We aimed to study the expression of CD24 and CD133 in colorectal cancer and normal adjacent tissues to assess a relationship between these markers and clinic-pathological characteristics and patient’s survival.Cancer stem cells are a group of tumor cells that have regeneration and multi-order differentiation capabilities. Expression of CD24 and CD133 was studied in a paraffin block of colorectal cancer and normal tissues near tumors with the immuneohistochemical method in patients who were referred to Imam Khomeini Hospital in Sari.  A total of 50 samples  with a mean age of 67.57±13.9 years old with range 28-93 years, included 3 mucinous carcinoma and 47 adenocarcinoma. Expression of CD133 marker was negative in 29 cases and positive in 21 cases. Expression of CD24 in tissue near tumor cells was found in 30% of available samples. The relationship between expressing CD24 with treatment (surgery and chemotherapy) was significant and its relationship with patient’s survival was insignificant statistically. However, there was a clear difference as mean survival age of patients based on CD24 expression was 26.64±18.15 for negative cases and 41.75±28.76 months for positive cases. CD24 and CD133 expressions and their co-expression with other clinic-pathological factors were not significant. During this study, the relationship between CD24 and treatment type was significant. To confirm this result, various studies with high sample numbers and other stem cell markers are recommended.  Colorectal cancer is a second to third common cancer and one of the major leading causes of cancer death in both females and males in the West. Colorectal cancer is responsible for more than 500,000 deaths annually throughout the world ,2. In IrIn recent years, a concept named cancer stem cells (CSCs) has been prevalent. CSCs, which have stem cell characteristics  and differentiation) was first expressed in 1997 for blood malignancies and solid tumor of colon cancer -5.Identifying these cells in colon cancer “colorectal cancer stem cell (CO-CSC)” was conducted with expression of some cell surface markers in various studies -6. CanceCD24 is a cell surface marker with mono-chain sialoglycoprotein and 24 KD molecular weights expressed in many solid tumors such as cancer stem cells in pancreas cancer. CD24 isCD133 or prominin-1 gene codes, which is a membrane glycoprotein on chromosome 4p15.32, has a role in connecting stem cells to their sites, maintaining polarity, as well as movement and cell-cell or cell-matrix interferences. The relationship between expressions of this marker and clinico-pathologic characteristics of patients were reported, including gender, microscopic grade stage, depth of invasion survival, resistance to chemotherapy, and involvement of lymph node -13.The 5-year survival rate for colorectal cancer patients is 50% in dependent from treatment which inPatients and samples In this study, paraffin blocks of 50 patients with colorectal cancer were selected. Patients had referred to Imam Khomeini hospital in Sari during 2001-2012. Verification and completion of clinico-pathologic data were done using hematoxylin and eosin (H&E) stained pathologic slides, pathology record, hospital file and contact with patients. Clinico-pathologic parameters were age, gender, location, type of tumor, depth of invasion, lymph node metastasis and distant metastasis (including liver), tumor differentiation, vascular, lymphatic invasion, treatment, recurrence, survival (time in a month) and current condition . Patients who had chemotherapy or family polypus history or IBD before surgery were excluded. All samples were fixed in neutral buffered formalin 10% and paraffin blocked. All cut sections from both tumor and adjacent normal colorectal mucosa were selected and stained immunohistochemically.Immunohistochemistry staining procedureTissue cut sections with 3-4 micrometer thickness were prepared and stained by H&E. After eliminating paraffin by immersing in Xylene, watering with descending ethanol concentration , tissues were placed in buffer TRIS-EDTA (pH=9) and transferred to microwave with a maximum power reaching to melting point. After microwave power was reduced to 40%, samples were kept for 15 minutes and were placed in room temperature for 15 minutes. After washing with tap water and TBS buffer, DAKO pen was used to determine the periphery (extent) of tissue and placed in a wet room and incubated using detection kit for mouse monoclonal antibody (Anti human CD24 100 µg and Anti CD133 100 µg) with 1/100 dilution for one night in room temperature. Then, samples were covered by peroxides for 30 minutes, and chromomeric reaction was conducted with a relevant kit. Positive control kits for CD24 and CD133 were tonsil lymph tissue. Our negative control was the tissue that was covered with just buffer. Samples were placed in room temperature and hematoxylin was used as background stain. Prepared specimens were studied semi-quantitatively by two expert pathologists using light microscope (Labomed LX400) in low and high powers . All cytoplasmic and membranous staining were observed and scored. In the case of CD24 marker, staining was less and more than 5% of tumor cells considered negative and positive, respectively ,12 Figu. For CD1Statistical analysis 2, Fisher exact test, student and paired T-test. P-value below 0.05 was significant. Patients’ data and immunohistochemistry results were performed by SPSS software (IBM SPSS statistics 20.0.1) using XIn 50 patients, mean age was 64.57±13.9 years with range 28-93 years old. Twenty-five patients were male. The mean age of men and woman was 65.71±12.11 and 63.48±15.62 years, respectively. There was no significant difference between two groups (p=0.58). Tumor type in 47 subjects was adenocarcinoma  and in 3 subjects were mucinous carcinoma (just in men). However, there was no significant relationship between gender and tumor pathology (p >0.05). It is worth mentioning that the mean age of patients suffering from adenocarcinoma and mucinous carcinoma were 65.37±13.8 and 52.33±10.7 years old, respectively. No significant difference was identified between two groups.CD24 expression in 29 cases was negative. Mean age of patients with negative marker was 63.24±12 and in patients with positive marker was a 63.24±16.3 year, which was not statistically significant. Based on pathologic findings, expression of CD133 was positive in 45 patients. In 19 cases (38%), both markers were positive. Markers expression has been summarized in Chart-1. Mean age of negative CD133 patients was 65.5±14.1 years and there was no significant difference between both groups (p=0.94). Mean age of patients with positive and negative expressions was 63.4±17.1 and 65.3±11.7 years old, and no significant difference was evident between two groups. Frequency distribution of gender, based on CD24 in negative group was 40% and in positive group was 51.1%, and no significant difference was observed between both groups. Frequency distribution of gender had no statistically significant difference based on the expression of two markers expression.Regarding the type of tumor (adenocarcinoma /mucinous carcinoma), there was no significant relationship between tumor nature and expression of CD24 and CD133. The mean size of tumors in all studied patients was 4.57±1.94 cm with a 1-10cm range. The relationship between tumor sizes and the expression of these two markers was not significant. Most tumors were in right colon (54%) and rectum (38%). Thirty-one cases (62%) were well differentiated, 14 cases (28%) were moderately differentiated and 3 cases were poorly differentiated. CD24 and CD133 co-expression based on tumor differentiation was not significantly different. Regarding CD24, 17 cases in negative and 16 cases in positive groups (58.2% versus 76.2%) and regarding CD133, 4 cases of negative and 29 cases of positive groups were well differentiated, and showed no significant difference. Interestingly, in co-expressed and non-co-expressed groups, differentiation was not significantly different. No significant relationship between these markers and depth of invasion was observed. In addition, their co-expression showed no significant relationship. Among 50 patients, 20 patients (40%) had lymph node metastasis, and in this regard lymph node metastasis showed no significant difference between the markers and their co-expression as well (P>0.05). Based on results, 31(62%), 12(24%) and 5 (10%) patients were in M0, M1A and MIB stages (the condition of two cases was unknown), respectively. According to data, frequency of distant metastasis based on both co-expression and expression of CD24 and CD133 alone had no significant difference. Among 50 studied patients, 11 patients  had liveIn evaluating results regarding disease stage, 5 (10%), 16(32%), 4(8%), 3(6%), and 13(26%) patients were in stage I, IIA, IIB, IIIA, and IIIB stage, respectively (9 patients had not known stage). Regarding the markers, the disease stage had not a significant difference. In 10 patients (20%), blood vessel invasion was observed and in 27 patients (54%). Regarding CD24, blood vessel invasion was not significant. This held for CD133 and co-expression of CD24 and CD133. In 7 patients (14%), lymphatic vessel invasion was seen (condition of 13 patients was unknown), but no significant difference based on co-expression of two markers was observed.According to the treatment method, 9(18%), 30(60%), 3(84%) patients treated with surgery, surgery and chemotherapy, as well as surgery along with other methods, respectively. Eight patients (16%) underwent surgery accompanied by other unknown treatments. Interestingly, the relationship between CD24 marker with treatment was seen in cases who underwent both surgery and chemotherapy (P=0.04). However, this relationship was not obtained in the CD133 positive cases and co-expression of both markers. The mean survival time for patients from the time of diagnosis was 33.83±24.66 months with a range of 1-120 months. Although there was no statistically significant difference between both groups, this 14-month-time difference in survival is considerable. Recurrence was found in 10(20%) patients after primary treatment (it was not known in 11 patients). Recurrence occurred in 28.6% and 22.2% of negative and positive CD24 patients, respectively. However, the difference between two groups was not significant. Regarding the CD133, recurrence was seen in 20% and 26.5% negative and positive patients and no significant difference was observed. Recurrence took place in 3 co-expressed and 7 non-co-expressed patients, which was insignificant in the analysis. During the study, 20 patients (40%) were died due to colorectal cancer and 21 patients (42%) were alive (condition of 9 patients was not available). No significant difference based on marker expression was obtained. Summary of results regarding the relation between clinicopathologic criteria and expression of markers was shown in Cancer stem cell (CSC) has been used as a new concept in scientific articles since 1997 , 5-7. ThFew studies showed increased expressions of CD24 and CD133 as a primary event in colorectal carcinogens -18. Up tIn animal studies, Baumann used the flow cytometry method in cancer cells of rat and reported the influence of CD24 in tumor growth (tumor size) and metastasis . Iinuma  Regarding the role of CD24 in intercellular junctions, it is claimed that its expression may enhance metastasis ,24. MoreIn their studies, Weichert  and Lee In this study, we found positive (cytoplasmic and membrane) expression of CD24 related to treatment (both surgery and chemotherapy), and somehow survival although it was not significant statistically. On the other hand, there was no relationship between CD133 expressions and clinico-pathologic factors in colorectal cancer. Furthermore, co-expression of these two markers had no significant effect on tumor characteristics. This indicates by evaluating the expressions of these markers on larger samples of colorectal cancer, we can help pre-awareness of patients suffering from colorectal cancers and come up with valuable information. This will also help to improve anti-stem cells treatment strategies.


The aim of the study is to analyze IABP use in patients undergoing cardiac surgery included in the ARIAM Registry of Cardiac Surgery.t test as needed, establishing the level of statistical significance at 95%.An observational, retrospective, multicenter study of all patients undergoing cardiac surgery included in the ARIAMANDALUCIA database of Cardiac Surgery from March 2008 to July 2012. We used the chi-square test and Student P = 0.0001. OR 8.1, 95% CI (6.4 to 10.3)). Besides mortality was higher, the later IABP was implanted the higher the mortality rate was . The ICU length of stay was 9 ± 22 days while the hospital length of stay was 21 ± 28 days. In patients who needed IABP, the ICU stay was higher than for those who did not need it  whereas there was no difference in hospital stay .Of the 8,026 patients who underwent cardiac surgery during the study period, BCIAO was implemented in 358 (4.5%) of them. In total, 65.4% were male. Surgical times in those patients where IABP was implanted were 146 ± 81 minutes and 90 ± 66 minutes . The in-surgery room mortality was 4.7%, 30-day mortality in these patients was 40.2%. Patients in whom IABP was implanted had a mortality rate eight times higher than those who did not require it during surgery or postoperatively (40.2% vs. 8.4%, The intra-aortic balloon pump was used by 4.5% of surgeries performed during the study period and in patients with an increased risk of perioperative complications, estimated by EuroSCORE. ICU length of stay was higher in patients requiring IABP, with no differences in overall hospital stay. Mortality rate was 40% higher, and increases with the delay in the implantation.


Early childhood caries (ECC) is the most common chronic disease in young children. Its reported prevalence varies greatly across China. This systematic review aimed to explore the epidemiological characteristics of ECC in mainland China from 1987 to 2013. In total, 102 articles were included. The pooled national prevalence and care index (ft/dmft%) for ECC were 65.5% and 3.6%, respectively. The overall ECC prevalence declined from 77.9% during 1987–1994 to 56.4% during 2010–2013. The pooled ECC prevalence for children aged 1–6 years was 0.3%, 17.3%, 40.2%, 54.4%, 66.1%, and 70.7%, respectively. There was no significant difference in prevalence between boys (59.1%) and girls (58.9%); and the care index was also similar (8.1% versus 7.7%). Slightly higher ECC prevalence was observed in rural areas (63.5%) compared with urban areas (59.5%) ; but a much higher care index was reported in urban children (6.0%) than their rural counterparts (1.6%) . The 2006–2013 map of ECC prevalence among 5-year-olds showed wide geographic variations across China. Four adjacent provinces, including Sichuan, Chongqing, Hubei, and Shaanxi, constituted the areas with the lowest ECC prevalence in mainland China. Dental caries, progressive damage to teeth caused by bacteria, is a widespread chronic disease that affects 60–90% of school-aged children and the vast majority of adults in most industrialized countries14The disease burden of childhood ECC varies substantially worldwide. In some European countries, ECC is not common, with prevalence ranging from 11.4% among 3–6 year olds in SwedenChina is the most populous and rapidly developing country in the world. The number of children under 6 years of age in China reached 130 million in 2010171927To date, there are no systematic reviews published on ECC prevalence and care index in mainland China. In particular, some important questions still remain unanswered: what is the overall prevalence and treatment level for ECC in mainland China? Are there temporal and spatial distribution trends? Is there a correlation between ECC prevalence with age, gender and location of residence ? To advance our understanding of these issues, it is necessary to use all of the available evidence from China to explore the epidemiological characteristics of this disease over the past30 years. Hence, we conducted a systematic review of the published literatures on the prevalence and treatment status of dental caries among children aged 1–6 years in mainland China . The aim of this evaluation was to provide evidence to inform health programme planning and policy-making for caries prevention and treatment among children.A total of 11,776 publications were identified, and 102 eligible articles were included in the meta-analysis; of which, 100 were written in Chinese and two in English . There w27The pooled overall prevalence of ECC in mainland China was 65.5%  and 58.9% (95% CI: 54.9–62.9%), respectively. The gender difference was not statistically significant . In the 31 studies that stratified prevalence of ECC in 5-year-olds by gender, the prevalence for boys and girls aged 5 years was 67.5% (95% CI: 64.3–70.8%) and 68.7% (95% CI: 65.4–72.0%), respectively , and the difference was not statistically significant .A total of 30 articles reported ECC prevalence in children aged 1–6 years from both urban and rural areas. The pooled prevalence of ECC in rural and urban China was 63.5% (95% CI: 52.1–75.0%) and 59.5% (95% CI: 47.8–71.2%), respectively. The RR for rural versus urban areas was 1.08 . Five distribution zones on the map with different colours were created based on the prevalence of ECC . The geoA total of 44 studies reported the overall care index for ECC from 1988 to 2012. The pooled overall care index for ECC was 3.6% . The lowest overall care index was 3.1% estimated in 2005–2009, and the highest was 4.3% in 2010–2013. The care index at age 5 was reported in 29 studies. There appeared to be an increase in care index from 3.1% in 2005–2009 to 4.9% in 2010–2013. However, the temporal trend was not significant due to insufficient information .The pooled national care index for ECC for children aged 3–6 years was 2.8%, 4.0%, 4.0%, and 4.3%, respectively. There were no data reported for children under age 2 . SpecifiA total of 23 articles reported the care index for ECC for 3–6 year olds in both genders. The pooled care index was 7.7% for boys (95% CI: 6.6–8.7%) and 8.1% for girls (95% CI: 7.0–9.2%). Statistically, the likelihood of receiving care was higher in girls than in boys , Sichuan (38.9%), Hubei (40.9%) and Shaanxi (48.2%), constituted an area of low ECC prevalence. Potential reasons may include a traditional lifestyle and the dietary habits prevailing in these regionsThis study has several limitations. First, heterogeneity is unavoidable in a meta-analysis, especially in a meta-analysis of cross-sectional studiesIn conclusion, this systematic review presents important epidemiological characteristics of the oral health status among children in mainland China over the last 30 years. Our analysis reports a declining trend of ECC over time. However, the prevalence of ECC is still at a relatively high level, while the care index is extremely low. This study provides important evidence for effective interventions to improve the prevention and treatment of ECC in Chinese children.This study was conducted according to the preferred reporting items for systematic review and meta-analyses (PRISMA) checklist  online.A comprehensive search strategy was first developed by a research team owning experts on health informatics, dentistry, clinical medicine, epidemiology and statistics; then a pilot search was conducted by the first and second author independently to test the strategy. The final search strategy was subsequently confirmed based on the pilot and group discussion. Peer-reviewed articles were searched in the following English and Chinese databases from the date of establishment to July, 2014: PubMed (1966-), Embase (1974-), Chinese Biomedical Literature Database (CBM) (1978-), Chinese National Knowledge Infrastructure database (CNKI) (1979-), Chinese Wan Fang database (1990-), and Chongqing VIP database (1989-), using the key terms ‘caries’, ‘prevalence’, ‘epidemiology’, and ‘China’. Additionally, a manual search was applied to the reference lists of all of the eligible articles.Studies were included if they (1) were conducted in mainland China ; (2) were cross-sectional surveys using random sampling; (3) were based on children under 6 years old from the general population rather than a specific group; (4) were conducted at city-level or above; (5) reported sufficient information on the prevalence or care index of ECC. Care index is defined as the proportion of filled teeth to the total number of teeth that are decayed, missing or filled (ft/dmft%)35A tool adopting from the Reporting of Observational Studies in Epidemiology (STROBE) guidelineTwo authors screened articles and extracted data independently. Any disagreement was resolved by consensus or the third author. We contacted the corresponding authors for further or missing information when necessary. The following information was extracted from each eligible study: (1) publication details including author(s) and year of publication; (2) design of study, including study location and period, sampling method, sample size, and characteristics of the participants; (3) details of targeted indicators, such as diagnosis criteria, examiners, number of cases, number of decayed, missing and filled teeth (dmft), and number of filled teeth (ft), which were subgrouped by year, age, gender, urban/rural area, and province where available.2-statistics. A random effects model was adopted in the case of significant heterogeneity (I2 > 50% or P < 0.1); otherwise, a fixed-effects model was used. If high heterogeneity was observed, subgroup analysis was conducted to explore possible factors including survey year, age distribution, gender, location, and province. The discrepancies between boys and girls, and urban and rural areas were compared using the RR and 95% CI. To reflect spatial distribution of ECC, pooled prevalence estimates for ECC in 5-year-old children in each province during 2006–2013 were entered into the ArcGIS software 10.0 to form a prevalence map. Potential publication bias was assessed by funnel plots and Begg’s test; the result was considered to be significant if P ≤ 0.05.Meta-analyses were performed by Cochrane Review Manager (RevMan) 5.1and STATA software 11.1 . The pooled estimates and 95% confidence intervals for each indicator were calculated by pooling the data from each study. Statistical heterogeneity was detected by Q-test and Iet al.51To obtain country-level estimate, we employed a qualified approach that weights prevalence estimates in each province according to the number of children present in the provincesHow to cite this article: Zhang, X. et al. Prevalence and care index of early childhood caries in mainland China: evidence from epidemiological surveys during 1987–2013. Sci. Rep.6, 18897; doi: 10.1038/srep18897 (2016).


Nigeria has a high burden of children living with HIV and tuberculosis (TB). This article examines the magnitude of TB among children receiving antiretroviral treatment (ART), compares their ART outcomes with their non-TB counterparts and argues that addressing TB among children on ART is critical for achieving the 90–90–90 targets.This was a facility-based, retrospective analysis of medical records of children aged <15 years who were newly initiated on ART between 2011 and 2012. Structured tools were used to collect data. STATA software was used to perform descriptive, survival and multivariate analyses.p<0.01) and had delayed ART initiation (p<0.05). The cotrimoxazole and isoniazid prophylaxes were provided to 87.9 and 0.8% of children, respectively. The rate of new TB cases was 3 (2.2–4.0) per 100 person-years at six months and declined to 0.2 (0.06–1.4) per 100 person-years at 24 months. TB infection , malnutrition , delayed ART initiation  and age less than 1 year at ART initiation  were associated with death. Additionally, patients with TB  and children below the age of 1 at ART initiation  were more likely to be lost to follow-up (LFU).A total of 1142 children with a median age of 3.5 years from 20 selected facilities were followed for 24 months. Of these, 95.8% were assessed for TB at ART initiation and 14.7% had TB. Children on ART were more likely to have TB if they were aged 5 years or older (Children on ART with TB are less likely to survive and more likely to be LFU. These risks, along with low isoniazid uptake and delayed ART initiation, present a serious challenge to achieving the 90–90–90 targets and underscore an urgent need for inclusion of childhood TB/HIV in global plans and reporting mechanisms. Tuberculosis (TB) is common in countries that have a high burden of children living with HIV and high under-five mortality . Yet, TBIn Nigeria, the proportion of notified TB cases with HIV-positive test results was 22% in 2013 . High mo3 or %CD4+≤25 for children aged 24 to 59 months and ≤350 cells/mm3 for children aged 5 years and older  and 618 (54.7%) being males. Approximately 16.4% of those initiating ART were less than one year of age, 44.9% were between one and five years of age, 25.4% were aged between five and ten years and 13.2% were aged 10 years and above. At baseline, 50.1% (507/1002) of children with CD4 results were severely immunocompromised and 12.1% were malnourished. The most-used regimen at ART initiation was AZT/3TC/NVP (75.5%) followed by AZT/3TC/EFV (13.1%). Only 1.1% of patients were initiated with ritonavir-boosted lopinavir-based regimens.p<0.01) and had delayed ART initiation (p<0.05). There was no significant statistical difference in severe immunosuppression between children with and without TB in the final multivariate regression model (p>0.05) despite its independent association with TB following bivariate analysis (The TB status was assessed and determined in 1094 (95.8%) patients. Of these, 161 (14.7%) were diagnosed as having active TB. In multivariate regression adjusting for other factors, children on ART were more likely to be diagnosed with TB if they were five years old or above at ART initiation  followed by AZT/3TC/ABC (19.4%) and AZT/3TC/NVP (18.8%). The cotrimoxazole prophylaxis was provided to 87.9% at ART initiation, including 95.7% (154/161) of those with TB. The median age at cotrimoxazole initiation was 4.7 years (IQR: 1.6–7.8) and 2.8 years (IQR: 1.3–6.4) for those with and without TB, respectively. Only 0.8% (7/933) of non-TB children were reported as receiving INH prophylaxis.At the 24 month follow-up, 7.3% (68/933) of non-TB children developed TB, with 50 (73.5%) of these new TB cases occurring in the first six months. The rate of new TB cases was 3 per 100 person-years (95% CI: 2.2–4.0) at six months which declined to 0.9 (0.5–1.5) and 0.2 (0.06–1.4) per 100 person-years at 12 and 24 months, respectively.p<0.01) and LFU (p<0.05) were higher among children on ART who had been diagnosed with TB compared to non-TB children. The rates of LFU and death at 24 months among TB co-infected children were 12.8 (95% CI: 8.8–18.6) and 8 (5.1–12.8) compared to 8.7 (7.3–10.6) and 2.3 (1.6–3.2) per 100 person-years for non-TB children.The differences in retention, LFU and deaths at the 6, 12 and 24 month follow-up periods among children on ART with and without TB is shown in p=0.001) and at increased risk to being LFU compared to those without TB  that seek to ensure that 90% of children living with HIV know their HIV status, 90% of children infected with HIV are receiving ART and 90% of those on treatment are virally suppressed . From thTB and delayed ART initiation have been previously found to be associated with poorer virological response and increased mortality –42, suppMalnutrition was also identified as a factor associated with mortality. Other studies have established the association between food insecurity and incomplete viral suppression among children on ART , emphasiThe results of this study are limited by several factors. Random selection of facilities was not done in order to ensure that a wide and diverse representation of facilities were included. Data gaps due to incomplete medical records and poor linkage between ART and TB registers were common, though efforts were made to fill in data gaps from alternative sources of data in the facilities, such as clinical charts or laboratory registers. Due to diagnostic challenges, TB diagnosis was likely underestimated in some facilities. Despite these limitations, the findings have been consistent with other reports and have provided preliminary proof of high burden and poorer outcomes for children on ART with TB compared to non-TB children on ART, which may hamper progress towards the 90–90–90 targets.TB among children on ART presents a major challenge to achieving the global targets of 90–90–90 requiring global attention. Despite diagnostic challenges, Nigeria has demonstrated that childhood TB screening is effective in identifying prevalent and incident TB cases at different intervals of HIV treatment. However, uptake of INH prophylaxis continues to remain low. Higher levels of mortality, higher rates of LFU and delayed initiation of ART among children with TB need to be addressed to assist progress towards the 90–90–90 targets. These findings underscore the urgent need for inclusion of TB/HIV co-morbidity among children in global plans and reporting mechanisms.


Gloydius halys caucasicus is venomous, viviparous pit viper of northwest of Iran. In this research, the spermatic reproductive parameters of this taxon were studied. Twenty six male snakes were collected from Takht-e-Soleiman region between September and October, 2010. Findings revealed that male snakes with body length of 45.07 ± 2.83 cm and body weight of 51.50 ± 10.42 g, and right and left gonads volume of 0.12 ± 0.03 mL and 0.06 ± 0.01 mL are mature ones and sperms concentration in first, middle and final regions of vas deferens duct were, 22.30 ± 19.34 ×106 mL-1, 30.34 ± 11.55 ×106 mL-1, and 37.65 ± 16.46×106 mL-1, respectively. The sperms motility at three regions of duct were 60.53%, 62.07%, and 60.00% and percentage of immotile sperms in these regions were 39.46%, 37.92%, and 39.84%, respectively. Percentage of morphologically normal sperms was 69.23 ± 10.57% and abnormal sperms was 30.76 ± 10.57%; including 12.69 ± 5.25% spiral tailed, 7.33 ± 4.37% coiled tailed and 4.16 ± 2.51% folded tailed sperms. Percentage of live sperms in the first, middle and final regions of duct were 55.76 ± 10.77%, 58.84 ± 12.77%, and 57.69 ± 9.91%, respectively and percentage of dead sperm in these regions were 44.23 ± 10.77%, 41.15 ± 12.77%, and 42.30 ± 9.91%, respectively. Results suggested, mature sperms with acceptable reproductive quality could be collected from Gloydius halys caucasicus snake of Iran between September and October.Reproductive parameters evaluation is considered as helpful tool for gene bank formation in ecological and economically important animals species.  It is widely distributed in different parts of Iran1-3 and its habitat is in provinces of Tehran, Gilan, Mazandaran, Golestan, North Khorasan, Razavi Khorasan and Semnan .1,2 Often in temperate zone the mating season of snakes is temporally dissociated from the time of fertilization. Similarly, in males, the mating season is often temporally dissociated from spermatogenesis. In temperate zone pit vipers of North America, estrus, the time when females signal that they are receptive to males, occurs at some time during vitellogenesis. In these pit vipers, vitellogenesis initiates in the late summer or fall. In tropical pit vipers the vitellogenic cycle is continuous (no winter pause) and the mating season occurs at some time during vitellogenesis.4 Snakes display considerable variability in reproductive traits among species5,6 exhibiting a wide range of mating systems and male and male reproductive behaviors.7, 8 Saint-Girons has described four patterns of male reproduction.10, 11 and all of them have estimated this parameter in a subjective manner. Only one study has thoroughly described the basic sperm dynamic parameters, velocity and motility of a snake species. 12 It is widely accepted that the regulation of sperm motility and fertilizing ability depends on the inter action of several factors, among which temperature is of vital importance.13 Still, the effect of temperature on sperm dynamic parameters in ectotherms in genera14,15 and reptiles in particular10, 16 remains poorly understood due to the lack of consistent evidence. In a research done in Brazil it was shown that the female Gloydius halys caucasicus ovulated in the beginning of spring and mated with male snakes. At the time of ovulation and pregnancy, level of progesterone increased and serum level remained high. Pregnancy lasted for four months, until the end of summer. The average number of neonates was 3 to 9.17, 18 Thus spermatogenesis began from the end of spring in Caucasian snake, and its climax was in the summer, and continued until autumn.19 Summer spermatogenesis takes place more often in snake living in temperate and cold regions.Gloydius halys caucasicus), of the Iran was studied. It is among dangerous and poisonous snakes and used for production of polyvalence antitoxin for snake bites. The enzymes existing in its poison are also of the pharmaceutical importance.21, 22The Caucasian pit viper21 More comprehensive scientific information about snakes of Iran could be found in the books: ''Snakes of Iran’’ by Mahmud Latifi,1,2 ''Mar Shenakht'' by Farzanpey24 There is no information about spermatic parameters of snakes in Iran as yet. In our research the reproductive parameters of sperm in Iran's Caucasian snake have been studied for the first time.This research contributes to conservation of special and rare species in the country and helps to identify and make optimal use of this reptile in the laboratory research.25 The sperm samples were collected from September to October .26 The snakes were kept in cages  with a temperature of 20-30 °C, 10/14 hr light/dark and fed every second week. Snake was anesthetized, using subcutaneous injection of 1% lidocaine  around the cloacae. The anesthetic was diluted to a total volume of 1.0 mL in normal saline. The total volume of anesthetic was divided between four injection sites (0.25 mL per site) anterior to the cloacae.11 Snakes were sacrificed by injection of 1.0 mL ethanol  to each optical cavity. The body length of snakes was measured from the tip of their snout to the vent by ordinary measuring tape.27 The testes and vas deferens were removed, then the dimensions of their right and left gonads including lengths, widths and heights were measured by caliper and volumes were calculated by Cha formula.28 The vas deferens was divided into three parts of first, middle, and final. Each part was separately placed in Petri dishes, and cut into pieces by sterilized blade and scissors, and immediately kept in vials in 1mL PBS for 45 minutes. During this period the gonads were weighted . Sperm motility, morphology, vitality, and concentration were studied. The sperm cells were counted using a hemocytometer under 400× and concentration of sperm was calculated according to Rashidi et al.29 In order to study the sperm motility, four grades were considered: A (quick progressive in straight paths), B (slow progressive in straight or not straight paths), C (motile in place), D (immotile).30-32 Morphology was evaluated by observing 100 sperm cells under microscope at 1000×, and finally the survival period of sperm cells was measured in laboratory temperature of 23 ± 2 °C. Twenty six Caucasian male snakes were collected from Takht-e-Soleiman . All procedures were carried out in accordance with ISIRI 7216-2 animal ethical guidelines.t-test for comparison of two values at P < 0.0001 and one way ANOVA where three values were compared at P < 0.05.Statistical analyses were performed by Student P < 0.0001 and P = 0.001, respectively. Average weight of the animals, body length, length of tail, details of testes  the length of right and left reproductive ducts were measured and have been shown in P < 0.0001. Sperms concentration and vitality in first, middle and final parts of vas deferens, motility, morphology of sperms have been presented in P > 0.05), percentages of live and dead sperms were not statistically different in three parts of duct at P > 0.05, percentages of motile & immotile sperms (P > 0.05).Length of left vas deferens duct was longer than right one at e sperms  differenGloydius halys caucasicus which is an important step in the development of assisted reproduction in snakes. The concentration of sperm obtained in our experiment were within ranges of results obtained for checkered garter and Crotalus durissus terrificus snakes using similar methods.33-35 Morphologically, 75.70% normal sperm cells reported in corn snakes' ejaculates,10 was different from our results (69.23%). The percentage of normal morphology in corn snakes is comparable to that of the ram, bull, boar, stallion, and buck.36 In mammals, a small range of morphologic abnormalities are considered normal in a healthy animal. Among the commonly measured semen parameters, morphologic abnormalities have the greatest negative correlation to fertility of farm animals, and heat stress is one of the main causes of sperm abnormalities in these animals. The effect of heat stress on reptile spermatozoa has not yet been assessed. Reptiles are ectotherms and depend on their environmental temperature to regulate their core temperature. It would not be unexpected for a Gloydius halys caucasicus snake to experience a 2.7-5.5 °C difference in body temperature in a given day. Although it has not been evaluated, one might expect that semen production would diminish and sperm abnormalities would increase in snakes that experience a large drop in body temperature. Genetics and exogenous drugs have also been associated with sperm cell abnormalities in higher vertebrates. This study represents basic values of the reproductive potential of Iranian 31 criteria and up to 86% according to strict criteria).35 Sperm morphology gives information for the function of the reproductive tract and is a predictor of animal's fertility potential. Generally it seems that Caucasian snakes in Iran with SVL 45.07 ± 2.83 cm and testis volume of right 0.12 ± 0.03 mL and left, 0.06 ± 0.01 mL, is mature,36 like other snakes in cold and mild regions, have got summer spermatogenesis. 19In normal fertile individuals up to 50% of sperm cells can have morphological defects (up to 70% according to WHOThe findings represent the qualitative and quantitative values of sperms of Iran's Caucasian snake from late September to early October, however in order to obtain higher quality sperms, more work is needed to be carried out.


Drosophila melanogaster. For this purpose, six graded concentrations ranging from 1 to 6 μg/ml were selected, which are below the reported residual values (up to 14 μg/ml) of the chemical. 1st instar larvae were fed with these concentrations up to the 3rd instar stage and accordingly hemolymph smears from these larvae were prepared for differential hemocyte count. Three types of cells are found in Drosophila hemolymph, namely, plasmatocytes, lamellocytes and crystal cells. Plasmatocyte count was found to decrease with successive increase in treatment concentrations. Crystal cells showed an increasing trend in their number. Though the number of lamellocytes was very low, a bimodal response was noticed. Lamellocyte number was found to increase with the initial three concentrations, followed by a dose dependent reduction in their number. As hemocytes are directly linked to the immune system of fruit flies, fluctuations in normal titer of these cells may affect insect immunity. Hemocytes share homologies in their origin and mode of action with the immune cells of higher organisms including man. Thus the present findings suggest that immune cells of humans and other organisms may be affected adversely under chronic exposure to Acephate.Acephate, an organophosphate (OP) pesticide, was used to investigate the effects of its chronic exposure on hemocyte abundance in a non-target dipteran insect  Each and every category has its own specific way through which it acts on target pests. Organophosphates and carbamates work by blocking the activity of acetyl cholinesterase in the synaptic cleft  foliar pesticide is widely utilized for lepidopteran insects in agricultural fields of tomatoes, lettuce, potatoes and carrots. Like other organophosphates, it blocks mainly the activity of acetylcholine esterase in synaptic clefts. But this chemical has also been reported to shorten the developmental duration in a dipteran insect An organophosphate insecticide Acephate  was used for the present study.Drosophila melanogaster were used as experimental organism to assess the toxicological impact of Acephate on hemocyte abundance. Larvae were reared in Standard Drosophila medium (SDM) following the established laboratory rearing techniques  for 10–15 min. After fixation, the smear was stained with Giemsa  for 20–30 min. Stained slides were washed in distilled water and examined under compound microscope at 400X magnification.3rd instar larvae) per treatment were prepared and examined. Slides for control group were also counted for comparison purpose.Triplicate slides  inside them. They usually take dark Giemsa stain. Crystal cells are involved in melanin synthesis.These are rare but large, flat and oval to elongated cells with prominent nuclei. They are non-phagocytic and their cytoplasmic constituents are relatively sparse. They encapsulate large pathogens which cannot be phagocytosed by plasmatocytes.p<0.05 was considered statistically significant.All data collected were considered and subsequently mean and standard error of means were calculated using MS Excel 2007. Student’s t test was carried out for analysis of the data to examine the significance in variations in each treatment category compared to their control counterpart. Changes in plasmatocyte count were found to represent a decreasing trend. Untreated larvae showed 91.27±2.13% of plasmatocytes in hemolymph smear followed by 83.35±1.54%, 82.68±3.07% and 77.54±1.81% for treatment concentrations of 1, 2 and 3 μg/ml, respectively.With further increase in treatment concentrations to 4, 5 and 6 μg/ml, further reduction in the count of these cells was noted representing 75.29±0.79%, 70.43±0.51 and 66.67±1.44% of total hemocytes .In contrast to plasmatocyte count, crystal cells exhibited an increasing trend in relative abundance with successive increase in treatment concentrations. Control sets revealed about 7.18±2.09% crystal cells. With graded chemical concentrations of 1, 2 and 3 μg/ml, a respective increase in crystal cell count of 15.75±1.63%, 17.25±3.34% and 17.16±0.79% was recorded. Larvae reared in 4, 5 and 6 μg/ml concentrations constituted about 20.87±0.57%, 25.84±0.67% and 32.11±1.58% of total hemocytes .Though lamellocyte numbers were very few in hemolymph smears, fluctuations in their relative abundance were observed. Lamellocytes in the control set constituted about 0.73±0.28% of total hemocytes. Chronic exposure for several days to three initial concentrations  of Acephate resulted in increased respective lamellocyte count of 0.86±0.23%, 1.15±0.21 and 5.29±1.17%. Interestingly, with further increase in treatment concentrations to 4, 5 and 6 μg/ml, the relative abundance of these cells was found to decrease to 3.83±0.71%, 3.71±0.52% and 1.22±0.14%, respectively .rd instar larvae of D. melanogaster. Plasmatocyte abundance was found to decrease under chemical stress. Comparable results were reported by Qamar and Jamal (Dysdercus cingulatus. Other organophosphates like methylparathion and monocrotophos were also noticed to decrease plasmatocyte abundance in Rhynocoris kumarii (George & Ambrose, et al., et al., The present work revealed the effect of chronic exposure to Acephate on relative abundance of hemocytes in the hemolymph of 3nd Jamal  with theCulex pipiens was found to possess higher serine protease activity than non resistant strains (Gong et al., et al., Crystal cell number showed an increasing trend in abundance with successive increase in treatment concentrations. Melanization has been considered as an important tool to minimize damage from physical, chemical and pathogenic stresses (Hamilton & Gomez, et al. (A similar kind of response for plasmatocytes and crystal cells was previously reported by Rajak et al.  where thet al., Lamellocyte number is known to increase under pathogenic stress (Evans rd instar larvae of the fruit fly. The findings indicate the involvement of apoptotic induction, mitotic failure and the effect on the hematopoietic mechanism of this insect. The hematopoietic system of Drosophila shares a high degree of homology with that of human beings, hence the study points toward the undesired alarming outcome of irrational use of such chemicals in the environment.The present study explored that, whether Acephate at selected concentrations interferes with normal hemocyte number of 3


To evaluate estrus induction response and fertility including plasma progesterone and biochemical profile following use of three standard hormonal protocols in anestrus crossbred cows.The study was carried out on 40 true anestrus and 10 normal cyclic cows. 10 anestrus cows each were treated with standard intravaginal controlled internal drug release (CIDR) device, Ovsynch (GPG) protocol, and Norgestomet ear implant with fixed-time artificial insemination (FTAI). 10 anestrus cows were kept as untreated control while 10 cows exhibiting the first estrus within 90 days postpartum without any treatment served as normal cyclic control. Blood samples were obtained from treated cows on day 0, 7, 9 (AI) of treatment and day 21 post-AI, and from control groups on the day of AI and day 21 post-AI for estimation of plasma progesterone, protein, cholesterol, calcium, and inorganic phosphorus profile.4) concentrations in anestrus cows studied on day 0 (initiation), 7 , 9 (FTAI) of treatment and on day 21 post-AI revealed that the values on day 7 and 21 were significantly (p<0.01) higher than other two periods in all three groups. The concentrations were significantly (p<0.05) higher in conceived than non-conceived group on day 21 post-AI in CIDR (4.36±0.12 vs. 1.65±0.82 ng/ml) and Ovsynch (4.85±0.62 vs. 1.59±0.34 ng/ml), but not in Norgestomet ear implant (4.50±0.53 vs. 3.02±1.15 ng/ml) or normal cyclic group (5.39±0.67 vs. 3.13±0.37 ng/ml). The cholesterol and protein levels were significantly higher, but not the calcium and phosphorus, in normal cyclic control than in anestrus groups. The influence of treatment days and pregnancy status was not significant for any of the biochemical constituents in any of the groups.The use of CIDR, Ovsynch, and Norgestomet ear implant protocols resulted in 100% estrus induction with conception rates at induced estrus of 60%, 50%, and 50%, and the overall of three cycles as 80%, 80%, and 70%. In untreated anestrus control (n=10), only three cows exhibited spontaneous estrus within 90 days of follow-up and conceived giving the first service and overall conception rates of 66.66% and 30.00%, respectively. In normal cyclic control (n=10), the conception rates at first and overall of three cycles were 50% and 80%. The overall mean plasma progesterone (POvsynch and/or CIDR synchronization protocol can be effectively used to improve fertility up to 80% in anestrus cows, as compared to 30% in anestrus control, combined with plasma progesterone to delineate the reproductive status before and after treatment. The optimum reproductive efficiency of these animals is equally important for economic productivity. Infertility is one of the pathological conditions qualified as disease of production. It is widespread in modern farming, especially in crossbred animals. Infertility negatively affects productivity and return on investment of the farmers. Anestrus forms the major condition constituting about 2/3d cattle . Variousd cattle . Hormonad cattle . The vard cattle ,2,6.The progesterone hormone is responsible for stimulation of cyclicity, follicular development and also for maintenance of pregnancy. The plasma protein, cholesterol and minerals profile denote the nutritional status of animals and are related with their fertility ,7. The cThe comparative studies involving the use of different estrus induction/synchronization protocols at a time under an identical environment in crossbred cows are, however, meager and are mostly based on clinical response only without plasma progesterone or biochemical evaluation -9. HenceThe prior approval from the Institutional Animal Ethics Committee was obtained for use of farmers animals in this study.viz., CIDR, Ovsynch and Norgestomet ear implant, n=10 each) with fix-timed artificial insemination (FTAI) [This study was carried out during November, 2013 to March, 2014 under middle Gujarat agro-climatic condition. 40 postpartum (>90 days) anestrus crossbred cows and 10 normal cyclic cows of the average body condition score were selected from villages of Amul and Panchamrut milk-shed areas of Gujarat. The cows were screened gynaeco-clinically for their reproductive status. Detailed history and rectal palpation findings were recorded. Anestrus cows were confirmed by palpating small smooth inactive ovaries per rectum twice 10 days apart. All the selected cows were dewormed using ivermectin, 100 mg S/C and were supplied with multi-mineral boluses one bolus daily for 7 days. They were randomly subjected to following three estrus induction/synchronization protocols (n (FTAI) ,7,10.2α) 25 mg . Injection GnRH 10 µg  was administered I/M on day 9 and FTAIs were performed twice on day 9 and 10, as shown in In 10 true anestrus crossbred cows, CIDR  was inserted intravaginally on day 0. It was removed on day 7 together with I/M injection of prostaglandin F2α  was inserted S/C in the outer face of the ear-base together with 2 ml Crestar injection I/M  on day 0. The implant was removed on day 7 together with I/M injection of 25 mg PGF h later . Signs oAnother 10 anestrus cows were kept as anestrus control without hormone therapy and 10 normal cyclic cows that expressed spontaneous estrus within 90 days postpartum and inseminated served as normal cyclic control group. Cows in spontaneous or induced estrus were inseminated using good quality frozen-thawed semen. Animals detected in estrus subsequent to FTAI were re-inseminated up to two cycles and in non-return cases pregnancy was confirmed rectum 60 days of last AI.2α administration, on day 9 - induced estrus/FTAI  and on day 21 post-AI. Blood sampling for control groups was done on the day of spontaneous estrus if any, and on day 21 post-AI. The samples were centrifuged at 3000 rpm for 15 min and plasma separated out was stored deep frozen at −20°C with a drop of merthiolate (0.1%) until analyzed.All the hormonally treated/untreated true anestrus and normal cyclic cows were studied for their reproductive status and plasma progesterone and biochemical profile. For this, jugular blood samples were collected in heparinized vacutainers 4 times from true anestrus animals, i.e., on day 0 - just before treatment (on diagnosis), on day 7 - at the time of PGFet al. [125), antibody coated tubes and standards were procured from Immunotech, France. The sensitivity of the assay was 0.1 ng/ml. The intra-and interassay coefficients of variation were 5.4% and 9.1%, respectively. The concentrations of plasma total protein, total cholesterol, calcium and inorganic phosphorus were determined by standard procedures and assay kits procured from Analytical Technologies Pvt. Limited, Baroda, on chemistry analyzer.Plasma progesterone profile was estimated using the standard radio-immuno-assay (RIA) technique of Kubasic et al. . LabeledThe data on estrus response, conception rate (by Chi-square test) and plasma profiles of progesterone and biochemical constituents were analyzed statistically through analysis of variance  using on2α injection. The conception rates obtained at induced estrus in cows under these three protocols were 60.00%, 50.00%, and 50.00%, respectively, with corresponding overall pregnancy rates of three cycles as 80.00%, 80.00%, and 70.00%. Results were better with CIDR and Ovsynch protocols as compared to Norgestomet ear implant . In the normal cyclic control group (n=10), the conception rates at the first cycle and overall of three cycles were 50.00% and 80.00%, respectively, with mean service period of 98.77±6.84 days (All the cows (100%) under CIDR, Ovsynch, and Norgestomet ear implant protocols exhibited induced estrus with varying intensity similar to normal cyclic control group within 42-72 h from the time of PGF implant . In untr.84 days .et al. [The mean estrus induction intervals of 58.27±1.93, 64.13±1.33, and 54.00±1.17 h observed in cows under CIDR, Ovsynch and Norgestomet ear implant protocols compared favorably with the previous reports in anestrus cows  and buffet al. .et al. [et al. [et al. [et al. [et al. [The first service conception rate of 50.00% with Norgestomet ear implant in the present study is comparable with the earlier result of Nak et al.  as 41.40 [et al.  as 50.00 [et al.  as 66.66 [et al.  reported [et al.  with Nor [et al. , and 36. [et al. , respectet al. [et al. [et al. [et al. [et al. [et al. [With Ovsynch and Norgestomet ear implant, Nak et al.  reported [et al.  also rep [et al.  reported [et al.  reported [et al. , which m [et al. . Althoug [et al.  in Synch [et al.  in anestFurther, around 30% conception rates were obtained at the second and third cycle in anestrus cows induced to cycle, which is near to normal cycling cows (40%and 33%). This proved that all the protocols induced and synchronized the estrus and then established normal cyclicity in treated animals, resulting into conceptions in subsequent cycles like normal breeding cows. These observations further supported the previous observations on the use of similar protocols in anestrus cows and buffaloes by many workers ,10,15,222α injection in each protocol and made them pregnant within a period of 10-12 days (95-100 days postpartum) in comparison to 149.52±5.67 days of service period recorded in untreated control group, indicating a huge curtailment (around 1.5-2.0 months) in the waiting period of anestrus animals to evince estrus and become pregnant. The pooled conception rates of three treatment protocols obtained (76.66%) in anestrus cows indicated the positive contributory role of handling the problem of acyclicity in cows, nearly at par with normal cyclic control cows (80.00%).Thus, estrus could be induced in true anestrus cows within 2-3 days from the day of PGF2α was injected. Thereafter, the mean progesterone levels dropped suddenly and significantly within 48 h of PGF2α injection and/or implant removal to the basal values coincident to induced estrus when FTAIs were done. These levels again increased significantly (p<0.01) on day 21 post-AI in all the groups  due to estruses being ovulatory with development and maintenance of corpus luteum (CL) and the establishment of pregnancy in some animals. In normal cyclic control group also the mean plasma progesterone concentration was the lowest (0.43±0.17 ng/ml) on the day of spontaneous estrus/AI, which rose significantly (p<0.05) on day 21 post-AI (4.98±0.45 ng/ml) due to the establishment of pregnancy in four cows in that cycle.The mean levels of plasma progesterone recorded on day 0, 7, 9 (AI) of treatment and on day 21 post-AI in anestrus cows under CIDR, Ovsynch, and Norgestomet ear implant protocols, and on the day of AI and day 21 post-AI in the normal control group are presented in 4 on the day of beginning of treatment protocol helped delineate the reproductive and endocrine status of the animals and thereby predicting the possible response to the therapy. The higher plasma P4 recorded on day 21 post-AI in conceived cows of all the groups was due to establishment of pregnancy and maintenance of CL function, while significantly low yet variable plasma P4 noted on day 21 post-AI in non-conceived cows could be due to their return to next estrus at varying intervals on account of probable irregular or long cycle length, early embryonic mortality after day 17 or uncoordinated, unexplained hormonal changes in some of them. These findings corroborated with the observation of Nakrani et al. [et al.[The mean plasma progesterone concentrations in conceived and non-conceived groups in all three treatment protocols and in normal cyclic control group were found to be statistically similar on day 0, 7 and even on day 9 (AI), but on day 21 post-AI, the conceived cows had significantly higher mean plasma progesterone concentrations as compared to non-conceived ones only in CIDR (4.36±0.12 vs. 1.65±0.82 ng/ml) and Ovsynch (4.85±0.62 vs. 1.59±0.34 ng/ml) protocols . These f. [et al. using No4 profile on day 7 of treatments in the present study with CIDR and Ovsynch protocols (4.97±1.68 and 3.75±0.47 ng/ml) over initial (0 day) values, with sudden drop to almost basal values on induced estrus within 48-60 h after PG injection (The mean plasma progesterone levels obtained on the day of initiation of CIDR and Ovsynch treatments in the present study corroborated with the earlier findings in zebu and crossbred cows ,9,10,25.njection , has alsnd day post-AI than that of pre-treatment level in GnRH treated anestrus buffaloes have been documented [et al. [et al. [et al. [et al. [et al. [et al. [The results of biochemical analysis did not reveal significant variations in plasma total cholesterol, total protein, calcium, and inorganic phosphorus profiles between days and periods of the treatment in any of the groups or between conceived and non-conceived cows. Although the cholesterol concentration was non-significantly higher, and protein was lower in conceived as compared to non-conceived cows. However, the pooled values of cholesterol and protein were significantly higher in normal cyclic cows than in anestrus cows of CIDR and Ovsynch groups . Similarcumented , and thecumented . The higcumented ,29,30. P [et al.  in cross [et al.  in Gir c [et al.  and Sava [et al.  in buffa [et al.  opined t [et al.  also opiet al. [et al. [A non-significant influence of CIDR, Ovsynch and/or Norgestomet ear implant protocols in anestrus cows and buffaloes on calcium and phosphorus levels  as obseret al.  obtainedet al. . Savalia [et al.  did not  [et al. ,34,35.From the results, it can be inferred that the hormonal protocols used, particularly Ovsynch and CIDR protocol, improved conception rates in anestrus crossbred cows under field conditions, and also influenced the plasma progesterone profile significantly, but not the biochemical profile, in a manner of normal cyclic animals, hence can be used by the practicing veterinarians in anestrus rural crossbred cows to improve their reproductive efficiency and thereby the farmers economy.AJD planned and designed the study. The experiment was conducted by AJD, BBN, KKH and JAP, while laboratory work was done by BBN, KKH and RGS. All authors participated in data analysis, preparation of draft of the manuscript, and read and approved the same.


Objectives: This paper reports on the findings of a population-based study to evaluate the relationship between atmospheric fine particulate matter (PM2.5) levels and hospital admissions for chronic obstructive pulmonary disease (COPD) in southwestern Taiwan over a three-year period, 2008–2010. Methods: Data on hospital admissions for COPD and PM2.5 levels were obtained from the National Health Insurance Research database (NHIRD) and the Environmental Protection Administration from 2008 to 2010, respectively. The lag structure of relative risks (RRs) of hospital admissions for COPD was estimated using a Poisson regression model. Results: During the study period, the overall average hospitalization rate of COPD and mean 24-h average level of PM2.5 was 0.18% and 39.37 μg/m3, respectively. There were seasonal variations in PM2.5 concentrations in southwestern Taiwan, with higher PM2.5 concentrations in both spring (average: 48.54 μg/m3) and winter (49.96 μg/m3) than in summer (25.89 μg/m3) and autumn (33.37 μg/m3). Increased COPD admissions were significantly associated with PM2.5 in both spring (February–April) and winter (October–January), with the relative risks (RRs) for every 10 μg/m3 increase in PM2.5 being 1.25 (95% CI = 1.22–1.27) and 1.24 (95% CI = 1.23–1.26), respectively, at a lag zero days . Lag effects on COPD admissions were observed for PM2.5, with the elevated RRs beginning at lag zero days and larger RRs estimates tending to occur at longer lags . Conclusions: In general, findings reveal an association between atmospheric fine particulate matter (PM2.5) and hospital admissions for COPD in southwestern Taiwan, especially during both spring and winter seasons. Chronic obstructive pulmonary disease (COPD) has been reported to be a leading cause of morbidity and mortality and will become the third leading cause of death by 2030 worldwide . COPD re2.5, defined as particulate matter with an aerodynamic diameter less than 2.5 μm) present more potential threats to human health, because they can easily penetrate into the small airways and the alveoli of the lung [2.5 has been associated with increased hospital admissions for multiple causes, including for all respiratory causes [2.5 is associated with increased respiratory disease hospitalizations and mortality [2.5 on COPD alone in Asia are limited. On the other hand, so far, studies on the lag effects and seasonal variability of PM2.5 on COPD are rare, and conflicting results have been reported in different countries and regions. Ko et al. [2.5 may increase the risk of inpatient admissions for COPD, and the relationship between particulate matter (PM) and respiratory disease-related hospital admissions differed by season [et al. [2.5 on COPD could be explained by variation in exposure patterns and seasonal differences in the air pollution mixture [Epidemiological studies have reported that air pollution has been associated with excessive respiratory ,11 and cthe lung . Recently causes ,13,14,15y causes ,12,16,17y causes  in Northortality ,20. PM cortality . Howevero et al.  pointed y season . Chen et [et al.  found th [et al. ,23. The  mixture ,22,23.2.5 levels over the southwestern Taiwan. On the other hand, the Central Mountain Range is aligned north-south in Taiwan, and this can influence the geographical and meteorological conditionals in many locations throughout Taiwan, and might further affect the differences of air pollution in the recirculation and accumulation of air pollution over Taiwan. Yang and Kao [2.5 annual mean concentrations ranged between 15.1 μg/m3 and 46.0 μg/m3 in Taiwan, and there are higher PM2.5 concentrations in southern Taiwan . The concentrations of PM2.5 in southwestern Taiwan were high in 2008–2010 compared to other administrative districts in Taiwan [2.5 in southwestern Taiwan in 2014 (ranging between 29.6 and 34.7 μg/m3) are higher than those in other areas in Taiwan  [2.5 on COPD have rarely been investigated in southwestern Taiwan. The objective of this study was to assess the impact of atmospheric PM2.5 levels on hospital admissions for COPD among residents in southwestern Taiwan, over a three-year period, 2008–2010, based on a population-based study.Taiwan is located under the lee side of the East-Asian winter and summer monsoons, sea-land breezes (SLBs) and the Northeastern Monsoon (NEW), which play important roles in the recirculation and accumulation fine particulate matter over southern Taiwan ,25. In s and Kao  indicaten Taiwan . Accordictively) . HoweverThis study has been approved by the Institutional Review Board (IRB) of Chang Gung Medical Foundation (IRB No. 101-3077C). A population-based study was conducted using a subset of medical claims data from the National Health Insurance Research Database (NHIRD) obtained from the National Health Insurance (NHI) program in Taiwan (Registered Number: NHIR-NHIRD 101-533). The NHI program, which provides compulsory universal health insurance in Taiwan, has operated since 1995 and had an initial coverage rate of 90% of the Taiwanese population. At the end of 2008, over 99% of the Taiwanese population were enrolled in the NHI program . The NatCases of COPD were ascertained by the service claim for patients who had at least one inpatient visit with a primary diagnosis of COPD  Code 490, 491, 492 and 496). Hospitalization rates for men and women  population aged ≥40 years were estimated and adjusted by annual age-specific population. Daily counts of hospital admissions for COPD were extracted from the medical insurance file (NHIRD) for the period of 2008–2010. The analyses covered 4 municipalities , which are located in southwestern Taiwan. The inpatient visits were further analyzed by four seasons  .2.5 concentrations data during 2008–2010 were obtained from the Environmental Protection Administration’s (EPA) Taiwan Air Quality Monitoring Network (TAQMN) [2.5 was measured by seven stations only. The Chiayi station , located in Chiayi City, is an urban site. The Xingan  and Puzi stations , located in Chiayi County, are rural sites. The Annan  and Tainan stations , located in Tainan City, are urban sites. The Xinying  and Shanhua stations , located in Tainan County, are rural sites. The 24-h average levels of PM2.5 were calculated from hourly PM2.5 concentrations and for further analysis.For each day, hourly PM (TAQMN) . Accordi2.5 using the relative risk (RR) and their 95% confidence intervals (95% CI). We examined lag effects of different days, including same-day lag (Lag 0); and cumulative lags by 2 (average of Lags 0 and 1), 3  to 6 days (average of Lags 0–5) [p < 0.05. Statistical analysis was performed using statistical software .Hospitalization rates from 2008 to 2010 for the population were estimated and adjusted by annual age-specific population. Additionally, Poisson regression was performed to determine the association between COPD inpatient visits and the levels of PMags 0–5) . The cri2.5. During the study period, the daily average concentration for PM2.5 was 39.37 μg/m3. There was a mean of 8.73 daily hospital admissions for COPD in southwestern Taiwan over the study period.There were 9568 admissions for chronic obstructive pulmonary disease from January 2008 to December 2010 with an average annual hospitalization rate of 0.18% for the population aged ≥40 years in southwestern Taiwan, 0.26% for male and 0.10% for female. There were more male than female hospitalizations, with male-to-female ratios ranging from 2.00 to 3.50 by age . Table 22.5 and the relative risks (RRs) estimates of PM2.5 on hospital admissions for COPD. There were obvious seasonal variations in PM2.5 in southwestern Taiwan, with higher PM2.5 concentrations in both spring (average daily mean concentrations: 48.54 μg/m3) and winter (49.96 μg/m3) than in summer (25.89 μg/m3) and autumn (33.37 μg/m3). In addition, COPD hospital admissions showed elevated relative risks (RRs) beginning at Lag 0  and the highest RRs at Lags 0–5  in both spring and winter, with the RRs for every 10 μg/m3 increase in PM2.5 being 1.51 (95% CI = 1.46–1.55) and 1.54 (95% CI = 1.50–1.58), respectively. It seems, then, that COPD admissions have stronger associations with PM2.5 concentrations after a delay of a few days.2.5) on hospital admissions for COPD in Southwestern Taiwan. The results showed that PM2.5 had a positive association with hospital admissions for COPD in both spring and winter seasons, with different magnitudes in terms of the relative risks and the number of lag days. In addition, the results indicated that PM2.5-induced effects varied by season. The observed seasonal differences on PM2.5 concentrations and effect estimates might be explained by variations in meteorological parameters, the chemical composition of PM2.5 and exposure patterns [2.5 levels and chemical constituents in the current study.The present study investigated the effects of atmospheric fine particles  and humid, and most people spend more time indoors. This might explain the reduced effects of PM2.5 exposure in patients with COPD in summer in southwestern Taiwan. Nevertheless, the seasonal pattern of PM effects on respiratory diseases requires further study.In this study, increased risks of COPD admissions associated with ambient PMaohsiung . Our finaohsiung . However [et al.  assessed2.5 on COPD admissions, which was not addressed in the previous studies in Taiwan [2.5 levels and COPD hospital admissions for no lag days (Lag 0) up to five days prior to hospitalization, with the most statistically-significant effect being at Lags 0–5. The lag pattern of PM2.5 on COPD admissions in the current study was similar to that reported in the Hong Kong study [et al. [2.5 constituents and hospital admissions in Denver, Colorado, and also found that larger effect estimates for respiratory hospital admissions tended to occur at longer lags. The lag effect of PM2.5 on respiratory hospitalizations may vary by PM2.5 constituents. PM2.5 chemical constituents are varied by pollution source and season. Particulate matter with elemental and organic carbon showed more obvious immediate effects than did sulfate and nitrate [Furthermore, this study has provided additional information on the lag effect of PMn Taiwan ,23. The ng study . Kim et  [et al.  explored nitrate . Furtheret al. (2015) indicated that short-term exposure to the 10 μg/m3 increment of daily ambient PM2.5 is associated with a 3.1% (1.6%–4.6%) and a 2.5% (1.5%–3.5%) increase in COPD hospitalizations and COPD mortality, respectively [et al. reported that an increase of 10 μg/m3 PM2.5 results in an elevation of 0.69% and 1.32% with a lag of three days for respiratory mortality and morbidity, respectively [et al. (2015) conducted a meta-analysis in the Chinese population and reported that a 10 μg/m3 increase in PM2.5 was associated with a 0.75% (95% CI: 01.39%–1.11%) increase in mortality due to respiratory disease [et al. conducted a study in Hong Kong and reported that the relative risk for COPD admissions for every 10 μg/m3 increase in PM2.5 was 1.031 (with a cumulative lag of 0–5) [2.5. The relative risks for COPD admissions for every 10 μg/m3 increase in PM2.5 at lag zero days were 1.25 (in spring) and 1.24 (in winter), respectively. One potential explanation for this discrepancy might be that most published studies lack effect estimates stratified by season. Kim et al. (2012) found that chemical constituents of particulate matter air pollution have seasonal variations in Denver, USA [et al. (2006) investigated the organic components in PM2.5 in the urban atmosphere in Beijing and found that different organic compounds presented apparently different seasonal characteristics [Li et al. 15 indicaectively . Lu et a disease . Ko et aver, USA . He et aeristics . Seasona2.5 in this current study were 39.37 and 37.16 μg/m3, respectively, compared to the levels of 29.99 and 27.06 μg/m3 in Taipei  in 2006–2010 [2.5 in this current study were also higher than those in Hong Kong (mean and median levels of 35.7 and 31.7 μg/m3) in 2000–2004 [2.5 on COPD and their seasonal variations in southwestern Taiwan have never been reported up to the present. In summary, findings from the present study can be helpful to illustrate the health effects of PM2.5 on respiratory diseases in Taiwan and to alert the government to pay closer attention to managing air pollution.The mean and median levels of PM006–2010 . The mea000–2004 . Further000–2004 . Some st000–2004 ,23. Howe2.5 data from ambient air monitoring sites to represent individual exposure to fine particulate matter. Previous studies have suggested that exposure misclassification from using stationary PM data might lead to diminishing the accuracy of exposure-response estimates compared to personal exposures, thus potentially weakening associations with stationary PM [Exposure measurement error is an inherent limitation of epidemiology studies of disease and the environment . In the onary PM ,23,38.2.5 effects on COPD. Second, other personal risk factors for COPD hospital admissions, including smoking habit, socioeconomic status and the detailed information, such as the data of pulmonary function tests, were not available from the NHIRD dataset in the current study. Third, personal PM2.5 exposure pattern and levels may affect personal exposure and were not taken into account in this study.Several potential limitations should be taken into consideration. Fist, this study analyzed medical claims data during 2008–2010, and it is too relatively short term to observe the temporal trend for PM2.5) has an association and lag effect on hospital admissions for COPD in Southwestern Taiwan, especially during both spring and winter seasons. Seasonal variation of the PM2.5 exposure patterns and levels may play an important role to induce the variations of effect estimations on COPD. Further long-term and large-scale studies are required to analyze the temporal trends on the effects of PM2.5 for respiratory diseases in Taiwan.In conclusion, atmospheric fine particulate matter (PM


The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening.The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein.Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+  as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology).HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18 genotyping could represent a more accurate methodology for primary cervical cancer screening in comparison to liquid-based cytology, especially in older women. Low coverage and poor patient compliance, subjective interpretation of results, in conjunction with factors concerning public health and legal issues, are some of the possible reasons for the substandard results that have been noted in cytology-based screening  was significantly higher than the specificity of HPV testing [90.3% (95% CI 89.3%–91.2%)] (P<0.05). The specificity difference (ASCUS+ cytology—14HR HPV testing) was 0.07 (95% CI: 0.06–0.08). Testing for types 16&18 only had similar diagnostic accuracy indices to cytology. Positivity for the combination of the two tests (co-testing), that is positivity for either ASCUS+ or 14HR HPV testing, resulted in reduced specificity , which was higher than the sensitivity of cytology [ASCUS+: 53.7% (95% CI 37.4%–69.3%)] to a statistically significant degree (cificity .P<0.05). The specificity difference (ASCUS+ cytology—14HR HPV testing) was 0.07 (95% CI: 0.06–0.08). Testing for type 16 only had similar diagnostic accuracy indices to cytology. Testing for types 16&18 only had at least equal accuracy compared to cytology for CIN3+ but not in a statistically significant degree. The combination of the two tests (co-testing) resulted again in reduced specificity (At the threshold of CIN3+ HPV testing had a sensitivity of 100% (95% CI 76.8%–100.0%), which was higher than the sensitivity of cytology [ASCUS+: 64.3% (95% CI 35.1%–87.2%)] but the difference did not reach statistical significance. The sensitivity difference (ASCUS+ cytology—14HR HPV testing) was -0.36 (95% CI: -0.61–-0.07). In contrast, the specificity of cytology  was significantly higher than the specificity of HPV testing [89.7% (95% CI 88.7%–90.6%)] (cificity .P<0.0001).With the exception of 14HR HPV testing, which has a sensitivity of 100% in both cases, changing the threshold from CIN2+ to CIN3+ has led to an increase in sensitivity and NPV for all tests examined. The largest increase in sensitivity, from 58.5% (95% CI 42.1%–73.7%) to 78.6% (95% CI 49.2%–95.3%), was noted for HPV 16 or 18 positivity. PLR was slightly increased if the threshold was CIN3+ for all tests apart from positivity for the 14HR HPV types, and NLR as well as PPV markedly decreased. Comparison of the diagnostic accuracy of the cobas HPV test to cytology with the McNemar’s test revealed a statistically significant difference , but also in 86% more cases of CIN2+ detected (22 cases with ASCUS+ vs 41 cases with ASCUS+ and/or 14HR HPV positive). The number of colposcopies needed to detect a CIN2+ after a positive cytology or positive 14HR HPV testing or both (co-testing) was 11.1 [41 CIN2+ detected after performance of 454 colposcopies (454/41)], whereas the respective number for 14HR HPV testing alone was 10.0 (409/41) and for cytology (ASCUS+) alone was 6.4 (141/22).HPV infection in general, and especially asymptomatic or transient HPV infections are more common in younger age groups compared to older, whereas the opposite is the case for the prevalence of high-grade cervical lesions (CIN2 or worse) ,40. TherFinally, By combining genotyping with cytology, testing positive for HPV16 or ASCUS+ cytology, as well as testing positive for HPV16 or HPV18 or ASCUS+ cytology had a markedly higher sensitivity of 75.6% (95% CI 65.4%–88.8%) and 92.6% (95% CI 91.8%–93.5%) respectively than ASCUS+ alone, the former being marginally significant and the latter being clearly significant and the highest among all test combinations. By replacing ASCUS+ by LSIL+ or HSIL+, combined with HPV16 testing alone or HPV 16 or HPV 18 or both, a gradual decrease in sensitivity was noted, although all rates were not statistically significantly different than ASCUS or worse cytology alone. Testing positive for HPV 16 or HPV 18 or both, and additionally ASCUS+ cytology would significantly increase PPV and PLR for the histological identification of CIN2+, in comparison to ASCUS+ alone [32.4% (95% CI 17.3%–47.5%) and 46.4 (95% CI 25.1–85.9) vs 10.4% (95% CI 6.3%–14.6%) and 11.2 (95% CI 8.2–15.4) respectively]. Similar significant increase in PPV and PLR in HPV 16/18 cases is noted when ASCUS+ is replaced by LSIL+ compared to ASCUS+ cytology alone [39.1% (95% CI 19.2%–59.1%) and 62.3 (95% CI 28.6–135.7) vs 10.4% (95% CI 6.3%–14.6%) and 11.2 (95% CI 8.2–15.4) respectively].Detection of HSIL or worse cytology or HPV 16/18 genotyping for the triage of HPV positive women has a slight, not statistically significant increase of sensitivity and PPV than ASCUS or worse cytology alone [68.3% (95% CI 54.0%–82.5%) vs 53.7% (95% CI 37.4%–69.3%) and 17.4% (95% CI 11.5%–23.2%) vs 10.4% (95% CI 6.3%–14.6%)]. However, considering that the PPV of HSIL alone is 46.7% (95% CI 21.4%–71.9%), testing positive also for HPV 16 or 18 or both would result in an almost similar PPV of 50.0% (95% CI 10.0%–90.0%), whereas the sensitivity would drop from 17.1% (95% CI 5.6%–25.6%) to 7.3% (95% CI 0.0%–15.3%)]. According to that, the addition of HPV 16/18 genotyping to HSIL cytology would not improve diagnostic accuracy concerning triaging HPV positive women.Finally, no statistically significant changes in relative risk between the different combinations of triage tests used to detect CIN2 or worse cases among HPV-positive women were noted.Many things have changed since 1943 when Papanicolaou and Traut published their paper on the role of the vaginal smear in cervical cancer diagnosis , leadinget al. taking into account the follow-up data of 176,464 women from 4 European studies have shown that HPV DNA-based screening alone provides a 60–70% better protection against cervical cancer compared to cytology . Sensitivity rates follow similar trend when ASCUS or worse is replaced by LSIL or worse cytology. Testing positive for HPV 16 or HPV 18 or both and additionally ASCUS+ cytology would also significantly increase PPV and PLR for the identification of CIN2+, in comparison to ASCUS+ alone [32.4% and 46.4 vs 10.4% and 11.2 respectively]. Regarding cytology of HSIL or worse, the addition of HPV 16/18 genotyping would result in reduced sensitivity and similar PPV, a sign for the relatively low additive value of 16/18 genotyping in HPV-positive women if a HSIL+ cytology result is present.As a conclusion, triaging of HR HPV positive women with HPV 16/18 genotyping alone has a similar or slightly better performance than using LBC alone, whereas combining cytological indices with HPV 16/18 genotyping results in an even more sensitive method to triage HPV-positive women than either genotyping or cytology alone. The latter, however, can only be achieved at the cost of performing two different diagnostic procedures, (i.e.) HPV 16/18 genotyping and LBC, additionally to the initial HPV testing. On the contrary, genotyping for HPV 16/18 using the cobas HPV test as alternative to cytology for triaging HPV-positive women, can be performed by one sole diagnostic procedure offering concurrently HPV testing and HPV 16/18 genotyping results.et al. using cobas was 10% [The superior sensitivity of HPV testing is an established finding in the literature, but it has also been shown that there are variations in performance depending on the HPV testing method used and the study population especially between continents . Therefo was 10% . It is oThe design of our study, in some aspects and in a much smaller scale, is similar to that of the larger ATHENA trial and, based on the respective results, we drew similar conclusions. As weaknesses of our study one can notice the relatively small sample of women tested, as well as the fact that it is more subject to verification bias since double screen negatives were not offered colposcopy, apart from a small fraction of the NILM and HPV-negative women used for verification bias adjustment. Taking in mind that this percentage of double negative women tested was relatively small, there were no cases of CIN2+ found in the random sample of double negative women that had colposcopy, making actual adjustments of the results not necessary. This could cause an overestimation of the HPV test sensitivity but should not affect the relative difference between HPV testing and cytology. Apart from that, the lack of histological verification in all HPV-positive or/and cytology-positive women by using the presence of abnormal colposcopic impression as a prerequisite for the performance of biopsies, as well as the relatively high number of HPV-positive or/and cytology-positive women lost to colposcopy, represent additional limitations in our study, which could cause a diminished identification of women with high-grade disease. This fact could probably affect to some extent the final accuracy of the two screening tests.As strengths of our study one could note the use of LBC, the fact that all women enrolled had both, HPV DNA testing and cytology performed, that cytology examinations were performed at the corresponding cytology laboratories of the hospitals cooperating in the study reflecting real-life routine laboratory conditions, that cytologists weren’t aware of the HPV DNA test results, nor were molecular biologists of the cytology results, that colposcopy and histology were conducted in each of the cooperating hospitals by experienced specialists and pathologists were not aware of the HPV DNA test result. As an additional strength of the present study one could consider the sub-analysis performed for women aged below and above 30, in order to identify the best option for starting age of cervical cancer screening, by assessing the performance of HPV DNA testing with genotyping as well as of LBC for the two age groups.In summary, our study is the first multicenter trial in Greece evaluating the performance of HR HPV DNA testing with concurrent HPV 16/18 genotyping in comparison to LBC for primary cervical cancer screening. According to our results from the first screening round, HPV DNA testing has much better sensitivity than cytology to identify high-grade cervical lesions (CIN2+ or CIN3+), with slightly lower specificity. Changing the starting age of screening from 25 to 30 years, leads to significant increase on both, specificity and PLR of HPV DNA testing, advocating for its optimal use in women over 30 years of age. Additionally, genotyping for HPV 16 or HPV 18 or both in order to detect high-grade lesions has similar sensitivity and specificity to ASCUS or worse cytology in young (25–29 years) as well as in older women (30–55 years). In conclusion, HPV DNA testing with concurrent individual genotyping for HPV 16, HPV 18 or both, could represent a more accurate methodology for primary cervical cancer screening in comparison to liquid-based cytology.


Beta lactams are the most commonly used group of antimicrobials worldwide.Escherichia coli isolated from nosocomial infections in Cotonou, Benin.The presence of extended-spectrum lactamases (ESBL) affects significantly the treatment of infections due to multidrug resistant strains of gram-negative bacilli. The aim of this study was to characterize the beta-lactamase resistance genes in Escherichia coli strains were isolated from various biological samples such as urine, pus, vaginal swab, sperm, blood, spinal fluid and catheter. Isolated bacteria were submitted to eleven usual antibiotics, using disc diffusion method according to NCCLS criteria, for resistance analysis. Beta-lactamase production was determined by an acidimetric method with benzylpenicillin. Microbiological characterization of ESBL enzymes was done by double disc synergy test and the resistance genes TEM and SHV were screened by specific PCR.ESBL phenotype was detected in 29 isolates (35.5%). The most active antibiotic was imipenem (96.4% as susceptibility rate) followed by ceftriaxone (58.3%) and gentamicin (54.8%). High resistance rates were observed with amoxicillin (92.8%), ampicillin (94%) and trimethoprim/sulfamethoxazole (85.7%). The genotype TEM was predominant in ESBL and non ESBL isolates with respectively 72.4% and 80%. SHV-type beta-lactamase genes occurred in 24.1% ESBL strains and in 18.1% of non ESBL isolates.Eschericiha coli in Cotonou. It demonstrated also high resistance rate to antibiotics commonly used for infections treatment. Continuous monitoring and judicious antibiotic usage are required.This study revealed the presence of ESBL producing  Escherichia coli and Klebsiella species, they have also been described in other genera of the Enterobacteriacea  and OT-2-R [5′-TAATTGTTGCCGGGAAGCTA-3′] which amplified a 465 bp fragment [The presence of beta-lactamase genes, fragment ; for thefragment .2 (1.5 mM), (volume) dNTPs (200 μM), (volume) primer (0.5 mM), 0.2 μL of Taq DNA polymerase . An Biometra thermal cycler was used for the amplification. The cycling conditions were the following:Amplification reactions were performed in a volume of 30 μl containing 4 μl of supernatant, (volume) PCR buffer (1x), MgClTEM: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s , extension at 72°C for 1 min;SHV: initial denaturation at 96°C for 15 s, followed by 30 cycles of denaturation at 96°C for 15 s, annealing at 50°C for 15 s, extension at 72°C for 2 min. Both PCR programs were followed by a final extension step of 10 min.Amplified PCR products were separated on 1.5% agarose gels, stained with ethidium bromide and visualized under UV illumination.-value < 0.05 was considered significant.Data were analyzed with Epi Info® version 3.5.4. Differences in antibiotic susceptibility among different groups were statistically analyzed by the Fisher exact test. An associated PEscherichia coli isolates were included in this study. The majority of isolates (72 strains: 85.7%) were from urine samples. Specimens from pus represented 4.76% of the isolates, followed by vagina swab and spinal liquid with 2.38% each. Isolates from sperm and catheter were 1.2% each.A total of 84 nosocomial The antibiotics susceptibility test revealed that the most efficient antibiotics were imipenem (96.4% as susceptibility rate) followed by ceftriaxone (58.3%) and gentamicin (54.8%). High resistance rates were observed with amoxicillin (92.8%), ampicillin (94%), and trimethoprim/sulfamethoxazole (85.7%). Resistance to amoxicillin/clavulanic acid was 85.7% with a high rate of intermediate resistance (46.7%). We observed homogeneity in the resistance profile of ESBL-producers which were multi drugs resistant with at least a resistance to 8 antibiotics out of the 11 tested  were positive using either cefotaxime or ceftriaxone. Among these ESBL-producers, 21 were from urinary tract infection (UTI) and 8 from other infection sites. The other infection sites seem to be disproportionally represented than UTI (27.6% versus 14%). This fact could be explained with reduced number of samples drawn from non-urinary infections.Comparison of susceptibility rates of ESBL producers with those of ESBL non producers showed similar values for amoxicillin, ampicillin and imipenem. No statistically significant difference between the two groups was observed Table . For allTEM 65 (77.4%) and blaSHV 17 (20.2%). These genotypes occurred singularly in 54 isolates (64.3%) with 51(94.4%) for blaTEM and 3 (5.6%) for blaSHV. Both TEM and SHV genes were present in 14 isolates (16.7%) and absent in 16 isolates (19%) including 10 non ESBL strains and 6 ESBL strains.According to the PCR, the genotypes TEM and SHV were distributed as follow: blaOut of the 29 isolates with ESBL phenotype, the PCR analysis revealed 72.4% strains with the genotype TEM, 24.1% with the genotype SHV and 5 strains harbored both genes. Figure As shown in Table Escherichia coli were predominantly responsible of UTI [Our study was carried out in three hospitals in Cotonou, Benin. Of the 84 isolates tested, 72 (85.7%) were from urinary tract infections (UTI). This finding is similar to results previously described by several studies -16. Thise of UTI ,18.E. coli isolates was found to imipenem (96.4%). In 2007 we have showed that the resistance rate to imipenem was 5% in ESBL producers and 2% in non ESBL isolates [et al. in Rwanda [E. coli isolates to imipenem at rates of 98.4%, 96.7% and 92.5% respectively. This finding may be due to the stability and the high activity of carbapenem against most beta-lactamases.The highest susceptibility of n Rwanda . Studiesn Rwanda  and Nigen Rwanda  reportedHigh resistance rates were founded to commonly used antibiotics in Benin such as ampicillin (97.6%), amoxicillin (95.2%) and trimethoprim/sulfamethoxazole (86.9%). The observed high rates may be due to uncontrolled consumption, consequence of easy access to inefficient and cheap antibiotics.et al. [et al. reported 48% and 16% resistance to ciprofloxacin in ESBL and non ESBL E. coli from nosocomial infections, respectively [Among the tested fluoroquinolones, only 8.3% of the isolates were sensitive to ciprofloxacin. Norfloxacin was more efficient with 44.6% of sensitive strains, but lower than the result reported by Thakur ectively . Our surLower resistance rates to ciprofloxacin, usually prescribed in uncomplicated UTI and other infections, have been reported in Nepal (45%) , in IndiE. coli strains from nosocomial infections [E. coli isolates from out-patients [High resistance rate to amoxicillin/clavulanate acid was also observed 85.7%). This finding is in agreement with previous investigations in Benin. In 2007, we reported resistance rates of 97.5% in ESBL and 53% in non ESBL %. This fWe showed in this study that 87% of isolated strains were positive for beta-lactamase detection by acidimetric method indicating that the most important mechanism of resistance to beta-lactam antibiotics is the production of beta-lactamase.The high prevalence of ESBL-producing strains (35.5%) was similar to those generally shown in developing countries.E. coli. In 2007, Ahoyo et al. showed a prevalence of 22% in isolates from various nosocomial infections. In 2004, we founded that 14.8% of E. coli strains isolated from urinary tract infection (UTI) were ESBL [Our finding is very close to the prevalence ESBL reported in India (35.45%)  and simiere ESBL , in Cameere ESBL .Among the ESBL-producers, the other infection sites seem to be disproportionally represented than UTI (27.6% versus 14%). This fact could be explained with a small number of samples drawn from non-urinary infections.The comparison between ESBL producing strains and non ESBL showed that ESBL-producers were significantly more resistant to cephalosporins, quinolones, aminosides, trimethoprim/sulfamethoxazole and amoxicillin/clavulanic acid than non-ESBL producers. The genes encoding ESBLs are usually located in transferable plasmids that may also carry other resistance determinants, such as those for resistance to aminoglycosides, tetracyclines, chloramphenicol, trimethoprim, sulphamides, and quinolones ,32.E. coli. The remaining 6 ESBL strains which were non TEM and non SHV could harbor CTX-M genes. Widespread dissemination of these genes has been described in Africa and elsewhere [The genotype TEM was predominant in ESBL and non ESBL isolates with respectively 72,4% and 80%. This fact was generally observed in lsewhere ,25,31.Out of the 65 strains harboring TEM genes, 59 showed positive result for the detection of enzyme production using hydrolysis of penicillin. Of the 17 SHV positive strains, 15 (80.95%) were positive for this test.et al. who found no significant association between genotypes TEM and SHV and susceptibility to cefotaxime and ceftriaxone [In this study, the phenotypic screening for ESBL was realized by resistance to cefotaxime or ceftriaxone. The presence of genotype TEM and SHV could not predict the resistance pattern to these cephalosporins. Our finding is consistent with the results reported by Maina triaxone .Escherichia coli in clinical isolates from several hospitals in Cotonou. The use of some first line treatment antibiotics such as penicillin and trimethoprim/sulfamethoxazole seems inappropriate. Antibiotics resistance surveillance and the determination of molecular characteristics of ESBL isolates are primordial to ensure the judicious use of antimicrobial drugs.This study reveals the presence of ESBL producing