Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite’s DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% – 99% similarity with each other and 95.15% – 100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis.Little is known about latent infection and molecular characterisation of  Neospora caninum is a worldwide protozoan having a variety of animal hosts . However, studies have indicated that the Nc-5 gene is one of the most highly sensitive and specific for the detection of neosporosis  in Tehran, Iran, by polymerase chain reaction (PCR) using primers specific for the Nc-5 gene.Until now, different genes such as internal transcribed spacer sequences, 18S-like ribosomal DNA , and  & Ellis ; GoodsweA total of 330 samples from healthy slaughtered sheep (180 hearts and 150 brains) were purchased from an abattoir in Vavan (located in the vicinity of Tehran) from April to September 2014. The animals tested originated from different counties , all of which are located between 50 km and 200 km from Tehran. These locations have hot summers and moderate winters. No clinical signs such as fever, lymphadenitis, nasal and ocular discharges, or jaundice were recorded in any of the animals before slaughter.et al. The whole brain and heart of each sheep were individually rinsed with distilled water, packaged, and refrigerated. Approximately 200 g – 250 g of different segments of brain and heart were homogenised with a pestle and mortar in liquid nitrogen, and DNA was extracted using a phenol–chloroform extraction method as described in our recent report  and Np6plus (5’-CTCGCCAGTCCAACCTACGTCTTCT-3’)  were included. Amplification was performed with initial denaturation for 5 minutes at 94 °C, followed by 40 cycles at 94 °C for 40 seconds (denaturation), annealing at 62 °C in the first round, and 56 °C in nested PCR for 40 seconds, extension at 72 °C for 40 seconds, and final extension at 72 °C for 10 minutes. PCR products were electrophoresed on a 1.5% agarose gel stained with safe stain  and visualised under ultraviolet trans-illumination.Nested PCR was conducted using specific primers for the Pfu DNA polymerase , extracted from the gel  according to the manufacturer’s protocols. Then the products were sequenced in the forward and reverse directions by Sequetech    were amplified with a master mix containing Neospora caninum DNA was detected in 13 out of 330 sheep samples (3.9%). The infection rates in the heart and brain samples were 6.7% (12/180) and 0.7% (1/150), respectively. Four nucleotide sequences of the Nc-5 gene with a length of 227 bp (N. caninum deposited in GenBank (Appendix N. caninum specimens deposited in GenBank (N. caninum isolated from sheep (DQ077661) in the UK and 96.9% – 99.1% similarity with N. caninum isolated from sparrows (Passer domesticus) in Iran. Interestingly, one of our samples (KR106181) showed 100% similarity with N. caninum isolated from wolves (Canis lupus) (KF649846) in the United States.f 227 bp  were subf 227 bp  and 3 anAppendix . Phyloge GenBank . Analysiet al. et al. et al. et al. N. caninum has been reported in a range of 1.1% – 8.3% of sheep from the west of Iran  slaughtered goat kids in Romania  in Brazil. Interestingly, both positive samples were isolated from brain samples, whereas all heart and tongue samples were negative . Our results also indicated that in sheep the heart is more susceptible to N. caninum infection than the brain.Although an association between ovine abortion and neosporosis has been reported in different studies  and four reverse  oligonucleotide primers that derived from the Nc-5 genes were compared for the detection of neosporosis in experimentally infected mice. Among 19 combinations of forward and reverse primers, the Np21/Np6, Np7/Np6, and Np2l/Np4 primer pairs were able to detect at least 10 pg genomic DNA with a specific single band  (Kaufmann et al. Nc-5 gene has been used as a highly sensitive and specific gene for detection of neosporosis (Almerıa et al. et al. et al. et al. et al. Nc-5 gene for sensitive and specific detection of neosporosis in the current study.In the current study, we used the . et al. , who comN. caninum sequences deposited in GenBank (Appendix N. caninum with the Nc-5 gene (Auriemma et al. et al. et al. et al. et al. et al. N. caninum sequences deposited in GenBank. It therefore seems that the Nc-5 gene is not a suitable biomarker for phylogenetic analysis and discrimination of genetic diversity for N. caninum. Instead, this gene is rather a highly sensitive and specific biomarker for the diagnosis of neosporosis. The use of ribosomal DNA, ITS-1, and recently microsatellites have been recommended for discriminating between N. caninum isolates (Al-Qassab et al. We also sequenced four positive samples for phylogenetic analysis. We found that our sequences displayed similarity levels of 96% – 99% with each other  and 95% Appendix . In compN. caninum infection in sheep in Iran. It can be expected that in future these results will contribute to revealing the role of latent N. caninum infection in the epidemiology of neosporosis in sheep.Taken together, the results of this study provide molecular and epidemiological information about latent


To investigate the incidence, causes, occurrence time, and range of wound and outcomes of wound dehiscence in patients treated by penetrating keratoplasty (PK) or lamellar keratoplasty (LK).  We retrospectively reviewed medical records of keratoplasty in Shandong Eye Hospital from January 2006 to June 2017. Thirty-one eyes of 30 patients had sustained wound dehiscence (WD) after surgical treatment. The surgical type, causes, occurrence time, extent of the wound, treatment, and outcomes were recorded. p < 0.05). Twenty-seven eyes  had trauma-induced dehiscence. The mean range of dehiscence was 5.5 o'clock. The vision ranged from 20/50 to light perception after wound suture. The eyes receiving LK had fewer serious complications than PK.  The study population consisted of 26 men and 4 women. The mean age at the occurrence of WD was 44.6 years old (range: 12–78 years), and the mean time from keratoplasty to WD was 45.9 months (range: 1–204 months). WD occurred in 23 eyes  after PK and 8 eyes  after LK ( Compared with LK, PK seems to be more prone to result in wound dehiscence. The WD after LK may be less severe. The visual acuity after treatment of WD can be worse in the eyes with PK than LK. Corneal diseases represent the second leading cause of blindness globally. Keratoplasty is the major surgical procedure for visual restoration of corneal blindness. Corneal wound dehiscence (WD) is not an uncommon complication after keratoplasty. Although its incidence is relatively low, compared with other complications , WD may From January 2016 to June 2017, 3017 keratoplasties were performed in Shandong Eye Hospital, including 1385 PK surgeries, 1632 LK surgeries, and 75 endothelial transplantations. The patients were 1988 males and 1029 females. We retrospectively reviewed the medical records of these patients and recorded the characteristics, risk factors, and outcomes of corneal WD in patients with different surgical approaches. Data collected included patient age and gender, indication for keratoplasty, surgical procedures, duration between keratoplasty and WD, causative events for WD, size of dehiscence, treatment procedures, and vision outcomes after surgical repair.p < 0.05 was considered statistically significant.All patients with WD were given eye shields or glasses to protect the eyes before treated with emergency corneal graft surgery. Patients who did not have eye content exposure or other serious complications underwent original corneal graft repair under topical anesthesia or general anesthesia. In the eyes with iris or vitreous prolapse, corneal graft rejoint surgery was performed, and complicated fundus surgery was combined if needed. WD was sutured using 10-0 nylon sutures. Postoperatively, systemic and intravenous antibiotics were administered, as well as topical antirejection drugs. All data analyses were performed using SPSS statistical software . Quantitative data are presented as the mean ± standard deviation (range). A value of Thirty-one eyes from 30 patients (1.0%) suffered WD after keratoplasty, including 26 males (86.7%) and 4 females (13.3%). The age span of the patients with WD was between 12 and 78 years old with the mean age being 44.6 ± 18.3 years old . The patp=0.01). The major indications for PK among these patients were fungal keratitis , herpes simplex virus keratitis , keratoconus , and bullous keratopathy . The common indications for LK included keratoconus , fungal keratitis , interstitial keratitis , and ocular chemical injury   after PK and 8 eyes  after LK. The incidence of WD after PK and LK was 1.66% and 0.49%, respectively  . The mea, 12.5%) .As shown in p > 0.05). And there is also no statistical significance (p > 0.05) in PK or LK. Two cases were excluded in that we did not know the location of WD. The mean range of dehiscence was 5.7 o'clock in all the eyes, 5.1 o'clock in the eyes with LK, and 5.9 o'clock in the eyes with PK. WD covered 1–3 clock hours in 6 eyes (20.7%), 4–6 clock hours in 11 eyes (37.9%), 7–9 clock hours in 11 eyes (37.9%), and 10–12 clock hours in one eye (3.5%).Slit lamp examination showed that the corneal fissure was located in the corneal graft-host interface. Nearly one-third (10/31) of the eyes had sutures in place after trauma. 37.5% of the eyes with LK had sutures, while 30.4% of the eyes with PK had sutures in place. The suture technique used in the keratoplasty was interrupted suture. The mean range of dehiscence was 5.5 o'clock in the eyes with sutures, while 5.2 o'clock in the eyes without sutures. There is no significant difference in wound dehiscence (p > 0.05).Four patients after PK and 2 after LK had wound disruption of 1 to 3 clock hours. Seven after PK and 4 after LK had wound disruption of 4 to 6 clock hours. Nine after PK and 2 after LK had wound disruption of 7 to 9 clock hours. One after PK had disruption of 10–12 clock hours. The wound dehiscence encompassed the inferior temporal quadrant in 4 eyes (26.7%), inferior nasal quadrant in 6 eyes (40.0%), superior nasal quadrant in 9 eyes (60.0%), and superior temporal quadrant in 10 eyes (66.7%). The wound dehiscence with 180° or more occurred in 14 eyes (48.3%) with 12 eyes in PK and 2 eyes in LK. And the incidence of extensive wound dehiscence is not different between PK and LK , lens expulsion or dislocation in 15 eyes (48.4%), and extrusion of vitreous in 11 eyes (35.5%). In the eyes treated by PK, the complications were iris prolapse in 4 eyes, lens expulsion or dislocation in 15 eyes, and extrusion of vitreous in 10 eyes. In the eyes treated by LK, the complications were iris prolapse in 1 eye and extrusion of vitreous in 1 eye. The lens in one eye and the vitreous in the other eyes were not seen clearly. The extrusion of the lens and vitreous mainly occurred in the patients with an extent of wound disruption ≥ 6 o'clock hours .The duration between the occurrence of corneal graft dehiscence and therapy was 2 to 72 hours. Among 31 eyes of 30 patients, 31 eyes, including 22 eyes after PK and 8 eyes after LK, just had the graft repaired, and only 1 eye after PK was treated with combined anterior chamber angioplasty surgery because of flat anterior chamber.p > 0.05). In addition, patients with LK were less likely to suffer lens loss (p < 0.05). Although LK patients had less extrusion of the lens (p > 0.05) and vitreous (p > 0.05), there was no significant difference. Furthermore, there was no difference in the range of WD between PK and LK (p > 0.05).Final visual acuity was 20/200 or better in 12 eyes (40%), better than hand motions (HM) to 20/200 in 11 eyes (36.7%), HM to light perception (LP) in 7 eyes (23.3%), and unknown in one eye. In the follow-up period, BCVA was improved in 19 eyes (65.5%), including 16 eyes with PK and 3 eyes with LK, unchanged in 9 eyes (31.0%), including 6 eyes with PK and 3 eyes with LK, and decreased in one eye with LK (3.5%). Patients after treatment of LK achieved better final visual acuity than those after PK, but the final visual acuity and the recovery of visual acuity were of no statistical significance . We could get a primary conclusion that LK, compared with PK, was less liable to lead to WD. Retaining the posterior corneal stroma during LK can better recover vision and lower postoperative complications.In the current study, the type of operation was found to be an important factor of the occurrence of WD. The incidence of WD was 1.66% after PK and 0.49% after LK (WD may result in many serious ocular complications including iris prolapse, crystalline or intraocular lens expulsion or dislocation, and extrusion of vitreous. As previously reported, lens expulsion or dislocation was associated with poor prognosis and the final visual acuity. We noticed that PK patients tended to suffer more severe complications from WD. The reason may be that the cornea still remained a part of the autologous corneal tissue after LK and was protected with the help of full thickness of the Descemet membrane , 18.Once WD occurs, the degree of injury would directly affect the patient prognosis. It was reported that in patients with poor prognosis after injury, only 1/3 to 1/2 of patients had visual acuity of 20/200 . In our In conclusion, WD is a risk factor for patients undergoing corneal transplant. Compared with LK, PK seems to be more prone to result in wound dehiscence. The WD after LK may be less severe. The visual acuity after treatment of WD can be worse in the eyes with PK than LK. To reduce the incidence of WD after corneal transplantation, the patient's condition needs to be comprehensively analyzed before selecting appropriate surgical approaches, regular postoperative follow-up is important, and the protective awareness of the patient and family members should be improved.


Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide, and it particularly affects people living with human immunodeficiency virus (HIV). This study aimed to estimate the prevalence of HPV and to describe HPV genotypes in HIV-1 infected women in Lomé, Togo. From September 2014 to September 2015, a cross-sectional study was conducted in two treatment and care centers for people living with HIV: the Centre Hospitalier Universitaire Sylvanus Olympio and the non-profit organization ‘Espoir Vie Togo’. Women living with HIV-1, aged 18 years and older, receiving a combination antiretroviral therapy for at least 12 months, and who gave their informed consent to participate in the study were recruited. Cervical swabs were collected using a cytobrush, and cells were stored in a preservative solution. HPV testing was performed using e-BRID equipment. Blood samples were collected for CD4+ count using a flow cytometer and for HIV viral load using polymerase chain reaction. A total of 221 HIV-1 infected women were enrolled. The prevalence of any type and oncogenic HPV was 22.2%, 95% confidence interval (95% CI): [17.1–28.2] and 16.7% (95%CI: 12.3–22.3), respectively. The most prevalent genotypes were: 18 (8.6%), 68 (4.1%), and 62/81 (2.7%). Only 1.3% (3/221) of participants were infected with HPV16. In regression analysis, no factor was associated with HRHPV.This study showed the diversity of circulating HPV genotypes in Togo. Programs of HPV vaccination and early detection of benign or precancerous lesions should be implemented to reduce cancer-related comorbidities. Human Papillomavirus (HPV) infection is the most common sexually transmitted virus worldwide , 2. HPV A cross-sectional study was carried out over a period of 13 months (from September 2014 to September 2015) in two leading treatment and care centers for PLWHIV in Lomé: the Centre Hospitalier Universitaire Sylvanus Olympio  and the non-profit organization ‘Espoir Vie Togo’.Women living with HIV-1, aged 18 years and older, receiving a combination antiretroviral therapy (cART) for at least 12 months, and who gave their informed consent to participate in the study were recruited. The first-line treatment included two nucleoside reverse transcriptase inhibitors (NRTIs), Lamivudine (3TC) + Zidovudine (AZT) combined with a non-nucleoside reverse transcriptase inhibitor (NNRTI), Efavirenz (EFV) or Nevirapine (NVP).Since no data on HPV infection were available in Togo, the sample size calculation was based on the following assumptions: an expected prevalence of HPV infection in HIV infected women of 60% in Burkina Faso , neighboA standardized questionnaire was used in a face-to-face interview to record participants’ socio-demographic characteristics and medical history.A cervical swab was obtained using a cytobrush to collect cells at the junction area between the endocervix and the exocervix. Cells were then collected in a preservative solution . The sample was transported and stored at room temperature (10–30°C) for two to five days before manipulation at the Laboratoire de Biologie Moléculaire et Immunologie (BIOLIM) of the Faculté des Sciences de la Santé of the Université de Lomé .3 after one year of treatment were described as having inadequate HIV virological control.Two blood samples were drawn for counting CD4+ T-cell and measuring HIV viral load. These samples were transferred to the BIOLIM laboratory at 4°C within 4 hours. CD4+ count was performed immediately, then plasma was collected after centrifugation on the second tube, aliquoted, and stored at -80°C for HIV viral load measurement. Women living with HIV and having an HIV viral load greater than or equal to 10 000 copies / mmScreening for HPV infection was performed after amplification by Polymerase Chain Reaction (PCR) and hybridization of HPV deoxyribonucleic acid (DNA) molecules. The PCR Mix and Phire Hot Start II DNA Polymerase  kit was used for amplification. The resulting PCR product was then hybridized using the optimized HPV Direct Flow CHIP kit  following the protocol provided by the manufacturer on the controller (e-BRID System of Hospitex Diagnostics). This system allows screening and genotyping of 18 HR-HPV  and 18 LR-HPV  . The HIVThe data collected were cleaned, coded, and entered into a Microsoft Excel database developed for this purpose. Data analysis was performed using STATA software version 14.1 . Results were presented as proportions for categorical variables and as medians for quantitative variables. The prevalence of HPV infection, for example, has been presented with its 95% confidence interval. Comparison of categorical variables was performed with the Chi2 test or the exact Fisher test, and comparison of quantitative variables was carried out with Student's t-test or the analysis of variance test. Regression analyses were performed to identify factors associated with HRHPV infection. The significance was set at 5%.The study is a project of the Ministry of Health of Togo. It was approved by the “Comité de Bioéthique pour la Recherche en Santé (CBRS)”  from the Ministry of Health of Togo (n°751/2014/MS/CAB/DGS/DPLET/CBRS). Participants provided written consent prior to participation and authorizations from the Directors of selected medical centers were obtained before conducting the study.3. HIV viral load was greater than or equal to 10 000 copies / mm3 in 57.9% of women living with HIV : [29–45]. The majority (79.6%) had at least a secondary education, and 50.7% were living with a partner. The median CD4+ count was 346 cells / mm3 (IQR: [233–456]), and the majority (76.5%) of HIV-infected women had a CD4+ count less than 500 cells / mmwith HIV .The prevalence of HPV infection was 22.2%, 95% confidence interval (95% CI): [17.1–28.2]. HPV infection was not associated with age (p = 0.136). More than one-third (37.1%) of women aged 50 years and older, and 17.5% of women aged 20 to 29 had HPV infection. The remaining sociodemographic factors were not associated with HPV infection (p>0.05). Only inadequate HIV virological control was associated with HPV infection (p = 0.017) .The prevalence of HR-HPV was 16.7% (95% CI: 12.3–22.3). This prevalence was significantly higher among women aged 20 to 29 years , 30 to 39 years , and those over 50 years  (p = 0.04) . Among HThe most prevalent genotypes were: 18 (8.6%), 68 (4.1%), and 62/81 (2.7%). Only three (1.3%) women were infected with HPV16. Single and multiples infections were observed in 71.4% (35/49) and 28.6% (14/49) of HPV infected participants, respectively. Of the 37 HR-HPV infected women, 78.4% (29/37) had a single infection and 21.6% (8/37) had multiple infection. Among LR-HPV infected women, 60.0% (9/15) had a single infection and 40.0% (6/15) had multiple infection. Of the eight identified oncogenic strains, the Cervarix and Gardasil-9 vaccines covered two strains for 64.9% and four strains for 75.7% of the participants, respectively.Genotypes 18, 68, and 62/81 were more frequently identified in women with CD4+ count below 500 cells/mm3 (p>0.05) . Among wIn univariate analyses, none of the explanatory variables were associated with HRHPV. Therefore, multivariate analyses were not conducted .This study is one of the firsts conducted on HPV infection in Togo where cervical cancer is the second leading cause of cancer related death in women after breast cancer . Other sHPV prevalence was estimated at 22.2%. This prevalence is one of the lowest reported in sub-Saharan Africa. According to a meta-analysis by Clifford et al. in 2006, the prevalence of HPV infection was estimated at 56.6% in Africa, 31.1% in Asia, 32.4% in Europe, 31.4% in North America, and 57.3% in Central and South America among women living with HIV . In the Despite the low HPV prevalence, oncogenic strains remained more prevalent as reported in other studies in the general population or in PLWHIV , 25–27. The distribution of the majority of genotypes differs greatly from one geographical area to another, with genotypes 35 (15.7%), 16 (14.2%), 18 (11.4%) and 58 (11.4%) in Côte d’Ivoire; 16 (30.4%), 35 20.3%), 53 (19.6%) and 18 (18.2%) in South Africa and 6 (40%), 40 (14%), 16 (12%), and 52 (9%) in Tunisia , 32. Wit.3%, 53 , and field-related characteristics . In the cervix, the majority of HPV is spontaneously eliminated within one to two years. This immune response is often moderate and delayed, leading to the onset and persistence of the infection. Immunosuppression is strongly associated with the development of HPV infection , 24. Theutations –40. To rThis study had several limitations with regards to study design and biological procedures. First, recruitment was difficult because of the low acceptability of cervico-vaginal specimens, hence a possible selection bias should not be excluded. Also, the generalizability of findings of this hospital-based study should be done with caution, since women recruited in our study could be different from those in the community, leading to an underestimation or overestimation of HPV prevalence. Finally, since we conducted a cross-sectional study, we could not infer causality between HPV and HIV as we did not record incident HPV cases among HIV infected women. However, with this study we were able to describe the diversity of HPV genotypes circulating in HIV-infected women.Some biological samples were unusable due to the lack of sufficient biological material for amplification and genotyping of the virus. The technique used in this study is simple, fast, and less expensive but needs to be improved. Washing the cells in the buffer had a limit for some samples that could not be amplified and genotyped. Prior extraction of the virus DNA from these samples or a complexation reaction of the inhibitors would allow its optimization. Also, the analysis of cytopathological data was not performed in this study to confirm or refute the presence of precancerous and cancerous lesions of the cervix. In Togo, despite existing national recommendations, visual inspection with Lugol’s iodine (VILI) and visual inspection with acetic acid (VIA) are not yet available in HIV care settings. Women screened positive for HPV were referred for colposcopy and gynecology for follow-up as recommended by national guidelines.3. HPV vaccination and early detection of HPV infection in women living with HIV would reduce this comorbidity. Behavioral, cytological and even cohort studies are required in order to identify factors associated with the HPV low prevalence. In addition, it will be necessary to intensify the sensitization and to integrate the cervical cancer screening into routine practices.HPV prevalence was 22.2% among HIV infected women in Togo. HR-HPV genotypes 18 and 68 were more common in women with HIV viral load greater than or equal to 10 000 copies/mmS1 File(XLSX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Ulva rigida) in the reproductive success and biochemical composition of the Pacific oyster (Crassostrea gigas) during broodstock conditioning. Five nutritional regimes were tested: 100% macroalgae (diet 1), 50% macroalgae+50% microalgae (diet 2), 25% macroalgae+75% microalgae (diet 3) and 100% microalgae (diet 4). An unfed group was used as a negative control. The microalgae blend was composed of 33% Isochrysis galbana and 67% diatoms . Gonadal maturation was reflected in the physiological condition of the individuals. All treatments, except diet 1, showed an increase in condition index and were fully matured at the end of the trial, with the best physiological condition observed in oysters fed diet 3 and diet 4. Protein and total lipid content increased during the conditioning period, whereas glycogen content decreased. Oysters conditioned with diet 3 had higher protein and total lipid content and lower glycogen content than the other treatments. In addition, diet 3 showed the highest percentage of viable veliger larvae. The current study demonstrated that it is possible to replace 25% of microalgae with macroalgae in the broodstock conditioning, minimizing the operative cost in bivalve hatcheries.The current study evaluated the microalgae replacement by dry macroalgae (This article has an associated First Person interview with the first author of the paper. Summary: The use of an alternative diet with 25% microalgae replacement by dry macroalgae is beneficial during broodstock conditioning and allows the operation costs of bivalve hatcheries to be minimized. Crassostrea gigas , a native species from northeast Asia, was introduced worldwide, mainly to prevent a crisis resulting from the massive decline of indigenous populations and to sustain aquaculture industries  with nutritional value    and for itamins) . The int species  and comm species  and abal species .C. gigas, the present study replaced the live microalgae with commercial dry macroalgae U. rigida at various substitution levels . A microalgae blend consisting of diatoms  and a flagellate Isochrysis galbana clone T.ISO was used as a positive control. This diet was formulated based in C. angulata. Reproductive output of C. gigas and the biochemical composition  of diets and of oysters during conditioning were evaluated. The aim is to find an alternative broodstock conditioning diet that maximizes fecundity and oocyte quality, being suitable to be used in commercial hatcheries.To evaluate the effect of substitution of live microalgae on broodstock conditioning of The nutritional composition of the diets  are presented in F=2422.549, d.f.=3, P<0.05; total lipids, ANOVA, F=3940.955, d.f.=3, P<0.05). While the total carbohydrates presented a decrease with the increase of the inclusion of microalgae in the diet .In general, the percentages of proteins and total lipids decrease with increasing inclusion of macroalgae in the diet , 20% of males were in early gametogenesis (stage I) and 10% in late gametogenesis (stage II); 60% of individuals were females in early gametogenesis and 10% of the oysters exhibited male and female gametes, hence were considered as hermaphrodites.In unfed group, there was a slight regression in gonadal development from week 0–2, with 20% of oysters at resting stage (stage 0). However, females showed slow gonadal development, with 10% of females at stage II and 40% at stage I, while males remained in the same gonadal stages. At week 5, it was observed that there was a development in gonadal maturation, with high intra-heterogeneity in maturation stages with 20% at resting stage, males 10% at early gametogenesis and 20% at late gametogenesis, females 10% at early gametogenesis, 10% at late gametogenesis and 20% at maturation (stage III). It was also observed in 10% of hermaphrodite individuals.Oysters fed with 100% macroalgae (diet 1) exhibited, at week 2, a gonadal development similar to the unfed group. Nevertheless, the 100% macroalgae group showed a higher percentage of males at stage II (20%), whereas there were 10% in stage I. At week 5 20% of females were at early gametogenesis, 20% at late gametogenesis and 40% were mature females, while males were at early gametogenesis (10%) and late gametogenesis (10%). At week 11 an increase in gonadal development as well as a homogeneity in maturation stages were observed, where all females (75%) were mature (stage III) while all males (25%) were at stage II.At week 2, from all oysters fed with 50% macroalgae and 50% microalgae (diet 2), 40% of females were at stage II, while 10% were at stage I. The same pattern was observed in males, i.e. 30% were at stage I and 20% at stage II. At week 5 there was a slight regression with 10% of individuals at resting stage. Nevertheless, all males (40%) were at stage III while females were at stages II and III (20% in each), with 10% of hermaphrodites. At week 11, all females were mature (stage III), whereas 10% of males were at stage I and 10% at stage III.Oysters fed diet 3 and diet 4 showed a faster maturation in two weeks of conditioning than the other treatments. Males fed diet 3 were all at stage II (20%), whereas 10% of males from diet 4 were at stage I and 10% at stage II. Females from diet 3 group, were at stage I (20%) and stage II (60%), whereas in diet 4 group, 60% of females were in late gametogenesis and 20% were mature females (stage III). At week 5, all animals fed with diet 3 were mature at stage III. A higher percentage of mature males (stage III) was observed in diet 4 group (60%), while 20% females were in the maturation stage and 20% were in the spawning and reabsorbing stage (stage IV). At the end of the conditioning period, both diet 3 and diet 4 groups were composed of 25% of mature males (stage III) and females, 50% were at stage III and 25% stage IV.d.f.=4; P=0.021). Condition index of oysters fed with diet 1 showed a continuous decrease until the end of the conditioning period . At week 5 and 11, significant differences between diet 1 and diets 2, 3 and 4 were observed . At week 5, condition index of the other dietary treatments showed a general increase . This pattern was also observed also at week 11. At weeks 5 and 11, diet 4 showed the highest value of condition index, followed by diet 3 . At week 5 and 11 significant differences were observed between the diet 4 group and the other dietary groups, with the exception of diet 3 group at week 11.The condition index of oysters from all treatments  decreaseThe analysis of biochemical composition of broodstock revealed a highly heterogeneous response to the different diets, where proteins were the predominant compound of the individuals, followed by total lipids and then by glycogen .Table 2−1 AFDW to 549.46±61.20 µg mg−1 AFDW. In general, an increase in protein content in all dietary groups was observed. From week 2, the most pronounced increase in protein content was observed in diet 4 group, which was significantly different from the other dietary treatments , whereas a less pronounced increase was observed in diet 3 group. The unfed group were significantly different from the diet 1 and diet 2 groups . At week 5 (T5), oysters fed with diet 3 and 4 exhibited a decrease in protein content, with the diet 3 group exhibiting significant differences from the remaining dietary groups . Both groups have recovered at the end of the conditioning period (T11), with diet 3 group exhibiting the most remarkable increase. The remaining treatments showed an increase in all sampling times.Protein content ranged from 151.57±10.32 µg mg−1 AFDW at the beginning of the trial and 6.61±2.23 µm mg−1 AFDW at the end. After two weeks of conditioning, there was a decrease in glycogen content in all dietary groups, with exception of diet 1 group, which revealed a significant increase when compared with the other treatments . At week 5 (T5), unfed and diet 3 groups showed an increase in glycogen content, while the other treatments decreased. Unfed group was significantly different from diet 2 and diet 4 , whereas diet 3 and diet 4 were different from each other . At week 11, glycogen content decreased in all treatments, where the diet 3 group showed the lowest glycogen content. Significant differences were observed in diet 1 when compared with diet 2  and diet 3 .Glycogen content showed an opposite pattern of that observed in proteins content, in general, glycogen content decreased during the conditioning period. Glycogen varied between 67.71±11.92 µm mgd.f.=4, P<0.001). At week 5, total lipids were significantly different in the unfed and diet 4 groups when compared to the other groups . At the end of conditioning period, there was no significant differences between dietary groups.Total lipid content revealed an irregular pattern during the conditioning period, with an accentuated increase from week 0 to week 2. At week 5, total lipids decreased in oysters fed diet 1 and diet 2, while increased in oysters fed diet 4. At the end of the conditioning period, diet 1 and diet 2 groups showed a small increase and diet 4 a decrease in total lipids. Total lipid content remained almost constant in the diet 3 group from week 2 to week 11. At week 2 (T2), total lipids in diet 4 group was significantly different from all treatments, except from diet 3  and between the unfed group and diet 1 and 4 . At week 5, diet 3 group was significantly different from all other groups . At the end of the conditioning period (week 11), no differences were detected between groups.In general, total energy increased during conditioning, except in the diet 3 group , which showed a decrease in energy content at week 5 (T5). This decrease coincided with the lowest protein content observed. However, at week 11 diet 3 group exhibited the highest total energy content. At week 2, significant differences were observed between the diet 3 group and diets 1, 2 and 4 , the condition index showed a positive correlation with glycogen content  and a negative correlation with total lipids . On the other hand, total lipid and protein content were positively correlated  and both were strongly correlated with total energy . Glycogen and total lipid content were negatively correlated . Condition index of oysters fed with 100% macroalgae (diet 1) (Table S1B) showed a negative correlation with protein content  and with total energy , and a positive correlation with glycogen content . Protein content exhibited a positive correlation with total lipids and total energy , while glycogen and protein content demonstrated a negative correlation . Total lipids and total energy were positively correlated .Correlations between parameters are presented in the Supplementary data. In the unfed group . In oysters conditioned with diets 2, 3 and 4, protein content was positively correlated with total lipids  and with total energy . Glycogen content was negatively correlated with protein content , with total lipids  and with total energy . In diet groups 2, 3 and 4, total lipids and total energy showed a positive correlation .For the remaining diets, the condition index did not show a correlation with the other parameters =−2.592, P=0.027].Digestive enzymes activities (amylase and lipase) are presented in Lipase activity also showed an increase from week 0 to week 11 in all dietary groups. Oysters fed with 100% macroalgae showed the highest increase in lipase activity, followed by oysters conditioned with 25% macroalgae+75% macroalgae. Oysters conditioned with 100% microalgae exhibited the lowest lipase activity at the end of the conditioning period. No significant differences were observed in lipase activity.Spawning success and larval parameters are expressed in t-test, t (15)=2.413, P=0.029].The number of eggs released by females ranged between 6.29 to 31.8 million, with the highest number of eggs being released by individuals conditioned with diet 3  whereas the unfed group and diet 4 group exhibited the lowest number of released eggs. Since only one female spawned in the unfed group, it was not possible to compare with the remaining groups. Therefore, the only significant differences were detected between oysters fed diet 3 and diet 4 .Fertilization rate was similar between all treatments, with oysters conditioned with diet 4 exhibiting a higher fertilization rate, while individuals in diet 2 group exhibited the lowest fertilization rate. These two groups were significantly different from each other  in 100 g of sample).−1 of AFDW).For each diet, oysters previously sampled and stored at −20°C were defrosted and opened. The entire soft body was separated from the shell and homogenized in an ice bath. Proteins, glycogen and total lipid content were determined by standard methods. The modified Lowry method  was used−1), glycogen (17.2 KJ g−1) and total lipids (33 KJ g−1)  for further analysis of protein, lipase and amylase. Aliquots were stored at −80°C until analysis.Hepatopancreas were homogenized in 1:10 of extraction buffer . After being centrifuged at 7000 An initial protein quantification was needed since amylase and lipase digestive enzymes were calculated in relation to the amount of protein in the tissue.Protein quantification was performed following the folin-phenol method, according to Amylase activity was measured from the increase in reducing maltose by the hydrolysis of α-D  glucosidic bond in polysaccharides and stained with 3, 5-dinitrosalicylic acid (DNS), as described by Lipase activity was performed by using ρ-nitrophenyl substrate as described by Results are expressed as mean±s.d. (s.d.). Depending on the violation or not of the normality and homogeneity of variance assumptions, one-way analysis of variance (ANOVA) or Kruskal–Wallis nonparametric tests were applied to compare condition index and biochemical composition of diets and broodstock among nutritional regimes. These analyses were performed separately for each sampling time . Whenever applicable, Tukey's, Dunn's or Tamhane's T2 post-hoc tests  were applied to identify the differences.For each diet, Spearman Rank Order correlation was used to determine the degree of association between parameters .t-test, comparing all diets with diet 4 (positive control). Significance level was set as P≤0.05 for all statistical tests. Statistical analyses were undertaken using Sigmaplot 12.5 statistical package.Statistical differences regarding to digestive enzymes results, as well as in the number of eggs released, fertilization and D-veliger larvae development were determined by performing a parametric Student's


Background: Cluster headache (CH) is a relatively uncommon primary headache disorder. Few studies in our region described CH. We aimed to study demographics, clinical characteristics and treatment modalities of CH patients referred to Headache Clinic in Kuwait.Materials and Methods: This cross-sectional study included all CH patients who are referred to headache clinic. The diagnosis of CH based on the CH diagnostic criteria of the Headache Classification Committee of the IHS, 3rd edition (beta version) (ICHD-3-beta). The demographics, clinical characteristics and treatment modalities of CH patients were recorded.Results: Forty six patients were diagnosed with CH which constituted 1.7% of all headache patients and 0.1% of all visits to Neurology clinic. Male:Female ratio was 15.3:1. Mean age was 40.1 ± 10.7 years and mean age at onset was 29 years (20–49). Family history was positive in 6.5%. Smoking was seen in 63.0% of patients while 6.5 % reported alcohol intake. CH was episodic in 84.4% and chronic in 15.2%. Seasonal predilection was seen in all patients; the most frequent season was Autumn 47.8%. The mean duration of the cluster bouts was 6 weeks (2–12 weeks). The mean duration of the attack was 60 min (15–180 min). Number of attacks per day ranged from 2 to 10 attacks with a mean of 5 attacks/day. The median of attack severity by Visual Analog Scale (VAS) was 8.5 (7–10). The time taken to diagnose the patients ranged from <1 year to 12 years with a mean of 4 years. Chronic CH have a statically significant relation with smoking (P = 0.036), older age (P = 0.027), and longer time taken to diagnosis (P = 0.026) compared to episodic CH.Conclusion: Our results reported that there is higher proportion of males compared to females and less positive family history. Smoking was a significant risk factor for chronicity in addition to advanced age, higher age at disease onset and a longer time taken till diagnosis. Cluster headache (CH) is a relatively uncommon yet incapacitating primary headache disorder classified together with other similar conditions as Trigeminal Autonomic Cephalalgias (TAC) . The disThe attacks of headache tend to cluster in bouts, lasting between 15 and 180 min, recurring from every other day to multiple times per day for weeks or months during active cluster bouts, followed by periods of remission . In episThe pain associated with CH can be excruciating and has been described as the most severe pain known to humans , 8. AccoFew studies in our region described the prevalence and the clinical characteristics of CH and no studies were published from the Arabian Gulf region including Kuwait , 11.We aimed to study demographics, clinical characteristics and treatment modalities of CH patients referred to our Headache Clinic at the only tertiary neurology center in Kuwait during the period from January 2016 to September 2018.This cross-sectional study included all CH patients who are referred to our headache clinic at the only tertiary neurology center in Kuwait. We reviewed the patient's data registry to identify those with the diagnosis of CH from January 2016, till September, 2018. The patients were contacted and scheduled for reassessment in our headache clinic. The diagnosis of CH was re-challenged and confirmed by headache specialist. All the patients that met the CH diagnostic criteria of the Headache Classification Committee of the IHS, 3rd edition (beta version) (ICHD-3-beta) , age at the onset, age at diagnosis, associated cranial autonomic features, other associated features  during attacks and treatment modalities were all recorded. The severity of headache was estimated using a Visual Analog Scale (VAS) to indicate the maximum intensity of pain by asking the patients to make a mark on a 10-cm line labeled with “no pain” at the zero end and “the worst pain possible” at the other , 13.This study was approved by ethical committee of Kuwait ministry of health. Written informed consent was obtained from all participant before using their information.t-test. SPSS for Windows, Version 16.0, was used for statistical analyses with the significance level set at P = 0.05.All measurements were reported as mean ± SD. Categorical variables were compared using the Chi-square test, and continuous variables were compared using Student's A total of 2,782 headache patients were evaluated at our Headache Clinic during the period between January 2016 till September 2018. Forty-six patients were diagnosed with CH which constitute 1.7% of all headache patients  and 0.1%A total of 46 patients were included, 43 [94.5%] were males and 3 [6.5%] were females, giving a M:F ratio of 15.3:1. Mean age of the patients was 40.1 ± 10.7 years (range 23–69 years). Mean age at disease onset was 29 years (range 20–49 years). Kuwaiti nationality 32 [69.6%] was the most prevalent in our cohort. Twenty-nine [63%] patients were smokers, while only 3 patients reported alcohol intake 6.5%. Family history of CH in a first or second degree relative was reported in only 3 [6.5%] patients .Most of our patients had episodic CH 39 [84.8%], while only 7 [15.2%] had chronic CH, interestingly all the chronic patients were smokers. CH was strictly unilateral in all our patients with equal right and left side affection. Pain was predominantly affecting peri-orbital and temporal region 43 [93.5%], frontal 2 [4.3%], and occipital 1 [2.2%]. Pain intensity was described by the majority as being severe in 41 [89.1%] patients and moderately-severe in 5 [10.9%] patients. VAS for pain severity was used in all our patients, the score ranged from 7 to 10 with a median of 8.5. Character of pain was throbbing in 35 [76.1%] patients, followed with stabbing 6 [13%], burning 4 (8.7%), and boring in only one 2.2% patient. The duration of the cluster bouts ranged from 2 to 12 weeks with a mean of 6 weeks. The duration of the attack ranged from 15 to 180 min with a mean of 60 min. The number of attacks per day ranged from 2 to 10 attacks with a mean of 5 attacks/day. Regarding the frequency of cluster periods, the majority had one bout per year; 25 [54.3%] patients, every 2 years in 10 [21.7%], every 3 years in 2 [4.3%], every 3 months in 4 [8.7%], every month in 4 [8.7%], and every 6 months in only one (2.2%) patient. Seasonal predilection was observed in our patients; the most frequent season was Autumn 22 [47.8%] patients, followed with Winter 15 [32.6%], Spring 7 [15.2%], and the least frequent was Summer 2 [4.3%] patients. These cluster periods occurred mainly in October, December, September, and November .The time taken to diagnose ranged from <1 to 12 years with a median of 4 years. The total duration of the disease among our patient ranged from 2 to 30 years with a mean of 10 years. Among cranial autonomic features, rhinorrhea and nasal congestion were found in all 46 [100%] patients, tearing was found in 45 [97.8%] patients, conjunctival injection in 44 [95.7%], ptosis and miosis were found in 10 (21.7%), while facial sweating in only 4 [8.7%] patients. Other associated features during the clusters were nausea with or without vomiting in 28 (60.9%) patients, photophobia and phonophobia in 23 [50%] patients, of which 20 [43.5%] patients reported it as unilateral. Agitation during the attacks was reported by 41 [89.1%] patients. Aural fullness was reported by 28 [60.9%] patients.Neurological examination between the cluster attacks was normal in all patients 100%. All patients underwent magnetic resonance (MR) imaging of the brain; 41 [89.1%] patients were normal, non-specific white matter changes (WM) were reported in 4 (8.7%) patients, and one patient 2.2% had incidental sellar cyst.In our cohort, some patients received the diagnosis of another type of headache in addition to CH, 8 [17.4%] patients had migraine, 4 [8.7%] patients had tension-type headache and one patient 2.2% had mixed type of migraine and tension headache. Two patients were diagnosed with secondary headache; one patient had a sellar cyst and another one had foreign bodies in the skull and left maxillary sinus during the Gulf War .P = 0.027).We studied the relationship between different headache characteristics and chronicity in CH. Age of chronic CH patients (48.3 ± 11.3) was higher than episodic CH patients (38.7 ± 10.1), the difference was statistically significant (P = 0.036). Smoking was reported by 100% of chronic cases compared to 56.4% of episodic cases which was statistically significant (P = 0.036). The mean time taken till diagnosis was 4 years in episodic CH and 6 years in chronic CH which was statistically significant (P = 0.026)  compared to episodic CH patients; 27 years (20–49) with a statistically significant difference (= 0.026) .Treatment was individualized for each patient. During acute attacks, all our patients received high-flow oxygen. Steroids either Dexamethasone or Prednisone were given to 45 [97.8%] patients. Triptans were given to 37 [80.4%] patients; sumatriptan being the most commonly used by 32 (69.6%) patients. Forty patients 87% reported using non-steroidal anti-inflammatory drugs (NSAIDs) during acute attacks. For preventive treatment, the most common drug used was topiramate in 24 [52.2%] patients followed by verapamil in 22 [47.8%] and lithium in one patient 2.2%. Other drugs used included Tricyclic antidepressants (TCA) in 4 [8.7%] patients and valproic acid in 1 [2.2%]. Greater occipital nerve block (GONB) and Botulinum toxin (BTX) were given to 6 [13.0%] patients each. Only one patient 2.2% in our cohort underwent Percutaneous radiofrequency ablation (RFA) of the sphenopalatine ganglion (SPG) due to poor response to medical treatment .CH data from our region in general are scarce in literature and no data were previously published from gulf countries , 11.Prevalence of CH in our cohort was 1.7% of all headache patients evaluated in our clinic which matches with the prevalence found in other clinic-based studies from Egypt 1.7% , China 1CH attacks affect patients in their most productive years with negative results on their quality of life , 15. TheCH was more common in males than in females in our cohort with male-to-female ratio of 15.3:1 which is higher than any figure published before. The male-to-female ratio is variable in literature being generally higher in Asian studies and lower in Western studies ranging from 14:1 to 2.5:1 , 22. In The high male to female ratio in our cohort may be related to the conservative nature of our population with low smoking prevalence among women and alcohol being prohibited. Another possible explanation can be related to the climate and latitude effect on CH which is not well studied so far.Smoking is considered a significant risk factor for developing and triggering CH , 23. In Prevalence of CH in the same family is variable in different studies. We reported 6.5% of our patients have history of headache in at least one of their family members. Positive history reported in 1.9–6.7% in Bharar et al patients , other sThe majority of our cases had episodic CH 84.4% which is in agreement with Japanese studies that reported a low prevalence of chronic CH (1.2–2.9%) , while oAll of the patients had a strictly unilateral headache with no side predominance, Xie et al. and Wang et al. reported right-sided predominance , 21, othSeasonal predilection is common in CH which is considered a circadian-based syndrome . In our It is well known that CH is one of the worst headaches and this was agreed upon by 89.1% of our patients who reported having severe rather than moderately severe headache with a mean VAS of 8.5. These results were similar to another clinic-based study from China where the mean VAS was 8.2 .The associated autonomic symptoms which are the defining feature of CH were found in all of our patients similar to other studies. Rhinorrhea 100% and nasal congestion 100% were the most reported symptoms in all of our patients followed by conjunctival injection and lacrimation while ptosis and miosis were the least reported. Lacrimation was the most reported associated autonomic symptom in some studied , 34, andPhotophobia and/or phonophobia were reported in half of our patients and interestingly most of them reported it being unilateral at the side of pain which was previously reported by Irimia et al. .The mean time taken till accurate diagnosis in our cohort was 4 years. In a Dutch study the median time before a proper diagnosis of CH was 3 years , KlapperTreatment of CH in our cohort followed an individualized approach. During acute attacks, all our patients received high flow oxygen with good response. The majority also received steroids 79.8% either oral or parenteral during the attacks and reported using over the counter non-steroidal anti-inflammatory drugs. Sumatriptan was the commonest triptan to be used as it has both oral and subcutaneous route of administration. Topiramate was the commonest prophylactic treatment used followed by verapamil. BTX injection and GONB were both given to some of our patients who showed inadequate response to medical treatment and were found to be a safe and effective option in several studies with good outcome –41. RFA-Our study had some limitations being a hospital-based study including: the small sample size and the lack of impact of CH on our patient's occupation and quality of life. Our cohort is small because CH is uncommon primary headache disorder in addition that it might be misdiagnosed as sinus headache by general practioner before referral to our specialized headache clinic. Health service in Kuwait start in primary care, then refractory cases are referred to secondary and tertiary services.This study has the strength of providing data from Kuwait where the prevalence of CH could be different from that in the general population, perhaps as a consequence of traditions and religion .This study describes the demographic and clinical characteristics of CH in a neurology outpatient in Kuwait. Our results coincided with other clinical studies published in many aspects, including the prevalence and clinical characteristics of CH. However, it was different in the highest proportion of men and in a less positive family history. We found that smoking is a significant risk factor for chronicity in patients with CH in addition to advanced age, higher age at disease onset and a longer time taken till diagnosis. Further community-based studies are needed in the gulf region to give better understanding of CH in this part of the world.Data are available at neurology department, Ibn Sina hospital, Kuwait.Our research was carried out according to ethical guidelines of Kuwait Ministry of Health.All authors have read and approved the submitted manuscript. Our manuscript has not been submitted elsewhere nor published elsewhere.JA-H designed the study and reviewed the manuscript. II performed data collection, statistical analysis and drafted the manuscript. DY performed data collection and drafted the manuscript. SA drafted, criticized and reviewed the manuscript. PG reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ontario, Canada introduced a publicly-funded 13-valent pneumococcal conjugate vaccine (PCV13) for infants in 2010, replacing the 10-valent  and the 7-valent  conjugate vaccine programs; a 23-valent pneumococcal polysaccharide vaccine (PPV23) has been available for older adults since 1996. We examined the epidemiology and serotype distribution of invasive pneumococcal disease (IPD) in Ontario in the context of provincial immunization programs.We included confirmed IPD cases reported in Ontario between 2007 and 2017. We grouped serotypes according to Ontario’s current immunization program  and calculated incidence rates  using population data.Between 2007 and 2017, annual incidence of IPD in Ontario ranged between 7.3 and 9.7/100,000 per year. Measures of illness severity were high throughout the period of surveillance. After PCV13 program implementation in 2010, incidence due to PCV13 serotypes decreased significantly across all age groups, with the greatest reductions in children <5 years and adults ≥65 years. Conversely, incidence due to PPV23 unique serotypes increased significantly between 2007 and 2017, with the greatest increases observed in adults 50–64 years  and ≥65 years . Similar increases were observed in incidence due to non-vaccine-preventable serotypes among all age groups, except infants <1 year. Within specific serotypes, incidence due to serotypes 3  and 22F  increased significantly between 2007 and 2017, while incidence due to serotypes 19A and 7F decreased significantly during the PCV13 period (2010–2017).Eight years after PCV13 implementation in Ontario, our data suggest both direct and indirect effects on serotype-specific incidence in young children and older adults. However, overall provincial rates have remained unchanged, and IPD continues to be a severe burden on the population. The rising incidence of IPD due to PPV23 unique and non-vaccine-preventable serotypes, and the growing burden of serotypes 3 and 22F, require further study. Streptococcus pneumoniae, is associated with severe morbidity and mortality, particularly in young children and the elderly [S. pneumoniae recognized worldwide, a subset of which cause the majority of disease in Canada [Invasive pneumococcal disease (IPD), caused by  elderly ,2. It is elderly . There an Canada ,3. The in Canada ,5. Howevn Canada . IPD conn Canada .In 2010, Ontario, Canada (population approximately 14 million) introduced a publicly-funded three-dose 13-valent pneumococcal conjugate vaccine (PCV13) program for infants at 2, 4 and 12 months, replacing a four-dose 10-valent conjugate vaccine program  and a four-dose 7-valent conjugate vaccine program  ,9.S. pneumoniae serotypes in Ontario between 2007 and 2017, in the context of the provincial immunization programs.With the PCV13 program now in place for eight years, there is an opportunity to assess the long term effect of the program on serotype distribution. We examined the epidemiology of IPD and the distribution of circulating Health Protection and Promotion Act, all public health units (PHUs) in Ontario are responsible for case management and reporting of IPD [S. pneumoniae or detection of S. pneumoniae DNA from a normally sterile site  [Under the g of IPD . PHUs cldle ear) . PHUs reS. pneumoniae from sterile sites to the National Microbiology Laboratory (NML) via Public Health Ontario (PHO) Laboratory for serotyping, and results are reported to the PHUs and entered into iPHIS. The exception is the Toronto Invasive Bacterial Diseases Network (TIBDN), which covers a surveillance population of approximately 4 million [S. pneumoniae identification. These isolates are serotyped at the TIBDN laboratory, the Alberta Provincial Public Health Laboratory or NML [In Ontario, most diagnostic laboratories forward isolates of  million . Hospitay or NML . Resultsy or NML , we extry or NML .We grouped IPD cases by associated vaccine type. Serotypes covered by the conjugate vaccines  were designated as ‘PCV13’  . SerotypU test. We grouped Ontario’s 35 PHUs into seven regions for geographic analyses. We calculated incidence rates per 100,000 population using population estimates (2007–2016) and projections (2017) obtained from Statistics Canada [p-values less than 0.05 as statistically significant. When assessing immunization status, we included only documented doses of a pneumococcal vaccine product administered at least 14 days prior to disease onset.We conducted descriptive analyses using SAS Enterprise Guide 7.1 . We used case-level data from iPHIS to describe demographic characteristics and clinical outcomes of IPD. We defined ‘infants’ as persons under one year of age, and ‘older adults’ as persons 65 years of age and older. We evaluated changes in median age over the study period using the Mann-Whitney s 2007–20 and projOntario Agency for Health Protection and Promotion Act, SO 2007, c 10) and not research, and therefore research ethics committee approval was not required.This manuscript reports on routine surveillance activities carried out under provincial legislation (p = 0.20). We observed geographical variations in incidence within the province, with the highest annualized incidence rates in the North West and North East regions , and the lowest in the Toronto and Central East regions . The median age of cases was 61 years (range: less than one day-104 years) and males had a higher rate of disease than females (Table 1).Between 2007 and 2017, 12,377 confirmed cases of IPD were reported in Ontario, representing an annualized incidence rate of 8.4 per 100,000 population. The annual incidence rate ranged between 7.3 and 9.7 per 100,000 population, however no statistically significant trend was observed over the 11-year period (Table 1). Immunization status was known for only 37.6% of cases , of whom 67.9% (3160/4654) were reported as having not received any pneumococcal vaccine product.Of the 12,377 cases, hospitalization was reported for 73.9%, and was high across all ages . Hospitalization was highest in older adults (76.9%) and infants (75.3%). The provincial case fatality ratio (CFR) during this period was 11.8%. CFR varied greatly across age groups; the CFR in those 50–64 years (10.9%) and older adults (17.2%) were respectively 2.7 and 4.3 times higher than the 4.0% CFR of infants. The annualized death rate in older adults was seven times that of infants (p = 0.004). Significant reductions in age-specific incidence were observed in infants (p = 0.02) and children 1–4 years old (p = 0.03), reaching a low of 4.1 and 7.3 per 100,000 population (respectively) in 2015 . However, rates in these age groups have increased since 2015, reaching 16.1 (infants) and 11.8 (1–4 years olds) per 100,000 population in 2017. A significant decrease in incidence was also observed in older adults between 2007 and 2017 (p = 0.01). When stratifying this age group further, a significant decrease was observed among those 65–74 years (p = 0.03) and those 85 years and older (p < .001); no statistically significant trend was observed in those 75–84 years old (p = 0.15) . There were also no significant trends in the annual incidence of IPD in those aged 50–64 years old over the study period (p = 0.44). Incidence in the 5–49 year age group have been low and stable , with an annualized rate of 3.2 per 100,000 population during the study period .The annual median age of cases increased from 60.4 years in 2007 (IQR: 41.1–75.1 years) to 63.5 years in 2017 (IQR: 48.0–75.9 years) (p = 0.007). Incidence peaked at 4.4 per 100,000 population in 2010, the year the PCV13 program was introduced, and decreased to 2.0 per 100,000 population in 2015, representing a 54.6% reduction within six years; incidence has since increased to 2.3 per 100,000 population in 2017 . After stratifying by age group, we observed significant decreasing trends in incidence due to PCV13 serotypes among all age groups (p < .001) during the PCV13 program period (2010 to 2017). We saw the largest reductions in children–specifically, infants between 2010 and 2014  and children 1–4 years old between 2009 and 2015 . However, incidence due to PCV13 serotypes in both these age groups has risen in recent years . In adults aged 50–64 years, incidence due to PCV13 serotypes decreased by 44.1% in the first three years after PCV13 program implementation ; annual incidence has since stabilized in this age group . Incidence due to PCV13 serotypes in older adults has declined significantly during the PCV13 program period, from a peak of 12.0 per 100,000 population in 2010 to 4.8 per 100,000 population in 2017 (p < .001), representing a 60.0% reduction in eight years .Serotype information was available for 78.6% of cases . Of these, 44.7% were due to serotypes covered by the PCV13 vaccine, 29.5% due to serotypes unique to PPV23 and 25.8% due to non-vaccine-preventable serotypes. Overall provincial incidence due to PCV13 serotypes decreased between 2007 and 2017 (p < .001) and non-vaccine-preventable serotypes (p < .001) over the study period . When stratified by age group, these increases disproportionately affected older age groups. We observed variability in annual incidence due to serotypes unique to PPV23 in children under five years, with a significant increasing trend in infants (p = 0.002), but no significant trend in children aged 1–4 years (p = 0.13) . Increases also occurred in adults aged 50–64 years, from 1.4 to 3.5 per 100,000 population between 2007 and 2017 (p < .001) . However older adults had the greatest increase in incidence due to serotypes unique to PPV23, with incidence more than tripling between 2007 and 2017  . We noted similar age-specific trends for non-vaccine-preventable serotypes; significantly increasing trends were observed in all age groups (p < .001) except infants (p = 0.58). In particular, increases in incidence due to non-vaccine-preventable serotypes was greatest in older adults  .We noted significantly increased overall incidence due to serotypes unique to PPV23 . Serotype 19A disproportionately affected younger populations, accounting for 26.6% of cases under five years with available serotype information (214/806). Annual incidence due to serotype 19A peaked in 2010  and declined to 0.47 per 100,000 population in 2017 (p < .001), representing a 66.9% reduction in eight years. We observed a similar pattern for incidence due to serotype 7F, which decreased from 1.42 to 0.10 per 100,000 population between 2010 and 2017, a 93.0% reduction in eight years (p < .001). We also noted increasing trends in incidence due to both serotype 3 and 22F between 2007 and 2017; incidence due to serotype 3 increased from 0.42 to 0.98 per 100,000 population (p < .001), and incidence due to serotype 22F increased from 0.31 to 0.72 per 100,000 population (p < .001) . Among non-vaccine-preventable serotypes, 23A was the most commonly identified serotype (4.0%) .In all, 70 different serotypes were reported during the study period. Of the 9,725 cases with available serotype information, the most commonly identified serotypes were 19A (13.1%), 3 (10.5%), 22F (9.8%) and 7F (9.2%), all four of which are vaccine-preventable and accounted for 42.6% of these cases; other reported serotypes individually accounted for less than 4.2% of cases with available serotype information during the entire eleven year period , aligning with previous findings of increased incidence in northern populations and among indigenous populations ,18.S. pneumoniae to adults) following the introduction of the PCV13 vaccine [As illustrated in our analysis, the direct effect of Ontario’s pediatric PCV13 vaccine program, and the indirect effect of herd immunity, is clear in the PCV13-specific trends. The incidence of IPD due to serotypes covered by the PCV13 vaccine was rising rapidly in Ontario prior to PCV13 program implementation in 2010, notwithstanding the implementation of a universal PCV7 program in 2005; previous studies in Ontario also observed the high burden of serotypes 19A and 7F in the province during this pre-PCV13 program period [ vaccine ,22. Whil vaccine –25. The In contrast to evidence that the PCV13 vaccine has impacted the epidemiology of most PCV13 serotypes, it appears to have had no impact on incidence due to serotype 3, which continues to rise. This trend has also been observed in several other jurisdictions –29 and iIncidence due to serotypes unique to PPV23 and non-vaccine-preventable serotypes has risen continuously during the study period, across age groups but most noteworthy in older age groups. The increase in incidence due to serotypes unique to PPV23 may be due to poor vaccine coverage, lower vaccine effectiveness or vaccine failure. While Ontario-specific coverage estimates for adults are not available, the Public Health Agency of Canada has estimated that coverage for the PPV23 vaccine among Canadian adults 65 years and older was 41.6% in 2016, well below the national target of 80% . StudiesDespite the effectiveness of the PCV13 program in Ontario, our analysis shows high illness severity among cases, particularly among young children and older adults. Severity is particularly pronounced in the older age groups, with the majority of cases, hospitalizations and deaths occurring in those 50 years and older, as noted elsewhere ,38,39. WS1 Fig). Improving the collection of these variables would allow for a more thorough understanding of IPD and more informed public health action in Ontario.There are some limitations with this study. IPD cases are likely underreported in Ontario’s passive surveillance system due to variety of reasons, including health care seeking behaviours and variations in clinical testing. While the degree of underreporting in Ontario is unknown, it is expected that our data is likely skewed towards severe cases and the true burden is higher than we have estimated due to the nature of passive surveillance of invasive disease. On the other hand, the proportion of cases reported to have been hospitalized may be underestimated if hospitalization status was not available or reported to the public health unit. Small numbers, particularly in the infant age group, may cause unstable trends and should also be interpreted with caution. Certain variables are also underreported. Completeness of immunization history for cases in iPHIS is low, which provides challenges in interpreting trends, limits the ability to interpret vaccine effectiveness and limits comparisons of serotype-specific trends between vaccinated and unvaccinated populations, which is necessary to inform Ontario’s immunization program. In addition, as the change from PCV10 to PCV13 occurred concurrently with the schedule change from four to three doses, we are not able to determine the impact of either change independently. Risk factor information is also lacking, but is necessary to understand the impact of socio-demographic factors and comorbidities on IPD risk. Serotype data is also missing for 21.4% of cases, which limits generalizability of the observed trends. However, substantial improvements to completeness of serotype data were made over time during the study period  and suboptimal vaccination coverage are potentially contributing factors. The shifting epidemiology and serotype distribution observed in this analysis highlights the importance of ongoing and robust surveillance, which can monitor circulating serotypes, detect serotype replacement, and inform Ontario’s pneumococcal immunization strategy.S1 Figa Cases with non-typeable serotypes were included in the non-vaccine-preventable category (n = 25). Abbreviations: PCV13: 13-valent pneumococcal conjugate vaccine; PPV23: 23-valent pneumococcal polysaccharide vaccine.(TIF)Click here for additional data file.


LTFU) while on antiretroviral therapy (ART). We compared adolescent and adult LTFU after ART initiation to quantify the impact of age, pregnancy, and site‐level factors on LTFU.Adolescence and pregnancy are potential risk factors for loss to follow‐up , older adolescents  and adults (≥20 years) from 2000 to 2014 at 52 health facilities affiliated with the International epidemiology Databases to Evaluate AIDS (IeDEA) East Africa collaboration. We estimated cumulative incidence  of LTFU (no clinic visit for ≥6 months after ART initiation) and identified patient and site‐level correlates of LTFU, using multivariable Cox proportional hazards models for all patients as well as individual age groups.We used routine clinical data for patients initiating ART, including 2496 YA, 2955 OA and 132,936 adults. Of these, 55%, 78% and 66%, respectively, were female and 0.7% of YA, 22.3% of OA and 8.3% of adults were pregnant at ART initiation. Cumulative incidence of LTFU at five years was 26.6% (24.6 to 28.6) among YA, 44.1% (41.8 to 46.3) among OA and 29.3% (29.1 to 29.6) among adults. Overall, compared to adults, the adjusted hazard ratio, aHR, (95% CI) of LTFU for OA was 1.54 (1.41 to 1.68) and 0.77 (0.69 to 0.86) for YA. Compared to males, pregnant females had higher hazard of LTFU, aHR 1.20 (1.14 to 1.27), and nonpregnant women had lower hazard aHR 0.90 (0.88 to 0.93). LTFU hazard among the OA was primarily driven by both pregnant and nonpregnant females, aHR 2.42 (1.98 to 2.95) and 1.51 (1.27 to 1.80), respectively, compared to men. The LTFU hazard ratio varied by IeDEA program. Site‐level factors associated with overall lower LTFU hazard included receiving care in tertiary versus primary‐care clinics aHR 0.61 (0.56 to 0.67), integrated adult and adolescent services and food ration provision aHR 0.93 (0.89 to 0.97) versus nonintegrated clinics with food ration provision, having patient support groups aHR 0.77 (0.66 to 0.90) and group adherence counselling aHR 0.61 (0.57 to 0.67).A total of 138,387 patients initiated LTFU compared to YA and adults. Interventions to prevent LTFU among older adolescents are critically needed, particularly for female and/or pregnant adolescents.Older adolescents experienced higher risk of  Women initiating ART solely for prevention of mother‐to‐child transmission of HIV (PMTCT) rather than for clinical indications were excluded from this analysis. Based on the age of ART initiation, age groups were defined as young adolescents (10 to 14 years), older adolescents (15 to 19 years) or adults (≥20 years).2.3A total of 52 health facilities representing six programmes in Kenya, Tanzania, and Uganda were included in this analysis. These sites represented a mixture of private and public facilities; primary, secondary and tertiary levels of care; and urban and rural healthcare facilities. In most sites, adolescents and adults were seen in the same clinic. However, models of service delivery varied by site 2.4As part of routine clinical care, patient characteristics  and clinical status , ART regimen and outcomes  were captured on paper‐based forms at enrolment and follow‐up visits. Data clerks within the programme entered these data into a site‐specific electronic medical record system and conducted quality and logic checks following standardized local data cleaning protocols. Data were deidentified and merged across sites by IeDEA East Africa regional data managers who performed additional data cleaning and validation. The date of database closure varied with three sites leaving the IeDEA consortium during the latter half of 2010, one site contributing data through March 2012, and the remaining sites contributing data through mid to late 2014. A structured site assessment was completed at each site in 2009 and 2014, and gathered information on location and type of clinic, as well as the type of services offered 2.5The outcome of interest was LTFU following ART initiation. Patients were considered LTFU if they did not have a clinic visit for ≥6 months prior to database closure and were not documented as having died  or transferred out. Patients who had no return visit after ART initiation were randomly imputed days of follow‐up between one and fourteen days, to allow for inclusion of their data in the survival analysis. The imputation model followed a simple uniform distribution for the days of follow‐up. The patient‐level variables of interest were age, CD4 count and WHO stage, calendar year and pregnancy status, all at ART initiation. Using age at ART initiation, patients were categorized as young adolescents 10 to 14 years (YA), older adolescents 15 to 19 years (OA) or adults ≥20 years. CD4 count at ART initiation was defined as the most proximal CD4 count obtained within three months prior to ART initiation and up to seven days after. WHO stage at ART start was defined as the maximum WHO stage documented prior to ART initiation or the first documented within 60 days after the start of treatment. In a number of cases, CDC class was used to reflect disease severity. This was mapped to WHO stage in these analyses as follows: CDC class N→WHO stage 1, A→2, B→3 and C→4. Pregnancy at ART initiation was defined as a recorded pregnancy within two months before or after ART initiation date; or a documented estimated or actual date of delivery (according to available data) which resulted in an actual or estimated date of conception which in turn identified a pregnancy prior to ART‐initiation.We compared demographic characteristics by age group using descriptive statistics. We estimated the cumulative incidence of LTFU, using the Aalen & Johansen estimator which accounts for competing risks 3Between 2000 and 2014, 211,362 patients age 10 and older were enrolled into East Africa programs, of whom 138,387 (65. 5%) initiated ART. Of these, 2496 (1.8%) were YA, 2955 (2.1%) were OA and 132,936 (96.1%) were adults. The majority of patients starting ART were female, including 54.7% of YA, 77.8% OA and 66.2% adults  of LTFU among OA was 1.54 (95% CI 1.41 to 1.68) Table . By contThe hazard of LTFU in the overall study population varied widely among IeDEA programs. In comparison to the largest programme (AMPATH), all other programmes had lower hazard of LTFU, except the Tanzania NACP programme   had particularly high risk of being LTFU. This analysis highlights the need to develop, test and assess interventions designed to retain older adolescents on ART, particularly girls and women initiating ART while pregnant.None. The authors report no conflicts of interest.HNB, BE, ANK, CTY, KWK and EJA contributed to the conception of the analysis idea; BSM, ANK, HNB, CTY, SA, HF, EL and JS contributed to data collection and cleaning; ANK, BSM and CTY analysed the data; HNB, BE, ANK and CTY wrote the manuscript; HNB, BE, ANK, CTY, KWK, BSM, EJA, SA, HW, EL and JS reviewed and contributed to the manuscript; BE gave overall technical oversight for the analytic process and manuscript writing.


At week 52, 11 of the 13 patients completed the study, and all 11 had a sustained improvement. The mean (SD) MRC sum score was 85.6 (8.7) at the baseline, and 90.6 (12.8) at week 52. The mean grip strength was 39.2 (30.0) kPa at the baseline and 45.2 (32.8) kPa at week 52. Two patients dropped out because of adverse event (dysphagia) and decision of an investigator, respectively. Three patients developed coronary spasm, dysphagia, or inguinal herniation, reported as the serious adverse events, but considered not related with the study drug. The other adverse effects were mild and resolved by the end of the study period. Our results show that maintenance treatment with 1.0 g/kg IVIg every 3 week is safe and efficacious for MMN patients up to 52 weeks. Further studies are required to investigate optimal dose and duration of maintenance IVIg for MMN.Intravenous immunoglobulin (IVIg) therapy is currently the only established treatment in patients with multifocal motor neuropathy (MMN), and many patients have an IVIg‐dependent fluctuation. We aimed to investigate the efficacy and safety of every 3 week IVIg (1.0 g/kg) for 52 weeks. This study was an open‐label phase 3 clinical trial, enrolling 13 MMN patients. After an induction IVIg therapy (0.4 g/kg/d for 5 consecutive days), maintenance dose (1.0 g/kg) was given every 3 weeks for 52 weeks. The major outcome measures were the Medical Research Council (MRC) sum score and hand‐grip strength at week 52. This trial is registered with  Four retrospective studies have described MMN patients who have received repeated IVIg therapy over several years, and suggested that it may be used as the long‐term treatment option for MMN.22.1http://clinicaltrials.gov, number NCT01827072.This study was a multi‐center, single‐arm open phase 3 trial conducted at 11 Japanese tertiary hospitals. The study procedures were in accordance with the Declaration of Helsinki, and Japanese Good Clinical Practice criteria, and approved by the internal review board of each hospital. This study is registered with A total of 13 patients with definite or probable MMN according to the EFNS/PNS clinical diagnostic criteria were enrolled.2.2The study design and trial profile are shown in Figure For neurological assessment, MRC sum score, hand‐grip strength, and Guy's Neurological Disability Scale (GNDS) score2.3The outcome measures were MRC sum score, hand‐grip strength score, and GNDS score. Difference in the mean scores at week 1 and week 52 was calculated. These measures were not distinguished as primary and secondary because the number of MMN patients included in this trial was small. Safety assessments included any adverse event during the study period.2.4Analyses were performed by the full analysis set which included all patients who received the drug at least once. Missing data of MRC sum score, hand‐grip strength, and GNDS score were input by data at the last visit (the last observation carried forward method). Confidence interval (CI) was calculated using the Clopper‐Pearson exact method for both periods. The target number of 10 patients was set in view of the rarity of MMN. All analyses were performed with the statistical software package SAS, release 9.2 . Statistical comparison was not performed because of the small number of patients included.33.1The study was conducted from June 2013, and ended in June 2015. A total of 15 patients were screened. Two patients were ineligible, and the remaining 13 were enrolled  of the 13 patients experienced adverse events (95% CI: 64.0%‐99.8%). Table % of the n = 1), dysphagia (n = 1), and inguinal hernia (n = 1). None of them was considered to relate with IVIg.Three patients experienced serious adverse events, including coronary artery stenosis  every 3 week resulted in sustained clinical improvement for 52 weeks. The results were supported by sequential findings of MRC sum score, grip strength, and GNDS score. The maintenance IVIg therapy was not associated with clinically significant adverse effects. Our results show the long‐term efficacy and safety of the maintenance IVIg.In previous retrospective studies, a variable maintenance IVIg regimens, such as 1.0 g/kg every 2 to 4 weeks, or 2 g/kg every 1 to 2 months, were used dependent on patients' condition. Whereas disease activity, immunoglobulin metabolism, and response to treatment are presumably different among patients with MMN, the optimal dose and interval may be determined according to patients' situation. Nevertheless, this study revealed uniform regular maintenance IVIg administration was successful in almost of the MMN patients enrolled. The regimen used in this trial can be an option to achieve sustained remission in MMN at least for 52 weeks.In patients with chronic inflammatory demyelinating polyneuropathy (CIDP), approximately 25% of patients have long‐lasting remission without immunological treatment,A recent clinical trial for CIDP using the same maintenance dose (1.0 g/kg) and interval (every 3 weeks) for 52 weeks has also shown similar long‐term efficacy.In conclusion, 52‐week maintenance IVIg therapy appears to be safe and efficacious to prevent a relapse for MMN patients. The longer‐term safety and efficacy, and need of dose adjustment should be investigated in future studies.


To the editor: We have read the interesting article by Eyre DW et al. describing a Neisseria gonorrhoea (NG) FC428 clone detected in two female cases who developed infection following contact with United Kingdom (UK)-resident men linked to travel to Ibiza, Spain. These cases provide evidence that the FC428 clone’s transmission is occurring within Europe. The widespread distribution of this clone threatens the effectiveness of gonorrhoea treatment [reatment .As both cases had contact with the same sexual network in Ibiza, an exhaustive search of other probable NG cases was developed by the national and local public health authorities. During 2018, 82 cases of gonorrhoea were diagnosed in Ibiza; 80% (n = 66) were men with a median age of 29 years (interquartile range (IQR): 25–36) and 20% (n = 16) were women with a median age of 32.5 years (IQR: 27.5–37.5). Forty percent of the cases (n = 33) were foreigners, but none of them were tourists. Among the foreigners, 26 were men, 20 were from Europe  and 12 were from Latin America. The most common clinical presentation was urethritis among men (62/66) and cervicitis among women (15/16).A positive culture was confirmed in 90% (n = 74) of the cases; 91% (n=60/66) of cultures were positive among men and 87% (14/16) among women. Of the 82 NG isolates tested, 26.8% showed resistance to penicillin (intermediate susceptibility), 2.4% to ciprofloxacin and 11% to both antibiotics. Resistance to ciprofloxacin and levofloxacin was found in 11% (n = 9) of isolated strains, and 15.8% (n = 13) showed additional resistance to penicillin. Two strains showed resistance to ciprofloxacin, levofloxacin and cefuroxime. None of the isolated and tested strains showed resistance to ceftriaxone. In Ibiza there are two hospitals, one public and one private. The notification of gonococcal cases are compulsory in both hospitals. No positive cases were diagnosed in the private hospital in 2018, and all previously described cases were diagnosed in the public hospital.The incidence rate of gonococcal infection has increased in Spain since 2001. From 2001–13, the annual change was 11.7% : 9.6–13.9) and from 2013–17 it was 26.3% (95% CI: 20.7–32.2). In 2017, the Balearic Islands had one of the highest incidence rates in Spain, with 41.79 cases per 100,000 population [In 2011, the first two cases of ceftriaxone-resistant NG were reported in Catalonia, Spain. Both cases were men who have sex with men (MSM). One of the cases was diagnosed with urethral discharge by a general practitioner. He was treated with 100 mg of doxycycline twice a day for 7 days. The other case was his partner and was asymptomatic, with no signs of proctitis or urethritis. He was treated with levofloxacin for 7 days. In both cases, ceftriaxone-resistant NG strains were isolated ; these wSpain is one of the participating countries in the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) . In its


Puccinia coronata f. sp. avenae (Pca) is a major production constraint of oat in North America, Europe, and Australia. There are over 100 genes effective against one or more Pca races, but only a handful of seedling resistance (Pc) genes have been mapped to a known chromosomal location. The goal of the present study was to use linkage mapping to identify the genomic location of the Pc53 gene, and to produce a list of linked SNPs with potential as molecular markers for marker assisted breeding. The Pc53 gene was placed on the linkage group Mrg08 at 82.4 cM using F5-derived recombinant inbred lines (RILs) from a cross between the Pc53 carrier 6-112-1-15 (PI 311624) and the susceptible cultivar Otana. The map location was validated using RILs from a cross between 6-112-1-15 and the Pc50 differential line. Single nucleotide polymorphism marker GMI_ES02_c14533_567 was the closest to Pc53. A major seedling resistance gene ‘PcKM’ and QTL QcC.Core.08.1, QCr.Core.08.2, QCr.Core.08.3 and QCr.cdl9-12D were previously reported on Mrg08. QPc.Core.08.1 and PcKM were mapped to within 1 cM of Pc53; but previous virulence studies have indicated separate identities. The chromosomal location of Pc53 and SNPs linked with it will facilitate the utilization of Pc53 in oat breeding programs.Crown rust disease caused by the fungus  Puccinia coronata f. sp. avenae (Pca) is a major production constraint of oats (Avena sativa L.) in North America [Rhamnus spp.) in North America [A. sterilis [A. sterilis-derived resistance gene, Pc39. Since then, genes Pc38, Pc39, Pc48, Pc68 and Pc94 have been deployed in cultivars such as: Dummont, AC Medallion, AC Assiniboia, AC Pinnacle, and Leggett with widespread use [Crown rust caused by the fungus  America . The dis America , 3. The  America . There a America . Deploym America –6. Over sterilis . ‘Fidlerread use . Unforturead use .Pc53 is one of the many Pc genes identified in oats. It was originally identified from the wild oat species, A. sterilis, in the Mediterranean region of Israel . Despite the occurrence of virulence to Pc53, the frequency of virulence against this gene in North America had been consistently low compared to most Pc genes [Pc genes , 10, 11,Pc genes have been mapped to a known chromosomal location of oat [Pc38 [PcKM/Pc45 [Pc58 [Pc68 [Pc71 [Pc91 [Pc94 [Pc genes has created multifaceted challenges in oat research. First, it has limited the utilization of genomic tools in oat breeding. Second, in the continuous search for new sources of crown rust resistance, the lack of information on genomic location restricts investigations as to novelty to the conventional virulence test methods which require tremendous amounts of time and resources. For example, Gnanesh et al. [PcKM’ in the oat cultivar ‘Morton’ in field and greenhouse studies; but disease reactions of differential lines and molecular marker data suggested that PcKM may be Pc45. Another study by Martens et al. [Pc54 could be allelic or closely linked with gene Pc35; while Leonard et al. [Pc35 and Pc54 on different linkage groups. An association mapping study of elite germplasm identified QTL conditioning crown rust resistance (Qpc.CORE.05) in the same region as Pc71 [Pc genes. The current study complements other studies which have mapped crown rust resistance genes, and will improve the availability to oat researchers of information on chromosomal location of Pc genes.Only a handful of the nearly 100 n of oat ,12,13. Tat [Pc38 , PcKM/Pc45 [Pc58 , Pc68 [158 [Pc68 , Pc71 [168 [Pc71 , Pc91 [691 [Pc94 . Absences et al.  and Simos et al.  had sugg as Pc71 . The nov as Pc71 ,22 couldPc53 on the oat consensus linkage map of Chaffin et al. [Pc53.The objectives of the present study were to: 1) map n et al.  using SN5-derived recombinant inbred lines (RILs) was developed from a cross between the Pc53 differential line, Clinton/6-112-1-15 (Pc53) and the susceptible cultivar Otana, developed by ARS-USDA, Aberdeen, ID (CI 5345/Zanster (CI 5345/2*Overland)). The map location of Pc53 was validated using 125 F5-derived RILs from a cross between the Pc53 and the Pc50 differential line (Pc50), (CAV 2643/4*Pendek). Both mapping populations were developed by single seed descent at the Small Grains and Potato Germplasm Research Facility of the USDA-ARS in Aberdeen, Idaho.A population of 149 FPca race LGCG designated according to Chong et al. [5:6 RIL, and the parents, were sown in 3.8 cm diameter ‘containers’ containing a 3:2:2 (v:v:v) mix of sand, peat moss, and vermiculite and maintained in a greenhouse adjusted to 22°C and 18 hour photoperiod. Each RIL was planted in three containers, for a total of 15 seed evaluated per line. Two weeks after planting, seedlings were inoculated with uredinia of the respective race suspended in Soltrol170 isoparaffin  using a motorized sprayer  and a small atomizer . Plants were left in the open for one hour to dry, and then transferred to a dew chamber set at 20°C with no light. After 18 hours in the dew chamber, plants were placed in a growth chamber adjusted to 20–22°C with an 18-hour photoperiod. Disease reactions on the first and second leaves were recorded 14 days after inoculation as IT on a 0 to 4 scale where 0–2 are considered resistant reactions, while 3–4 are susceptible reactions [Seedling tests were carried out on both RIL populations (Pc53 x Otana and Pc53 x Pc50) at the Small Grains and Potato Germplasm Research Facility of the USDA in Aberdeen, Idaho in 2017–2018. g et al.  was usedeactions .5 plants of the two populations (Pc53 x Otana and Pc53 x Pc50) and parents following a protocol by Anderson et al. [High quality DNA was isolated from Fn et al.  with mod5:6 families. The goodness-of-fit of the observed disease reaction to the expected segregation ratio of 1:1 for a single gene was tested using Pearson’s Chi-squared (X2) distribution analyses.Individual RILs in each population were classified based on the IT produced by the FSNP markers that were polymorphic between parents of each population, and with less than 10% missing data were selected. Of these, only SNPs that were assigned a location on the oat consensus map of Chaffin et al.  were usePc53 gene were compared with the rice (Oryza sativa L.) genome (https://www.ncbi.nlm.nih.gov) using the search function ‘sequence(BLAST) search’ to identify candidate genes for Pc53.The sequences of the single nucleotide polymorphism markers linked with the 5:6 families showed that the Pc53 x Otana and Pc53 x Pc50 progeny segregated at a ratio of 79R:71S and 66R:59S. Based on Chi-square tests both populations fit 1R:1S Mendelian ratio with X2 of 0.32 and 0.39, and P-value of 0.572 and 0.531, respectively. The segregation ratio shows that the single-seed descent procedure worked as expected without introducing any selection bias.Race LGCG produced compatible ITs of 3 to 4 on the susceptible parent (Otana) and incompatible ITs of; and; 1 were observed on the resistant parent Pc53. Race NBTG produced ITs of 0 and; 1 on the Pc53 and Pc50 differential lines, respectively. Phenotyping of FPc53 gene was localized at 2.4 cM distal to the SNP markers GMI_ES01_c28412_66 and GMI_ES02_c12737_306 (Pc53 were GMI_DS_LB_9591 (2.8 cM), GMI_ES02_c3359_447 and GMI_ES02_c3359_678 (3.2 cM), GMI_ES02_c8737_267 and GMI_ES02_c24327_173 (7.9 cM), and GMI_ES02_c8277_506 (9.1 cM). All the SNP markers linked to Pc53 were located on the linkage group Mrg08 of the oat consensus map (23)  .Pc53. The 569 polymorphic markers were assigned to 8 linkage groups. Linkage analysis revealed that Pc53 was on the same map location as GMI_ES02_c14533_567 at 46.6 cM of the linkage group  with ≥80% sequence homology to rice loci. Rice chromosome 4 loci LOC107276041 (31764063), LOC_Os04g0661600 (33740410), and LOC_Os04g56620 (33761378) had sequence identity of 90, 80, and 84%, respectively to these three oat SNPs. The former rice locus encodes an uncharacterized protein, and the latter two loci encode the molybdopterin biosynthesis protein CNX1. The oat SNPs closest to the Pc53 gene in the Pc53 x Otana population  did not have significant sequence homology to a rice locus.According to Oliver et al.  Mrg08 ofPc53 was originally identified from the A. sterilis accession 6-112-1-15 collected in northern Israel. This gene has not been used in cultivar development apart from some component lines of the Iowa multilines [Pc53 is present in the Pca populations of North America [Pc53 has never been greater than 5% of all isolates collected. In contrast, 65–95% of isolates collected in the same period were virulent against the widely deployed genes Pc38, Pc39, Pc63, Pc67, and Pc71 [Pc53 gene may have sufficient effectiveness to be useful in variety development, particularly when deployed in combination with other Pc genes. America ,11. HowePcKM’, and the QTL QCr.cdl9-12D were reported on Mrg08, as were the GWAS QTL QcC.Core.08.1, QCr.Core.08.2, and QCr.Core.08.3 [QPc.Core.08.2 and QPc.Core.08.3 were proximal to GMI_ES02_c3359_447 and GMI_ES15_c6639_318, while Pc53 was distal to both markers, suggesting that the two QTL may not be related to Pc53. Similarly, QCr.cdl9-12D was proximal to GMI_DS_LB_9591, GMI_ES02_c3359_678 and GMI_ES17_c19330_272, and distal to GMI_ES17_c3134_489, GMI_ES01_c14013_124 and GMI_ES17_c12269_176, while Pc53 was distal to the former three and proximal to the latter three SNPs.QTL conditioning crown rust resistance in oats have been previously mapped to the Mrg08 linkage group. A major seedling resistance gene ‘ore.08.3 ,21,22. QPc53 was mapped to within 1 cM of the reported locations of QPc.Core.08.1 and PcKM, suggesting that they may be linked or allelic. Based on crown rust virulence profiles over the period of their field trials, Esvelt-Klos et al. [Pc45 and Pc53 as possible candidate genes for the QTL QPc.Core.08.1. The Pc45 gene has been widely used in North American oat breeding and is expected, therefore, to be abundantly represented in the CORE panel. It is less likely that the QTL identified in the CORE germplasm originated from Pc53 as this gene is not known to be in the pedigrees of released cultivars apart from the Iowa multi-lines ([https://triticeaetoolbox.org/), which were not included in the CORE association mapping panel. In addition, virulence analyses of differential lines and molecular marker data have suggested that PcKM might be Pc45 [Pc45/PcKM and Pc53 we examined previous virulence analyses reports. Admassu-Yimer et al. [Pca race ‘MGBH’ produced an IT of 0; on Pc45 and an IT of 3–4 on Pc53. However, the present study placed Pc53 at the same linkage group and map location as PcKM [Pc45 and Pc53 may be components of a complex of genes in this region conditioning resistance to crown rust. Such phenomena are not uncommon in oats. A good example is the resistance of TAM-O-301 to crown rust, which is conferred by complex of Pc58 genes [et al. [A. sativa.s et al.  suggeste be Pc45 . To under et al.  reported as PcKM . We hypo58 genes . Gregory58 genes  and Wise [et al.  also repAvena sativa genome, limiting our ability to exhaustively catalog positional candidate genes for Pc53. However, sequence homology of SNPs linked to Pc53 are consistent with synteny to a region of rice chromosome 4 spanning over 10 Mbp (https://www.ncbi.nlm.nih.gov). A smaller region of synteny corresponding to the region between GMI_ES02_c14533_567 and GMI_ES02_c3359_678 spans close to 2 Mbp and contains over 600 gene annotations. Genes of interest in that region include LOC9269204 disease resistance protein RGA2, and the innate immune system associated LOC4336849 secretory carrier-associated membrane protein 6. In the future, access to an annotated oat genome sequence may enable the identification of positional candidate genes for Pc53 with sufficient confidence to advance as targets for cloning to further study the mechanisms of crown rust resistance in oats.There is currently no sequence available for the Pc53 conferring race-specific crown rust resistance in oat is linked with SNP markers placed on the oat consensus linkage group Mrg08. This is the first report of the chromosomal location of Pc53. The SNPs linked with it may facilitate the use of this gene in oat breeding programs. In the long term, mapping of oat crown rust resistance genes will contribute towards a more complete understanding of oat genomics and enable researchers to utilize molecular tools to develop new oat cultivars with desirable traits.In summary, we found that