Adjuvant treatment with Gliadel wafers may prolong overall survival (OS) for malignant glioma patients without increasing toxicity. In Japan, the long-term OS of these patients treated with Gliadel 7.7 mg implants has not been studied. We evaluated OS and prognostic factors that might affect OS in Japanese patients with malignant glioma who received the Gliadel 7.7 mg implant.This observational, long-term, postmarketing surveillance was an extension of a previous surveillance. Data were collected through case report forms at 2 and 3 years after Gliadel implant. Up to 8 Gliadel wafers (61.6 mg of carmustine) were placed over the tumor resection site. Primary endpoints were OS and prognostic factors that may influence OS.P = .002), lower resection rate , recurrence , and concomitant radiotherapy  were identified as significant prognostic factors.Among the 506 patients analyzed, 62.6% had newly diagnosed disease, and 37.4% had recurrent disease; 79.1% had glioblastoma histological type and 79.6% had World Health Organization Grade IV disease. Patients received a median of 8 wafers. The median OS was 18.0 months; OS rates were 39.8% and 31.5% at 2 and 3 years, respectively. Age ≥65 years (hazard ratio [HR]: 1.456; This study confirmed the 2- and 3-year OS of Japanese malignant glioma patients with varied backgrounds after Gliadel implant. With a careful interpretation of indirect comparisons with previously reported data, the results suggest that prognosis could be improved with Gliadel implants.NCT02300506 Gliadel wafers plus standard of care yielded survival benefits for malignant glioma.Age, resection rate, recurrence, and radiotherapy may predict prognosis.To our knowledge, this study is the first to conduct a large-scale analysis of Gliadel use and its effect on the long-term overall survival of Japanese patients with malignant glioma. The present results show that overall survival was prolonged for these patients. Potential prognostic factors determined herein can help identify patients who may benefit the most from therapy with Gliadel wafer implants concomitant with temozolomide and radiotherapy.1 Primary malignant brain and other central nervous system (CNS) tumors have an average annual incidence of 7.08 cases/100 000 persons.2 Collectively, these tumors are referred to as malignant gliomas and are graded based on their malignant behavior according to the World Health Organization (WHO) Grades I–IV.3 Reportedly, the median survival of patients with high-grade malignant glioma (glioblastoma [GB] and anaplastic astrocytoma [AA]) is 8–18 months.2 GB is the most common and most malignant glioma subtype  and has a relative 5-year survival rate of approximately 5%–16%.4Globally, gliomas account for approximately 81% of primary malignant intracranial tumors.5 However, efficient delivery of drugs to the brain and the CNS is challenging; thus, new drug delivery methods are needed. Carmustine  is a nitrosourea alkylating agent that exerts its antitumor effect by alkylating DNA and RNA. The carmustine wafer implant  is a biodegradable copolymer (prolifeprospan 20) impregnated with carmustine. This method allows for the controlled-release delivery of carmustine within the confines of the brain tumor environment.6–9The most common initial approach and the standard of care for malignant gliomas is maximal safe surgery, followed by radiotherapy, and in some cases, chemotherapy.10 The median OS was 13.9 months for the Gliadel wafer-treated group and 11.6 months for the placebo-treated group (log-rank P = .03 stratified by country), with a 29% reduction in the risk of death in the treatment group.10 The results of a recently conducted retrospective study11 and a systematic review12 suggested adjuvant radiotherapy with concomitant and adjuvant temozolomide , and Gliadel wafers may contribute to improving OS without increasing toxicity. In patients with recurrent malignant gliomas, the median OS of the 110 patients who received carmustine polymers was 31 weeks compared with 23 weeks for the 112 patients who received only placebo polymers .13 More recently, a phase 1/2 clinical trial was conducted in Japan to evaluate the efficacy, safety, and pharmacokinetics of Gliadel implants in 16 patients with newly diagnosed malignant gliomas and 8 patients with recurrent malignant gliomas. Patients with newly diagnosed malignant gliomas achieved OS rates of 100.0% and 68.8% at 12 and 24 months, respectively.14A phase 3 study conducted in 14 countries, including the United States, Germany, France, the United Kingdom, Scotland, Finland, and Israel, suggested a prolongation of overall survival (OS) in newly diagnosed patients with malignant glioma who received Gliadel wafer implants.15 However, the evaluation period included in our previous surveillance was too short to evaluate the long-term OS in patients with Gliadel wafer implants. Therefore, we conducted a postmarketing surveillance of long-term observation to investigate the prognosis of patients with malignant glioma and prognostic factors of these patients based on extended observation of patients who were surveyed and whose case report forms (CRFs) were collected in our previous surveillance.Based on the available clinical evidence, the marketing approval for Gliadel as adjuvant therapy for malignant glioma was obtained in September 2012 in Japan, and the implant was marketed from January 2013. Thus far, data on the long-term OS of patients who received Gliadel wafers as adjuvant therapy for malignant glioma in Japan are limited. Therefore, we conducted a postmarketing surveillance to investigate the long-term OS of malignant glioma patients with Gliadel wafer implants in Japan. Previously, we described the safety results of 558 patients who received Gliadel wafers as adjuvant therapy for malignant glioma after the market launch of Gliadel on January 9, 2013 to July 10, 2013.15 This study was an extension of the previous surveillance, and it was conducted between April 1, 2014 and March 31, 2017. The total observation period after Gliadel wafer placement was 3 years.A postmarketing surveillance was previously conducted by Eisai Co., Ltd, which examined all patients with malignant glioma who received treatment with Gliadel since its launch in accordance with the principles of Good Post-Marketing Study Practice (GPSP) in Japan.GPSP, an authorized guideline for postmarketing surveillance, does not require the establishment of an institutional review board or ethics committee. However, the data in this surveillance were collected with due consideration of the Declaration of Helsinki and data integrity, in compliance with the personal information protection law in Japan. Patients who participated in the previous surveillance, agreed to participate in the current study, and provided signed informed consent were enrolled using a central registration system from the previous surveillance sites.CRFs were collected at the end of the second and third years after receiving the Gliadel implant. The data from the CRFs were recorded by attending physicians. According to the size and shape of the tumor resection cavity, up to 8 wafers of Gliadel, impregnated with 61.6 mg of carmustine, were used to cover the brain tumor resection site.The endpoints were OS and prognostic factors (patient background factors at baseline and factors after Gliadel implant) that may be associated with OS outcomes. The OS was defined as the period from the day of Gliadel implant placement until death. For patients who survived past the final observation, the OS period was censored by the day of the final confirmation of survival. For these analyses, OS results were stratified by patient background characteristics, and OS was the responding variable. Analyses of OS and prognostic factors were performed in all patients and patients with newly diagnosed or recurrent glioma.16 The present study investigated prognosis after Gliadel implantation in clinical practice; therefore, a prospective evaluation using the strictly defined Stupp protocol was not conducted. Instead, we performed an exploratory subgroup analysis of the long-term prognosis in subgroups under similar conditions to those in the Stupp protocol. This analysis included patients ≤70 years/>70 years of age with newly diagnosed GB who had received concurrent temozolomide and concurrent radiotherapy. A separate analysis was also conducted that included patients ≤70 years of age with newly diagnosed GB, a resection rate ≥95%, and who had received concurrent temozolomide and radiotherapy.In Japanese real-world clinical practice, concurrent temozolomide and radiation are usually performed as standard of care after surgery; the Stupp protocol is listed as a Grade A recommendation in the Japanese guidelines.The planned sample size of this study was up to 560 Gliadel recipients as this was the number of patients whose CRFs were collected in the previous surveillance when this study was planned. For patient disposition, data were obtained regarding the number of patients for whom a CRF was obtained.Patient background data were summarized using distributions for categorical data and descriptive statistics  for quantitative data. The OS was summarized using descriptive statistics . The rate of events (deaths) among censored subjects was calculated. The survival rates at each time point with 95% confidence intervals (CIs) were calculated by the Kaplan–Meier method, and Kaplan–Meier curves were plotted to show OS rate at each time point. The same analyses were performed using populations stratified by resection rate , age , and WHO classification . These stratification factors were applied to both the newly diagnosed and recurrent subgroups. Prognostic factors that may be associated with OS outcomes were investigated using a Cox proportional hazards model. More specifically, Cox regression analyses  were conducted for the following patient demographic factors to identify the factors that were associated with OS outcomes: sex, age , weight (continuous variable), history of allergy, past medical history, complications , primary or recurrent glioma, resection rate , histopathological diagnosis, number of tumors, number of implanted wafers, Karnofsky Performance Status immediately before implantation of this drug (10–70 vs 80–100), concomitant anticancer agents for malignant glioma, concomitant radiotherapy for malignant glioma, use of a fixation agent for Gliadel, recurrence of glioma after implantation of Gliadel, and elimination of remnant by 30 days after Gliadel implantation. All analyses were performed using 2 populations (stratified by newly diagnosed and recurrent Grade IV glioma). The significance level was set at 5%. All statistical analyses were conducted using SAS System Release 9.3 (SAS Institute Japan Ltd).Of the 561 patients enrolled in this study , 507 patients at 203 sites were enrolled between April 1, 2014 and March 31, 2017. One patient was excluded from the OS analysis for being counted twice.Patient background characteristics are given in The majority of patients (68.0% [344/506]) received 8 Gliadel wafers. The median number of wafers placed was 8 wafers. The percentage of patients who underwent a second placement of Gliadel was 6.9% (35/506).Concomitant therapies after Gliadel placement are also given in The median OS for all patients was 18.0 months . The OS OS in patients with newly diagnosed glioma according to subgroup is given in When stratified by age (<65 and ≥65 years), the median OS was 25.6 months for non-elderly patients  and 16.5 months for elderly patients . The OS rate was 53.2% and 35.8%, respectively, at 2 years and 44.8% and 25.0%, respectively, at 3 years.When stratified by WHO Grade, the median OS was not reached (95% CI: 32.2–NE) for patients with Grade III WHO classification (51/317 patients) and 18.0 months for patients with Grade IV WHO classification (266/317). The OS rate was 72.8% and 39.4%, respectively, at 2 years and 64.7% and 30.2%, respectively, at 3 years.When stratified by type (exploratory), the OS rate at 2 years in patients with GB (264/317 patients), AA (18/317), and anaplastic oligodendroglioma/anaplastic oligoastrocytoma  was 39.8% (95% CI: 33.3–46.2), 48.3% (95% CI: 22.5–70.1), and 87.1% (95% CI: 65.0–95.7) and that at 3 years was 30.5% (95% CI: 24.3–36.9), 48.3% (95% CI: 22.5–70.1), and 76.2% (95% CI: 51.5–89.5), respectively.An exploratory subgroup analysis to evaluate prognosis after Gliadel implant was performed in patients who had received concomitant temozolomide and radiotherapy, which is the standard of care after surgery in clinical practice in Japan. When these patients were extracted and stratified by age (≤70 and >70 years), those aged ≤70 years had a median OS of 23.4 months and a survival rate of 48.9% at 2 years and 40.0% at 3 years . FurtherOS in patients with recurrent glioma according to subgroup is given in When stratified by age (<65 and ≥65 years), the median OS was 15.9 months for 136/189 patients <65 years and 13.2 months for 53/189 patients ≥65 years. The OS rate was 34.4% and 23.5%, respectively, at 2 years and 27.0% and 16.8%, respectively, at 3 years.When stratified by WHO Grade, the median OS was 32.3 months for patients with Grade III gliomas (52/189 patients) and 13.4 months for patients with Grade IV gliomas (137/189). The OS rate was 58.7% and 21.2%, respectively, at 2 years and 45.0% and 16.5%, respectively, at 3 years.When stratified by type (exploratory), the median OS was 13.4 months (95% CI: 11.1–15.1) for patients with GB (136/189 patients), 25.5 months (95% CI: 8.0–NE) for patients with AA (21/189), and not reached (95% CI: 21.5–NE) for patients with AO/AOA (22/189). The OS rate was 21.4% (95% CI: 14.4–29.2), 53.8% (95% CI: 29.9–72.8), and 73.9% (95% CI: 47.8–88.3), respectively, at 2 years and 16.6% (95% CI: 10.2–24.4), 41.5% (95% CI: 19.3–62.6), and 53.3% (95% CI: 27.0–74.0), respectively, at 3 years.P = .002); lower resection rate ; histopathological diagnosis of AA , AO , and AOA ; number of Gliadel wafers used ; concomitant radiotherapy ; and recurrence .Prognostic factors evaluated for association with OS outcomes in all patients who received Gliadel implants are given in P = .001), past medical history , lower resection rate , concomitant chemotherapy , and recurrence after Gliadel placement .For patients with newly diagnosed Grade IV glioma, factors that were significantly associated with OS outcome after multivariate analysis (with selection by the backward elimination method) were age ≥65 years  and recurrent disease .For patients with recurrent Grade IV glioma, factors that were significantly associated with OS outcome after multivariate analysis (with selection by the backward elimination method) were lower resection rate 13 than that reported for patients with newly diagnosed gliomas. A recent study in Japan reported that the OS rate was 68.8% at 24 months in patients with newly diagnosed gliomas and 25% in those with recurrent glioma.14 Our results suggest a beneficial effect of Gliadel wafers (perioperative) when administered concurrently with the standard of care on the survival of patients with malignant gliomas in real-world clinical practice.Previous international studies reported that implantation with Gliadel wafers provided relevant survival benefits (median OS of approximately 13–14 months) to patients with newly diagnosed malignant glioma.4 When considering those findings, we think that our study confirmed a good postoperative prognosis for malignant glioma in a similar patient population. We also conducted an exploratory subgroup analysis of patients in our study who had similar background characteristics to patients in the EORTC–NCIC trial. In this trial,18 adult patients aged 18–70 years with newly diagnosed GB who received standard of care with temozolomide and postoperative radiotherapy (the Stupp protocol) showed an OS rate of 27.2% (95% CI: 22.2–32.5) and 16.0% (95% CI: 12.0–20.6) at 2 and 3 years, respectively. Patients with complete resection showed OS rates of 38.4% (95% CI: 29.4–47.3) and 21.4% (95% CI: 14.3–29.6) at 2 and 3 years, respectively. Patients included in our exploratory analysis had newly diagnosed GB, had received concomitant temozolomide and radiotherapy, and were ≤70 years of age. An additional subgroup analysis of these patients according to resection rate (≥95%) was also conducted. We note that these exploratory subgroup analyses revealed that 2- and 3-year OS rates were 48.9% (95% CI: 40.4–56.8) and 40.0% (95% CI: 31.8–48.2), respectively, and were numerically more favorable in patients with newly diagnosed GB who received concomitant temozolomide and radiotherapy; the same could also be said when patients were further stratified by tumor resection rate (≥95%) . As this is not a direct comparison and the treatment environment between a clinical trial and postmarket clinical practice is quite different, careful interpretation is required. In the recurrent GB subgroup of this study, the median OS was 13.4 months and the 2- and 3-year OS rates were 21.4% and 16.6%, respectively. Previously conducted international clinical trials investigating postoperative Gliadel placement for patients with recurrent GB have reported a median OS of 35.3–50.3 weeks (8.1–11.6 months).20 Our findings, which are comparable to those previously reported, suggest that postoperative Gliadel placement may improve the prognosis in Japanese patients with recurrent GB, which is generally considered to have a poor prognosis.4In patients who received adjuvant treatment with Gliadel for malignant glioma in this study, a subgroup analysis revealed that a good prognosis was observed in newly diagnosed patients with a high resection rate (>95%), aged <65 years, and WHO Grade III tumors. Additionally, a Japanese registry study of patients with malignant glioma showed a relatively high survival rate in patients with a high resection rate and lower WHO Grade tumors.P = .007; AO, HR: 0.224, P < .001; AOA, HR: 0.264, P = .022) and concomitant radiotherapy . We speculate that survival was longer in patients with less aggressive tumors compared with those with more aggressive Grade IV gliomas, thus explaining the association with histological subtype. Regarding concomitant radiotherapy, it was considered that survival benefit might be prolonged due to the therapeutic benefit of this treatment.In patients who received adjuvant treatment with Gliadel for malignant glioma in the present study, multivariate analysis (step-down method) suggested that several variables were determinants of OS in all patients. Variables that indicated a good prognosis were histological subtype , lower resection rate , number of Gliadel wafers implanted , and recurrence of glioma after Gliadel wafer implantation . In the case of age, we considered that the general decline in the physical health of elderly patients was associated with shorter survival. We also considered that a higher resection rate indicated greater effectiveness of surgery, which would likely prolong survival. Importantly, resection rate was identified as a significant factor affecting OS in all 3 analyses . The trend of a prolonged OS with increased resection rate in patients with newly diagnosed GB has been reported previously.21 More wafers may have been implanted in patients with high tumor burden and wider tumor resection areas, suggesting that the number of Gliadel wafers was a significant prognostic factor affecting OS. This is a tentative hypothesis as preoperative tumor burden was not investigated. Regarding recurrence, progression of the primary disease was presumably more rapid in patients with recurrence compared with those without, leading to an association between recurrence and a shorter survival time.Variables that indicated a poor prognosis were age ≥65 years  and tumor mass were suggested to be important predictors of long-term prognosis.This study had several limitations, such as those inherent to drug use surveys, including the heterogeneity of treatment of patients with recurrent disease and lack of randomization and comparator groups. The results have limited generalizability to other ethnic populations.In conclusion, we confirmed the 2- and 3-year OS of Japanese patients with malignant glioma after Gliadel implant (7.7 mg) in the clinical practice setting. Until now, long-term data in Japanese patients treated with Gliadel implants have been limited. Although this study included patients with varied backgrounds, the results suggest that prognosis could be improved with a Gliadel implant.


An absolute change in LCI between a stable condition period and PEx was 1.05 (±1.92) units, which corresponds to a relative change of 11.48% (±18.61) of the baseline. The relative decrease in FEV1 was −9.22% (±12.00) and the z-score was −0.67 (±1.13). After the PEx treatment, FEV1 increased by 11.05% (±9.04) on average, whereas LCI decreased by 1.21 ± 1.59 units on average, which represented 9.42% ± 11.40 compared to the value at the beginning of PEx. (4) Conclusions: The change in LCI captures a higher proportion of events with functional impairment than FEV1 in school-age children with CF.(1) Background: Pulmonary exacerbation (PEx) is one of the main factors affecting the quality of life and life expectancy in patients with cystic fibrosis (CF). Our study aimed to evaluate the change in selected pulmonary function parameters, including lung clearance index (LCI), in patients with CF diagnosed with PEx. (2) Methods: We enrolled 40 children with CF aged 6–17. They performed spirometry and multiple breath nitrogen washout (MBNW) tests during a stable condition period at the beginning and the end of intravenous antibiotic treatment. (3) Results: LCI increased by 65% and FEV Advances in the clinical care of patients with cystic fibrosis (CF) in the past six decades have significantly improved health outcomes . The natHowever, pulmonary exacerbation (PEx) is still one of the main factors affecting the quality of life and life expectancy of individuals with CF. It remains an important clinical event in the course of cystic fibrosis that increases the risk of lung transplant and mortality .1) is the main parameter used in assessing and monitoring CF lung disease. It is also used for diagnosing and detecting treatment response of PEx , in patients with 5–9.99% decrease, it was 4.69 ± 18.22% [−10.79–39.67%], and in patients with ≥10% decrease, 19.23 ± 21.04% [−12.28–60.66%].Under stable conditions, LCI ≥ 7.91 was reported in 37 (92.5%) patients, the median was 11.04 [8.9–13.7]. Upon admission, the mean (SD) LCI was 12.70 (±3.10) units . An incrp < 0.001) by 1.21 ± 1.59 units on average, which represented 9.42% ± 11.40 compared to the value at the beginning of PEx. Spirometry parameters FEV1 and FVC increased from 79.48 ± 13.19% pred. to 90.53 ± 15.44% pred. (p < 0.001), and from 92.98 ± 13.02% pred. to 98.68 ± 13.26, respectively (p < 0.001). After the PEx treatment, FEV1 increased by 11.05% (±9.04) on average. FEV1 and LCI changes between the start and end of PEx treatment are presented in 1, FVC, and LCI during the stable condition, PEx, and after antibiotic therapy for the study population are outlined in After antibiotic therapy, overall LCI decreased significantly  had mild lung disease (FEV1 > 80% pred) and four (10%) had moderate lung disease (40% pred ≤ FEV1 ≤ 80% pred). An exacerbation was diagnosed based on Fuchs criteria. A decrease in pulmonary function (FEV1) by 10% or more from a previously recorded value was observed in only 16 (40%) subjects. Most of the children did not fulfill Fuchs criteria regarding spirometry outcomes. The relative decrease in FEV1 during exacerbation was −9.22% (±12.00) and the z-score was −0.67 (±1.13). After the treatment of PEx, FEV1 increased by 11.05% (±9.04) on average. This observation confirms that in children with early lung disease, spirometry is not sensitive enough to diagnose PEx.In our study, based on spirometry performed during a stable period, most subjects  in stable condition had LCI > 7.91 and only 3 (7.5%) subjects had LCI < 7.91.According to the new reference values reported for MBNW outcomes measured on the same equipment , for healthy Caucasian school-aged children, the ULN for LCI is 7.91. In our study, most of the patients  more than 15%, respectively. A change in LCI as an increase of 10% or more from baseline was the better indicator of PEx than FEV.5% more fibrosis . HoweverWhen comparing LCI during the stable condition and start of antibiotic therapy in 14 subjects (35%), we observed a decrease in this parameter. Some hypotheses could explain the heterogeneity of the LCI results. The overall heterogeneity of ventilation on the LCI will be influenced by the persistent abnormalities of the respiratory tract caused by fibrotic and destructive processes and modifiable differences in inflammation and mucus retention escalating, especially during PEx ,26. ThesLCI as a sensitive marker of ventilation heterogeneity is increasingly used for the detection of treatment effects of variable interventions ,43. Howe1 [Rayment et al. investigated pulmonary function parameters to monitor treatment response in PEx in preschool CF children. They analyzed LCI changes during lower respiratory tract symptoms relative to a recent clinically stable measurement. In this study, LCI increased from baseline in both treated (mean relative change +23.8%) and untreated symptomatic visits (mean relative change +11.2%). A significant antibiotic treatment effect was noticed when LCI was used as the outcome measure (average treatment effect −15.5%) but not for the z-score FEV1 .Our study, as well as those mentioned in the Discussion, indicate the need for further research into LCI use in diagnosing PEx in children with cystic fibrosis. This sensitive parameter could be useful in the diagnosis of PEx and in monitoring children with early lung disease during routine clinical surveillance. The MBNW technique could be helpful especially in young patients who often cannot perform spirometry and adolescents with mild lung disease. Longitudinal analysis is needed to assess LCI potential as a criterion of PEx diagnosis in children with CF and as an outcome measure in detecting treatment response.It is very important to integrate MBNW into routine practice with patients with early lung disease. LCI seems to be a good parameter that can help clinicians diagnose PEx in children with cystic fibrosis and monitor response to the treatment.From the authors’ perspective, LCI is likely to become a valuable tool in clinics especially in children with preserved lung function to identify PEx. In young children (3–5 years old) who cannot perform spirometry, MBNW is the only lung function test that can help in recognizing PEx. In older children, this technique has still been a complementary tool to spirometry. However, there remain some limitations for wider use, particularly the skills and time it takes to obtain reliable maneuvers in children and particularly in those younger than 6 years of age. We are aware that a small group of children with cystic fibrosis were enrolled in our study. Therefore, we cannot generalize results to the whole population. We think that the recommended cut-off for LCI relative change should be 15%. However, an increase above 11% may indicate the need for careful observation and clinical decision-making, such as improving physiotherapy, checking adherence, or starting antibiotic therapy. This study has several methodological limitations. The overall sample size is limited. Outcomes were based on unstandardized clinical treatment decisions . The treatment effect observed in pulmonary function parameters was attributed to antibiotic therapy, but treatment for PEx will also often include the modification of inhaled medications and aggressive chest physiotherapy. These factors could have contributed to the observed treatment effect. The study is focused on school children; however, this narrow group characteristic may not be generalizable to the entire CF population.In the era of better care of patients with cystic fibrosis, there is increasing interest in using the MBNW technique for detecting early changes in lung disease.1 in school-age children with CF during PEx. As FEV1 changed significantly during PEx, compared to baseline, and improved with antibiotic therapy, LCI shows a greater promise in clinical practice, particularly in younger patients with mild disease.Our study results suggest that the change in LCI captures a higher proportion of events with functional impairment than FEVAs we gain more insight into how changes in LCI can be interpreted to guide clinical decisions, MBNW testing may become an integral part of the clinical management of children with cystic fibrosis.


Neonatal brain injuries in England: population-based incidence derived from routinely recorded clinical data held in the National Neonatal Research Database. Arch Dis Childhood – Fetal and Neonatal Edition 2018;103:F301–6.Gale C, Statnikov Y, Jawad S, The authors have identified an error in data extraction that resulted in the following: (1) the inclusion of babies with Grade 1 HIE who did not receive therapeutic hypothermia; (2) the exclusion of babies with central nervous system infection after 48 hours after birth. A data transcription error resulted in incorrect numbers of exclusions. Therefore the following sections are corrected as outlined below.The results section of the Abstract should be as follows: ‘In 2010, the lower estimate of the rate of ‘Brain injuries occurring at or soon after birth’ in England was 3.99 and the upper estimate was 4.66; in 2015, the rate was 4.52 (4.36 to 4.68). For preterm infants, the population incidence in 2015 was 24.45 (23.12 to 25.85) and for term infants 2.91 (2.78 to 3.05). Hypoxic ischaemic encephalopathy was the largest contributor to term brain injury and intraventricular/periventricular haemorrhage was the largest contributor to preterm brain injury.’The first and second paragraph of the Results section of the manuscript are corrected to ‘The number of infants admitted to neonatal units contributing data to the NNRD increased from 64 375 in 2010 to 88 785 in 2015; the annual rate of brain injuries after exclusions in England in 2010 was between 3.87 and 4.44 per 1000 live births; in 2015 to the rate was 4.52 (95% CI 4.36 to 4.68) per 1000 live births ; annual ‘The annual rate of brain injuries among term infants  in England in 2015 was 2.91 (95% CI 2.78 to 3.05) per 1000 live term births; data for term infants born over the period 2010 to 2015 are presented in brain injuries that occur at or soon after birth, and the source of the data, the NNRD which is formed from routinely recorded clinical information makes it necessary to consider how incidence rates of individual conditions we report compare with other published data. The annual incidence rates for moderate and severe HIE of between 1.6 and 1.9 per 1000 live births are consistent with other reported rates of neonatal encephalopathy of between 0.77–3.8 per 1000 live births in low neonatal mortality regions such as the United Kingdom and the United States of America. When considering neonatal intracranial haemorrhage, published data commonly reported incidence separately for term and preterm infants. A 30-year-old, single centre study from the USA reported a regional incidence of 2.7 per 10 000 live births for symptomatic intracranial haemorrhage in term infants, which is comparable with the population incidence of 1.5 to 1.9 per 10 000 term births that we report. For preterm infants born at 22–31 weeks gestational age, comparable population-level incidence data for intraventricular/periventricular haemorrhage from the national French EPIPAGE cohort are 3.8% for grade 3% and 3.3% for grade four intra/periventricular haemorrhage. In the same gestational age band, we report annual incidence rates between 5.8% and 6.1% for a composite including grades 3 and 4 intraventricular/periventricular haemorrhage. We report annual incidence rates for neonatal or perinatal stroke of between 0.11 and 0.15 per 1000 live births, which is similar to the estimated minimum incidence rate of 0.10 per 1000 live births reported by a prospective, population-based study from Canada. The annual incidence of neonatal central nervous system infection we report of 0.56 to 0.82 per 1000 live births is similarly in agreement with the population-level incidence rate for neonatal meningitis in England and Wales of 0.39 per 1000 live births (1996–1997) reported by the British Paediatric Surveillance Unit (BPSU). Similarly, annual rates of bilirubin encephalopathy reported here of between 0.3 and 1.2 per 100 000 live births are comparable with BPSU population surveillance rates of 0.9 per 100 000 live births (2003–2005). EPIPAGE 2 (2011) report an incidence among 23 to 34 gestational week infants of 1.8% for cystic periventricular leukomalacia; in the same gestational age group, we report annual rates of between 1.13% and 1.27%. Finally population-based studies of neonatal seizures over the last 30 years report incidence rates between 1.8 and 3.5 per 1000 live births, results that are again in keeping with our annual rates of 1.9 to 2.2 per 1000 live births.’The third paragraph of the Discussion section of the manuscript is corrected to: ‘The novelty of the measure The following 4 tables  are corr


Blood urea nitrogen (BUN) affects the reproduction of dairy cows, but it is challenging to measure routinely. Milk urea nitrogen (MUN) is a useful proxy for BUN, but the effects of MUN and climatological factors on the reproduction efficiency of dairy cows in the subtropics, which is characterized by extreme temperature and humidity indices, are lacking. The present study investigated the effects of MUN and climatological factors on the reproduction efficiency of Holstein Friesian and Jersey cows in South Africa. The results confirm that MUN influences the reproduction efficiency of dairy cows in the subtropics. High MUN, relative humidity, and maximum daily temperatures compromise the reproduction of Holstein Friesian cows. Jersey cows have a lower threshold MUN concentration compared to Holstein Friesian cows, but they are not adversely affected by high humidity or temperatures.n = 1177) and Jersey cows (n = 3305) in different seasons in the subtropics. Threshold values for MUN on the reproduction of dairy cows in the subtropics remain controversial due to complex environmental interactions, especially with high environmental temperatures. A retrospective analysis was conducted of data obtained from the National Milk Recording scheme of the Agricultural Research Council (ARC) in South Africa. The results confirm that MUN influences the reproduction of dairy cows in the subtropics. MUN concentrations exceeding 18.1 ± 4.28 mg/dL in Holstein Friesian cows and 13.0 ± 4.70 mg/dL in Jersey cows extended the inter calving period (ICP), and decreased RP% and RI. Jersey cows have a lower threshold MUN concentration compared to Holstein Friesian cows, but they are not adversely affected by high humidity or temperatures, while Holstein Friesian cows are.This study investigated the effects of MUN and climatological factors on the inter calving period (ICP), reproductive performance (RP%), and reproductive index (RI) in Holstein Friesian ( Conception and reconception rates of high-producing dairy cows remain problematic in modern dairy production systems , especiaSince BUN cannot be measured reliably and routinely, milk urea nitrogen (MUN) is generally used as a proxy of BUN because this non-invasive measurement can be easily obtained. MUN correlates significantly with BUN r = 0.88) ,7,8, and8 ,7,8, aAs the protein to energy ratio increases, so does MUN . Non-proThe results are contradictory with regard to the effect of breed on MUN concentrations. One study reported a higher MUN concentration in Holstein Friesian cows than in Jersey cows . AnotherSeasonal variations in MUN influence cow reproduction . The nex), minimum (Tn), and average (TAv) temperatures, and minimum (RHn) maximum (RHx) and average humidity RHAv), which were calculated from the hourly data. The cows included in this retrospective analysis were representative of at least one complete lactation cycle and in 1st, 2nd, and or 3rd parity with monthly composite milk samples . Records of a total of 1177 Holstein Friesian and 3305 Jersey cows that complied with the criteria described above were included in the analyses.The Animal Ethics committee of the University of Pretoria (EC171101-156) approved this research. Data consisting of ca. 10,000 records were collected from two Holstein Friesian and two Jersey herds in the Gauteng region of South Africa from 2006 to 2008, during which time no extreme climatic conditions occurred. Dairy farms were selected based on (1) their participation in the National Milk Recording scheme of the Agricultural Research Council (ARC) in South Africa; (2) on the basis of being representative of typical commercial dairy farms using total mixed rations (TMRs) of which the nutritional data were available for comparative purposes; and (3) being close to an official South African weather station to obtain accurate daily weather data. Weather data consisted of daily maximum (Thttps://www.sanas.co.za/ (accessed on 10 June 2021)); and International Committee for Animal Recording (ICAR), (https://www.icar.org/ (accessed on 10 June 2021)) for butterfat %, protein %, lactose %, milk urea nitrogen (mg/dL), and somatic cell count (cells/mL) (https://www.merieuxnutrisciences.com/za/raw-milk-testing-sa (accessed on 10 June 2021)). The data were captured in Microsoft Excel and checked for accuracy before statistical analyses. The target variables selected were the reproductive characteristics, namely inter calving period (ICP), reproductive performance (RP%), and reproductive index (RI). Inter calving period was calculated as the number of days between consecutive calving dates. Reproductive performance (RP%) was calculated to describe the reproduction success of the dairy cows. It was calculated as follows:Seasons were defined as summer (December–February), autumn (March to May), winter (June to August), and spring (September to November). Composite milk samples were analyzed by Mérieux Nutrisciences South Africa  is the age at last calving in days, which was calculated by using the average number of calvings  of the cow and correlating it with the breed and then calculating the average age of the cow in months (Age(M)) as described in . Age in RP% and RI were used to obtain more accurate and practical estimates of reproduction efficiency. Summary statistics and frequency distributions were calculated and the data were checked for missing values and outliers. The normality of variables was tested by plotting histograms and employing the Shapiro–Wilk test in IBM SPSS statistics version 27. The effects of fixed effects, random factors, and repeated measures on the target variables were analyzed using the general linearized mixed models analysis procedure in SPSS version 27, with breed (Holstein Friesian or Jersey), season , parity , and climatological variables as fixed factors and dairy herd and year (2006–2008) as random factors, and including milk composition data as repeated variables. Days in lactation were used as a covariate to correct for the stage of lactation because milk recording data were obtained monthly with cows in different stages of lactation at every recording. Records of animals with incomplete data were omitted, which yielded a final usable dataset of 8110 cow observations in this study. The linearized mixed models analysis procedure was also repeated within breeds due to the known differences between the Holstein Friesian and Jersey breeds in terms of certain milk production characteristics and their genetic differences in terms of adaptability in warm and humid environments by including herd as a random factor and using the same fixed factors and repeated measures as described above.p < 0.05 and p < 0.01. Variance components were estimated using maximum likelihood (ML) and restricted maximum likelihood (REML) methods, and the relationships among variables were estimated through Pearson product-moment correlations (r2). A difference was considered significant at a level of p ≤ 0.05 and p < 0.01.The effects of main factors, random factors, and repeated measures on reproduction parameters were tested using the significance of coefficients in the general linearized mixed models procedure at The climatological conditions in the Gauteng region during the period of the study for Holstein Friesian and Jersey herds are presented in Av > 50%) in spring and summer, mild conditions in autumn, and about six weeks of cold to very cold and dry (RHAv < 30%) conditions in winter.The climatological data indicate that the dairy herds sampled are located in a subtropical region. The climate in Gauteng reflects warm and high humidity . This analysis revealed a coefficient of 8.04 (std. error = 1.152), indicating that the average daily milk yield of Holstein Friesian cows was 8,04 kg/day higher (p < 0.01) compared to Jersey cows. The butterfat content of milk was higher in composite milk samples from Jersey cows compared to Holstein Friesian cows, which agrees with previous results [p < 0.01) in samples from Holstein Friesian cows compared to those from Jersey cows, which agrees with previous results [The results from the mixed models analysis confirmed the known difference between Holstein Friesian and Jersey cows in terms of total daily milk yield  due to other significant fixed and residual effects . In Holstein Friesian cows, it was reported that a MUN <14 mg/dL may indicate an insufficient dietary protein intake [A fair assumption was made about somatic cell counts and udder health based on previous research findings . The aven intake .p < 0.04) in Jersey cows and 0.039 mg/dL per 1 L increase in milk yield in Holstein Friesian cows (p < 0.04). In contrast, MUN decreased by 1.056 mg/dL per 1 MJ increase in ME content. RDP influenced MUN concentrations (p < 0.05) by a 0.051 mg/dL increase in MUN per 1% increase in RDP%. Parity also influenced MUN, with milk samples from cows in the 1st parity containing 0.54 mg/dL more MUN (p < 0.01) compared to samples from the 2nd and 3rd parities. In Holstein Friesian cows, the effect of parity was more pronounced than in Jersey cows, with cows in the 1st parity containing 2.145 mg/dL more MUN (p < 0.01) than samples from cows in the 2nd or 3rd parity.In milk samples from both breeds, MUN was influenced by the average daily milk yield, which was associated with an increase in MUN by 0.016 mg/dL per 1 L increase in milk yield (n = 2352) and 3rd (n = 3660) parities, compared to the 1st parity (n = 1854)  .p < 0.01), respectively, compared to those in the 3rd parity. MUN increased the ICP by 1.03 days per 1 mg/dL exceeding 14.3 mg/dL threshold (p < 0.01), while CP increased ICP by 4.6 days per 1% increase in CP exceeding 17.1% in dairy cows on average (p < 0.01). In contrast, ICP was shortened by 5.5 days per MJ ME exceeding 10.9 MJ ME. The inclusion of climatological variables in the mixed models analysis indicates that ICP is extended by 2.87 days with each 1 °C increase above an average daily temperature of 17.5 °C and extended by 0.94 days for each 1% increase in daily RHAv exceeding 53.14% (p < 0.01). In previous studies on dairy cattle in the subtropics, it was indicated that high environmental temperatures and humidity adversely affected their reproduction [Breed did not significantly affect the inter calving period (ICP) of dairy cows in this study and herd was not a significant random factor in the variance components model. ICP was influenced by season, parity, MUN, RDP, CP, and ME . Cows thoduction ,35. Heatoduction .p < 0.01). MUN and CP decreased the RP% of dairy cows by 0.1% per 1 mg/dL increase in MUN exceeding 14.3 mg/dL and 0.81 % per 1% increase in CP exceeding 17.1% (p < 0.01), respectively. However, RDP and ME improved RP% by 0.37% per 1% increase in RDP exceeding 56.4% (p < 0.01) and 0.85% per 1 MJ increase in ME exceeding 10.9 MJ in the TMR (p < 0.05), respectively. Climatological factors adversely affected RP%, with a 0.42% decrease in RP% per 1 °C increase in average daily temperature exceeding 17.5 °C and 0.11% decrease in RP% per 1% increase in RHAv exceeding 53.1% (p < 0.01).Similar to ICP, reproduction performance (RP%) did not differ between breeds but was influenced by parity, season, MUN, CP%, ME, and climatological factors as outlined in p < 0.01), seasons (p < 0.05), and parity (p < 0.01). The RI of Jersey cows was 4.9 units higher (p < 0.01) compared to that recorded for Holstein Friesian cows in this subtropical region of South Africa. RI was higher for cows that calved in summer compared to winter, spring, or autumn (p < 0.01), while the RI of cows that calved in spring, winter, and autumn did not differ. RI was higher for the 1st parity than the 2nd parity (p < 0.01), and lowest in the 3rd parity cows (p < 0.01). Climatological factors did not affect RI, but RDP and ME improved RI (p < 0.01) by 0.04 and 0.1 units each, respectively. This finding confirms the long-term benefits of improved nutrition on reproduction success as measured over the first three parities in dairy cows.Reproduction index (RI) is a longer-term measure of reproduction efficiency since it considers parity. RI was influenced by the fixed effects, namely breed (p = 0.630).The effects of season on reproduction parameters within the Holstein Friesian and Jersey breeds are presented in p < 0.01), parity (p < 0.01), total milk yield (p < 0.01), MUN (p < 0.05), CP (p < 0.01), ME (p < 0.05), and RHAv (p < 0.01). The ICP of Holstein Friesian cows that calved in summer was shorter compared to those that calved in other seasons, and those that calved in winter was longer (p < 0.01). ICP was respectively 39 and 28 days shorter in the 1st and 2nd parity cows compared to those in the 3rd parity (p < 0.01). An increase in daily milk yield and MUN extended the ICP by respectively 0.9 days (p < 0.01) and 0.8 days (p < 0.05). In contrast, ICP decreased by 11.8 days and 7.9 days respectively per unit increase in dietary CP and ME (p < 0.01). ICP was extended by 1.44 days per 1% increase in daily RHAv exceeding 54.21% (p < 0.01). The effects of daily minimum, maximum, or average temperatures on the ICP of Holstein Friesian cows were not significant.The ICP of Holstein Friesian cows was affected most by season (p < 0.01) compared to cows that calved in winter, spring, or autumn. There was no difference between spring and autumn, but both were higher than in winter. RP% was 5.1% higher for 1st compared to 2nd parity cows, and 3.7% higher for 2nd compared to 3rd parity cows (p < 0.01). Similar to ICP, RP% was lower  and 0.12% lower (p < 0.05) per 1 mg/dL increase in MUN. Of the climatological factors, only RHAv was significant, which caused a decrease of 0.18% per 1% increase in daily RHAv exceeding 54.2%. The effects of RI responded similarly to RP%, but were also affected by environmental temperatures, notably, RI decreased by 0.02% per 1 °C increase in maximum daily temperature exceeding 24.8 °C.The RP% of Holstein Friesian cows that calved in summer was respectively 5.2% 2.6%, and 2.4% higher (p < 0.01). ICP was respectively 41.1 and 13.2 days shorter in the 1st and 2nd parity cows compared to those in the 3rd parity (p < 0.01). An increase in daily milk yield and MUN extended ICP in Jersey cows by respectively 0.7 and 1,0 days (p < 0.01). Increasing RDP above 54.3% decreased ICP by 4.5 days (p < 0.01). Jersey cows did not respond significantly to RHAv, but ICP was extended by 3.1 days for each 1 °C decrease in minimum daily temperature (Tn) below 9.4 °C.Seasonal effects were less significant in Jersey cows in this study, with the ICP being shorter only for cows calving in summer compared to the other seasons (p < 0.01). RP% decreased by 5.4% from 1st to 2nd and 1.8% from 2nd to 3rd parity in Jersey cows. RP% decreased by 0.11% per 1 L increase in average daily milk yield (p < 0.01), but was unaffected by MUN concentration. RI was not influenced by season in Jersey cows. RHAv did not affect RP% or RI, but minimum daily temperatures decreased RP% in Jersey cows by 0.54% per 1 °C below 9.4 °C .RP% responded in a similar way as ICP in Jersey cows, indicating a small but significant increase in reproduction performance for cows calving in summer compared to other seasons  per 1 mg/dL increase in MUN exceeding 14.3 mg/dL. In contrast, ICP was shortened by 11.8 days and 7.9 days, respectively, per unit increase in dietary CP and ME (p < 0.01). The average MUN concentration of Holstein Friesian cows in the present study was less than the proposed threshold value, namely <20 mg/dL for dairy cows previously proposed [The effects of MUN and climatological factors on reproduction parameters of Holstein Friesian and Jersey cows in the subtropics were investigated. The Holstein Friesian and Jersey cows included in this study had a similar calving number (2.23 ± 0.81) and reproduction parameters in both breeds were influenced to a varying degree by the fixed factors parity, season, daily milk yield, MUN, RDP, ME, and climatological factors. In both breeds, ICP was extended by an increase in daily milk yield as well as an increase in the MUN content of milk. In Holstein Friesian cows, the ICP was extended by 0.8 days (proposed .Av (p < 0.01), while the effects of daily minimum, maximum, and average temperatures on the ICP of Holstein Friesian cows were not significant. RH caused a decrease of 0.18% in RP% per 1% increase in daily RHAv exceeding 54.2%. RI in Holstein Friesian cows also exhibited a significant decrease of 0.02% per 1 °C increase in maximum daily temperature exceeding 24.8 °C. Other researchers have also reported seasonal effects on conception rates in dairy cows [However, during summer, Holstein Friesian cows frequently experience heat stress in many regions in South Africa. ICP was extended by 1.44 days per 1% increase in daily RHiry cows . Heat stIn the subtropics, heat stress occurs in dairy cows when temperatures exceed 23.8 °C . A decreThe ICP, RP%, and RI were less affected by season or climatological factors in Jersey cows. Similar to Holstein Friesian cows, Jersey cows showed an increase in ICP and decrease in RP% and RI in cows with a higher daily milk yield and MUN content of composite milk samples. However, increasing RDP above 54.3% decreased ICP in Jersey cows by 4.5 days. RH and high maximum temperatures did not adversely affect the reproduction parameters of Jersey cows. In contrast to Holstein Friesian cows, Jersey cows were more sensitive to cold rather than warm conditions, for example, the ICP of Jersey cows was extended by 3.1 days for each 1 °C decrease in minimum daily temperature below 9.4 °C.It follows that MUN may significantly affect the reproduction efficiency of Holstein Friesian and Jersey cows as the concentration of MUN increases above threshold values, as demonstrated by the significant effect of MUN on ICP between and within breeds. These breeds responded adversely to MUN concentrations exceeding the threshold values of 18.1 mg/dL in Holstein Friesian and 12.9 mg/dL in Jersey cows. Since the negative effect of MUN on the ICP of Jersey cows was observed at MUN concentrations below 14 mg/dL, it follows that Jersey cows may have a lower threshold for MUN in terms of possible adverse effects on reproduction efficiency compared to Holstein Friesian cows. It follows that both protein inclusion, RDP, and the derivative, MUN, should be considered in diet formulations for Jersey cows.Av > 54.3%) and maximum daily temperatures (TX > 24.8 °C), while Jersey cows were sensitive to minimum temperatures decreasing below 9.4 °C. These findings suggest different MUN threshold values for different dairy cattle breeds in different seasons. Jersey cows are generally smaller, better adapted to the subtropics, and have a lower genetic capacity for milk production than Holstein Friesian cows. This may explain the variation in responsiveness of dairy cows to RDP and negative effects on reproduction parameters due to its conversion to BUN. Approximately 12.54 g of urinary nitrogen is excreted per unit of MUN [The reproduction performance of Holstein Friesian cows was affected more by climatological factors such as the relative humidity  and high maximum temperatures (>24.8 °C), while Jersey cows were adversely affected by minimum daily temperatures below 9.4 °C.


Schistosoma spp. DNA for the diagnosis of intestinal schistosomiasis and a sequence of the human β-actin gene as internal control. Firstly, the qPCR was standardized and next it was evaluated for diagnosis and cure assessment of intestinal schistosomiasis in the resident individuals in Tabuas and Estreito de Miralta, two locations in Brazil endemic for intestinal schistosomiasis. The qPCR assay results were compared with those of the Kato-Katz (KK) test, examining 2 or 24 slides, Saline Gradient (SG) and “reference test” (24 KK slides + SG). The cure assessment was measured by these diagnostic techniques at 30, 90, and 180 days post-treatment. In Tabuas, the positivity rates obtained by the qPCR was 30.4% (45/148) and by “reference test” was of 31.0% (46/148), with no statistical difference (p = 0.91). The presumed cure rates at 30, 90, and 180 days post-treatment were 100, 94.4, and 78.4% by the analysis of 24 KK slides, 100, 94.4, and 78.4% by the SG, and 100, 83.3, and 62.1% by the qPCR assay. In Estreito de Miralta, the positivity obtained by qPCR was 18.3% (26/142) and with “reference test” was 24.6% (35/142), with no statistical difference (p = 0.20). The presumed cure rates were 93.3, 96.9, and 96.5% by the KK, 93.3, 96.9, and 100% by the SG, and 93.3, 93.9, and 96.5% by the qPCR at 30, 90, and 180 days post-treatment, respectively. This study showed that the diagnostic techniques presented different performance in the populations from the two districts  and reinforces the need of combining techniques to improve diagnosis accuracy, increasing the detection of individuals with low parasite burden. This combination of techniques consists an important strategy for controlling the disease transmission.The laboratorial diagnosis of the intestinal schistosomiasis is always performed using Kato-Katz technique. However, this technique presents low sensitivity for diagnosis of individuals with low parasite burden, which constitutes the majority in low endemicity Brazilian locations for the disease. The objective of this study was developed and to validate a real-time PCR assay (qPCR) targeting 121 bp sequence to detect  Schistosoma mansoni, the only species with established transmission. Despite the prevalence and parasite burden having decreased over the years after implanting the preventive measurements of the Schistosomiasis Control Program, in 1975, the disease still occurs in Brazil. Intestinal schistosomiasis is currently found in low, moderate, and high endemicity areas of 19 Brazilian federal units . This test became available in 2003 and seems to be more sensitive than the Kato-Katz technique when applied in areas highly endemic for S. mansoni  obtained from the liver of Swiss albino mice 60 days after infection with 150 cercariae using QlAamp DNA Mini and Blood Mini Handbook , following the manufacturer`s protocol.To contorn this limitation, genomic DNA was extracted from adult S. mansoni fecal samples collected from children resident in non-endemic area who had negative results by Kato-Katz technique. The total DNA was extracted using the QIAamp DNA Stools Mini Kit , according to the manufacturer’s recommendations and following the protocols of DNA Isolation from Stool for Pathogen Detection and DNA Isolation from Large Amounts of Stool. The DNA concentration and A260/A280 absorbance ratio was measured in a Nanodrop ND-1000 spectrophotometer  to ensure the efficiency of the DNA extraction and to verify the purity of the DNA obtained.As negative controls we used DNA extracted from three negative S. mansoni (GenBank: M61098). A forward 5’-CCA TCT ACG AGG GGT ATG-3’ and reverse 3’-GGT GAG GAT CTT CAT GAG GTA-5’ primers, and the 56-JOE/CCT GCG TCT GGA CCT GGC TG/[3BHQ1] probe were designed to amplify and detect a 92 pb of the human β-actin gene (GenBank: AY582799.1) as internal control  in DEPC treat H2O]. Next, we tried the human β-actin gene primers at 0.1, 0.15, and 0.2 μM and human β-actin gene probe at 0.1, 0.25, and 0.5 μM in a simplex qPCR assay using DNA extracted from negative S. mansoni fecal samples diluted 1:5 in linear acrylamide solution. In this way, the best qPCR protocol was defined as:A forward 5′-CCG ACC AAC CGT TCT ATG A-3′ and reverse 5′-CAC GCT CTC GCA AAT AAT CTA AA-3′ primers and a 5′-6[FAM]/TCG TTG TAT CTC CGA AACCAC TGG ACG/[3BHQ1] probe were designed to amplify and detect a 90 bp fragment of a highly repetitive 121 bp sequence of  control Figure 1 control  and subm® Universal PCR Master Mix , S. mansoni specific primers at 0.1 μM, 5′-6[FAM]—[3BHQ1] probe at 0.25 μM, β-actin specific primers at 0.15 μM and 56-JOE—[3BHQ1] probe at 0.25 μM, BSA 0.01 μg/μl, MgCl2 at 2 μM and 4 μl of DNA diluted 1:5 in linear acrylamide solution. Two controls were used for each reaction, a positive control (PCR mix plus DNA extracted from adult worms) and a negative control consisting of PCR mix (No Template Control). The assays were performed in duplicate using microplates  sealed with adhesive film  on the StepOnePlus™ Real-Time PCR System  under the universal cycling program with 45 cycles and annealing temperature of 60 °C. Based on a standard curve produced with serial dilutions of S. mansoni DNA, samples presenting Ct ⩽ 42 were classified as positives. Samples that did not presented internal control JOE (β-actin Probe) amplification were retested and a new DNA sample was reextracted when necessary.The reaction was performed with a final volume of 25 μl containing 12.5 μl of TaqManExtraction and amplification protocols were performed in different rooms to minimize the possibility of contamination. All experiments were performed in a laminar flow chamber, previously irradiated with ultraviolet light, and employing only sterile disposable products, including barrier tips.E), Slope, and R2, following recommendations of Johnson et al. , according to the Kato-Katz technique. The repeatability test was measured by the coefficient of variation (CV) by retesting four times the same samples in a single assay (intra-assay test). The reproducibility test was measured by the coefficient of variation (CV) of retesting positive control containing 38 ng, 3.8 ng, 380 pg, 38 pg, 3.8 pg, 380 fg and 0.38 fg of genomic DNA of adult worms diluted 1:5 in linear acrylamide solution in three different days.The repeatability test was carried out using six DNA samples extracted from human feces  as recommended by the Brazilian Ministry of Health.The stools samples were provided by the participants at day 0 and examined using the Kato-Katz and Saline Gradient techniques in both locations. The participants who presented ® produced by BioManguinhos-Fiocruz . Twenty-four slides of the same stools sample, which correspond to the 1,000 mg of feces, were examined to perform a quantitative comparison between the parasitological tests. Infection intensity was calculated by the number of S. mansoni eggs found in 24 slides, resulting in eggs per gram of feces (epg). According to WHO (S. mansoni infection intensity is classified as light (1–100 epg), moderate (101–400 epg), and high (>400).The fecal samples from the residents of Tabuas and Estreito de Miralta were submitted to the Kato-Katz technique using the Helm-Test® tube (15 ml), after which 20% formaldehyde was added to the sediment obtained (approximately 2 ml of sediment). The final solution was examined in an optical microscope. All sediment was examined, the helminth eggs were counted, and the S. mansoni eggs were separated in two preparations (500 mg + 500 mg) representing eggs per gram of feces (epg).The Saline Gradient technique was performed according to the protocol published by Coelho et al. . Fecal sThe DNA of 1,000 mg fecal samples obtained from residents of Tabuas and Estreito de Miralta was extracted using the QIAamp DNA Stools Mini Kit , according to the manufacturer’s recommendations and following the protocols of DNA Isolation from Stool for Pathogen Detection and DNA Isolation from Large Amounts of Stool. The DNA concentration was measured by absorbance at 260 nm in a Nanodrop ND-1000 spectrophotometer . The A260/A280 absorbance ratio was analyzed to verify the purity of the DNA obtained.The qPCR assay was performed using fecal samples according to the conditions standardized. The DNA samples that did not present amplification for the human β-actin gene were retested for ensure the efficiency of DNA extraction and PCR-amplification.The database was built in Microsoft Office Excel 2007 spreadsheets and analyzed using GraphPad Prism version 6.0  or Open Epi software version 3.0 . The posThe use of human samples was approved following the standards of the Ethical Review Committee of the IRR/FIOCRUZ, Brazil (CEPSH 03/2008) and National Committee of Ethical Research  in accordance with the Brazilian legislation (RDC 466/2012). The written informed consent was obtained from all the participants/parents or guardians before collecting the samples.S. mansoni DNA was detected up to the seventh dilution, which corresponds to 0.38 fg. Moreover, repeatability and linearity in the standard curve were obtained up to Ct 41 .A standard curve was constructed and the analytical sensitivity assay showed that the Ancylostoma duodenale, Ascaris lumbricoides, and Fasciola hepática adult worms. The results showed no unspecific amplifications when using genomic DNA in the qPCR assay. The repeatability test presented acceptable Ct variations in the four replicates of three S. mansoni negative and three S. mansoni positive samples. In this assay the coefficient of variation (CV) were 1.74, 2.18, and 2.33% for the target (FAM probe) and 0.69, 0.48, and 0.40% for internal control (JOE probe). Likewise, the reproducibility test presented Ct consistent, resulting CV ranging from 1.6 to 5.4%.The analytical specificity was assessed using DNA from In Tabuas, 84.5% (148/175) of the population participated of this study. From these, 73 were females and 75 males, aged between 1 and 86 years. Ninety-six individuals were residents in the Tabuas district and 52 in the Ribeirão de Tabuas, an adjacent location. The reasons for non-participation in the study were: 1) refusal; 2) insufficient biological sample for performing all techniques, and 3) health reasons.The S. mansoni eggs in their stools, producing a positivity rate of 29.0%. Likewise, the SG technique showed higher positivity rates in the age range from 10 to 19 (57.1) followed by 20–29 years (50%) (The SG technique detected 43/148 participants with rs (50%) Figure 3p = 0.04), although without statistical difference compared to SG (p = 0.70). The positivity rate obtained by the qPCR assay was 30.4%, represented by 45/148 positive participants. All samples (148/148) presented amplification of the internal control. The highest positivity rates for this assay occurred in the age ranges from 10 to 19 (52%) and 30 to 39 years (50%)  and SG were combined and the positivity increased to 31.0% (46/148), with a statistical difference regarding the previous KK positivity (rs (50%) Figure 3S. mansoni, the two combined techniques detected 37 positive participants for other parasites, 16 (10.8%) positive for hookworms, eight (5.4%) for Enterobius vermicularis, eight (5.4%) for Ascaris lumbricoides, six (4%) for Giardia sp., 20 (13.5%) for Entamoeba coli, two (1.4%) for Trichuris trichiura, one (0.68%) for Taenia sp., and two (1.4%) for Hymenolepis nana. Of the participants infected with S. mansoni, six presented co-infection with hookworms and E. vermicularis.Besides Considering the results of the KK technique with two slides as a definitive diagnostic, the qPCR presented 95.7% sensitivity (95% CI: 79–99.2), 81.6% specificity (95% CI: 73.9–87.4), and 83.8% accuracy (95% CI: 77–88.9). Considering the results of the KK technique with 24 slides, the qPCR presented 96.7% sensitivity (95% CI: 83.8–99.4), 87.2% specificity (95% CI: 79.9–92.0), and 89.2% accuracy (95% CI: 83.2–93.2).Considering the SG results, the qPCR presented 81.4% sensitivity (95% CI: 67.3–90.2), 90.5% specificity (95% CI: 83.3–94.7), and 87.8% accuracy (95% CI: 81.6–92.2). Based in the results from the “reference test,” the qPCR presented 82.6% (95% CI: 69.3–90.9), 93.1% (95% CI: 86.5–96.6), and 89.8% (95% CI: 83.9–93.7) of sensitivity, specificity, and accuracy rates, respectively Table 2.S. mansoni by the qPCR assay, 22 were consistent with the KK technique (two slides), one presented positive KK and negative qPCR results (with positive amplification of human β-actin gene), and 23 were positive only by the qPCR (Kappa index: 0.56). On the other hand, 30 participants were consistent with the KK technique (24 slides), one presented positive KK and negative qPCR results (with positive amplification of human β-actin gene), and 15 were positive only by qPCR (Kappa índice: 0.72). Furthermore, there is a negative correlation between the microscopic egg counts (epg) and Ct (r: −0.404) obtained by the KK technique (24 slides) and qPCR assay, respectively.In the crosstabulation between SG and qPCR, eight participants presented positive results with the SG technique and negative with the qPCR (with positive amplification of human β-actin gene). On the other hand, 10 participants presented negative results with the SG technique but were positive with the qPCR. Thirty-five positive and 95 negative results were consistent between the SG technique and qPCR assay (Kappa index: 0.71). Forty-five individuals were positive by the qPCR, of which seven cases were not detected by the “reference test.” In contrast, eight cases were positive by the “reference test” and were not detected by the qPCR (with positive amplification of human β-actin gene), resulting in a Kappa index of 0.76.S. mansoni eggs, one of which was diagnosed by both techniques (KK and SG) and two identified separately by each parasitological technique. qPCR diagnosed six positive participants at 90 days. Therefore, the presumed cure rates at 90 days post-treatment were 94.4% for the KK and SG techniques and 83.3% for the qPCR assay. At 180 days post-treatment, new stool samples were collected from 37 participants for cure assessment, of which eight were positive for S. mansoni eggs by both parasitological techniques and 14 were positive for S. mansoni DNA by the qPCR. The presumed cure rates were 78.4% for the KK and SG techniques and 62.1% for the qPCR assay.In Estreito de Miralta, 87.1% (142/163) of the population was included in this study, of which 77 were females and 65 males, aged from 01 to 86 years, 96 residents of the Estreito de Miralta and 46 of Serra Verde, an adjacent location. The reasons for non-participation in the study were the same as those considered for the Tabuas location.S. mansoni eggs, with a positivity rate of 18.3% concentrated at ages from 10 to 19 (51.9%) and 20 to 29 years (50%) (The positivity rates obtained by the KK technique were 9.2% (13/142), 10.5% (15/142), 11.3% (16/142), 12% (17/142), 16.2% (23/142), and 19.7% (28/142) using 1, 2, 3, 6, 12, and 24 slides, respectively Table 5.p = 0.32) and SG (p = 0.20) positivity rates. The positivity rate obtained by the qPCR assay was equal to the one obtained with the SG technique (18.3%), represented by 26/142 positive participants. The higher positivity rates with qPCR occurred in the age ranges from 10 to 19 years (44.4%)  and SG techniques. The positivity increased to 24.6% (35/142), with no statistical difference regarding the previous KK ( (44.4%) Figure 4Enterobius vermicularis, one (0.7%) for Ascaris lumbricoides, and three (2.1%) for Hymenolepis nana. Five participants presented co-infection with S. mansoni and hookworms and two with S. mansoni and E. vermicularis.Other intestinal parasites were also detected by both parasitological techniques. The KK and SG techniques detected 14 (9.9%) individuals positive for hookworms, eight (5.6%) for The qPCR results were crosstabulated with the parasitological techniques and the results are presented in the The crosstabulation of the SG and qPCR results showed that 126 were consistent and 16 discordants. Among the discordant results, eight were qPCR positive and SG negative and eight were qPCR negative (with positive amplification for the human β-actin gene) and SG positive (Kappa index: 0.62). There were 21/142 qPCR and “reference test” discordant results, of which six presented positive qPCR and negative “reference test” results. In contrast, 15 individuals presented negative qPCR (with positive amplification of human β-actin gene) and positive “reference test” results, resulting in a Kappa index of 0.56 Table 7.S. mansoni infection by the KK, SG techniques and qPCR assay, resulting in a presumed cure rate of 93.3%. In the evaluation 90 days post-treatment, of the 35 participants positive for S. mansoni who were treated with praziquantel, 33 were sampled to evaluate cure assessment. The KK and SG techniques detected two positive participants, one by each technique. The qPCR assay diagnosed both participants as positive for S. mansoni DNA. The presumed cure rates were 97% measured by the KK and SG techniques and 93.3% by the qPCR assay.In the follow-up for cure assessment 30 days post-treatment, new stool samples were collected from 30/35 positive participants. This evaluation showed two participants diagnosed as positive for The follow-up at 180 days post-treatment consisted of 29/35 samples for new stool samples for the last cure assessment. This evaluation showed only one participant diagnosed with intestinal schistosomiasis by the KK technique and qPCR, resulting in a presumed cure rate of 96.5% Table 8.S. mansoni and the human β-actin gene to diagnose the intestinal schistosomiasis and to secure the optimal conditions of amplification, respectively.Despite the parasitological technique presenting the best cost-benefit conditions, the assessment of more sensitive techniques is essential for an efficient diagnosis in endemic areas. Current scenarios show that molecular tests are a promising tool for diagnosis and cure assessment of intestinal schistosomiasis in individuals of low parasite burden , 32, 33.S. mansoni DNA, which corresponds to approximately 0.00065 times its genome, that contains ~580 fg of DNA  and SG techniques (p = 0.79) and like the “reference test,” with no statistical difference (p = 0.91). Espírito-Santo et al. .The sensitivity of the qPCR was high (96.7%) but the specificity was low (87.2%) when the KK (24 slides) results were taken as reference. Only one participant positive with eggs diagnosed by the Kato-Katz technique was not identified by the qPCR assay, which can be explained by the absence of eggs in the sample examined. The qPCR detected 15 positive participants not identified by the Kato-Katz, by examining 24 slides. This discordance was probably due to the limitation of parasitological technique for detecting parasite eggs in stools samples from residents of low endemicity areas, where most of the carriers present low parasite burden (<100 epg). In these cases, the PCR assay detects more cases of infection than the evaluations of many slides by the Kato-Katz technique, suggesting that it can be a useful diagnostic tool. In contrast, the sensitivity rates were lower (81.4 and 82.6%) and the specificity rates were high (90.4 and 93.4%) when the SG or “reference test” were considered as a reference. Thus, the accuracy ranged from 87.8 to 89.8%, with no statistical difference , Saline Gradient, and Helmintex techniques. The qPCR assay presented sensitivity of 91.4%, specificity of 86.9%, and Kappa index of 0.71, when the results of the three parasitological techniques were considered as a “reference test.” Moreover, the qPCR assay diagnosed 86.9% of the participants with very low parasite burden (<12 epg) while the POC-CCA® diagnosed 50.8% , a portion of a mitochondrial gene (nad1) and cell-free parasite DNA (cfDNA) detection by Droplet Digital PCR (ddPCR) has shown to be applicable to the diagnosis of schistosomiasis , 42. AlsSchistosoma ITS2 applied in a population from Senegal (n = 197) and Kenia (n = 760), high and low endemicity areas, respectively, presented 13–15% more positivity regarding the KK technique (two slides) of a single stool sample  is small . Althougl sample . MoreoveS. mansoni eggs in the stool or with qPCR at 90 and 180 days post-treatment. Similar data were found in a study performed in the residents from the Pedra Preta community, in the municipality of Montes Claros, Minas Gerais, Brazil  using the KK technique (24 slides) and Ct obtained by the qPCR assay. In contrast, a qPCR assay for the quantitative detection of S. mansoni and S. haematobium DNA in stool samples in the Senegal population showed significant correlation between the qPCR Ct values and microscopic eggs counts for both Schistosoma species  and qPCR assay (p = 0.76). In this district the positivity rate increases constantly according to number of slides examined, disagreeing with the behavior shown in Tabuas. In Estreito de Miralta all participants presented low parasite load and possibly this fact influenced the correlation between the number of slides and positivity rates  was high than the SG technique , the high positivity rates for schistosomiasis were found in participants aged from 10 to 19 years, followed by participants aged between 20 and 29 4. BurlaS. mansoni continue to eliminate eggs and contribute for maintaining the transmission of the disease in the area. This evidence emphasizes the urgent need for a more sensitive diagnostic method for surveilling schistosomiasis cases in low transmission areas , as is performed in the Brazilian Schistosomiasis Program Control. Thus, approximately 50% of the individuals infected with on areas .Schistosoma DNA obtained with the qPCR assay. It is a consensus that the specificity of any diagnostic test benefits when it is applied in individuals from low endemicity areas. Also, it is well established that the sensitivity of a diagnostic test benefits when it is applied in individuals from high endemicity areas.Apparently, the sensitivity rates of the qPCR assay  were impaired and the specificity  were favored when considering the KK (24 slides) and SG techniques or “reference test” as true results. These data indicate the influence of individual parasite burden in the performance of the diagnostic techniques used. Some authors reported that false negative results obtained by PCR may be due to a few factors such as inhibition of the amplification reaction by fecal compounds or DNA degradation during transportation of the sample from the field , 43. HowIn contrast to the presumed cure rate observed in the population from Tabuas, the presumed cure rates in Estreito de Miralta were high and more consistent in the follow up at 30, 90, and 180 days post-treatment, with no statistical difference Table 8.S. mansoni eggs evaluated by the qPCR assay after undergoing treatment with the recommended dose of praziquantel. In contrast, all participants in Estreito de Miralta were classified as low parasite burden carriers, with less therapeutic failure during the cure assessment performed in this study. Based in these data, we can to suggest that the transmission force of the parasite in Tabuas is higher than that in Estreito de Miralta.In conclusion, the diagnostic techniques presented different performance in the populations from two districts . In Tabuas, the positivity rate was higher, with the participants presenting low, moderate, and high parasite burdens and a considerable percentage of participants positive with The qPCR was an acceptable diagnostic tool, with added value to microscopy in low endemicity areas for the diagnosis of intestinal schistosomiasis in fecal samples, which makes it particularly useful in low transmission areas, and consequently, in post-treatment settings. Moreover, the qPCR can be multiplexed for the diagnosis of other intestinal parasites making the assay more useful. On other hand, because of the relative high costs, the qPCR assay is not cost-effective for the routine diagnosis of schistosomiasis in endemic countries. However, the qPCR assay has become less expensive over time, with increase of the number of research centers with qPCR infrastructure. Thus, the qPCR assay can be used in the routine diagnosis of helminth infections in countries of low economic power.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LMVS aided with the field work and parasitological techniques, and performed the molecular techniques and analyzed the results. CS assisted with the qPCR assay and analyzed the results. ÁAO aided with the enrollment of the participants, assisted with the parasitological techniques, and assisted with the field work. NFFC assisted with the enrollment of the participants and field work. LG assisted with the qPCR assay and critically reviewed the manuscript for intellectual content. AR supported with the study design and critically reviewed the manuscript for intellectual content. PMZC supported with the study design and critically reviewed the manuscript for intellectual content. EO assisted with the study design, data analysis, drafted the manuscript, and critically reviewed the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.o 03/2012. EO is supported by CNPq-Brazil .This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001; DECIT/CNPq program 2012 #404405/2012-6; PROEP/Pesquisa Clínica Program MCT-CNPq/FIOCRUZ NThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Weight loss is most severe in patients with >20%TBSA with an initial gain of weight due to fluid resuscitation. These findings led the American Burn Association to propose new quality measures for burn-injury admissions, including weight loss from admission to discharge. We aim to assess how our institution’s outcomes adhere to the proposed measures and if our findings correlate with previously described results.Burn patients with >20%TBSA since 2016. Three groups were established based on %TBSA: 20-29% (Group 1), 30-39% (Group 2), and >40% (Group 3). We assessed weight changes from admission to discharge and performed a multivariate analysis to account for age, sex, number of surgical procedures, and hospital length-of-stay (LOS).A retrospective review was conducted for adult patients admitted to our institution with burn injuries of >20%TBSA showed 11 patients with %TBSA of 20-29%, 10 patients with %TBSA of 30-39%, and 19 patients with %TBSA of >40%. When comparing groups 1 and 2, we saw significantly more weight loss in group 2 over the course of admission without a significant change in total hospital LOS. The average %weight loss for group 1 was 1.46%, 8.36% for group 2, and 10.56% for group 3. No patients in group 1 had a weight loss >15%. For group 2, patients with weight loss >15% had a significantly longer LOS and underwent significantly more surgical procedures during their admission. For group 3, most patients that experienced weight loss >20% did not have a longer LOS but did require more surgical procedures during their course of admission.Data from 40 patients suffering burn injuries of >20%TBSA do suffer significant weight changes, likely due to extreme metabolic disturbances. Our data suggests that an increased length of stay is not a significant driver for weight loss changes between patients with %TBSA of 20-29 and 30-39, suggesting other pathophysiologic mechanisms in play. Our data supports the idea that patients with %TBSA>40 are a unique subset of patients, requiring specialized nutritional protocols and metabolic analysis.Analysis of the data demonstrates that patients with


Determining chronic lymphocytic leukemia (CLL) prognosis using the International Prognostic Index markers such as TP53 and immunoglobulin heavy-chain variable region gene mutation in a resource-limited setting is difficult to achieve because of cost and equipment unavailability. The aim of this study is to determine prognostic factors easily available to hematologists in low- or medium-income countries.This was a retrospective study conducted at the University of Port Harcourt Teaching Hospital, Nigeria. Data were retrieved from CLL patient records from January 2004 to December 2019 (15 years). Data collected were analyzed using SPSS software version 25.9/L were significant poor prognostic markers . Thirty-five (76.1%) received chemotherapy, and they had a better median survival than those who did not (26 v 17.5 months). The most common regimen used was cyclophosphamide, vincristine, and prednisolone for 15 (42.9%) patients.A total of 46 records were reviewed, with a median age of 55 years and a male:female ratio of 1:1.2. All patients were symptomatic at presentation, with splenomegaly (91.3%), anemia (82.6%), and lymphadenopathy (76.1%) predominating. About 89.1% of the patients presented at Binet stage C and/or high-risk Rai (Rai stages III and IV) with 10.9% presenting at Binet stage B and/or intermediate-risk Rai (Rai stage II). Only 13% of the patients had immunophenotyping done with 6.5% being done for the Matutes CLL score. The 5-year overall survival (OS) was 15.7% with a median survival of 26 months. WBC count and absolute lymphocyte count (ALC) > 100 × 109/L were poor prognostic markers. Patients who received chemotherapy had a better OS.WBC count and ALC > 100 × 10 Chronic lymphocytic leukemia (CLL) is a clonal B cell malignant disease with attendant lymphocytosis characterized by progressive accumulation of clonal B cells in the blood, bone marrow, lymph nodes, and spleen permitting easy diagnosis from the peripheral blood. The clinical features of CLL at presentation are related to the accumulation of these leukemic cells.Key ObjectivePrognostic factors such as del(11q), del(17p)/TP53, and immunoglobulin heavy-chain variable region gene mutation are unavailable in many resource-limited settings. What other available factors could aid prognostication in patients diagnosed with Chronic Lymphocytic Leukemia?Knowledge Generated9/L at presentation had a poor prognostic outlook when compared with those with WBC count and ALC < 100 × 109/L .Patients with WBC count and absolute lymphocyte count (ALC) > 100 × 10RelevanceA majority of patients could not afford novel drugs such as ibrutinib, obinutuzumab, or venetoclax; hence, older management protocols such as CP (chlorambucil and prednisolone), CHOP , and CVP  were used. Patients on CHOP appeared to have a better prognostic outlook.2 It is a slowly progressive disease, with an 81.7% 5-year survival rate.1 It accounts for 17%-20% of all hematologic malignancies in Nigeria with 2-6 per 100,000 new cases diagnosed annually.4 CLL accounts for one quarter of the new cases of leukemia with an age-adjusted incidence of 4.5 per 100,000 persons in the United States.1 The incidence varies widely across geographical locations, with Asia having a 5-10-fold lower prevalence.6 It is more common in males, mostly seen in the elderly (age > 65 years) with some familial tendency being alleged.7CLL is predominantly a disease of the elderly, with a median age of 65-72 years at diagnosis.9, and clonality confirmed by immunophenotyping (IMPT). Staging of the tumor is usually done at diagnosis, using Rai or Binet staging,2 both of which are used for prognosis and decision on the commencement of therapy. The system uses lymphadenopathy, organomegaly, and cytopenias (anemia and thrombocytopenia) to establish prognostic groups that can be used to predict median survival. The Rai and Binet staging systems were the first prognostic markers to affect disease management. Although still widely used, they do not predict disease progression or response to therapy. There are numerous ongoing efforts to identify and characterize additional prognostic markers at the cellular and molecular level.8Diagnosis requires peripheral blood lymphocytosis, an absolute lymphocyte count (ALC) of ≥ 5 × 102-microglobulin (β2m), thymidine kinase, and soluble CD23 have all been described as independent prognostic markers in CLL.10 Other newer prognostic factors include mutational status of the immunoglobulin heavy chain variable region (IGVH) genes and surrogate markers such as CD38 and zeta-associated protein (ZAP)-70 gene expression, telomerase length, and telomerase activity.11 In a resource-limited setting like Nigeria, most of these investigations cannot be done and hence the need to investigate the usefulness of some clinical and laboratory parameters in assessing the prognosis and survival of patients with CLL.Serum β12Not all patients require treatment at diagnosis. Indications for treatment include a rapid doubling time, worsening cytopenias and symptomatic nodal or extranodal disease. Treatment options include the use of alkylating agents, purine analogs, anti-CD20 monoclonal antibodies, Bruton Tyr kinase inhibitors, and BCL-2 inhibitors.The aim of this study was to identify some clinical and laboratory prognostic markers for patients with CLL in a resource-limited setting.9/L and peripheral blood film showing predominantly mature lymphocytes with a Matutes score of 4 or 5 (if IMPT was done). Those with ≥ 10% prolymphocytes on blood film or Matutes score ≤ 3 were excluded. Data collected were analyzed using SPSS software version 25 . The results were expressed in charts and tables. A P value of < 0.05 (at 95% CI) was taken as statistically significant.This was a retrospective study conducted at the University of Port Harcourt Teaching Hospital. Data were retrieved from the files of all patients diagnosed with CLL from January 2004 to December 2019. Inclusion criteria were asymptomatic and symptomatic patients with ALC > 5 × 10P = .35).A total of 46 patients  were diagnosed with CLL from January 2004 to December 2019 (15 years). There were 21 (45.7%) males and 25 (54.3%) females with a male to female ratio of 1:1.2. The median age at presentation for all the cases was 55 years . Twenty-four patients (52.2%) were ≤ 55 years at presentation. Females had a lower median age at presentation (55 years) compared to the males (58 years); however, this was not statistically significant . The most common symptoms were splenomegaly seen in 42 (91.3%) of the cases, followed by anemia  and lymphadenopathy . A little over a quarter of them  had fever. The mean spleen size was 14 cm (± 6.7 cm). Of the 42 patients with splenomegaly, 6 (14.3%) had a spleen size > 20 cm . One patient presented with asplenia following splenectomy for hypersplenism. Figure 9/L , 88.7 × 109/L , and 131.5 × 109/L (12-392 × 109/L), respectively. Using the PCV and platelet count cutoff from the Rai staging criteria, there were 33 patients (71.7%) with anemia and 18 (39.1%) with thrombocytopenia. Eighteen patients (39.1%) presented at Rai stage 4, whereas 23 (50%) presented at Rai stage 3 and 5 (10.9%) at Rai stage 2. None of the patients presented at Rai stage 0 or 1 or Binet stage A, 41 patients (89.1%) presented at Binet stage C, and 5 (10.9%) at Binet stage B.The median packed cell volume (PCV), total WBC count, ALC, and platelet count were 24% , 101.0 × 10IMPT was done for only six patients 13.0%); half of them  had only CD20 done, whereas the other three (50%) were tested using the Matutes CLL score. All the six cases who had IMPT done were CD20+  received chemotherapy and 11 (23.9%) did not receive chemotherapy . The mean duration of treatment for all regimens was 4.2 months (± 3.2 months). The major first-line chemotherapy used was cyclophosphamide, vincristine, and prednisolone (CVP) for 15 patients (42.9%), followed by chlorambucil and prednisolone (CP) for 11 (31.4%). Only three (8.6%) patients had rituximab with cyclophosphamide, hydroxodaunorubicin, oncovin, and prednisolone (R-CHOP). Fludarabine, cyclophosphamide, and prednisolone (FCP) was received by two patients (Table % receiveP = .8). Patients who had CHOP had the longest median survival of 48 months (Table v 13 months); however, this was not statistically significant (P = .3). Survival rate of younger patients (≤ 55 years) was better than patients of age > 55 years (29.9% v 11.08%), P = .9. Patients with massive splenomegaly (> 20 cm) had a shorter median survival (6 months) compared with those with splenomegaly < 20 cm (26 months), although this was not statistically significant, P = .3.Patients on chemotherapy were grouped into those who received older forms of therapy  and those who received newer therapy, which were either FCP or R-CHOP . Patients who received older therapy had a median survival of 26 months, whereas those who had newer therapy had a median survival of 38 months, although this was not statistically significant  had a significantly lower 5-year survival rate of 13.3%  compared with those with WBC count < 100 × 109/L  whose 5-year OS was 18.2% , which was significant (P = .013)  had a significantly lower median survival of 6 months compared with those with ALC < 100 (41 months), which was also significant (P = .021).Patients with WBC count > 100 × 109/L n = 4, 52.2% Patients who received chemotherapy had a higher median survival of 26 months with a 5-year survival rate of 14.7%, those without chemotherapy had a median survival of 17.5 months, and 5-year survival rate was zero. The median follow-up duration for all patients was 5 months .2 Salawu et al13 showed a median age of 60 years in Ile-Ife, South-West Nigeria. However, western studies have shown a median age of between 65 and 72 years.14 More than half of our patients with CLL were young (< 55 years) compared with those in the United States and Europe where only about 5%-11% of cases are younger than 55 years.15 Although the epidemiology of CLL both in Nigeria and globally generally shows a male predominance,16 our study showed a slight female preponderance. A female preponderance has also been reported in our environment.17The median age at diagnosis of CLL was 55 years, showing a younger age at presentation when compared with other studies.18 This could be due to the poor health seeking behavior and the low socioeconomic status of the populace. This is further buttressed by the long median duration of symptoms. More than 90% of our patients had splenomegaly, which is higher than the results found by Basabaeen et al in Sudan and Salawu et al in Nigeria .19 Patients with massive splenomegaly had a lower median survival, albeit not statistically significant.No patient was asymptomatic, unlike in western studies where 25%-50% of patients are asymptomatic at presentation.9/L, representing a high tumor burden, possibly associated with bone marrow suppression . The presence of anemia and thrombocytopoenia explains why a majority had a high-risk Rai (III and IV) and Binet C at the time of presentation.Slightly more than half of the patients presented with WBC count and ALC > 100 × 102Only 13% of our patients could afford the cost of IMPT, of which half of them tested for the Matutes CLL score  and the remaining half were tested for CD20 only. IMPT should be performed routinely as part of the workup for diagnosis of CLL as this aids confirmation of diagnosis and excludes other lymphoid neoplasms whose blood or marrow morphology may mimic CLL.20 in which the median survival from the time of diagnosis was 12.3  years for patients diagnosed in 2001 or earlier and 13.3  years for patients diagnosed between 2002 and 2008, whereas the median survival was not achieved in those diagnosed between 2009 and 2017. This short median survival may be due to misdiagnosis of patients who may have had other more severe lymphoproliferative neoplasms, since IMPT was not routinely performed. Our study showed that young patients < 55 years had a better OS than those > 55 years, even though it was not statistically significant. This is similar to studies where patients younger than 55 years had better OS than older patients.15 The better survival in the younger age group (despite having more poor prognostic markers such as ZAP-70 expression or unmutated immunoglobulin heavy-chain variable status) could be due to their fewer comorbidities and shorter time to treatment, when compared with the older age group.15 The females in our study had a slightly younger median age than males (55 years v 58 years), which may have contributed to the better median survival than the males (26 months v 13 months).The median survival was 26 months, which is only slightly above 2 years. This is poor when compared with some western studies like the one conducted by Weide et al,9/L had a statistically significant better median survival than those with higher WBC counts or ALC, suggesting that a high tumor burden at presentation is a poor prognostic feature. This is validated by other studies that showed similar results.21 However, Madu et al showed that there was no association between the ALC and median survival.14 The hematocrit, platelet count, and Rai and Binet staging did not have any statistically significant impact on OS in our patients, unlike several other studies where these factors were associated with a shorter OS, with the exception of Salawu et al who noted that their patients with anemia had a better median OS.22Patients with WBC count and ALC < 100 × 1013 Patients with Binet-A had a longer median OS of 100 months; stage B patients, 55 months; and stage C patients, 45 months.23 Our patients with Rai stages II, III, and IV had the median survival of 41, 26, and 11 months, respectively. None of our patients presented in Binet-A, but patients with Binet-B had a better median survival than those at stage C.The later the stage at presentation using Rai or Binet, the shorter the median survival. Since our patients presented late, this was not statistically significant in our findings but might have been due to not having patients who presented with Rai 0 or I or Binet-A, which is yet to be compared. Other studies have shown a similar pattern in median survival, with it declining at later stages of presentation .v 17.5 months). A majority of our patients were not exposed to newer regimens because of nonavailability and financial implications. Although CVP was the most used older regimen, patients who received CHOP had the best median survival. This is similar to the 1989 findings by the French Cooperative Group on CLL.24 In recent years, CLL treatment has evolved. First-line regimens now include novel drugs such as BTK inhibitors (ibrutinib), newer anti-CD20 monoclonal antibodies (obinituzumab), or BCL-2 inhibitors (venetoclax).12 Therefore, comparatively speaking, our five patients who had the newer regimens did not actually receive novel therapy, and this may explain the poor survival in our patients with CLL compared with other climes. Those five patients in the newer therapy group (FCP and R-CHOP) had better median survival unlike the older therapy group (38 v 26 months); however, patients on CHOP had the longest median survival (Table Patients who received chemotherapy had a higher median survival than those who did not (26 9/L are poor prognostic markers. Patients who received chemotherapy had a longer median survival. It is recommended that full blood count is done for all patients for earlier detection of CLL, which may affect survival.In conclusion, while striving for better diagnostic and therapeutic availability and reduced costs, there is a need to use the limited available investigations and drugs at our disposal to patients with prognosticate CLL in our environs. Our patients presented at a younger median age of 55 years. At presentation, WBC count and ALC > 100 × 10