and Abura, a community with seasonal malaria transmission. Genomic DNA was extracted from the DBS and used in polymerase chain reaction (PCR)-based genotyping of the P. falciparum. The msp 1 and msp 2 genes were successfully amplified from 73.8% (344/466) and 82.5% (385/466) of the P. falciparum positive samples respectively. The geometric mean MOI in Abura ranged between 1.17 (95% CI: 1.08–1.28) and 1.48 (95% CI: 1.36–1.60) and was significantly lower  than that determined in Obom, where the geometric mean MOI ranged between 1.82 (95% CI: 1.58–2.08) and 2.50 (95% CI: 2.33–2.678) over the study period. Whilst the msp 1 R033:MAD20:KI allelic family ratio was dynamic, the msp 2 3D7:FC27 allelic family ratio remained relatively stable across the changing seasons in both sites.Out of a total of 787 samples that were collected from the two study sites, 59.2% (466/787) tested positive for msp 1 rather than msp 2 due to the constantly changing relative intra allelic frequencies observed in msp 1 and the fact that the dominance of any msp 2 allele was dependent on the transmission setting but not on the season as opposed to the dominance of any msp 1 allele, which was dependent on both the season and the transmission setting.This study shows that seasonal variations in parasite diversity in these communities can be better estimated by The online version of this article (10.1186/s12879-018-3350-z) contains supplementary material, which is available to authorized users. P. falciparum infections for informed interventions to be instituted [P. falciparum in high transmission areas with some infected people harboring multiple clones of the parasites. On the contrary, people living in low transmission areas tend to have low genetic diversity with a high proportion being monoclonal infections [The high prevalence of malaria in Ghana has resulted in a majority of adults and children older than 5 years attaining a semi-immune status due to past and continuous exposure to different parasite clones . Repeatestituted , 13. Thefections –17. Tranfections , 19. Thufections .merozoite surface protein 1 , merozoite surface protein 2  and the glutamate-rich protein  [msp 1 and msp 2 genes helps to measure MOI and transmission in a population. Although only a limited number of loci can be examined at a time and some minor parasite populations missed by conventional PCR [msp genotyping followed by agarose gel electrophoresis as their parasite diversity analysis tool [The most common and widely used tool for the estimation of parasite diversity is PCR based genotyping of a gene with high diversity such as 1035300) –25. The onal PCR  comparedonal PCR . Many resis tool –31.P. falciparum circulating in the middle belt of Ghana has been extensively characterized [The genetic diversity of cterized , 33. Howcterized , 31 or icterized , 35. ThiPlasmodium falciparum parasite prevalence in Obom has been noted to be higher than in Abura [The study was conducted in community primary schools in two areas of southern Ghana; Obom, a semi-rural community in the Ga South Municipality of the Greater Accra Region with high and perennial malaria transmission and Abura, a semi-urban community in the Cape Coast Municipality of the Central Region with low and seasonal malaria transmission . In 2010in Abura , 39.Fig.P. falciparum parasites. The same children were followed up during subsequent sampling visits.After obtaining parental consent, 237 children between the ages of 6 and 12 years were recruited in a multiple cross sectional study; 83 from Obom and 154 from Abura. Filter paper (Whatman® 3 mm) blood spots (DBS) were prepared from ~ 50 μL of finger pricked blood that was collected in April, June and October 2015 as well as in January 2016. The filter paper blots were air-dried and stored desiccated at room temperature. None of the children sampled exhibited any signs or symptoms of clinical malaria at the time of blood draw and were classified as either healthy with no PCR detectable parasites or asymptomatic, harboring PCR detectable g for 1 min), the extracted DNA was transferred into a labelled 0.6 mL microcentrifuge tube and then stored at − 20 °C.Total genomic DNA (gDNA) was extracted from the dried blood spot (DBS) using the Chelex extraction procedure , 41. BriP. falciparum [2, 200 nM dNTP mix, 200 nM rPLU5 and rPLU6 primer set  gene was used to detect lciparum . The 15 8S rRNA gmsp 1 block 2 and msp 2 block 3 were amplified using a protocol adapted from the WHO protocol for malaria parasite genotyping [2, 200 nM each of forward and reverse primers for both msp 1 and msp 2  for msp 1 KI and msp 2 FC27 alleles, MRA-155G (HB3) for msp 1 MAD20/msp 2 FC27 alleles and MRA-200G (R033) for msp 1 RO33/msp 2 3D7 alleles) were included in each set of PCR reactions. PCR products were resolved for an average of 50 min at 120 V on a 2% agarose gel stained with 0.5 μg/mL ethidium bromide. After the electrophoresis, the gels were visualized under UV trans-illumination using a Toyobo FAS-III gel doc system and then analyzed.The polymorphic regions of notyping  and siminotyping , 44. Allmsp 1 or the two msp 2 allelic family PCR reactions. A sample containing a clonal infection produced a single product after the three msp 1 PCR reactions as well as a single product after the two msp 2 reactions. A simplified method of estimating multiplicity of infection (MOI), where MOI of a sample was taken as the highest number of PCR products (amplicons) obtained after the family specific msp 1 or msp 2 PCR amplifications was used. Multiplicity of infection was generalized and reported as the geometric mean MOI for infections in the group of children from one study site at each time point.A sample containing a multiclonal infection produced more than one amplified fragment after either the three msp 1 and msp 2 family alleles was calculated as the ratio of the number of PCR products obtained for each family to the total number of gene-specific PCR products identified. The MOI for msp 1 or msp 2 in a sample was estimated as the total number of distinct msp 1 or msp 2 PCR amplicons detected in that sample, the total MOI of a sample is the highest number of PCR fragments obtained after both msp 1 and msp 2 genotyping [msp 1 and msp 2 allele at the different time points. Statistical significance was defined as p value ≤ 0.05 unless otherwise stated.The geometric mean, column statistics, unpaired T test and One-way analysis of variance were obtained using GraphPad Prism v5.0. The distribution of the different allelic families was presented as proportions. The frequency of notyping , 45. A cp = 0.8, Unpaired T test) from the mean age of 9.10 years in Abura . Asymptomatic parasite carriage over the course of the study was 41% (197/481), ranging between 13.7% (13/95) and 64% (89/139) in Abura and 88%, ranging between 80% (64/80) and 96.4% (80/83) in Obom .At enrolment, there were 46.5 and 45.3% male children in Obom and Abura respectively. The mean (SEM) age of the children in Obom was 8.96 years, which was not significantly different (ra Table . Out of msp 1 or msp 2 gene amplifications was considered as the number of independent parasite clones co-existing within an infection and as the multiplicity of that infection. The msp 1 and msp 2 genes were successfully amplified from 73.8% (344/466) and 82.5% (385/466) of the P. falciparum positive samples respectively. The samples yielded amplicons compared to the 57.4% of asymptomatic samples that yielded products in Abura. The msp 2 amplification efficiencies were similar in both sites, 83% (223/269) in Obom and 82.2% (162/197) in Abura  in Obom and 82.2% (162/197) in Abura  than the lowest geometric mean MOI of 1.82 (95% CI 1.58–2.08) at the same site, which occurred in January 2016  .The highest number of fragments obtained after either the msp 1  and msp 2 (3D7 and FC27) allelic families. Each of the multiple msp 1/msp 2 PCR products was considered as a different parasite. The msp 1 and msp 2 alleles were characterized based on the size of the PCR products using 50 bp and 100 bp DNA ladders as size standards. Variations in the sizes of the largest and smallest PCR products within each family specific PCR was noted both msp 1 and msp 2 family members in the two study sites. The approximate size of the smallest and largest alleles of msp 1 was 120 bp and 350 bp respectively for parasites from Obom and 150 bp and 320 bp respectively for parasites from Abura. No obvious trends were observed in the diversity of the smallest and largest sized PCR fragments obtained after msp 1 genotyping in both study sites across the various time points. However, in Obom the largest sized msp 2 fragment, the 400 bp variant persisted from April through to January while in Abura, no trend in the diversity of the largest and smallest sized msp 2 PCR fragments were observed across the time points . The proportion of parasites belonging to the K1 family reduced from 38.8% in April 2015 to 24.3% in July 2015, increased again in October 2015 to 48.8% and reduced slightly to 43.7% in January 2016. The parasites belonging to MAD20 allelic family increased steadily from April (22.4%) to July 2015 (42.8%), reduced to 27.1% in October 2015 and 27.7% in January 2016  in both Obom  and Abura   Fig. . Thus thmsp 2 family ranged from 57% in July 2015 to 43.3% in January 2016, while the proportion of parasites belonging to the FC27 family ranged from 43% in July to 56.7% in January 2016 with slight variation between the seasons  or Abura   Fig. , d. ThusP. falciparum and the multiplicity of P. falciparum infections over changing malaria seasons in one community with perennial and high malaria transmission and another with seasonal and low malaria transmission. The most prevalent circulating parasite clones at different time points in different transmission settings were also identified.This study sought to characterize the genetic diversity of p > 0.05, Dunn’s Multiple comparison test). This is to be expected in perennial transmission settings, where mosquito vectors and malaria transmission only vary slightly between seasons [In Abura a lot children were lost to follow up at between July and October 2015  as well as the dominance of any msp1 allele across the seasons and between the two transmission settings Primers used for Additional file 2:Plasmodium falciparum parasite carriage in study participants. (DOCX 15 kb)


Skimmia laureola was determined by GC and GC-MS. Twenty-eight components were identified, accounting for 93.9% of the total oil. The oil is mainly composed of monoterpenes (93.5%), of which monoterpene hydrocarbons and oxygenated monoterpenes represent 11.0% and 82.5%, respectively. Sesquiterpenes constitute only 0.3% of the total oil. Linalyl acetate is the main component (50.5%), with linalool (13.1%), geranyl acetate (8.5%) and cis-p-menth-2-en-1-ol (6.2%) as other principal constituents. The essential oil showed a significant antispasmodic activity, in a dose range of 0.03–10 mg/mL. The essential oil also possesses antibacterial and antifungal activities against some pathogenic strains. The phytotoxic and cytotoxic activities were also assessed.The composition of the essential oil from leaves of  Skimmia laureola (DC.) Decne (Rutaceae) is an aromatic shrub distributed from northern China to the Northern Himalayas; its leaves give off an aromatic smell when crushed. Traditionally, the plant is used in folk and cultural practices: the smoke of dried leaves is considered useful to ward off evils , with either those of the literature  strains, purchased from Schazo Laboratories  and Schering Pharmaceutical Industries Ltd.Lahore, Pakistan). Bacterial strains were first cultured on nutrient broth and incubated for 24 h. Nutrient agar was melted, cooled to 40 °C and poured into sterilized Petri dishes. Four-height hold bacterial culture was spread on the surface of nutrient agar. The experiment was repeated thrice turning the plate 60° between each streaking. About l00 µL of 3 mg/mL of essential oil dissolved in DMSO was then added to the wells. Other wells were supplemented with DMSO and 10 µg imipenem, used as positive and negative control, respectively. The plates were then incubated for 24 h at 37 °C. The plates were then observed for zones of inhibition, measuring the inhibition zones by using a caliper, mm/inch reading scale, precision 0.05 mm. All the experiments were conducted in triplicate.Antibacterial activity was measured by the agar well diffusion method, against four Gram (+) [Tricophyton longifusis , Candida albicans (ATCC 2091), Fusarium solani (ATCC 11712), Microsporum canis (ATCC 111622), Aspergillus flavus (ATCC 32611), and Candida glabrata (ATCC 90030). Twenty four mg of essential oil was dissolved in 1 ml sterile DMSO serving as a stock solution. Four mL of Sabouraud dextrose agar (SDA) growth media was transferred to each screw capped tube, under sterile conditions and autoclaved at 121 °C for 15 min. These tubes were then allowed to cool to 50 °C and 400 µg/mL test sample were added to the SDA tubes, which were then allowed to solidify at room temperature. DMSO and miconazole were used as negative and positive controls, respectively. The tubes were incubated at 28 ± 1 °C for 7 days. Cultures were observed twice weekly during incubation. Growth in the media was estimated by measuring the linear growth (mm) by .using a caliper, mm/inch reading scale, precision 0.05 mm. The percentage inhibition of fungal growth was calculated as follows:The antifungal activity was assayed against six fungal strains: Lemna minor as test species, as previously reported [2PO4 (680), KNO3 (1515), Ca(NO3)2·4H2O (1180), MgSo4·7H2O, H3BO3 (2.86), MnCl2·4H2O (3.62), FeCl3·4H2O (5.40), ZnSO4·5H2O (0.22), CuSO4·5H2O (0.22), Na2MoO4·2H2O (0.12), EDTA (11.20), were added to each flask. Other flasks (three for each test) were supplemented with the medium alone, to serve as a negative control. To each flask ten plants of L. minor with 2–3 fronds were transferred. The flasks were kept under about 12 h day light conditions. Plants were observed daily and on the seventh day the numbers of fronds were counted. The phytotoxic activity of the essential oil was evaluated using reported . FifteenThe cytotoxic activity of the essential oil of was tested using the brine shrimp assay as previously reported . About 2Animals Bye-Laws 2008 of the University of Malakand (Scientific Procedures Issue-1) and fed on standard diet and tap water. The animals were kept in fasting condition 24 h prior to the start of the experiments with free excess to water. Rabbits of either sex with (1.0–1.4 kg) were used. The animals were kept for 14 days before starting the experiments at the Animal House of the Department of Pharmacy, University of Malakand , under standard conditions mentioned in the g pressure. On attaining a reproducible response, test samples at the doses of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0, and 10.0 mg/mL essential oil were applied to the bath solution. The experiments were repeated thrice. For the determination of the possible mode of action, the tissue was pretreated with a high concentration KCl (80 mM) to cause depolarization and keep the tissue in sustained contraction. The essential oil was then applied in cumulative manner to obtain a dose dependent curve and relaxation. Intestinal responses data were recorded using a Force Transducer  attached with a Power lab . Data were recorded at a range of 20 mv. Experiments on rabbit’s jejunum preparations were carried as previously reported . Slaughtt test was used at 95% confidence interval. p values less or equal to 0.05 were considered as statistically significant.Chart 5 (AD Instruments) was used to interpret the graph tracings. Student Skimmia essential oils. The oil showed antibacterial activity against some of the tested bacterial strains and such activity could be, at least in part, explained by the high percentage of linalyl acetate. On the other hand, the essential oil showed a good antifungal activity against some strains. Of interest seems the activity against Candida albicans, considering the pathogenicity of this microorganism. Data about the phytotoxicity of the essential oil agree with the available literature, that ascribes to the volatile fractions antigerminative and allelopathic properties. The muscle relaxation caused by S. laureola essential oil suggests that the possible mode of action is either by blocking the release of stored calcium from the sarcoplamic reticulum or by blocking the calcium channels. The data obtained can be useful in explaining the traditional uses of the plant.The data obtained contribute to a phytochemical knowledge of the essential oil of an important flavor species, widely used in traditional medical practices. The chemical pattern of the essential oil agrees with the composition of other


Echinococcus granulosus. We investigated the presence of E. granulosus-specific DNA in the serum of CE patients by detecting the cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) mitochondrial genes. Serum and formalin-fixed paraffin embedded (FFPE) cyst tissue samples of 80 CE patients were analyzed. The extracted DNA of samples was submitted to PCR amplification of cox1 and nad1 genes, and products were sequenced and genotyped. Nineteen  serum and 78  FFPE cyst tissue samples were successfully amplified with at least one gene. Echinococcus DNA was detected in the sera of 15.0% (95% CI: 8.8–24.4) and 10.0% (95% CI: 5.2–18.5) and in cyst tissue of 91.3% (95% CI: 83.0–95.7) and 83.8% (95% CI: 74.2–90.3) of 80 patients by cox1 and nad1 gene, respectively. Four genotypes of E. granulosus were distinguished in the CE patients, with predominance of genotype G1, followed by G3, G2, and G6. The finding of E. granulosus DNA in 23.8% of serum samples from CE patients confirmed that E. granulosus releases cell-free DNA into the circulatory system, but quantities may be inadequate for the diagnosis of CE. Genotype G1 predominance suggests the sheep-dog cycle as the primary route of human infection.Cystic echinococcosis (CE) is a worldwide zoonotic disease caused by the larval stage of  Echinococcus granulosus sensu lato, a complex of four species and ten genotypes classified according to the host range and genetic diversity: E. granulosus sensu stricto (G1 to G3), Echinococcus equinus (G4), Echinococcus ortleppi (G5), and Echinococcus canadensis (G6 to G10) . Patients were informed of the study objectives and gave written informed consent for their blood and tissue samples to be used for research.Serum and cyst tissue samples of 80 patients who had undergone echinococcosis cyst removal surgery in Milad Hospital, Tehran, from April 2015 to December 2017, were included in the study. After radical surgery, cyst tissue samples were fixed in 10% formalin. Macroscopic observations were recorded, and samples were embedded in paraffin according to routine histological procedures. Five μm sections were stained with hematoxylin and eosin and examined by light microscopy.The DNA was extracted from 200 μl of serum by QIAamp DNA Blood Minikit  according to the manufacturer’s instructions. Five 10 μm sections were cut from each embedded cyst tissue sample, and excess paraffin was trimmed. The prepared sections were submitted to the DNA extraction procedure of GeneRead DNA FFPE Tissue Kit  according to manufacturer’s instructions. The obtained genomic DNA of cyst and serum samples was stored at -20°C until analysis.E. granulosus was detected by PCR amplification of two mitochondrial genes, cox1 and nad1, in patient sera and cysts. The 400 bp fragments of cox1 and 450 bp of nad1 genes were amplified by primers as described by Bowles [The DNA of y Bowles  and Shary Bowles , respecthttp://blast.ncbi.nlm.nih.gov) to compare similarity with sequences in GenBank database. Sequences were deposited in GenBank under accession numbers LC476594–LC476659 for cox1 and LC476660–LC476714 for nad1. The final sequences of each samples were aligned with reference sequences for each genotype to determine E. granulosus genotype in MEGA 7 (www.megasoftware.net). A concatenated sequence of each sample was obtained by combining the cox1 and nad1 sequences. The phylogenetic tree was created with MEGA 7 software using neighbor-joining algorithms with evolutionary distances calculated by the Kimura-2 parameter method and a bootstrap value of 1000.PCR products were purified from agarose gel using the MinElute gel extraction kit , sequenced in both directions using forward and reverse primers , and read by Chromas software . The forward and reverse sequences of each sample were aligned and assembled using DNASIS MAX  and BLAST searched (The 80 participants comprised 28 (35%) males and 52 (65%) females, aged 7 to 76 years with a mean of 39 years. The largest number of subjects fell into the 31–45 year age range with the fewest in the ≤15 years category . Cyst loE. granulosus in cysts confirmed CE  (The (n = 80) . Nineteecox1 fragments were successfully sequenced in 59 FFPE cyst and 11 serum samples, and nad1 in 51 FFPE cyst and eight serum samples  or G3 (AJ237634 and FJ796214) genotype sequences, so they were designated as G2/G3. One sample was not successfully sequenced. Of thirteen samples identified as G2/G3 genotypes with nad1, cox1 determined ten samples as G1, two as G2, and one as G3.The  samples . The genand nad1 . Three sTehran Province contributed the highest number of participants to this study. The genotype distribution according to the patient province of residency is shown in cox1 sequencing multiple alignments of 50 G1-genotype isolates were grouped into 11 patterns according to the single nucleotide polymorphisms of isolates compared with published sequences for the G1 genotype  and 10.0% (95% CI: 5.2–18.5) of serum samples based on the cox1 and nad1 gene, respectively. This finding may be a result of a low level of DNA filtration through the cyst wall. The DNA of E. granulosus may be more detectable in blood early in infection when the oncosphere is migrating through the circulatory system or when the cyst wall is not completely developed. However, patients undergoing CE surgery are usually in late stages with a large cyst having a thick impermeable wall that inhibits DNA release. Chaya and Parija [Molecular analysis of sera and cyst tissue of patients with CE confirmed by surgery and histology detected E. granulosus cell-free DNA (cfDNA) in serum. Due to the highly fragmented character of cfDNA [Plasmodium spp. [Entamoeba histocytica [Toxoplasma gondii [Schistosoma spp. [Strongyloides stercoralis [Both target mitochondrial genes in our study were amplified in large DNA fragments (400 and 450 bp), which might reduce the chance of detecting of cfDNA , 16, it ium spp. , 18, Enttocytica , Toxoplaa gondii , 21, Schoma spp. –24, and rcoralis .cox1 and nad1, respectively. The obtained results from cyst tissue samples were in agreement with previous reports of 91.0% [cox1 gene and 85.0% by the nad1 gene [The quantity and quality of DNA are crucial to obtaining an accurate result in PCR. Among FFPE cyst samples of 80 CE patients, 91.3% (95% CI: 83.0–95.7) and 83.8% (95% CI: 74.2–90.3) were amplified, and 80.8% (95% CI: 70.3–88.2) and 74.6% (95% CI: 63.1–83.5) successfully sequenced for of 91.0%  by the cad1 gene . It is pad1 gene  stated tE. granulosus  responsible for human surgically-treated CE in Iran, with genotype G1 predominating, followed by G3, G2, and G6. Genotype G1 is the most prevalent genotype worldwide [The sequencing and phylogenetic analyses revealed four genotypes of orldwide , 28–34, orldwide –37. Thisorldwide , which wIn our samples, the most prevalent genotype after G1 was G3, in agreement with previous studies of human CE in Iran , 32 and E. granulosus sensu stricto genotype, G2, is found worldwide [cox1 sequencing analysis in three inhabitants of Tehran. Previous analysis of this locus has resulted in human G2 reported only in a single patient from Kerman [nad1 gene sequence analysis was not able to distinguish between G2 and G3 in the fragment sequenced in the present study. This agrees with recent studies by Kinkar et al. [cox1 and nad1  worldwide, its low occurrence in E. granulosus endemic areas exerts a minor influence on human health [E. granulosus sensu stricto is rare [Genotype G6 was detected in one case of liver and one of lung CE. The results of this study agreed with the suggestion that, although genotype G6 is the second most common causative agent of human CE after the n health , 45. How is rare . Studies is rare  and Birj is rare  of southE. granulosus genotypes based on the partial cox1 and nad1 mitochondrial genes using sequences of insufficient length to separate the G1-G3 complex [E. granulosus, providing a basis for comparing our findings.A limitation of this study was the identification of  complex , 38, 44.E. granulosus in 23.8% of serum samples from CE patients confirmed the presence of cfDNA released from the hydatid cyst. Although, due to the low quantity of detectable DNA in the serum, the test may be inadequate for the diagnosis of CE, it might be a starting point for further research into tracing smaller fragments of E. granulosus DNA to accelerate the diagnosis of the CE, particularly for screening high-risk individuals in endemic areas. The predominance of genotype G1 could confirm that the main transmission route of human infection is through the sheep-dog cycle.The finding of DNA specific to S1 Table(DOCX)Click here for additional data file.


Background and Purpose: Decision algorithms for large vessel occlusions in the anterior circulation remain unconfirmed for acute basilar artery occlusion (aBAO). The aim of this study was to investigate procedural parameters, patient characteristics, functional outcome, and survival in dependency of the time window to recanalization from symptom onset. Furthermore predictors of outcome were identified.Materials and Methods: Retrospectively 231 patients with aBAO treated with endovascular treatment (EVT) between November 2008 and February 2019 were identified in a prospectively maintained single center stroke database. Baseline characteristics such as age, cardiovascular risk factors, NIHSS at admission, pre-interventional PC-ASPECTS, periprocedural parameters such as time to recanalization, duration of treatment, extent of reperfusion, collateral status, and occlusion patterns, as well as post-interventional 24 h NIHSS and post-interventional ICH were evaluated. Target variables were mRS at 90 days and mortality over 90 days.Results: Good outcome (mRS 0–2) was attained in 29.5% (n = 68) of patients, overall mortality was 36.8% (n = 85). In mulitivariate analyses patients with time to reperfusion beyond 6 h had a more than half fold decreased chance of good outcome [OR 0.47 95% CI (0.23–0.95) p < 0.05]. The odds for good outcome were reduced by almost 2/3 if post-interventional imaging revealed intracerebral hemorrhage [OR 0.28 95% CI (0.08–0.98)]. Unfavorable outcome was noted in 100% (n = 14) of patients with symptomatic ICH. Risk for death was reduced by more than 80% if collaterals were present [0.16 95% CI (0.03–0.87)] and if recanalization was successful (TICI 2b-3) [OR 0.19 95% CI (0.05–0.78)]. The odds for survival were 5-fold higher in patients with no post-interventional hemorrhages present [OR 5.35 95% CI (2.2–1.58)].Conclusion: This study might contribute to explaining the ambiguous findings regarding the validity of the 6 h time window in aBAO, suggesting that collateral status impacts the odds of survival in the time window to recanalization beyond 6 h. In our study recanalization within 6 h from symptom onset was associated with good outcome. Successful recanalization (TICI 2b-3a) was necessary for good outcome and survival, post-interventional ICH was highly associated with unfavorable outcome. This might ease the decision making for EVT. The overwhelmingly positive trial results favoring mechanical thrombectomy over thrombolysis have represented one of the most important advances in stroke care for decades. However, there are still numerous questions that need to be answered. The positive trials all enrolled patients with anterior circulation strokes and therefore, the advantage of mechanical thrombectomy in large vessel thromboembolic occlusion in the posterior circulation is yet to be determined. Acute basilar artery occlusion (aBAO) is one of the most devastating subtypes of stroke with a mortality rate of more than 30% following mechanical thrombectomy (mTE) , 2. The We retrospectively identified all consecutive patients in our prospectively maintained single center stroke database who had undergone EVT for an aBAO between November 2008 and February 2019. Inclusion criteria included proven aBAO on CT or MR angiography, treatment with or without IV rtPA, use of accepted endovascular treatment strategies with either stent-retrievers, aspiration or a combination of both. No limit was placed on the admission NIHSS or the age of the patient. Exclusion criteria were if patients had a basilar artery occlusion due to an aneurysm, dissection, in-stent thrombosis, non-occlusive thrombi, and failed attempts as well as manifest brain tissue damage not compatible with life or spontaneous recanalization in aBAO. In total 46 patients were excluded. Endovascular treatment is always our first line treatment for an aBAO and was attempted regardless of age, time from stroke onset and unknown stroke onset. Patients had either been admitted directly to our comprehensive tertiary care neurological center or had arrived as inter-hospital transfers from secondary care hospitals in surrounding areas after undergoing baseline imaging (computed tomography or magnetic resonance imaging). All patients underwent an mTE using either stent-retrievers or aspiration.Baseline characteristics including demographics (age and sex), cardiovascular risk factors including smoking, cholesterol levels, hypertension, diabetes mellitus, and the NIHSS at admission were recorded. Furthermore, an Alberta Stroke Program Early CT Score (PC-ASPECTS) for the posterior circulation was derived from each patient's CT or MRI scan by an independent reviewer (JR) not party to any of our patient information, according to the method described in literature  was recorded and categorized into up to 6 h and over 6 h, with the latter group including patients with unknown time of onset and last seen well beyond 6 h.All patients were treated in the neuro-angiography suites under general anesthesia and were therefore sedated and intubated irrespective of their clinical status at admission. The devices used to achieve recanalization were at the discretion of the operator. Procedures were performed via the right common femoral route as standard using a 6F sheath. A brachial access route was used in patients if femoral access was not successful. Balloon guide catheters were not used as standard due to the location of the occlusions in the posterior circulation. The standard guide catheters included 6F Guider Softip (Boston Scientific) and 5F Envoy (Cordis). The microcatheters used in mechanical thrombectomy were either RapidTransit or Prowler Select Plus ; Trevo pro 18 MC (Stryker) or Velocity (Penumbra), The standard microguidewires were Synchro2 0.014” (Stryker) or pORTAL (phenox). Navien A+ 0.058” ID (Medtronic) and SOFIA Plus 0.070” ID (Microvention) were used for aspiration thrombectomies. Mechanical thrombectomy was performed with Solitaire FR (Medtronic) or pRESET (phenox). The earliest cases in the subset (2008–2009) used pCRC (phenox) or BONnet (phenox)devices.The procedure time was defined as groin puncture time to final angiographic run. The date of treatment was recorded to test for any longitudinal effect on the clinical outcome.The site of the arterial occlusion was defined in a binary way with either “patent” or “occluded” assigned to each artery, encompassing occlusion of the proximal, middle, distal third, or whole of the basilar artery as per standard nomenclature . The preEach branch and segment of the basilar artery was reviewed as above and its patency or lack thereof noted. Both the occlusion sites and collateral status were obtained retrospectively from the angiographic DSA images by two independent reviewers  who were not party to the 90-day mRS. Successful recanalization was defined as TICI 2b-3. Any intraoperative or periprocedural complications were recorded.Intracranial hemorrhage (ICH) after EVT was assessed by CT and/or MRI and was rated as “asymptomatic” or “symptomatic.” “Symptomatic” was defined according to ECASS II criteria . Each daAll post-interventional CT or MRI images were reviewed by an independent reviewer (JR), blinded to our patient information.Post-interventional treatment management included admission to the neuro-intensive care unit or hyper acute stroke unit with routine follow-up imaging comprising either CT or MRI scans at 24–36 h and a 24 h NIHSS if possible. The mRS was assessed 24 h after EVT, at discharge, and at 90 days. The 90-day mRS was evaluated either within a follow-up clinic appointment or via telephone by trained research nurses or members of the neurological team.Continuous variables were expressed as mean ± SD or median (interquartile range [IQR]) and categorical variables as numbers (percentage). Fisher's exact test was used in univariate analysis to determine whether functional outcome was independent of each variable tested. The odds ratio was outputted for each variable calculated.Survival and functional outcome analysis for multiple variables was performed for both the ≤ 6 h time window and the >6 h time window. For the >6 h group the collateral status was drawn up . At thisAUC-ROC curves were calculated for admission NIHSS and 24 h NIHSS, pre-interventional PC-APECTS as per CCT or cMRI.Wilcoxon matched pairs signed rank test was performed to test for a difference in the median NIHSS improvement (assessed at admission and 24 h after admission) stratified by mRS 0–2 and mRS 3–6.Reliability of interobserver agreement was calculated to enable assessment of occlusion patterns and collateral status using Cohen's kappa. Value ranges from 0–0.20 as slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1 as almost perfect agreement .Any data sets with discrepancies were reviewed in a group with mutual agreement achieved in each case.A separate multivariate analysis (logistic regression) was performed for 87 patients to analyze for separate influence of early neurological improvement (assessed in NIHSS improvement) on functional outcome and mortality.To analyze for separate influence of different covariates  on functional outcome and mortality at 90 days logistic regression models were applied.p < 0.05.Level of significance was set at The program Stata/IC 13.1 for Windows  was used for the statistical analysis.n = 85), favorable outcome (mRS 90d 0–2) was achieved in 29.4% (n = 68). The median (min. – max.) age was 75 (19–110) years, with the majority of patients being male . Pre-and post-interventional NIHSS at 24 h was available for 87 (100%) patients. 56 (64%) patients had a NIHSS ≥9. Out of these 56 patients 32.1% (n = 17) had early neurological improvement <9. The median NIHSS at admission was 14 [(0–42) n = 155]. Median 24 h NIHSS was 6 [(0–33) n = 110]. The median time to recanalization from symptom onset was 6.4 h (2.5–20.4). Only a minority of 31 patients (9.4%) received iv rtPA as the vast majority  received treatment beyond 6 h from symptom onset. In the latter group, stroke onset had not been witnessed for 81 (35.1%) patients.We identified 231 patients (100%) that met our inclusion criteria. Mortality at 90 days was 36.8%  a score for pre-interventional PC-ASPECTS had to be reached via CCT.Pre-interventional PC-ASPECTS with MRI were attained for 62 (26.8%) patients. For the majority of patients . One hundred ninety seven patients (85.3%) had collaterals. One hundred eighty three patients (79.3%) had at least one PCOMA. Thirty five patients (15.1%) were being supplied via the PICA-superior cerebellar artery (SCA) pathway. In the over 6 h group, 133 patients (57.6%) had a collateralization via at least one PCOMA while in the under 6 h TTR subgroup, 50 patients (21.6%) had at least one PCOMA.Collateral status and pre-interventional stroke burden depicted by pre-interventional PC-ASPECTS was inconsistent regarding prediction of functional outcome and mortality. The median PC-ASPECTS with CCT was 10 irrespective of collateral status. This finding is due to the technical weakness of CCT for acute stroke in posterior fossa.In cMRI Median PC-ASPECTS was eight if collaterals were present and 7.5 with no collaterals. This marginal effect was most likely caused by the small number of pre-interventional cMRI scans.n = 161) followed by occlusion of the middle third (17.3% n = 40) and the proximal third (3.4% n = 8). It was most common for one third of the basilar trunk to be occluded (45.6% n = 151), while occlusion of two thirds was seen in 25.1% of cases (n = 58). Total occlusion of the basilar trunk was recorded in 22 patients (9.5%)  .1.4 h (0.9–2.3). In 38.4% (n = 88) of cases, treatment was longer than 1 h but shorter than 2 h. In 26.2% (n = 60) of patients, treatment procedures lasted over 2 h. There was no effect regarding prediction of functional outcome and mortality.The median duration of treatment was n = 179) whereas aspiration was used as the primary method of mTE in 15.2% (n = 35).Stent retrievers were used for 77.5% of patients (n = 221).In 64.5% of patients, mTICI 2b-3 was achieved after 1 passage. At the end of the procedure, mTICI 2b-3 was achieved in 93.5% of patients (n = 17), followed by Solitaire stent retrievers (25% n = 9), pBONNET stent retrievers (22.2% n = 8) and a few pREset devices (5.6% n = 2). In 2011 to 2013, pREset devices were used in most cases  followed by Solitaire (6.7% n = 7) and pBONNET devices  another 1% were other devices.From 2008 to 2010, the pCRC device was predominantly used for mTE (47.2% n = 39) the pREset device was used followed by SOFIA Plus aspiration catheters (38.5% n = 35). In 14.2% of cases (n = 13) pREset in combination with SOFIA Plus aspiration catheters were used. In 4.3% (n = 4) of procedures, other stent retriever devices were used.From 2014 to 02/2019, in most cases (42.8% n = 20) and symptomatic ICH was seen in 6% (n = 14) of patients.Asymptomatic ICH occurred in 8.7% (Good Outcome mRS 90 days 0–2 vs. 3–6.n = 25 10.8%) was associated with a good outcome (p = 0.02) and diabetes mellitus (n = 61) was associated with poor outcome (p = 0.001) (p = 0.7) nor hypercholesterolaemia had an effect on clinical outcome (n = 155) of ≥28 had a good functional outcome  with poor clinical outcome (mRS 3–6) showed no gender effect . A posit= 0.001) . Neither outcome . The loc outcome . Further outcome .n = 110), as none of these patients assessed with NIHSS ≥ 23 achieved a good functional outcome .Out of 87 patients with baseline and 24 h NIHSS 31 patients (35.6%) experienced good outcome and 56 patients (64.4%) had an unfavorable outcome. The median 24 h NIHSS for the patient group with favorable outcome was two compared to 20 in the group with unfavorable outcome. The difference in median of pre-interventional and 24 h NIHSS was significant (p < 0.001). AUC for each was >0.8.NIHSS at admission and at 24 h had a very good and significant discriminative power regarding good functional outcome in AUC-ROC analysis (ROC p = 0.025) compared to patients with a TTR of <6 h (p = 0.043)  . The pro= 0.043) . There w= 0.043) .Surprisingly, the odds of a good functional outcome were reduced by 70% if PCOMA was absent through a distal occlusion in the basilar artery. However, the proximal or middle third being occluded did not have any effect on good functional outcome, this finding is most likely due to a small sample size .Post-interventional hemorrhage decreased the likelihood of good functional outcome by more than 70% This latter effect was particularly pronounced in patients with symptomatic post-interventional intracranial hemorrhages (sICH) as 100% did not achieve good functional outcomes .Multivariate analysis revealed both the time to recanalization and ICH to be predictors for functional outcome at 90 days. The likelihood of a good outcome was reduced by half if TTR was >6 h and by more than 70% if post-interventional hemorrhage was found .Patients treated from 2011 to the beginning of 2019 had an almost fourfold likelihood of a good outcome in comparison to patients treated in the earlier period of 2008–2010 .p < 0.001]. Latter finding should be interpreted with caution as the NIHSS was more likely missing for patients with mRS 90d 3–6.In a separate multivariate analysis with 87 patients NIHSS improvement was an independent predictor for good functional outcome [OR 1.28 (1.14–1.45) In bivariate analysis Risk of death over 90 days was high if the NIHSS had a value of 12–22 points, both for NIHSS at admission and at 24 h after admission. In each case, there was a 6-fold risk compared to patients with an NIHSS of 0–4 .Adjusted for 90-day mortality, the odds of death were reduced by more than half if collaterals were present. Collateral status did matter in the TTR over 6 h subgroup. The odds were reduced by 65 percent when compared to patients with no collaterals .If PCOMA was absent, the likelihood of death was 3.4 times as high as for patients with bilateral PCOMA present . PatientRegarding occlusion patterns observed in the ≤ 6 h TTR the likelihood for death was reduced to more than 90% in comparison to total occlusion if partial occlusion was noted. This effect was not observed in the >6 h TTR .For patients in the TTR >6 h subgroup, we observed that likelihood of death was increased by a factor of 2.4 compared to the TTR within 6 h group .Looking at TICI scores, patients with a TICI of 2b-3 had a 80% lower probability of dying in comparison to patients with a TICI of 0–2a .If post-interventional hemorrhage occurred, these patients had an over 4-fold probability of dying. This effect was even more pronounced in patients with symptomatic hemorrhage .p < 0.009) compared to patients treated in the years 2008–2010  (008–2010 .p < 0.001), collateral status (p = 0.03), and TICI score (p = 0.02)  . In a seIn our study on aBAO certain trends correlating to good functional outcome and to mortality over 90 days were observed.In this respect, the 6 h TTR and post-interventional hemorrhage were most predictive for functional outcome at 90 days. In bivariate analysis, collateral status, diabetes mellitus, smoking status, year of treatment, admission NIHSS and 24 h NIHSS could predict functional outcome.As regards mortality, in multivariate analysis the strongest predictive power was found for collateral/PCOMA status, post-interventional hemorrhage, and TICI score. In bivariate analysis, the following variables were associated with mortality over 90 days: collateral status , devices used, year of treatment, sICH, admission NIHSS and 24 h NIHSS. The observed mortality rate was in line with other reports , 2.The success of multiple recent trials –22 has lAntithetically, there are findings from a large single center report with 184 patients by Strbian et al.  that sugIn our study, patients with recanalization within <6 h from symptom had the best outcome. This finding is in line with the BASICS registry.Now the reasonable question for the ambiguous findings regarding the TTR arises. Subsequently potential causes shall be discussed.It is widely accepted that pial collaterals are important in preserving brain tissue in anterior circulation strokes. However, published literature for aBAO in this context is scarce.A study  with 149In our study, we could observe the pronounced impact of collateral status on functional outcome and mortality, in particular in the >6 h TTR group. This might be due to the fact that collaterals become more vital the longer a vessel occlusion is present.In terms of occlusion patterns it is a surprising finding that the presence of collaterals when the distal third is occluded is associated with both a good functional outcome and with reduced mortality. This might be pathophysiologically explained by head loss in the basilar artery and the subsequent suction effect from the anterior circulation via PCOMA. The missing effect of collaterals on other occlusion patterns is most likely a sample size issue. Moreover, distal occlusions were most frequently seen.Another controversial debate in aBAO concerns thrombus length, functional outcome and survival. In a small study  with 32 In our study, the extent to which the basilar artery was occluded did not predict functional outcome but could predict mortality if applied to the ≤ 6 h time window to treatment. Latter finding should be interpreted with caution as only three patients had a total occlusion.In our study, successful reperfusion conveyed through a TICI score was a separate predictor for mortality. This finding, that patients with successful recanalization had a lower mortality rate, is in line with current literature . A limitRangaraju et al.  found thThe results regarding early neurologic improvement were inconclusive due to a small sample size. Nevertheless a trend toward NIHSS improvement and functional outcome as well as mortality was observed.In current literature, pre ASPECT scores for the posterior circulation are the subject of discussion. All share the view that native CCT is not suitable for evaluation of the PC-ASPECT. As in our study the vast majority received pre-interventional CCT pre-interventional PC-ASPECT scores were not predictive of functional outcome. Nevertheless, in the small number of patients who has a pre-interventional cMRI, a trend for predicting functional outcome using the PC-ASPECT score could indeed be seen. This latter trend is in line with current findings in literature , 36.As recently published, symptomatic intracranial hemorrhage is associated with an unfavorable outcome , 38. In Furthermore, the year of treatment showed an impact on both functional outcome and mortality in bivariate analysis. This can be ascribed to the old fashioned pCRC devices still pre-dominantly used in mechanical thrombectomies in the period 2008–2010.Furthermore, various patient characteristics are currently being discussed as possible predictors of outcome. A positive smoking status is being considered as a predictor of good outcome and is also known as the smoking paradox , 39. In In our cohort, diabetes mellitus was a predictor for poor functional outcome in bivariate analysis. Most reports , 39, 41 Limitations of this study are the retrospective single center design. A strength of this study is its sample size, to date the highest to be published from a single center.Our data suggest that the time window to treatment, collateral status, reperfusion status, extent of occlusion and ICH in aBAO can predict functional outcome and mortality.The most important finding of this study is that the time window to successful treatment seems to be dependent on collateral status and extent of occlusion. These findings may serve as an explanation for the ambiguous findings regarding the validity of the 6 h time window in aBAO. They might also be helpful for decision making regarding EVT in aBAO, especially as an increasing number of patients are being referred from primary or secondary care hospitals to specialized neuro centers where time from symptom onset is often considered when deciding how to best treat an aBAO. Furthermore, baseline NIHSS, NIHSS at 24 h and post-interventional ICH were good predictors for mRS at 90 days.The datasets generated for this study are available on request to the corresponding author.The study was approved by the ethics committee of the Regional Medical Board Baden-Württemberg, project number F-2012-077.Patient consent was not obtained as data were analyzed anonymously.JR and HH contributed to the conception and design of the work, literature search, analysis and interpretation, and article drafting. MA, VH, PB, HB, and HH contributed to critical revision. All authors gave final approval of the version to be published.MA and PB have proctoring and consulting agreements with phenox GmbH. HH is co-founder and shareholder of phenox GmbH and femtos GmbH. JR, VH, and HB report no potential conflict of interest.


Organizing pneumonia (OP) is a rare disease that is often easily misdiagnosed as a malignancy. The diagnosis of OP can prove quite challenging. Patients typically receive treatment with high-dose corticosteroids. Relapse is common if corticosteroid treatment is reduced or stopped. However, given that long-term corticosteroid treatment often results in significant side-effects, the aim of this study was to discuss the diagnosis and surgical treatment of OP.The medical records of 24 patients with pathologically diagnosed OP between October 2007 and January 2019 were retrospectively reviewed. All patients underwent thoracic computed tomography (CT) and transbronchial biopsy or CT-guided percutaneous needle aspiration. We analysed the clinical manifestations, radiological findings, diagnostic methods, treatment, and follow-up outcomes of all patients.n = 16; 66.6%), and the most common radiological finding was consolidation  on thoracic CT. The diagnosis of OP was made by transbronchial biopsy in 11 patients (45.8%), and percutaneous needle aspiration biopsy in 13 (54.2%). We performed 11 wedge resections, 9 segmentectomy, and 4 lobectomies. Twenty patients underwent video-assisted thoracoscopic surgery (VATS), and 4 underwent thoracotomy. Complete lesion resection was obtained in all patients, and all patients were discharged from the hospital between 5 and 11 days after surgery. The mean follow-up period was 59.1 ± 34.5 (range: 2–134) months. Residual lesions or local or distant recurrence were not observed.In total, 24 patients with OP were identified. The study included 17 (70.8%) men and 7 (29.2%) women, and the mean age was 61.25 ± 11.33 years (range: 31–82). The most common symptom was cough (OP is a rare disease, and the exact aetiology remains unclear. Preoperative diagnosis is difficult to achieve despite the use of transbronchial biopsy or CT-guided percutaneous needle aspiration. Complete surgical resection represents an effective method for the treatment of OP. Organizing pneumonia (OP) is a common histopathological response to injury in the lung, demonstrating inflammatory intra-alveolar infiltration that leads to fibrosis on pathology . OP can The histopathological pattern of OP typically reveals granulation tissue plugs within the alveoli, alveolar ducts, and small airways (Masson bodies) , 4. ThesThe medical records of 24 patients diagnosed with OP from October, 2007 to January, 2019 in our hospital were reviewed. All patients had no history of malignancy, and haven’t undergone chemotherapy or radiotherapy. All patients were subject to thoracic computed tomography (CT) scans. Diagnosis of OP was made by transbronchial biopsy or CT-guided percutaneous needle aspiration. The demographic data, symptoms, thorax CT findings, diagnostic methods, and treatments were recorded in all cases. All patients were followed between November 2007 and April 2019. This retrospective study was approved by the Institutional Review Board (IRB) of the Weihai Municipal Hospital, and permission was obtained from all participants.Only descriptive statistics are provided. In descriptive statistics, frequency and percentages were used for categorical variables, and mean ± standard deviation values were used for continuous variables. All recorded data statistical analyses were performed using SPSS 20.0 software.Between 2007 and 2019, we retrospectively evaluated 24 patients (17 men and 7 women) with a diagnosis of OP. The mean age of the patients was 61.25 ± 11.33 years (range: 31–82). The most common symptom was cough, sputum, fever, shortness of breath and chest pain  patients. Increased erythrocyte sedimentation rate (ESR) values were noted in 6(25%) patients. Increased C-reactive protein values (CRP) were noted in 8(33.3%) patients, and neutrophilic leucocytosis was noted in 5(20.8%) patients. Pulmonary function test (PFT) was normal in 20 (83.3%) of our patients. A restrictive pattern was observed in 2(8.3%) patients, and an obstructive pattern was determined in 1(4.2%) patients. The main radiological manifestation was a consolidated, mass-like lesion and ground glass opacity (GGO)  patients, percutaneous needle aspiration in 13 (54.2%)  minutes, the blood loss was 83.50 ± 69.45 (20–400) ml, the postoperative hospitalization time was 3.67 ± 1.13 (3-7) days, and the thoracic tube extubation time was 2.50 ± 0.92 (2-5) days (Table The mean follow-up period of the patients was 59.1 ± 34.5 months (range 2 to 134 months). There were no obvious discomfort and complication after discharge. One patient was lost to follow-up. Patients are currently still being followed.OP is rare. The exact aetiology of this inflammatory reaction remains unclear . OP doesPatients may remain asymptomatic in 30 to 70% of cases . Common There are no specific radiological signs for OP. The most typical imaging pattern of OP is a migratory bilateral patchy alveolar infiltrate , 18, 19.Spontaneous remission of OP is rare, therefore, the diagnosis of OP typically requires treatment if the patient is symptomatic , 22. OP OP is often found in the shadows of lung cancer , 33. TheComplete lesion resection with preservation of uninvolved pulmonary parenchyma remains the fundamental goal in the surgical treatment of OP. If surgical management confirmed, small-range pneumonectomy should be performed, such as segmentectomy or wedge resection, to preserve as much of the functional lung tissue as possible. Hilar lymphadenectomy cannot be recommended as a routine procedure for the treatment of OP. For larger lesions or the mass is located at the hilum, lobectomy can be performed. The frozen pathological sections should be examined during the operation to avoid extending the resection. In our study, 24 patients were treated by surgery. Three patients were treated by surgery because the family history of lung cancer, 7 had not responded to corticosteroid therapy, and 14 cannot tolerate the side effect of steroid. We performed 11 wedge resections, 9 segmentectomy, and 4 lobectomies. We performed 4 lobectomies because those mass is large and close to the hilum of the lung, and wedge resection is very difficult. Among these procedures, VATS was utilized in 2083.3%) cases, and the postoperative hospitalization time was 3.67 ± 1.13 [.3% casesIn conclusion, the exact aetiology of OP remains unclear. Hormone therapy should be considered after a clear diagnosis of OP. We can consider surgical treatment when the patient cannot tolerate the side effect of steroid or had not responded to treatments. At surgery, small-range pneumonectomy should be performed and to preserve as much of the functional lung tissue as possible. Complete surgical resection is a safe effective method for the treatment of OP.


In Germany, patients receiving oral anticoagulation (OAC) are often treated by general practitioners (GPs), and large proportions of patients receive vitamin K antagonists (VKAs). The quality of OAC in German GP practices, differences between various practices, and improvement potential through implementation of case management, have not yet been investigated satisfactorily.Based on results of a cluster-randomized controlled trial, we aimed to assess whether OAC quality can be improved, any variations between practices exist and determine practice- and patient-level factors.The PICANT trial (2012–2015) was performed in 52 GP practices in Hesse, Germany. Adult patients with long-term indication for OAC received best practice case management in the intervention group. International normalized ratio (INR) values were recorded from anticoagulation passes. The Rosendaal method was used to calculate Time in Therapeutic Range (TTR) at patient level, and mean pooling to obtain center-specific TTR (cTTR) at practice level. The quality of OAC was assessed by TTR and cTTR. Linear model analyses were used to investigate associations between practice−/ patient-level factors and TTR.p = 0.670). The cTTR averaged 75.1%  in the intervention versus 74.3%  in the control group (p = 0.668). At practice level, the TTR was significantly lower in practices with a male physician and certification in quality management. At patient level, the TTR was significantly higher in patients with moderate to high compliance, in men, and in patients that performed self-management. The TTR was significantly lower in patients with certain comorbidities, and who were hospitalized.Inclusion of 736 patients (49.6% intervention and 50.4% control patients); 690 (93.8%) received phenprocoumon. Within 24 months, the TTR was 75.1% (SD 17.6) in the intervention versus 74.3% (SD 17.8) in the control group  contains supplementary material, which is available to authorized users. Oral anticoagulation (OAC) is indicated for a variety of conditions. Long-term antithrombotic treatment with OAC reduces the risk of thromboembolic events in patients with atrial fibrillation (AF). AF is a common condition, with an estimated 10 million patients suffering from it in Europe in 2014 ) number of participating patients per practice was 14.0 (1.6), while in the control group it was14.3 (1.5). Details on the screening process and characteristics of the sample have been described elsewhere , 30.In brief, practices and patients in the intervention and control groups showed similar characteristics  in the intervention group vs. 74.3%  in the control group (p = 0.668). Figure Data on INR measurements were available for 688 patients, 344 from each randomization group. The standard target ranges were 2.0–3.0 for 678 (98.5%) patients and 2.5–3.5 for the remaining 10 (1.5%). GP-based target ranges were more variable with 2.0–3.0 being the most common (657 (95.5%) patients), followed by 2.5–3.5 (11 (1.6%) patients) and 2.0–4.0 (10 (1.5%) patients). For the full list of ranges documented in the anticoagulation passes see Table p = 0.787). The mean cTTR was 75.3%  in the intervention group vs. 74.8%  in the control group (p = 0.780).The analyses using “GP-based” INR target ranges showed similar results: Within 24 months, the mean TTR was 75.3% (SD 17.2) in the intervention vs. 74.8% (SD 18.0) in the control group  were not statistically significantly associated with the TTR  during the study period. Factors such as age and educational attainment were not significantly associated with TTR  had left the study before it ended either because of death, or the patient’s decision to cancel participation.2  patients changed their anticoagulant medication during the course of the study for a variety of reasons. Thus, oral anticoagulation with a given drug could not be monitored over the entire study period in these patients. This fact also reflects the changes in antithrombotic therapy resulting from increased approval of DOACs during the course of the trial. Despite the overall large sample size, the variation in the cTTR between GP practices must be assessed cautiously in light of the limited sample size per practice. Finally, an important limitation in the interpretation of the results are the small values for R.8% patieAs the quality of OAC was generally high, the intervention resulted in no statistically significant improvement. However, variation between the practices indicates optimization potential in some of them. Nevertheless, a repeatedly demonstrated association between patient-level factors and the TTR underlines the importance of bearing in mind those patient characteristics that may make it difficult to achieve high quality therapeutic outcomes.Additional file 1:Knowledge test for GPs. The additional file shows an English version of the knowledge test for GPs which was developed for the PICANT study. It was used to evaluate the level of knowledge of participating GPs about oral anticoagulation therapy. (DOCX 220 kb)


Accidental occupational injuries to health care workers (HCWs) continue to have a significant problem in the healthcare system. Thus, the aim of this study was to assess prevalence of needle sticks and sharp injury and associated factors among health care workers working in Central Zone Tigray northern Ethiopia.The prevalence of needle stick and sharp injury in the past 12 months preceding the study and entire job were 25.9% and 38.5% respectively. Nearly one-third (31%) of the injuries occurred in emergency unit and 122 (71.3%) of the materials caused injury were used on patients. Practice of needle recap, ever used cigarette in last 12 months, training, work hours > 40 per week, job dissatisfaction and work experience less than 5 years were found factors significantly associated with needle stick and sharp injury for health care workers. The magnitude of Needle stick and sharp injury is high in the study area. Policy makers should formulate strategies to improve the working condition for healthcare workers and increase their adherence to universal precautions. Needle stick and Sharp injuries are a piercing body trauma caused by a sharp medical equipment that was used to screen, diagnose, treat or follow a patient’s disease conditions . AccidenSub-Saharan African countries have the highest share of HIV prevalence and more than 90% of occupational exposure . FurtherThe factors associated with NSSI were socio-demographic factors , environmental factors  and other behavioral factors including recapping needles, Use of personal protective equipments (PPE), not complying with standard operating procedures –21. HoweA facility based cross-sectional study was conducted from February 6, to April 15, 2017 in selected health care facilities of central zone of Tigray which is located approximately 1024 km away from Addis Ababa, Ethiopia. It owns three urban and nine rural districts with a total population of 1,352,557: . In this zone there are 3 general hospitals, 1 comprehensive specialized referral hospital, 6 primary hospitals, 54 health centers and around 200 health posts with 1582 health care workers. All health professionals and auxiliary workers at governmental health care facilities of central zone of Tigray were the source population. From the selected health care facilities, all health care professionals who perform medical procedures on a daily basis, auxiliary workers who worked for at least 1 year and above were included in the study.The sample size was determined using Open-Epi version 2.3.1 software by taking 19.1% prevalence from previous studies on needle stick and other sharp injury [An anonymous, structured, interview questionnaire was developed after reviewing relevant literatures that reviewed key variables as well as earlier studies on NSSI among health care workers , 18, 20.p value ≤ 0.2 in bi-variable analysis were transferred to a multi-variable analysis to control the effect of confounding. Finally, the 95% CI with p-value of ≤ 0.05 was considered as statistically significant.The pre coded responses were entered in Epi-info version 3.5.3, and then exported to SPSS software version 21 for windows. We have used descriptive statistics to explore the entire variables of the study. Both bi-variable and multi-variable binary logistic regressions were used to identify risk factors for NSSI; variables with From a total of 456 selected health care workers, 444 participated giving a response rate of 97.4%. The mean age of the respondents was 34 (SD ± 9.6) years. The majority of the respondents 213 (48%) were diploma holders in education. The mean time of thier work experience was 11.72 (SD ± 9.8) years  were concerned about the health risks of NSSI, of whom the risk was perceived as high in 325 (73.2%) respondents. One hundred sixty-seven (37.6%) respondents had recapped the needle at least once in 12 months preceding the study, and 76 (45.5%) respondents had recapped the needle using their two hands. Nearly all respondents (96.8%) knew the diseases transmitted through needle stick and sharp injuries. The reported prevalence of substance use was 10 (2.3%), 82 (18.5%), 600 (13.5%) and 6 (1.4%) for khat, alcohol, cigarette and other substances respectively , 25.9% (95% CI of 21.8.0 to 30.2), and 38.5% (95% CI of 34 to 43) respectively. Majority 122 71.3%) were exposed at the day time . Health care and auxiliary workers who ever smoked cigarette in the last 12 months were nearly four times more likely to experience needle sticks and sharp injury than those who did not smoke in the last 12 months . Study participants with work experience of less than 5 years were 4.5 times more likely to experience needle sticks and other sharp injury than those with experience of greater than five years  . This finding is also similar with the studies conducted in Bale, Hawassa and Addis Ababa, Ethiopia , 19, 25.Respondents who work for greater than 40 h per week were 16 times more likely to experience NSSI than those who were working less than or equal to 40 h per week . This is similar with the findings from Gondar and Addis Ababa , 25. StuIn conclusion, this study revealed that one-fourth of the respondents had needle stick and sharp injury at least once per year; still the magnitude of NNSI is high in the study area. Practice of needle recap, cigarette smoking, lack of training on occupational health and safety, more working hours per week, job dissatisfaction and less work experience were found to be the major identified risk factors. Therefore, policy makers and health care planners should formulate strategies to improve the working condition for healthcare workers and increase their adherence to universal precautions.The accuracy of the past experince of the rspondents with regard to the ocurence of the NNSI mighthave been affected by recall bias.Additional file 1: Table S1. Socio-demographic characteristics of health care and auxiliary workers at central zone of Tigray, northern Ethiopia, 2017 (n = 444).Additional file 2: Figure S1. A bar graph which shows job category of health care and auxiliary workers in central Zone Tigray, northern Ethiopia, 2017.Additional file 3: Figure S2. A bar graph which shows Current working department of health care and auxiliary workers in central Zone Tigray, northern Ethiopia, 2017.Additional file 4: Table S2. Participants behavior and working environment of health care and auxiliary workers at central zone of Tigray, northern astern Ethiopia, 2017.Additional file 5: Table S3. Needle and Sharp handling method of health care and auxiliary workers at central zone of Tigray, northern Ethiopia, 2017.Additional file 6: Figure S3. A bar graph shows place where needle sticks and sharp injury occurred among health care workers in central Zone Tigray, northern Ethiopia, 2017.Additional file 7: Figure S4. A bar graph which shows the reason for the occurrence of needle sticks and sharp injury among health care and auxiliary workers in central Zone Tigray, northern Ethiopia, 2017.


Hymenoscyphus tetracladius  is a common aquatic hyphomycetous fungus. Here, we report the draft genome sequence of H. tetracladius strain NNIBRFG329, which comprises 41.8 Mb in 20 scaffolds with an overall G+C content of 46.95% and an N50 value of 3.973 Mb. Hymenoscyphus tetracladius  is a common aquatic hyphomycetous fungus. Here, we report the draft genome sequence of H. tetracladius strain NNIBRFG329, which comprises 41.8 Mb in 20 scaffolds with an overall G+C content of 46.95% and an N50 value of 3.973 Mb. Hymenoscyphus tetracladius  is well known as one of the aquatic fungi that contribute to leaf decomposition in streams  and was preprocessed using Trimmomatic v0.30 for paired-end sequencing (H. tetracladius NNIBRFG329 consisted of 20 contigs with an N50 value of 3.973 Mb and 428.53-fold coverage. The total length of the assembled genome was 41,846,809 bp with a G+C content of 46.95%. The maximum and minimum scaffold lengths were 7,110,143 bp and 2,165 bp, respectively. This draft genome sequence will support the identification of genes related to leaf litter decomposition by aquatic fungi in freshwater ecosystems and contribute to genetic research studies of the fungi.NNIBRFG329 was isolated from plant litter as previously described . Genomicquencing  and Nextquencing . The reaquencing  and SOAPquencing . The draH. tetracladius NNIBRFG329 has been deposited in GenBank under accession number PYIV00000000. The SRA accession number is SRR7800875. This paper describes the first version of the genome.The draft genome sequence of


Salmonella spp. is one of the most important zoonotic diseases and transmits to human through raw food animal products including poultry meat. Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are the most important strains that infect human. This study was conducted to evaluate the contamination rate of poultry carcasses with S. Enteritidis and S. Typhimurium using multiplex PCR assay.Salmonellosis caused by Salmonella isolates were identified by multiplex-PCR. Three sets of primer pairs tagreting invA gene for Salmonella genus, prot6 gene for entritidis serovar and fliC gene for Typhimurium serovar were used.100 samples were selected during the summer and fall of 2010 by cluster sampling method from 10 broiler flocks, which were slaughtered in a poultry abattoir located in Mashhad suburb. After culturing the samples in enrichment and selective media and obtaining suspected colonies, DNA was extracted and Salmonella was 14% (14/100) which 43% (6/14) of them were identified as S. Enteritidis and 36% (5/14) identified as S. Typhimurium, respectively.The contamination of poultry carcasses with S. Enteritidis and S. Typhimurium is relatively high in poultry carcasses.Results of this study indicated that the risk of zoonotic diseases created by  Salmonella is a Gram-negative, facultative anaerobic bacteria, belonging to the family of Enterobacteriaceae  of the samples  due to settings . MDR Salocessing .Polymerase Chain Reaction (PCR) is a valuable method for investigating food-borne outbreaks and pathogens identification . PCR proSalmonella are intestinal tract, skin and feathers of chickens, which may develop along the processing line , citrate, lysine decarboxylase, urease and indole tests  and incubated at 37°C for 24 h. Amount of 1 mL of each re-enriched sample was transferred into 9 mL of selenite cystine broth (Bio-Rad) and incubated at 37°C for 24 h. Following incubation, a loopful of each culture was streaked onto brilliant green agar (Bio-Rad) which was incubated at 37°C for 24 h. Presumptive le tests .Salmonella spp. by biochemical tests were cultured overnight on nutrient agar. Then, DNA extraction was performed using boiling method , 0.5 μl Taq DNA polymerase (Fermentas), 2 μl extracted DNA and 0.5 μl each primer. Amplification reaction and cycling condition were performed in a thermal cycler  as follows: an initial incubation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and a final extension period for 10 min at 72°C , 1.25 μl deoxynucleotide triphosphate (10 mM), 1.5 μl MgCl at 72°C . AmplifiinvA gene were used for detection of the genus Salmonella and primers of prot6e-6 and prot6e-5 which were specific for prot6E gene were used to identify S. Enteritidis and Fli15 and Tym primers which were specific for fliC gene were used to identify S. Typhimurium (S. Enteritidis (ATCC-13076) and S. Typhimurium (ATCC: 14028) were used as positive controls and sterile distilled water as negative control.Primers of S139 and S141 which were specific for himurium . S. EnteSalmonella colonies on selective and non-selective media, 14 samples were positive (14%) and confirmed by PCR (a 284-bp fragment of the invA gene). From 14 isolates, 6 were identified as S. Enteritidis (a 185-bp fragment of the prot6e gene) (S. Typhimurium (a 559-bp fragment of fliC gene) using multiplex PCR assay (Using culture method and based on the morphology of 6e gene)  and 5 asCR assay .S. Enteritidis or S. typhimurium.The remaining 3 isolates were not approved as Salmonella genus isolation obtained in this study was 14% using culture method and multiplex PCR. Jamshidi et al.  in fresh chicken and beef meat samples in retail outlets of Tehran  of slaughtered birds was found after stunning, and the highest contamination rate was found before chilling (52%)  .S. Enteritidis and S. Typhimurium are the main serovars implemented in controlling risk factors of salmonellosis because they are transmissible from parent to the chickens leaving the hatchery (S. Entritidis was detected in 43% (6/14) and S. Typhimorium in 36% (5/14) of the samples and the remaining three isolates (21%) were probably belonged to other serovars which were not evaluated in the current study. Ulloa et al.  of the samples using traditional culture methods and in 38 (19%) of the samples by PCR method, when culture and PCR results were combined, the pathogen detection increased to 45 out of the 198 samples (23%)  .Salmonella spp. infection for humans. The poultry industry has to focus on implementing control measures in order to reduce the spreading of the pathogen in the production processes.In conclusion, results of study showed chicken carcasses are potential vehicles of


This case series of three children reports clinical features and chromosomal abnormalities seen in a craniofacial clinic. All presented with orofacial cleft, developmental or intellectual disability, and dysmorphism. Emanuel syndrome or supernumerary der (22)t, the prototype of complex small supernumerary marker disorders, was seen in one child. Duplication 4q27q35.2 with concomitant deletion 21q22.2q22.3 and duplication 12p13.33p13.32 with concomitant deletion 18q22.3q23 seen in the remaining two children are not reported in literature. Maternal balanced translocation was established in both of these children. Orofacial cleft (OFC) is a common congenital anomaly with a prevalence of 1 in 600 live-births. It includes cleft lip, cleft lip with palate (CLP), and cleft palate (CP). Chromosomal etiology accounts for 6% of all children with OFC –4. OlderGenomic DNA was extracted from whole blood treated with EDTA by standard protocol using protein precipitation solution . DNA concentration and purity were measured by biospectrometer . Purified DNA was dissolved with nuclease free water and stored at −20°C until further processing. The CMA was performed with whole genome scanning panel  according to manufacturer's protocol utilizing 200 ng of DNA per sample in an accredited laboratory. This panel is incorporated with ~300,000 single nucleotide polymorphism probes with dose sensitivity of >800 genes. The resolution was 30kb for copy number variations and higher for regions of known cytogenetic importance. Data output was analyzed using GenomeStudio and KaryoStudio provided by the manufacturer.Karyotype was performed on metaphase chromosomes obtained from peripheral lymphocytes and twenty Giemsa banded (450-500 level) spreads were studied. The International System for Human Cytogenetic Nomenclature was used for reporting . SnapshoParental consent for photography and diagnostics was obtained for all the children presented in this series upon approval by the study institute ethics committee.The infant, first born male, was seen at ten months of age with DD and seizures. There was no parental consanguinity. He had microcephaly and central hypotonia. Dysmorphism included unilateral ptosis, deep-set eyes, low set ears, CP, and micrognathia . MagnetiThis male child born of nonconsanguineous parentage presented with failure to thrive at four months of age. There was sibling death with congenital heart disease. He had microcephaly and spasticity. Dysmorphism included downward slant, prominent nose, ear anomalies, right CLP, retrognathia , and rocThis 9-year-old boy born of nonconsanguineous parentage had intellectual disability with normal neurologic examination. He had hypertelorism, wide eyebrows, narrow nasal root, everted lower lip, short ears, short neck , and opeES (Proband 1) is the prototype of complex and small supernumerary marker disorders. The most common non-Robertsonian rearrangement in humans is the translocation between chromosomes 11 and 22 as these chromosomes have palindromic AT repeats that are vulnerable to breaks and recombination during meiosis. ES results from 3:1 mal-segregation of usually the maternal balanced carrier state as in our case , 10. ThePartial trisomy 4q A is usually seen with autosomal monosomy; however, concomitant partial monosomy 21 is not reported in literature. Rinaldi et al.  describePartial monosomy of 18q is associated with OFC with 18q22.3q23 as the critical region . CongeniTo conclude, this series compiles some of the rare and unreported chromosomal abnormalities that include duplication 4q27q35.2 with concomitant deletion 21q22.2q22.3 and duplication 12p13.33p13.32 with concomitant deletion 18q22.3q23. The report also substantiates 4q25q31.3 as a critical locus for renal anomaly and 18q22.3q23 for OFC. In a craniofacial clinic it may be important to identify subtle dysmorphism and DD to establish a chromosomal etiology.