Although environmental and human behavioral factors in countries with Zika virus (ZIKV) outbreaks are also common in Nigeria, such an outbreak has not yet been reported probably due to misdiagnosis. The atypical symptoms of malaria and ZIKV infections at the initial phase could leverage their misdiagnosis. This study randomly recruited 496 malaria-suspected patients who visited selected health institutions in Adamawa, Bauchi, and Borno states for malaria tests. These patients’ sera were analyzed for ZIKV antibodies using ELISA and plaque reduction neutralization tests (PRNT) at 90% endpoint. About 13.8% of Zika virus-neutralizing antibodies (nAb) did not cross-react with dengue, yellow fever, and West Nile viruses suggesting possible monotypic infections. However, 86% of the sera with ZIKV nAb also neutralized other related viruses at varied degrees: dengue viruses (60.7%), West Nile viruses (23.2%), yellow fever virus (7.1%) and 39.3% were co-infections with chikungunya viruses. Notably, the cross-reactions could also reflect co-infections as these viruses are also endemic in the country. The serum dilution that neutralized 90–100% ZIKV infectivity ranged from 1:8 to 1:128. Also, our findings suggest distinct protection against the ZIKV between different collection sites studied. As indicated by nAb, acute ZIKV infection was detected in 1.7% of IgM-positive patients while past infections occurred in 8.5% of IgM-negatives in the three states. In Borno State, 9.4% of IgG neutralized ZIKV denoting past infections while 13.5% were non-neutralizing IgM and IgG indicating other related virus infections. The age, gender, and occupation of the patients and ZIKV nAb were not significantly different. ZIKV nAb from samples collected within 1–7 days after the onset of symptoms was not significantly different from those of 7–10 days. A wider interval with the same techniques in this study may probably give better diagnostic outcomes. ZIKV nAb was significantly distinct among recipients and non-recipients of antibiotic/antimalaria treatments before seeking malaria tests. The inhibiting effect of these drugs on ZIKV infection progression may probably contribute to the absence of neurological disorders associated with the virus despite being endemic in the environment for several decades. Also, protection against ZIKV as marked by the nAb was different among the vaccinated and unvaccinated YF vaccine recipients. Thus, the YF vaccine may be a good alternative to the Zika vaccine in resource-constrained countries.The cryptic ZIKV infections underscore the need for differential diagnosis of malaria-suspected febrile patients for arboviruses, especially the Zika virus. The absence of systemic surveillance for the virus is worrisome because of its association with neurological disorders in newborns. Co-infections with other arboviruses may impact adversely on the management of these diseases individually. Aedes (Stegomyia) Africana [Flavivirus of the family Flaviviridae [Aedes mosquitoes but transmission through sexual [Zika virus (ZIKV), a re-emerging virus infection was first isolated in 1947 from a rhesus macaque in the Zika forest of Uganda, Africana . ZIKV haiviridae . It is th sexual  and blooh sexual  have beeh sexual , and Senh sexual , New Calh sexual , and Eash sexual  in 2014 h sexual . Brazil h sexual . In 2016h sexual . The sitOverall, routine surveillance for endemic arboviruses in Nigeria {including dengue (DENV) –17, yellNigeria is one of the 32 countries in Sub-Saharan Africa that account for almost 93% of malaria death worldwide [Nigeria is located between latitude 9.081999 and longitude 8.67527 and covers an area of 923,769 square kilometers  with a projected population of 210,277,910 in 2022 . North-eThis study randomly recruited 496 patients with febrile illness who visited the selected hospitals to request a malaria test between April and August 2018 and endorsed the consent forms. A brief structured questionnaire was designed for each patient. Their socio-demographic characteristics and other parameters were collected using the structured questionnaire . Althougn = sample size, P = prevalence, Q = 1 − P, Z = Z score at 95% Confidence intervals, e = margin of error. Using the past prevalence rate of ZIKV IgM of 6%  [The sample size was estimated using the formula:al 2016) , the minThe designed consent form was attached to the proposal submitted for ethical approval at each selected hospital. The research ethical clearance approval letters were obtained from the Research and Ethics Committee of ATBUTH , State Specialist Hospital (SSH), Maiduguri, Borno State (ref no. SSH/GEN/641/Vol.1 dated 14 February 2018) and Federal Medical Centre, Yola (ref no FMC/YO/001/Vol.1 dated May 2018). The approved consent form was randomly given to 250 participants from each of the three states, to read, endorse and return if they agreed to be recruited for the study. Once minimum of 200 of the participants in each state endorsed the forms, we recruited them for the study. This include parents of children below five years who endorsed the forms on behalf of their children.Sera samples were qualitatively analyzed for human ZIKV IgM antibody according to the manufacturer protocol . Briefly, 50ul of undiluted serum  was treated with 100 ul of HRP-conjugate and incubated for 1 h at 37°C. After appropriate washing, 50 ul of chromogen A and B were added to each well and incubated for 15 min at 37°C before the reaction was stopped. The plate was read at an optical density (OD) of 450 nm within five minutes after the reaction was stopped. The average of the positive control well was typically ≥1.0; the average of the negative control well was typically ≤0.15. The Cut-off values were the average of the negative control well + 0.15 in the samples tested. The cut-off OD units of ≥ 200 were considered positive.Sera samples were analyzed using Sandwich Complex ELISA kits  according to the manufacturer’s protocol. Briefly, each diluted serum (1:40), including positive, and negative controls, and the patient’s serum was incubated with the ZIKV antigen in a coated microtiter plate. The plate was incubated at room temperature for 10 minutes. Enzyme conjugate was added to the wells after appropriate washings and incubated at room temperature for 10 min. The plates were washed and incubated with the chromogen at room temperature for 5 minutes before the termination of the reaction. The plate was read at an OD of 450 nm within one hour after the reaction was stopped. The cut-off OD units of ≥ 200 were considered positive.5 cells/ well and incubated at 37°C for 24–48 hours or until it reached 70–80% confluence. Each serum sample was diluted 1:8 to 1024 using in-house diluent (IHD) prepared with PBS supplemented with Penicillin/Streptomycin (22%), Gentamycin  and Fungizone (0.02%). An equal volume of ZIKV virus stock  at a concentration of 100 PFU/ml was added to each of the diluted serum and incubated at 37°C for 1 hour. Each serum dilution–virus mixture was prepared in duplicate and three controls including a virus dose control (100 PFU virus plus diluent only), a cell control (diluent and cell only) and negative virus control were tested simultaneously. After incubation at 37°C for 1 hour, the virus-serum mixture was added to BHK-21 cells and incubated for 1 hour at 37°C. The plates were rocked gently every 15 minutes for uniform distribution of the inoculum. Different concentrations  of overlay medium: Carboxyl methylcellulose (CMC) salt (Low viscosity) (Sigma) was tested using 100 PFU/ml of the virus to determine the best consistency. The best consistency of CMC was obtained at 6% concentration. Therefore, the overlay medium was prepared by mixing an equal volume of 12% CMC with EMEM supplemented with 4% fetal bovine serum (FBS) . 1 ml of the overlay medium was added to each of the 24 wells and incubated at 37°C for 10–14 days. Thereafter, each well was fixed with 500 ul of 10% formaldehyde solution for 30 minutes after removing the overlay and stained with 500 ul of 0.5% crystal violet (Sigma) for 20–30 minutes. The percentage of plaque reduction by the specific antibody was calculated using the formula: 100-(Number of plaques in sample/Number of plaques in control) x 100. The validity of the test was determined by the virus control having a minimum of 50 plaques and cell control and positive sera having no plaques at all. The PRNT titer was defined as the reciprocal of the serum dilution that reduced the number of plaques by 90% (PRNT90) [The PRNT was performed as previously described for the dengue virus with few modifications 36). Each serum sample was inactivated at 56°C for 30 minutes and stored at -80°C. BHK-21 cells were grown in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 2% Penicillin/Streptomycin, and 1% HEPES and 2.5% sodium bicarbonate. The cells were seeded in 24-well plates at a density of 1 x 10(PRNT90) , 36. Mod. Each se90 results) and other variables in the three states. The fitness of the model to correctly predict the PRNT results (positive and negative) by the independent variables was tested and found significant using the Omnibus test of model coefficient and insignificant with the Hosmer and Lemeshow test. Overall, the percentage accuracy in the classification was 87% with 100% specificity (true negative) but 0% sensitivity (True positive).The data was analyzed using Statistical Product and Service Solutions (SPSS), Version 25.0, from IBM SPSS. We used the mean standard deviation/standard error of the mean and percentage when appropriate for the patients’ characteristic description. Age groups and occupations were compared using the Pearson χ2 or Fisher’s exact test for categorical variables when the expected count is less than 5. Additionally, Kruskal-Wallis H test and Mann-Whiteney U test were used to determine the difference between ZIKV neutralizing antibodies and age, states, gender, treatment with antibiotics/antimalaria, urban/rural dwellers, intervals of sample collection and onset of symptoms and yellow fever vaccination status. Binary Logistic Regression in SPSS was used to obtain the odds ratios and confidence intervals for the association between ZIKV infection /immunity  and Adamawa  (Table 5). The significant difference between the three states was further confirmed using Krustal- Wallis H test (test-statistic = 63.102 (p< 0.001) , 35 (36.5%), and 24 (12.0%) in Adamawa, Bauchi and Borno respectively had ZIKV neutralizing antibody (nAb) by PRNT< 0.001) . The oddAbout 9 samples at a dilution of 1:128 neutralized 90–100% of the virus infectivity while 16 did the same at 1:64, 13 at 1:32, 11 at 1:16, and 18: 1:8 , 9 (13.8%) did not cross-react with other arboviruses tested while 56 (86%) did so at varying degrees: DENV (60.7%), WNV (23.2%) and YFV (7.1%). These cross-reactions are more likely to be co-infections. Similarly, 39.3% (22/56) of the patients were co-infections of Zika (a Flavivirus) and Chikungunya (an Alphavirus) viruses. .2 = 1.16, df = 1, p = 0.180; OR = 1.35, Cl 95% = 0.79–1.44)  of the patients tested were urban dwellers while only 15.7% were rural. Among urban dwellers, 11.5% and 12.2% had ZIKV IgM and nAb respectively. Among the rural dwellers, 15.4% ZIKV IgM and 17.9% nAb were detected . The typ79–1.44)  and this79–1.44) . However2 = 1.17, df = 7, p = 0.310; OR = 1.16, Cl 95% = 0.85–1.59) ZIKV IgM increased with increasing age up to 60 years and thereafter decreases. Overall, age was not significantly associated with ZIKV nAb  and occupation  were not significantly associated with ZIKV nAb  and those at child bearing age of 15–40 years (Category 2). In category 1, 9.8% were IgM and 14.2% had nAb. However, in category 2, 16.2% had ZIKV nAb. Among males aged 15–40 years, 10.7% had ZIKV nAb . Overall, gender . This observation was also confirmed using Mann-Whitney U test   .2 = 6.21, df = 1, p = 0.014; OR = 1.45, Cl = 1.11 to 1.88) (The majority (59.7%) of the patients received treatment with either antimalaria and/ or antibiotics before seeking a laboratory test for malaria parasites. Among the treated, 14.2% had ZIKV IgM and 10.8% were nAb. Similarly, 9.5% and 16.4% of untreated had ZIKV IgM and nAb respectively . Treatmeto 1.88) . This dito 1.88) .2 = 6.01, df = 1 p = 0.015). The difference was further confirmed with Mann- Whitney U test  (YF vaccination status in Adamawa state was higher (53.5%) than in Bauchi (25.0%) and Borno (7.5%) . Overall = 0.15) .The results of this study have demonstrated the misdiagnosis of ZIKV infections for malaria in Nigeria and underscore the importance of screening malaria-suspected cases for arboviruses.To estimate the burden of ZIKV in Nigeria, both ELISA IgM/IgG and PRNT were used.90 positivity with high antibody titre amidst co-infections may be confounded by the concept of ‘original antigenic sin’ (antibody to the earlier virus may be higher than the new) [In this study, 13.1% had ZIKV nAb which indicated both acute and past infections but only 1 (1.7%) IgM in the three states had nAb, indicating acute infection while 11.9% were infections by other flaviviruses . In Bornthe new) . Additiothe new) , 40. Thethe new) –43. Overthe new) –47. Thisthe new) . Thus, tthe new)  resultinthe new) . We spec2 = 1.53, df = 1, p = 0.229). However, 16.2% of females at childbearing age of 15–40 years having ZIKV nAb is alarming. The potential of ZIKV to cause stillbirth and other neurological disorders in newborn necessitates active surveillance especially in females of childbearing age. ZIKV nAb from samples collected between 1–7 days and 7–10 days after the onset of symptoms were not significantly associated  and the untreated had more ZIKV nAb than those treated . This observation corroborated a report that some antibiotics  and antimalarial  are effective treatments for many viral diseases [A significant number of the patients (59.7%) tested received treatment with antibiotics/anti-malaria before the malaria parasites tests. This practice is common in the country because every febrile illness is presumed and immediately treated as malaria and or bacterial infections. In this study, ZIKV nAb and treatment with antibiotics/antimalaria  were sigdiseases  includindiseases . We spec2 = 6.08, df = 1 p = 0.015) between YF vaccine recipients and ZIKV nAb was observed. Our findings corroborated a previous study that demonstrated significant ZIKV–related microcephaly in the Northeast region of Brazil where YF vaccination coverage was the lowest [The low population immunity against YFV as evidenced by 73.5% of patients who did not receive the YF vaccine explains its periodic epidemics in the country . In thise lowest . The stre lowest  has opene lowest  has provThe limitations of this study include our inability to test the samples from Bauchi and Adamawa for IgG due to the high cost of these kits. Also, PRNT was performed on ZIKV-positive sera diluted 1:8–1024 while other co-infecting flaviviruses were not diluted beyond 1:8 due to insufficient quantity of the samples.90 allowed the detection of true ZIKV infections. Co-infections with other arboviruses  may confound the management of these diseases individually. ZIKV infections in females at childbearing age are warning signs for congenital Zika syndrome. Treatment with anti-malaria/antibiotics may ameliorate recovery from ZIKV. ZIKV nAb among samples collected within 1–7 and 7–10 days were not significantly different. YF vaccination may cross-protect against ZIKV. This study underscores the need for differential diagnosis of suspected malaria febrile patients for arboviruses, especially Zika virus. More affordable diagnostics in the market will ultimately facilitate the timely detection of these viruses before they spread, cost lives, and become difficult to control in resource-constrained countries.ELISA IgM and PRNT were used to unmask ZIKV infections from malaria-suspected febrile patients in Adamawa, Bauchi and Borno states at different prevalence rates. ZIKV IgM, IgG, and PRNT performed only on Borno samples allowed the detection of both acute and past infections. The species specificity of PRNTS1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(JPG)Click here for additional data file.S2 Fig(BMP)Click here for additional data file.S3 Fig(BMP)Click here for additional data file.S4 Fig(JPG)Click here for additional data file.S5 Fig(JPG)Click here for additional data file.


This article has been corrected at the request of the authors to edit data that had typographical errors in the initial published version:In Table 6, column 3, rows 3-6 have been updated from:0.930.970.750.65To: 0.290.191.751.65The corresponding data in the abstract has also been updated accordingly. The authors deeply regret that these errors were not identified and addressed prior to publication.


There have been a number of advances in the treatment paradigm of pancreatic ductal adenocarcinoma (PDAC) over the past two decades. Medical and surgical approaches to PDAC have continued to evolve. The individual effects of these changes have been investigated. Understanding the implementation of these changes over time and its effect on patient survival is important. While patient survival from PDAC has been reported to have modest improvements over the decades, the National Cancer Database offers the ability to study survival trends. We aim to evaluate survival trends in patients diagnosed with PDAC in the United States.p < 0.001). Median survival improved from 7.2 months to 10.1 months for patients treated without surgery during the same years (p < 0.001). On multivariable analysis, the hazard ratio for death was estimated to multiply by 0.975 per year for patients treated with surgery and 0.959 per year for patients treated without surgery (p < 0.001). This increase in survival in the setting of evolving care validates continued efforts aimed at improving survival for patients with this devastating disease.Over the last two decades, there have been many reported advances in the clinical management of pancreatic ductal adenocarcinoma (PDAC). We sought to evaluate changes in survival for patients diagnosed with PDAC between 2004 and 2017. The National Cancer Database was queried for patients diagnosed with PDAC between 2004 and 2017. There were 55,401 patients who underwent surgery and 109,477 patients who underwent non-surgical treatment for PDAC between 2004 and 2017. Patients were categorized into four groups by year of diagnosis. Median survival improved from 15.5 months to 25.3 months for patients treated with surgery between the years 2016 and 2017 compared with between 2004 and 2007 ( Pancreatic ductal adenocarcinoma (PDAC) remains a deadly malignancy with an 11% five-year survival . Even afIn the last two decades, there have been significant changes in the treatment paradigm of PDAC. Neoadjuvant chemotherapy has become more commonplace for even resectable PDAC . The cheThe effect of many of these changes on survival for patients with PDAC has been individually evaluated and, while there are likely numerous factors not listed here that have further contributed to patient outcomes, the overall trends in survival for patients treated for PDAC have not been well described. We aimed to use the National Cancer Database (NCDB) to evaluate changes in survival for patients diagnosed with pancreatic ductal adenocarcinoma between 2004 and 2017.The NCDB participant user file for patients with pancreatic malignancies diagnosed between 2004 and 2017 was queried for patients with pancreatic ductal adenocarcinoma (PDAC). The NCDB is a joint project of the Commission on Cancer of the American College of Surgeons and the American Cancer Society. It includes data from more than 1500 commission-accredited cancer programs in the United States. The data used in the study are derived from a deidentified NCDB file. The American College of Surgeons and the Commission on Cancer have not verified, and are not responsible for, the analytic or statistical methodology employed or the conclusions drawn from these data by the investigator. The database includes about 70% of all newly diagnosed cancers in the United States . BecausePatient characteristics and socioeconomic variables included facility type and location, age, sex, race, ethnicity, insurer, median income and percentage of population without a high school degree for the ZIP code in which the patient resides, distance from hospital, rural/urban residence, and Charlson–Deyo comorbidity index. Oncologic factors included tumor location, tumor size, stage, days from diagnosis to treatment, lymph nodes, margin status, neoadjuvant therapy, receipt of chemotherapy, readmission, and mortality. Survival data were available for patients between 2004 and 2017. Patients were grouped into four groups by year of diagnosis: 2004–2007, 2008–2011, 2012–2015, 2016–2017. The primary outcome of interest was survival. In patients undergoing surgery, survival was defined as time from surgery to death or most recent follow-up. In patients who did not undergo surgery, survival was defined as time from diagnosis until death of most recent follow-up.Statistical analysis was conducted using the R statistical software package . Descriptive statistics by time period were created for the variables of interest. Since interest was in assessing trends over time, the time periods were treated as ordered categories and associations with time were assessed using Spearman correlation tests (continuous variables) or logistic regression models , with the factor as outcome and the time period as predictor. Kaplan–Meier methods were used for estimating survival by year of diagnosis.Mixed-effects Cox regression was used to estimate the effect of year of diagnosis (treated as a continuous variable) on survival when controlling for patient and hospital factors. Surgical and non-surgical patients were analyzed in separate models. A random effect for hospital was included in both models to adjust for the clustering of observations on center. Variables missing ≤5% of cases were imputed with mean, median, or mode. Variables missing in >5% but <10% of cases were not imputed; instead, a “missing” category was added to these variables. Tumor size and postoperative length of stay were excluded due to >10% missingness. For surgical patients, fixed factors were year of diagnosis, facility type, facility location, age, sex, race, ethnicity, insurer, median income and percentage of population without a high school degree for the zip code in which the patient resides, distance from hospital, rural/urban residence, Charlson–Deyo comorbidity index, primary site, histology, stage, number of lymph nodes examined, number of nodes positive, receipt of chemotherapy, T stage, N stage, and days from diagnosis to first treatment. For non-surgical patients, fixed factors were year, facility type, facility location, age, sex, race, ethnicity, insurer, median income and percentage of population without a high school degree for the zip code in which the patient resides, distance from hospital, urban/rural residence, Charlson–Deyo comorbidity index, primary site, histology, stage, number of nodes examined, number of nodes positive, receipt of chemotherapy, and days from diagnosis to first treatment. For both the surgical patient and non-surgical patient modeling, the natural log of distance, nodes examined, and nodes positive were used in the model because these variables were highly skewed. In order to generate survival curves using the Cox regression model, we selected a patient with the average characteristics of the study population. The average characteristics were represented by either the mean or mode, depending on whether the variable was categorical or continuous. p < 0.001). The sex of patients did not significantly change (p = 0.072). As the percentage of patients being treated at an academic center decreased from 52.4 to 49.3% (p < 0.001), the rate of patients treated at a comprehensive community cancer center increased from 25.9 to 27.4% (p < 0.001). The rate of patients insured by Medicare increased from 49.7 to 53.3% (p < 0.001) and the percentage of privately insured patients decreased from 42.7 to 37.8% (p < 0.001). The percentage of patients living in zip codes with median income <USD 38,000 increased from 12.8 to 16.2% (p < 0.001) and patients living in zip codes with median income >USD 63,000 decreased from 41.7 to 38.7% (p < 0.001). Patients living in zip codes where >21% of the population had not achieved high school education increased from 15.1 to 18.5% (p < 0.001). Patients living in zip codes where < 7% of the population had not achieved high school education decreased from 37.5 to 27.0% (p < 0.001).There were 55,401 patients who underwent surgical resection for PDAC between 2004 and 2017 . Averagep < 0.001), while Charlson–Deyo comorbidity index scores >3 rose from 1.8 to 4.8% (p < 0.001). The vast majority of patients had stage 1 or stage 2 disease across all years. The number of examined lymph nodes increased from 11.7 ± 8.5 to 18.9 ± 10.4 (p < 0.001) The percentage of patients receiving chemotherapy increased from 63.2 to 81.7% (p < 0.001). Length of stay decreased from a median of 9 days to a median of 7 days. Mortality at 30 days, 90 days, 1 year, and 2 years all decreased significantly (p < 0.001).Patients with a Charlson–Deyo comorbidity index score of 0 decreased from 68.1 to 64.6% (p < 0.001). p < 0.001). Median survival increased from 15.5 : 15.2–15.9) months to 25.3 (95%CI: 24.7–26.1) months from the years 2004 to 2007 to 2016 to 2017.Kaplan–Meier analysis of survival in patients undergoing surgery for PDAC was performed . Patientp < 0.001) per year. Hazard ratios for death by stage can be found in Controlling for other variables as outlined in the methods, the hazard ratio for death is estimated to multiply by 0.975 . In contrast to surgical patients, the rate of patients receiving treatment at an academic center increased from 39.2 to 42% (p < 0.001) and the rate of patients being treated at a comprehensive community cancer center decreased from 35.4 to 33.5% (p < 0.001). Patients insured by Medicare increased from 48.1 to 53.4% (p < 0.001), whereas privately insured patients decreased from 43.5 to 35.6% (p < 0.001). The percentage of patients living in zip codes with median income <USD 38,000 increased from 12.8 to 17.6% (p < 0.001). The percentage of patients living in zip codes with median income >USD 63,000 decreased from 42.4 to 37.8% (p < 0.001). Patients living in zip codes with a percentage of the population with less than high school education of >21% increased from 15.4 to 19.7% (p < 0.001). Patients living in zip codes with a percentage of the population with less than high school education of <7% decreased from 37.7 to 26.0% (p < 0.001).There were 109,477 patients who received non-operative treatment for pancreatic ductal adenocarcinoma between 2004 and 2017 . Age at p < 0.001). p < 0.001). Median survival increased from 7.2 (95%CI: 7.1–7.3) months to 10.1 (95%CI: 9.9–10.2) months from the years 2004 to 2007 to 2016 to 2017.Kaplan–Meier analysis of survival in patients treated medically for PDAC was performed . There wp < 0.001) per year. Hazard ratios for death by stage can be found in After adjusting for covariates, the hazard ratio for death is estimated to multiply by 0.959  database found pancreatic cancer incidence increased 1.03% per year between 1974 and 2014. In the same study, mortality increased 2.22% per year . A similSurgical outcomes improved in our study era, including a 2.3-day reduction in hospital length of stay. This finding may be attributable to ERAS protocols and minimally invasive pancreatic resections, which have become more common. Multiple studies have shown that ERAS protocols can lead to decreased length of stay, with similar readmission rates for patients undergoing pancreas surgery . Any effFor patients treated without surgery, there were significant increases in survival in both Kaplan–Meier analysis and multivariable analysis. The majority of these patients had locally advanced or metastatic disease at diagnosis. This diagnosis has poor prognosis and the results of our study suggest there is much work to be conducted for improving survival for these patients. Results of trials using FOLFIRINOX regimens were published during our study period and have demonstrated improvement in survival for patients with advanced disease ,22. It iThis retrospective review of a national dataset carries a number of limitations. First, the NCDB captures 70% of newly diagnosed cancers ; this leThe results of our study suggest improving survival for patients with PDAC whether treated with surgery or medical therapy. Future studies to understand which factors are responsible for improved survival are warranted. While these data validate the efforts of physicians and scientist, they also demonstrate a significant need for continued work to improve outcomes for patients. Nevertheless, this overall improvement in survival is a significant achievement towards better helping patients with this deadly disease.


The proportion of patients with persistent airflow limitation significantly decreased from 26.7% at baseline to 12.6% at year 1 (p < 0.0001), 14.4% at year 2 (p < 0.0001), 15.9% at year 3 (p = 0.0006), 12.7% at year 4 (p = 0.0047), and 12.2% at year 5 (p = 0.0011). In patients with asthma managed according to GINA recommendations, asthma symptoms control and lung function improved after 3 months and the improvement was sustained over 5 years.Symptoms control remains challenging for most patients with asthma. This study was conducted to evaluate the level of asthma symptoms control and lung function over 5 years of GINA  implementation. We included all patients with asthma who had been managed following GINA recommendations at the Asthma and COPD Outpatient Care Unit (ACOCU) of the University Medical Center in Ho Chi Minh City, Vietnam from October 2006 to October 2016. Of 1388 patients with asthma managed following GINA recommendations, the proportion of patients with well-controlled asthma significantly improved from 2.6% at baseline to 66.8% at month 3, 64.8% at year 1, 59.6% at year 2, 58.6% at year 3, 57.7% at year 4, and 59.5% at year 5 ( The Global Initiative for Asthma (GINA) has published recommendations to help physicians diagnose and manage patients with asthma properly . These r1 (forced expiratory volume in one second) increased from 71.8% to 79.7% of predicted values [Since 2000, we have implemented the GINA strategy at the Asthma and COPD Outpatient Care Unit (ACOCU) of the University Medical Center in Ho Chi Minh City (UMC-HCM). We have transferred this model of ACOCU to other hospitals, especially district-level hospitals throughout Vietnam. A one-year follow-up study at 4 ACOCUs in Ho Chi Minh City showed that the proportion of patients with GINA-defined well-controlled asthma rose from 1.0% to 36.8%, and average FEVThis is a retrospective, observational, and real-world follow-up study. This study was conducted at the ACOCU of UMC-HCMC, a tertiary teaching hospital in southern Vietnam. This unit functions as a model for asthma management in the community in Vietnam . All doc2-agnosit (LABA)  or montelukast [® spirometer  before and 15 min after inhalation of 400 microgram of salbutamol. The spirometry maneuvers met the American Thoracic Society/European Respiratory Society quality standard criteria [Patients with asthma had been managed by attending doctors following GINA recommendations. In short, patients with stable asthma had been prescribed asthma controllers based on a stepwise approach depending on their level of symptom control and spirometry results . The asttelukast . At the criteria . Spiromecriteria . At eachcriteria , asthma Eligible subjects were all patients with asthma who were older than 12 years of age attending the ACOCU of UMC-HCM from October 2006 to October 2016. Patients were included if they fulfilled all the following criteria: a definitive diagnosis of asthma had been previously established, based on clinical symptoms such as recurrent attacks of cough, wheezing, chest tightness, and shortness of breath in the early morning or when exposed to the triggering factors; asthma symptoms had occurred more than one year before the enrolment; the first visit to the unit must have taken place from October 2006 to October 2011; and having had at least 2 visits to the unit. Patients were excluded from the study if they had any of the following criteria: pregnancy; breast-feeding; other chronic diseases that affected the quality of life such as active pulmonary tuberculosis, all types of cancer, congestive heart failure, kidney failure, and liver failure.Data were extracted from patients’ medical records which were kept in two forms: the electronic form had been saved on the server of the hospital and the paper form had been kept in a storage room. All the patients were followed up for 5 years and were censored when they stopped to attend the unit or until October 2016. During the follow-up period, patients might have several visits which might not take place at the predetermined time; consequently, only the visit which was closest to the predetermined time was chosen. The times of follow-up visits were preset at 3, 6, 12, 24, 36, 48, and 60 months after enrolment for the seven visits  following baseline assessment.Data of all patients were extracted from the electronic medical records using an in-house extraction program. Paper-based medical records of all patients who met the inclusion criteria were retrieved from the storage room. Data from the electronic medical records were extracted on a spreadsheet of an Excel file. Missing data from the electronic medical records were looked for in the paper-based medical records to fill out the variables on the Excel file.1 and/or PEF (peak expiratory flow) are included in the GINA 2006 [1/FVC  < 0.70 and FEV1 < 80% of predicted values [Study variables included clinical and spirometry data as well as asthma therapies. Level of asthma symptoms control was classified according to the criteria of GINA 2006  for visiINA 2006  but are d values . The perd values ,15. High1 < 60% or on a high dose of ICS between each follow-up visit and the baseline visit was made using McNemar’s test. Comparisons of the number of annual asthma-related exacerbations or hospitalizations between each follow-up visit and the baseline visit were examined by using paired Wilcoxon signed rank test. Comparisons of spirometry parameters between each follow-up visit and the baseline visit were made by paired Student-t-test. Differences between groups were examined using a Chi-square test or Wilcoxon test, as appropriate. A p-value < 0.05 was considered statistically significant. Statistical analysis was done by using JMP 9.0.2 software . Some results of this study were previously published in abstract form [Comparison of the proportion of patients with well-controlled asthma or with persistent airflow limitation or with FEVact form .Among 4871 patients with asthma extracted from the electronic medical records, 1388 patients (28.5%) met all the inclusion criteria . The agep < 0.0001 for all comparisons between each of the follow-up visits and the baseline visit)  . The impe visit) .There was no relation between baseline characteristics and the proportion of patients with well-controlled asthma at year 1 or at year 5 , 14.4% at year 2 (p < 0.0001), 15.9% at year 3 (p = 0.0006), 12.7% at year 4 (p = 0.0047), and 12.2% at year 5 (p = 0.0011) (1 < 60% significantly decreased from 18.5% at baseline to 7.4% at year 1 (p < 0.0001), 7.3% at year 2 (p < 0.0001), 9.2% at year 3 (p = 0.0006), 7.3% at year 4 (p = 0.0073), and 6.5% at year 5 (p = 0.0455) .The proportion of patients with persistent airflow limitation significantly decreased from 26.7% at baseline to 12.6% at year 1 ( 0.0011) . Similar 0.0455) . The difp < 0.0001), 31.7% at year 2 (p < 0.0001), 35.6% at year 3 (p < 0.0001), 39.5% at year 4 (p < 0.0001), and 27.2% at year 5 visit (p < 0.0001)  significantly decreased from 79.2% at baseline to 31.4% at year 1 ( 0.0001) .p = 0.0178); having allergic rhinitis (p = 0.0059); BMI < 25 kg/m2 (p = 0.0219); having co-payment by health insurance (p = 0.0462); receiving fluticasone propionate (p = 0.0111) , 436 (31.4%), 309 (22.3%), 197 (14.2%), and 128 (9.2%), respectively . In the  0.0111) . In mult 0.0111) .1 < 60% fell significantly. These improvements occurred after 3 months and were sustained over 5 years of asthma management. These results corroborate the findings from our previous study which showed that the levels of asthma control and lung function improved after 3 months and were sustained over one year [1 started to improve within days, and reached a plateau after around 2 months [This real-world study showed that if patients with asthma were managed appropriately, the proportion of well-controlled asthma rose significantly, lung function increased significantly, and the proportion of persistent airflow limitation or of FEV2 months . The imp2 months .1 and/or PEF as one of the criteria) than GINA 2016 criteria [The finding that there was no difference in baseline characteristics between patients with well-controlled and patients with uncontrolled or partly controlled at year 1 or at year 5 suggests that about 60% of patients with asthma in the real world could be well-controlled if they were managed appropriately over time by attending physicians who followed GINA recommendations. This is in line with the results of a post-hoc analysis of the GOAL study which showed that the proportion of patients achieving well-controlled asthma according to GINA 2016 criteria ranged from 40–64% after one year of receiving ICS/LABA . Well-cocriteria  or in 20criteria  or 13.9%Most (96.8%) patients with asthma in this study received ICS/LABA as asthma controllers at the baseline visit because 93.2% of patients were moderate or severe persistent asthma. The reasons why no patient received stand-alone ICS at the baseline visit may be as follows: patients with asthma only visited the clinic when they were severe enough that attending doctors had to initiate asthma controllers at step 3; stand-alone ICS was not available at the clinic when attending doctors prescribed asthma controllers; and attending doctors might prefer ICS/LABA combination to stand-alone ICS. The finding that fluticasone propionate was prescribed as an asthma controller twice more than budesonide in this study was due to the availability of ICS molecules at the clinic and the preference of attending doctors. Montelukast was used as an add-on controller for patients with asthma on steps 3–5 or as a stand-alone controller for asthma patients with asthma on steps 1–2. In this study, the dosage of ICS/LABA was stepped down over time as recommended by the GINA strategy . The proThe proportion of patients who attended follow-up visits fell significantly after one year. This finding is in line with a finding from a national pharmacy database study in the United States . Of the 1 < 60% is associated with an increased risk of exacerbations [There are several strong points in this real-world study. Spirometry performed at the baseline visit and at follow-up visits helped us to diagnose asthma more accurately and to evaluate treatment response more objectively . One of rbations  and rapirbations , indepenrbations . This firbations . The fivrbations .This study has some limitations. First, there were missing data because this is a retrospective and data-based extraction study. However, with the within-subject analysis of outcomes over the follow-up time, the results are still valid. Second, we do not know what happened to those without follow-up visits. However, there was no difference in baseline characteristics except for allergic rhinitis between patients who attended and patients who did not attend the follow-up visit at year 1. This finding suggests that if patients with asthma continue receiving appropriate asthma treatment, their level of asthma control will be sustained over time, regardless of their clinical characteristics.In conclusion, closer detection and individualized follow-up of patients with asthma by providing them with an appropriate treatment according to GINA recommendations improved asthma symptoms controls and lung function after 3 months and the improvement was sustained over 5 years. This improvement should favorably impact asthma outcomes and alleviate its overall current burden worldwide.


Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory infection (ALRI) in children, causing frequent outpatient visits and hospitalizations. Our study aimed to describe the clinical and direct economic burden of ALRI hospitalizations related to RSV in children in Spain and the characteristics of patients and their episodes. In this retrospective study, ALRI hospitalizations in children aged < 5 years for 2015–2018 were reviewed using anonymized administrative public hospital discharge data from Spain. Three case definitions were considered: (a) RSV-specific; (b) RSV-specific and unspecified acute bronchiolitis (RSV-specific and bronchiolitis); and (c) RSV-specific and unspecified ALRI (RSV-specific and ALRI). The study reported a mean of 36,743 yearly admissions potentially due to RSV, resulting in a mean annual cost of €87.1 million. RSV-specific codes accounted for 39.2% of cases, unspecified acute bronchiolitis for 20.1%, and other unspecified ALRI codes for the remaining 40.6%. The mean hospitalization rate per 1,000 children was 55.5 in the first year of life, 16.0 in the second, and 5.4 between 24 and 59 months. A considerable proportion of cases occurred in children under two years old (> 80.4%) and even during the first year of life (> 61.7%). Otherwise healthy children accounted for 92.9% of hospitalizations and 83.3% of costs during the period. Children born preterm accounted for 1.3% of hospitalizations and 5.7% of costs. The findings revealed that RSV still contributes to a high burden on the Spanish health care system. Children under one year of age and otherwise healthy term infants accounted for most of the substantial clinical and economic burden of RSV. Current evidence potentially underestimates the true epidemiology and burden of severe RSV infection; thus, further studies focusing on the outpatient setting are needed.The online version contains supplementary material available at 10.1186/s12879-023-08358-x. Respiratory syncytial virus (RSV) is one of the most common seasonal infections and a major public health burden , 2. It iCurrent treatment strategies for RSV focus on the management of high-risk children, and supportive care remains the mainstay therapy , 8, 11. Haemophilus influenzae type b disease ) and/or median (interquartile range [IQR]), as appropriate. Statistical analyses were carried out using IBM SPSS Statistics 19.0.A total of 110,229 RSV-specific and ALRI hospitalizations were identified during the three analyzed epidemic seasons, of which 43,328 (39.3%), 35,744 (32.4%), and 31,157 (28.3%) occurred during the 2015/2016, 2016/2017, and 2017/2018 seasons, respectively  . A considerable proportion of cases occurred during the first year of life (61.7% in RSV-specific cases and ALRI and 83.4% mean in RSV-specific cases)  of RSV-specific and ALRI cases were observed in children born at term and 102,437 (92.9%) in children with no known predisposing risk factors for severe RSV infection. Heart disease (3.3%) was the most frequently reported risk factor, followed by neuromuscular disorders (2.5%), congenital disorders of the respiratory system (0.8%), Down syndrome (0.7%), congenital musculoskeletal anomalies (0.5%), bronchopulmonary dysplasia (0.4%), immunodeficiency (0.2%), and cystic fibrosis (0.1%)  days and the median 4.0 days (2.0–6.0). The mean LoS was higher in children with a risk factor and in those born preterm or with low birth weight Table .Table 3MA total of 52,884 (48.0%) RSV-specific and ALRI cases reported a respiratory severity marker. Invasive mechanical ventilation was used in 1,017 (0.9%) cases, non-invasive ventilation in 4,018 (3.6%), and oxygen supplementation in 26,999 (24.5%). In RSV-specific cases, a respiratory severity marker was reported in 22,467 (52.0%) hospitalizations. Invasive mechanical ventilation was used in 474 (1.1%) cases and non-invasive mechanical ventilation in 2,140 (4.9%). Oxygen supplementation was used in 11,160 (25.8.%) cases.Overall, 154 deaths were reported among 110,229 RSV-specific and ALRI cases, resulting in a mean in-hospital case-fatality rate of 0.14%. Similarly, 39 deaths among 43,247 RSV-specific cases led to an in-hospital case-fatality rate of 0.09%. All deaths were observed during the first two years of life, mostly among children with at least one risk factor (72.7%). The in-hospital case-fatality rate was higher among children with at least one risk factor—RSV-specific cases and ALRI with at least one risk factor (112 [1.4%]) versus those without a risk factor (42 [0.0%])—but was overall relatively low. The median age of children who died during an RSV-specific and ALRI hospitalization was 6.0 months (2.5–18.2). Preterm birth, low weight at gestational age, and exposure to tobacco accounted for 25 , 27 (2.1%), and 2 (2.4%) fatalities in RSV-specific and ALRI cases, respectively.The mean yearly total direct medical cost to the NHS for RSV-specific and ALRI hospitalizations in children aged < 5 years during the study period was €87.1 million, ranging from €75.8 million in season 2017/18 to €99.1 million in season 2015/16. Otherwise healthy children accounted for mean yearly hospitalization costs of €72.5 million (83.3%). Children born preterm accounted for 5.7% of costs.The mean NHS hospitalization cost per patient stood at €2,406 SD €3,203) in RSV-specific cases and ALRI. The mean cost per patient was higher in children under two years of age  than in those aged 24–59 months . The mean hospitalization cost per patient was also higher in children with a risk factor , as detailed in Table ,203 in RUsing an administrative data review from 2015 to 2018, we found a high burden in Spain of hospitalizations potentially due to RSV in children aged < 5 years, especially during the first year of life. Most hospitalizations were observed in previously healthy children and in children born outside the RSV season.To our knowledge, this is the first study reporting the burden of ALRI hospitalizations potentially related to RSV in Spain including children aged < 5 years across three seasons and distinct RSV case definitions.The three definitions were designed to maximize the likelihood of identifying RSV cases, starting with hospitalizations classified using RSV-specific codes only, then adding unspecified acute bronchiolitis codes to the previous (RSV-specific and bronchiolitis), and, finally, adding other unspecified ALRI codes (RSV-specific cases and ALRI). Case definitions were based on findings from Cai et al. .The RSV-specific and bronchiolitis and the RSV-specific and ALRI definitions identified 1.5 and 2.5 more potential RSV cases, respectively, than RSV-specific codes alone. RSV-specific case and ALRI were estimated to represent a mean yearly cost for the NHS of €87.1 million, with RSV-specific cases accounting for 39.7% of these costs, unspecified acute bronchiolitis for 16.9%, and the other unspecified ALRI codes for the remaining 43.4%.The RSV-specific and ALRI case definition captured a higher share of cases in children aged 24–59 months than the other two case definitions. The higher share of children aged 24–59 months in our RSV-specific and ALRI definition may be a result of the different RSV infection complications across ages. Hall et al. observed that bronchiolitis was the most frequent diagnosis in RSV-positive inpatients aged < 12 months (reported in 85% of cases), while in children aged 24–59 months, pneumonia (51%) and asthma (60%) were the most frequent diagnoses . For SpaThe extent to which this difference can be explained by the inclusion of non-RSV cases in the RSV-specific and ALRI definition or by a bias in RSV testing and codification toward younger children cannot be ascertained by this study . Given iThe RSV-specific and bronchiolitis case definition identified more potential RSV cases while maintaining a similar demographic profile to the one obtained through the RSV-specific case definition. Furthermore, this case definition has been consistently reported as having high specificity and is frequently used in studies aimed at assessing the RSV burden , 21, 37.The ability of our RSV-specific and ALRI definition to detect true RSV cases and exclude false RSV cases is expected to be more sensitive to local testing and coding practices and should be tested for Spain in future studies. Cai et al. 2020) reported 0.90 (95% CI: 0.85–0.94) specificity using this algorithm for children aged < 5 years in Germany 20 report.Across all case definitions, hospitalization rates decreased as age increased. Most admissions occurred in children under two years old  and, particularly, during the first year of life (61.7%–86.1%). The results are consistent with multiple retrospective studies from Europe that have consistently reported the highest RSV hospitalization rate in the first year of life , 38–41. We observed a mean in-hospital case-fatality rate of 0.09% in RSV specific, 0.07% in RSV-specific and bronchiolitis, and 0.14% in RSV-specific and ALRI cases. For the equivalent case definition, we report a lower case-fatality rate than the 0.14% reported in a previous epidemiological survey in Spain in hospitalizations due to RSV bronchiolitis in children aged < 5 years of age . AnotherThe mean LoS reported in our study is similar to that reported in previous findings, ranging from 4.0 to 5.7 days , 48, 49.The mean cost per patient was also 2.6 times higher in patients with comorbidities than in those without comorbidities, and higher in children born preterm or with low birth weight. The results are consistent with published literature , 51.In terms of total cases, however, hospitalization costs were primarily driven by previously healthy term children and those aged less than two years across all RSV case definitions.Approximately half of the hospitalized children in our study were born outside the November–March period (53.8% to 56.1%), in line with the results reported for infants hospitalized with laboratory-confirmed RSV in the Valencia region . As the Finally, although most RSV hospitalizations were observed between December and February, in line with published dates for RSV peaks in temperate Northern Hemisphere countries , 43, 44,The major limitation of the present study is that it relies on data from an administrative database without linkage to RSV laboratory testing, thus not quantifying the proportion of the reported unspecified ALRI cases caused by RSV and not other infectious agents. The used case definitions were based on algorithms tested in another geography , and theThe study is based on extrapolated administrative data, subject to coding errors or missing information. Laboratory data or drug administration data  were not available. The study period included two ICD systems (ICD-9-CM and ICD-10-ES). Data on oxygenation procedures, prematurity diagnosis, weight, and tobacco exposure may have been understated. Case counts from July to August for each season were not included in the reported incidence rates to reduce the risk of including non-RSV cases. This corresponds to an exclusion of 0.2% of RSV-specific cases, 0.8% of RSV-specific and bronchiolitis cases, and 2.2% of RSV-specific and ALRI cases observed between January 2015 and December 2018.Importantly, this study does not consider the full burden of RSV, as it includes only the burden of potential NHS RSV hospitalizations. An important burden of RSV is expected to be driven by cases treated in the outpatient setting , 28, 36.The findings of this study highlight that RSV represents a major burden in Spain, since it is linked with significant morbidity, particularly in young infants. Previously healthy children accounted for more than 90% of hospitalizations and 80% of hospitalization costs. The study revealed a substantial, underestimated burden of RSV, which is higher in patients aged below one year of age. The results of the BARI study showed that all infants are at risk of acquiring an RSV infection with severe complications, which are unpredictable and cause hospitalization in children without previous risk factors. There is a need to continue efforts to improve surveillance of RSV to support the introduction of prevention strategies in the future.Additional file 1: Listing S1. List of analyzed comorbidities and ICD9/10 codes used. Listing S2. List of ICD-9-MC and ICD-10-ES codes used as “severity markers”. Listing S3. Evolution in the incidence rate of hospitalizations by respiratory syncytial virus definition and age group.


BackgroundLong-term metformin treatment in individuals with type 2 diabetes mellitus causes vitamin B12 insufficiency, which is typically neglected, undetected, and under-treated. A severe deficit may cause life-threatening neurological problems. This study assessed the prevalence of vitamin B12 deficiencies among T2DM patients and its factors at a tertiary hospital in the Tamil Nadu district of Salem.Materials and MethodsThis is an analytical cross-sectional study conducted in a tertiary care hospital in the Salem district, Tamil Nadu, India. Patients with type 2 diabetes mellitus who were prescribed metformin at the outpatient department of general medicine took part in the trial. Our research instrument was a structured questionnaire. We used a questionnaire containing information on sociodemographic characteristics, metformin use among diabetic mellitus patients, diabetes mellitus history, lifestyle behaviors, anthropometric measurement, examination findings, and biochemical markers. Prior to administering the interview schedule, each participant's parents provided written informed consent. A thorough medical history, physical exam, and anthropometric examination were performed. Data were entered in Microsoft Excel  and analyzed using SPSS version 23 .ResultsAmong the study participants, we diagnosed nearly 43% of diabetes cases in participants between the ages of 40-50 years, while we diagnosed 39% aged under 40 years. Nearly 51% had diabetes for 5-10 years, while only 14% had diabetes for over 10 years. In addition, 25% of the study sample had a positive family history of type 2 diabetes. Nearly 48% and 13% of the study group had been on metformin for 5-10 years and >10 years, respectively. The majority, 45%, were found to take 1000 mg of metformin per day, whereas just 15% take 2 g per day. In our study, the prevalence of vitamin B12 insufficiency was 27%, and nearly 18% had borderline levels. The duration of diabetes mellitus, the duration of metformin intake, and the dose of metformin were statistically significant  among the variables associated with diabetes mellitus and vitamin B12 deficiency.ConclusionThe results of the study show that a deficiency in vitamin B12 increases the likelihood that diabetic neuropathy would worsen. Therefore, individuals with diabetes who take larger dosages of metformin (more than 1000mg) for an extended period must have their vitamin B12 levels monitored often. Preventative or therapeutic vitamin B12 supplementation can mitigate this issue. Hyperglycemia is a symptom of the metabolic disorder diabetes mellitus, which is characterized by difficulties in the breakdown of carbohydrates, lipids, and proteins. Patients with diabetes are deficient in absolute or relative insulin. Diabetes mellitus is a significant noncommunicable disease whose prevalence has exploded during the past two decades . In the According to a recent report by the World Health Organization (WHO), India has the highest number of diabetics in the world, surpassing China . Diabete In the "biguanide" class, metformin is an anti-hyperglycemic medication. Metformin decreases blood glucose levels and is the cornerstone of treatment for type 2 diabetes . MetformCobalamin is an important B vitamin that is water soluble. Vitamin B12 plays a significant role in normal brain function , DNA synthesis, and red blood cell (RBC) synthesis . VitaminMetformin inhibits calcium-dependent channels in the ileum, hence preventing the absorption of vitamin B12 in the ileum. Several studies have shown that extended use of metformin causes vitamin B12 insufficiency through this mechanism -16. A seThe literature on vitamin B12 deficiency is also devoid of findings, and they have undertaken few investigations on the frequency of vitamin B12 deficiency in Tamil Nadu. This study assessed the frequency of vitamin B12 deficiencies among T2DM patients and its causes at a tertiary hospital in the Tamil Nadu district of Salem.Study setting and study periodWe conducted this study as an analytical cross-sectional study in a hospital of tertiary care in the Tamil Nadu district of Salem. Participants in the study were type 2 diabetics using metformin who visited the General Medicine outpatient department. We conducted the study from November 2020 to April 2021, six months.Sample size estimationWe estimated the sample size based on a 2012 study by Reinstatler et al. in the United States, which found a prevalence of vitamin B12 deficiency of 5.8% . We calcSelection criteriaThe inclusion criteria were patients having type 2 diabetes for at least two years, aged between 30 and 75 years, free of urinary tract infection complaints, taking metformin up to the maximum daily dose of 2,000 mg, and willing to participate in the study. Patients with type 1 diabetes, type 2 diabetes mellitus patients on insulin therapy or mixed therapy (insulin with metformin or other hypoglycemic drugs), pregnant women, patients with pernicious anemia or a history of malabsorption syndrome after gastrointestinal surgery, patients with chronic hepatitis, and those with hypothyroid disorders were excluded from the study.Ethics clearanceBefore conducting the research, we got approval from the Institutional Ethics Committee of Medical College and Hospital (No: VMKVMC&H/ IEC/20/08). The informed consent was drafted in the local language and was based on ICMR criteria. Before beginning the study, we received approval from our institution's Ethics Committee.Data collection procedureWe adopted a semi-structured questionnaire to get data from study participants through interviews. We used a questionnaire containing information on sociodemographic characteristics, metformin use among diabetes mellitus patients, diabetes mellitus history, lifestyle behaviors, anthropometric measurement, examination findings, and biochemical markers. After receiving consent, a single investigator performed the interview, and their responses were documented on the questionnaire. Each participant was interviewed for 10 to 15 minutes. A thorough medical history was taken and after collecting blood samples, biochemical analyses were conducted.Data analysisData were entered in Microsoft Excel  and analyzed using SPSS v. 23 . Descriptive and analytic statistics were used for our data analysis. We carried out a descriptive analysis and presented qualitative data as frequencies and percentages as well as means and standard deviations for qualitative data. The odds ratio was determined, and the chi-square test was employed for statistical analysis .About 47% of samples were of patients aged 40-50 years, while only 12% were over the age of 60. The respondents' average age was 49.93 ± 9.58 years. In our survey, there was a preponderance of men (55%), and only 45% were females. Around 16% of the population was illiterate, while 31% had completed middle school and 22% had completed high school. Regarding occupation, 25% were unemployed, while 40% and 24% were engaged in unskilled and semi-skilled occupations, respectively , and 4% had coronary artery disease.Around 43% of diabetic patients were diagnosed between the ages of 40 and 50, whereas 39% of diabetic patients were diagnosed before the age of 40. Nearly half had diabetes mellitus for 5-10 years, while only 14% had diabetes for beyond 10 years. In addition, 25% of the study sample had a positive family history of type 2 diabetes. Estimated 48% and 13% of the study group had been on metformin for 5-10 years and > 10 years, respectively. Around 45% of patients were taking 1000 mg of metformin per day, whereas just 15% were taking 2 g per day. About 45% of responders on metformin report taking 500 mg twice daily, while 39% were taking 500 mg three times daily.In our study, 27% of participants had vitamin B12 deficiency, approximately 18% had borderline levels, and the mean vitamin B12 level was 308.6 ± 6115.58 pmol/L. Around 27% of respondents were deficient in vitamin B12 (148 pmol/Litre), whereas 55% were normal (> 221 pmol/litre)  among the variables related to diabetes mellitus and vitamin B12 deficiency. We described the association between variables related to diabetes mellitus and vitamin B12 deficiency among the study respondents in Table In our study, variables significantly correlated with serum vitamin B12 values were duration of diabetes mellitus , duration of metformin use , daily dose of metformin , and hemoglobin . We found no significant correlation for other variables . We showed the correlation between serum vitamin B12 value and study variables in Table In our study, the mean age of the respondents was 49.93 ± 9.58 years. The mean age was comparable to our study in studies by Raizada et al., Agarwal et al., Baidya et al., Miyan et al., and Aroda et al., in which the mean age was 50.1 ± 11.5 years, 51.98 ± 5.17 years, 51.37 ± 4.49 years, 51.16 ± 14.64 years, and 51.2 ± 10.1 years, respectively ,23-26.We have observed male preponderance (55%) in our study, and females made up only 45%. Similarly, we noted male preponderance in studies by Reinstatler et al., Baidya et al., Chattopadhyay et al., and Singh et al., in which males made up 50.3%, 52%, 57%, and 57.1%, respectively ,24,27,28In our study, the mean duration of metformin medication was 6.42 ± 3.37 years, whereas, in studies by Ahmed et al., Nervo et al., Sato et al., Baidya et al., Kanyal et al., and Roy et al. the mean years of metformin use were 9.6 ± 6.8 years, 4 years, 2.8 ± 3.3 years, 5±0.8 years, 3.23 ± 1.17 years, and 2.05 ± 0.39 years respectively ,29-33.Nearly 45% were taking 1000 mg/day of metformin and just 15% were taking 2 g/day in the present study. In the study by Krishnan et al., 83.9% of patients were taking over 1000 mg of metformin per day . In reseIn our study, 27% of participants had a deficiency of vitamin B12, and nearly 18% had levels close to the upper limit. In publications by Ahmed et al., Krishnan et al., Baidya et al., and Tal et al., the prevalence was found to be 28.1%, 28.3%, 22%, and 24%, respectively ,34,36. OIn our study, vitamin B12 deficiency was significantly associated with age over 45, alcohol intake, diabetes duration of seven years, overweight/obese BMI category, duration of metformin use for seven years, and a daily dose of over 1000 mg. We noticed similar findings in studies by Aroda et al., al-Hamdi et al., Ahmed et al., Krishnan et al., Reinstatler et al., Ko et al., and Raizada et al. in which vitamin B12 deficiency was associated with the duration and daily dose of metformin therapy, respectively, in these studies ,34,35,37Using proton pump inhibitors and H2 blockers, as well as the duration of metformin therapy, was found to be significantly associated with vitamin B12 deficiency in the Damiao et al. study . In the In our study, vitamin B12 deficiency was negatively correlated with the duration and daily dose of metformin therapy and positively correlated with hemoglobin values. We observed comparable results in studies by Sato et al., Agarwal et al., Nayyar et al., and Kanyal et al. ,29,32,42LimitationsOur study had a few limitations. First, the study was conducted with a smaller number of samples because of a lack of resources. However, the results might be generalized to a comparable population in a comparable setting. We did not perform nerve conduction investigations and neuropathy screening because of logistical difficulties. A cross-sectional examination of causal relationships and risk estimates is not feasible. A future analytic investigation (case-control or cohort study) in the study population can circumvent this constraint. Patients on combined therapy (metformin plus other medicines) were excluded, which restricts the generalizability of the study. These patients may experience a higher incidence of B12 deficiency and/or neuropathy (or lower).According to the findings of the study, a large proportion of the sample group was deficient in vitamin B12. This study also demonstrates that there are gaps in the identification and treatment of vitamin B12 deficiency in the study area, as it is frequently undiagnosed. The longer duration and higher daily dose of metformin were the risk factors for vitamin B12 insufficiency. A vitamin B12 shortage increases the likelihood that diabetic neuropathy may worsen. Therefore, monthly vitamin B12 measurement is required for diabetic patients on long-term, high-dose metformin. Preventative or therapeutic vitamin B12 supplementation can mitigate this issue.


Meliponula ferruginea propolis from 10 wooden hives (n = 49). The samples were collected from five different locations comprising the entrance, involucrum, pillars, pots and sealant. Principal component analysis showed that there is an intra-hive variation in phytochemical content and RSA. Phenolic content constituted the highest phytochemical content in all the locations. The sealant and entrance had the highest amounts of phytochemicals compared to the involucrum, pillars and pots. Further analysis of propolis extracts by gas chromatography-mass spectrometry revealed occurrence of different compounds such as monoterpenoids, hydrocarbons, triterpenoids and alkaloids. Hydrocarbons were common in all parts while monoterpenes and triterpenes were present in the entrance. The findings of our study indicates that there is an intra-hive variation in propolis of M. ferruginea and hence this information will provide further insight into better understanding of stingless bee propolis.Research on stingless bee products has increased in recent years, and of particular interest is propolis because of its biological activities such as antioxidant and antimicrobial. However, there is paucity of information regarding intra-hive variations in the biochemical composition and biofunctional properties of this propolis. In this study, we investigated the phytochemicals and radical scavenging activity (RSA) of  Feral honeybees inhabiting tree cavities also use propolis to smooth the inside of the cavity [Propolis is a resinous and complex phytocompound collected by bees from living plants, to serve various physical and physiological functions in a beehive including construction and adaptation of their nests . Feral he cavity . It alsoe cavity  and in ee cavity . The bene cavity . The bioe cavity . These be cavity ,6. TheseStingless bees also produce propolis which has a different name, cerumen  and it iMeliponula ferruginea belongs to subgenus Axestotrigona with the species being first described by Lepeletier in 1836 and recently by Michener in 2000 [M. ferruginea has the following parts: entrance tube, nest cavity, batumen and involucrum layer, storage pots, brood combs and drainage tube [The stingless bee  in 2000 . They ar in 2000  and are  in 2000 . In cont in 2000 . The basage tube . Batumenage tube , while iM. ferruginea propolis indicate that they do not have preference to the kind of resins they use for nest construction [M. ferruginea propolis. This study investigated the phytochemical composition and RSA of propolis from different locations in the hive. The findings of our study will help to further understand stingless bee propolis and add more knowledge to the scarce information regarding African stingless bees.The chemical composition of propolis depends on the botanical sources, the availability of plant resins to bees and the bees' preferences . Some sttruction  and henctruction . Moreove. 2. 2.1. 2.1.12CO3), ethanol.Folin-Ciocalteu's reagent, gallic acid, sodium carbonate (Na. 2.1.23), sodium nitrite (NaNO2), sodium hydroxide (NaOH).Quercetin, aluminium chloride (AlCl. 2.1.32SO4), methanol.Linalool, chloroform, sulfuric acid (H. 2.1.43), 1, 10-phenanthroline hydrochloric acid (HCl) and liquid nitrogen.Colchicine, ferric (III) chloride .. 2.1.6Dichloromethane.All purchased from Sigma-Aldrich (Kobian Kenya Ltd.) and were of analytical grade.. 2.2n = 49) were collected directly from 10 wooden M. ferruginea hives at icipe- Nairobi Duduville campus meliponary . The hives were opened using a hive tool and the sealant and entrance were scraped off and wrapped in aluminium foil. The other parts, namely, involucrum, pillars and pots were gently cut with sterile scalpel and were wrapped in aluminium. All these parts are indicated in Propolis samples  in a 15 ml Falcon tube and left to soak at room temperature for 72 h. They were vortexed at 3000 rpm for 3 min after every 24 h. After the extraction, they were centrifuged at 600 rpm for 2 min and the supernatant decanted and stored at −80°C.For gas chromatography-mass spectrometry (GC-MS) analysis, the extraction was done using dichloromethane by dissolving 100 mg of the propolis samples from the different locations of three hives in 10 ml of dichloromethane. The resulting mixture was vortexed at 3000 rpm for 2 min and then incubated for 24 h at room temperature. They were then filtered through glass wool and the filtrate further diluted with dichloromethane by taking 0.1 ml of it in 1.9 ml of dichloromethane.. 2.4. 2.4.13) colorimetric assay was carried out as described [2 in a test tube. After 5 min, 0.3 ml of 10% AlCl3 was added to the mixture and left for another 1 min before adding 2 ml of 1 M NaOH and finally 2.4 ml of distilled water was topped-up. The absorbance was measured using a spectrophotometer  at 510 nm against a reagent blank with water instead of the sample and quercetin was used to generate a calibration curve (20–200 µg ml−1), For the quantification of total flavonoid content (TFC), an aluminium chloride , The Folin–Ciocalteu method was used to quantify total phenol content (TPC) as described . Briefly. 2.4.33 in 0.5 M HCl and 1 ml of 1, 10-phenanthroline (0.05 M) prepared in 50% ethanol and water mixture (v/v) was added to 1 ml propolis extract, and the mixture incubated for 30 min in a hot water bath at 70°C. The absorbance of red coloured complex was measured at 510 nm against reagent blank. This was done in triplicate for each sample. Alkaloid contents were determined using a standard curve of colchicines (0.1–1.5 mg ml−1), prepared by dissolving 20 mg of colchicine in 10 ml of a 50% mixture of ethanol and water (v/v) and diluted to the required concentration, The total alkaloid content (TAC) of the propolis extracts was determined using the 1, 10-phenanthroline method as described previously . In brie. 2.4.42SO4. The supernatant was decanted gently to leave a reddish brown precipitate at the bottom and 1.5 ml of absolute methanol added and vortexed again until the precipitate dissolved completely. Each sample was assayed in triplicate. The same procedure was repeated for the standard using different concentrations of linalool (10 mg ml−1–500 mg ml−1), The total terpenoid content (TTC) was quantified using the colorimetric method . This wa. 2.5−1), The DPPH assay was carried out as described . Briefly. 2.6−1. The oven temperature was programmed at 35°C where it was held for 5 min followed by a gradual increase at the rate of 10°C min−1 to 280°C where it was held at an isothermal state for 10 min. The transfer line temperature was at 280°C while the temperature of the ion source was 230°C and the ionization energy was 70 eV. Fragment ions analysed in scan mode over 40–450 m/z mass range and MS quadrupole temperature was maintained at 150°C.GC-MS analysis of propolis extracts was carried out using a GC  directly coupled with spectrometer . Chromatographic separations was achieved by an HP5-MSI capillary column, 30 m in length × 0.25 mm in diameter, 0.25 µm film thickness  immobilized with 5% phenyl methyl silicone as the stationary phase. An aliquot (2 µl) of diluted propolis sample was injected into the GC-MS instrument, in the splitless mode, using an auto sampler  at an injector temperature of 270°C.The sample was then transported by Helium gas (99.99% purity) as the carrier gas at a flow rate of 1.2 ml min. 2.7factoextra 1.0.5 and ggplot2 3.1.1 [post-hoc Dunn's test after confirming that the data for the parameters were not normally distributed. A table of means and boxplots were also prepared using package ggplot2 3.1.1.The R working environment v 3.5.0 (R Core Team 2019) along with packages t2 3.1.1 ,26 was uThe relative integration of each identified peak was determined using the ChemStation integrator. To eliminate potential column, or solvent contamination, blank runs were conducted and subsequently analysed. Detected peaks were initially identified through a comparative analysis of mass spectral data against reference spectra published by library–MS databases: National Institute of Standards and Technology (NIST) available versions of 2005, 2008 and 2011 as well as by considering retention times. When authentic standards were available, the compound identifications were definitively confirmed by comparing their fragmentation patterns and retention times, with those of commercially available standards.The data presented had a similarity structure compound estimate (similarity index) ≥90% . Predict. 3. 3.1p < 0.0001 . This is in accordance with the phytochemical data where the same have high total content of the phytochemicals.Radical scavenging activity (% RSA) is the percentage of DPPH radical molecules that has been reduced to stability rendering it non-reactive. From the box plots , there i. 3.1.2Analysis of GC-MS chromatograms of the five hives with samples representing each location showed more than a hundred peaks indicating the presence of various compounds . These compounds belong to different classes such as hydrocarbons, terpenoids, alkaloids, triterpenoids, fatty acid and phenols . The num. 4. 4.1Geniotrigona thoracica propolis of 29.1 mg g−1 GAE and 19.78 mg g−1 QE [−1 GAE and 0.16 to 3.39 mg g−1 QE TFC are in the range of our results including pillars, pots and cerumen [Varying amounts of phytochemicals illustrate the complex chemical composition of propolis as a mixture of plant resins and bee secretions . In sting g−1 QE . The phyg g−1 QE . In comp cerumen .Trigona sp. propolis extracts [Apis mellifera propolis extract has values ranging from 6.2 to 9.8 mg/100 g which is lower compared to our results [Monoterpenes and sesquiterpenes are among the terpenoids that make up the volatile components of the propolis and are known to produce the aroma given off by the propolis . Terpenoextracts . Indian  results . Alkaloi results . These p. 4.2−1 in our study promoted varying antioxidant activity, with the highest being 80%. The higher the percentage recorded means the extract has a higher antioxidant activity. Our results are in accordance to those that have been reported in different stingless bee species [Tests for RSA such as the DPPH assay has been extensively applied to determine the RSA of extracts and pure substances . Its sta species . Propoli species . Plant a species . These a species . We adop. 4.3In this study, the propolis samples had varying compound profiles as reported in other studies . There w. 5M. ferruginea propolis exhibits intra-hive variation in accordance to its location in the hive. This is corroborated by the total phytochemical content and GC-MS analysis of the propolis extracts. The difference can be attributed to the various functions of propolis in the hive leading to different resins being incorporated. The different compounds also result in varying RSAs. Our work provides additional information to the existing knowledge on stingless bee propolis since it is, to our knowledge, the first report on stingless bee intra-hive variation in phytochemical composition and RSAs of M. ferruginea propolis.The outcome of our study suggests that the biochemical content of


The generic dispensing ratio (GDR)-the number of generic fills divided by the total number of prescriptions-is a standard performance metric on which pharmacy benefit designs and their managers are routinely evaluated. Higher GDRs are considered important because they consistently produce lower prescription drug costs.To (a) quantify the relationship between GDR and gross pharmacy expenditures and (b) distinguish pharmacy cost savings realized from brand-to-generic conversion from those due to brand drug utilization decreases.This study was a longitudinal, retrospective analysis of paid pharmacy claims and insurance eligibility information for 548 employers covering nearly 14 million members. Data were from the period January 1, 2007, through December 31, 2009, aggregated quarterly. In a linear fixed effects model controlling for plan membership demographics and time trends, percentage changes in gross pharmacy expenditures per member per quarter (PMPQ) were associated with changes in GDR. A second model estimated the association of GDR with gross pharmacy cost, holding total drug utilization constant. All claims counts were adjusted to 30-day equivalents, and expenditures were log-transformed.Mean generic claims PMPQ increased by 18.4% during the study period, from 2.01 in 2007 Q1 to 2.38 in 2009 Q4. Conversely, brand claims PMPQ decreased by 21.0%, from 1.76 in 2007 Q1 to 1.39 in 2009 Q4. As a result, mean GDR per plan increased by 9.8 percentage points or a relative change of 18.2%, from 53.9% in 2007 Q1 to 63.7% in 2009 Q4. Over the 3 years, average gross pharmacy costs PMPQ increased by 14.0% from $242 to $276. The relationship between GDR and gross pharmacy expenditures, estimated in the linear fixed effects multivariate models, varied depending upon whether or not total utilization was controlled. In the first model, which did not control for total utilization, each percentage point increase in GDR was associated with a 2.5% reduction in gross pharmacy expenditure. Holding total utilization constant, the reduction in gross pharmacy expenditure for each percentage point increase in GDR was 1.3%.Prescription drug cost savings are realized with increases in GDR. During 2007-2009, each 1 percentage point increase in GDR was associated with a drop of 2.5% in gross pharmacy expenditures. Slightly more than one-half of the savings was derived from the lower drug prices enjoyed with brand-to-generic conversions. The remaining savings, however, were attributed to reduced brand drug utilization. Pharmacy benefit managers and plan sponsors should exercise care to ensure that increases in GDR do not represent reductions in appropriate medication use.


Mental illnesses affect one in eight people in the world according to the WHO in 2019. They are a leading cause of morbidity and a major public health problem. Stigma harms the quality of life of people with mental illness.Our study aimed to evaluate the association of mental illness stigma with socio-demographic characteristics in Tunisian students.This is a cross-sectional study conducted on Tunisian students who anonymously completed a form circulated online through the groups and social network pages related to each academic institution. The form was containing an Arabic validated version of the “Mental Health Knowledge Schedule” (MAKS) and the “Reported and Intended Behaviour Scale” (RIBS) along with a sociodemographic questionnaire.-3) and RIBS (p<10-3). In addition, we found greater knowledge of mental illness and less discrimination among participants with a psychiatric history (p=0.013 and p<10-3 respectively) and among those who had previously attended a stigma awareness session (p=0.020 and p=0.002 respectively). We also noted higher behaviour scores among people with substance use (p<10-3) and lower scores among people with religious beliefs (p=0.009).We have included 2501 Tunisian students with a sex-ratio Male/Female of 0.37. The mean age was 21.57 (±2.55) ranging from 17 to 42 years. Participants’ fields of study were: Science and Technology (58.7%), Literature , Economics and management (15.8%), and Arts (4.8%). Among them, 17.1% had a history of family psychiatric disorders and 17.6% had a psychiatric disorder. Besides, 20.9% of the students were using tobacco and 75.6% of them were religious. We also found that 26.7% of participants had previously attended an awareness session. Several determinants had a statistically significant association with the stigma of mental illness in our study population. We noted that females had higher mental health knowledge scores (p=0.001), while males had higher behavior scores (p=0.002). Moreover, students in the scientific and literary fields had higher scores on both MAKS (p<10Our results show a multiplicity of factors related to mental illness stigma that we can target in anti-stigma strategies. Addressing stigma is a long-term effort; small and large-scale interventions should be considered and evaluated on an ongoing basis to strive for a better future.None Declared