The Burden of Respiratory Syncytial Virus in Children Under 2 Years of Age in a Rural Community in Maharashtra, India.

BACKGROUND: Globally, respiratory syncytial virus (RSV) is a common cause of acute lower tract infection (LRTI) in children younger than 2 years of age, but there are scant population-based studies on the burden of RSV illness in rural communities and no community studies in preterm infants. METHODS: Active surveillance of LRTI was performed in the community and hospital setting for the population of 93 tribal villages in Melghat, Central India, over 4 respiratory seasons. A nasopharyngeal swab was obtained from cases presenting as a severe LRTI for molecular analysis of respiratory pathogens including RSVA and B. RESULTS: High rates of RSV-associated LRTI were found in preterm and term infants beyond 6 months of age, extending into the second year of life. Community severe RSV LRTI rates for 0-11 months of age was 22.4 (18.6-27.0)/1000 child-years (CY) and the hospital-associated rate was 14.1 (11.1-17.8)/1000 CY. For preterm infants, these rates were 26.2 (17.8-38.5)/1000 CY and 12.6 (7.2-22.0)/1000 CY. Comparable rates in the first 6 months were 15.9 (11.8-21.4)/1000 CY and 12.9 (9.3-18.0)/1000 CY in term infants and 26.3 (15.4-45.0)/1000 CY and 10.1 (4.2-24.2)/1000 CY for preterms. The single RSV B season had higher incidences of RSV LRTI in every age group than the 2 RSV A seasons in both preterm and term infants. There were 11 deaths, all term infants. CONCLUSIONS: Studies restricted to the healthcare settings significantly underestimate the burden of RSV LRTI and preterm and term infants have comparable burdens of disease in this rural community.

Mortality From Respiratory Syncytial Virus in Children Under 2 Years of Age: A Prospective Community Cohort Study in Rural Maharashtra, India.

BACKGROUND: Although respiratory syncytial virus (RSV) is the most important viral cause of lower respiratory tract infection deaths in infants, there are few data on infant community deaths caused by RSV. METHODS: This was an active surveillance of children younger than 2 years of age in 93 villages, 5 primary health centers, and 3 hospitals serving these villages. Village health workers and counselors at the health facilities monitored all lower respiratory tract infections (LRTIs) in consented subjects. Children with severe, or very severe LRTIs and all who died, had nasopharyngeal swabs collected for detection of RSV by molecular methods. RESULTS: In the 12 134 subjects, there were 2064 episodes of severe LRTIs and 1732 of very severe LRTIs, of which 271 and 195, respectively, had RSV. Fifteen of 16 (94%) children with RSV died of LRTIs, 14 in the community and 1 in the hospital. The case fatality ratios for severe RSV LRTIs in the first 6 months of life were 3/52 (7.1%) and 1/36 (2.8%) in the community and hospital, respectively. Of those with very severe LRTIs in the community, 17.6% died. There were no very severe RSV LRTI hospital deaths. The adjusted RSV LRTI mortality rates ranged from 1.0 to 3.0/1000 child-years (CY) overall, and 2.0 to 6.1/1000 CY, accounting for 20% of the LRTI deaths and 10% of the postneonatal infant mortality. CONCLUSIONS: Community deaths from RSV account for the majority of RSV LRTI deaths, and efforts at prevention should be preferentially directed at populations where access to care is limited.

Population-based otoscopic and audiometric assessment of a birth cohort recruited for a pneumococcal vaccine trial 15-18 years earlier: a protocol.

INTRODUCTION: A cohort of 12 000 children in the Philippines who had enrolled in a 2000-2004 (current ages 16 to 20 years) Phase 3 11-valent pneumococcal conjugate vaccine for the prevention of radiographically confirmed pneumonia are now being asked to participate in a separate study (expected completion date September 2021) to assess the cohort's current long-term audiometric and otologic status. This new study would allow assessments of the utility of the pneumococcal vaccine in conferring its protective effects on the long-term sequelae of otitis media (OM), if any. Lack of trained local healthcare providers in otolaryngology/audiology and testing equipment in Bohol, Philippines, necessitates the development of a distinct methodology that would lead to meaningful data analysis. METHODS AND ANALYSIS: Reliable data collection and transfer are achieved by a US otolaryngologist/audiologist team training local nurses on all procedures in a didactic and hands-on process. An assortment of portable otolaryngologic and audiologic equipment suitable for field testing has been acquired, including an operating otoscope (Welch-Allyn), a video-otoscope (JedMed), a tympanometer with distortion product otoacoustic emission measurements (Path Sentiero) and a screening audiometer (HearScreen). Data will then be uploaded to a Research Electronic Data Capture database in the USA.Tympanometric and audiologic data will be codified through separate conventional algorithms. A team of paediatric otolaryngology advanced practice providers (APPs) have been trained and validated in interpreting video otoscopy. The protocol for classification of diagnostic outcome variables based on video otoscopy and tympanometry has been developed and is being used by APPs to evaluate all otoscopy data. ETHICS AND DISSEMINATION: The study was approved by the Research Institute of Tropical Medicine, Alabang, Manila, Philippines, and the institutional review board and the Colorado Multiple Institutional Review Board of the University of Colorado School of Medicine, Aurora, Colorado, USA.Research results will be made available to children and their caregivers with abnormal audiologic outcomes, the funders and other researchers. TRIAL REGISTRATION NUMBER: ISRCTN 62323832; Post-results.

Trivalent influenza vaccination randomized control trial of pregnant women and adverse fetal outcomes.

INTRODUCTION: The purpose of this study was to evaluate the association of influenza vaccine during pregnancy and adverse fetal outcomes. Preventing fetal death, low birth weight, small for gestational age birth and preterm birth are important potential effects of antenatal maternal influenza immunization for which there are conflicting data. MATERIALS AND METHODS: A double-blind, randomized, placebo-controlled clinical trial of trivalent inactivated influenza vaccine was conducted in South Africa from March 2011 until after the 2012 influenza season when the infants born had reached the age of 24 weeks. Mothers were administered the vaccine or placebo during pregnancy at a gestation of 20 to 36 weeks. A comparison of rates of fetal death, low birth weight, small for gestational age birth, and preterm birth, between vaccinated and placebo groups was made. Fetal outcome differences between the groups were measured using Student's t-tests, vaccine efficacy with 95% confidence intervals, and Poisson regression for incidence rates. All analyses except fetal death excluded mothers who were administered vaccine or placebo after 34 weeks gestational age. RESULTS: There were 2116 HIV-uninfected pregnant women age 18 to 38 years in the trial; 2005 infants were born to mothers where vaccine or placebo had been administered ≥ 14 days prior to delivery, and there were 6 miscarriages and 23 stillbirths. There was no significant vaccine efficacy (with [95% confidence interval]) on fetal death (-21.2% [-150.8, 41.4]), low birth weight (-11.1% [-42.3, 12.5]), small for gestational age birth (-9.9% [-35.6, 11.0]), or preterm birth (-21.3% [-60.5, 8.3]). Neither was vaccine efficacy demonstrated when the analysis was restricted to infants of mothers who were exposed to an influenza season (1832 outcomes available). CONCLUSION: We did not find a beneficial effect of trivalent inactivated influenza vaccine during pregnancy on adverse fetal outcomes.

Otitis media related hearing loss in Indonesian school children.

OBJECTIVE: There are scant recent studies from low middle-income countries that investigate the impact of otitis media (OM) on hearing loss (HL) in school children. METHODS: This was a prospective epidemiological survey conducted by otorhinolaryngologists and audiologists in a sample of 7005 public school children (6-15 years) from 6 urban and rural sub-districts, in Indonesia. Children with otoscopic abnormalities or who failed a hearing-screening test conducted at school, underwent diagnostic audiometry and tympanometry. RESULTS: OM was detected in 172 children (2.5%), acute otitis media - AOM (17%), otitis media with effusion - OME (15%), and chronic suppurative otitis media - CSOM (67%). The overall rate of HL in the school children was 181/10,000, which was almost three-fold higher in rural (273/10,000) than urban areas 92.6/10,000. OME accounted for much of the mild HL, while CSOM accounted for most of the moderate HL. There was a significantly higher rate of OM related HL in rural areas (116.2/10,000), than in urban areas (47.4/10,000), p = 0.002. OM related disabling HL was found at a rate of 44.2/10,000, mostly due to CSOM (37.1/10,000). CONCLUSION: Otitis media contributed to 57% of all HL in school children, and posed a significant burden on Indonesian school children. Most of the disabling HL was due to CSOM. Efforts to find these children and offer ear and hearing care are important.

Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study.

BACKGROUND: We have previously estimated that respiratory syncytial virus (RSV) was associated with 22% of all episodes of (severe) acute lower respiratory infection (ALRI) resulting in 55 000 to 199 000 deaths in children younger than 5 years in 2005. In the past 5 years, major research activity on RSV has yielded substantial new data from developing countries. With a considerably expanded dataset from a large international collaboration, we aimed to estimate the global incidence, hospital admission rate, and mortality from RSV-ALRI episodes in young children in 2015. METHODS: We estimated the incidence and hospital admission rate of RSV-associated ALRI (RSV-ALRI) in children younger than 5 years stratified by age and World Bank income regions from a systematic review of studies published between Jan 1, 1995, and Dec 31, 2016, and unpublished data from 76 high quality population-based studies. We estimated the RSV-ALRI incidence for 132 developing countries using a risk factor-based model and 2015 population estimates. We estimated the in-hospital RSV-ALRI mortality by combining in-hospital case fatality ratios with hospital admission estimates from hospital-based (published and unpublished) studies. We also estimated overall RSV-ALRI mortality by identifying studies reporting monthly data for ALRI mortality in the community and RSV activity. FINDINGS: We estimated that globally in 2015, 33·1 million (uncertainty range [UR] 21·6-50·3) episodes of RSV-ALRI, resulted in about 3·2 million (2·7-3·8) hospital admissions, and 59 600 (48 000-74 500) in-hospital deaths in children younger than 5 years. In children younger than 6 months, 1·4 million (UR 1·2-1·7) hospital admissions, and 27 300 (UR 20 700-36 200) in-hospital deaths were due to RSV-ALRI. We also estimated that the overall RSV-ALRI mortality could be as high as 118 200 (UR 94 600-149 400). Incidence and mortality varied substantially from year to year in any given population. INTERPRETATION: Globally, RSV is a common cause of childhood ALRI and a major cause of hospital admissions in young children, resulting in a substantial burden on health-care services. About 45% of hospital admissions and in-hospital deaths due to RSV-ALRI occur in children younger than 6 months. An effective maternal RSV vaccine or monoclonal antibody could have a substantial effect on disease burden in this age group. FUNDING: The Bill & Melinda Gates Foundation.

Effectiveness of Palivizumab in High-risk Infants and Children: A Propensity Score Weighted Regression Analysis.

BACKGROUND: Infants with premature birth ≤35 weeks gestational age, chronic lung disease of prematurity and congenital heart disease are at an increased risk for lower respiratory tract infections and hospitalization from respiratory syncytial virus (RSV), which has been shown in randomized trials to be prevented by palivizumab. However, palivizumab effectiveness (PE) has not been studied in a large clinical setting. METHODS: A multicenter study among high-risk US and Canadian children younger than 24 months hospitalized with lower respiratory tract infection and whose nasopharyngeal aspirates were tested for human metapneumovirus (HMPV) and RSV were the subjects of the trial. We conducted a test-negative case-control study in these subjects to determine PE. We used an inverse propensity score weighted (IPSW) multiple logistic regression model to adjust PE. RESULTS: Palivizumab was used in 434 (51%) of 849 eligible children. RSV was identified in 403 (47%) children. The unadjusted PE was 43% [95% confidence interval (CI), 34%-51%)]. After IPSW adjustment, the adjusted PE was 58% (95% CI, 43%-69%). Palivizumab prevented intensive care unit admissions (PE, 62%; 95% CI, 35%-78%). PE for 29-35 weeks gestational age and ≤6 months of chronologic age without chronic lung disease of prematurity or congenital heart disease was 74% (95% CI, 56%-85%). CONCLUSIONS: Using a test-negative case-control design with RSV molecular detection, palivizumab is shown to prevent RSV hospitalizations and intensive care unit admissions in high-risk infants.

Cost of management of severe pneumonia in young children: systematic analysis.

BACKGROUND: Childhood pneumonia is a major cause of childhood illness and the second leading cause of child death globally. Understanding the costs associated with the management of childhood pneumonia is essential for resource allocation and priority setting for child health. METHODS: We conducted a systematic review to identify studies reporting data on the cost of management of pneumonia in children younger than 5 years old. We collected unpublished cost data on non-severe, severe and very severe pneumonia through collaboration with an international working group. We extracted data on cost per episode, duration of hospital stay and unit cost of interventions for the management of pneumonia. The mean (95% confidence interval, CI) and median (interquartile range, IQR) treatment costs were estimated and reported where appropriate. RESULTS: We identified 24 published studies eligible for inclusion and supplemented these with data from 10 unpublished studies. The 34 studies included in the cost analysis contained data on more than 95 000 children with pneumonia from both low- and-middle income countries (LMIC) and high-income countries (HIC) covering all 6 WHO regions. The total cost (per episode) for management of severe pneumonia was US$ 4.3 (95% CI 1.5-8.7), US$ 51.7 (95% CI 17.4-91.0) and US$ 242.7 (95% CI 153.6-341.4)-559.4 (95% CI 268.9-886.3) in community, out-patient facilities and different levels of hospital in-patient settings in LMIC. Direct medical cost for severe pneumonia in hospital inpatient settings was estimated to be 26.6%-115.8% of patients' monthly household income in LMIC. The mean direct non-medical cost and indirect cost for severe pneumonia management accounted for 0.5-31% of weekly household income. The mean length of stay (LOS) in hospital for children with severe pneumonia was 5.8 (IQR 5.3-6.4) and 7.7 (IQR 5.5-9.9) days in LMIC and HIC respectively for these children. CONCLUSION: This is the most comprehensive review to date of cost data from studies on the management of childhood pneumonia and these data should be helpful for health services planning and priority setting by national programmes and international agencies.

Otitis Media and Its Sequelae in Kenyan Schoolchildren.

BACKGROUND: The goal of this study was to obtain representative Kenyan data on the point prevalence of acute otitis media (AOM) and its sequelae (otitis media with effusion [OME] and chronic suppurative otitis media [CSOM]), a major cause of preventable hearing loss in children in developing countries. In Africa, there are limited studies on the prevalence of AOM and its sequelae in children. METHODS: Study subjects were children aged 2 to 15 years and were enrolled from randomly selected preprimary and primary schools. After parental or guardian consent, subjects had a questionnaire administered, otoscopy and tympanometry were done, and audiometry was performed on those with ear problems detected on these examinations. RESULTS: A total of 9825 (75%) children was from rural schools. The prevalence of CSOM was 15 of 1000, OME was 15 of 1000, and AOM was 7 of 1000 children. Rural Rift Valley schoolchildren had the highest prevalence of CSOM (24 of 1000) compared with other regions (12 of 1000; P < .0001). Ear discharge occurred before 3.5 years in 50% of 901 children with ear discharge. A history of ear discharge was associated with abnormal tympanograms (odds ratio [OR], 11.9-19.2) and mild-to-severe hearing loss (OR, 21.6-38.6), even in children without ear disease (OR, 10.7-24.4). CONCLUSIONS: The burden of AOM sequelae in Kenyan preschool and schoolchildren is significant, and it occurs mostly in the first 4 years of life. By preventing early recurrent AOM, pneumococcal vaccination might partly avert nonreversible sequelae.

Intravenous acyclovir and renal dysfunction in children: a matched case control study.

OBJECTIVES: A cluster of children receiving intravenous (IV) acyclovir for meningoencephalitis developed acute renal failure in April-May 2008, which prompted a retrospective case-control study to determine the rate of and risk factors for acute nephrotoxicity during IV acyclovir treatment in children. STUDY DESIGN: The percentage decrease in glomerular filtration rate in children receiving IV acyclovir who had ≥ 1 creatinine measurement after acyclovir initiation from October 2006 to January 2009 was classified as renal risk, injury, or failure according to modified Pediatric Risk Injury, Failure, Loss, End-Stage Renal Disease criteria. Univariate and multivariate matched analyses were conducted to identify risk factors contributing to nephrotoxicity. RESULTS: In the selected study group, renal dysfunction was seen in 131 of 373 (35%) treatment courses studied: 81 of 373 (22%) risk, 36 of 373 (9.7%) injury, and 14 of 373 (3.8%) failure. Most renal dysfunction occurred within 48 hours of the initiation of acyclovir. Renal function returned to the normal range but not to baseline in most cases during the follow-up period. Risk factors for renal dysfunction included acyclovir dose >15 mg/kg (OR 3.81, 95% CI 1.55-9.37) for risk; cumulative exposure greater than calculated cumulative exposure based on 500 mg/m(2)/dose (OR 6.00, 95% CI 1.95-18.46) for injury; and age >8 years (OR 21.5, 95% CI 2.2, >1000) and ceftriaxone coadministration (OR 19.3, 95% CI 1.8, >1000) for failure. CONCLUSIONS: Nephrotoxicity associated with IV acyclovir is common and necessitates renal function monitoring. Risk factors include greater dose, older age, and concomitant ceftriaxone administration. Outside the neonatal period, renal dysfunction may be minimized by dosing IV acyclovir below thresholds associated with nephrotoxicity (ie, ≤ 500 mg/m(2)/dose or ≤ 15 mg/kg/dose), particularly in older patients.

Otitis media in indonesian urban and rural school children.

BACKGROUND: Although the epidemiology of otitis media is well-known in industrialized countries, the extent of otitis media in developing Asian countries, especially in south East Asia is not well studied. METHODS: To define the burden of otitis media and its sequelae in children 6-15 years of age, we enrolled elementary and junior high school children in 6 areas in rural and urban Indonesia. Randomly selected schools and classrooms were selected. All children were administered a questionnaire and had ear examinations, pneumatic otoscopy and screening audiometry. Children with any abnormality on examination or with a relevant history underwent diagnostic audiometry and tympanometry, if indicated. RESULTS: Of the 7005 children studied, 116 had chronic suppurative otitis media (CSOM), 30 had acute otitis media and 26 had otitis media with effusion. 2.7% of rural children had CSOM compared with 0.7% of urban children (P < 0.0001). The rates per 1000 of CSOM in rural Bali and Bandung were significantly higher (75 and 25, respectively) than in the rest of Indonesia (P < 0.05). In rural Bali, the rate per 1000 children of inactive CSOM was 63 in 6- to 9-year-old children, compared with 37 in children aged 13-15 years. Concomitantly, the rates of tympanosclerosis were 7 and 26/1000, respectively, in these age groups. CONCLUSIONS: In Indonesia, the prevalence of CSOM is relatively high with most disease occurring in rural areas. The high rates in rural Bali with early progression to tympanosclerosis suggest a significant burden of potentially vaccine preventable illness.

Incidence of acute mastoiditis in Colorado children in the pneumococcal conjugate vaccine era.

BACKGROUND: Acute otitis media is among the most common reasons young children seek medical care, with Streptococcus pneumoniae the most common pathogen. Despite introduction of heptavalent pneumococcal conjugate vaccine (PCV7) in 2000, recent experience suggests an increase in complications of acute otitis media, particularly acute mastoiditis. METHODS: We performed a retrospective review of acute mastoiditis in children from 1999 to 2008 using inpatient data from the Colorado Hospital Association and the Children's Hospital Colorado. The study included patients with documentation of acute mastoiditis or mastoidectomy and excluded those with chronic mastoiditis, chronic otitis media or cholesteatoma. RESULTS: The annual incidence of acute mastoiditis in children <2 years/100,000 population was 11.0 in 2001 before decreasing to 4.6 in 2002 and 4.5 in 2003. The incidence then increased to 12.0 in 2008 (total N = 242). The proportion of S. pneumoniae isolates nonsusceptible to penicillin increased from 0% (0/16) between 1999 and 2004 to 38% (5/13) between 2005 and 2008 (P = 0.03). CONCLUSIONS: The incidence of acute mastoiditis in Colorado children <2 years of age exhibited a dynamic pattern from 1999 to 2008: a significant decline early after introduction of PCV7 that paralleled initial vaccine uptake, followed by an increase in subsequent years to pre-PCV7 levels. Replacement with non-PCV7 pneumococcal serotypes and increased pneumococcal antibiotic resistance may be responsible for the increase in incidence to pre-PCV7 rates. Surveillance of mastoiditis incidence, pathogen distribution and resistance patterns following introduction of 13-valent PCV is warranted.

Pathogen chip for respiratory tract infections.

Determining the viral etiology of respiratory tract infections (RTI) has been limited for the most part to specific primer PCR-based methods due to their increased sensitivity and specificity compared to other methods, such as tissue culture. However, specific primer approaches have limited the ability to fully understand the diversity of infecting pathogens. A pathogen chip system (PathChip), developed at the Genome Institute of Singapore (GIS), using a random-tagged PCR coupled to a chip with over 170,000 probes, has the potential to recognize all known human viral pathogens. We tested 290 nasal wash specimens from Filipino children <2 years of age with respiratory tract infections using culture and 3 PCR methods-EraGen, Luminex, and the GIS PathChip. The PathChip had good diagnostic accuracy, ranging from 85.9% (95% confidence interval [CI], 81.3 to 89.7%) for rhinovirus/enteroviruses to 98.6% (95% CI, 96.5 to 99.6%) for PIV 2, compared to the other methods and additionally identified a number of viruses not detected by these methods.

Global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis.

BACKGROUND: The global burden of disease attributable to seasonal influenza virus in children is unknown. We aimed to estimate the global incidence of and mortality from lower respiratory infections associated with influenza in children younger than 5 years. METHODS: We estimated the incidence of influenza episodes, influenza-associated acute lower respiratory infections (ALRI), and influenza-associated severe ALRI in children younger than 5 years, stratified by age, with data from a systematic review of studies published between Jan 1, 1995, and Oct 31, 2010, and 16 unpublished population-based studies. We applied these incidence estimates to global population estimates for 2008 to calculate estimates for that year. We estimated possible bounds for influenza-associated ALRI mortality by combining incidence estimates with case fatality ratios from hospital-based reports and identifying studies with population-based data for influenza seasonality and monthly ALRI mortality. FINDINGS: We identified 43 suitable studies, with data for around 8 million children. We estimated that, in 2008, 90 million (95% CI 49-162 million) new cases of influenza (data from nine studies), 20 million (13-32 million) cases of influenza-associated ALRI (13% of all cases of paediatric ALRI; data from six studies), and 1 million (1-2 million) cases of influenza-associated severe ALRI (7% of cases of all severe paediatric ALRI; data from 39 studies) occurred worldwide in children younger than 5 years. We estimated there were 28,000-111,500 deaths in children younger than 5 years attributable to influenza-associated ALRI in 2008, with 99% of these deaths occurring in developing countries. Incidence and mortality varied substantially from year to year in any one setting. INTERPRETATION: Influenza is a common pathogen identified in children with ALRI and results in a substantial burden on health services worldwide. Sufficient data to precisely estimate the role of influenza in childhood mortality from ALRI are not available. FUNDING: WHO; Bill & Melinda Gates Foundation.

Using the Phenogen website for 'in silico' analysis of morphine-induced analgesia: identifying candidate genes.

The identification of genes that contribute to polygenic (complex) behavioral phenotypes is a key goal of current genetic research. One approach to this goal is to combine gene expression information with genetic information, i.e. to map chromosomal regions that regulate gene expression levels. This approach has been termed 'genetical genomics', and, when used in conjunction with the identification of genomic regions (QTLs) that regulate the complex physiological trait under investigation, provides a strong basis for candidate gene discovery. In this paper, we describe the implementation of the genetical genomic/phenotypic approach to identify candidate genes for sensitivity to the analgesic effect of morphine in BXD recombinant inbred mice. Our analysis was performed 'in silico', using an online interactive resource called PhenoGen (http://phenogen.ucdenver.edu). We describe in detail the use of this resource, which identified a set of candidate genes, some of whose products regulate the cellular localization and activity of the mu opiate receptor. The results demonstrate how PhenoGen can be used to identify a novel set of genes that can be further investigated for their potential role in pain, morphine analgesia and/or morphine tolerance.

Quantitative trait locus mapping for acute functional tolerance to ethanol in the L x S recombinant inbred panel.

BACKGROUND: Acute functional tolerance (AFT) develops shortly after ethanol administration, and is determined as the change in brain or blood ethanol concentration (BEC) measured at 2 behavioral or physiological endpoints. Acute functional tolerance studies in some rodent strains support a long-held hypothesis that more sensitive strains develop more within-session tolerance. We used the new, 74-strain L x S recombinant inbred (RI) panel, developed from inbred long-sleep (ILS) and inbred short-sleep (ISS) strains, to revisit this hypothesis and to map quantitative trait loci (QTLs) for AFT. We report replication of QTL regions reported by earlier studies of AFT and preliminary application of a coarse single nucleotide polymorphism map analysis to limit QTL intervals for subsequent candidate gene hypotheses. METHODS: Acute functional tolerance was assayed using a test of ataxia: loss and regain of balance on a stationary wooden dowel. Following an initial dose of 1.75 g/kg, BEC was measured at initial loss (BEC(0)) and regain of balance (BEC(1)). A second injection (2.0 g/kg) was administered and blood taken at the second regain of balance (BEC(2)). Acute functional tolerance was calculated as a difference score in 2 ways: (1) between BEC at the 2 successive regains of balance (AFT(1)), or (2) as the difference in BEC at final regain and at initial loss of balance (AFT(2)). We mapped QTLs for BEC(0), a measure of initial sensitivity, and both AFT scores. RESULTS: All 4 parental strains (LS, SS, ILS, and ISS) developed tolerance, replicating previous published reports. There were significant sex effects for 3 of these strains. The L x S panel showed a 128-fold range in tolerance, with a few strains showing negative tolerance (sensitization). The ISS surpassed the next highest RI strain by 55% and was more than 4 times greater than SS. Heritability estimates for both AFT measures were close to 0.25 for both sexes. One significant QTL accounting for approximately 18% of phenotypic variance (V(P)), on chromosome 12 (AFT(1)), and 1 suggestive QTL (16% V(P)), on chromosome 16 (AFT(2)), were identified. These QTLs replicated regions reported in other studies. A multiple QTL model incorporating the effects of all significant interacting QTLs was developed, explaining almost 60% of V(P). The chromosome 12 region was further investigated by haplotype analysis, which identified many nonpolymorphic regions within the confidence interval, and possible candidate genes in the polymorphic regions. CONCLUSIONS: Both SS and ISS developed greater AFT, assessed by both methods, than LS and ILS; this difference was significant in virtually all sex by strain comparisons. In the L x S RI, there was no correlation between initial sensitivity, measured by BEC at initial loss of balance, and either measure of AFT, on a stationary dowel. These results indicate that in this model system, initial sensitivity does not predict tolerance. Several QTLs for tolerance were identified; candidates in the narrowed chromosome 12 region, which has been reported in 2 other mapping studies, merit additional study.

QTL mapping for low-dose ethanol activation in the LXS recombinant inbred strains.

BACKGROUND: Most mouse quantitative trait loci (QTLs) for behavioral traits have been mapped using populations of mice derived from C57BL/6J (B6) and DBA/2J (D2). It is also important to identify QTLs for behavior in populations derived from other progenitors. We report results from QTL mapping for low-dose (ethanol) locomotor activation (LDA) using the recently developed LXS recombinant inbred (RI) strains, derived from Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS) progenitors. The LXS RI panel has additional genetic variation, and greater power due to a larger number of strains, compared with other RI panels and strain crosses. METHODS: Mice were tested using a 3-day protocol in which activity levels were monitored for 15 minutes each day. On day 1, baseline activity was recorded; on day 2, mice were injected with saline before testing; and on day 3, mice were injected with 1.8 g/kg ethanol and tested. RESULTS: Several suggestive QTLs were found, on chromosomes 2, 3, 4, 7, 8, 12, and 13; 3 of these QTLs were sex-specific. CONCLUSIONS: Two apparently novel LDA QTLs were identified, on chromosomes 4 and 8. The other QTLs appear to replicate previously identified LDA QTLs. These replicated QTLs will be pursued in subsequent studies designed to identify candidate genes.

Confirmation and fine mapping of ethanol sensitivity quantitative trait loci, and candidate gene testing in the LXS recombinant inbred mice.

In previous studies, we have mapped quantitative trait loci (QTLs) for hypnotic sensitivity to ethanol using a small recombinant inbred (RI) panel and a large F(2) backcross. Alcohol sensitivity is a major predictor of long-term risk for alcoholism. We remapped hypnotic sensitivity using a new set of 75 RI strains, the LXS, derived from Inbred Long Sleep and Inbred Short Sleep strains. We expected to improve mapping resolution in the QTL regions and to identify novel QTLs for loss of the righting reflex due to ethanol. We used three common mapping algorithms (R/qtl, QTL Cartographer, and WebQTL) to map QTLs in the LXS, and we compared the results. Most mapping studies use only a single algorithm, an approach that may result in failure to identify minor QTLs. We confirmed most of our previously reported QTLs, although one major QTL from earlier work (Lore2) failed to replicate, possibly because it represented multiple linked genes separated by recombination in the RI strains. We also report narrowed confidence intervals, based on mapping with a new genetic resource of more than 4000 polymorphic single-nucleotide polymorphism markers. These narrowed confidence intervals will facilitate candidate gene identification and assessment of overlap with human regions specifying risk for alcoholism. Finally, we present an approach for using these RI strains to assess evidence for candidate genes in the narrowed intervals, and we apply this method to a strong candidate, the serotonin transporter.

Genetics of body weight in the LXS recombinant inbred mouse strains.

This is the first phenotypic analysis of 75 new recombinant inbred (RI) strains derived from ILS and ISS progenitors. We analyzed body weight in two independent cohorts of female mice at various ages and in males at 60 days. Body weight is a complex trait which has been mapped in numerous crosses in rodents. The LXS RI strains displayed a large range of weights, transgressing those of the inbred progenitors, supporting the utility of this large panel for mapping traits not selected in the progenitors. Numerous QTLs for body weight mapped in single- and multilocus scans. We assessed replication between these and previously reported QTLs based on overlapping confidence intervals of published QTLs for body weight at 60 days and used meta-analyses to determine combined p values for three QTL regions located on Chromosomes 4, 5, and 11. Strain distribution patterns of microsatellite marker genotypes, weight, and other phenotypes are available on WebQTL (http://www.webqtl.org/search.html ) and allow genetic mapping of any heritable quantitative phenotype measured in these strains. We report one such analysis, correlating brain and body weights. Large reference panels of RI strains, such as the LXS, are invaluable for identifying genetic correlations, GXE (Gene X Environment) interactions, and replicating previously identified QTLs.

Confirmation and genetic dissection of a major quantitative trait locus for alcohol preference drinking.

OBJECTIVE: In previous work, we created congenic strains that carry the DBA/2IBG (D2) region for alcohol preference on chromosome 2, on an otherwise C57BL/6IBG (B6) background. Here, we report construction and testing of interval-specific congenic recombinant strains (ISCRSs) for the purpose of narrowing the quantitative trait loci (QTL) interval. METHODS: ISCRSs were derived by identifying mice that carry recombination events in the D2 interval, during the backcrossing for congenics. Recombinant mice were backcrossed to B6, and progeny that carry the reduced chromosome 2 region were tested for its effect on the alcohol preference phenotype. RESULTS: We developed multiple ISCR strains, which spanned the QTL interval. Three of these showed the D2 phenotype of reduced alcohol consumption. The overlap of two of these strains reduced the QTL interval from 66.8 to 3.5 Mb. A third positive ISCRS suggests the possibility of a second, linked QTL. CONCLUSIONS: Use of ISCRSs can narrow a QTL region to a few Mb. This reduced interval size will facilitate identification of candidate genes, through bioinformatics, gene expression, and DNA sequencing strategies. Potential difficulties, including reduced power as a result of variable phenotypes or small effect size, are discussed.