Setting. Ophthalmology Department, Military University Hospital in Prague (Czech Republic).  To evaluate a 12-year follow-up of myopic patients after iris-fixated phakic intraocular lenses (IF pIOLs) implantation.  Single-center retrospective cohort study.  We describe the results of a cohort study that included 85 eyes of 46 myopic patients who underwent implantation of Verisyse myopia, Veriflex, and Verisyse myopia toric  intraocular lenses. Refractive functions and adverse events were assessed preoperatively, at 6 months, and 1, 2, 5, and 12 years after IF pIOL implantation.  Mean spherical equivalent was measured as −9.37 ± 2.87 D, 0.14 ± 0.61 D, and −0.42 ± 1.08 D, preoperatively, at 6 months and 12 years postoperatively, respectively. There was a significant reduction in the cylinder after surgery. At 12 years postoperatively, 90% of eyes had uncorrected distance visual acuity (UDVA) of 20/40 and 64% of 20/20. The safety index was 1.10 for the whole postoperative follow-up period. We found cataract formation in 3 eyes (3.5%). The endothelial cells loss (EC loss) directly caused by IF pIOL implantation was 6.0%, 8.10%, 12.8%, and 11.9%, at 1, 2, 5, and 12 years, respectively. In our cohort, 95% of eyes lost a higher percentage of EC than would be expected from a physiological loss at 12 years postoperatively. We found a significant negative interaction between preoperative pachymetry and EC loss, indicating that the lower pachymetry leads to a faster decline in endothelial cells density (ECD). IF pIOL re-enclavation was found in 28% of eyes. 7% of subluxations were caused by trauma. The mean time of nontraumatic re-enclavation was 6 years postoperatively.  The study confirmed the advantages of IF pIOL implantation due to rapid visual recovery and stable visual function over the 12-year follow-up and also showed the influence of lower corneal pachymetry regarding EC loss. Phakic intraocular lens (pIOL) implantation has been a powerful solution for moderate and high myopia for more than 30 years. It helps patients who may have some contraindication to excimer laser surgery to be glasses independent. The first models of the pIOLs implanted in the 1980s and 1990s were angle-supported anterior chamber pIOLs. Because of unacceptable complication rate, especially corneal endothelial cells loss (EC loss) from the pIOL's proximity to the corneal endothelium, they were consequently removed from the market.EC loss was also the problem with the Worst–Fechner iris-fixated IOLs (IF pIOLs), a coplanar single-piece PMMA pIOLs that were enclavated in the folds of midperipheral iris stroma, a relatively immobile portion of the iris. The first implantation of IF pIOL in myopic patient occurred in 1986. By the end of the 1990s, a newly designed, safer model of PMMA IF anterior chamber pIOLs was introduced. First, a foldable single-piece Artisan (Ophtec BV)/Verisyse  made from Perspex CQ-UV appeared on the market. Subsequently, a foldable variant of an IF anterior chamber pIOL Artiflex (Ophtec BV)/Veriflex  composed of hydrophobic polysiloxane was designed. The optic vault measured approximately 0.87 mm anterior from the iris and provided good clearance for the anterior lens capsule and the endothelium of the cornea .During the past 10 years, several long-term reports about IF pIOLs were published. The EC loss and cataract formation were found to be more frequent than under physiological conditions. Detection of basic risk factors showed to be important for correct indication and minimization of postoperative complications. Most of the publications reported 5-year results –8, but oWe describe the results of our cohort study that includes a group of 85 eyes in 46 myopic patients, 13 men (28%) and 33 women (72%). The patients underwent implantation of IF pIOLs from January 2005 to November 2010, and we were tracking their refractive results and complications over 12 years. All surgeries were performed by one surgeon in the Department of Ophthalmology, Military University Hospital, Prague (Czech Republic). The study was approved by the Medical Ethics Committee at University Medical Centre in accordance with tenets of the Declaration of Helsinki.2, ACD (anterior chamber depth) from endothelium (the distance between endothelium and anterior surface of the clear lens) >2.9 mm, iridocorneal angle ≥30°, no anomaly of iris or pupil and mesopic pupil size <6 mm. One eye (1.2%) of 1 patient underwent a keratoplasty prior to pIOL implantation. Seven eyes (8.2%) in 5 patients had a previous scleroplasty procedure, and their refraction had been stable for more than 2 years before the IF pIOL was implanted. Preventative laser photocoagulation was carried out in one patient (2 eyes) to treat lattice degeneration to decrease the risk of retinal detachment.We strictly adhered to the following inclusion criteria. All patients had to be older than 18 years of age and their refractive error had to be stable at least for 1 year. The IF pIOL implantation was carried out only in patients where excimer laser surgery was not indicated for the correction of existing ametropia. We respected the following criteria in the operated eye: ECD  >2300 cells/mmAs for exclusion criteria, we excluded glaucoma and IOP >21 mmHg, active disease in the anterior segment, recurrent or chronic uveitis, any form of cataract, preexisting macular pathology or abnormal retinal condition, as well as any systemic disease   D and mean cylinder 1.37 ± 1.44  D. Postoperative MRSE and cylinder are shown in p < 0.01). The cylinder was stable 2 years after surgery. There was a statistically significant change 5 years after the surgery of 0.86 ± 0.51 D (p < 0.05). 12 years after the implantation, the cylinder was again without statistically significant changes compared to the previous value.There was a statistically significant reduction of cylinder 6 months after IF pIOL implantation, 0.70 ± 0.48 D (p < 0.05). The mean postoperative CDVA (0.98 ± 0.12) was also statistically better than the mean preoperative CDVA values (p < 0.01). The follow-up mean CDVA results showed statistically significant improvement 5 years after the surgery, 1.00 ± 0.07 (p < 0.05). With respect to UDVA, we noted statistically significant worsening between 5 and 12 years after the surgery, 0.93 ± 0.17 and 0.86 ± 0.21, respectively (p < 0.01).Uncorrected (UDVA) and corrected (CDVA) distance visual acuity, efficacy index (EI), and safety index (SI) are common parameters to assess the effect of the iris-claw IOLs implantation. Visual acuity was measured in the Snellen decimal scale and is summarized in We had to proceed with excimer laser surgery (photorefractive keratectomy-PRK) for residual refractive error in 1 eye (1.2%), one year postoperatively. There was a progression of myopia >−1.0 D in 3 eyes (3.5%) of 2 patients.Efficacy is commonly reported as the cumulative percentage of eyes within the visual acuity range , 16. TheSafety is commonly reported as the change in visual acuity preimplantation vs. visual acuity postimplantation , 16. In The incidence of secondary angle-closed glaucoma (SACG) was 4.7% in the early postoperative period. Due to the intraocular pressure elevation, we had to enlarge iridotomies in 2 eyes (2.3%) of one patient just after the primary IF pIOL implantation and in one eye (1.2%) 2 months after the IF pIOL re-enclavation. One eye (1.2%) experienced an attack of acute pupillary block glaucoma on the first night after the surgery. Iridotomies had to be enlarged 20 hours after the surgery, but pupilloplegia remained. It was rectified later with a pupilloplasty procedure. There was no pigment dispersion glaucoma in our cohort.p=0.957). All values are summarized in From the long-time perspective, the patients remained without statistically significant changes in intraocular pressure after the IF pIOLs surgery. This is supported by the values before and 5 years after the implantation, of 14.8 ± 3.0 mmHg and 15.2 ± 3.0 mmHg, respectively  and with iridotomy in 1 eye (1.2%). Any sign of optics decentration did not occur, and there was no need of miotic eye drops application.2. We recorded ECD at 1, 2, 5, and 12 years after the implantation, with values of 2430 ± 312 cells/mm2, 2369 ± 262 cells/mm2, 2175 ± 298 cells/mm2, 2091 ± 312 cells/mm2, respectively  suffered trauma and underwent subsequent re-enclavation.During the postoperative 5- and 12-year follow-up, 93% and 95% of the eyes lost a higher percentage of EC than the expected physiological loss, respectively, in our cohort. According to the AAO Task Force guideline for standardized reporting on EC loss in studies of pIOLs , we repop=0.006), indicating that the lower pachymetry leads to a faster decline in ECD. The longitudinal decrease in ECD was not significantly related to gender (p=0.425), degree of myopia (p=0.449), or keratometry (p=0.520). Higher baseline ACD values have shown to be indicative of slower EC loss. However, the effect was slightly above the selected significance level (p=0.057).Next, we tested the association between baseline risk factors and long-time EC loss. We found a significant inverse relationship between preoperative pachymetry and EC loss  .Refractive stability for at least 1 year is one of the main conditions before pIOL implantation [Because we worked with younger patients, we calculated IF pIOL on the side of slight hypermetropia. There was a statistically significant shift into myopia 12 years after surgery, −0.42 ± 1.08 D. There was also a statistically significant reduction in the cylinder at 6 months after IF pIOL implantation. For the rest of the 12 years postoperatively, the cylinder remained stable without statistically significant changes.We proceeded with excimer laser correction of residual refractive error in one eye (1.2%) at 1 year postimplantation. We elected PRK because Güell et al. hypothesized that laser in situ keratomileuses in an eye with anterior chamber or IF pIOL might induce contact between the corneal endothelium and the pIOL when the microkeratome (or applanation of femtosecond laser at the present time) is used , 22.We closely monitored the efficacy of this method. UDVA of 20/40 or better was found in 99% of patients at 1 year and 90% of patients at 12 years after surgery. UDVA of 20/20 or better was noted in 77% of patients at 1 year and 64% of patients 12 years after surgery. The EI remained stable at 12 years after IF pIOL implantation. The safety of IF pIOL implantation was high (99%) and stable at 12 years after surgery. Just one eye (1.2%) lost one Snellen line of BDVA. The postoperative value of the SI was 1.10 and was also stable for 12 years postoperatively.Our results are in good agreement with other studies which showed successful refractive results of IF pIOLs implantation , 23, 24.There is a danger of secondary glaucoma due to the pigment dispersion or pupillary block in the early postoperative period . The pigTo prevent pupillary block, an iridotomy or iridectomy is done in the eyes with IF pIOLs. Like Monteiro et al. , we had In keeping with the literature, we did not find any long-term statistical change of intraocular pressure after surgery , 24, 27.Jiang et al.  found thSubluxation of IF pIOLs can occur spontaneously or after trauma. Spontaneous haptic disenclavation is usually connected with iris depigmentation and iris atrophy . Peres-SCataract development has been noted after IF pIOLs implantation. Several factors may be involved including surgical trauma, age, pIOL-crystalline lens touch (including intermittent contact during accommodation), myopia, the biocompatibility of the pIOL, change in the blood-aqueous barrier, and chronic subclinical inflammation . Most ca2 for patients aged 21 to 25 years, >2650 cells/mm2 for patients aged 26 to 30 years, >2400 cells/mm2 for patients aged 31 to 35 years, >2200 cells/mm2 for patients aged 36 to 45 years, and >2000 cells/mm2 for patients aged more than 45 years [As in many other studies, our inclusion criteria were based on a minimum amount of preoperative ECD , 10, 16.45 years . These c45 years , 16.2 in 3% of cases. We had to carry out one IF pIOL explantation (1.2%) due to corneal endothelium decompensation 14 years after the initial surgery.We found that the total chronic EC loss that was significantly higher than the expected physiological dropout. We measured corrected EC loss to demonstrate the EC loss directly caused by the IF pIOL presence. The values were 6.0%, 8.1%, 12.8%, and 11.9%, at 1, 2, 5, and 12 years postoperatively. During the postoperative 5- and 12- year follow-up, 93% and 95% of the eyes lost a higher percentage than the expected physiological loss, respectively. In the period 5 and 12 years after IF pIOL implantation, 15% and 20% of eyes had an EC loss of ≥25% of the preoperative value, respectively. In the 12-year follow-up, we noticed an ECD of <1500 cells/mm2 in 3.9% and 4% in the myopic and toric group. In 6% of eyes in the myopic group and 4.8% of eyes in the toric group, excessive EC loss or corneal decompensation resulted in IF pIOL explantation after 11.9 and 7.4 years, respectively [2 and 1 phakic lens was explanted due to a severe decrease of the endothelial density (862 cells/mm2) [Jonker et al. calculated the total chronicle EC loss in 507 eyes of 289 patients receiving the Artisan myopia® or Artisan Toric ® (Ophtec B.V.) IF pIOL. Like us, he reported the EC loss corrected for physiologic EC loss (0.6% per year), at 5.2% and 7.5% from 6 months to 5 years, and 10.9% and 15.8% from 6 months to 10 years, respectively. Ten years after implantation, ECD had decreased by ≥ 25% in 7.9% and 6.3%, whereas the ECD was <1500 cells/mmectively . Galvis lls/mm2) . The patlls/mm2) .2, a critical ECD of 1500 cells/mm2 will be reached at 39, 28, and 15 years after the implantation, respectively [Some papers try to predict the mean time from initial surgery to IF pIOL explantation. Bouheraoua et al. performed a linear model analysis of the 5-year follow-up to present a model that describes endothelial cell loss as a linear decrease. This model predicted that for patients with preoperative ECD of 3000, 2500, and 2000 cells/mmectively . Such a ectively .As we mentioned above, the general aim is to evaluate each patient for basic risk factors to avoid unexpected postoperative EC loss. We tested the correlation between postoperative EC loss and basic factors like gender, degree of myopia, keratometry, pachymetry of the cornea, and ACD. In line with some previous studies, we did not find any significant relationship between gender, age, degree of myopia, and either basic keratometry . MoreoveOur outcomes describe, for the first time, the inverse correlation between the baseline corneal pachymetry and the EC loss at 12 years postoperatively. We noted slower EC loss in patients with thicker corneas and faster EC loss in those with thinner corneas. The most likely explanation of this finding could be just the effect of eye rubbing. The corneas with lower pachymetry have a stronger tendency to be deformed than the corneas with higher pachymetry. Shallowing of ACD could lead to contact between IF pIOL and endothelium. This finding corresponds with Galvis's suggestion  that theIn conclusion, this study confirms the advantages of IF pIOLs implantation due to rapid visual recovery and stable visual function in the 12-year follow-up. This method is safe, effective, and predictable in the long term and seems to be the better alternative to laser corneal refractive surgery for younger patients with high myopia and the ability to accommodate. Patients with IF pIOLs have to be regularly monitored because of the risk of EC loss and cataract formation, which is higher than in the general population. We proved the relationship of lower corneal pachymetry to increased EC loss. In addition to keeping in mind the safety limits of ECD (corrected to the age) and ACD, we recommend focusing on corneal thickness measurement prior to IF pIOL implantation.


This study was aimed to identify risk factors associated with unfavorable outcomes  in patients hospitalized in Galicia with COVID-19 pneumonia.Retrospective, multicenter, observational study carried out in the 8 Galician tertiary hospitals. All Patients admitted with confirmed COVID-19 pneumonia from 1st of March to April 24th, 2020 were included. A multivariable logistic regression analysis was performed in order to identify the relationship between risk factors, therapeutic interventions and the composite outcome variable.A total of 1292 patients  were included. Two hundred and twenty-five (17.4%) died and 327 (25.3%) reached the main outcome variable. Age [odds ratio (OR) = 1.03 : 1.01–1.04)], CRP quartiles 3 and 4 [OR = 2.24 (95% CI: 1.39–3.63)] and [OR = 3.04 (95% CI: 1.88–4.92)], respectively, Charlson index [OR = 1.16 (95%CI: 1.06–1.26)], SaO2 upon admission [OR = 0.93 (95% CI: 0.91–0.95)], hydroxychloroquine prescription [OR = 0.22 (95%CI: 0.12–0.37)], systemic corticosteroids prescription [OR = 1.99 (95%CI: 1.45–2.75)], and tocilizumab prescription [OR = 3.39 (95%CI: 2.15–5.36)], significantly impacted the outcome. Sensitivity analysis using different alternative logistic regression models identified consistently the ratio admissions/hospital beds as a predictor of the outcome [OR = 1.06 (95% CI: 1.02–1.11)].These findings may help to identify patients at hospital admission with a higher risk of death and may urge healthcare authorities to implement policies aimed at reducing deaths by increasing the availability of hospital beds. In late 2019, a new coronavirus  was identified in a number of patients presenting with pneumonia in Wuhan, Hubei province, China, and since then, virtually all countries have been affected by the pandemic. The severity of coronavirus disease-2019 (COVID-19) ranges from mild symptoms of upper respiratory tract infection to severe pneumonia and, although most reported cases are at the mild end of the spectrum, 15% of infected patients develop viral pneumonia and 1–5% of them die : 59.5–79.3)]. 2 was 63.9 ± 15.6 mmHg [median: 64 (IQR: 55–74)] and mean SaO2 was 92.1 ± 8.1% [median: 94 (IQR: 92–96)]. Two hundred and sixteen (16.7%) patients presented with respiratory failure.Mean days from symptom onset to admission was 7.5 ± 5.1 . The most common symptoms were : fever  and azithromycin (500 mg daily for 5 days) were employed in 91.9% and 79.2% of the patients, respectively . It deseOf the total 1292 patients, 225 (17.4%) died, 149 (11.5%) were transferred to the Intensive care Unit and received mechanical ventilation and 327 (25.3%) either died or needed mechanical ventilation (main composite outcome variable). Median time from admission to mechanical ventilation was 2.0 days  and the median of the hospital stay was 10 days .2 upon admission [OR = 0.93 (95% CI: 0.91–0.95)], hydroxychloroquine prescription [OR = 0.22 (95%CI: 0.12–0.37)], systemic corticosteroids prescription [OR = 1.99 (95%CI: 1.45–2.75)], and tocilizumab prescription [OR = 3.39 (95%CI: 2.15–5.36)].The predictors of mortality or need for mechanical ventilation in the multivariable analysis were as follows : age [od2, hydroxychloroquine and tocilizumab, similar to those found for the main composite outcome variable.We carried out an additional analysis using in-hospital mortality as a secondary outcome variable. Results are shown in The results of the sensitivity analysis for the main outcome variable are shown in We found a 17.2% in-hospital mortality in this cohort, formed by patients admitted with COVID-19 pneumonia in 8 tertiary Galician hospitals during the peak of the outbreak in March and April 2020. Among 126,137 patients hospitalized for COVID-19 from March to July 2020 in the USA, 15% of patients  died during the index hospitalization . Du et aData on COVID-19 pneumonia mortality during the second wave are lacking and the scarce available information suggests that mortality rates have declined, at least in developed countries, but this observation could be explained by the fact that second-wave infections tended to affect younger people and it cannot be attributed with certainty to therapeutic advances –19. Anot2, high CRP values, preexistence of comorbidities and the use of systemic corticosteroids and tocilizumab. Hydroxychloroquine prescription was associated with a favorable outcome.We identified predictors for poor outcome  in patients admitted with Covid-19 pneumonia: old age, low SaOIncreased age has been repeatedly associated with adverse outcome , 22 and In this study we have found that anti-inflammatory drugs (systemic corticosteroids and tocilizumab) negatively impact on prognosis, even after adjusting for other potential risk factors. In the earlier period of the pandemic, some influential experts advised not to use systemic corticosteroids in patients with COVID-19 pneumonia, relying on the lack of evidence, the negative results observed in influenza pneumonia and impaired clearance of SARS-CoV . Ever siIn contrast, we have found a protective effect of hydroxychloroquine on death or need for mechanical ventilation, whilst investigators of the Recovery Trial reported that results convincingly exclude any significant mortality advantage of hydroxychloroquine in hospitalized COVID-19 patients .Therefore, our results seem to be counterintuitive and two explanations can be put forward to account for these differences. The first one is obvious: in our population, corticosteroids and tocilizumab worsen the prognosis of patients with COVID-19 pneumonia. In fact, we have performed an exhaustive regression analysis containing significant covariates in an attempt to simultaneously adjust for their effect with the hope of isolating the impact of the intervention. The second explanation has to do with the inherent drawback of retrospective studies: the existence of potential bias and/or confounders. It is important to note that the captured data reflect the patients´ clinical condition at the moment of admission, but this situation changes over time, with some patients improving and some deteriorating, and this surely influences the physician´s decision to prescribe or not a given drug. Since this study was not performed under a controlled condition and treatments were not randomly assigned, we acknowledge that confounding by indication may still persist. Indeed, anti-inflammatory treatment was prescribed in patients with a more severe clinical condition and statistical adjustment for known confounders may not suffice to arrive at 2 groups that have truly the same prognosis at baseline . With hydroxychloroquine it happened exactly the opposite: it was given to patients with less severe disease.Interestingly, we found that hospital stress was associated with a higher risk of unfavorable outcomes in sensitive analysis, applying different regression schemes  Tables. The study’s strengths include the large sample size and the homogeneity of the included population . Besides, the fact that all Galician hospitals share the same model of electronic medical record enabled us to include every patient admitted to hospital during the study period. Baseline demographic, clinical and laboratory characteristics, alongside the presence of significant comorbidities were individually and exhaustively studied. We also evaluated the effectiveness of viral and host-targeted medications. The main limitation is its retrospective character leading to missing data. Moreover, as aforementioned, studies with retrospective design are not the most accurate to evaluate the impact of treatment on prognosis and survival, due to the possible presence of unnoticed and uncontrolled confounders. Another limitation lies in the unavailability of certain variables that would have been of interest for the aim of our study, such as body mass index.In conclusion, we identified several predictors of mortality  among a large cohort of COVID-19 pneumonia patients consecutively admitted to hospitals in Galicia (Spain) during the first outbreak of the epidemic. These findings offer a valuable insight into the characteristics and outcome of patients hospitalized with COVID-19 pneumonia. Our results also suggest that countries have the opportunity to implement policies aimed at reducing deaths by increasing the availability of hospital beds. Results concerning the efficacy of treatments should be interpreted with caution and more well-designed prospective studies are needed to clarify the role of the different therapies in this clinical context.S1 Appendix(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableModel 1: enter variables method; Model 2: forward automatic selection method.(DOCX)Click here for additional data file.S4 TableModel 1: enter variables method; Model 2: forward automatic selection method.(DOCX)Click here for additional data file.


Helicoverpa armigera (Hub.) is a destructive pest of the tomato (Lycopersicon esculentum Mill) crop in Pakistan. Although insecticides are the primary management strategy used to control H. armigera, most of them are not effective due to considerable toxic residual effects on the fruits. Nonetheless, H. armigera is rapidly evolving resistance against the available pesticides for its management. This situation calls upon the need of alternative management options against the pest. Different plant extracts have been suggested as a viable, environment-friendly option for plant protection with minimal side effects. Furthermore, the plant extracts could also manage the insect species evolving resistance against pesticides. This study evaluated the efficacy of different plant extracts  against H. armigera. Furthermore, the impact of the plant extracts on growth and yield of tomato crop was also tested under field conditions. The results revealed that all plant extracts resulted in higher mortality of H. armigera compared to control. Similarly, the highest plant height was observed for the plants treated with the plant extracts compared to untreated plants. Moreover, the highest tomato yield was observed in plants treated with plant extracts, especially with neem seed (21.013 kg/plot) followed by pepper extract (19.25 kg/plot), and garlic extract 18.4 kg/plot) compared to the untreated plants (8.9 kg/plot). It is concluded that plant extracts can be used as eco-friendly approaches for improving tomato yield and resistance management of H. armigera. Lycopersicon esculentum Mill) is an important and the most popular vegetable crop, grown and eaten around the globe. Tomato ranks second among major vegetables grown in Pakistan [th largest tomato producer in the world. However, country’s tomato production is low than developed countries in the world. Insect pests’ infestation is the major threat reducing tomato yield in Pakistan. Tomato crop is more likely to be attacked by several insect species from seedling to fruiting. Helicoverpa armigera (Hub.) is a destructive pest of tomato crop in Pakistan causing significant yield losses that worth 5 billion US$ annually [Tomato  at Entomological Research Area, Department of Entomology, University of Agriculture Faisalabad, Pakistan , during August 2018 and 2019. The mean temperature ranged from 28.6 to 34.5 °C and mean relative humidity ranged between 43.9 and 64.3% during the study period. The plant-to-plant and row-to-row distances were kept 30 and 65 cm, respectively. There were five rows and each row consisted of ten plants in each experimental unit. The size of each experimental unit was 26 mThe experiment was arranged according to completely randomized design (CRD) with three replications. Four botanical extracts  were applied and their efficacy was checked 3 and 7 days after application. In total, there were five treatments .Neem seed, turmeric, garlic and marsh pepper were bought from local market , washed and dried under shade for 7 days. The dried plant parts were separately grinded into fine powder. For each plant a 10% stock solution was prepared by mixing 100 grams of powder in 1 liter of distilled water in a flask. The mixture was shaken properly, placed at room temperature for 24 hours and then filtered using a plain cloth to remove impurities.H. armigera was recorded after 3 and 7 days of plant extracts application.For the application of plant extracts, tomato plants were sprayed twice at fifteen days interval using Knapsack sprayer after first fruiting. At the time of spray, polythene sheet was hanged around each plot to protect the adjacent plots from the drift effect. The infestation of Mature tomato fruits were harvested from each experimental unit. Number of damaged fruits and weight of all harvested tomato fruits was recorded for each experimental unit. Total yield of each experimental unit was calculated by adding yield of tomato fruits per harvesting. Plant height in all the experimental units was measured using meter rod. The population reduction percentage was calculated by counting the number of insects in treated and untreated plots before and after the application of plant extracts.The collected data on measured traits were analyzed to infer the differences among growing seasons through paired t test, which indicated that year effect was non-significant. Therefore, data of both growing seasons were pooled for further analysis. The data were tested for normality using Shapiro-Wilk normality test, which indicated that data had a normal distribution. Hence, original data were used for further analysis. One-way analysis of variance (ANOVA) was used to test the significance in the data. The means were compared by least significant difference post hoc test at 5% probability where ANOVA indicated significant differences. The dataset of the manuscript has been given as 2,14 = 114.08, P<0.01) altered plant height of tomato plants  affected total yield of tomato plants. The highest yield (21.013±0.67 kg/plot) was noted for neem seed extract which was followed by marsh pepper extract and garlic extract, where yield per plot was 19.25±0.56 kg and 18.4±0.58 kg, respectively. The lowest yield (8.9±0.58kg) per plot was recorded in control treatment of the study  affected the population of H. armigera three and seven days after treatment after both rounds of spray (Different plant extracts significantly (Fof spray . The higof spray .The highest population reduction percentage (62.93 ± 1.38%) was recorded for neem seed extract after seven days of spray. The population reduction percentage after seven days in marsh pepper extract, garlic extract and turmeric extract was 59.22 ± 1.51%, 53.08 ± 1.08% and 40.683 ± 2.19%, respectively. The lowest population reduction after 7 days of application in first round of spray was 10.00 ± 3.33%, recorded for control treatment .2,14 = 165.97, P<0.01) affected the population of H. armigera in second round of spray at three days after treatment. The highest population reduction percentage (49.383 ± 2.69%) was observed for neem seed extract followed by marsh pepper and garlic extract, where population reduction was 48.237 ± 1.78% and 44.7 ± 1.79%, respectively. The lowest population reduction (5.55 ± 1.92%) was recorded in control treatment of the study  affected the population of H. armigera seven days after the treatment during second round of spray. The highest population reduction percentage (73.69 ± 2.07%) was recorded for neem seed extract, followed by marsh pepper extract, garlic extract and turmeric extract, where population reduction was 67.077 ± 2.73%, 57.897 ± 2.01% and 50 ± 2.00%, respectively. The lowest population reduction (15.557 ± 1.93%) was recorded for control treatment of the study Click here for additional data file.


For 10 consecutive years, the ESPN/ERA-EDTA Registry has included data on children with stage 5 chronic kidney disease (CKD 5) receiving kidney replacement therapy (KRT) in Europe. We examined trends in incidence and prevalence of KRT and patient survival.We included all children aged <15 years starting KRT 2007–2016 in 22 European countries participating in the ESPN/ERA-EDTA Registry since 2007. General population statistics were derived from Eurostat. Incidence and prevalence were expressed per million age-related population (pmarp) and time trends studied with JoinPoint regression. We analyzed survival trends using Cox regression.Incidence of children commencing KRT <15 years remained stable over the study period, varying between 5.5 and 6.6 pmarp. Incidence by treatment modality was unchanged over time: 2.0 for hemodialysis (HD) and peritoneal dialysis (PD) and 1.0 for transplantation. Prevalence increased in all age categories and overall rose 2% annually from 26.4 pmarp in 2007 to 32.1 pmarp in 2016. Kidney transplantation prevalence increased 5.1% annually 2007–2009, followed by 1.5% increase/year until 2016. Prevalence of PD steadily increased 1.4% per year over the entire period, and HD prevalence started increasing 6.1% per year from 2011 onwards. Five-year unadjusted patient survival on KRT was around 94% and similar for those initiating KRT 2007–2009 or 2010–2012 .We found a stable incidence and increasing prevalence of European children on KRT 2007–2016. Five-year patient survival was good and was unchanged over time. These data can inform patients and healthcare providers and aid health policy makers on future resource planning of pediatric KRT in Europe.The online version contains supplementary material available at 10.1007/s00467-021-04928-w. After decades of continuous growth, the incidence of kidney replacement therapy (KRT) for stage 5 chronic kidney disease (CKD 5) in European adult patients has slightly declined during the last decade, whereas the increase in prevalence has started to slow down and patient survival has improved .CKD 5 etiologies in children are notably different from those in adults, as kidney failure at pediatric age is mostly caused by congenital or hereditary disorders rather than by diabetes or hypertension which are the most common causes in adults , 3. DespFor pediatric patients from the USA, the incidence of KRT decreased by 21% since 2004 after a long period of increasing incidence rates, whereas the prevalence was stable over the last decade . FurtherWith 10 consecutive years of data collection on European pediatric KRT patients within the ESPN/ERA-EDTA Registry, we are now able to report on trends in the incidence, prevalence, treatment modality, and patient survival from 2007 to 2016.Patient data were extracted from the ESPN/ERA-EDTA Registry, a population-based registry that collects data on individual pediatric patients on KRT in Europe on an annual basis , 10. CurData were categorized as follows: age at initiation of KRT (for incidence) or at the 31st of December of each year  , sex , treatment modality , peritoneal dialysis (PD), or kidney transplantation), cause of CKD 5 (categorized according to the ERA-EDTA coding system adapted for children) [Incidence rates per million age-related population (pmarp) were calculated as the number of new cases on KRT in each calendar year divided by the mid-year age-related general population. General population characteristics were extracted from the Eurostat database .Prevalence was calculated as the number of cases being alive and treated with KRT on the 31st of December of each calendar year and expressed as pmarp using the mid-year general population.To evaluate time trends in the observed incidence and prevalence, the JoinPoint regression program provided by the Surveillance Research Program of the US National Cancer Institute was used. The annual percentage change (APC) was computed using Poisson regression provided by the JoinPoint regression program, as described previously by Kramer et al. . In shorPatient survival probabilities were defined as the probability that a person has survived up to a specific time point, e.g., 1, 2, or 5 years after commencing KRT or after receiving a first kidney transplant, and causes of death were classified according to the ERA-EDTA Registry coding system, whereas cardiac failure, cardiac arrest/sudden death, myocardial ischemia and infarction, and cerebro-vascular accidents were combined as cardiovascular mortality . PatientThe annual number of kidney transplants performed (pmarp) between 2007 and 2016 was calculated, as well as the 1-, 2 -, and 5-year graft survival, defined as being alive with a functioning kidney transplant at 1, 2, or 5 years after transplantation, respectively.The Cox proportional hazard regression model was used to calculate patient and graft survival probabilities, while accounting for confounders. To compare adjusted 5-year survival between two periods of initiating KRT (2007–2009 and 2010–2012), adjusted hazard ratios were calculated using Cox proportional hazard regression models. Adjustments were made for the confounding effects of country, sex, cause of CKD 5, and age and treatment modality at start of KRT.Analyses were performed using SAS 9.4 statistical software package  and JoinPoint version 4.7.0.0.P values of 0.05 were considered statistically significant.Two-sided A total of 4459 patients aged 0–14 years from 22 European countries initiated KRT between 2007 and 2016. The yearly incidence of patients under 15 years commencing KRT by country fluctuated considerably from year to year   and metabolic disorders  leading to CKD 5, whereas the incidence rate of unknown or missing causes of CKD 5 increased significantly by 5.2% annually (95% CI: 1.1 to 9.4) between 2007 and 2016. Although not statistically significant, we observed a tendency towards decreasing numbers of patients commencing KRT due to vasculitis and hemolytic uremic syndrome (HUS). The incidence of KRT due to other causes remained stable over the study period . Overall 1-, 2-, and 5-year patient survival were 97.6% (95% CI: 97.1–98.1), 96.4% (95% CI: 95.8–96.9), and 94.4% (95% CI: 93.6–95.2), respectively. Similar adjusted survival probabilities were found (Table P < 0.001).For patients commencing KRT in the period 2007–2009, the 5-year unadjusted survival probability was 93.9% (95% CI: 92.6–95.2), whereas it was 93.3% (95% CI: 91.8–94.7) for patients commencing KRT between 2010 and 2012. After adjustment for age, sex, cause of CKD 5, and treatment modality at start, the patient survival at 1, 2 (data not shown), and 5 years did not differ between the periods . The causes of death differed by period  performed annually was stable and varied between from 3.1 to 3.9 pmarp .Overall unadjusted graft survival was 94.4% (95% CI: 93.5–95.3) at 1 year and 89.1% (95% CI: 87.8–90.5) at 5 years post-transplantation. Graft survival varied by year between 90.8% (95% CI: 87.3–94.4) and 97.0% (95% CI: 94.8–99.2) at 1 year and between 87.1% (95% CI: 83.2–91.2) and 94.1% (95% CI: 91.2–97.0) at 5 years after transplantation, but did not change over time Table .Table 6UOverall unadjusted patient survival after transplantation was 98.8% (95% CI: 98.4–99.3), 98.5% (95% CI: 97.9–99.0), and 97.2% (95% CI: 96.3–98.1) at 1, 2, and 5 years post-transplantation, respectively.In this ESPN/ERA-EDTA Registry study, we found a stable incidence and an increasing prevalence of nearly 2% per year in European pediatric KRT over the last decade. The first mode of KRT was stable over time, with the majority of patients initiating KRT on dialysis (both HD and PD 40%), and 20% of patients received a pre-emptive kidney transplant. Patient survival was good and did not change over time, but we found a shift in causes of death. Patients who commenced KRT before 2010 died more often from infectious causes, whereas patients commencing KRT later were more likely to die from cardiovascular disease.A previous ERA-EDTA Registry study reported a stable incidence for patients initiating KRT at an age below 19 years of age between 1997 and 2006 . SimilarIn adults, lifestyle-related diseases such as hypertension, diabetes, and obesity are important risk factors for CKD . AlthougIn accordance with findings in European adults  and in AWe observed an increasing trend in prevalence for all age groups and for all treatment modalities. Throughout the study period, the prevalence for both dialysis modalities was similar, whereas the number of patients living with a functioning transplant was about 4 to 5 times larger than the number of patients treated with dialysis. We did not find any further improvement in patient survival, but as long as the number of new CKD 5 cases is higher than the numbers of deaths, the prevalence rate will continue to grow.Overall, 1- and 5-year patient survival on KRT were 98% and 94%, respectively, and are similar to or slightly higher than rates reported in other high-income countries . We founThe improvement in patient survival of pediatric KRT has possibly reached its ceiling, but mortality remains at least 30 times higher than in the general pediatric population .Patients initiating KRT between 2007 and 2009 died more often from infections, whereas cardiovascular disease was the leading cause of death for patients initiating KRT between 2010 and 2012. Although others also reported cardiovascular disease and infections to be the leading causes of death in pediatric KRT patients , 20–22, We did not find an overall trend in the number of kidney transplants performed. Also the graft survival remained stable over time. The prevalence of kidney transplantation increased over the decade as the incidence of kidney transplantation continues to outweigh the number of patients whose kidney graft fails. One-year graft survival remained stable over the study period ranging from 91.5 to 97.3%. Overall graft survival was 94% at 1 year and 89% at 5 years and thereby slightly higher than in registry studies from Australia and New Zealand (83%) and the USA (86% for living donor kidneys and 79% for deceased donor kidneys) .As with every KRT registry, a limitation of our study is that only CKD 5 patients treated with KRT are included, and the numbers presented in this study do not reflect pediatric patients with CKD 5 not (yet) on KRT. Furthermore, only countries that participated in the ESPN/ERA-EDTA Registry for the entire study period of 2007 to 2016 are included in the current study, and our study might not be fully representative for the whole of Europe. However, our study is the first one to provide data on trends in incidence, prevalence, and rates of kidney transplantation and patient survival for European pediatric KRT patients over a 10-year period.In conclusion, we found a stable incidence and an increasing prevalence of European children on KRT over the past decade. Five-year patient survival was good and did not change over time. Also, the annual number of kidney transplantations performed did not change. These data can be used for future resource planning of pediatric KRT in Europe.ESM 1(DOCX 14 kb)


After the publication of the original article , the autLR and ACCT, the accuracy symbols have been swapped. The right sentences are: “ACCT were on average lower than ACCLR (Table 3) and more variable . This resulted in a much higher coefficient of variation for ACCT , particularly for ADG (41.06 vs. 7.79%) and OSS (37.07 vs. 10.24%).”In the first paragraph of page 5, where we compare ACCThe related Table 3, Fig. 1 and the discussion around those results are correct.


Aspergillus niger LFP-1 grown on pomelo peels as a sole carbon source under solid-state fermentation (SSF). The purification process begins with the concentration of crude enzyme using ammonium sulfate precipitation and followed by purification using anion-exchange column chromatography (DEAE-Sephadex) and subsequently using gel filtration column chromatography (Sephadex G-100). On the other hand, the molecular weight of the purified enzyme was determined through SDS-PAGE. The findings revealed the crude enzyme was purified up to 75.89 folds with a specific activity of 61.54 U/mg and the final yield obtained was 0.01%. The molecular mass of the purified pectinase was 48 kDa. The optimum pH and temperature were 3.5 and 50°C, respectively. This enzyme was stable at a range of pH 3.5 to 4.5 and a relatively high temperature (40°C–50°C) for 100 min. The Km and Vmax were found to be 3.89 mg/mL and 1701 U/mg, respectively. Meanwhile, pectin from citrus fruit and the metal ion (Co2+) were the best substrate and inducer to enhance pectinase yield, respectively.In the present study, pectinase was produced by local fungal isolate,  Aspergillus niger LFP-1 grown on pomelo peels as a sole carbon source under solid-state fermentation.Pectinase was successfully produced by local fungal isolate, Crude enzyme was purified up to 75.89 folds with a specific activity of 61.54 U/mg and the final yield obtained was 0.01%.2+) were the best substrate and inducer to enhance pectinase yield, respectively.Pectin from citrus fruit and the metal ion  . Since pAspergillus niger using pomelo peel as a substrate via SSF was purified and characterised by accessing the optimal temperature and pH. Meanwhile, the molecular mass of the enzyme was determined as well as Michaelis-Menten constant.The purification and characterisation of the enzyme are crucial since optimal fermentation conditions and enzyme recovery can reduce the production cost  prior to its use in SSF using an electrical grinder .A. niger LFP-1 provided from the stock fungal cultures of the Industrial Biotechnology Research Laboratory (IBRL), School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysia was used for pectinase production throughout the study. The fungal culture was maintained on potato dextrose agar (PDA) slant nourished with 1.0% citrus pectin (w/v) at 30°C for 7 days (7 spores/mL) into 250 mL Erlenmeyer flasks containing 5 g pomelo peels powder (0.75 mm particle size) which previously moistened with 4 mL of a solution containing 1.2% ammonium nitrate and autoclaved at 121°C for 15 min. The flasks were incubated at room temperature for 7 days under static conditions. After a week of cultivation, the cultures were harvested by mixing with 50 mL of distilled water  using a column of DEAE Sephadex . An amount of 0.4 mL of the concentrated enzyme was loaded to DEAE-Sephadex  which was previously washed and equilibrated with 0.05 M citrate buffer (pH 4.4). After equilibration, the enzyme was eluted with the same buffer and 3 mL of the sample was then collected. The active fraction was then pooled and collected together for the subsequent purification step.Subsequently, the active fractions from the previous step were loaded to the top of the Sephadex-100 column which was pre-equilibrated 0.05 M citrate buffer (pH 4.4). The fractions were eluted with the same buffer at the flow rate of 0.41 mL/min. The fractions of each purification steps were assayed for enzyme activity.The molecular weight of purified pectinase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme was treated with sample buffer  Glycerin, 10% (v/v) (2-Mercaptoethanol) and incubated at 95°C for 5 min in a Thermomixer Incubator . After running, the gel was stained with 0.25% Bromophenol blue followed by de-staining and drying. The authentic broad range protein molecular weight markers  contains standards molecular weight of 10 kDa, 15 kDa, 35kDa, 50 kDa, 75kDa, 100 kDa, 150 kDa, and 225 kDa was used.The effect of incubation temperature  on pectinase activity was determined by incubation of the reaction mixture in 50 mM, Pectin-citrate buffer (pH 4.4) for 30 min at each temperature. Meanwhile, the thermal stability of the purified pectinase was evaluated at various incubation temperature degrees  from 10 to 100 min . The residual activity was assayed by the standard method.The effect of reaction pH on pectinase activity was accessed using citrate buffer (pH 2.0–4.0), potassium-phosphate buffer (pH 5.0–8.0) and glycine-NaOH buffer (pH 9.0). The reaction mixture was incubated for 30 min at 50°C. The pH stability of the purified pectinase was determined by pre-incubation of the enzyme at various pH range (pH 2.0–9.0) for 100 min. The residual activity was measured by the standard method.The substrate specificity of purified pectinase towards various substrates including xylan, citrus pectin, potato dextrose agar (dextrose), D-galacturonic acid, and starch was evaluated. The substrates were incubated with the purified enzyme in 50 mM citrate buffer (pH 4.4) for 30 min at an optimal temperature. The relative activity of pectinase was determined for each substrate using the standard methods in which pectin from citrus was set as a control.A. niger pectinase was evaluated by incubation of the purified enzyme with 20 mM metal ions and reagents including sodium dodecyl sulphate (SDS), ethylenediamine tetraacetic acid (EDTA), NaCl, KCl, CaCl2.2H2O, FeSO4, FeCl3, MgSO4 and AgNO3. The residual activity was determined using the standard method under optimum conditions.The influence of chemical compounds on the activity of m and Vmax values of the enzyme were assessed by measuring the reaction velocity at different substrate concentration and the citrus pectin was used as a substrate. A stock solution of citrus pectin with a concentration of 10 mg/mL was firstly prepared in citrate buffer (pH 4.5). The stock was subsequently diluted by a volume of citrate buffer to make a serial dilution with the final concentration of citrus pectin in between 2 mg/mL to 10 mg/mL. The pectinase activity was determined using the previously standard assay. The relationship between velocity (enzyme activity) and substrate concentration was plotted using GraphPad Prism 8 software. The Michaelis-Menten constant values (Km and Vmax) were calculated using non-regression linear.The KOne-way analysis of variance (ANOVA) and Duncan Multiple Range Test (DMRT) with PASW Statistics 18 version has been used to analyse the significant difference of the mean of experimental data. Five percent (5%) confidence level or α = 0.05 has been used to test all experimental data. All measurements were determined in triplicates.A. niger LFP-1 was previously isolated from rotten orange nearby Industrial Biotechnology Research Laboratory (IBRL) building, Universiti Sains Malaysia, Pulau Pinang is a filamentous fungus which forms filaments and resembles the structure of a plant. A. niger LFP-1 with smooth and colourless conidiophores and spores, whereas A. niger LFP-1 colonising and penetrating the substrates. Fungal strain, A. niger LFP-1 that utilising pomelo peels as a substrate under SSF. Crude pectinase was harvested from the filamentous fungus, A. niger LFP-1 was determined under denaturing conditions using SDS-PAGE . 10 molecular weight versus relative mobility (Rf) was plotted to determine the more accurate molecular mass of purified pectinase and The molecular weight of the purified pectinase of A. niger LFP-1 was accessed using a pH range of 2.5 to 5.5 and the result is shown in The influence of pH value (pH 1.5–8.5) on purified pectinase activity was determined in the study. The affinity of purified pectinase towards substrates was evaluated as shown in A. niger LFP-1 is summarised in Table 2. The enzyme has reached its maximum activity by pretreating with Co2+ in which, the relative activity was shot up to 190.13 ± 0.3% compared to the control. Besides, the pectinase was enhanced by several metal ions and reagent with higher activity achieved when the enzyme was pre-incubated with Fe3+ (125.04 ± 0.8%), Ca2+ (116.02 ± 1.1%), Na+ (103.42 ± 2.2%), NH4+ (103.42 ± 1.2%), EDTA (115.39 ± 0.2%) and 2-Mercaptoethanol (114.26 ± 1.6%). On the other hand, the enzyme activity was significantly reduced by the presence of Mn2+, Al3+, Ba+ retaining only about 38.19 ± 1.1%, 28.47 ± 2.1% and 6.58 ± 1.2%, respectively. It is noteworthy that Ag+ and SDS were completely inhibited pectinase activity whereby no activity was observed.The impact of various metal ions and reagents on the pectinase activity produced by m and Vmax values of pectinase were assessed by measuring the reaction velocity at different concentration of the citrus pectin. The relation between velocity and the substrate concentration was analysed with non-regression analysis. The results showed the regression coefficient (R2) was 0.9970 which indicated the concentration of the substrate and the enzyme activity was positively correlated  and organic acids such as citric and gluconic acids . The purification of pectinase has several series of methods such as salt/solvent precipitation, gel filtration chromatography, ion-exchange chromatography  whereas the smaller molecules can access the higher number of pores and retain longer in the column . Howevertography . HoweverA. niger LFP-1 grown in pomelo peels as a substrate and sole carbon source was approximately 48 kDa. Similar observation reported by Rhizomucor pusillus A13.36 was estimated to be in the range of 43.5 kDa–47 kDa. The finding was in agreement with the previous study that reported the molecular weight of microbial pectinases to fall within the size in the range of 30 kDa–70 kDa , studying pectinase from A. niger URM4645 grown on forage palm in SSF. They revealed that the enzyme activity was stable at 50°C and deactivated at higher temperature. The unstable enzyme activity at higher temperature might be due to hydrolysis of peptide bonds, destruction of disulfide bonds, deamination and oxidation of the amino acid side chains of protein molecules  observed at 45°C and 55°C . Howeverolecules .A. niger LFP-1 while pH 4.5 showed appreciable enzyme activity  was strongly inhibited the pectinase activity. Similar observation reported by + was completely inhibited exo-polygalacturonase activity produced by A. niger MTCC 478. The formation of homogalacturonan cross-link by a cation such as Mg2+ decreased the availability of the substrate to the enzyme or the binding of cation to an acid amino side chain involved in catalysis of the substrate might be the reason on the inactivation effect of the metal ion on enzyme activity  as a result of its highest Vmax. The finding revealed that a small quantity of the enzyme will digest a considerably high amount of substrate.The kinetics parameter is crucial in enzymatic reaction since it describes the efficiency of the enzyme. The present study demonstrated that the pectinase from A. niger LFP-1 which was isolated from orange peels was successfully purified. The biochemical properties demonstrated by the pectinase enzyme suggested that this particular enzyme is a potential candidate for various biochemical reactions in the industrial biotechnological process, especially in the juice and food industry.Pectinase from


Salmonella enterica serovar Typhi, remains a considerable public health threat in impoverished regions within many low- and middle-income settings. However, we still lack a detailed understanding of the emergence, population structure, molecular mechanisms of antimicrobial resistance (AMR), and transmission dynamics of S. Typhi across many settings, particularly throughout the Asia-Pacific islands. Here we present a comprehensive whole genome sequence (WGS) based overview of S. Typhi populations circulating in Papua New Guinea (PNG) over 30 years.Typhoid fever, a systemic infection caused by S. Typhi isolates collected between 1980–2010 demonstrated that the population structure of PNG is dominated by a single genotype (2.1.7) that appears to have emerged in the Indonesian archipelago in the mid-twentieth century with minimal evidence of inter-country transmission. Genotypic and phenotypic data demonstrated that the PNG S. Typhi population appears to be susceptible to former first line drugs for treating typhoid fever , as well as fluoroquinolones, third generation cephalosporins, and macrolides. PNG genotype 2.1.7 was genetically conserved, with very few deletions, and no evidence of plasmid or prophage acquisition. Genetic variation among this population was attributed to either single point mutations, or homologous recombination adjacent to repetitive ribosomal RNA operons.Bioinformatic analysis of 86 S. Typhi in this setting.Antimicrobials remain an effective option for the treatment of typhoid fever in PNG, along with other intervention strategies including improvements to water, sanitation and hygiene (WaSH) related infrastructure and potentially the introduction of Vi-conjugate vaccines. However, continued genomic surveillance is warranted to monitor for the emergence of AMR within local populations, or the introduction of AMR associated genotypes of  Salmonella enterica serovar Typhi, is a systemic infection common to many low- to middle-income settings. While the population structure of S. Typhi has been genetically characterised using whole genome sequencing in many endemic countries throughout Sub-Saharan Africa and South Asia, we are still lacking a detailed understanding for many regions including those among the Asia-Pacific islands. Genomic surveillance of isolates spanning 30 years demonstrated a population structure of S. Typhi in Papua New Guinea (PNG) dominated by a single genotype (2.1.7) that emerged in the mid-twentieth century, is genetically homogeneous, and sensitive to a wide range of antibiotics commonly used in the treatment of typhoid. There was little evidence of inter-country transmission and the setting appeared free of S. Typhi genotypes commonly associated with AMR e.g. H58 (genotype 4.3.1). These data suggest that former first line drugs , fluoroquinolones, third generation cephalosporins and macrolides all remain viable options for controlling typhoid in addition to the introduction of Vi-conjugate vaccines and improvements to water, sanitation and hygiene (WaSH) related infrastructure. Routine molecular surveillance is necessary to monitor for introduced or emerging AMR to inform treatment guidelines and intervention strategies.Typhoid fever, caused by  Salmonella enterica serovar Typhi[S. Typhi is transmitted faeco-orally, usually through contaminated food and water and therefore constitutes a major public health threat in many low- to middle-income countries (LMIC), particularly in impoverished regions where hygiene and sanitation infrastructure is limited[Typhoid fever is a systemic infection of the bacterium var Typhi. Each yevar Typhi. S. Typhs limited.S. Typhi, with multidrug-resistance  emerging from the 1970s onwards[gyrA, gyrB, and parC[S. Typhi populations resistant to all orally administered antimicrobials except for azithromycin have emerged in Pakistan outbreaks[S. Typhi populations[Antimicrobial chemotherapy has become a mainstay in the treatment of typhoid fever, with death rates declining from 20–30% to 1% following the introduction of antimicrobials,5. Howevs onwards, followeoutbreaks,11, and pulations–15.Papua New Guinea (PNG) has been recognised as a high burden setting for typhoid since the mid-1990s when the incidence rate was reported as 1,208 cases per 100,000 population, amongst the highest rates in the world at that time. AlthougS. Typhi appeared endemic to PNG, with many appearing unique to the country, and some also observed in Southeast Asian countries. Further, 80.6% of isolates from 1986–1989 belonged to a single phage type (D2) [S. Typhi from PNG appeared free of both detectable AMR genes and plasmids, and have suggested that genome plasticity might be common. However, despite these early observations, only two locally collected S. Typhi sequences from PNG have been reported on in detail[S. Typhi in PNG. Here we combine data on 41 novel isolates collected locally in PNG between 1992 and 2010, with those data sequenced previously from return travellers, to provide a 30-year overview (1980–2010) of S. Typhi in PNG.Early molecular studies –20 reveaype (D2) ,20, whicype (D2) , have alin detail. A furthEthical approval for this study was granted by the PNG Institute of Medical Research Institutional Review Board (1609), the PNG Medical Research Advisory Board (MRAC 16.43) and the Federation University Human Research Ethics Committee (A17-074).PNG is a tropical country situated approximately 6° south of the equator in the Western Pacific region. It is a LMIC, with a large proportion of its ~9 million inhabitants living in regional and remote areas of the country. Health and development indicators are low by regional and global standards, with ~40% of people having access to improved water supply and <20% with access to adequate sanitation,23. In 2The PNG Institute of Medical Research (PNGIMR) is headquartered in Goroka, the capital of Eastern Highlands Province. The institute has been responsible for the majority of studies that have been conducted on typhoid fever in PNG.S. Typhi that were sequenced as part of this study (n = 41) were obtained from the PNGIMR culture collection. All isolates had been preserved by freeze-drying in glass ampules. The content of each ampule was resuspended in nutrient broth, and inoculated onto nutrient agar (incubated for 18–24 hours at 35°C). PCR was conducted on isolates using previously described methods [S. Typhi.Isolates of  methods  to confiS1 Table. AST was performed on S. Typhi isolates using agar breakpoint dilution and interpreted using Clinical and Laboratory Standards Institute (CLSI) breakpoints as described previously[Salmonella) that has been operated by the Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL)[Antimicrobial susceptibility testing (AST) was carried out for n = 40 (88.9%) of travel-associated cases for chloramphenicol, ampicillin, sulfamethoxazole, trimethoprim, nalidixic acid, tetracycline, kanamycin, gentamycin, and spectinomycin as detailed in reviously. Phage t(MDU PHL).Isolated colonies (1–3) were suspended in sterile water to 0.5 McFarland, and 100 μl of the suspension was used for DNA extraction with the DNeasy Blood and Tissue Kit , following the manufacturer’s instructions. Extracted genomic DNA (gDNA) was then subjected to indexed whole genome sequencing on an Illumina HiSeq 2500 platform at the Wellcome Sanger Institute to generate paired end reads of 150 bp in length as described previously.Three isolates were selected for additional long-read sequencing using the Nanopore MinION R9 device which was carried out as described previously. To geneS. Typhi isolates collected in this study together with n = 45 from a previous study[S1 Table), were aligned to the CT18 reference genome (GenBank accession AL513382) [https://github.com/katholt/reddog). RedDog maps reads to the reference genome with Bowtie2 (v2.2.9)[S. Typhi genotypes (~80% 2.1.7.1 and ~20% 2.1.7.2) and was subsequently removed from further phylogenomic analyses. A core SNV alignment was generated by concatenating alleles with high-quality consensus base calls (phred scores >20), for all SNV sites meeting this criteria in >95% of genomes (representing the 95% ‘soft’ core of the S. Typhi genome). This alignment was filtered to exclude SNV called in previously defined [S1 Fig).For Single Nucleotide Variant (SNV) analysis, paired end reads for n = 41 ous study (S1 TablL513382)  using th v2.2.9), before  (v2.2.9) to ident, before  defined ,32 proph defined , which whttps://github.com/katholt/genotyphi/) was used together with RedDog Bowtie2 alignment (BAM) files to infer genotypes for all sequences analysed in this study using SNVs that were defined in the extended S. Typhi genotyping framework[S. Typhi population was dominated by a single genotype , necessitating the completion of a reference genome for the PNG genotype 2.1.7 population to allow for high resolution phylogenomic analyses. Three PNG genotype 2.1.7 genomes were selected for Nanopore long read sequencing and hybrid assembly (see S1 Table), with the oldest of these (MDUST348 from 1985), selected as a reference sequence for lineage-focused SNV analyses and phylogenetic inference using the mapping-based approach described above. For this newly completed PNG genotype 2.1.7 reference genome , repetitive regions were identified using the ncumer command in MUMmer (v3.23) [https://phaster.ca/)[S2 Table) were then filtered from the inferred PNG genotype 2.1.7 alignment, with any further recombination removed with Gubbins as described above. The resultant recombination filtered alignment of PNG genotype 2.1.7 S. Typhi sequences (n = 83) also included alleles from representative non-genotype 2.1.7 sequences as outgroups for phylogenetic tree rooting (detailed in S3 Table), and this final alignment  was then used to infer the final phylogenies shown in Figs S1 Table) from previous studies[Fig 2; 184 sequences including outgroups). An interactive version of the resulting global context clade 2.1 phylogeny is available at: https://microreact.org/project/icGPbF_O8.GenoTyphi   and intester.ca/). These rs studies,38 was iS. Typhi sublineages were manually extracted from RedDog allele tables using R. SNVs causing non-synonymous mutations in highly conserved genes were prioritized for sublineage definitions. SNV distances were calculated from the core genome SNV alignment using snp-dists .Unique SNVs defining PNG genotype 2.1.7 S. Typhi  were derived from the gene presence/absence matrix output from the RedDog mapping pipeline using the MDUST348 PNG 2.1.7 S. Typhi reference genome completed in this study.Assessments of the gene conservation across PNG genotype 2.1.7 S. Typhi in PNG, we used multiple methods. Initially we used TempEst (v1.5.1) [ as a function of sampling time. The heuristic residual mean squared method was used for the regression analysis with the best-fitting root option selected. The resultant data from these analyses were visualised in R.To investigate the temporal signal and emergence of genotype 2.1.7 (v1.5.1)  to assesS1 Table), the precise year of isolation was not available, however, as they were known to have been collected between 2009–2010 their date of isolation was estimated with BEAST2 using a tip date sampling prior specifying a uniform distribution between 2009–2010. Models were fit using both constant-coalescent population size and Bayesian skyline tree priors, together with either a strict clock model or a relaxed  clock model, to identify the model combination that best fit the data. For all model and tree prior combinations, a chain length of 10,000,000 steps sampling every 5,000 steps was selected[S3 Fig) indicated that the data displayed temporal structure [In order to estimate divergence times we analysed the sequence data with BEAST2  (v2.4.7) selected. Prelimi selected suggeste selected,42. Thestructure .https://github.com/zadyson/PNG_STyphi_2022). The online tool Microreact [https://microreact.org/project/CYcTnSmjT.For the final analysis reported here, a single independent run conducted with a chain length of 10,000,000 states, sampling every 5,000 iterations, was subjected to removal of the first 10% of steps as burn-in with LogCombiner (v2.4.7). Maximumcroreact  was usedS1 Table). The resultant de Brujin assembly graphs were then resolved into single contigs where necessary through manual curation with Bandage (v0.8.1)[Hybrid Illumina-Nanopore genome assemblies were generated with Unicycler (v0.4.7)  for thre (v0.8.1), and ann (v0.8.1). Nucleot (v0.8.1) (vSnapsh (v0.8.1).For analysis of prophage regions detected with PHASTER  in hybrigyrA, gyrB, and parC that are known to reduce susceptibility to fluoroquinolones in S. Typhi [acrB mutations associated with azithromycin resistance in typhoidal Salmonella[described above).The mapping based allele typer SRST2  (v0.1.8)S. Typhi , and acralmonella, were deS1 Table. Genome assemblies for completed sequences of traveller isolates MDUST348, MDUST255, and MDUST305 were deposited in GenBank under accession numbers GCA_001173525, GCA_001122625, and GCA_001134605, respectively (see S1 Table for details).Raw read sequences for the 41 novel isolates from PNG have been deposited in the European Nucleotide Archive under project PRJEB20541; and individual accession numbers are listed in S. Typhi collected locally in PNG and sequenced for this study, with n = 45 sequences from a previous study [S1 Fig), and revealed that the population structure of S. Typhi during this period was dominated by a single genotype, 2.1.7  which has been present for at least 25 years. Only one other genotype was detected; clade 4.1  .We combined sequence data from n = 41 us study  that ori, members of which are separated by a median pairwise distance of ~33 SNVs. We designated these as lineage I (genotype 2.1.7.1) and lineage II (2.1.7.2). In our data, genotype 2.1.7.1 was more common with n = 66 (78.6%) members, compared to n = 18 (21.4%) 2.1.7.2 members. Interestingly, 2.1.7.2 was not observed after 1998. Genotype 2.1.7.1 can be identified by the presence of a synonymous marker SNV C1503T in gene STY4417 (position 4286788 in CT18), and genotype 2.1.7.2 can be identified by the presence of a synonymous marker SNV C540T in gene STY4106 (position 3967063 in CT18). These genotypes have been added to the GenoTyphi scheme available at http://github.com/katholt/genotyphi)[The population structure of genotype 2.1.7 in PNG can be subdivided into two main lineages enotyphi),35,56.Methods) were detected among any of the S. Typhi sequences from PNG, indicating this population is sensitive to commonly used antimicrobials. Susceptibility testing data was available for n = 40 (88.9%) traveller isolates, all of which tested susceptible to all drugs tested (see Methods and S1 Table).No known acquired AMR genes, nor mutations associated with either decreased ciprofloxacin susceptibility or azithromycin resistance (see  interactive phylogeny available at: https://microreact.org/project/CYcTnSmjT). This analysis yielded a local substitution rate of 0.49 SNVs per genome per year (95% HPD 0.32–0.66) or 1.1x10-7 genome-wide substitutions per site per year (95% HPD 7.3x10-8–1.5x10-7). These data showed temporal structure [Methods and S3 Fig), which were slightly lower than published estimates for S. Typhi for genotype 4.3.1 in Nepal [-7, 95% HPD1.5x10-7–2.2x10-7)[−7, 95% HPD 1.0x10−7 to 1.8x10−7)[Fig 1. PNG 2.1.7 sublineages appear to have emerged contemporaneously in PNG in the mid-late 1970s, with the MRCA of 2.1.7.1 estimated circa ~1976 (95% HPD 1963–1985) and the MRCA of 2.1.7.2 around the same time or potentially slightly later, circa ~1979 (95% HPD 1971–1984).We applied Bayesian phylodynamic analysis to the set of PNG 2.1.7 sequences to estimate the dates of emergence for genotype 2.1.7, and its sublineages 2.1.7.1 and 2.1.7.2 (tructure  to suppo2.2x10-7), and gloS. Typhi to its nearest relatives in the pathogen population, we inferred a phylogeny of all available genome sequences belonging to its parent clade 2.1 ( interactive phylogeny available at: https://microreact.org/project/icGPbF_O8). Genotype 2.1.7 isolates were mostly from PNG . Within genotype 2.1.7, PNG sequences formed a distinct cluster (further subdivided into lineages 2.1.7.1 and 2.1.7.2), with a small number of distant relatives in India (three genotype 2.1.7 isolates separated from PNG isolates by a median pairwise distance of ~64 SNVs) and Vietnam (one sequence separated by median ~41 SNVs). The only non-PNG isolate clustering within the PNG sublineages was a single 2.1.7.1 sequence from India , which was intermingled in the phylogeny with the PNG isolates. This sequence was separated by a distance of ~3 SNV from its closest PNG relative suggestive of transmission of this genotype from PNG to India on at least one occasion. The other (i.e. non-2.1.7) isolates from clade 2.1 originated almost exclusively from other islands in the Indonesian archipelago , suggesting that the PNG 2.1.7 group emerged from a local pathogen population that had been established in the region for some time.In order to better understand the relationship of PNG genotype 2.1.7 Methods, we completed three PNG genotype 2.1.7 genomes to facilitate high resolution analysis of this clone. Three 2.1.7 genomes from PNG S. Typhi were selected for completion; two were the oldest sequences from each sublineage available . We also completed a recent sequence of the most common genotype 2.1.7.1 (sequence MDUST255 isolated in 2010). The three completed genomes were markedly similar in size, differing by 4.4 kbp . Further, pairwise nucleotide sequence comparisons revealed high levels of genetic similarity , and evidence of genome plasticity with multiple large-scale inversions observed adjacent to repetitive ribosomal RNA (rrn) operons .As described in S4 Fig). Very few deletions were observed, and were not restricted to a particular sublineage. Three sequences, two of genotype 2.1.7.1 (MDUST329 and UJ 212G) and one of 2.1.7. 2 (MDUST332) appeared to have lost the Salmonella pathogenicity island 7 (SPI7) locus known to encode genes for Vi polysaccharide capsule formation, type IVB pili, and bacteriophage SopE. Such deletions are known to occur in the laboratory and did not appear to affect the phage types previously determined for the PNG 2.1.7 traveller isolates as all were of phage type D2. Besides the deletion of prophage SopE within SPI7, no other prophage deletions were apparent.Gene content was highly conserved among both PNG 2.1.7 sublineages . The inversion was located in the prophage tail gene module carrying homologues of prophage ST35 genes (from reference sequence CT18) encoding for a tail fibre (STY3691), tail fibre assembly chaperone (STY3692), alternate tail fibre tips (STY3693 and STY3694), a site-specific invertase (STY3695), and major tail tube filament structural gene (STY3696)[Examination of detected prophage elements amongst the three completed prophage sequences revealed a smaller ~1.8 kbp inversion within an ST35-like prophage in the genome sequence of MDUST255 ((STY3696). The inv(STY3696).S. Typhi (genotype 2.1.7) which emerged in the Indonesian archipelago in the mid-twentieth century before differentiating into two sublineages in the mid-late 1970s, with genotype 2.1.7.2 no longer appearing common after 1998. These findings correlate well with the broad epidemiology of typhoid fever in PNG. Typhoid fever was sporadically reported in PNG in the 1960s, becoming more commonly diagnosed in the 1970s and then becoming endemic in the PNG highlands in the 1980s [S. Typhi 2.1.7 sublineages correspond to the approximate time when typhoid fever became endemic. S. Typhi genotype 2.1.7 appears to have persisted within PNG since this time, and was dominant among both returning travellers and locally detected cases over the course of 30 years of genomic surveillance. We observed little evidence of genetic variation among the genotype 2.1.7 population, with infrequent gene deletions , no obvious evidence of prophage or plasmid acquisitions , as well as fluoroquinolones, third generation cephalosporins, and macrolides. The AMR sensitivity of the PNG 2.1.7 genotype and the long term stability of this population is in sharp contrast to many other endemic settings, e.g. South Asia where infections are commonly driven by H58 genotypes[These findings demonstrate that the vast majority of typhoid fever cases in PNG are caused by a single lineage of he 1980s ,58. Thuseletions  Fig, no genotypes,60, and genotypes,11, and genotypes–15,61 haS. Typhi into India . However, it is possible that this individual may also have visited PNG as well as India in their travels with only the later recorded. The limited international movement of S. Typhi in and out of PNG may partially explain why AMR has not been observed in this setting, in the form of the arrival of H58 or other drug resistant genotypes of S. Typhi.We observed limited evidence of inter-country transmission, with no obvious recent introductions into PNG from outside. A small number of genotype 2.1.7 isolates were found outside PNG in India and Vietnam, but these were mostly distantly related to the PNG lineages and we observed only one possible case of transmission of PNG genotype 2.1.7 Shigella from PNG and the Pacific region revealed this pathogen to have resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole, with resistance more common in bacteria isolated since 2010. Resistance to cephalosporins (ceftriaxone) and ciprofloxacin was not detected in Shigella from PNG[Shigella, resistance to first-line antibiotics could emerge in other gastrointestinal pathogens in PNG, but exposure, and therefore resistance to, cephalosporins and second-generation quinolones (e.g. ciprofloxacin) is unlikely.Among PNG 2.1.7 populations, the lack of emergence of AMR may be influenced by a lack of local selective pressures related to antimicrobial usage. Tracking antimicrobial usage is challenging in PNG, but evidence of sub-optimal administration of antibiotics has been reported. Approxi from PNG. On the S. Typhi genomes sequenced previously from PNG by Baddam and colleagues [S. Typhi population [rrn operons , rather than a diverse range of genotypes or the acquisition of mobile genetic elements such as prophages or plasmids consistent with previous reports[rrn operons have been described previously in S. Typhi and are driven by homologous recombination [S. Typhi[rrn operons are relatively common in S. Typhi[The two lleagues  were genpulation  appears s reports,19. Rearbination ,65. This[S. Typhi, and our S. Typhi, and app S. Typhi. We also S. Typhi. Such va S. Typhi,69.S. Typhi. There is no ongoing routine surveillance conducted in PNG, nor is there routine blood culture for diagnosis of febrile illness. Consequently, locally collected isolates are obtained primarily through targeted studies. Most isolates analysed in this study originated from Eastern Highlands Province, where the PNG Institute of Medical Research is headquartered and is thus the site of most studies. Eastern Highlands Province is one of 22 provinces in the country , thus there is a lack of geographical distribution of isolates. Given most isolates originate from targeted studies, the temporal distribution of isolates is intermittent. Moreover, the lack of ongoing surveillance precluded contemporary isolates (from the past 10 years) being collected and thus included in this study. However, as locally collected and travel associated cases both demonstrated a lack of AMR and similar genotype frequencies, in line with limited previously published sequences[S. Typhi population in this setting.This study is not without limitations, primarily relating to the challenges associated with obtaining clinical isolates of sequences, these cS. Typhi populations in PNG appear sensitive to a diverse range of antimicrobials with former first line drugs, fluoroquinolones, third generation cephalosporins and macrolides all remaining viable treatment options prior to the introduction of Vi-conjugate vaccines and improvements to water, sanitation and hygiene (WaSH) related infrastructure. However, despite this, further ongoing surveillance is required to monitor for the potential emergence or introduction of AMR S. Typhi in this setting.Overall, our data highlight that unlike other recently examined settings in Kenya, Zimbabwe and other regions of Sub-Saharan Africa, as well as South Asian settings in India, Nepal, Bangladesh and Pakistan ,60,70–73S1 FigS. Typhi genotype. Square nodes indicate the position of the reference sequence and other completed genomes.Tip colours indicate the source of the sequence as per the inset legend. Branches are labelled by (PDF)Click here for additional data file.S2 FigFig 1.Tip colours indicate the source of the sequence as per the inset legend, and branches are labelled by genotype. Square nodes indicate the position of the reference sequence and other completed genomes. A Bayesian dated tree inferred using the same alignment and year of isolation is shown in (PDF)Click here for additional data file.S3 Fig(A) Tempest regression of root-to-tip distances as a function of time, with the root of the tree selected using heuristic residual mean squared. Each point represents a tip in the maximum likelihood phylogenetic tree shown in S2 Fig. The slope is a crude estimate of the annual substitution rate for the SNV alignment, the x-intercept corresponds to the age of the root node, and R2 is a measure of clock-like behaviour among the data. (B) Date randomisation test results with the right most box plot showing the posterior substitution rate estimate from the SNV alignment of the data with the correct sampling times, and the remaining 20 boxplots showing the posterior distributions of the rate estimate from replicate runs where the dates were subjected to randomisation. The data are considered to have strong temporal structure if the estimates using the correct dates do not overlap with those where the dates were randomised.(PDF)Click here for additional data file.S4 FigBranches are labelled by genotype. Coloured square nodes indicate the position of the reference sequence and other completed genomes. Heatmap shows the presence or absence of all annotated genes in the reference sequence as per the inset legend. Features of interest are highlighted with red boxes and labels.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Preterm related complications are the single largest direct cause of neonatal deaths throughout the world, responsible for 35% of the world’s neonatal death (1.1 million deaths/year). In Ethiopia preterm related complications are still the leading cause of neonatal mortality. Identifying the hazard time to death and predictors of mortality play an important role to decrease preterm mortality. Therefore, this study aimed to determine the survival and predictors of mortality among preterm neonates admitted to neonatal intensive care unit of Addis Ababa public hospitals, Ethiopia, 2021.An institutional based prospective follow up study was conducted among 358 preterm neonates admitted to selected public hospitals of Addis Ababa, Ethiopia from February 12 to May 12, 2021. Systematic random sampling was used to recruit each sample and data was collected prospectively using structured questioner. Epi-data version 4.6 and STATA version 16 was used to data entry and analysis respectively. Kaplan Meier failure curve, Log rank tests were computed. Schoenfeld residual test was used to check overall model fitness. Cox proportional hazards models were fitted to identify independent predictors of preterm mortality.At the end of this cohort, 125(34.9%) of the neonates died, with incidence rate of 36.4/1000 (CI: 0.031–0.044) person-day with the median time to death of 6 days. Born from antepartum hemorrhage mother , lack of Kangaroo mother care , unable to start feeding with in 24 h of admission , apnea  and dehydration  were the identified predictors of time to death.The first 7 days of admission was the hazard time to death with median time of 6 days. Being born to antepartum hemorrhage mother, lack of Kangaroo mother care, unable to start feeding with 24-h, Apnea and dehydration were the predictors of time to death. Therefore, intervention that focuses on the identified predictors could have a paramount effect to prolong time to death and reduce preterm mortality.The online version contains supplementary material available at 10.1186/s12887-022-03176-7. Preterm neonates are newborns delivered before 37 completed weeks’ (259th day) of gestation, counting from the first day of the last menstrual period –3. BasedGlobally, 2.4 million neonatal deaths were recorded in 2019 . One third of all neonatal deaths occur within the first day of life and close to three-quarters occurring within the first week of life . Among tIn Africa half of the neonatal deaths are from preterm babies caused by only preterm complications and sub-Saharan African countries loses approximately 290,000 neonates in each year due to preterm complication . The higEthiopia is the 4th ranked country in the world which has highest neonatal mortality next to India, Nigeria and Pakistan . Almost Furthermore, Ethiopia as a country accepts initiatives to decrease preterm mortality and much is already being done but neonatal death related to preterm complication is still the leading cause of neonatal mortality . KnowingThe study was conducted in Addis Ababa, the capital and largest city of Ethiopia. The city has twelve public hospitals. From these hospitals, the study was conducted in five randomly selected public hospitals of Addis Ababa. These are Tikur Anbessa Specialized Hospital (TASH), Gandihi Memorial Hospital (GMH), Ras-Desta Damtew Memorial Hospital (RDDMH), Yekatit-12 Hospital Medical College (Y12-HMC) and St. Peter Specialized Hospital (SPSH). An institutional based prospective cohort follow up study was conducted from February 12–May 12, 2021.Neonates who need medical care are often put in special units in the hospital called the neonatal intensive care unit. Respiratory support, antibiotic treatment, blood product transfusion, fluid and electrolyte management, phototherapy, thermal stabilization are among the common treatment given in neonatal intensive care unit . CPAP isEarly administration of optimal nutrition to preterm birth survivors lowers the risk of adverse health outcomes and improves cognition in adulthood for preterm and low birth weight neonates. In preterm and low birth weight neonates the following are recommended, enteral feeding is safe and may be preferred to parenteral nutrition; early, fast, or continuous enteral feeding yields better outcomes compared to late, slow, or intermittent feeding respectively; routine use of nasogastric tubes is not advisable; preterm infants can be fed while on ventilator or continuous positive airway pressure; routine evaluation of gastric residuals and abdominal girth should be avoided, expressed breast milk is the first choice for feeding preterm infants due to its beneficial effects on cardiovascular, neurological, bone health, and growth outcomes; the second choice is donor pasteurized human milk, optimizing weight gain in preterm infants prevents long-term cardiovascular complications .All preterm neonates admitted to neonatal intensive care unit (NICU) in selected public hospitals of Addis Ababa are sources of population and all preterm neonates admitted to NICU of selected public hospitals in Addis Ababa in the study period  were study population.All alive neonates admitted to NICU by the diagnosis of preterm in selected public hospital with in the study period were included whereas preterm neonates diagnosed with major congenital anomaly  were excluded.Sample size was calculated by using the STATA version 16 considering the formula N = E/P (E) by taking the hazard ratio and the proportion from previously conducted studies resulted final sample size of 365 by taking the hazard ratio (1.74) of the variables gestational age less than 32 weeks  . Independent variables include:- Maternal socio demographic related predictors; , maternal obstetrical and medical predictors; multiple pregnancies, preterm prolonged rupture of membrane (PPROM), mode of delivery, preeclampsia, abruption placenta, antenatal care (ANC) follow up, steroid administration, hypertension, DM, human immune virus/ acquired disease syndrome (HIV/AIDS) and sepsis), Preterm demography related predictors , preterm admission diagnosis and new problem diagnosed in the follow up  and treatment and health service related predictors; , nurse to patient ratio and feeding).Appropriate for gestational age (AGA),The percentile of birth weight to gestational age is 10 and 90%) . Died, pData collection tool was adapted from different studies with some modification , 17, 23.Data entry was made by the principal investigator. Preterm death was the event of interest coded as “1” and “0” for censored. Time to death was calculated in days using the time interval between the time of admission and the time of death. Data was entered and cleaned by using EPi Data version 4.6 and transported to STATA version 16 for analysis. Multicollinearity test was done and the variable chorioamnionitis was omitted by the software (STATA) due to multicollinearity with oligo/polyhydramnios.P-Value> 0.05). This means that, the Kaplan Meier (KM) failure curve was not statistically different with respect to categories of significant covariates or we have no enough evidence to say that there has difference on failure curve. On the other hand, variables like, birth weight, Gestational age,1st and 5th minute APGAR scores, nurse to patient ratio, apnea, dehydration, necrotizing enterocolitis (NEC), hospital acquired infection (HAI) and preeclampsia/eclampsia has statistically significant result of log-rank test with . This means at 95% confidence, there had difference in hazard time to preterm mortality among the categories . P-value < 0.05 was considered as statistically significant association. Finally, the results of the study were presented with text, graph and table.Bi-variable cox regression was first fitted and those independent variables having During the study period, there were a total of 593 preterm admission, of those 43 were excluded due to congenital anomaly. A total of 365 neonates with their index mothers were involved initially but 7 preterm neonates were diagnosed with the exclusion criteria after they had been recruited to the study. Response was obtained from 35898.1%) participants out of these preterm neonates, males were 190 (53.07%) and females were 168 (46.9%). The mean age of the mother was 27.4 ± 4.7SD years. The maximum and minimum maternal ages found in this study were 18 and 40 respectively. In this study 89(24.8%) mothers had no formal learning, 90(25.1%) primary education, 31(8.7%) secondary education 79(22.1%) technical/vocational and 69(19.3%) higher educational level. Most 313(87.4) mothers are living in urban. More than half 221 (61.73%) of the neonates were admitted at the age of less than 24 h and the rest 137 (38.27%) were admitted at the age of 24 and above. The smallest weight and the youngest gestational age recorded in this study were 600 g and 27 weeks respectively  PPROM and 32 (8.9%) had antepartum hemorrhage (APH)  index mothers of this study had ANC follow up, of whom around three quarter 238(66.5%) of them had four or more antenatal visit whereas only 39(10.9%) of the mothers have less than four antenatal visits. Around quarters 100 (27.9%) of the mothers had multiple pregnancies, the rest 258(72.1%) had single pregnancy type. From the total index mothers,144(40.2%) had took steroid of them 74(51.4%) got full doses. Almost all 349(97.5%) of the mother had delivered in health institution and only 9(2.5%) gave birth at home. Nearly half of 188(52.5%) the neonates were born via spontaneous vaginal delivery (SVD) and 161(45%) of them were via c-caesarian section (C/S), the rest 9(2.5%) were born instrumentally. Significant majority 316(88.3%) of the mothers had risk for preterm delivery. Around one-third 109(30.4%) of the mothers were preeclamptic and /or eclamptic, 22(6.2%) had oligo/polyhydramnios% index mMost 313(87.4%) neonates were appropriate for gestational age, only 42 (11.7%) and 3(0.8) of preterm neonates were SGA and LGA respectively. In this study early onset neonatal sepsis (EONS) was found in significant majority of the neonates 309(86.8%) followed by RD 275 (76.8%). More than half 192(53.6%) of the neonates were hypothermic at admission (Table Majority 277(77.4%) of the neonate’s had developed new medical problem during the follow up. Of those new medical problems observed during the follow up, 170(47.5%) of the neonates developed hyperbilirubinemia, 98(27.3%) preterm develop HAI, 87(24.3%) develop NEC, 120(33.5%) develop apnea, 136(38%) develop thrombocytopenia and 84(23.5%) were diagnosed for dehydration (DHN)  were cared by greater than 1 to 2 patient nurse ratio in their hospital stay. Among those preterm babies 232(64.8%) had received CPAP for respiratory support the rest 126(35.2) didn’t get CPAP. Initiation of CPAP in nearly half of the neonates 123(53%) was after admission and only 2(0.8%) of neonates had started CPAP in delivery room before transportation to NICU. The most widely used CPAP type in this study was homemade (homegrown) CPAP. Significant majority 337(94.1%) of preterm neonates had received antibiotics. Among neonates who didn’t start feeding within the first 24 h of admission, 50 (87.7%) were died and only 7(12.3%) survived  of the neonates died with incidence rate of 36.4/1000 neonates-day  died in the first 7 days after admission, 8 (6.4%) died before 1 days after admission, 48(38.4%) between one and 3 days from admission, above 3 days up to 7 days of admission 49 (39.2%) and the rest 20(16%) of the neonates died between 7 and 28 days of life (above 7 days of admission). The total incidence rate was 36.4/1000 (CI: 0.031–0.044) neonate - days observation.The mean, median and standard deviation of time to death of the entire cohort was 9.6, 6 and 8.7 days respectively. The minimum follows up times observed in this cohort was 2 h and the maximum was 28 days. There were no survivors observed in the cohort of less than 28 weeks of gestational age and the survivors of those preterm neonates having less than 1000 g were only two from a total of 15 preterm neonates.The cumulative failure of preterm neonates was low in the first day after admission, which increases as follow up time increases up to 28 days of age. At the end of first day of hospital stay the failure was 3.6%, at the end of 3rd day 15.88% which increase to 33.9% at the end of 7 days and 45.5% on 28 day of follow up. As length of hospital stay increases the hazard of time to death will increase  (95%CI: 1.3–4.7)  . The incidence rate was 10.6/100 and the difference was statistically significant with p-value 0.000 . This was statistically significant with p-value of 0.000  with (95%CI: 2.4–14.4). The incidence rate was 11.5/100 to neonates with KMC and 51.3/100 neonates without KMC. This was statistically significant with p-value less than 0.25 were transported to multi-variable cox regression which yields Table p value< 0.05. This indicates that preterm neonates who developed apnea had 2.4times (CI: 1.3–4.7) shorter time to death compared to those preterm with no apnea during the follow-up. Similarly preterm neonates who developed dehydration during the follow up had 2.33time (CI: 1.3–4.3) shorter time to death than those who didn’t develop dehydration. Preterm neonates born to APH mother had 3.1times (CI: 1.4–6.6) shorter time to death compared to their counter parts. Preterm neonates who hadn’t got care of KMC had 5.8times (CI: 2.37–14.33) shorter time to death than those neonates who had got KMC during the follow up period. Preterm neonates who didn’t start feeding with in 24 h of admission had 6.4times shorten hazard of time to death than their counter parts.After bi-variable cox regression, variables having p-value > 0.05 . This result was similar with the finding of studies done in Jimma university specialized hospital and Bahr Dar university hospital 34.9% . The posHowever, the finding of this study was higher than the study conducted in China and Iran the 1.9%  and 9.1%The incidence density rate of death in this study was 36.4/1000 neonate-day observation. This finding was lower than the result found in a study conducted in TASH (39.1/1000) neonate-day observation . This diThis study showed that the highest hazard time was the first 7 days of admission in which majority of the deaths (84%) were encountered. This finding was almost similar with study done in Iran 84.3% , Kenya 8In this study the survival of preterm neonates born at the gestational age of bellow 28,28–32, 32 + 1–33 + 6,34–36 + 6 weeks was 0,38,68 and 89% respectively. This finding was almost similar with the study conducted in Bahir Dar university hospital . This siIn this study, being born from mother having APH was predictors of time to death of preterm mortality which increases the hazard by 3.1times (AHR: 3.1) compared to their counter parts. This was similar with the study conducted in Tanzania . This isApnea was one of the significant predictor identified in this study which shorten the hazard of time to death by 2.4times (AHR: 2.4). This is supported by the scientific evidence that preterm neonates are vulnerable for intra ventricular hemorrhage (IVH), sepsis and anemia causing for apnea (or it could be apnea of prematurity a diagnosis common in premature babies) . In factPreterm neonates developing dehydration during the follow up period were 2.3times (AHR: 2.33) fasten the time to death compared to neonate who didn’t develop dehydration. The possible practical explanation for this reason could be the fact that preterm neonates are vulnerable for excessive water loss due to thin skin and high coverage of water to body mass index. In addition to this, preterm neonates are vulnerable for different medical problems like sepsis and neonatal jaundice which causes dehydration during pathological process and at the time of treatment cascades . SimilarUnable to start feeding with in the first 24 h of admission 6.4times (AHR: 6.4) fasten the hazard time to death compared to their counter parts. This finding was relatively similar with studies conducting in Uganda , Axum UnPreterm neonates without KMC had 5.8 times shorter time to death compared to neonates who had got KMC. This finding was similar with the study conducted in Uganda Hospital , Axum UnThis study was conducted prospectively in multicenter institutions by excluding major congenital anomaly. Therefore, it is more representatives and gives qualified data by avoiding the possible missed deaths due to chart incompleteness commonly encountered problem in retrospective studies. Since the study period was only 3 months, it may mask seasonal variability. In this study all preterm neonates were considered as they have equal chance of mortality and survival regardless of their gestational age.The incidence rate of preterm mortality was 36.4/1000 neonate-day observation with median time to death of 6 days. Being born to APH mother, lack of KMC, apnea and dehydration during the follow up period, inability to start feeding within 24 h of admission were the identified predictors of time to death. Acting timely on the possible prevention and management of the identified predictors were recommended to prolong time to death and improve survival of preterm neonates.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.Additional file 8.Additional file 9.Additional file 10.Additional file 11.Additional file 12.