Bartonella species are human pathogens but data on their global distribution and epidemiology are limited. The aims of the study were to: 1) determine the prevalence of Bartonella infection in rodents and fleas; 2) identify risk factors for Bartonella infection in rodents; and 3) characterize the Bartonella genotypes present in these rodent and flea populations.Bartonellae are intracellular bacteria, which can cause persistent bacteraemia in humans and a variety of animals. Several rodent-associated Bartonella DNA, which was detected in 57 individuals , of three rodent species  using a qPCR targeting the ssrA gene. Considering R. rattus individuals only, risk factor analysis indicated that Bartonella infection was more likely in reproductively mature as compared to immature individuals . Bartonella DNA was also detected in 53 of 193 Xenopsylla cheopis fleas (27.5%: 95% CI 21.3–34.3) collected from R.rattus individuals. Analysis of ssrA and gltA sequences from rodent spleens and ssrA sequences from fleas identified multiple genotypes closely related (≥ 97% similar) to several known or suspected zoonotic Bartonella species, including B. tribocorum, B. rochalimae, B. elizabethae and B. quintana.Spleen samples collected from 381 rodents representing six different species were tested for the presence of ssrA and gltA sequences obtained from rodent spleens and ssrA sequences obtained from fleas reveal the presence of a diverse set of Bartonella genotypes and increase our understanding of the bartonellae present in Tanzanian. Further studies are needed to fully characterise the prevalence, genotypes and diversity of Bartonella in different host populations and their potential impacts on human health.The  Bartonella are fastidious, Gram-negative, vector-borne bacteria with worldwide distribution. Bartonella species are known to infect mainly erythrocytes and endothelial cells of various mammals, such as humans, cats, dogs, ruminants, wild rabbits and rodents [Bartonella species and that ectoparasites such as fleas, ticks, sand flies, and lice are key vectors of Bartonella infection [ rodents . Epideminfection ,3.Bartonella species have been identified as zoonotic pathogens. To date there are roughly 45 Bartonella species and subspecies that have been designated [Bartonella are the cause of human infections in various regions of the world, particularly in areas where humans are in close contact with rodents [Bartonella in rodents and of the role of Bartonella species in human disease in Tanzania is limited.In recent years, an increasing number of signated . Several rodents . HoweverBartonella infection in humans can range from mild [Bartonella species include endocarditis [Bartonella infections hard to diagnose clinically. This contributes to a poor understanding of the current distribution and relative importance of infections caused by this pathogen. The challenges of identifying the causes of non-specific febrile illness are demonstrated by previous research conducted in Moshi, northern Tanzania, where a study of patients admitted to hospital with febrile illness revealed that a range of zoonotic pathogens were responsible for roughly a quarter of the hospital admissions [Clinical manifestations of rom mild  to life rom mild ,11. Knowcarditis , myocardcarditis , fever acarditis , intraoccarditis , meningicarditis . This ramissions . Howevermissions –10,17,18Bartonella are widely used due to their greater sensitivity and ease of use in comparison to culture and serology based approaches. Real-time PCR assays are recommended for primary screening of Bartonella species followed by confirmatory assays, using either conventional or real-time PCR and sequencing [Bartonella species in rodents using conventional PCR detection methods for multiple gene targets. In northern Tanzania, a study performed in Mbulu, a rural district in northern Tanzania, detected Bartonella in 41% of indigenous rodents using gltA and rpoB PCR targets [gltA sequences from these samples revealed the presence of multiple genotypes similar to known Bartonella species, including B. elizabethae, B. tribocorum, B. birtlesii and B. queenslandensis [Molecular detection and typing methods for quencing ,20. In Aquencing , Kenya [quencing , South Aquencing , the Demquencing ,25 and Uquencing  have preBartonella species are present in Tanzania and may contribute to the burden of human febrile illness in northern Tanzania. However, the distribution and epidemiology of Bartonella in Tanzania is largely unknown. The aims of this study were to use molecular diagnostic tools to estimate the prevalence of Bartonella infection in rodents and their fleas sampled in northern Tanzania. Specifically, we aimed to: 1) determine the prevalence of Bartonella infection in rodents and fleas using quantitative real-time polymerase chain reaction (qPCR); 2) identify risk factors for Bartonella infection in rodents; and 3) use sequencing of the gltA and ssrA genes to characterize the Bartonella genotypes present in these rodent and flea populations.Ethical approval for the study was granted by the Tanzania Commission for Science and Technology (COSTECH 2012-471-ER-2005-141 & 2015-71-NA-2011-199); Kilimanjaro Christian Medical Centre (KCMC) Ethics Committee (535 & 537); National Institute of Medical Research (NIMR), Tanzania (NIMR/HQ/R.8a/Vol.IX/1499 & NIMR/HQ/R.8a/Vol.IX/1522); Tanzania Wildlife Research Institute (TAWIRI); University of Glasgow College of Medical, Veterinary and Life Sciences Ethics Committee (200120020), and University of Glasgow Faculty of Veterinary Medicine Ethics and Welfare Committee (01a/13 & 02a/13). Written consent for study participation was obtained for each participating household, using forms translated into Swahili. Rodent sampling was performed in accordance with the UK Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 and American Veterinary Medical Association Guidelines for the Euthanasia of Animals ,28.Xenopsylla cheopis fleas were selected for DNA extraction and Bartonella testing based on their known contribution to Bartonella transmission [X. cheopis collected, DNA was extracted from one (if only one X. cheopis present on that host) or two (if more than one X. cheopis present on that host). Where multiple X. cheopis were collected from the same rodent, selection of individual fleas for DNA extraction was opportunistic.Rodent spleen samples and ectoparasites were obtained from a cross-sectional study conducted to explore the role of rodents in the epidemiology of leptospirosis and other zoonoses in the Kilimanjaro region of northern Tanzania . Rodentssmission . For eacDNA was extracted from approximately 10 milligrams (mg) of spleen tissue using the DNeasy Blood and Tissue Kit spin-column protocol for DNA purification from tissues . DNA from spleen tissues was eluted in 100μl of AE buffer and quantified using a Nano-Drop spectrophotometer . DNA from individual whole fleas was also extracted using the DNeasy Blood and Tissue Kit, following the protocol for purification of total DNA from ticks and eluted in a final volume of 65μl AE buffer. For all extractions, a no-template extraction control (PCR-grade water) was included for every 20 samples and DNA extracts were stored at -20°C prior to testing. DNA extracts from spleens were diluted in 20μl of AE buffer to a standard DNA concentration of 10–50 ng/μl for PCR testing, to minimize the potential for PCR inhibition due to the high concentration of host DNA in the rodent spleen extracts. Due to the lower concentration of DNA in extracts from fleas, these were tested directly from extraction concentrations.Bartonella genus-specific real-time PCR assay (qPCR) targeting the transfer-mRNA ssrA gene, using a previously published protocol [Bartonella DNA, equivalent to < 3 genomic copies per reaction when tested against four Bartonella species  [ssrA-F (5ˈ-GCTATGGTAATAAATGGACAATGAAATAA-3ˈ), ssrA-R (5ˈ GCTTCTGTTGCCAGGTG-3ˈ) and 6-carboxyfluorescein FAM-labelled probe (5ˈ-ACCCCGCTTAAACCTGCGACG-3ˈ-BHQ1) were used to amplify an ssrA gene fragment of approximately 300bp. qPCR reactions were carried out in 20 μl volumes comprised of 10 μl QuantiNova Probe PCR mix (Qiagen), 0.8 μl of each primer (10 μM) and probe (5 μM), 2.6 μl nuclease-free water and 5 μl DNA template. Positive control (rodent tissue DNA extract obtained from a previous study [Bartonella with closest similarity to B. tribocorum), extraction controls and no-template controls were included in each qPCR run. Assays were performed on a Rotor-Gene Q/6000 (Qiagen) with manufacturer recommended thermocycling conditions as follows: 95 °C for 2 minutes, followed by 45 cycles of 95 °C for 5 seconds and 60 °C for 5 seconds. A qPCR run was considered valid when all negative controls showed no evidence of amplification and the positive controls amplified with a Ct value of < 40. Extracts were tested in duplicate and considered positive when amplification was recorded in one or more test wells with a Ct value ≤40.DNA extracts from rodent spleens and fleas were screened using a protocol . The oriabethae) . The prius study  positivessrA gene was performed on all DNA extracts from both rodent spleens and fleas that were positive in the ssrA qPCR, based on a previously published protocol [2, 0.5 μl dNTPs (10 μM)  and 0.5 μl of each primer ssrA-F and ssrA-R at 10 μM [ssrA assay Ct value. Nuclease-free water was used to make up the total reaction volumes as needed. Amplifications were performed with the following conditions: 94 °C for 2 minutes, followed by 40 cycles of 94 °C for 15 seconds, 60 °C for 60 seconds, and 72 °C for 30 seconds, and then a final extension step of 72 °C for 3 minutes. Positive and negative controls were included in each PCR run. PCR products were visualized by electrophoresis in a 1.5% agarose gel stained with GelRed . A sample was considered positive if a clearly defined DNA band of approximately 300 bp was visible in the gel and confirmed as Bartonella by sequencing of the product. To confirm and characterize the genotypes of Bartonella detected, amplicons with the expected size were purified using either a QIAquick PCR or Gel Extraction Purification Kit (Qiagen). Sequencing was performed at Source Biosciences  using the same primers as for the detection PCR [For sequencing, conventional PCR amplification of the protocol . Each PCat 10 μM . Templattion PCR . SequencgltA gene was performed on all rodent spleen DNA extracts, based on a previously published protocol [5ˈ-GGGGACCAGCTCATGGTGG-3ˈ) and BhCS1137.n (5ˈ- AATGCAAAAAGAACAGTAAACA-3ˈ) [Bartonella by sequencing of the product. Purification, sequencing and BLAST analyses were conducted as for the ssrA gene.Conventional PCR amplification of the protocol . Each PCAACA-3ˈ) , and 5 μssrA and gltA gene fragments. For each gene, sequences were aligned using the ClustalW algorithm, implemented in MEGA 7.0 [ssrA analysis, rodent spleen and flea sequences from this study were aligned with reference ssrA sequences from cultured Bartonella species downloaded from GenBank  were excluded from phylogenetic analysis for both MEGA 7.0 . The modoutgroup . For anaTanzania , Kenya [Tanzania , the DemTanzania ,25 and UTanzania  that incoutgroup .ssrA qPCR test status (qPCR positive vs negative) and implemented using the package ‘lme4’ [R. rattus only, given the dominance of this species. Explanatory variables considered in the GLMM for rodent ssrA qPCR status included host sex and reproductive maturity (mature or immature), which were determined based on external sexual characteristics [ssrA qPCR test status was the only explanatory variable evaluated in the GLMM and the village identification variable was included as a random effect. Initial maximal multivariable models were created including all candidate explanatory variables and likelihood ratio tests were used to compare candidate models and guide model selection.Statistical analyses were performed in R . Exact be ‘lme4’ . For roderistics . Trappineristics . The vilR. rattus) ; African pygmy mice ; multimammate mice , spiny mice  and striped bush squirrels . Of the rodents tested, 219 individuals (57.5%) were female. The majority of all sampled rodents  were classified as sexually mature based on external sexual characteristics. A total of 265 of the 381 rodents (69.6%) were trapped during wet season sampling. A total of 513 fleas were collected from 153 of the 381 (40.2%) rodents included in the study. Flea species identified were Xenopsylla cheopis (n = 306), Echidnophaga gallinacea (n = 204) and Ctenocephalides felis (n = 3). DNA extracts from a total of 193 Xenopsylla cheopis collected from 124 rodents  were available for Bartonella testing using ssrA.Spleen samples from a total of 381 rodents were available for testing. The majority , were from black rats ( rattus) . Other rBartonella DNA was detected by ssrA qPCR in a total of 57 of 381  rodents screened , with reproductively mature individuals more likely to be ssrA qPCR positive . None of the other candidate variables evaluated  were significantly associated with R. rattus ssrA qPCR test status. The breakdown of rodents trapped by village is given in screened . Samples. rattus  were allBartonella DNA was detected by ssrA qPCR in 53 of 193  X. cheopis flea extracts. All ssrA qPCR positive flea extracts were collected from R. rattus individuals. The positive control used in ssrA qPCR runs to test flea extracts had a mean Ct value of 32. Logistic regression analysis identified a relationship between flea ssrA qPCR test status and the ssrA qPCR test status of the rodent that each flea was collected from . X. cheopis fleas collected from ssrA qPCR positive rodents were more likely to themselves be ssrA qPCR positive .ssrA gene target were obtained from 45 of 57 rodents spleen samples  and 39 of 53 ssrA positive X. cheopis fleas. From the 237bp ssrA sequences amplified, 12 unique genotypes (1s to 12s) were identified in the sequences from rodent and flea populations combined (B. tribocorum reference sequence from strain GDHL73 (GenBank accession number MF765681); 2) group Bs: genotype 6s was unique but clustered with a B. elizabethae reference sequence (GenBank accession number MF765617); 3) group Cs: genotypes 7s to 10s clustering with a sequence from B. tribocorum strain GDHL25 (GenBank accession number MF765675); 4) group Ds: genotype 11s clustering with a B. rochalimae reference sequence (GenBank accession number MF7651); and 5) group Es: genotype 12s from P. flavovittis, which was mostly closely related to a B. quintana reference sequence (GenBank accession number HG518998). The distribution of ssrA genotypes between trapping villages  were present in rodents trapped at multiple villages and provides no evidence of spatial segregation of the genotypes in this rodent population. Data on ssrA genotypes were available for fourteen pairs of X. cheopis fleas and R. rattus hosts (n = 11 R. rattus including three from which two X. cheopis were collected and tested). For seven pairs the ssrA genotype detected in fleas and rodent hosts were identical, but in the other seven pairs the genotypes differed. At the group level, 11 flea and host pairs had sequences from the same ssrA group and three pairs differed.Full length sequences of the combined ; Fig 2. villages  show thagltA gene were obtained from 17 rodent spleen DNA extracts . To be conservative, the alignment was pruned to the length of the shortest sequence (283 bp) and three unique genotypes were identified in this fragment  to a B.elizabethae sequence (GQ225710). Eight sequences (genotype 3g) collected from M. natalensis (GenBank accession number MK906045) and from R. rattus (GenBank accession number MK906043) were identical to a sample identified in a R. rattus from an urban site in Kenya .Full length sequences of the fragment . The corAs to Cs . Eight sn B29297 , GenBankquence GQ5710. EigBartonella genotypes among rodents and their fleas in a previously uncharacterised region of northern Tanzania . Rattus rattus was the most common rodent species trapped and showed a high Bartonella prevalence defined by ssrA qPCR, which is consistent with the global distribution of Bartonella species in Rattus [R. rattus, the probability of qPCR positivity was higher in reproductively mature individuals as compared to immature individuals, consistent with other studies (performed in the USA) that have found an association with age [ssrA and gltA gene fragments revealed a variety of genotypes and the majority of sequences obtained showed greatest similarity to B. tribocorum and B. elizabethae reference sequences, both of which have been isolated in humans with febrile illness [B. rochalimae and B. quintana were also identified based on ssrA sequencing. These species were not detected by sequencing of the gltA, indicating reduced sensitivity of the gltA conventional PCR for detection of Bartonella species as compared to the ssrA qPCR. This is consistent with the findings of a previous study [This study reveals substantial variation in n Rattus . Within with age ,44. Sequ illness . Sequencus study .ssrA qPCR was used to estimate prevalence in rodents and fleas, and the sequencing of ssrA and gltA PCR products to assess genetic variation and characterise the Bartonella detected. The overall prevalence of Bartonella (15%) detected in rodents using the ssrA qPCR was lower than has been detected in many comparable studies of global rodent populations [gltA assay for prevalence determination [gltA prevalence of 41% was detected [gltA to test invasive and indigenous rodent populations found variable prevalence across species, with higher prevalence in indigenous species (60% in Arvicanthus niloticus and 61% in Cricetomys gambianus), but low prevalence (1.4%) was recorded in invasive R. rattus [Bartonella prevalence determined by culture varied by rodent species [R. rattus only, the prevalence seen in this study and previous African studies reveals consistently lower prevalence in comparison to R. rattus sampled in Asia and tested using ssrA qPCR methods  X. cheopis tested were positive for Bartonella DNA [Bartonella in R. rattus and X. cheopis observed in this study are consistent with several other studies conducted in Africa. It has been argued that this pattern of lower Bartonella prevalence in African R. rattus populations could be attributed to host escape during colonization [The ulations ,45, inclmination ,47. In adetected . A Ugand. rattus . Similar species . Consideg. 32.5% ). The prnization . FurtherssrA genotypes were identified that were most similar to reference sequences of B. tribocorum and B. elizabethae (Groups As to Cs), with only one genotype (10s) showing an identical match to a published B. tribocorum sequence in GenBank. However, since all of the reference sequences that were most similar were from a single study in China [Bartonella found in Tanzania, Moreover, B. elizabethae and B. tribocorum share identical published ssrA sequences in Genbank and our results show clustering in the phylogenetic tree (ssrA fragment. The other two Bartonella species identified were: 1) a single sequence (11s) with greatest similarity (98%) to B. quintana obtained from a sample from a P. flavovittis host; and 2) a sequence obtained from multiple samples of R. rattus and X. cheopis that showed an identical match to B. rochalimae, emphasising the diversity of Bartonella present in rodents in Tanzania. To the best of our knowledge, this is the first report of molecular detection and characterization of Bartonella species in rodents and their associated ectoparasites in Africa using the ssrA gene target. The scope for comparison with other sequences is thus limited, as there is currently little reference material on ssrA sequences from Bartonella sampled elsewhere, particularly in Africa.Phylogenetic analysis of sequences from rodents and their fleas revealed high concordance of sequences between hosts and ectoparasites. Overall, 10 distinct in China  it is imtic tree , so the gltA gene has been widely used to study Bartonella globally. Phylogenetic analysis of sequences from 17 gltA PCR products from this Tanzanian rodent population  showed the highest similarity to reference sequences of B. elizabethae, which has multiple published sequences in Genbank, including many from east Africa. The association of sequences similar to B. elizabethae with Rattus spp. in this study is consistent with similar findings from Malaysia [B. elizabethae has also been identified in different rodent species in Africa [gltA sequences were amplified from R. rattus and M. natalensis in our study, suggesting possible transmission between different rodent species in the Tanzania site or a shared common source of infection. Identical sequences were also identified previously in R. rattus sampled at both rural  and urban  sites in Kenya [Bartonella could be found in rodents in Kenya and Tanzania. However, these comparisons are based on short sequences of a single gene target only. Longer sequences from multiple genes and greater sampling effort across the region would be required to robustly confirm sharing of genotypes to trace source populations or determine patterns of host connectivityIn contrast, the Malaysia  and ThaiMalaysia . B. elizn Africa ,26. Idenin Kenya . This suBartonella species have been identified as important causes of human febrile illness in several global settings but there has been little investigation of the impact of bartonellosis upon human health, in Africa particularly. The finding of Bartonella genotypes that are most similar to B. elizabethae, B. rochalimae and B. quintana reference sequences in rodents trapped in and around households in Moshi, Tanzania, and the fleas collected from these rodents, indicates the possibility that Bartonella infection may be responsible for an as yet unknown proportion of febrile illnesses in this region. Efforts are needed to determine the clinical impact of bartonellosis in this region and increase awareness about Bartonella and other zoonotic pathogens among physicians and health care workers, especially where the cause of large proportions of febrile illness remains unknown. Our results also demonstrate that molecular detection tools can be effectively used for surveillance and diagnostic of zoonotic pathogens in resource limited settings.Several studies of zoonotic disease have shown that a variety of pathogens account for high proportions of febrile illness in northern Tanzania but that considerable proportions remain unexplained ,52. Bart


This cohort study examines the association of attention-deficit/hyperactivity disorder (ADHD) with teenage pregnancy among women and girls in Sweden. Is attention-deficit/hyperactivity disorder (ADHD) associated with increased risk of teenage birth?This nationwide cohort study of 384 103 women and girls in Sweden who gave birth for the first time between 2007 and 2014, including 6410 women and girls with ADHD, found that teenage deliveries occurred at a significantly higher rate among women and girls with ADHD than among those without ADHD (15.2% vs 2.8%).This study suggests that women and girls with ADHD may have an increased risk of giving birth as teenagers compared with their unaffected peers. Attention-deficit/hyperactivity disorder (ADHD) is associated with a plethora of adverse health outcomes throughout life. While Swedish specialized youth clinics have carefully and successfully targeted risk of unplanned pregnancies in adolescents, important risk groups, such as women and girls with ADHD, might not be identified or appropriately assisted by these interventions.To determine whether women and girls with ADHD are associated with increased risk of teenage birth compared with their unaffected peers and to examine the association of ADHD with risk factors for adverse obstetric and perinatal outcomes, such as smoking, underweight or overweight, and substance use disorder.This nationwide cohort study included data from 6 national longitudinal population-based registries in Sweden. All nulliparous women and girls who gave birth in Sweden between January 1, 2007, and December 31, 2014, were included. Data analyses were conducted from October 7, 2018, to February 8, 2019.Women and girls treated with stimulant or nonstimulant medication for ADHD  in the Swedish Prescribed Drug Register between July 1, 2005, and December 31, 2014.Maternal age at birth. Secondary outcome measures were body mass index, smoking habits, and psychiatric comorbidities.Among 384 103 nulliparous women and girls aged 12 to 50 years who gave birth between 2007 and 2014 included in the study, 6410 (1.7%)  were identified as having ADHD. The remaining 377 693 women and girls without ADHD  served as the control group. Teenage deliveries were more common among women and girls with ADHD than among women and girls without ADHD . Compared with women and girls without ADHD, those with ADHD were more likely to present with risk factors for adverse obstetric and perinatal outcomes, including smoking during the third trimester , body mass index less than 18.50 , body mass index more than 40.00 , and alcohol and substance use disorder .This study found that women and girls with ADHD were associated with an increased risk of giving birth as teenagers compared with their unaffected peers. The results suggest that standard of care for women and girls with ADHD should include active efforts to prevent teenage pregnancies. The clinical context may render outcomes vulnerable to low power and with limited generalizability to community populations. Furthermore, studies using self-reported measures may be sensitive to recall bias. However, a 2017 Danish nationwide cohort study5 that addressed these limitations found increased likelihood of teenage parenthood among individuals with ADHD compared with those without.Adolescents with ADHD are at increased risk of externalizing and risk-taking behaviors compared with their unaffected peers and more often engage in risky sexual behavior, such as earlier initiation of sexual activity and more sexual partners.12 Risks for the children include perinatal morbidity and mortality, low socioeconomic status, and low quality of life.16 In Sweden, teenage birth rates have decreased from 15.3% of all births in 1973 to 2.4% in 2014,17 one of the lowest rates internationally.19Teenage pregnancies are associated with several long- and short-term adverse outcomes for both parents and children. Young parents are at risk of low educational attainment, single habitation, and use of public assistance.6To our knowledge, the prevalence of teenage pregnancies in women and girls with ADHD has not been fully explored from a clinical perspective. Given the easy access to counseling and contraception, Sweden represents an ideal setting for investigating teenage births in women and girls with ADHD. Independent of socioeconomic status, all women and girls with ADHD have access to adequate contraception and counseling. Thus, this large-scale epidemiological study was designed to explore the prevalence of birth in young women and teenage girls with ADHD and to address modifiable risk factors associated with adverse obstetric and perinatal outcomes, such as smoking, body mass index (BMI), and substance use disorder in these women and girls.20 The Swedish National Board of Health and Welfare provided data from the Swedish Medical Birth Register, the Patient Register, the Swedish Prescribed Drug Register, and the Cause of Death Register. Statistics Sweden provided data from the Education Register and the Total Population Register.This nationwide cohort study was based on data from 6 Swedish national population-based registries. The personal identity number assigned to every Swedish citizen at birth or immigration facilitated information linkage across registries.21 Information was compiled from the standardized antenatal care records, at the first antenatal visit , and in gestational week 32 for smoking habit.22 Information on year and month of birth and gestational length at birth was collected from standardized birth records.The Medical Birth Register includes 98% of all births in Sweden, including prospectively collected clinical variables, demographic data, and information on reproductive history, as well as complications during pregnancy, birth, and the neonatal period.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10)23 diagnoses, with full national coverage since 1987.24 The Patient Register has also covered specialized outpatient visits since 2001. Both registers captured information from 2001 to 2014. The Prescribed Drug Register contains information on drug identity using Anatomical Therapeutic Chemical (ATC) classification codes and dates of prescriptions for the entire Swedish population since July 2005.25 The Cause of Death Register provides information on dates of all registered deaths since 1958. The Education Register contains information on highest level of education achieved of all Swedish individuals and the Total Population Register contains information on the identity of all residents born in Sweden since 1932.The Patient Register includes information on dates of hospital admissions and STROBE) reporting guideline. Data analyses were conducted from October 7, 2018, to February 8, 2019.This study was approved by the ethical review board in Uppsala, Sweden. Patient data confidentiality was assessed and approved by the National Board of Health and Welfare, Statistics Sweden, and the ethical review board before deidentified data from national registers were disclosed to the research team. The ethical review board waived informed consent because the exposure was within normal clinical practice and informed consent is not feasible in nationwide register-based data linkages. The study was designed, conducted, and reported adhering to the Strengthening the Reporting of Observational Studies in Epidemiology  and nonteenagers (women aged ≥20 years). Body mass index  was calculated based on information on weight and height at first antenatal visit and categorized according to the World Health Organization as less than 18.50, 18.50 to 24.99, 25.00 to 29.99, 30.00 to 34.99, 35.00 to 39.99, and more than 40.00. Information on smoking was captured at the first antenatal visit and in gestational week 32, and ongoing smoking, regardless of quantity, was coded as yes; no smoking was coded as no.ICD-1023 at least twice before the index pregnancy were considered to fulfill diagnostic criteria for comorbid bipolar disorder (ICD-10 codes F30-F31), schizophrenia or other psychotic disorder (ICD-10 codes F20-F29), emotionally unstable personality disorder (ICD-10 code F60.3), or substance use disorders (ICD-10 codes F10-F16 or F18-F19). As mood and anxiety disorders are mostly diagnosed in primary care, we used dispensed prescription of antidepressant treatment (ATC classification code N06A) from the Prescribed Drug Register as a proxy for indexing past episodes. Furthermore, as substance use disorder may vary over time, we analyzed presence of substance use disorder in 12 months preceding estimated date of conception, and only required 1 diagnostic occasion in this period. At least 1 filled prescription of ADHD treatment (ATC classification code N06BA) in the year prior to the estimated date of conception and during pregnancy were used to define ADHD treatment during the year preceding pregnancy and during pregnancy, respectively.We collected information on psychiatric comorbidities from the Patient Register between January 1, 2001, and December 31, 2014. Women and girls who received a diagnosis according to Data on maternal education in 2014 were collected from the Education Register. Maternal education was categorized as less than 10 years completed, 10 to 12 years completed , and more than 12 years completed . Data on maternal country of birth were collected from the Total Population Register. Maternal country of birth was classified as Nordic , European, or other.Characteristics of the population were described according to exposure with absolute and relative frequencies. Logistic regression models were used to estimate the magnitude of the associations between age at first pregnancy, risk factors for adverse obstetric and perinatal outcomes, psychiatric comorbidities, and ADHD diagnosis, presented as odds ratios (ORs) with 95% CIs. Missing data were excluded. As the emphasis in this study was on the total burden of disease, no adjustments were made. In addition, we refrained from any adjustment on any of the outcomes by ADHD treatment during the year preceding pregnancy, as we would not be able to ascertain whether participants had stopped taking their medication when planning a pregnancy or not.P values were 2-tailed, and statistical significance was set at less than .05.Furthermore, differences in risks factors for adverse obstetric and perinatal outcomes  were explored in distinct age-at-birth subgroups among women and girls with ADHD (<20 years vs ≥20 years). All analyses were performed using SPSS statistical software version 25.0 (IBM). The total cohort included 384 103 women and girls, including 6410 women and girls with ADHD  and 377 693 women and girls without ADHD . The sociodemographic and clinical variables of women and girls in the cohort are presented in In addition, compared with women and girls without ADHD, women and girls with ADHD were more likely to present with risk factors for adverse obstetric and perinatal outcomes, including being underweight , obese , or smokers. Smoking during the first trimester was reported by 25.9% of women and girls with ADHD compared with 5.6% of women and girls without ADHD . Furthermore, 20.2% of women and girls with ADHD continued to smoke when entering the third trimester compared with 3.8% of women and girls without ADHD . In addition, 48.6% of women and girls with ADHD had 10 years of education or less compared with 14.5% of women and girls without ADHD, and 93.2% of women and girls with ADHD were born in Nordic countries compared with 79.2% of women and girls without ADHD.This population-based cohort study examined the association of ADHD with age at first childbirth and associated medical and psychiatric risks. Our results showed an increased likelihood for teenage childbirth in women and girls with ADHD. Pregnant women and girls with ADHD presented with a number of medical and psychiatric comorbidities, among which substance use disorder was the most common.16 Consequently, our findings argue for an improvement in the standard of care for women and girls with ADHD, including active efforts to prevent teenage pregnancies and address comorbid medical and psychiatric conditions. In addition, antenatal care should focus on adequate measures to reduce effect of obstetrics risk factors in these women and girls. Unfortunately, owing to a lack of understanding and specific research addressing sex differences, ADHD in women and girls is still underrecognized, misdiagnosed, and, once appropriately diagnosed, suboptimally treated.28Although teenage pregnancies are a rare occurrence in women and girls in Sweden with or without ADHD, as suggested by an overall rate of teenage deliveries of 3.0% in this study, women and girls with ADHD were associated with a 6-fold increased risk for teenage birth compared with women and girls without ADHD and contributed 8.4% to all teenage births. This is evident despite the widespread availability of contraception in Sweden. Becoming a mother at such early age is associated with long-term adverse outcomes for both women and their children.Some important but unexplored hypotheses that may explain our results are that women and girls with ADHD receive inadequate contraceptive counseling, inadequately respond to counseling, fail to access or act on counseling, or experience more adverse effects from hormonal contraceptives. As to contraceptive counseling, Swedish youth clinics have made counseling and contraception easy to access at low cost for this population. However, collaboration between psychiatric care clinics for youths and specialized youth clinics is needed to specifically address and provide adequate care, including contraception, for women and girls with ADHD. Thus, it is possible that a lack of transdisciplinary knowledge and a subsequent team effort failure may be associated with the high proportion of teenage births among women and girls with ADHD. However, even if provided with such assistance, women and girls with ADHD may be less likely to act on or implement such counseling owing to their disorder and its associated deficits in self-regulation.29 and an increasing proportion of younger women and girls abstain from hormonal contraceptives owing to a fear of future adverse effects.30 Previous placebo-controlled randomized clinical trials have suggested minor mood disturbances with combined hormonal contraceptive use in healthy women,33 especially among women with a history of mental health conditions.34 As women with chronic psychiatric conditions are excluded from most clinical trials on hormonal contraceptives, these studies provide limited guidance in selecting contraceptives most suitable for this group. Further studies on contraceptives in women and girls with ADHD are needed.Adverse mental health effects from hormonal contraceptives are increasingly reported by young users,35 However, while our findings may imply that the obstetric risk factor profile is somewhat normalized with increased age at first birth, this may also be due to reverse causation, ie, women and girls with ADHD with a lower symptom load and high functioning may simply choose to delay their first birth.Our study found that young women and girls with ADHD were more likely to present with a number of risk factors for adverse obstetric and perinatal outcomes, such as underweight and smoking. Our findings suggest that delaying first childbirth until after age 20 years may be advantageous for women with ADHD in terms of the risks associated with smoking and underweight for the offspring. This is in line with the current conceptualization of ADHD as a neurodevelopmental disorder with delayed brain maturation associated with age-inappropriate symptoms of disinhibiting, risk taking, and impulsivity.36 Indeed, studies controlling for confounding by indication by using individuals as their own comparisons during periods with and without treatment suggest that stimulant medication may reduce or even ameliorate relevant adverse outcomes, such as educational failure,38 and decrease risk for unwanted outcomes associated with ADHD, such as substance use disorder,39 motor vehicle crashes,40 suicidal behavior,41 and criminal behavior.42 While it may be hypothesized that not using stimulant medication is associated with increased risk of unplanned pregnancy, this study cannot confirm any such association. Low rates of ADHD drug treatment may simply be owing to some women discontinuing psychotropic medication when planning for pregnancy.Pharmacological ADHD treatment in the year preceding the first pregnancy was not frequent in any of the age groups. Stimulant drug treatment has been associated with a decrease of core symptoms and adverse outcomes associated with ADHD.ICD-10 or Diagnostic and Statistical Manual of Mental Disorders43 diagnoses. However, Swedish national guidelines state that medication should be reserved for ADHD treatment when other supportive interventions have failed,27 indicating that our proxies most likely underestimated the incidence of ADHD and identified the most severe cases. Thus, while our definition of exposure probably could not avoid false-negatives, we considered bias due to false-positives more unlikely. Also, it should be noted that our findings did not rule out the possibility of an association of age at first pregnancy with more refined ADHD-related neurocognitive deficits. To further explore this, studies using more detailed measures of ADHD symptom severity are warranted. Additionally, as in all observational studies, we could not fully rule out selection bias due to a lack of intact information on exposure and outcome variables.Our study has limitations. The ascertainment of ADHD diagnosis was based on prescribed medication unique for the treatment of ADHD rather than 5 suggesting increased likelihood of teenage parenthood among women and girls with ADHD by showing that women and girls with ADHD were associated with a 6-fold increased risk of giving birth as teenagers compared with their unaffected peers. Furthermore, our results expanded the findings from the prior Danish study5 by suggesting that women and girls with ADHD are a significant and underrecognized group with several obstetric risk factors and comorbid medical and mental health concerns, among which substance use disorder was the most common. Standard of care in women and girls with ADHD should include active efforts to prevent teenage pregnancies to reduce long-term adverse consequences for both mothers and children. Transdisciplinary collaboration between psychiatric clinics for youths and specialized youth clinics, as well as further studies on tolerability of hormonal contraception in women and girls with ADHD, are warranted to provide adequate care and suitable contraception for youth with neurodevelopmental disorders.In conclusion, our data replicated the findings from a 2017 Danish cohort study by Ostergaard et al


R genes and rice improvement strategies.Understanding of genetic diversity is important to explore existing gene in any crop breeding program. Most of the diversity preserved in the landraces which are well–known reservoirs of important traits for biotic and abiotic stresses. In the present study, the genetic diversity at twenty-four most significant blast resistance gene loci using twenty-eight gene specific markers were investigated in landraces originated from nine diverse rice ecologies of India. Based on phenotypic evaluation, landraces were classified into three distinct groups: highly resistant (21), moderately resistant (70) and susceptible (70). The landraces harbour a range of five to nineteen genes representing blast resistance allele with the frequency varied from 4.96% to 100%. The cluster analysis grouped entire 161 landraces into two major groups. Population structure along with other parameters was also analyzed to understand the evolution of blast resistance gene in rice. The population structure analysis and principal coordinate analysis classified the landraces into two sub–populations. Analysis of molecular variance showed maximum (93%) diversity within the population and least (7%) between populations. Five markers viz; K3957, Pikh, Pi2–i, RM212and RM302 were strongly associated with blast disease with the phenotypic variance of 1.4% to 7.6%. These resistant landraces will serve as a valuable genetic resource for future genomic studies, host–pathogen interaction, identification of novel  Oryza sativa L.) is the staple food for more than half of the world’s population [Rice  genes is the most economically viable and environmentally friendly choice for the control of this disease. Resistance is generally conferred by either major R genes that provide complete protection against few races of the pathogen or minor genes, which conferred partial protection [The rice crop is affected by several diseases, of which blast disease caused by the fungus nd roots . The funportance . Owing tportance . Use of portance . Though portance . The utiR genes, and more than 350 QTLs for resistance to rice blast have been identified, and 27 have been molecularly cloned and characterized viz., Pib, Pb1, Pita, Pi9, Pi2, Pizt, Pid2, Pi33, Pii, Pi36, Pi37, Pikm, Pit, Pi5, Pid3, Pid3–A4, Pikh, Pish, Pik, Pikp, Pia, PiCO39, Pi25, Pi1, pi21, Pi50 and Pi65(t) [R genes/alleles and minor resistance genes in landraces in which resistance has co–evolved along with fungus over thousands of years. Genetically diverse rice landraces are one of the most important sources for major blast resistance to be introgressed into rice cultivars for the control of the blast [R genes into popular rice cultivar through marker assisted selection [To date, more than 100  Pi65(t) ,12,13. T Pi65(t) . Hence, he blast . Though election ,17, therR gene in diverse elite germplasm through DNA markers and differential blast races is an essential step in ensuring the accuracy in utilization of R gene in MAS for different rice breeding program [viz. Andhra Pradesh, Himachal Pradesh, Haryana, and Tamil Nadu resulted in severe yield losses in the farmer’s fields [With the discovery and fine mapping of blast resistance genes, many PCR–based markers have been developed and employed in the mining of blast resistance genes in the diverse elite germplasm which contain untapped resources of discrete alleles. The potential of elite germplasm will remain unknown unless the efforts are initiated to screen them intended for their possible use and function ,19,20. A program . To dates fields  which neA set of 161 diverse Indian rice landraces obtained from the National Gene Bank, ICAR–National Rice Research Institute, Cuttack were used in this study was. These landraces were collections of nine states of India with diverse ecologies . The detth day after sowing or the spreader row/checks achieved 85% of the disease symptom. Reactions of each landrace for leaf blast were scored as per the Standard Evaluation System (SES) of IRRI, (2002). The test entries with 0–3 scores were graded as highly resistant (HR), 4–5 as moderately resistant (MR), and 6–9 as susceptible (S). Whenever differences observed in score between the replications, the higher value was considered for scoring.A total of 161 landraces from all over India, representing nine states was screened for their leaf blast resistance under natural condition in the Uniform Blast Nursery (UBN) at the experimental farm of ICAR–NRRI, Cuttack (85°55′48″ E longitudes and 20°26′35″ N latitude) as described in Yadav et al . The leaLocation severity index (LSI) was calculated using following formula:Young leaf tissues of all the test entries were collected from two weeks old seedlings and stored at –80°C for genomic DNA extraction. Genomic DNA was isolated using the cetyltrimethyl ammonium bromide (CTAB) method  with sliPib, Pb1, Pita, Pi9, Pi2, Pizt, Pid2, Pi33, Pi36, Pi37, Pikm, Pit, Pi5, Pi54, Pish, Pik, Pikp, Pia, Pi25, Pi1, pi21, Pi56, Pi65(t) and Piz. A total of twenty eight functional/linked markers corresponding to the twenty four R genes were used for screening of the blast resistance genes. The details of the markers used in the present study are shown in The entire set of 161 landraces were genotyped using markers specific to 24 different blast resistance genes viz. 2, 1X Taq buffer and 1U of Taq DNA polymerase . The PCR program was conducted as: initial denaturation at 94°C for 5 min; followed by 35 cycles of denaturation for 30 sec at 94°C, primers annealing for 30 sec at different temperatures , 0.2 μM of primers, 1.5 mM of MgCleratures , and eloThe amplified PCR products of 28 markers were scored based on the presence (1) or absence (0) to create binary matrix for each marker. The genetic distance and similarity coefficients were estimated using the binary matrix of 28 markers. Major allele frequency, gene diversity and Polymorphism information content (PIC) value of each marker were estimated using Powermarker program Ver3.25 software . An unweP–value was observed in <5% of the permutations for the most significant polymorphism in a region.The hypothesis of genetic association between blast resistance genes and blast disease was tested through a general linear model (GLM) function using TASSEL5 software . The TASST were performed using the GenAlEx version 5.0 software [Population structure analysis of 161 landraces based on 28 markers was investigated using STRUCTURE version 2.3.4  using thsoftware .Based on the blast disease scoring data for consecutive two seasons in the UBN, 161 landraces were categorized into three groups; twenty one  were highly resistant (score 0–3), seventy  exhibited moderate resistance (score 4–5) and seventy  showed susceptible reaction (score 6–9) . The locR genes varied from 8.69 to 100%. The number of positive alleles of R genes varied from 5 to 19 in the landraces. Among the studied landraces, Biran from Tripura was found to contain highest number of the R genes (19) with positive allele. Three landraces (1.86%), showed positive loci for eighteen R genes, seven (4.34%) for seventeen R genes, ten (6.21%) for sixteen R genes, nineteen (11.80%) for fifteen R genes, twenty three (14.28%) for fourteen R genes, twenty four (14.90%) for thirteen R genes, twenty eight (17.39%) for twelve R genes, fourteen (8.69%) for both eleven and ten R genes, thirteen (8.07%) for nine R genes, four (2.48%) for eight R genes, three (1.86%) for seven R genes and one landrace (Jaya) from Gujarat contained positive allele for only five R genes, respectively  selected for the present study were distributed over the eight chromosomes . The genectively .Pish gene was estimated by visualization of PCR product with the linked markers, RM6648 and RM5811. The average gene frequency was found to be 10.55% with a range from 8.69% (RM5811) to 12.42% (RM6648). The estimation of Pit R gene localized on chromosome 1 was determined through visualization of the presence of 733 bp and 530 bp amplicons corresponding to the tk59–1 and tk59–2 markers, respectively. Ninety seven landraces were found to be positive for a Pit gene with 60.24% gene frequency. The SSR markers, RM302 and RM212 were used to detect positive alleles of a Pi37 gene with a gene frequency of 21.11% and 14.90%, respectively for the corresponding markers. The marker, Pb28 could amplify rice blast Pib gene with a fragment size of 388 bp. The gene frequency of Pib was found to be 26.70%. The recessive blast resistance gene pi21 located on chromosome 4 was detected by using the InDel marker, pi21_79–3. It was observed that only eight landraces were found positive for the pi21 gene with a gene frequency of 4.96%. The functional marker, Pi9–i was able to detect the presence of Pi9 gene and twenty five landraces were found positive with a gene frequency of 15.52%. Similarly, the presence of Pi2 gene was noticed using the functional marker Pi2–i. Interestingly, only twenty landraces were found positive for the Pi2 gene with a gene frequency of 12.42%. The functional (dln2) and linked (RM564) markers were used to determine the presence of Pid2 and Pi25 genes, respectively. It was revealed from this result that the presence of Pid2 gene in 151 landraces and Pi25 gene in 47 landraces with the gene frequency of 98.75% and 29.19%, respectively. The SNP primers, z56592, and zt56591 were used to ascertain the presence of Piz and Piz–t genes, respectively, which demonstrated the presence of Piz in 158 landraces and Pizt in 137 landraces. Surprisingly, landraces positive for the Piz–t gene were noted to be positive for the Piz gene. The rice blast R genes, Pi33 and Pi36 on chromosome 8 was amplified using the linked marker RM72 and RM5647, respectively which showed the presence of Pi33 in 15 landraces with a gene frequency of 9.31% and Pi36 in 99 landraces having a gene frequency of 61.4%. Eighty three landraces were found to harbour the blast resistance gene Pi5 located on chromosome 9 which was amplified with the marker, 40N23r. Likewise, the presence of Pi56(t) gene was observed in seventy two landraces determined by using InDel marker CRG4_2.The presence of Pik and Pik–p genes can be verified by the markers, K39512 and K3957 producing the PCR amplicon size of 112 bp and 148 bp, respectively. Based on these markers, 161 landraces were found to carry Pik where as Pik–p were distributed in 156 landraces. Uses of the InDel marker, Pikm, showed its presence with a fragment size of 619 bp in 97 landraces while the functional marker, Pikh, detected this gene in 118 landraces amplifying the fragment size, 216 bp. Further, the broad spectrum blast resistance gene Pi1 was noticed using two linked markers, RM1233 and RM224 in 61 and 82 landraces respectively, whereas 48 landraces were found positive for both the markers. The PCR based amplification of the neck blast resistance gene Pb1 showed its presence in seventy nine landraces with a gene frequency of 49.06%. The InDel and STS marker; SNP_3, and Pia–STS were used for the detection of Pi65(t) and Pia genes, respectively. The present study showed the presence of Pi65(t) gene in 89 landraces with a gene frequency of 55.27%, whereas, Pia was distributed in 123 landraces with a gene frequency of 76.39%. The presence of broad spectrum Pita/Pita–2 on chromosome 12 was detected by visualization of 1,042 bp with YL155/87 primer pairs. The Pita/Pita–2 gene was present in eighty seven landraces showing the gene frequency of 54.0%. In all the PCR amplification, HR-12 was used as a negative control  owing to its monomorphic nature in all the landraces in the present study. In contrast, the markers such as YL155/YL87 (Pita/Pita–2), 40N23r (Pi5), RM26998 (Pb1), RM224 (Pi1), CRG4–2 (Pi56(t)) and SNP_3 (Pi65(t)) were found to be more informative to study the genetic diversity as they showed highest PIC value of 0.3749  to 0.37  with an average value of 0.25. The gene diversity varied from 0 (K39512) to 0.49 . The PIC value of marker K39512 for f 0.3749 .For assessment of the genetic distance, 28 genic/linked markers were used to construct a dendrogram by using the UPGMA method of pooled 28 marker data led to the segregation of 161 landraces into two distinct major groups. The dendrogram was divided in to two major clusters, I and II . Major cSimilarly, sub–cluster IB clutched 94 landraces and further grouped into five sub–clusters IB–1, IB–2, IB–3, IB–4 and IB–5. Sub–cluster IB1 contained 30 landraces, with the highest number (six) of resistant landraces. No resistant landraces falls in sub–clusters IB–2 though it consisted of fifteen genotypes. Twenty eight landraces were found in the sub–cluster IB–3 including four (14.29%) resistant landraces. Further, sub–cluster IB–4 and IB–5 consisted of fourteen and seven landraces respectively; none of them were resistant. No resistant landraces were found in sub–cluster IB–4 whereas IB–5 consisted of two (28.57%) resistant landraces. This result shows that the landraces Biran from Tripura state possessing a maximum number of resistance genes (nineteen) falls in the sub–cluster IB–1 whereas a landrace, Jaya of Maharashtra collection carrying the minimum number of resistance genes (five) showing resistant reaction was found in the sub–cluster IA–1; this shows the ability of all 28 markers for discriminating the landraces of maximum and minimum number of resistance genes. Surprisingly, resistant landraces were almost equally distributed in two major clusters. The highly resistant landraces viz: Champe, Badda atte belonged to major cluster II, while landrace Khasa found in major cluster I. This result gives a clear picture that the landraces of the genetically similar forms a group together whereas the landraces belonging to same ecologies did not show any grouping among them.R genes  showed a significant association to the blast disease  among them.Genetic association analyses of the blast resistant genes were calculated using the GLM model of TASSLE5 software to assess the significant association with the blast disease scores. Among twenty eight markers used for association study, only five markers  for five  disease ; the assST) among the populations are listed in the ST estimate was highest between the highly resistant and susceptible while it was least between the highly resistant and moderately resistant populations. Based on the estimated value of fixation indices, it is indicated that there is weak population structure and they are not also isolated genetically from each other.An AMOVA analysis was conducted to assess the existence of genetic diversity within and between the populations. Based on the disease score, 161 landraces were divided into three populations: HR (21), MR (70) and S (70). Further, it was observed that highest diversity (94%) exists within population, whereas least (6%) between population ; Fig 4. R gene data. The PCoA analysis showed a scattered plot where the first two axes explained variations of 15.66% and 9.56%, accounting to total of 25.22% of genetic variation. The scatter plots have also clearly distributed the resistant landraces mostly in second and forth quadrants whereas moderately and susceptible landraces were distributed in all the four quadrants  or the combination of major R gene and major quantitative trait loci or minor gene interactions [A few landraces like Jaya possessed only five ractions .R genes intriguingly showed susceptible reaction (Score 7). Similarly, Biran landrace from Tripura state was found positive for nineteen R genes and displayed moderate resistance which corroborated the findings of Yadav et al. [R genes presence in these landraces or resistant breading down due to mutations occurred in the R genes or evolution of new pathogen races.On the other hand, some landraces such as Erava pandy, Basmati(s), Maichakca, Gujuri, Kajal champa, Maichakca and Red binni found to be carrying fourteen or more v et al. , where fv et al. ,36 whichR genes for blast resistance can be used in diverse elite genetic resources [R genes in elite germplasm to know the presence of blast resistance genes [R genes from 5 to 19. Likewise, the genetic frequency of the twelve major blast resistance genes varied from 0 to 100% in NRVs and 6 to 97% in North East and Eastern germplasm for nine blast resistance genes [The availability of linked molecular markers for detection of esources ,39,40,41ce genes ,43,44. Ice genes .Pish gene was detected in fourteen (12.42%) and twenty (8.69%) landraces using linked marker RM58118 and RM664, respectively. Similarly, Pit gene was reported in the ninety seven landraces with a gene frequency of 60.24%. A similar study was carried out by Yang et al. [Pit gene in 14 (3.9%) among 358 rice accessions. The result of Pi37 gene showed its gene frequency of 21.11% and 14.90%, respectively corresponding to the linked markers, RM302 and RM212. Interestingly, pi21 gene was found positive only in eight landraces exhibiting the gene frequency of 4.96%. The Pib gene was found in 43 landraces which was also detected in NRRI released varieties, North East and Eastern germplasm and Manipur rice accessions [Pi9 gene was observed in twenty five (15.52%) landraces. Similarly, it was detected in 15 NRVs, 2 North East and Eastern Indian rice [Pi2 gene was detected in twenty (12.42%) landraces whereas, it was observed in 14 accessions of the tested 358 accessions [Pid2 and Pi25 genes showed its presence in 151 (98.75%) and 47 (29.19%) landraces, respectively. The presence of Piz and Piz–t genes was demonstrated in 158 and 137 landraces, respectively, which is concurrent with the previous reports where these two genes conferred partial resistance to the tested entries [The presence of g et al.  which shwenty 8.6% landraccessions ,47). Thewenty 8.6% landracian rice . Likewiscessions . The Pidwenty 8.6% landrac entries .R gene, Pi33 and Pi36 showed its presence in 15 (9.31%) and 99 (61.4%) landraces, respectively. Similarly, Pi33 gene was observed in 77 (40.10%) of tested accessions [Pi56(t) gene. The Pi5 gene was detected in eighty three (51.55%) landraces whereas previous studies showed its presence in 60 landraces from Karnataka, 4 from Manipur and 26 NRVs [Pik locus on chromosome 11 possessing at least five genes, Pik, Pikm, Pikh, Pikp, and Piks [Pik, Pik–p and Pikm genes appeared in 161 (100%), 156 (96.89%) and 97 (60.24%) landraces, respectively. Likewise, these genes were found to be present in maximum frequency in the NRVs [Pik gene was detected in the majority of the accessions tested [Pikh isolated from Tetep variety was scored in one hundred eighteen (73.29%) landraces. Similarly, this gene was detected in fifty–six (70%) NRVs and in another study it was observed in 18 and 52 accessions [Pi1 gene was scored in sixty one (RM1233) and eighty two (RM224) landraces. In another study, it was observed in 39 landraces and 20 NRVs [Pb1, Pi65(t) and Pia genes in seventy nine (49.06%), eighty (55.27%) and one hundred twenty three (76.39%) landraces, respectively. The broad spectrum Pita/Pita–2 was present in eighty seven (54%) landraces. Similarly, other workers reported its presence in 32.50%, 19.29%, 6.25% and 27% accessions [R genes and disease reaction.The rice blast cessions . Seventy 26 NRVs ,9. The mand Piks . The Piks tested ,9. The bcessions ,48. The cessions ,49. IntrR genes could differentiate the entire landraces into two sub–populations (SG1 and SG2) with only two admixtures. The landraces belonged to the SG1 and SG2 were observed to be concomitant with the two major clusters IB and IA, respectively. Interestingly, the cluster analysis could discriminate all the resistant and susceptible landraces from 161 landraces tested which is similar to the study of Yadav et al. [In the current study, cluster analysis categorized the 161 landraces into two major sub–clusters. It was observed that the genetically similar landraces of each major cluster were distinguished with landraces of diverse ecologies. Population structure analysis based on twenty eight markers corresponding to twenty four v et al. . The PCoAn AMOVA analysis indicated the existence of maximum diversity 94%) within population and minimum (6%) diversity between populations which is similar to NRRI released varieties [4% withinR genes such as Pik–p, Pikh, Pi2, Pi1 and Pi37. This result could exhibit a robust marker by utilization of associated markers to assess the genetic diversity of rice blast resistance genes in diverse germplasm. However, our study did not described the complete association between the phenotypic reaction and resistance gene(s) which could be explained by addition of more blast R gene markers or these landraces could to be tested for identification of novel R genes/alleles or QTLs that can be utilized in rice breeding programs.Association mapping is an important strategy for the discovery of novel genes for important traits and identification of potential donor for their utilization in rice improvement . The preR gene(s) and identification of potential donors for their use in rice breeding. Additionally, the genotyping and phenotyping data of 161 landraces generated in this study could be quiet useful to identify the novel blast R gene(s) using association mapping in a precise way.The phenotypic screening and molecular characterization of blast resistance genes will help in identification of potential germplasm for leaf blast. Our results offered an outline of the genotypic diversity of Indian landraces representing nine major rice growing states of India with diverse ecologies. Besides, the precise screening of leaf blast for the identification of resistance genes in landraces along with the identified associated marker could be used for the selection of parental materials and the development of resistant breeding lines. Identification of resistant landraces from diverse ecologies will help in better utilization of these landraces as a donor for improvement of existing varieties with blast resistance. Potential landraces for blast resistance could be utilized for mapping to discover novel blast S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Low birth weight (LBW) remains a major public health problem in developing countries, including Nepal. This study was undertaken to examine the association between LBW and maternal factors and antenatal care service utilization, in rural Nepal, using data obtained for a capacity-building and text-messaging intervention, designed to enhance maternal and child health service utilization among pregnant women, in rural Nepal (“MATRI-SUMAN”). The study used a clustered randomized controlled design and was conducted during 2015–2016. We investigated maternal and antenatal care service utilization determinants of LBW, using a logistic regression model. Of the four hundred and two singleton babies, included in the present study, seventy-eight (19.4%) had an LBW (mean (SD), 2210.64 (212.47)) grams. It was found that Dalit caste/ethnicity, illiteracy, manual labor, a female baby, and having more than four family members were significantly positively associated with LBW. In addition, mothers who did not visit an antenatal care (ANC) unit, visited an ANC < 4 times, did not take iron and folic acid (IFA), de-worming tablets, and mothers that did not consume additional food, during pregnancy, were more likely to have an LBW baby, than their counterparts. The MATRI-SUMAN intervention and availability of a kitchen garden at home, were found to reduce the risk of LBW. Nepalese child survival policies and programs should pay attention to these maternal and antenatal care service utilization factors, while designating preventive strategies to improve child health outcomes.  Low birth weight (LBW) has been defined as a birth weight <2500 grams . In 2013Numerous direct and indirect factors have been associated with LBW ,16,17. SFew community-based studies have assessed the risk factors of LBW in Nepal. In a recent Nepalese study on nationally representative samples, it was reported that 12% of infants had an LBW, and that mothers who did not attend antenatal care units, did not take iron tablets during pregnancy, and resided in the westernmost region of the country were at a significant risk of having an LBW baby . Given tWe used data from a capacity-building and text-messaging intervention that was developed to enhance maternal and child health service utilization among pregnant women, in rural Nepal (“MATRI-SUMAN”), which was conducted using a clustered randomized controlled design. Pregnant women aged 15–49 years, in their second trimester (13–28 weeks of gestation), from fifty-two clusters (wards) of six village development committees (VDCs), from the Dhanusha district, were selected for the MATRI-SUMAN trial, using multistage cluster sampling method. During the first stage of the MATRI-SUMAN trial, two healthcare facilities were selected as a primary sampling unit. In the second stage, six of the twelve VDCs, in the catchment areas of these two healthcare facilities, were selected, using a stratified random sampling method. In the intervention group, Female Community Health Volunteers (FCHVs) were trained in capacity development skills through reinforcement training, supervision, and monitoring of maternal and child health care services. Pregnant women received periodic health promotion texts about maternal and child health (MCH) components, via a mobile messaging service, while participants in the control group received the usual care. Details of the study design have been reported in a previously published paper . A total of four hundred and twenty-six pregnant women aged 15–49 participated in the MATRI-SUMAN trial. Of the four hundred and twenty-six participants interviewed at baseline, four hundred and thirteen of them were approached at postnatal follow-up and the remainder were not available for follow-up . Additionally, we were not able to measure the birth weights, within the stipulated time, for eight cases and three mothers had still births, giving us a sample of four hundred and two mothers with live birth babies. Among the four hundred and two cases, seventy-eight newborn babies had low birth weight and the remaining three hundred and twenty-four babies had normal birth weight. We included four hundred and two live birth cases for the statistical analyses . The criA low birth weight (LBW) was defined as one <2500 grams. Birth weights were classified as LBW (<2500 grams) or (ii) normal (≥2500 grams or more), and birth weight was the dependent variable in the present study. Independent variables examined were chosen, as previously described . Age wasp-value ≤0.1 were entered in a final multivariate logistic regression model with a backward elimination and the analysis results with a p-value of <0.05 was considered statistically significant. Statistical analysis was performed using the Statistical Package for Social Sciences version 21.0 .The Chi-square test (unadjusted) was used to examine the association between the LBW and the categorical independent variables of interest. All factors, significant by the unadjusted analysis, were included in the multiple logistic regression analysis to control for confounding effects. Unadjusted and adjusted odds ratios, with their 95% confidence intervals (CI), were reported. Variables having The study protocol was approved by the Nepal Health Research Council , the Ethics Committee of the Institute of Medical Sciences, Banaras Hindu University, India  and District Public Health Office, Dhanusha, Nepal (Ref. 2245). Additional ethical approval for the data analysis was obtained from the Institutional Review Board of Janaki Medical College, Nepal. The study objectives and procedures were explained, and a written informed consent was taken from each participant, before the data collection process began. Personal identifiers were removed before data analysis.p < 0.0001, 95% CI: 745.94–940.55). Regarding ANC service utilization, more than half (59.7%) visited four or more ANCs, 58.5% consumed recommended doses of iron and folic acid tablets, 81.6% were properly immunized for tetanus and diphtheria, 81.3% consumed de-worming tablets, 84.3% got adequate rest and sleep, and about one in four received additional food during pregnancy. Utilization of the antenatal care services during pregnancy , consumption of recommended doses of iron and folic acid (IFA), immunization for tetanus and diphtheria, consumption of deworming tablets, adequate rest and sleep, and additional food intake during pregnancy, were significantly associated with LBW .Mothers from the Dalit caste/ethnicity group were four-times more likely ) to have an LBW baby than mothers from the Adibasi/Janajati and upper-caste groups. Similarly, illiterate mothers were eight-times more likely ) to have an LBW baby than literate mothers. Mothers that performed manual work were at greater risk ) of having an LBW baby than mothers with an agricultural or service/business/household occupation. Female children were twice more likely ) to be born with an LBW than male children. Mothers who had more than four family members had higher odds of having LBW babies ) than mothers who had four or fewer number of family members. Mothers in the MATRI-SUMAN intervention area were less likely ) to have an LBW baby than mothers not in the intervention area. Access to a kitchen garden reduced the risk ) of having an LBW baby. Mothers who had not visited ANCs were five-times more likely ) to have an LBW baby, and mothers who visited an ANC, less than four times, were three-times more likely ) to have an LBW baby than the mothers who visited four or more times. Not taking IFA tablets during pregnancy increased the risk ) of an LBW baby. Mothers who did not consume de-worming tablets during pregnancy were three-times more likely ) to have an LBW baby. Finally, not taking additional food during pregnancy was found to increase the risk ) of delivering an LBW baby. We found a wide range of maternal factors, that is, the Dalit caste/ethnicity, illiteracy, occupation as a manual laborer, having four or more family members, and birth of a female child were significantly positively associated with an LBW, and that those with a kitchen garden and those who resided in the intervention area of the MATRI-SUMAN trial area were less likely to have LBW babies. In addition, lack of utilization of antenatal care services (as indicated by no ANC visit), fewer than four ANC visits, not consuming the recommended doses of IFA and de-worming tablets, and not consuming additional food during pregnancy, increased the risk of an LBW baby.Our study revealed that babies born to mothers from the Dalit caste/ethnic group were at a greater risk of having an LBW baby than those from the Adibasi/Janajati and upper caste/ethnic group. Several previous studies have also observed relations between ethnicity and LBW ,27,28. TIn the present study, maternal occupation as a manual laborer, four or more family members in a household, and birth of female child were found to increase the risk of LBW, while access to a kitchen garden or residing in the MATRI-SUMAN intervention area reduced the risk of LBW. Furthermore, we found that physical hard work was found to increase the risk of LBW babies, which concurred with the findings of two Indian studies and one Nepalese studies, which showed hard physical work during pregnancy increased the risk of LBW babies ,40,41. IPrevious study reported male babies are less likely to have LBW, compared to their female counterparts because the male chromosome confers advantages in the determination of birth weight . InteresWe also found that a lack of utilization of essential prenatal care services was linked to LBW. A number of previous studies are in agreement with our finding of a positive association between LBW and poor utilization of prenatal care services, as indicated by low numbers of ANC visits ,32,33,53Previous studies have reported that mothers employed in the agricultural sector  have no The present study was conducted to examine an important child healthcare problem faced by many developing countries and had a high response rate. However, our findings should be understood in the light of its pitfalls. First, data collection was conducted in a cross-sectional manner, which prevented investigation of causal relationships. Second, information on socioeconomic and maternal factors was self-reported, and thus, might be subject to bias. Third, although calibrated instruments were used, the different instruments were used to measure the facility-born and home-born (newborn) babies, and this may have influenced their birth weights. Fourth, this study was conducted in a small rural area of Nepal on a relatively small cohort, so special precautions should be taken to generalize our study findings. Nonetheless, a number of clusters from a rural community of Nepal was enrolled in the study, which we feel has policy implications.Child survival policies and programs in Nepal aimed at improving child health outcomes should pay more attention to maternal and antenatal care service utilization factors. In our opinion bespoke maternal and child health programs, such as MATRI-SUMAN, a capacity-building and text-messaging intervention designed to improve maternal and child health outcomes, should be promoted. We suggest future studies to explore the determinants of small-for-gestational-age and pre-term births, and the economic feasibility of MATRI-SUMAN interventions in Nepal and other south Asian countries.


N = 547) we examined the relation between maternal discipline and sensitivity in the preschool period and the occurrence of attention-deficit hyperactivity disorder (ADHD) symptoms in middle childhood, taking into account pre-existing child attention and executive function (EF) problems, and oppositional defiant disorder (ODD) symptom comorbidity. Maternal parenting was observed during a ‘do not touch task’ (positive and negative discipline) and a teaching task (sensitivity) at age 3. Parents reported on the occurrence of ADHD and ODD symptoms at age 8 using the Conners’ Parent Rating Scale. Attention and executive function problems were assessed using parent questionnaires at age 4. Important covariates such as harsh discipline and maternal depression were also taken into account. Maternal sensitivity significantly predicted later ADHD symptoms beyond pre-existing child attention and EF problems, and comorbid ODD symptoms. However, maternal negative and positive discipline did not significantly predict later ADHD symptoms over and above these covariates. This study demonstrates the importance of maternal sensitivity in the etiology of core ADHD symptoms above and beyond pre-existing child attention and EF problems, and comorbid ODD symptoms.In this longitudinal population-based cohort ( Attention-deficit hyperactivity disorder (ADHD) is the most common behavioral disorder in children and tends to persistently impact multiple areas of development into adulthood  and child  characteristics and how it serves as a ‘filter’ through which other family variables  influence child functioning, while taking into account the developmental stage of the child and the family  childhood has been demonstrated in several longitudinal population-based studies. For example, observed maternal restrictiveness in infancy is related to more impulsive behavior in later childhood . However, by controlling for initial levels of ADHD symptoms, the direction of effects becomes clearer and the contribution of parenting behaviors over and above existing child symptoms can be explored. For this reason, in the current study two well-known precursors of ADHD, EF and attention problems, are taken into account Barkley . These pThe aim of the current study is to examine the association between observed maternal sensitivity and discipline in the preschool period and ADHD symptoms in middle childhood in a non-clinical population. To study the influence of early parenting on later ADHD symptoms, early precursors of ADHD, specifically EF and attention problems, and comorbid ODD symptoms are taken into account. Additionally, we control for harsh discipline, to differentiate the association between normal variation in negative discipline and ADHD from effects of extreme or abusive discipline  to 3 (very much true). Higher scores indicate more ADHD-related behaviors. The scores on the ADHD subscale (12 items), for example ‘short attention span’ and Oppositional subscale (6 items), for example ‘loses temper’, were used for this study. Composite scores for ADHD and ODD were positively skewed and square root transformed to approach normality. In the current study sample, high internal consistencies were found for the total scale, ADHD subscale, and Oppositional subscale .The occurrence of ADHD and ODD symptoms was assessed with parent-report at age 8 using the Dutch version of the Conners’ Parent Rating Scale-Revised: Short Form  during a 2-min task (“Do not touch”) in which the mother prohibited the child to touch or play with a set of attractive toys. Maternal verbal and physical discipline strategies were coded in different categories: Commands, Support, and Physical Obstruction/interference . An overall maternal positive discipline score (i.e. Support and Supportive Presence) and an overall maternal negative discipline score  were created by standardizing and summing the scores. The positive discipline score was negatively skewed and the negative discipline score was positively skewed, both were transformed (resp. square root transformation and log transformation) to approach normality.Maternal discipline was observed at a laboratory visit around age 3 (n = 53) and for the etch-a-sketch task 0.79 (n = 55). An overall sensitivity score was created by reversing the Intrusiveness scales, standardizing the scores on the subscales (Z-score), and creating an average over both subscales and both tasks. Correlations between supportive presence and intrusiveness within and across teaching tasks ranged from r = 0.16 to 0.39.Maternal sensitivity was observed at age 3 during two 3- to 4-min teaching tasks (“Building a tower” and “Etch-a-Sketch”) that were too difficult to complete for the child independently. The “Building a tower” task contains a rod and a set of blocks that can be placed on the rod, but only in a particular order, as the blocks are like puzzle pieces that fit together in a certain way. The “Etch-a-sketch” is a drawing board with two knobs: if the child turns the left knob, a vertical line appears; if the child turns the right knob, a horizontal line appears. A maze was taped on the drawing board and the child needed to go from the start of the maze to the center of the maze, by alternately turning the two knobs. Mothers were instructed to help their child as usual when introducing a new toy. Maternal sensitivity was coded with the revised Erickson 7-point rating scales for Supportive Presence and Intrusiveness .The Dutch version of the Brief Symptom Inventory (Derogatis α = 0.51).Mothers reported on their use of harsh discipline at the 3-year follow-up with the Parent-Child Conflict Tactics Scales .To explore the effect of maternal parenting beyond pre-existing child attention and EF problems on later child ADHD, these potential precursors were added in a separate step in the regression model. Attention problems were assessed via parent-report on the five items of the attention syndrome scale of the Dutch Child Behavior Checklist 1.5–5 at age 3 .Executive function problems were assessed with the Dutch version of the Behavior Rating Inventory of Executive Function-Preschool Version at age 4 , maternal depressive symptoms (n = 24) maternal harsh discipline (n = 27), child attention problems (n = 28), and child EF problems (n = 32) we generated 20 imputed datasets by multiple imputation. Based on between-group comparisons for the five aforementioned variables with missing data and Little’s MCAR Test, we found that these date were missing (completely) at random. Analyses conducted with the imputed datasets (N = 547) yielded similar results compared to the analyses with the complete dataset (N = 515). Results with the imputed dataset are presented unless otherwise indicated.Due to missing data on maternal educational level  predicted ADHD symptoms at age 8, independent of pre-existing child attention and EF problems, comorbid ODD symptoms, maternal depressive symptoms, and background variables. Each model was composed of four blocks. In the first block, all covariates were entered. In the second block, child attention and EF problems were added. In the third block, comorbid ODD symptoms were added. In the final block, maternal parenting  was entered. Third, a regression analysis including all three parenting variables in the final block (and covariates in the previous blocks) was performed, to isolate the impact of the individual elements of parenting. Lastly, sex differences in the association between observed parenting and ADHD were explored, by examining interactions between parenting and child sex on ADHD symptomatology.First, bivariate associations among maternal parenting, child ADHD symptoms, covariates, and background variables were explored with Pearson’s correlations and t(545) = 4.45, p < 0.001. Furthermore, mothers used more positive discipline strategies toward girls than toward boys, t(545) = −3.26, p = 0.001, and fewer negative discipline strategies toward girls than toward boys, t(545) = 4.24, p < 0.001. Similarly, mothers were more sensitive toward girls than toward boys, t(545) = −2.08, p = 0.038. Maternal age was not associated with maternal parenting or child ADHD symptoms. Highly educated mothers used more positive discipline than mothers with a lower educational level, t(541) = −2.19, p = 0.029, and highly educated mothers were more sensitive than mothers with a lower educational level, t(541) = −4.16, p < 0.001.A sample description is presented in Table The bivariate correlations among the main variables and covariates are presented in Table Maternal sensitivity correlated with more positive discipline and less negative discipline at child age 3 years. There was a negative correlation between maternal positive and negative discipline. Furthermore, all maternal parenting measures were significantly correlated with attention and EF problems, and ADHD symptoms in the expected direction.p = 0.06) but not significant, after adjustment for the same covariates, pre-existing and comorbid symptoms . In the model with transformed variables (see Table p = 0.06).All regression models were rerun with untransformed variables to check whether transformation or non-normality affected the results. All analyses yielded similar results, except for the regression model with negative discipline as predictor of ADHD symptoms, controlled for covariates. In the model with untransformed variables, negative discipline significantly predicted ADHD symptoms  and repeated follow-up measurements of ADHD , allow for greater insight into the transactional relationship between changes in parenting and changes in ADHD symptoms over time. Also, with regard to potential precursors of ADHD, we included attention and EF problems, but not symptoms of hyperactivity-impulsivity. In addition, we could not control for a shared genetic risk, effects of parental ADHD symptomatology or parental EF problems, as alternative factors explaining variation both in parenting behavior and in child ADHD symptoms (Deater-Deckard Our findings illustrate the unique contribution of maternal sensitivity, beyond pre-existing attention and EF problems, and ODD comorbidity, in the development of attention and hyperactivity problems in children. Although a modest association between maternal sensitivity and child ADHD symptoms was found, this finding further increases the understanding of the etiology of core ADHD symptoms. Also, it provides an avenue for the incorporation of parental sensitivity into psychoeducation or parental guidance programs aimed at ADHD symptomatology.